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Chemical and biological investigation of the Antarctic red alga delisea pulchra

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Title:
Chemical and biological investigation of the Antarctic red alga delisea pulchra
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English
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Nandiraju, Santhisree
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University of South Florida
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Subjects / Keywords:
Antifouling
Secondary metabolites
Dimer
Furanones
Pulchralide
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Our interest in the red alga Delisea pulchra (=D.fimbriata) (Greville) Montagne 1844 (Rhodophyceae, Bonnemaisoniales, Bonnemaisoniaceace) was stimulated by its activity in the biosssays done at Wyeth Pharmaceuticals. Halogenated compounds from D. pulchra interfere with Gram-negative bacterial signaling systems, affect the growth of Gram-positive bacteria, inhibit quorum sensing and swarming motility of marine bacteria (inhibit bacterial communication). They also inhibit surface colonization in marine bacteria and exhibit antifouling properties against barnacle larvae and macroalgal gametes. Chemical investigation of D.pulchra collected near Palmer Station, Antarctica yielded three new dimeric halogenated furanones, pulchralide A-C (41-43), along with previously reported fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23) and halogenated ketone 40.
Thesis:
Thesis (MSci)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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by Santhisree Nandiraju.
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Title from PDF of title page.
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Document formatted into pages; contains 99 pages.

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oclc - 62791335
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Chemical and Biological Investigat ion of the Antarctic Red Alga Delisea pulchra by Santhisree Nandiraju A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Bill J. Baker, Ph.D. Edward Turos, Ph.D. Kirpal Bisht, Ph.D. Date of Approval: July 9 th 2004 Keywords: furanones, Pulchralide, dimer, secondary metabolites, antifouling @ Copyright 2004, Santhisree Nandiraju

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DEDICATION This thesis is dedicated to my beloved mother Mrs. Lakshmi Gouri, who motivated me to pursue this degree and accomplish my goals. I present this work as a token of appreciation and gratitude for a ll her efforts. I would also like to dedicate this thesis to my sisters for their encouragement and inspiration at all times.

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ACKNOWLEDGMENTS I wish to express my sincerest thanks to my adviser Dr. Bill J. Baker, for his wise counsel, viable guidance and constant encouragement and for ensuring the successful culmination of this thesis. I would like to thank Dr. James B. McClin tock and Dr. Charles D. Amsler at the University of Alabama, Birmingham for their help in the field work as well as in the laboratory. I would like to thank Dr. Steven Mullen at the University of Illinois, Urbana-Champaign, for the mass spectral data. I wish to thank Dr. Maya P. Singh from Wyeth Pharmaceuticals and Dr. Fred Valeriote from Ford hospital for their bioactivity data. I would like to acknowledge my committee members, Dr. Kirpal Bisht and Dr. Edward Turos for their encouragement and as sistance. I am thankf ul to Dr. Bakers students for their timely help during my resear ch work. Last but not the least I wish to acknowledge my friends and roommates for th e lighter moments, I have shared with them.

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i TABLE OF CONTENTS LIST OF FIGURES ...iii LIST OF TABLES .....vi LIST OF SCHEMES vii LIST OF ABBREVATIONS ...viii ABSTRACT.....x CHAPTER 1. INTRODUCTION 1.1. Marine Natural Products...1 1.2. Antarctic Ecology and Chemistry.....9 CHAPTER 2. CHEMICAL INVESTIGATI ON OF THE ANTARCTIC RED ALGA DELISEA PULCHRA 2.1. Introduction 2.2. Extraction and Isolation of Secondary Metabolites....................17 2.3. Characterization of Fimbrolide ( 21)...19 2.4. Characterization of Acetoxyfimbrolide ( 22 )...22 2.5. Characterization of Hydroxyfimbrolide ( 23)..25 2.6. Character ization of Halogenated ketone 40 2.7. Characterization of Pulchralide A ( 41 )...31 2.8. Characterization of Pulchralide B ( 42 )...39

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ii 2.9. Characterization of Pulchralide C ( 43 )...44 CHAPTER 3. DISCUSSION.49 3.1. Biological Impor tance of Halogenated Metabolites of Delisea pulchra 3.2. Origin of Halogenated Metabolites of Delisea pulchra ..51 3.3. Ecological Impor tance of Halogenated Metabolites of Delisea pulchra CHAPTER 4. EXPERIMENTAL..54 4.1. General Experimental Procedure 4.2. Bioassay of Pure Compounds.....56 4.3. Isolation of Secondary Metabolites from Delisea pulchra .....58 4.2.1. Spectral data of Fimbrolide ( 21 )..........59 4.2.2. Spectral data of Acetoxyfimbrolide ( 22).60 4.2.3. Spectral data of Hydroxyfimbrolide ( 23).61 4.2.4. Spectral data of Halogenated ketone 40 ...62 4.2.5. Spectral data of Pulchralide A ( 41)..63 4.2.6. Spectral data of Pulchralide B ( 42)..64 4.2.7. Spectral data of Pulchralide C ( 43)..65 REFERENCES..66 APPENDICES...73

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iii LIST OF FIGURES Figure 1. The Antarctic Red alga Delisea pulchra ..15 Figure 2. 1 H NMR spectrum of fimbrolide ( 21) (500 MHz, CDCl 3 ).19 Figure 3. 13 C NMR spectrum of fimbrolide ( 21) (125 MHz, CDCl 3 )...20 Figure 4. 1 H Chemical shift values of H-6 in Z and E isomers of fimbrolide ( 21) ..21 Figure 5. 1 H NMR spectrum of acetoxyfimbrolide ( 22) (250 MHz, CDCl 3 )...22 Figure 6. 13 C NMR spectrum of acetoxyfimbrolide ( 22) (62.5 MHz, CDCl 3 ).23 Figure 7. Acetoxyfimbrolide ( 22).24 Figure 8. 1 H NMR spectrum of hydroxyfimbrolide ( 23) (250 MHz, CDCl 3 )..26 Figure 9. 13 C NMR spectrum of hydroxylfimbrolide ( 23) (125 MHz, CDCl 3 )....26 Figure 10. Hydroxyfimbrolide ( 23)...27 Figure 11. 1 H NMR spectrum of halogenated ketone 40 (250 MHz, CDCl 3 )..28 Figure 12. 13 C NMR spectrum of halogenated ketone 40 (62.5 MHz, CDCl 3 ).29 Figure 13. Halogenated ketone 40.........30 Figure 14. 1 H NMR spectrum of pulchralide A (41) (500 MHz, CDCl 3 )..32 Figure 15. 13 C NMR spectrum of pulchralide A (41) (125 MHz, CDCl 3 ).32 Figure 16. DEPT-135 spectrum of pulchralide A ( 41) (125 MHz, CDCl 3 )..33 Figure 17. gCOSY spectrum of pulchralide A ( 41 ) (500 MHz, CDCl 3 )..34 Figure 18. 1 H1 H COSY correlations of pulchralide A ( 41).34

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iv Figure 19. gHMQC spectrum of pulchralide A ( 41 ) (500MHz, CDCl 3 )...35 Figure 20. gHMBC spectrum of pulchralide A ( 41 ) (500 MHz, CDCl3) Figure 21. KEY HMBC correlations determined in pulchralide A ( 41 )...36 Figure 22. Perspective view of X-ray crystal structure of pulchralide A ( 41)..37 Figure 23. Sterochemical assi gnments of pulchralide A ( 41 )...38 Figure 24. 1 H NMR spectrum of pulchralide B (42) (500 MHz CDCl 3 )..39 Figure 25. 13 C NMR spectrum of pulchralide B (42) (125 MHz, CDCl 3 ) Figure 26. DEPT-135 spectrum of pulchralide B ( 42) (125 MHz CDCl 3 )...41 Figure 27. gHMBC spectrum of pulchralide B ( 42) (500 MHz, CDCl 3 )...42 Figure 28. Key HMBC correla tion of pulchralide B ( 42).42 Figure 29. Pulchralide B C 2 dimer and pulchralide B meso dimer..43 Figure 30. 1 H NMR spectrum of pulchralide C (43) (500 MHz, CDCl 3 ).45 Figure 31. 13 C NMR spectrum of pulchralide B (43) (125 MHz CDCl 3 ) Figure 32. 1 H1 H COSY spectrum of pulchralide C ( 43) (500 MHz, CDCl 3 )..................46 Figure 33. Key 1 H1 H COSY correlation of pulchralide C (27)...47 Figure 34. stereo chemical as signments of pulchralide C ( 27 ).48 Figure 35. IR spectrum of fimbrolide ( 21)74 Figure 36. UV spectrum of fimbrolide ( 22)...74 Figure 37. LREI Mass spectrum of fimbrolide (23 )..75 Figure 38. IR spectrum of acetoxyfimbrolide ( 22 ) Figure 39. UV spectrum of acetoxyfimbrolide ( 22 )..76 Figure 40. LREI Mass spectrum of Acetoxyfimbrolide ( 22).76

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v Figure 41. IR spectrum of hydroxyfimbrolide ( 23 )...77 Figure 42. UV spectrum of hydroxyfimbrolide ( 23 ).77 Figure 43. IR spectrum of halogenated ketone 40 .78 Figure 44. UV spectrum of halogenated ketone 40 ...78 Figure 45. LREI Mass spectru m of halogenated ketone 40......79 Figure 46. IR spectrum of pulchralide A (41) Figure 47. UV spectrum of pulchralide A ( 41)..80 Figure 48. LREI Mass spectrum of pulchralide A ( 41).80 Figure 49. gCOSY spectrum of pulchrlalide B ( 42 ) (500 MHz, CDCl 3 )..81 Figure 50. gHMQC spectrum of pulchralide B (42) (500 MHz, CDCl 3 )..81 Figure 51. IR spectrum of pulchralide B (42) Figure 52. UV spectrum of pulchralide B ( 42)..82 Figure 53. LRESI Mass spectrum of pulchralide B ( 42)..83 Figure 54. gHMQC spectrum of pulchralide C ( 43 ) (500 MHz CDCl 3 )...83 Figure 55. gHMBC spectrum of pulchralide C ( 43 ) (500 MHz, CDCl 3 )..84 Figure 56. IR spectrum of pulchralide C (43) Figure 57. UV spectrum of pulchralide C ( 43)..85 Figure 58. LREI Mass spectrum of pulchralide C ( 43).85

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vi LIST OF TABLES Table 1. NMR data of pulchralide A ( 41) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz)..37 Table 2. NMR data of pulchralide B ( 42) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz)..43 Table 3. NMR data of pulchralide C ( 43) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz)..47 Table 4. Antimicrobial activity of pure compounds (100 g/disk) using the disk diffusion assay (Zone of I nhibition in mm)...50

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vii LIST OF SCHEMES Scheme1. Isolation of fimbrolide (21 ), acetoxyfimbrolide ( 22 ), hydroxyfimbrolide ( 23), halogenated ketone 40 pulchralide A ( 41), pulchralide B ( 42) and pulchralide C ( 43)

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viii LIST OF ABBREVATIONS [ ] specific rotation = 100 / lc CDCl3 deuterated chloroform CH2Cl2 dichloromethane chemical shifts DEPT distortionless enhancem ent by polarization transfer EtOAc ethylacetate the molar extinction coefficient in UV spectroscopy gCOSY gradient correlation spectroscopy gHSQC gradient heteronuclear single quantum correlation gHMBC gradient heteronuclear multiple bond connectivity HREIMS high resolution electron impact mass spectrometry or spectrum HRESIMS high resolution electrospray ionization mass spectrometry HPLC high performance liquid chromatography IR infrared J coupling constant n JCH n-bond hydrogen to carbon correlation (n = 2, 3 or 4) n JHH n-bond hydrogen to hydrogen correl ation (n = 2, 3 or 4) LREIMS low resolution electron impact mass spectrometry or spectrum

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ix LRESIMS low resolution electrospray ionization mass spectrometry max the wavelength at which maximum absorption occurs MeOH methanol m / z mass/charge for mass spectrometry NMR nuclear magnetic resonance UV ultraviolet

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x Chemical and Biological Investigation of the Antarctic Red Alga Delisea pulchra Santhisree Nandiraju ABSTRACT Our interest in the red alga Delisea pulchra (= D. fimbriata ) (Greville) Montagne 1844 (Rhodophyceae, Bonnemaisoniales, Bonnemaisoniaceae) was stimulated by its activity in the bioassays done at Wyeth Pharma ceuticals. Halogenated compounds from D. pulchra interfere with Gram-negative bacterial signa ling systems, affect the growth of Grampositive bacteria, inhibit quorum sensing an d swarming motility of marine bacteria (inhibit bacterial communication). They also inhibit surface colonization in marine bacteria and exhibit antifouling properties against barnacle larvae and macroalgal gametes. 2 Chemical investigation of D. pulchra collected near Palmer Station, Antarctica yielded three new dimeric halogenated furanones, pulchralides A-C ( 41-43), along with previously reported fimbrolide ( 21), Acetoxyfimbrolide ( 22), hydroxyfimbrolide ( 23) and halogenated ketone 40. The reported Compounds were characterized by comparison of their 1 H and 13 C NMR data with that previously published. Pulchralide A-C were characterized by both 1D ( 1 H NMR, 13 C NMR, DEPT, 1 H1 H COSY) and 2D (gHMQC, gHMBC) NMR techniques, supported by HREIMS/HRESIMS data. The absolute stereochemistry of Pulchralide A was dete rmined by a single crystal X-ray analysis.

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xi Significant antimicrobial activity was observed in acetoxyfimbrolide ( 22) and hydroxyfimbrolide (23), where as pulcharlide A (41) and fimbrolide ( 21) were weakly active. References 1. De Nys, R.; Steinberg, P. D.; Willemsen, P.; Dworjnyn, S. A.; Gabelish, C. L.; King, R. J. Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays, Biofouling 1995 8, 259-271.

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1 Chapter 1. INTRODUCTION 1.1. Marine Natural Products If indeed it is true that all life orig inated in the oceans, some organisms remained in the sea and others escaped ont o the land. All have captured ou r imagination and now in a retro-evolutionary sense, chemists are turni ng their gaze back from land to their watery origins. This has become possible with the advent of scuba diving equipment and deep sea submissible collection facilities. The recent chromatographic techniques and the wide-spread use of high-field Nuclear Magnetic Resonance (NMR) spectrometers have meant that the complete structural elucidation is now possible on small amounts of material. Because of these developments chemists have all found excitement and adventure in the discovery of secrets of organisms hidden beneath the sea. 1 The oceans cover more than 70% of the earths surface and the origin of all form s of life is supposed to have occurred in the sea. 2 Thus it is fair enough to conclu de that the biodiversity of the sea could be far greater than any other terrestrial ecosystem. Evidence coming from recent research on marine ecosystems like th e deep sea floor and the coral reefs does affirm it suggesting that their biodiversity is mu ch higher than that of tropical rain forests, a terrestrial ecosystem renown for its enormous biodiversity. 3

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2 Marine natural products are of enormous importance to chemists and pharmacologists, who are keen to discover bi ologically active lead molecules. Marine natu ral products chemistry has evolved from being the source of a handful of chemical curiosities to one of the most productive areas of natural products research. 1,4-9 The advent of sophisticated chromatographic techniques such as High Performance Liquid Chromatography (HPLC), Medium Pressure Liquid Chromatography (MPLC), Counter Curre nt Chromatography (CCC) and gel filtration, high-field NMR tec hniques such as Heteronuclear Multiple Bond Correlation (HMBC), Heteronuclear Multiple Quantum Correlation (HMQC), Correlation Spectroscopy (COSY) and Nuclea r Overhauser Enhancement Spectroscopy (NOESY) and mass spectrometric techniques coupled with X-ray crystallography have led to the structural eluc idation of substantially more complex and diverse natural products. This has led to an enhanced intere st in the isolation of biologically active natural products. Marine organisms have pr ovided natural product chemists with a rich source of unusual secondary metabolites. 10 It is the novelty and complexity of the compounds discovered from marine sources that assures the success of research in this area. There are many marine natural products that have no c ounterparts in the terrestrial world. Oceans are the reservoirs of many complex and unusual secondary metabolites. One of the most striking aspects of the field is its interdisciplinary nature; marine natural products chemists routinely collaborate with industrial pharmacologists, with marine biologists and ecologists whose interest and involvement is essen tial for meaningful progress in the field.

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3 Many marine organisms are soft bodied and mo ve slowly whereas some of them have a sedentary life style. Moreover, some of them do not have physical armaments like hard external shells or spicules. Such organisms need to defend themselves from predators, as they are vulnerable to predation and comp etition. Many marine invertebrates such as corals, sponges and ascidians (tunicates) use highly evolved chemical compounds for purposes such as reproduction, communicati on, and protection against predation, infection, and competition. 11 These bioactive chemical co mpounds may have antibiotic, anti-inflammatory, antiviral, cytotoxic, antitumor or antifungal properties. Some of these marine organisms have the ability to synthe size their own defensiv e secondary metabolite chemicals via de novo biosynthesis. Other organisms ha ve been found to derive their defensive chemicals by a symbiotic relationshi p or simply from their dietary sources. 12 With great biodiversity in th e marine realm, marine orga nisms produce a wide array of secondary metabolites with broad structural diversity. 12,13 Therefore, the likelihood of finding a bioactive molecule that would be a potential remedy to dreaded diseases like cancer or AIDS, from the marine realm is far greater. Interest on the part of chemists has been two fold: natural product chemists have probed marine organi sms as a source of new and unusual molecules, while synthetic chemists have followed by targeting these novel structures for development of new an alogues and new synthe tic methodologies and strategies. 14,15 More recent studies of marine or ganisms have focused on their potential applications, particularly to the treatment of human diseases and control of agricultural pests. 16 The original literature that has ama ssed over the years has been reviewed topically by Scheuer. 5-9,15,17-20 The task of tracking and ca taloging the steady stream of

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fascinating new structures has been done by John Faulkner in an annual survey. 10 More natural products have been reported from sponges than from any other marine invertebrate phylum and many of the most promising pharmaceuticals and agents for cell biological research were isolated from sponges. 21 The first isolation of a secondary metabolite from a marine organism was tyrian purple (6,6-dibromoindigotin) (1) from a marine mollusk, 1,22 identified in 1901. Tyrian purple was also the first marine natural product to be used for commercial applications. NHHNOBrBrO Tyrian purple (1) In the last few decades attention of many natural product drug discovery programs has been focused on the oceans and several drug candidates coming from different phyla of marine organisms have progressed into advanced phases of clinical trials. 23 Metabolites of the phylum porifera account for almost 50% of the natural products reported from marine invertebrates. 13 An unusual acetylinic fatty acid derivative taurospongin A (2) was isolated from an Okinawan sponge, Hippospongia sp. which inhibited both DNA polymerase and HIV reverse transcriptase enzymes. 24 HO3SNHOOOOO(CH2)13 Taurospongin A (2) 4

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Discodermalide (3), a polyketide isolated from marine sponge Discodermia dissolute, was found to be a potent antitumor agent. 25 It induces tublin polymerization similar to taxol. Structure-activity studies of discodermalide 26 resulted in synthetic analogues and derivates, which showed greater promise and versatility than taxol, a current anticancer drug derived from Pacific yew tree Taxus bravifolia. 27 OOHOHHOOHCONH2O Discodermalide (3) Papuamine (4), an antifungal pentacyclic alkaloid was isolated from a thin, red encrusting sponge Haliclona sp. Papuamine is formally derivable from a C22 unbranched hydrocarbon and 1,3-diaminopropane and inhibits the growth of the fungus Trichophyton mentagrophytes. 28 HNHNHHHH Papuamine (4) Soft corals also provide an excellent source for bioactive marine natural products. Eleutherobin (5), a novel natural product isolated from an Indopacific marine soft coral is believed to be as potent as taxol. 29 It inhibits microtubule depolymerization and thereby prevents division of cancer cells, which is the same mechanism of action as taxol. 30 5

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HHOOOOOAcOOHOHNNO Eleutherobin (5) Marine prostaglandins, first discovered in a gorgonian 31 have been isolated from other invertebrates and from red algae. Punaglandins, 32 halogenated antitumor eicosanoids, were isolated from the octocoral Telesto riisei. Punaglandin 3 (6) is characterized by C-12 oxygen and an unprecedented C-10 chlorine group and inhibits L1210 leukemia cell proliferation with an IC50 value of 0.02 g/mL. 32,33 OOClOAcOAcOHO Punaglandin 3 (6) Ecteinascidins, isolated from the colonial ascidian Ecteinascidia turbinata were not only cytotoxic but also found to be DNA interactive agents. 34,35 Ecteinascidin 743 (7) was very selective against breast cancer and melanoma and has advanced into phase II clinical trials. 36 6

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NHHOOOONNOOOOHOHHOHSHO Ecteinascdin 743 (7) Of the alkaloid metabolites reported from tunicates, eudistomins are the most interesting from a chemical point of view. The oxathiazepine-bearing eudistomin C (8), has been isolated from a colonial tunicate Eudistoma olivaceum. 37 The oxathiazepine-bearing eudistomins are potent antiviral agents. 37 NBrHOSOH2NNH Eudistomin C (8) Dolastatin 10 (9) isolated from the sea hare Dolabella auricularia, is a short polypeptide containing unique amino acids and showing microtubulin stabilization properties. 38 Dolastatins have also been isolated from a nudibranch and a cyanobacterium. Dolastatin analogues are in clinical trials. 39 7

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NOHNNOHOONNHOOHHHSN Dolastatin 10 (9) Isolated from the bryozoan Bugula neritina, bryostatin 1 (10) is one of the first drug candidates from the ocean to proceed into clinical trial stages. 40 Bryostatin 1, a complex polyketide which inhibits protein kinase C and thereby prevents cancer, is in Phase II clinical trails. 41 OOHOOOHHOOOOHOHOOOOOHHOHO Bryostatin 1 (10) 8

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9 1.2. Antarctic Ecology and Chemistry A few decades ago it was conventional wisdom that competition and predation among the marine species are most intense in tropical waters 12 and as a result the chemical ecology of the marine organisms dwelling in the tropical waters received more attention in drug discovery programs. 12 However, recent research on the organisms of the Antarctic benthos suggests that they are indeed th reatened by invertebrate predators and competitors such that they have evolved chemical defenses to ward them off. 12 The physical environment on the Antarctic benthos has been stable for more than 20 million years 42 a period sufficient for the biologi cally accommodated ecosystem whereby predation and competition are dominant forces determining the species composition and distribution. This would have given ample time to facilitate the evolution of the biogenetic pathways leading to bioactive secondary metabolites. 13 Also; the continent has been isolated from its lower latitude neighbo rs even longer. The factors such as physical stability and isolation are important for gene tic divergence and this has resulted in high levels of endemism in Antarctica. 13 An expedition undertaken to study the sea floor of McMurdo Sound Antarctica in 1980 revealed that the benthos under the ice is rich in marine life and is dominated by a dynamic co mmunity of sponges, soft corals, molluscs, tunicates and echinoderms. 42

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Many of these organisms are immobile and cannot move to less densely populated regions if the area they live in becomes over-grown with competitors. Hence, it is apparent that either these sessile organisms do not have predators or that they have some kind of a defense. Studies on bottom-dwellers of Antarctica showed that they do have many predators. These include swarms of the voracious Paramoera antractica, a one-centimeter long crustacean resembling a shrimp, and dense populations of sea stars. 12 It can be concluded that these organisms produce defensive chemicals to protect themselves from predators. Among chemical investigations done on Antarctic organisms to date, several bioactive molecules have been characterized. There are nearly 200 different secondary metabolites described from Antarctic organisms. 13 Sponges are the dominant macro-invertebrates found on the Antarctic benthos. The sponge Dendrilla membranosa produces defensive chemicals membranolide (11) and 9,11-dihydrogracilin A (12) which showed mild activity against Bacillus subtilus. 43,44 COOMeOOOHOAcOAcHH Membranolide (11) 9,10-dihydrogracillin (12) The Antarctic green sponge Latrunculia apicalis from McMurdo Sound has been shown to elaborate a series of iminoquinone pigments called discorhabdins typified by discorhabdin C (13). 44,45 In addition to significant feeding deterrence activity they are 10

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antibiotic and cytotoxic. 46 Variolin A (14), an unusual cytotoxic alkaloid, has been isolated from a bright red sponge Kirkpartrickia variolosa. 47 HNOBrBrO+NHHNNNNNNO-NH2HONH2++ Discorhabdin C (13) Variolin A (14) Leucetta leptorhapsis, the Antarctic rubber sponge, produces the acetogenin, rhapsamine (15), which bears the unusual 1,3-diaminoglycerol group. In addition to cytotoxicity, rhapsamine has antipredatory activity. 48 H2NHNOHNHH2NOH Rhapsamine (15) Sponges of McMurdo Sound Antarctica are subject to predation by sea stars, and it is considered to be a dominant ecological factor that might drive the production of defense chemicals. The bright yellow Antarctic sponge, Isodictiya erinacea, which lacks physical defenses (spicules and mucus) is one of the several chemically defended sponges in the region. Erebusinone (16), a yellow pigment found in the sponge, showed molt inhibition in crustaceans, 49 a possible strategy of chemical defense. 11

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Further investigation of I. erinacea showed the presence of purines and a nucleoside metabolite erinacin (17), which showed cytotoxicity. 50 NHOOHONNNHHNNHONH2HOO Erebusinone (16) Erinacin (17) The pteropod Cliona antarctica is a shell-less pelagic mollusc which blooms in each austral summer in McMurdo Sound and has an intriguing relationship with the amphipod Hyperiella dilatata, where the amphipod positions the mollusc on its dorsum and defends itself from predatory fish utilizing the defense chemicals of the mollusc. A bioassay guided fractionation of the mollusc afforded the feeding deterrent pteroenone (18). 51 OOH Pteroenone (18) Perknaster fuscus is the major predator of sponges, regulating the abundance of the potentially space-dominating sponge Mycale acerata. 42 Aqueous extracts from body wall tissues of P. fuscus showed cytotoxic activity in a fertilized echinoderm egg assay employing the Antarctic sea urchin Sterechinus neumayeri. 52 The cytotoxicity likely 12

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results from a novel tetrahydroisoquinoline alkaloid, fuscusine (19) which has been isolated from the tissues of body wall of P. fuscus. 53 NHNHNH2NHHOOH Fuscusine (19) The Antarctic nudibranch Tritoniella belli sequesters the secondary metabolite chimyl alcohol (20) from its diet, an Antarctic soft coral Clavularia frankliniana. Chimyl alcohol (20) was found to cause feeding inhibition in the omnivorous Antarctic sea star Odontaster validus. 54 HOO(CH2)15CH3OH Chimyl alcohol (20) The benthos of Antarctica has many organisms that need to be investigated for their chemistry and bioactivity. In addition there are many parts of underwater Antarctic Peninsula that have never been explored. Considering the already evident significant variations in chemistry of the species that have been studied from the regions of McMurdo Sound and Palmer Station, a comprehensive chemical and bioactivity investigation of these species in various parts of Antarctica is of considerable interest. 13

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14 Chapter 2. CHEMICAL INVESTIGATIO N OF THE ANTARCTIC RED ALGA DELISEA PULCHRA 2.1. Introduction Marine macroalgae have been a remark able source of chemical diversity, responsible for roughly 20% of compounds repo rted from marine sources. Compounds from macroalgae are characteristic of their biological origin: red algae (Rhodophyceae) produce largely polyhalogenated monoterpenes, sesquiterpenes and acetogenins. Brown algae (Phaeophyceae) produce primarily diterpenes but are also rich in phlorotannins and known for their prenylated quinones or hyd roquinones, and green algae (Chlorophyceae) produce sesquiand diterpenes and are known for their 1,4-dialdehydes. 10,55,56 10,55,56 Marine red alga from the family Bonnemaisoniaceae have been shown to produce a wide range of halogenated metabolites, 57 including butenones, 58 pyranones, 59 acetones, acrylic and acetic acids, 60 octenones 61 and from the genus, Delisea halogenated furanones. 62 Delisea species are somewhat special when compared to many other algal species, in their apparent ability to stave off colonization by common epiphytes and also to be a food source which is generally not pr eferred by obligate herbivores. 63

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Delisea pulchra (=D. fimbriata) (Figure 1) (Greville) Montagne 1844 (Rhodophyceae, Bonnemaisoniales, Bonnemaisoniaceae) has been the focus of prior ecological and bioactivity studies. 62 Halogenated compounds from D. pulchra interfere with Gram-negative bacteria signaling systems, affect the growth of Gram-positive bacteria, inhibit quorum sensing and swarming motility of marine bacteria (inhibit bacterial communication). They also inhibit surface colonization in marine bacteria and exhibit antifouling properties against barnacle larvae and macroalgal gametes. 63 15 Figure1. The Antarctic red alga Delisea pulchra (photograph courtesy: www.seaweed.ie ; John Huisman).

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A series of 2 ( 5H )-furanones, with structures 21 through 39 were isolated from D. pulchra collected over a wide geographic range. 64 OOBrR1OOR1R2R334 OH CH2I OCH3_______________________________R1 R2 R335 OH OCH3 CH2I36 OAc CH3 OCH337 OAc OCH3 CH338 OAc CHBr2 OCH339 OAc OCH3 CHBr2________________________________ 21 H H Br 22 OAc H Br 23 OH H Br 24 H Br H 25 OH Br H 26 OAc Br H 27 OH H IR1 R2 R3 28 OH I H 29 OAc H I 30 OH Br Br 31 OAc Br Br 32 OH H Cl 33 OH Cl HR2R3Br 16

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17 2.2. Extraction and Isolation of Secondary Metabolites The collection and extraction of Delisea pulchra from Palmer Station, Antarctica in 2001, yielded seven compounds, fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23), halogenated ketone 40, pulchralide A (41), pulchralide B (42) and pulchralide C (43). D. pulchra (800 g wet) was extrac ted thrice with 1:1 dichloromethane/methanol to yield 3.5 g of lipophilic extract and then extracted thrice with 1:1 methanol/water to yield 18.8 g of hydrophilic extract (Sch eme 1). The lipophilic extract showed a 12 mm zone of inhibition against two Gram-positive bacteria, methicillin-sensitive and -resisitant Staphylococcus aureus (MRSA and MSSA respectively) and an 8 mm zone of inhibition against a fungus Candida albicans. The lipophilic extract was subjected to silica gel flash column chromatography to yield 5 fractions. Fimbrolide (10 mg, 0.0012%) was obt ained from Normal Phase (NP) HPLC of fraction 1 (1:99 EtOAc/hexane), halogenated ketone 40 (40 mg, 0.005%) was obtained from NP HPLC of fraction 2 (5:95 EtoAc/ Hexane), acetoxyfimbro lide (13 mg, 0.0016%) and two new compounds, pulchralide B (3 mg, 0.00037%) and C (2.5 mg, 0.0003%), were obtained from NP HPLC of fraction 3 (1:9, 12:88, 15:75 EtOAc/hexane). Another new compound, pulchralide A (10 mg, 0. 0012%), and hydroxyfimbrolide (14 mg, 0.0017%) were obtained from the NP HPLC of fraction 5 (2:8 EtOAc/hexane).

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Scheme 1. Isolation of fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23), halogenated ketone 40, pulchralide A (41), pulchralide B (42) and pulchralide C (43). Delisea pulchra 800 g (Frozen and wet)1. Extracted with 3X 1:1 CH2Cl2:MeOH, 24 hr each2. Combine supernatents, filter and concentrate in vacuoLipophilic extract(3.5 g) 51.5 mg 50 mg 500 mg 384 mg 2.6 gNP silica gel coulmn2.5 cm X 45 cm gradient elutionfrom hexane to EtOAcHalogentatedKetone 40Fimbrolide (21)NP HPLCEtOAc/Hexanes(1:99)NP HPLC EtOAc/Hexanes(5:95)NP HPLCEtOAc/hexanes(10:95)NP HPLC EtOAc/hexanes(15:85)Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Acetoxy-fimbrolide (22)Pulchralide B (42)Pulchralide C (43)Pulchralide A (41)Hydroxy-fimbrolide (23)Hydrophilic extract (18.8 g) 18

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2.3. Characterization of Fimbrolide (21) Compound 21 was the first fraction to elute from NP HPLC of fraction 1 (Scheme 1). Compound 21 was found to be identical with fimbrolide 65 from the data discussed below. Fimbrolide (21) was obtained as colorless oil and was assigned a molecular formula of C 9 H 10 O 2 Br 2 deduced from LREIMS m/z 312/310/308 (1:2:1). The 1 H NMR spectrum (Figure 2) of fimbrolide in CDCl 3 showed signals at 6.27 (1H, s) corresponding to an olefinic hydrogen and to an aliphatic chain at 2.41 (2H, t, J = 7.5 Hz), 1.35 (4H, m) and 0.93 (3H, t, J = 6.5 Hz). 6.272 2.413 2.175 1.595 1.583 1.577 1.377 1.367 1.362 1.353 1.347 1.340 1.267 0.965 0.958 0.950 0.945 0.943 0.935 09 3 3 6 5 4 3 2 1 PP M Figure 2. 1 H NMR spectrum of fimbrolide (21) (500 MHz, CDCl 3 ). 19

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The 13 C NMR spectrum (Figure 3) of fimbrolide (21) showed signals corresponding to a lactone carbonyl at 166.3, a tetrasubstituted double bond at 150.2 and 134.1, a trisubstituted, enolic double bond at 130.0 and 91.1 and other signals corresponding to an aliphatic chain at 29.2, 25.2, 22.6 and 13.9. The infrared spectrum gave evidence for a carbonyl at 1787 cm -1 The UV max of 292 nm (log 4.92) suggested the presence of a conjugated ketone. Figure 3. 13 C NMR spectrum of fimbrolide (21) (125 MHz, CDCl 3 ) The structure of fimbrolide (21) was established by comparing the 1 H NMR, 13 C NMR, IR, UV, mass spectra and optical rotation values with those of the previously reported compound 65 For fimbrolide (Figure 4), the reported chemical shifts of the olefinic hydrogen established the configuration of the exocyclic double bond as Z in our isolate. 65 20

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OB r OHBrOBrOBrH6.246.56 Figure 4. 1 H NMR chemical shift values of H-6 in Z and E isomers of fimbrolide (21). 65 Fimbrolide (21) was weakly active in antimicrobial assays, with 7 mm hazy zone of inhibition (all antimicrobial assays done at 200 g/spot) MRSA and MSSA (no inhibition of other microbes tested). 21

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2.4. Characterization of Acetoxyfimbrolide (22) Compound 22 was eluted from the NP HPLC of fraction 3 as yellow oil (Scheme 1). Compound 22 was found to be identical with acetoxyfimbrolide 66 from the spectral and physical data discussed below. Acetoxyfimbrolide (22) was the major halogenated metabolite from our collections of Delisea pulchra. LREIMS m/z 370/368/366 (1:2:1) indicated a molecular formula of C 11 H 12 Br 2 O 4 The 1 H NMR spectrum of acetoxyfimbrolide (Figure 5), showed signals at 2.09 (3H, s) and 5.52 (1H, dd, J = 7.2, 7.0 Hz), indicative of an acetoxyl group on an aliphatic chain. 6.387 5.529 5.524 5.498 2.090 1.832 1.607 1.369 1.340 0.978 0.948 0.919 6 5 4 3 2 1 Figure 5. 1 H NMR spectrum of acetoxyfimbrolide (22) (250 MHz, CDCl 3 ). 22

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The 13 C NMR spectrum (Figure 6) of acetoxyfimbrolide (22) also indicated the presence of an acetate function ( 170.0 and 20.4) and an --unsaturated -lactone ( 163.6, 149.6 and 131.2). From the comparison of 1 H NMR and 13 C NMR spectra to published spectra 66 it can be concluded that acetoxyfimbrolide (Figure 7) differs from fimbrolide (21) only in the acetoxy substitution on the butyl chain. 170.006 163.653 149.602 131.234 130.402 93.376 68.098 33.577 20.466 18.391 13.505 160 140 120 100 80 60 40 20 PP M Figure 6. 13 C NMR spectrum of acetoxyfimbrolide (22) (62.5 MHz, CDCl 3 ). The 1 H NMR spectrum of acetoxyfimbrolide (22) showed the bromomethine ( 6.38, 1H, s) at a higher field than ( E )-acetoxyfimbrolide, 67 indicating that the exocylic double bond has a Z configuration. The absolute configuration of acetoxyfimbrolide, previously reported from X-ray analysis as 1'R and 5 Z was found to match with our isolate because of similar optical rotation value of 25 D +15.0 (c, 0.4 CHCl 3 ). 68 23

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OOBrOHB r O234561'2'3'4'78 Figure 7. Acetoxyfimbrolide (22). The IR spectrum of acetoxyfimbrolide (22) showed max of 1788 cm -1 (acetate carbonyl) and 1736 cm -1 (lactone carbonyl). Analysis of the UV spectrum, showed max 291 nm (log 4.94) which is similar to the absorption properties of ,-unsaturated -lactones compounds. 66 From the Palmer Station collections of Delisea pulchra antimicrobial activity was greatest in acetoxyfimbrolide, which showed potent activity with 25 and 24 mm zones of inhibition against MRSA and MSSA respectively, a 16 mm zone toward vancomycin-resistant Enterococci faecium (VREF) and a 17 mm zone using Candida albicans. It was modestly active against permeablized (a mutant strain with increased permeability to large molecular weight compounds) Escherichia coli (9 mm hazy zone). 24

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25 2.5. Characterization of Hydroxyfimbrolide (23) The most polar compound isolated in this study was compound 23, obtained from NP HPLC of fraction 5 (Scheme 1) as yellow oil. Compound 23 showed physical and spectral data similar to the reported hydroxyfimbrolide, 65 which is discussed below. LREIMS of hydroxyfimbrolide (23) m/z 328/326/ 324 (1:2:1) supported a molecular formula of C 9 H 10 O 3 Br 2 The 1 H and 13 C NMR resonances of hydroxyfimbrolide were very similar to those of acetoxyfimbrolide (22), in particular with reference to the furanone ring and the butyl chain, indi cating a closely rela ted structure. The 1 H NMR spectrum (Figure 8) of hydroxyfimbrolide showed a doublet at a 2.54 (1H, d, J = 9.5 Hz) probably resulting from an unus ual coupling of a hydroxyl proton (D 2 O exchangeable) to the hydroxy substituted pr oton. This suggests the presence of a hydroxyl substitution on the butyl chain in hydroxyfimbrolide compared to the acetoxy substitution in acetoxyfimbrolide. Comparison of the 1 H and 13 C NMR spectral (Figure 8 and 9) data set to that reported 65 for hydroxyl fimbrolide (Figure 10) confirmed their identity. The IR spectrum showed th e presence of a carbonyl group at 1736 nm -1 (lactone) and a broad hydroxyl group at 3457 nm -1 The UV max of 292 nm (log 4.90) demanded that the -lactone function was doubly conjugated.

PAGE 40

6.388 4.636 4.606 4.569 4.542 2.562 2.524 1.902 1.863 1.841 1.809 1.770 1.733 1.707 1.679 1.599 1.496 1.466 1.427 1.397 1.370 1.330 1.303 0.990 0.960 6 5 4 3 2 1 0 P P Figure 8. 1 H NMR spectrum of hydroxyfimbrolide (23) (250 MHz, CDCl 3 ). Figure 9. 13 C NMR spectrum of hydroxylfimbrolide (23) (125 MHz, CDCl 3 ). 26

PAGE 41

The assignment of a Z configuration of the double bond in hydroxyfimbrolide (23) was established by consideration of the chemical shift of H-6, which was upfield at 6.38 (1H, s). Hydroxyfimbrolide (23) was found to be 1'R and 5Z from its 25 D +15.0 (c, 0.4 CHCl 3 ), which matched the previously reported value. 68 OOBrOHHB r 234561'2'3'4'6.38 Figure 10. Hydroxyfimbrolide (23). Hydroxyfimbrolide displayed large zones of inhibition with Gram-positive bacteria (23 mm and 23 mm for MRSA and MSSA respectively), 14mm for VREF and a 25 mm zone against Candida albicans. It was modestly active against Escherichia coli (9 mm hazy zone). 27

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2.6. Characterization of Halogenated Ketone 40 NP HPLC of fraction 2 resulted in the isolation of compound 40 (40 mg, 0.005%) as a colorless oil. Compound 40 was found to be identical with the reported 69 Halogenated ketone from the physical and spectral analysis, which is discussed below. Analysis of mass spectrum showed a molecular formula of C 8 H 11 Br 3 O deduced from LREIMS m/z 304/306/308/310 (1:2:2:1). The 1 H NMR spectrum (Figure 11) of halogenated ketone 40 showed a methylene triplet at 2.79 (2H, t, J = 7.5 Hz), an aliphatic envelope at 1.70-1.33 (6H) and a methyl triplet at 0.94 (3H, t, J = 7.0 Hz). 2.822 2.792 2.763 2.176 2.152 2.104 1.708 1.679 1.650 1.587 1.363 1.336 0.968 0.941 0.913 3.0 2.5 2.0 1.5 1.0 0.5 PP M Figure 11. 1 H NMR spectrum of halogenated ketone 40 (250 MHz, CDCl 3 ). 28

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A signal at 197.4 in the 13 C NMR spectrum (Figure 12) of Halogenated ketone 40 has a ketone function and signals at 91.2 and 121.9 are indicative of a shielded, tetrasubstituted olefin. A pentyl chain is evident from 13 C NMR resonances at 40.7, 31.1, 23.2, 22.4 and 13.9.The infrared spectrum supports the assignment of a carbonyl at 1715 cm -1 The UV spectrum showed max of 220 nm (log 4.84) and 283 nm (log 4.03). 197.407 121.906 91.271 40.754 31.125 23.206 22.436 13.952 0 150 100 50 Figure 12. 13 C NMR spectrum of halogenated ketone 40 (62.5 MHz, CDCl 3 ). 29

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Halogenated ketone 40 (Figure 13) was identified by the matching of physical and spectral data set with those of the previously reported compound. 69 OBrB r B r 12345678 Figure 13. Halogenated ketone 40. 30

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31 2.7. Characterization of Pulchralide A (41) A second compound, 41, obtained from the polar fractio n 5 (Scheme 1) was found to be a new compound from its structural elucidation discussed below. Compound 41 was named after the species name of the red alga Delisea pulchra as Pulchralide A. Pulchralide A (41), molecular formula of C22H24Br4O8 (HREIMS m/z 734.8260. requires m/z 734.8262), was crystallized from ethanol as colorless needles. Pulchralide A displayed the characteristic aliphatic 1 H NMR signals (Figure 14) similar to those observed in acetoxyfimbrolide, in cluding the olefinic hydrogen at 5.18 (1H, s), an acetoxy substituted methine at 5.52 (dd, 1H, J = 6.5, 8.0 Hz), an acetate methyl at 2.11 (3H, s), signals corres ponding to the butyl chain at 1.91, 1.36 and 0.96 (3H, t, J = 7.5 Hz). But the olefinic pr oton is at higher field than in pulchralide A than in acetoxyfimbrolide, wher e it resonates at 6.38. The 13 C NMR spectrum (Figure 15) of pulchralide A (41) significantly differed from acetoxyfimbrolide (22). In particular, only two 13 C signals were observed in the olefinic region of pulchralide A, unlike four ol efinic signals found in acetoxyfimbrolide ( 149.6, 131.2, 130.4, 93.3). It lacked the tris ubstituted enolic double bond signals of acetoxyfimbrolide at 130.4. The heteroatom-bearing re gion suggested there were three heteroatom-bearing sp 3 carbons. The complete 1 H NMR and 13 C NMR assignments can be found in Table 1.

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5.539 5.526 5.523 5.510 5.181 2.114 0.980 0.966 0.951 5 4 3 2 1 P Figure 11. 1 H NMR spectrum of pulchralide A (41) (500 MHz, CDCl 3 ). Figure 15. 13 C NMR spectrum of pulchralide A (41) (125 MHz, CDCl 3 ). 32

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The 135 Distortionless Enhancement by Polarization Transfer (DEPT-135) spectrum of pulchralide A (41) (Figure 16) showed a methine at 39.5 suggesting the absence of an exocyclic double bond. It also showed the presence of the acetoxy bearing carbon at 64.6 as a methine, two methylenes at 33.8 and 16.7 and two methyl groups at 20.9 and 14.7. 64.676 39.525 29.682 16.795 14.751 7 0 60 50 40 30 20 PP M Figure16. DEPT-135 spectrum of pulchralide A (41) (125 MHz, CDCl 3 ). The planar structure was established by 2D NMR techniques. In the 1 H1 H Correlation Spectroscopy (COSY) spectrum (Figure 17) of pulchralide A (41), cross peaks were observed between H-1' and H-2', H-2' and H-3' and between H-3' and H-4' (Figure 18). Gradient Heteronuclear Multiple Quantum Correlation (gHMQC) spectrum (Figure 19) of pulchralide A showed the correlations of acetate substituted methine proton at 5.5 with C-1' ( 68.5) which suggested the substitution of acetoxy group at C-1' on the butyl 33

PAGE 48

side chain. Cross peaks were also observed between H-6 ( 5.18) and C-6 ( 43.9), as well as the acetate methyl Me-8 ( 2.11) and C-8 ( 20.8). Figure 17. gCOSY spectrum of pulchralide A (41) (500 MHz, CDCl 3 ). OOOOBrHBr1'2'3'4'682345 Figure 18. 1 H1 H COSY correlations of pulchralide A (41). 34

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5 4 3 2 1 PPM Direct Dimension 120 100 80 60 40 20 0 Figure 19. gHMQC spectrum of pulchralide A (41) (500MHz, CDCl 3 ). The significant correlations observed in gradient heteronuclear mutlitple bond correlation spectrum (gHMBC) spectrum (Figure 20) of pulchralide A (41) were between H-1' ( 5.5) and C-2 to C-4 and C-8 of the lactone ring (Table 1). Correlations were also seen for H-6 to C-5 and C-6. Because gHMBC spectra show only two and three bond correlations ( 2 J H 3 J H correlations), the correlation of H-6 to the C-6 (Figure 21) was the first clue that the metabolite was a dimer. The hypothesis that pulchralide A is a dimer was supported by mass spectral analysis which provided a molecular formula of C22H24Br4O8 (m/z 732/734/736/738 (1:4:6:4:1), HREIMS 734.8260, C 22 H 24 0 8 79 Br 3 81 Br requires 734.8262). Thus pulchralide A appears to be a dimer of acetoxyfimbrolide (22) and has not been previously reported. 35

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6 5 4 3 2 1 PPM Direct Dimension 180 160 140 120 100 80 60 40 20 0 Figure 20. gHMBC spectrum of pulchralide A (41) (500 MHz, CDCl 3 ). OOOOOBrBrBrBrHHOOO4'3'2'1'7823455a66a2a3a4a1'a2'a3'a4'a Figure 21. KEY HMBC correlations determined in pulchralide A (41). 36

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Table 1. NMR data of pulchralide A (41) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz). Position 1 H () 13 C () gHMBC 1'/1'a 5.52 (1H, dd, J = 6.5, 8.0 Hz) 68.5 C-7/7a, C-2/2a to C-4/4a, 6/6a 5.18 (1H, s) 43.9 C-6/6a, C-55a, C-4/4a 8/8a 2.11 (3H, s) 20.8 C-7/7a 2'/2'a 1.91 (2H, m) 33.8 C-1'/1'a, C-3'/3'a, C-3/3' 3'/3'a 1.36 (2H, m) 18.6 C-1'/1'a, C-4'/4'a 4'/4'a 0.96 (3H, t, J = 7.5 Hz) 13.8 C-2'/C-2'a C-3'/3'a 7/7a 170.0 2/2a 164.9 4/4a 139.7 3/3a 134.5 5/5a 90.4 The crystallization of the pulchralide A (41) has led to the determination of the absolute stereochemistry of pulchralide A by a single crystal X-ray analysis performed by Dr. Mike Zaworotko at University of South Florida, Tampa. (Figure 22 and 23). Figure 22. Perspective view of X-ray crystal structure of pulchralide A (41). 37

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OOOOOOOBrBrBrHHBrO Figure 23. Sterochemical assignments of pulchralide A (41). To ensure that the pulchralide A (41) is not an artifact, especially of photochemical origin, acetoxyfimbrolide (22) (1 mg), dissolved in chloroform was irradiated with UV and visible light for 24 hrs each. 1 H NMR spectra of the irradiated acetoxyfimbrolide did not show signs of dimer formation, supporting their biotic origin. Pulchralide A was modestly active in antimicrobial assays against MRSA and MSSA with 7 mm hazy zones of inhibition respectively. 38

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2.8. Characterization of Pulchralide B (42) Compound 42, obtained from the NP HPLC of fraction 3 (Scheme1) as white solid, was characterized as discussed below and found to be a new compound, which was named as pulchralide B. Pulchralide B (42) demonstrated many of the structural features of pulchralide A (41). The 1 H NMR spectrum (Figure 24) of pulchralide B lacked the acetoxymethine of pulchralide A, and a new allylic methylene signal was found at 2.43 (3H, t, J = 7.5 Hz), analogous to the allylic H-1 of fimbrolide (21). The 13 C NMR spectrum (Figure 25) of pulchralide B showed only two signals in the olefinic region, similar to pulchralide A, but did not show any signals indicative of an acetoxy group. Figure 24. 1 H NMR spectrum of pulchralide B (42) (500 MHz CDCl 3 ). 39

PAGE 54

The 13 C NMR spectrum (Figure 25) of pulchralide B showed only two signals in the olefinic region, similar to pulchralide A, but did not show any signals indicative of an acetoxy group. The DEPT-135 spectrum (Figure 26) of pulchralide B showed one methine carbon at 44.1, one methyl of the butyl side chain at 13.9 and three methylenes at 29.0, 25.5, and 22.4. Figure 25. 13 C NMR spectrum of pulchralide B (42) (125 MHz, CDCl 3 ). 40

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44.187 29.026 25.525 22.472 13.946 40 35 30 25 20 15 PP M Figure 26. DEPT-135 spectrum of pulchralide B (42) (125 MHz CDCl 3 ). The planar structure of pulchralide B (42) was deduced by interpretation of 2D NMR spectra. The gHMBC spectrum (Figure 27) of pulchralide B showed correlations of H-6 with C-6 (Figure 28), similar to that in pulchralide A (41) revealing that pulchralide B is also a dimer. The significant correlations (Table 2) observed in pulchralide B are the correlation of H-1' with C-3 indicating the attachment of the butyl side chain to the furanone ring. It also showed cross peaks between H-6 and C-5. Pulchralide B therefore is the symmetrical fimbrolide dimer, an assignment which is fully supported by the COSY and HMBC data sets and confirmed by mass spectral data, (m/z 41

PAGE 56

615/617/619/621/623 (1:4:6:4:1), [M + H] HREIMS 538.8894 [M + Br], C 18 H 20 O 4 79 Br 2 81 Br requires 538.8891). 6 5 4 3 2 1 0 PPM Direct Dimension 200 150 100 50 0 Figure 27. gHMBC spectrum of pulchralide B (42) (500 MHz, CDCl 3 ). OOOBrBrBrHHO1'3562'3'4'244a5a3a2a1'a2'a3'a4'a6a Figure 28. Key HMBC correlations of pulchralide B (42). 42

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Table 2. NMR data of pulchralide B (42) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz). Position 1 H () 13 C () gHMBC 6/6a 5.20 (1H, s) 44.1 C-6/6a, C-5/5a, C-4/4a 1'/1'a 2.43 (1H, t, J = 7.5Hz) 29.0 C-3/3a, C-4/4a, C-2/2a 2'/2'a 1.55 (2H, m) 25.5 C-1'/1'a, C-3'/3'a, C-3/3'a 3'/3'a 1.27 (2H, m) 22.4 C-1'/1'a, C-4'/4'a. 4'/4'a 0.93 (3H, t, J = 7.5Hz) 13.9 C-2'/2'a, C-3'/3'a 2/2a 167.4 3/3a 138.2 4/4a 137.1 5/5a 90.5 _____________________________________________________________________ Of the two possibilities that pulchralide B (42) is either a C 2 dimer or meso dimer (Figure 29), the possibility that it is a meso dimer was ruled out because of its optical rotation of [] 25 D +4.26 o (c 0.06, CHCl3). Hence pulchralide B is a C 2 dimer. OOOB r BrBrHHB r O OOOB r BrBrHHB r O Figure 29. Pulchralide B C 2 dimer and pulchralide B meso dimer. 43

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44 2.9. Characterization of pulchralide C (43) Compound 43, was the third compound, to be elut ed from NP HPLC of fraction 3 (Scheme1), whose structural determination as discussed below led to a new compound, named as pulchralide C. Pulchralide C (43), was obtained as white solid and was found to have molecular formula of C 20 H 22 O 6 79 Br 2 81 Br 2 (TOFMS m/z 700.8008, requires 700.8007). The 1 H NMR spectrum (Figure 30) of pulch ralide A has signals related to both acetoxyfimbrolide (22) and fimbrolide (21). A single acetoxy methyl group at 2.09 (3H, s) and an acetate substituted proton at 5.49 (1H, dd, J = 6.5, 8.0 Hz), evident in the 1 H NMR spectrum (Figure 30) confirm the re semblance to acetoxyfim brolide while the signal at 2.38 (1H, t, J = 7.5 Hz) is reminiscent of fimbrolide. The doublets at 5.38 (1H, d, J = 10.0 Hz) and at 5.13 (1H, d, J = 10.0 Hz) are indicative of the presence of olefinic bromo-methines. The 13 C NMR spectrum (Figure 31) of pulchralide C also showed peaks related to acetate group ( 170.3 and 20.6), peaks characteristic of two lactone carbonyls (167.0, 165.3) and one acetoxy substitution at 68.8. The 1 H and 13 C NMR assignments are illustrated in Table 3.

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Figure 30. 1 H NMR spectrum of pulchralide C (43) (500 MHz, CDCl 3 ). Figure 31. 13 C NMR spectrum of pulchralide C (43) (125 MHz CDCl 3 ). 45

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The planar structure of pulchralide C (43) was established based on analysis of 2D NMR data. Analysis of the 1 H1 H COSY spectrum (Figure 32) of pulchralide C (43) illustrated two bromomethine doublets, H-6 and H-6a, at 5.35 and 5.13 respectively, coupled with one another. This significant 1 H1 H COSY correlation (Figure 33) supports the evidence that pulchralide C is an unsymmetrical dimer. The gHMBC spectrum established a two bond correlation between H-6 and C-6/C-6a supporting the assumption that pulchralide C is an unsymmetrical dimer. 2 5 20 15 10 5 0 PPM Indirect Dimension 1 9 8 7 6 5 4 3 2 1 0 Figure 32. 1 H1 H COSY spectrum of pulchralide C (43) (500 MHz, CDCl 3 ). 46

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OOOOOBrBrBrBrHHO66a23455a4321'a2'a3'a4'a781a2a3a4a Figure 33. Key 1 H1 H COSY correlation of pulchralide C (43). Table 3. NMR data of pulchralide C (43) (CDCl 3 ) ( 13 C, 125 MHz; 1 H, 500 MHz) Position 1 H () 13 C () gHMBC 1' 5.49 (1H, dd, J = 6.5, 8.0 Hz) 68.8 C-2, C-3, C-7 6 5.38 (1H, d, J = 10.0 Hz) 47.7 C-5, C-6a 6a 5.13 (1H, d, J = 10.0 Hz) 44.4 C-5a, C-6 1'a 2.38 (1H, t, J = 7.5 Hz) 33.4 C-3, C-2'a. C-3'a 8 2.09 (3H, s) 170.3 C-1' 2' 1.96 (2H, m) 28.9 C-1a, C-3a, C-3 2' a 1.81 (2H, m) 25.4 C-1a, C-3a, C-3a 3' 1.34 (2H, m) 22.5 C-2a, C-4a, C-1a 3'a 1.32 (2H, m) 18.7 C-2a, C-4a, C-1a 4' &4' a 0.95 (6H, t, J =7.0 Hz) 13.8 C-3a/3a, C-2a/2a 2 167.0 2a 165.3 4 140.9 4a 139.6 3 135.5 3a 134.5 5 93.3 5a 88.1 The bromomethine protons couple with each other to form doublets with J = 10 Hz ( 5.35) and J = 10.0 Hz ( 5.13). The trans coupling constants for protons of a four membered ring like cyclobutane is 2-10. 67 This indicates that these two are trans to each 47

PAGE 62

other Figure 34 illustrates the sterochemical assignments of pulchralide C (43) based on the above considerations. OOOOOBrBrBrHHBrO245632a2'4'1'3'84'a6a4a5a2'a3'a1'a3a7 Figure 34. stereochemical assignments of pulchralide C (43). 48

PAGE 63

49 Chapter 3. DISCUSSION 3.1. Biological Importance of Ha logenated Metabolites of Delisea pulchra Our interest in Delisea pulchra is due to the antimicrobi al and antifungal activity of the organism we observed in the bioassay of the crude extract performed by Wyeth Pharmaceuticals, Pearl River, NY. Chemical investigation of D. pulchra resulted in the isolation of three new dimeric halo genated furanones pulchralide A-C (41-43) along with the previously reported fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23) and halogenated ketone 40. The structures of the new compounds were determined by 1D, 2D NMR and mass spectral data. The absolu te stereochemistry of pulchralide A (41) was established by single crystal X-ray cr ystallography. The structures of known compounds fimbrolide, acetoxy-fimbrolide, hyd roxyfimbrolide and halogenated ketone 40 were identified by comparing the physical and spectral data with those reported previously. The absolute stereochemistry of these compounds was already reported on the basis of chemical interconversio ns and X-ray and CD analyses. 68 Fimbrolide, acetoxyfimbrolide, hydroxyfimbrolide were found have a Z configuration based on the chemical shifts of their respective hydrogen atoms.

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50 The bioassay data (Table 4) indicates that acetoxyfimbrolide (22) and hydroxyfimbrolide (23) have potent antimicrobial act ivity against two Gram-positive bacteria, MRSA and MSSA and against one Gram-negative bacterium, VREF and mild activity against the fungus Candida albicans. Acetoxyfimbrolide and hydroxyfimbrolide showed modest activity against permeablized Escherichia coli Table 4. Antimicrobial acti vity of pure compounds (100 g/disk) using the disk diffusion assay (Zone of Inhibition in mm) _____________________________________________________________________ MSSA MRSA VREF Permeablized Candida Escherichia coli albicans _____________________________________________________________________ Acetoxyfimbrolide (22) 25 24 16 9:10H 17 Hydroxyfimbrolide (23) 23 23 14 9H 25 Fimbrolide (21) 7H 7H 0 0 0 Pulchralide A (41) 7H 6:7H 0 0 0 _____________________________________________________________________

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51 3.2. Origin of Halogenated Metabolites of Delisea pulchra While several dimeric furanones have been described from the genus Delisea, two of which bear the central cyclobutane of th e pulchralides, pulchralides A and C (41 and 43) are the first such dimers bearing the C-1 acetoxy group. Pulchralides B (42) and C demonstrated many of the structural features of pulchralide A and C and pulchralide C is the only unsymmetrical member of the gr oup. The cyclobutane dimers, pulchralide A could be derived from acetoxyfimbrolide (22), pulchralide B from hydroxyfimbrolide (23) and pulchralide C from both acetoxy-fimbrolide (22) and fimbrolide (21) by 2 s + 2 s cycloaddition reactions. 62 Molecules which are formed by enzyme mediated reactions are almost invariably optically active. 70 Examples of cyclobutane containing dimers are known. 70 To ensure that the dimers de scribed here were not artifacts, especially of photochemical origin, the solution of acetoxyfimbrolide was exposed to both UV and visible light for 24 hrs each with no discernable effects on the compound.

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52 3.3. Ecological Importance of Halogenated Furanones of Delisea pulchra Halogenated furanones from Delisea pulchra have various ecological and biological functions. The red macroalga (seaweed) D. pulchra is relatively free of surface colonization and the knowledge that it pr oduced unusual secondary metabolites, halogenated furanones or fimbrolides prompted an in-depth investigat ion of the role of these metabolites as inhibitors of su rface colonization. These compounds were encapsulated in vesicles in gland cells in the seaweed, providing a mechanism for delivery of the metabolites to the surface of the alga at concentrations which deter a wide range of prokaryote and eukaryote fouling organisms. 71 Inhibition in these studies was by a non-toxic and non-growth inhibitory mechanis m and the structural similarities between these algal metabolites and the bacterial signaling molecules, acylated homoserine lactones (AHLs), led to the hypothesis that furanones act as specific antagonists of AHL regulatory systems. 71 Furanones inhibit AHL-regulated phenotypes in a wide range of Gram-negative bacteria and their specific mode of action has now been demonstrated via both AHL phenotypic bioassays and targeted molecular systems. 71 For example, furanones shut down AHL-regulated swarming in Serratia liquefaciens, biofilm development in Pseudomonas aeruginosa and bioluminescence in both wild-type Vibrio fischeri and luminescence constructs (e.g. in Escherichia coli backgrounds). The use of natural products such as furanones, which target specific bacterial signaling and regulatory systems, represents a promis ing approach to inhibition of biofilm. 71

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53 Barnacles cause corrosion and make sh ips heavier and harder to steer. Antifouling paints that contain tin or copper stop barnacl es from attaching and leach metals into the sea and kill many non-target orga nisms. Antifouling furanones made by Delisea pulchra blocks bacterial communication systems and prevents bacter ial biofilms from developing on its surface. 63 This then stops barnacles from attaching. The seaweed metabolite may be used to replace the toxic chemicals in any environment that can be submerged. Research is now focused on further development of furanone based paints. 63 The geographic variation of halogenated metabolites of D. pulchra has also been the subject of study. 72 The large variability in c oncentrations of furanones, and the absence of any positive relationships between furanones, he rbivores and epiphytes, suggest that quantitative variation in furanones in D. pulchra is not driven by population-level selection or induction, but is more likely to be a result of sma ll-scale vari ation in environmental factors such as nutrients, lig ht and genetic differences among individual plants. 72

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54 Chapter 4. EXPERIMENTAL 4.1. General Experimental Procedures. Optical rotation was determined on a Rudolph Research Analytical AUTOPOL IV with a sodium lamp (589 nm) and 0.5 dm cell at 25c. Infrared spectra were obtained with Nicolet Avatar 320FT-IR in solid st ate. Ultraviolet-Visible experiments were measured on a Hewlett-Packard 8452A diode array UV/Vis spectrometer. 1 H and 13 C NMR, HMQC, HMBC and 1 H1 H COSY spectra were obtai ned on either a Varian 500 instrument operating at 500 MHz for 1 H NMR and 125 MHz for 13 C, or a Bruker Avance 250 instrument operating at 250 MHz for 1 H and 62.5 MHz for 13 C. The 13 C resonance multiplicities were determined by DEPT experiments. 1 H1 H correlations were determined by using gCOSY experiment optimized coupling constant (J HH of 7 Hz). One bond connectivities ( 1 J CH ) of 1 H13 C were determined via the 2D proton-detected gHSQC experiment. The interpulse dela ys were optimized for average 1 JCH of 120 Hz. Twoor three-bond heteronuclear multiple-bonds ( 3 JCH/ 2 J CH ) were recorded via the 2D proton detected gHMBC experiment optimized for a long range coupling constant (JCH of 7 Hz). High resolution mass spectra ESIMS, EI-MS and CIMS were obta ined on Micro mass 70-VSE spectrometer at the University of Illinois. HPLC was performed on Waters 510 equipped with a Waters 490E programmable mu ltiwavelength UV detector at 254 nm and

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55 Shimadzu LC-8A equipped with a multisolvent delivery system connected to a Shimadzu SPD-10A UV-VIS tunable abso rbance detector and/or an Alltech ELSD 2000 using a YMC-Pack ODS-AQ C-18 analytical co lumn, a Waters prepLC (25 mm X 30 cm) C18 column for reversed pha se, or Waters Sphereclone (250 X 10 mm) for normal phase. Flash column chromatography was performed on EM Science normal phase Silica gel 60 (200 400 mesh). Thin layer chromatogra phy (TLC) was carried out using Whatman normal phase silica gel 60 Partisil K6F, reversed phase silica gel 60 Partisil KC18F, and CNF254s plates 0.25 mm thickness. There were visualized by spraying with 5% phosphomolybdic acid in EtOH or heati ng and 2 % ninhydrin in BuOH/acetic acid (95:5). X-ray Crystal Structure Determination: Single crystals suitable for x-ray crystallographic analysis were selected following examination under a microscope. Single-crystal x-ray diffracti on data for the compounds were collected on a Bruker-AXS SMART APEX/CCD diffr actometer using Mo radiation ( = 0.7107 ). Diffracted data were corrected for Lorentz and polari zation effects and for absorption using the SADABS 73 program. The structure was solved by direct methods and the structure solution and refinement was based on |F| 2 All non-hydrogen atoms were refined with anisotropic displacement parameters whereas hydrogen atoms were placed in calculated positions when possible and given isotropic U values 1.2 times that of the atom to which they are bonded. All crystallographic calcu lations were conducted with the SHELXTL v.6.1 74 program package. 75

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56 4.2. Bioassay of Pure Compounds Bioassays for the pure compounds were done at Wyeth Pharmaceuticals, Ford Hospital and in the Marine Natural Products Lab at Univers ity of South Florida, using standard disk diffusion method against two Gram-positive bacteria, Methicillinresistant and -sensitive Staphylococcus aureus (MRSA and MSSA respectively) against two Gram-negative bacteria Vancomycin resistant Escherichia faecium (VREF) and permeablized Escherichia faecium and against one fungus Candida albicans. Antibiotic assays: In vitro antimicrobial activities against methicillin-sensitive (MSSA, strain 375) and -resistant (MRSA, strain 310) Staphylococcus aureus, vancomycin-resistant Enterococci faecium (VREF, strain 379), E. coli (strain 442), E. coli imp (strain 389, a mutant strain with in creased permeability to large molecular weight compounds) and Candida albicans (strain 54) were determ ined by agar diffusion method. Media used were Difc o nutrient agar (pH 6.8) for S. aureus, LB (Luria-Bertani) agar for E. faecium and E. coli, and YM agar for C. albicans. Assay plates (9x 9 Sumilon) were prepared by pouring 125 ml volume of agar medium (tempered at 50C) inoculated with an overnight broth cult ure of the test orga nisms (adjusted to approximately 10 6 cells per ml). Sample concentrations of 200 g in 10 L aliquots were spotted onto agar surface and the pl ates were incubated at 37C for 18h. 75

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57 The zones of growth inhibition were measured using a hand-held digital caliper. The zones of growth of inhibition were measured fr om the edge of the disk to the edge of the clear inhibition zone in mm, respectively. C ontrol disks were treated with solvent alone (MeOH or CHCl3). The bioassay data for th e pure compounds done at Wyeth Pharmaceuticals and at Marine natural products lab at University of South Florida can be found in Table 3.

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58 4.3. Isolation of Secondary Metabolites from Delise pulchra Plant Material. The algal material was collected at Palmer Station, Antarctica by scuba diving in November December of 2001. Identifications were made by C. Amsler and K.B. Iken. A voucher specimen is avai lable at the University of Alabama at Birmingham. The red alga Delisea pulchra is a reddish brown alga. The red alga D. pulchra (800 g wet) was extrac ted thrice with 1:1 dichloromethane/methanol to yield 3.5 g of li pophilic, and then extracted thrice with 1:1 methanol/water to yield 18.8 g of hydroph ilic extract. The lipophilic extract was subjected to silica gel flash column chroma tography to yield 5 fractions. Fimbrolide (21) (10 mg, 0.0012%) was obtained from NP HP LC of fraction 1 (1:99 EtOAc/hexane), halogenated ketone 40 (40 mg, 0.005%) was obtained from NP HPLC of fraction 2 (5:95 EtoAc/Hexane), acetoxyfimbrolide (22) (13 mg, 0.0016%) and two new compounds, pulchralide B (42) (3 mg, 0.00037%) and C (43) (2.5 mg, 0.0003%), were obtained from NP HPLC of fraction 3 (1:9, 12:88, 15:75 EtOAc/hexane). Another new compound, pulchralide A (41) (10 mg, 0.0012%), and hydroxyfimbrolide (23) (14 mg, 0.0017%) were obtained from the NP HPLC of fraction 5 (2:8 EtOAc/hexane).

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4.3.1. Spectral Data for Fimbrolide (21) OOB r HB r 34561'2'3'4'2 Fimbrolide (21) Fimbrolide (21): 10 mg, 0.0012%, colorless oil; 25 D +4.5 (c, 0.9 CHCl 3 ); IR v max 1787, 1213, 1042 cm -1 ; UV max 292 nm (log 4.92); 1 H NMR (250 MHz, CDCl3) integration, multiplicity, J (Hz), assignment): 6.27 (1H, s, H-6), 2.41 (2H, t, 7.5, H-1), 1.35 (4H, m, H-2, H-3), 0.93 (3H, t, 6.5, H-4); 13 C NMR (125 MHz, CDCl3) (assignment) 166.3 (C, C-2), 150.2 (C, C-4), 134.1 (C, C-3), 130.0 (C, C-5), 91.1 (C, C-6), 29.2 (CH, C-1), 25.2 (CH 2 C-2), 22.6 (CH 2 C-3), 13.9 (CH 3 C-4); LREIMS m/z 312/ 310/308 (1:2:1) for C 9 H 10 O 2 Br 2 59

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4.3.2. Spectral Data for Acetoxy Fimbrolide (22) OOBrOHB r O234561'2'3'4'78 Acetoxyfimbrolide (22) Acetoxyfimbrolide (22): 13 mg, 0.0016%, yellow oil; 25 D +47.0 (c, 1.5, CHCl 3 ); IR v max 1788, 1736, 1236, 1092 cm -1 ; UV max 291 nm (log 4.94) ; 1 H NMR (250 MHz, CDCl3) integration, multiplicity, J (Hz), assignment): 6.38 (1H, s, H-6), 5.52 (1H, t, 7.2, H-1), 2.09 (3H, s, H-8), 1.83 (2H, m, H-2), 1.35 (2H, m, H-3) 0.94 (3H, t, 7.5, H-4); The 13 C NMR (125 MHz, CDCl3) (multiplicity, assignment) 170.0 (C, C-7), 163.6 (C, C-2), 149.6 (C, C-4), 131.2 (C, C-3), 130.0 (C, C-5), 93.3 (C, C-6), 68.0 (CH, C-1), 33.5 (CH 2 C-2), 20.4 (CH 3 C-8), 18.3 (CH 2 C-3), 13.5 (CH 3 C-4); LREIMS m/z 370/368/366 (1:2:1) for C 11 H 12 Br 2 O 4 60

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4.3.3. Spectral Data for Hydroxyfimbrolide (23) OOBrOHHB r 234561'2'3'4' Hydroxyfimbrolide (23) Hydroxyfimbrolide (23): 14 mg, 0.0017%, yellow oil; 25 D +15.0 (c, 0.4 CHCl 3 ); IR v max 3457, 2959, 1736 cm -1 ; UV max 292nm (log 4.90); 1 H NMR (250 MHz, CDCl3) integration, multiplicity, J (Hz), assignment): 6.38 (1H, s, H-6), 4.58 (1H, dd, 6.7, 7.0, H-1), 2.54 (1H, d, 9.5, OH), 1.84 (2H, m, H-2), 1.42 (2H, m, H-3), 0.96 (3H, t, 7.5, H-4). The 13 C NMR (125 MHz, CDCl3) ( multiplicity, assignment); 165.5 (C, C-2), 149.9 (C, C-4), 133.6 (C, C-3), 129.7 (C, C-5), 93.7 (C, C-6), 67.7 (CH, C-1), 38.1 (CH 2 C-2), 18.8 (CH 2 C-3), 13.9 (CH 3 C-4) ; LREIMS m/z of 328/326/324 (1:2:1) for C 9 H 10 O 3 Br 2 61

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4.3.4. Spectral Data for Halogenated ketone 40 OBrB r B r 12345678 Halogenated ketone 40 Halogenated ketone 40: 40 mg, 0.005%, colorless oil; IR v max 1715, 1134,1085 cm -1 ; max 220 nm (log 4.84), 283 nm (log 4.03); 1 H NMR (250 MHz, CDCl3) integration, multiplicity, J (Hz), assignment): 2.79 (t, 2H, 7.5, H-4), (1.70-1.33, 6H, H-5 to H-7), 0.94 (3H, 7.0, H-8); 13 C NMR (125 MHz, CDCl3) (multiplicity, assignment): 197.4 (C, C-3), 91.2 (C, C-2), 121.9 (C, C-1), 40.7(CH 2 C-4), 31.1( CH 2 C-5), 23.2(CH 2 C-6), 22.4 (CH 2, C-7), 13.9 (CH 3, C-8); LREIMS m/z 304/306/308/310 (1:2:2:1) for C 8 H 11 Br 3 O. 62

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4.3.5. Spectral Data for Pulchralide A (41) OOOOOOOBrBrBrHHBrO245632a2'4'1'3'788a7a4'a6a4a5a2'a3'a1'a3a Pulchralide A (41) Pulchralide A (41): Colorless crystals; [] 25 D +6.0 (c, 0.25, CHCl3); IR vmax 1747, 1740, 2962, 1220 cm -1 ; UV max 252 nm (log 4.67); 1 H NMR (500 MHz, CDCl3) integration, multiplicity J (Hz), assignment): 5.58 (2H, dd, 6.5, 7.5, H-1, H-1a), 5.17 (2H, s, H-6, H-6a), 2.11 (6H, s, H-8, H-8a) 1.90 (4H, m, H-2, H-2a), 1.36 (4H, m, H-3, H-3a), 0.96 (6H, t, 7.5, H-4, H-4a); 13 C NMR (125MHz, CDCl3) (multiplicity, assignment): 170.0 (C, C-7, C-7a), 164.9 (C, C-2, C-2a), 139.7 (C, C-4, C-4a), 134.5 (C, C-3, C-3a), 90.4 (C, C-5, C-5a), 68.5 (CH, C-1, C-1a), 43.9 (CH, C-6, C-6a) 33.8 (CH 2 C-2, C-2a), 20.8 (CH 3 C-8, C-8a), 18.6 (CH 2 C-3, C-3a), 13.8 (CH 3 C-4, C-4a); EIMS m/z 732/734/736/738 (1:4:6:4:1); HREIMS 734.8260 (C 22 H 24 0 8 79 Br 3 81 Br requires 734.8262) 63

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4.3.6. Spectral Data for Pulchralide B (42) OOOB r BrBrHHB r O245632a2'4'1'3'4'a6a4a5a2'a3'a1'a3a Pulchralide B (42) Pulchralide B (42): Colorless oil; [] 25 D +4.2 (c 0.06, CHCl3); IR vmax 1788, 2958 cm -1 ; UV max 245 nm (log 5.20); 1 H NMR (500 MHz, CDCl3) (integration, multiplicity, J (Hz), assignment): 5.21 (2H, s, H-6, H-6a), 2.43 (2H, t, 7.5, H-1, H-1a), 1.56 (4H, m, H-2, H-2a), 1.35 (4H, m, H-3, H-3a), 0.94 (6H, t, 7.5, H-4, H-4a); 13 C NMR (125MHz, CDCl3) (multiplicity, assignment): 167.4 (C, C-2, C-2a), 138.2 (C, C-4, C-4a), 137.1 (C, C-3, C-3a), 90.5 (C, C-5, C-5a), 44.1 (CH, C-6, C-6a), 29.0 (CH 2 C-1, C-1a), 25.5 (CH 2 C-2, C-2a), 22.4 (CH 2 C-3, C-3a), 13.9 (CH 3 C-4, C-4a); EIMS m/z 615/617/619/621/623(1:4:6:4:1), [M + H]; HREIMS 538.8894 ([M + Br], C 18 H 20 O 4 79 Br 2 81 Br requires 538.8891). 64

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4.3.7. Spectral Data for Pulchralide C (43) OOOOOBrBrBrHHBrO245632a2'4'1'3'84'a6a4a5a2'a3'a1'a3a7 Pulchralide C (43) Pulchralide C (43): Colorless oil [] 25 D +11.6 (c, 0.06, CHCl3); IR v max 1786, 1741, 2961, 1234 cm -1 ; UV max 246 nm (log 5.16); 1 H NMR (500 MHz, CDCl3) (integration, multiplicity, J (Hz), assignment): 5.49 (1H, t, 7.5, H-1), 5.35 (1H, d, 10.0, H-6), 5.13 (1H, d, 10.0, H-6a), 2.38 (1H, t, 7.5, H-1a), 2.09 (3H, s, H-8), 1.89 (4H, m, H-2, H-2a), 1.34 (4H, m, H-3, H-3a), 0.95 (6H, m, H-4, H-4a); 13 C NMR (125MHz, CDCl3) (multiplicity, assignment): 170.3 (C, C-7), 167.0 (C, C-2), 165.3 (C, C-2a), 140.9 (C, C-4), 139.7 (C, C-4a), 135.5 (C, C-3), 134.5 (C, C-3a), 93.3 (C, C-5), 88.1 (C, C-5a), 68.8 (CH, C-1), 47.7 (CH, C-6), 44.4 (CH, C-6a), 33.4 (CH 2 C-2), 28.5 (CH 2 C-2a), 25.4 (CH 2 C-3), 22.5 (CH 2 C-3a), 20.6 (CH 3 C-8), 18.7 (CH 2 C-1), 13.87 (CH 3 C-4), 13.80 (CH 3 ,C-4a); TOFMS m/z 696.8047/698.8026/ 700.8008/702.7991/704.7977 (1:4:6:4:1, C 20 H 22 O 6 79 Br 2 81 Br 2 Na requires 700.8007). 65

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66 References 1. Scheuer, P. J. Chemistry of Marine Natural Products; Academic Press: New york, 1973. 2. Castro, P.; Huber, M.E. Prin ciples of marine science. Marine Biology; McGrawHill: New york, 2003. 3. Haefner, B. Drugs from the deep mari ne natural products as drug candidates. Drug Discov. Today 2003, 8, 536. 4. Scheuer, P. J. Marine Natural Products: Chemical and Biological Perspectives; Academic Press: New York, 1978; Vol. I. 5. Scheuer, P. J. Marine Natural Products: Chemical and Biological Perspectives; Academic Press: New York, 1979; Vol. II. 6. Scheuer, P. J. Marine Natural Products: Chemical and Biological Perspectives; Academic Press: New York, 1981; Vol. III. 7. Scheuer, P. J. Marine Natural Products: Chemical and Biological Perspectives; Academic Press: New York, 1982; Vol. IV. 8. Scheuer, P. J. Marine Natural Products: Chemical and Biological Perspectives; Academic Press: New York, 1983; Vol. V. 9. Fenical, W. H. Natural products chemistry in the marine environment. Science 1982, 215, 923-928. 10. Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1-48, and the previous articles in this series. 11. Paul, V. J. Secondary metabolites from marine organisms. Biomedical Importance of Marine Organisms; Fauntin, D. G. Ed.; California Academy of Sciences: San Francisco, 1988; Vol.13, pp 23-27. 12. McClintock, J. B.; Baker, B. J. Chemical ecology in Antarctic seas. Am. Scientist. 1998, 86, 254-263.

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69 35. Reinhart, K. L.; Holt, T. G.; Fregeau, N. I.; Stroh, J. G.; Kiefer, F.; Sun, L. H.; Li. Martin, D. G. Ecteinascidins 729, 743, 745, 759A, 759B, and 770: potent antitumor agents from the Caribbean tunicate Ecteinascidia turbinate. J. Org. Chem. 1990, 55, 4512-4515. 36. Bowman, A.; Twelves, C.; Hoekman, K.; Si mpson, A.; Smyth, J.; Vermorken, J.; Hoppener, F.; Beijnen, J.; Vega, E.; Jimeno, J.; Hanauske, A. Phase I clinical and pharmacokinetic studies of Ecteinascidin 743. Ann. Oncol. 1998, 9, 119-125. 37. Rinehart, K. L.; Kobayashi, J.; Harbour, G. C.; Hughes, R. G.; Scahill, T. A. Eudistomins C, E, K, and L, potent antiviral compounds containing a novel oxathiazepine ring from the Caribbean tunicate Eudistoma olivaceum. J. Am. Chem. Soc. 1984, 106, 1524-1526. 38. Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner, F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems R. J. The isolation and structure of a remarkable marine anim al antineoplastic constituent: dolastatin 10. J. Am. Chem. Soc. 1987, 109, 6883-6885. 39. Pitot, H. C.; McElroy, E. A., Jr.; Reid, J. M.; Windebank, A. J.; Sloan, J. A.; Erlichman, C.; Bagniewski, P. G.; Walker, D. L.; Rubin, J.; Goldberg, R. M.; Adjei, A. A.; Ames, M. M. Phase I trial of dolastatin-10 in patients with advanced solid tumors. Clin. Cancer Res. 1999, 5, 525-531. 40. Pettit, G. R.; Herald, C. L.; Doubeck, D. L.; Herald, D. L.; Arnold, E.; Clardy, J. Isolation and structur e of bryostatin 1. J. Am. Chem. Soc. 1982, 104, 6846-6848. 41. Philip, P. A.; Rea, D.; Thavasu, P.; Carmichael, J.; Stuart, N. S. A.; Rochett, H.; Talbot, D. C.; Ganesan, T.; Pettit, G. R.; Balkwill, F.; Harris, A. L. Phase I study of byostatin1: assessment of interleuki n 6 and tumor necrosis factor alpha induction in vivo. The cancer research campaign Phase I committee. J. Natl. Cancer Inst. 1993, 85, 1812. 42. Dayton, P. K.; Mordida, B. J.; Bacon, F. Polar marine communities. Am. Zool. 1994, 34, 90. 43. Molinski, T. F.; Faulkner, D.J. An antibacterial pigment from the sponge dendrilla membranosa. Tetrhedron Lett. 1988, 29, 2137-2138. 44. Baker, B. J.; Kopitzke, R.; Hamann, M.; McClintock, J. B. Chemical ecology of Antarctic marine invertebrates in McMur do Sound, Antarctica: Chemical aspects. J. Ant. U.S. 1993, 28, 132-133.

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70 45. Perry, N. B; Blunt, J. W.; McCombs, J. D.; Munro, M. H. G. Discorhabdin C, a highly cytotoxic pigment from a sponge of the genus Latrunculia. J. Org. Chem. 1986, 51, 5476-5478. 46. Yang, A.; Baker, B. J.; Grimwade, J. E.; Leonard, A. C.; McClintock, J. B. Discorhabdin alkaloids from the Antarctic sponge Latrunculia apicalis. J. Nat. Prod. 1995, 58, 1596-1599. 47. Trimurtulu, G.; Faulkner, D. J.; Perry, N. B.; Ettouati, L.; Litaudon, M.; Blunt, J .W; Munro, M. H. G.; Ja meson, G. B. Alkaloids from the Antarctic sponge Kirkpatrickia varialosa. Part 2: Variolin A and N (3')-methyl tetrahydrovariolin B. Tetrahedron 1994, 50, 3993-4000. 48. Jayatilake, G. S.; Baker, B. J.; McClintock, J. B. Rhapsamine, a Cytotoxin from the Antarctic Sponge Leucetta leptorhapsis. Tetrahedron Lett. 1997, 38, 7507-7510. 49. Moon, B. H.; Park, Y. C.; Baker, B. J.; McClintock, J .B. Stru cture and bioactivity of erebusinone, a pigment from Antarctic sponge Isodictya erinacea. Tetrahedron 2000, 9057-9062. 50. Moon, B. H.; Baker, B. J.; McClintock, J. B. Purine and nucleoside metabolites from Antarctic sponge Isodictya erinacea. J. Nat. Prod. 1998, 61, 116-118. 51. Yoshida, W. Y.; Bryan, P. J.; Baker, B. J.; McClintock, J. B. Pteroenone: a defensive metabolite of the abducted Antarctic pteropod Cliona Antarctica. J. Org. Chem. 1995, 60, 780-782. 52. Heine, J. N.; McClintock, J. B.; Slattery, M.; Weston, J. Biochemical and energetic composition, population biology, and chemi cal defense of the antarctic ascidian Cnemidocarpa verrucosa lesson. J .Exp. Mar. Biol. Ecol. 1991, 153, 1525. 53. Kong, F.; Harper, M. K.; Faulkner, D. J. Fuscusine, a tetrahydroquinoline alkaloid from sea star Perknaster fuscus antarcticus. Nat. Prod .Lett. 1992, 1, 71-74. 54. McClinctock, J. B; Baker, B. J.; Slattery, M.; Heine, J. N.; Bryan P. J.; Yoshida, W.; Davies-Coleman, M.T.; Faulkner, D. J. Chemical defense of common Antarctic shallow-water nudibranch Tritoniella belli Eliot (Mollusca: Tritonidae) and its prey, Clavularia frankliniana Rouel (Cnidaria: Octocorallia). J. Chem. Ecol. 1994, 20, 3361-3372.

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72 67. Silverstein, R.M.; Bassler, G.C.; Mo rrill, C.T. Proton magnetic resonance spectrometry, Spectrometric Identification of Organic Compounds; Ed.; John Wiley& Sons: New york1991. 68. Konig, G. M.; Wright A. D. Determina tion of the absolute configuration of a series of halogenated furanone s from the marine red alga Delisea pulchra. Helvetica Chimca Acta, 1995, 78, 758-763. 69. Rose, A. F.; Pettus, J. A, Sims, J. J. Is olation and synthesis of some halogenated ketones from the red seaweed Delisea fimbriata. Tetrahedron Lett, 1977, 22, 1847-1850. 70. Waler, R. P.; Faulkner, D. J.; Van Engen, D.; Clardy J. Sceptrin, an antimicrobial agent from the sponge Agelas sceptrum. J. Am. Chem. Soc. 1981, 103, 6772-6774. 71. Kjlleberg, S.; Steinberg, P. Surface warfare in the sea. Microbiology Today 2001, 28, 134-135. 72. Wright, J. T.; De Nys, R.; Steinber g, P. D. Geographical variation in halogenated furanones from the red alga Delisea pulchra and associated herbivores and epiphytes. Mar. Ecol. Prog. Ser. 2000, 207, 227241. 73. SADABS v2.02: Area-Detector Abso rption Correction. (1996) Siemens Industrial Automation, Inc.: Madison, WI. 74. SHELXTL 6.1, Bruker AXS Inc., Madison, WI, 2001. 75. Ankisetty, S.; Nandiraju, S.; Win, H.; Park, Y. C.; Amsler, C. D.; McClintock, J. B.; Baker, J. A.; Diyabalange, T.; Passeribu, A.; Singh, M. P.; Maiese, W. M.;Walsh, R.D.; Zaworotko, M. J.; Baker, B. J. Chemical investigation of predator-deterred macroalgae from the Antarctic peninsula. J. Nat. Prod. in press.

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73 APPENDICES

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Figure 35. IR spectrum of fimbrolide (21) 00.511.522.53200250300350400wavelengthabsorbance Figure 36. UV spectrum of fimbrolide (21) 74

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Figure 37. LREI Mass spectrum of fimbrolide (23) Figure 38. IR spectrum of acetoxyfimbrolide (22) 75

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Figure 39. UV spectrum of acetoxyfimbrolide (22) 00.20.40.60.811.21.4190240290340390wavelength, nmAbsorbtion Figure 40. LREI Mass spectrum of Acetoxyfimbrolide (22) 76

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Figure 41. IR spectrum of hydroxyfimbrolide (23) 77 Figure 42. UV spectrum of hydroxyfimbrolide (23) 18573 00.020.040.060.080.10.120.140.16190240 290 340390wavelen g th nmabsorption

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Figure 43. IR spectrum of halogenated ketone 40 00.20.40.60.811.2200250300350400wavelengthabsorbance Figure 44. UV spectrum of halogenated ketone 40 78

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Figure 45. LREI Mass spectrum of halogenated ketone 40 Figure 46. IR spectrum of pulchralide A (41) 79

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Figure 47. UV spectrum of pulchralide A (41) 00.050.10.150.20.250.30.350.40.450.5190240290340wavelenght, nmabsorption Figure 48. LREI Mass spectrum of pulchralide A (41) 80

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6 5 4 3 2 1 0 PPM Indirect Dimension 1 10 8 6 4 2 0 Figure 49. gCOSY spectrum of pulchrlalide B (42) (500 MHz, CDCl 3 ) 5 4 3 2 1 PPM Direct Dimension 140 120 100 80 60 40 20 0 Figure 50. gHMQC spectrum of pulchralide B (42) (500 MHz, CDCl 3 ) 81

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Figure 51. IR spectrum of pulchralide B (42) 00.20.40.60.811.21.41.61.8150200250300350400wavelengthabsorbance Figure 52. UV spectrum of pulchralide B (42) 82

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Figure 53. LREI Mass spectrum of pulchralide B (42) 70 60 50 40 30 20 10 0 PPM Indirect Dimension 1 9 8 7 6 5 4 3 2 1 0 Figure 54. gHMQC spectrum of pulchralide C (43) (500 MHz CDCl 3 ) 83

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70 60 50 40 30 20 10 0 PPM Indirect Dimension 1 9 8 7 6 5 4 3 2 1 0 Figure 55. gHMBC spectrum of pulchralide C (43) (500 MHz, CDCl 3 ) Figure 56. IR spectrum of pulchralide C (43) 84

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00.20.40.60.811.21.41.61.8150200250300350400wavelengthabsorbance Figure 57. UV spectrum of pulchralide C (43) Figure 58. LRESI Mass spectrum of pulchralide C (43) 85


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Chemical and biological investigation of the Antarctic red alga delisea pulchra
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ABSTRACT: Our interest in the red alga Delisea pulchra (=D.fimbriata) (Greville) Montagne 1844 (Rhodophyceae, Bonnemaisoniales, Bonnemaisoniaceace) was stimulated by its activity in the biosssays done at Wyeth Pharmaceuticals. Halogenated compounds from D. pulchra interfere with Gram-negative bacterial signaling systems, affect the growth of Gram-positive bacteria, inhibit quorum sensing and swarming motility of marine bacteria (inhibit bacterial communication). They also inhibit surface colonization in marine bacteria and exhibit antifouling properties against barnacle larvae and macroalgal gametes. Chemical investigation of D.pulchra collected near Palmer Station, Antarctica yielded three new dimeric halogenated furanones, pulchralide A-C (41-43), along with previously reported fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23) and halogenated ketone 40.
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