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Novel Structures and Activities of Secondary Metabolites from Floridian Marine Cyanobacteria

Permanent Link: http://ufdc.ufl.edu/UFE0022462/00001

Material Information

Title: Novel Structures and Activities of Secondary Metabolites from Floridian Marine Cyanobacteria
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Taori, Kanchan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Marine cyanobacteria are a rich source of structurally intriguing bioactive compounds. Many of these metabolites exhibit cytotoxic activity, antiproliferative activity and specific protease inhibitory activity. Marine cyanobacteria from Florida sea water of the genera Symploca (Pillars, Key Largo, Florida Keys) and Lyngbya (Summerland Key, Florida Keys) were extracted and investigated for their biomedical importance. Target-based screening of Lyngbya sp. was carried out resulting in the isolation of three new compounds along with already reported analog somamide B with specific serine protease inhibitory activity. Lyngbyastatin 7 and somamide B, analogs of dolastatin 13, were isolated with potent elastase-inhibitory activity. Elastase overactivity is involved in tissue destruction and inflammation characteristic of various diseases such as emphysema, adult respiratory distress syndrome, as well as cutaneuos wrinkling. Kempopeptin A with elastase and chymotrypsin-inhibitory activity and kempopeptin B with trypsin-inhibitory activity were also isolated. Overactivity of the serine proteases, trypsin and chymotrypsin, can lead to the gland destruction, resulting in acute pancreatitis. Their structures were elucidated by a combination of NMR techniques (1D and 2D) and absolute configurations were established by either chiral HPLC or by modified Marfey's analysis of the acid hydrolyzates. Their structural differences and distinct selectivity profile against three proteases (trypsin, chymotrypsin, and elastase) allowed deducing a structure-activity relationship (SAR). Most importantly, with its low-nanomolar activity lyngbyastatin 7 represents one of the most potent inhibitors of elastase. Selectivity for elastase over trypsin was conferred by a hydrophobic residue that binds to the enzyme's specificity pocket. Furthermore, the double bond in the same unit also conferred unprecedented selectivity over chymotrypsin, possibly due to stabilizing CH/? interaction specifically with elastase. Phenotypic screening of Symploca sp. was carried out resulting in the isolation of one novel cytotoxin, termed largazole. The structure of largazole was elucidated mainly by 1D and 2D NMR spectroscopy and the absolute configuration was established by chiral HPLC and Marfey's analysis of the degradation products. Largazole displayed differential growth-inhibitory activity against transformed versus nontransformed cells. The molecular target and pharmacophore of largazole was further investigated and described, suggesting it to be a potent class I HDAC inhibitor. Structure-activity relationship studies were carried out which revealed that the thiol group was the pharmacophore of the natural product.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kanchan Taori.
Thesis: Thesis (M.S.P.)--University of Florida, 2008.
Local: Adviser: Luesch, Hendrik.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022462:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022462/00001

Material Information

Title: Novel Structures and Activities of Secondary Metabolites from Floridian Marine Cyanobacteria
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Taori, Kanchan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Marine cyanobacteria are a rich source of structurally intriguing bioactive compounds. Many of these metabolites exhibit cytotoxic activity, antiproliferative activity and specific protease inhibitory activity. Marine cyanobacteria from Florida sea water of the genera Symploca (Pillars, Key Largo, Florida Keys) and Lyngbya (Summerland Key, Florida Keys) were extracted and investigated for their biomedical importance. Target-based screening of Lyngbya sp. was carried out resulting in the isolation of three new compounds along with already reported analog somamide B with specific serine protease inhibitory activity. Lyngbyastatin 7 and somamide B, analogs of dolastatin 13, were isolated with potent elastase-inhibitory activity. Elastase overactivity is involved in tissue destruction and inflammation characteristic of various diseases such as emphysema, adult respiratory distress syndrome, as well as cutaneuos wrinkling. Kempopeptin A with elastase and chymotrypsin-inhibitory activity and kempopeptin B with trypsin-inhibitory activity were also isolated. Overactivity of the serine proteases, trypsin and chymotrypsin, can lead to the gland destruction, resulting in acute pancreatitis. Their structures were elucidated by a combination of NMR techniques (1D and 2D) and absolute configurations were established by either chiral HPLC or by modified Marfey's analysis of the acid hydrolyzates. Their structural differences and distinct selectivity profile against three proteases (trypsin, chymotrypsin, and elastase) allowed deducing a structure-activity relationship (SAR). Most importantly, with its low-nanomolar activity lyngbyastatin 7 represents one of the most potent inhibitors of elastase. Selectivity for elastase over trypsin was conferred by a hydrophobic residue that binds to the enzyme's specificity pocket. Furthermore, the double bond in the same unit also conferred unprecedented selectivity over chymotrypsin, possibly due to stabilizing CH/? interaction specifically with elastase. Phenotypic screening of Symploca sp. was carried out resulting in the isolation of one novel cytotoxin, termed largazole. The structure of largazole was elucidated mainly by 1D and 2D NMR spectroscopy and the absolute configuration was established by chiral HPLC and Marfey's analysis of the degradation products. Largazole displayed differential growth-inhibitory activity against transformed versus nontransformed cells. The molecular target and pharmacophore of largazole was further investigated and described, suggesting it to be a potent class I HDAC inhibitor. Structure-activity relationship studies were carried out which revealed that the thiol group was the pharmacophore of the natural product.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kanchan Taori.
Thesis: Thesis (M.S.P.)--University of Florida, 2008.
Local: Adviser: Luesch, Hendrik.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022462:00001


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1 NOVEL STRUCTURES AND ACTIVITIES OF SECONDARY METABOLITES FROM FLORIDIAN MARINE CYANOBACTERIA By KANCHAN TAORI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY UNIVERSITY OF FLORIDA 2008

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2 2008 Kanchan Taori

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3 To my parents

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4 ACKNOWLEDGEMENT I would like to take this opport unity to thank m y research su pervisor, Dr. Hendrik Luesch, for his benevolent guidance, constructive criticis m, and constant encouragement. It was his vigilant supervision of the work that enabled me to give present shape to this work and publications that have or will come out of this thesis. I would like to thank our collaborator, Dr. Valerie Paul, for providing cyanobacterial extracts without which this work would not have been possible. I would like to thank my committee members, Dr. Margaret O. Jame s and Dr. Brian Law, for their support and availability. I owe special thanks to my parents and brothe rs, for all the unconditional support they have given me throughout my career. Needless to menti on, I would not have seen this day without the emotional support provided by my husband. I would also like to thank Jim Rocca, for a ssistance in obtaining NMR data, Dr. Jodie V. Johnson, Department of Chemistry, for car rying out LC-MS analyses, Dr. Jiyong Hong, Department of Chemistry, Duke University, fo r providing us with synthetic largazole and analogs, and Dr. Susan Matthew, for providing a synthetic standard of N,O -dimethyl-Lbromotyrosine. Special thanks go to my friends, Preeti Subhe dar and Manish Rangnekar for their constant support and encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGEMENT...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 LIST OF SCHEMES......................................................................................................................10 ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION AND BACKGROUND........................................................................... 13 2 SECONDARY METABOLITES FROM Lyngbya majuscula ...............................................16 2.1 Overview...........................................................................................................................16 2.2 Lyngbyastatin 7 and Somamide B.................................................................................... 16 2.2.1 Isolation and Structure Dete rm ination of Lyngbyastatin 7.................................... 17 2.2.2 Absolute Configuration..........................................................................................21 2.3 Kempopeptin A ................................................................................................................21 2.3.1 Isolation and Structure Dete rm ination of Kempopeptin A.....................................22 2.3.2 Absolute Configuration..........................................................................................23 2.4 Kempopeptin B.................................................................................................................26 2.4.1 Isolation and Structure Dete rm ination of Kempopeptin B.....................................26 2.4.2 Absolute Configuration..........................................................................................27 2.5 Biological activity ....................................................................................................... .....30 2.6 Structure-Activity Relationship (SAR).............................................................................31 2.7 Summary...........................................................................................................................32 3 SECONDARY METABOLITES FROM Symploca sp .........................................................34 3.1 Overview...........................................................................................................................34 3.2 Largazole..........................................................................................................................35 3.2.1 Isolation and Structure Determination of Largazole.............................................. 35 3.2.2 Absolute Configuration..........................................................................................36 3.3 Biological Evaluation.......................................................................................................39 3.3.1 Cell Viability Assays.............................................................................................. 39 3.3.2 HDACs as Molecular Targets................................................................................ 40 3.3.3 Structure-Activity Relationship (SAR)..................................................................44 3.3.4 HDAC Isoform Selectivity..................................................................................... 44 3.4 Summary...........................................................................................................................45

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6 4 EXPERIMENTAL SECTION................................................................................................46 4.1 General..............................................................................................................................46 4.1.1 Spectral Analysis.................................................................................................... 46 4.1.2 Amino Acid Standards........................................................................................... 47 4.2 Biological Material........................................................................................................ ...47 4.2.1 Lyngbya Collection .................................................................................................47 4.2.2 Symploca Collection ...............................................................................................48 4.3 Extraction and Isolation................................................................................................... .48 4.3.1 Isolation of Lyngbya Metabolites ...........................................................................48 4.3.2 Isolation of Symploca Metabolites .........................................................................49 4.4 Physical Data....................................................................................................................50 4.4.1 Lyngbya Metabolites ..............................................................................................50 4.4.2 Symploca Metabolites .............................................................................................51 4.5 Determination of Absolute Configuration........................................................................ 51 4.5.1 Lyngbyastatin 7 and Somamide B..........................................................................51 4.5.2 Kempopeptin A......................................................................................................52 4.5.3 Kempopeptin B.......................................................................................................53 4.5.4 Largazole................................................................................................................55 4.6 Testing for Biological Activity......................................................................................... 56 4.6.1 Protease Activity Assay.......................................................................................... 56 4.6.2 Cell Culture............................................................................................................57 4.6.3 General Cytotoxicity Assay.................................................................................... 57 4.6.4 Cell-free HDAC Enzymatic Assay with HeLa Nuclear Extract............................. 58 4.6.5 Cellular HDAC Activity Assay..............................................................................58 4.6.6 Enzymatic Assay with Recombinant Human HDAC1 or HDAC6........................ 59 4.6.7 Immunoblot Analysis............................................................................................. 59 APPENDIX: NMR SPECTRA...................................................................................................... 61 LIST OF REFERENCES............................................................................................................... 91 BIOGRAPHICAL SKETCH.........................................................................................................95

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7 LIST OF TABLES Table page 2-1 NMR Spectral Data for Lyngbyastatin 7 ( 1) in DMSO-d6...............................................19 2-2 NMR data for Both Conformers of Kempopeptin A (3) in DMSO-d6 (ratio 1:1) at 500 MHz (1H) and 150 MHz (13C)....................................................................................24 2-3 NMR data for Kempopeptin B ( 4) in DMSOd6 at 600 MHz (1H) and 150 MHz (13C).... 28 2-4 Protease Inhibitory Activity (IC50) from Metabolites Isolated from the Lyngbya sp. from Kemp Channel........................................................................................................... 30 3-1 NMR Spectral Data for Largazole ( 5 ) in CDCl3 (600 MHz)............................................37 3-2 Growth-Inhibitory Activity (GI50) of Natural Product Drugs........................................... 40 3-3 IC50 and GI50 Values for HDACs and Growth Inhibition (nM)........................................43 3-4 IC50 Values for HDAC1 and HDAC6 Inhibition (nM).....................................................45

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8 LIST OF FIGURES Figure page 1-1 Potent cytotoxic cyanobacterial metabolites, derivatives of which ar e in clinical trials... 14 2-1 Structures of lyngbyastatin 7 ( 1 ) and somamide B ( 2)......................................................16 2-2 Structure of kempopeptin A ( 3) .........................................................................................22 2-3 Structure of kempopeptin B ( 4) .........................................................................................26 2-4 SAR comparison of protease inhibitors from Lyngbya sp. ................................................31 3-1 Structure of largazole ( 5) ...................................................................................................35 3-2 ESI-MSn fragmentation pattern for largazole ( 5 )...............................................................38 3-3 Structural similarity be tween FK228 and largazole ( 5) and modes of activation.............. 41 3-4 Target identification for largazole..................................................................................... 42 3-5 Structures of synthe tic largazole analogs* ......................................................................... 43 A-1 1H Spectrum of Lyngbyastatin 7 ( 1 ) in DMSO-d6.............................................................62 A-2 13C Spectrum of Lyngbyastatin 7 ( 1 ) in DMSO-d6............................................................63 A-3 COSY Spectrum of Lyngbyastatin 7 ( 1) in DMS Od6......................................................64 A-4 ROESY Spectrum of Lyngbyastatin 7 ( 1) in DM SOd6....................................................65 A-5 TOCSY Spectrum of Lyngbyastatin 7 ( 1) in DM SOd6....................................................66 A-6 HMQC Spectrum of Lyngbyastatin 7 ( 1) in DM SOd6.....................................................67 A-7 HMBC Spectrum of Lyngbyastatin 7 ( 1) in DM SOd6.....................................................68 A-8 1H Spectrum of Somamide B ( 2) in DMSO-d6..................................................................69 A-9 1H Spectrum of Kempopeptin A ( 3) in DMSOd6.............................................................70 A-10 13C Spectrum of Kempopeptin A ( 3) in DMSOd6............................................................71 A-11 COSY Spectrum of Kempopeptin A (3) in DM SOd6.......................................................72 A-12 ROESY Spectrum of Kempopeptin A ( 3) in DM SOd6....................................................73 A-13 TOCSY Spectrum of Kempopeptin A ( 3) in DM SOd6....................................................74

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9 A-14 HSQC Spectrum of Kempopeptin A (3) in DM SOd6.......................................................75 A-15 HMBC Spectrum of Kempopeptin A ( 3) in DM SOd6.....................................................76 A-16 1H Spectrum of Kempopeptin B ( 4 ) in DMSO-d6.............................................................77 A-17 13C Spectrum of Kempopeptin B ( 4 ) in DMSO-d6...........................................................78 A-18 COSY Spectrum of Kempopeptin B (4) in DM SOd6.......................................................79 A-19 ROESY Spectrum of Kempopeptin B ( 4) in DM SOd6....................................................80 A-20 TOCSY Spectrum of Kempopeptin B ( 4) in DM SOd6....................................................81 A-21 HSQC Spectrum of Kempopeptin B (4) in DM SOd6.......................................................82 A-23 1H Spectrum of Largazole ( 5) in CDCl3............................................................................84 A-24 13C Spectrum of Largazole ( 5) in CDCl3...........................................................................85 A-25 COSY Spectrum of Largazole ( 5) in CDCl3......................................................................86 A-28 HMBC Spectrum of Largazole ( 5) in CDCl3 (optimized for nJCH = 3.5 Hz).....................89 A-29 NOESY Spectrum of Largazole ( 5) in CDCl3...................................................................90

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10 LIST OF SCHEMES Schem e page 2-1 Isolation Strategy for Lyngbya sp. (Non-Polar E xtract).................................................... 18 3-1 Isolation Strategy for Symploca sp. (Non-Polar E xtract)................................................... 34 3-2 Degradation Strategy to Liberate Chiral Subunits ............................................................. 39

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacy NOVEL STRUCTURES AND ACTIVITIES OF SECONDARY METABOLITES FROM FLORIDIAN MARINE CYANOBACTERIA By Kanchan Taori August 2008 Chair: Hendrik Luesch Major: Pharmaceutical Sciences Marine cyanobacteria are a ri ch source of structurally intriguing bioactive compounds. Many of these metabolites exhibit cytotoxic activity, antiproliferative activity and specific protease inhibitory activity. Marine cyanobacteria from Flor ida sea water of the genera Symploca (Pillars, Key Largo, Florida Keys) and Lyngbya (Summerland Key, Florida Keys) were extracted and investigated for their biomedical importance. Target-based screening of Lyngbya sp. was carried out resulting in the isolation of three new co mpounds along with already repor ted analog somamide B with specific serine protease inhibitory activity. Lyngbyastatin 7 and soma mide B, analogs of dolastatin 13, were isolated with potent elasta se-inhibitory activity. Elastase overactivity is involved in tissue destruction and inflammation characteristic of various diseases such as emphysema, adult respiratory distress syndrome, as well as cutaneuos wrinkling. Kempopeptin A with elastase and chymotrypsin -inhibitory activity and kempopep tin B with trypsin-inhibitory activity were also isolated. Overactivity of the serine proteases, trypsin and chymotrypsin, can lead to the gland destruction, resulting in acute pa ncreatitis. Their structures were elucidated by a combination of NMR techniques (1D and 2D) and absolute configurations were established by either chiral HPLC or by modified Marfeys analysis of the acid hydrolyzates.

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12 Their structural differences and distinct select ivity profile against three proteases (trypsin, chymotrypsin, and elastase) allowed deducing a structure-activity relationship (SAR). Most importantly, with its low-nanomolar activity lyng byastatin 7 represents one of the most potent inhibitors of elastase. Selectivity for el astase over trypsin was conferred by a hydrophobic residue that binds to the enzymes specificity pocket. Furthermore, the double bond in the same unit also conferred unprecedente d selectivity over chymotrypsi n, possibly due to stabilizing CH/ interaction specifically with elastase. Phenotypic screening of Symploca sp. was carried out resulti ng in the isolation of one novel cytotoxin, termed la rgazole. The structure of largazole was elucidated mainly by 1D and 2D NMR spectroscopy and the absolute configur ation was established by chiral HPLC and Marfeys analysis of th e degradation products. Largazole disp layed differential growth-inhibitory activity against transf ormed versus nontransformed cells. The molecular target and pharmacophore of largazole was further investigat ed and described, sugges ting it to be a potent class I HDAC inhibitor. Structureactivity relationship studies we re carried out which revealed that the thiol group was the pharm acophore of the natural product.

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13 CHAPTER 1 INTRODUCTION AND BACKGROUND Cyanobacteria (blue-green algae) are ubiquitous, photoautotrophic, prokaryotic m icrorganisms that originated 3.5 billions years ago.1 Some species of cyanobacteria like freshwater Microcystis spp. can grow as unicellular organi sms and secrete toxic compounds such as microcystins and nodularia, whereas some species of marine cyanobacteria like Lyngbya sp. can grow in filamentous units and produce a wi de array of chemical compounds; many of them are toxic and also contain irritable properties. Th e marine biosphere can be considered as one of Earths richest hab itats but underexplored as of yet. The greater the biodiversity in the marine biosphere, the greater the opportunity for discoveries which ultimately lead to potential targets for biomedical development.2 Cyanobacteria have been known to inhabit many di fferent and extreme environments as well as to participate in symbiotic relationships with se veral other invertebrates which may be indicative of their ability to produce a unique range of defensive metabolites.3 Marine cyanobacterial peptides are considered products of nature s own combinatorial biosynthesis. Marine cyanobacteria are well known to produce an inte resting array of compoun ds ranging from simple linear peptides to complex m acrocyclic peptides. Over 600 cy anobacterial peptides possessing diverse structural types have been reported from the prokaryotic marine cyanobacteria.4 They have been shown to biosynthesi ze a plethora of structurally distinct bioactive secondary metabolites that cover a wide range of pharm acological effects including antitumor, antiinflammatory, analgesic, antialle rgy, and antiviral activ ity, and specific inhibitors of enzymes.5 Some metabolites isolated from cyanobacteria with promising anticancerous activity have entered into either preclinical or clinical trials. For example crytophycin-52, a s ynthetic analogue of cryptophycin-16 (Figure 1-1) and dolastatin 10 which have been evaluated in phase I and II

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14 clinical trials. Dolastatin 10 (Figure 1-1), a linear peptide that was originally isolated from the sea hare Dolabella auricularia by Pettits group,7 displayed potent antimitotic activity, and was later shown to be a cyanobacterial metabolite.8 N H N O N O O O N O O H N S N Dolastatin10 O O O N H O HN Cl O O O Cryptophycin-1 Figure 1-1. Potent cytotoxic cyanob acterial metabolites, derivatives of which are in clinical trials L. majuscula is one of the most abundant benthi c marine filamentous cyanobacterium .9 A large number of structurally diverse bi oactive compounds such as curacins A-D,10 lyngbyastatins 1-2,11 microcolins A-C,12 majusculamides A-D,13 and malyngamides A-U14 have been isolated, exhibiting an array of biologica l activity including antibiotic, anticancer, antifungal, antiviral, cytotoxic, immunosuppressant and antimitotic activ ity. It has been known that toxins from L.majuscula such as lyngbyatoxins are harmful for human health,13b but some of the cytotoxins isolated from this species might be useful as antiproliferative drugs. It has also been known to produce inhibitors of serine proteases such as elastase, plasmin, thrombin, trypsin and chymotrypsin. These proteolytic enzymes ar e responsible for controlling many biological processes. Elastase has been implicated in pul monary emphysema and rheumatoid arthritis, and thrombin is important in the formation of blood clots.15 Plasmin is another serine protease that regulates blood coagulation and has been implicated in cardiovascular dis eases, such as stroke and coronary occlusion.3 Other proteases with biologica l importance are trypsin, and chymotrypsin which are important pancreatic enzymes. The biological importance of these enzymes indicated that the discovery of novel inhibitors of these enzymes may be of great

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15 benefit. Thus, we screened a collection of the cyanobacterium L. majuscula for specific protease inhibitory activity by target-based screening and isolated four new protease inhibitors with three distinct selectivity profiles. Marine cyanobacteria, especially of the genus Symploca, have recently been proven to be source of an array of structurally distinct and biologically active secondary metabolites such as symplostatins 1-3,16 dolastatin 10,7 tasiamides A and B,17 tasipeptins A and B,18 belamide A19 and most recently symplocamide A20 and malevamide E.21 Dolastatin 10 and its synthetic analog TZT-1027,22 have entered phase I or II clinical trial for use in an ticancer therapy. Thus, we screened cyanobacteria Symploca sp. in phenotype-based assays fo r their ability to inhibit cancer cell growth. We found one extract to be cytotoxic and then proceeded with the isolation and structure elucidation of a novel anti proliferative agent, largazole.

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16 CHAPTER 2 SECONDARY METABOLITES FROM Lyngbya majuscula 2.1 Overview More than 100 novel m etabolites originated from L. majuscula collected from various geographical locations as descri bed in the Introduction (chapter 1). In the search of novel protease inhibitors from marine cyanobacteria in Florida waters, we invest igated a collection of Lyngbya sp. with morphological characteristics of L. majuscula from a mangrove channel at Summerland Key in the Florida Keys. Chem ical investigation of an extract of Lyngbya sp. yielded one dolastatin 1323 analog, lyngbyastatin 7 ( 1),24 along with the already reported compound somamide B ( 2)25 with potent elasta se-inhibitory activity and kempopeptin A ( 3) with an elastaseand chymotrypsin-inhib itory activity and kempopeptin B ( 4 ) with trypsin-inhibitory activity. Their isolation, structure determinati on and biological activity are described below. Their structural differences and distinct select ivity profiles against th ree proteases (trypsin, chymotrypsin, and elastase) allowed deduci ng a structure-activity relationship (SAR). 2.2 Lyngbyastatin 7 and Somamide B Lyngbyastatin 7 (1) and som amide B ( 2) were isolated from th e marine cyanobacterium Lyngbya sp. and are structural analogs of dolastatin 13. Figure 2-1. Structures of lyngbyastatin 7 ( 1 ) and somamide B ( 2)

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17 2.2.1 Isolation and Structure Determination of Lyngbyastatin 7* A sample of the cyanobacterium Lyngbya sp. was collected from Kemp Channel, a mangrove channel to the southw est of Summerland Key in the Florida Keys. The sample was freeze dried and extracted with CH2Cl2MeOH (1:1). Fractionation by solvent partition and successive chromatographic steps using silica, C18 cartridges and finally reversed-phase HPLC afforded lyngbyastatin 7 ( 1) along with somamide B ( 2)25 as a colorless, amorphous solid. NMR data combined with a [M + Na] + peak at m/z 969.4710 in the HRESI/APCIMS of 1 suggested a molecular formula of C48H66N8O12. Analysis of 1H NMR, 13C NMR, HMQC, COSY, TOCSY, and HMBC spectra revealed the presen ce of valine, threonine, phenylalanine, N methyltyrosine, glutamine, hexanoic acid (Ha), 2-amino-2-butenoic acid (Abu) and 3-amino-6hydroxy-2-piperidone (Ahp) moieties (Table 2-1). HMBC and ROESY analysis (Table 2-1) and comparison of 1H and 13C NMR data revealed that the cyclic core structure for this compound is identical to that observed for lyngbyastatin 4.26 Furthermore ROESY correlations between Thr NH ( H 7.87) and Gln H-2 (H 4.39) and from Gln 2-NH ( H 8.07) to Ha H2-2 ( H 2.13) are consistent with the propose d structure shown for compound 1. Compound 1 is most closely related to the previously reported cyanobacterial metabolite somamide B ( 2), which differs from 1 only by the presence of a terminal butanoic acid (Ba) residue in the side chain instead of the hexanoic acid (Ha) residue in 1. ROESY cross peaks between the Abu methyl group and the Abu NH in compounds 1 and 2 unequivocally established the Z geometry of the Abu group. Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy

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18 Non-pola r ex t r ac t ( Lyngbya sp.) 24.1256g hexanes80:20MeOH-H2O 2.9286g 20.24g 1.6102g 18.520g 100%CH2Cl224.8mg 2% iPrOH 6.8mg 5% iPrOH 4.7mg 10% iPrOH 75.8mg 20% iPrOH 13.7mg 50% iPrOH 168.5mg 75% iPrOH 165mg 100%MeOH 538.3mg C18column 100%H2O 60mg 25%MeOH 0.9mg 50%MeOH 5.4mg 75%MeOH 45.3mg 100%MeOH 47.9mg 100%H2O 105mg 25%MeOH 1.4mg 50%MeOH 2.2mg 75%MeOH 13mg 100%MeOH 37.9mg Liquid-Liquidpartitioning Silicagelchromatography KempopeptinA( 3 )at tR30.2min(1.0mg) Lyngbyastatin7( 1 )at tR35.2min(7.4mg) SomamideB( 2 )at tR26.2min(1.2mg) Lyngbyastatin7( 1 )at tR35.2min(3.1mg) 100%CH2Cl2400mg 20%MeOH 23.2mg 60%MeOH 13mg 80%MeOH 10.5mg KempopeptinB( 4 )at tR25.2min(1.6mg)Reversed-phaseHPLC YMC-packODS-AQ,25010mm,2.0mL/min MeOH/H2Ogradientn -butanol H2O 40%MeOH 21.2mg C18column ReversedphaseHPLC YMC-packODS-AQ,25010mm,2.0mL/min MeOH/H2Ogradient Silicacolumn Scheme 2-1. Isolation Strategy for Lyngbya sp. (Non-Polar Extract)

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19Table 2-1. NMR Spectral Data for Lyngbyastatin 7 ( 1) in DMSOd6 Unit C/H no. H ( J in Hz)a C, mult.b COSYa HMBCa,c,d ROESYa,d Val 1 173.9, qC 2 4.71, br 56.1, CH H-3, NH 1 ( N -MeTyr) H-3, H3-4, H3-5, NH 3 2.08, m 30.9, CH H3-4, H3-5 1, 2, 4, 5 H-2, H3-4, H3-5, NH 4 0.86, d (6.8) 19.3, CH3 H-3 2, 3, 5 H-2, H-3, H3-5, N -Me ( N -Me-Tyr), H3-4 (Thr), NH 5 0.74, d (6.8) 17.5, CH3 H-3 2, 3, 4 H-2, H-3, H3-4, NH, N -Me ( N -Me-Tyr) NH 7.47, br d (8.5) H-2 H-2, H-3, H3-4, H3-5, N -Me ( N -Me-Tyr), H-2 ( N -Me-Tyr), 6-OH (Ahp) N -Me-Tyr 1 169.4, qC 2 4.88, d (11.7) 60.8, CH H-3a, H-3b 1, 3, 4 H-3a, H-3b, N -Me, H-5/9, H-5/9 (Phe), NH (Val), H-2 (Phe), H-3b (Phe) 3a 3.07, d (13.5) 32.8, CH2 H-2, H-3b 2, 5/9 H-2, H-3b, H-5/9, H-2 (Phe) 3b 2.69, dd (13.5, 11.7) H-2, H-3a 1, 2, 5/9 H-2, H-3a, H-5/9 4 127.8, qC 5/9 6.97, d (8.4) 130.5, CH H-5/9 4, 5, 7, 8 H-2, H-3a, H-3b, H-6/8, N -Me, H-2 (Phe) 6/8 6.75, d (8.4) 115.3, CH H-6/8 4, 7, 9 H-5/9, 7-OH, H-5/9 (Phe) 7 156.2, qC 7-OH 9.37, s 7, 5/9 H-6/8 N-Me 2.74, s 30.4, CH3 2, 1 (Phe) H-2, H-5/9, H3-4 (Val), H3-5 (Val), NH (Val) Phe 1 170.5, qC 2 4.73, dd (11.5, 4.4) 50.3, CH H-3a, H-3b 1, 3, 2 (Ahp) H-3a, H-3b, H-5/9, H-2 (N -Me-Tyr), H-3a ( N Me-Tyr), H-5/9 ( N -Me-Tyr), H-6 (Ahp) 3a 2.86, dd (13.7, 11.5) 35.3, CH2 H-2, H-3b 2, 4, 5/9 H-2, H-3b, H-5/9, H-6 (Ahp), 6-OH (Ahp) 3b 1.81, dd (13.7, 4.4) H-2, H-3a 2, 4, 5/9 H-2, H-3a, H-6 (Ahp), H-2 ( N -Me-Tyr) 4 136.7, qC 5/9 6.83, d (6.9) 129.4, CH H-6/8 3, 5/9, 7 H-2, H-3a, H-6/8, H-2 ( N -Me-Tyr), H-6/8 ( N Me-Tyr) 6/8 7.18, m 127.5, CH H-5/9 4, 6/8 H-5/9, H-7 7 7.14, m 126.3, CH H-6/8 5/9, 6/8 H-6/8 Ahp 2 168.9, qC 3 3.79, ddd (11, 9, 6) 48.2, CH H-4a, H-4b, NH 2, 4 NH, H-5/9 (Phe), H-3 (Abu), H-2 (Phe), H-4b, H-5b 4a 2.40, m 21.9, CH2 H-4b, H-5a, H-5b, H-3 H-4b, H-5a, 6-OH, NH 4b 1.56, m H-4a, H-5a, H-3 H-3, H-4a, NH

PAGE 20

20Table 2-1. Continued Unit C/H no. H ( J in Hz)a C, mult.b COSYa HMBCa,c,d ROESYa,d 5a 1.72, m 29.3, CH2 H-4a, H-4b, H-5b, H-6 H-5b, H-6, 6-OH 5b 1.55, m H-5a, H-6, H-4a H-3, H-5a, H-6, 6-OH 6 5.07, s 73.8, CH 6-OH, H-5a, H-5b H-5a, H-5b, 6-OH, H-2 (Phe), H-3a (Phe), H-3b (Phe) 6-OH 6.09, s H-6 H-4a, H-5a, H-5b, H-6, NH (Val), H-3a (Phe) NH 7.17, d (9) H-3, H-4a, H-4b, NH (Abu) Abu 1 162.8, qC 2 130.0, qC 3 6.51, q (6.9) 131.8, CH H3-4 1, 2, 4 H3-4, H-3 (Ahp) 4 1.48, d (6.9) 13.1, CH3 H-3 2, 3 H-3, NH, H-2 (Thr) NH 9.17, br s H3-4, H-2 (Thr), NH (Ahp) Thr 1 172.7,e, qC 2 4.53, br 55.7, CH NH (Abu), H-3, H3-4, H3-4 (Abu) 3 5.47, br 71.8, CH H3-4 H3-4, H-2 4 1.21, d (6.5) 18.1, CH3 H-3 2, 3 H-2, H-3, H3-4 (Val), h-2 (Gln), NH NH 7.87, br H-2 H-2 (Gln), H3-4 Gln 1 172.7, qC 2 4.39, ddd (8, 8, 6) 52.2, CH 1, 4 H-3a, H-3b, H2-4, H3-4 (Thr), NH (Thr), H2-3 (Ha) 3a 1.91, m 26.9, CH2 H-3b, H-2, H2-4 H-2 H-3b, H2-4 3b 1.71, m H-3a, H-2, H2-4 1, 2, 4, 5 H-2, H2-4, H-3a, 2-NH, H2-2 (Ha) 4 2.12, m (2H) 31.5, CH2 H-3a, H-3b 5 H-2, H-3b, 2-NH, 5-NHa 5 173.8, qC 2-NH 8.07, br s H-2 H-3b, H2 -4, H2-2 (Ha) 5-NHa 7.22, br s 5 H2 -4, H2-2 (Ha) 5-NHb 6.72, br s 4 Ha 1 172.5, qC 2 2.13, m (2H) 35.1, CH2 H2 -3 1, 3 H2 -3, H2 -4/5, H-3b (Gln), 2-NH (Gln), 5-NHa (Gln) 3 1.49, m (2H) 24.9, CH2 H2-2, H2 -4, H2 -5 2, 4, 5 H3 -6, H2-2, H2 -4/5, 2-NH (Gln), H-2 (Gln), H2 -4 (Gln) 4 1.27, m (2H) 30.9, CH2 H2 -3 3 H2-2, H2 -3, H2 -5, H3 -6 5 1.27, m (2H) 21.9, CH2 H3 -6 4, 6 H2-2, H2 -3, H2 -4, H3 -6 6 0.84, t (7.0) 13.9, CH3 H2 -5 4, 5 H2 -3, H2 -4/5 aRecorded at 500 MHz. b Recorded at 150 MHz. c Protons showing HMBC correlations to the indicated carbon. d Refers to nuclei within the same unit unless indicated otherwise. e No HMBC correlation observed. Carbon assigned to Thr unit base d on remaining unassigned signal in the 13C NMR.

PAGE 21

21 2.2.2 Absolute Configuration* The absolute configuration of th e amino acid residues in compounds 1 and 2 determined by modified Marfeys analysis 27 suggested that all the amino acids are in the L-form. The absolute configuration at C-3 of each Ahp residue was determined after CrO3 oxidation and acid hydrolysis. This reaction sequence liberated L-glutamic acid which permitted us to establish the configuration of th e Ahp residues as 3 S. It was found earlier for l yngbyastatin 4 that oxidation prior to hydrolysis increase s the yield of phenylalanine;26 this procedure again enabled us to clearly assign the 2 S configuration to each Phe residue in 1 and 2. Proton-proton coupling constants and ROESY correlations within the A hp residues of 1 and 2 (Table 2-1) suggested that the relative configuration and conformation of the Ahp moieties are identical to the one in symplostatin 2,16 somamide A25 and lyngbyastatin 4 26(3 S,6R ). For our compound 2 the 13C NMR and 1H NMR chemical shifts are equivalent to those reported for somamide B,25 suggesting that their relative conf igurations are identical and thus that these compounds are not diastereomers. And although we were unable to reliably detect an optical rotation for 2, the fact that lyngbyastatin 7 ( 1 ) and our compound 2 had the same absolute configuration based on Marfeys analysis and that optical rotation da ta for lyngbyastatin 7 ( 1 ) matched closely the data reported for somamide B indicated that compound 2 is indeed somamide B itself but not an enantiomer. 2.3 Kempopeptin A Kempopeptin A ( 3), was isolated from the same collection of Lyngbya sp. that had afforded lyngbyastatin 7 ( 1) and somamide B ( 2). Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

PAGE 22

22 Figure 2-2. Structure of kempopeptin A ( 3) 2.3.1 Isolation and Structure Determination of Kempopeptin A The non-polar extract (see 2.2.1) wa s partitioned with organic solvents followed by various chrom atographic steps using silica and C18 and ultimately reversed-phase HPLC to yield kempopeptin A ( 3) as a colorless, amorphous solid. It was shown to have the molecular formula of C50H70N8O13 as determined by HRESI/APCIMS based on a [M + Na] + peak at m/z of 1013.4965 (calcd for C50H70N8O13Na, 1013.4960). The presence of a peptide backbone was evident from the 1H NMR spectrum recorded in DMSO-d6 due to a tertiary amide N -Me 3H singlet at 2.75 and characteristic secondary amide NH resonances occurring as one 1H doublet at 7.06 and eight 0.5H doublets at 7.42, 7.43, 7.62, 7.75, 7.91, 8.12, 8.39, and 8.40. The differential integration was suggestive of conformers in only one part of the mo lecule (Table 2-2). The combination with 13C NMR, COSY, HMQC, HMBC, and TOCSY data revealed the presence of valine, N -methyltyrosine, phenylalanine, leucine, proline, two threonine resi dues, the modified amino acid 3-amino-6hydroxy-2-piperidone (Ahp) and an acetyl group, with si gnal doubling for the two threonine moieties, proline, the acetyl group and exchangeab le protons of valine and leucine residues. The sequence of the constituent amino acids of 3 was established with the aid of HMBC data as N Ac-ProThr-2Thr-1LeuAhpPhe N -Me-TyrVal (Table 2-2). An ester linkage was proposed

PAGE 23

23 based on an HMBC correlation between H-3 of Thr-1 ( H 5.38/5.39 for conformer 1/conformer 2) and the Val carbonyl ( C 172.1), forming the cyclic stru cture depicted for compound 3. The doubling of the 1H NMR signals in the side chain was attributed to restricted rotation around the N -acetyl prolyl amide bond based on ROESY cross-peaks between H-2 of proline ( H 4.52) and the acetyl protons ( H 1.83) for the cis isomer and between H-5b of proline (H 3.47) and the acetyl protons ( H 1.95) in the trans isomer. A 1:1 ratio of c is and trans isomers in DMSOd6 around the N -acetylprolyl bond was also reported for the most closely related metabolite, oscillapeptilide 97-B,28 which contains an isoleucine instead of the valine in the cyclic core and a glutamine rather than the threonin e-2 residue in th e side chain. 2.3.2 Absolute Configuration Analysis of the acid hydrolyzate of 3 using L-FDLA based Marfeys analysis 29 suggested that all regular and the N -methyl amino acid constituents in 3 have L-configuration. CrO3 oxidation was carried out followed by acid hydrolysis to establish the abso lute configuration at C-3 of the Ahp unit. This reaction liberated L-glutamic acid, thus estab lishing the configuration of the Ahp residue as 3S The relative stereochemistry within the Ahp moiety was determined based on ROESY peaks (Table 2-2), which were also observed for dolastatin 13 analogs symplostatin 2,16b somamide A25 and lyngbyastatin 424,26 and other related Ahp-containing protease inhibitors, includ ing oscillapeptilide 97-B,28 all of which possess 3 S,6R configuration. The assignment was also consistent with the n early identical NMR data for kempopeptin A ( 3) and oscillapeptolide 97-B, sugges ting that the relative configurat ion including conformation of the cyclic core are th e same for both compounds.

PAGE 24

24Table 2-2. NMR data for Both Conformers of Kempopeptin A ( 3) in DMSOd6 (ratio 1:1) at 500 MHz (1H) and 150 MHz (13C) Trans conformera Cis conformera Unit C/H no. H (J in Hz) C, mult. H (J in Hz) C, mult. HMBCb,c Key ROESYc Val 1 172.1, qC 172.1, qC 2 4.65, dd (9.2, 4.5) 55.8, CH 4.64, dd (9.5, 4.5) 55.8, CH 1, 3, 4, 5, 1 ( N -Me-Tyr) 3 2.04, m 31.8, CH 2.04, m 30.8, CH 2, 4, 5 4 0.85, d (6.5) 19.5, CH3 0.84, d (6.5) 19.3, CH3 2, 3, 5 N -Me ( N -Me-Tyr) 5 0.71, d (6.5) 17.2, CH3 0.70, d (6.5) 17.2, CH3 2, 3, 4 N -Me ( N -Me-Tyr) NH 7.43, d (9.2) 7.42, d (9.5) 1 ( N -Me-Tyr) H-2 (N -Me-Tyr), N -Me ( N -Me-Tyr), 6-OH (Ahp) N -Me-Tyr 1 169.1, qC 169.1, qC 2 4.89, dd (10.6, 1.5) 60.9, CH 4.89, dd (10.6, 1.5) 60.9, CH H-3a, N -Me, H-2 (Phe), H-5/9 (Phe), NH (Val) 3a 3.10, dd (, 10.6) 32.8, CH2 3.10, dd (, 10.6) 32.8, CH2 4, 5/9 H-2 3b 2.69, dd (, 1.5) 2.69, dd (, 1.5) 4, 5/9 4 127.5, qC 127.5, qC 5/9 6.99, d (8.5) 130.4, CH 6.99, d (8.5) 130.4, CH 3, 5/9, 7 N -Me, H-2 (Phe) 6/8 6.77, d (8.5) 115.3, CH 6.77, d (8.5) 115.3, CH 4, 6/8, 7 7 156.2, qC 156.2, qC 7-OH 9.35, s 6/8, 7 N -Me 2.75, s 30.3, CH3 2.75, s 30.3, CH3 2, 1 (Phe) H-2, H-5/9, H3-4 (Val), H3-5 (Val), NH (Val) Phe 1 170.4, qC 170.4, qC 2 4.73, dd (11.5, 4.3) 50.3, CH 4.73, dd (11.5, 4.3) 50.3, CH 1, 2 (Ahp), 6 (Ahp) H-3b, H-5/9, H-2 ( N -Me-Tyr), H-5/9 ( N -Me-Tyr), H-6 (Ahp) 3a 2.85, dd (.8, 11.5) 35.3, CH2 2.85, dd (.8, 11.5) 35.3, CH2 2, 4 3b 1.77, dd (.8, 4.3) 1.77, dd (.8, 4.3) 2, 4 H-2 4 136.7, qC 136.7, qC 5/9 6.82, d (7.0) 129.4, CH 6.82, d (7.0) 129.4, CH 3, 5/9, 6/8 H-2, H-2 ( N -Me-Tyr) 6/8 7.15, m 127.7, CH 7.15, m 127.7, CH 4, 7 7 7.12, m 126.2, CH 7.12, m 126.2, CH 5/9, 6/8 Ahp 2 168.9, qC 168.9, qC 3 3.61, m 48.6, CH 3.61, m 48.6, CH 2 H-4b, H-5a, NH 4a 2.37, m 21.7, CH2 2.37, m 21.7, CH2 H-4b, 6-OH, NH 4b 1.55, m 1.55, m H-3, H-4a 5a 1.66, m 29.0, CH2 1.66, m 29.3, CH2 H-3, H-5b, H-6, 6-OH 5b 1.54, m 1.54, m H-5a, H-6, 6-OH 6 5.05, br s 73.7, CH 5.05, br s 73. 7, CH H-5a, H-5b, 6-OH, H-2 (Phe) 6-OH 6.02, br s 6.02, br s H-4a, H-5a, H-5b, H-6, NH (Val) NH 7.06, d (9.1) 7.06, d (9.1) 1 (Leu) H-3, H-4a, H-2 (Leu)

PAGE 25

25Table 2-2 Continued. Unit C/H no. H (J in Hz) C, mult. H (J in Hz) C, mult. HMBCb,c Key ROESYc Leu 1 170.1, qC 170.1, qC 2 4.19, m 50.3, CH 4.19, m 50.3, CH H-3a, H3-5, NH, NH (Ahp) 3a 1.70, m 40.0, CH2 1.70, m 40.0, CH2 5, 6 H-2 3b 1.28, m 1.28, m NH 4 1.44, m 23.3, CH 1.44, m 23.3, CH NH 5 0.70, d (6.4) 20.9, CH3 0.70, d (6.4) 20.9, CH3 3, 4, 6 H-2 6 0.83, d (6.4) 21.5, CH3 0.83, d (6.4) 21.5, CH3 3, 4, 5 NH 8.40, d (8.7) 8.39, d (8.8) 1 (Thr-1) H-2, H-3b, H-4, H-2 (Thr-1), H-3 (Thr-1) Thr-1 1 169.2, qC 169.2, qC 2 4.58, dd (9.1, 2.1) 54.6, CH 4.60, dd (9.1, 2.0) 54.6, CH 1, 1 (Thr-2) H3-4, NH (Leu) 3 5.38, br q (6.4) 72.0 CH 5.39, br q (6.4) 72.0, CH 1 (Val) NH (Leu) 4 1.18, d (6.4) 17.7, CH3 1.17, d (6.4) 17.7, CH3 NH 7.62, d (9.1) 7.75, d (9.1) 1 (Thr-2) H-2 (Thr-2), H-3 (Thr-2), H3-4 (Thr2) Thr-2 1 170.7, qC 170.6, qC 2 4.30, dd (8.2, 4.1) 58.1, CH 4.39, dd (8.4, 4.2) 58.0, CH 1 NH (Thr-1) 3 4.03, m 66.5, CH 4.03, m 66.5, CH NH (Thr-1) 4 1.02, t (6.7) 19.2, CH3 1.04, t (6.8) 19.3, CH3 NH (Thr-1) OH 4.86, d (5.1) 4.96, d (5.4) NH 7.91, d (8.2) 8.12, d (8.4) 1 (Pro) H-2 (Pro) Pro 1 172.3, qC 172.1, qC 2 4.44, dd (8.4, 2.8) 58.9, CH 4.52, dd (8.6, 2.9) 60.1, CH NH (Thr-2) 3a 2.05, m 29.3, CH2 2.23, m 29.5, CH2 3b 1.90, m 1.93, m 4 1.89, m 24.3, CH2 1.76, m 24.1, CH2 5a 3.51, m 47.6, CH2 3.40, m 46.3, CH2 5b 3.47, m 3.38, m Ac 1 168.7, qC 168.5, qC 2 1.95, s 22.0, CH3 1.83, s 22.2, CH3 1 H-2d (Pro)cis or H2-5e (Pro)trans a Refers to restricted rotation around the N -acylprolyl amide bond. b Protons showing HMBC correlations to the indicated carbon. c Refers to nuclei within the same unit unless indicated otherwise. d Refers to cis isomer. e Refers to trans isomer.

PAGE 26

26 2.4 Kempopeptin B* A closely related cyclodepsipeptide, kempopeptin B ( 4), was isolated from the same collection of Lyngbya sp. that had afforded lyngbyastatin 7 ( 1 ), somamide B ( 2) and kempopeptin A ( 3). Figure 2-3. Structure of kempopeptin B ( 4) 2.4.1 Isolation and Structure Determination of Kempopeptin B The non-polar extract (see 2.2.1) wa s partitioned with organic solvents followed by various chrom atographic steps using silica and C18 and ultimately reversed-phase HPLC to yield kempopeptin B ( 4) as a colorless amorphous powder. The HRESI/APCIMS data showed a [M + H] + peak at m/z 993.4663 and an isotope peak of approximately equal intensity at m/z 995.4656, indicating the presen ce of one bromine atom and a molecular formula of C46H73BrN8O11 (calcd for C46H74 79BrN8O11, 993.4660). Five doublet NH proton signals in the amide range ( H 7.34, 7.68, 7.80, 7.82, 8.44) and one broad singlet for two primary amide protons ( H 7.60) in the 1H NMR spectrum suggested that 4 was a peptide. 1H NMR, 13C NMR, HSQC, COSY and TOCSY analysis revealed seven amino acid spin systems, one carboxylic acid unit, one N -Me ( H 2.72 s, C 30.2) as well as one O -Me group ( H 3.74 s, C 56.1), and a 1,3,4-trisubstituted phenyl ring (Table 2-3). Further NMR including Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

PAGE 27

27 HMBC analysis confirmed the presence of two valine units, threonine, isoleucine, lysine, N,O dimethyl-3-bromotyrosine, Ahp, and butanoic aci d (Ba) moieties, and provided most of the amino acid sequence (Table 2-3). The depsipeptide nature of 4 was proven by HMBC correlation across an ester linkage between H-3 of Thr ( H 5.49) and the carbonyl of Val-1 ( C 172.3). However, no HMBC was observed between th e NH of Ahp and the carbonyl of Lys, but a ROESY correlation between NH of Ahp ( H 7.34) and H-2 of Lys ( H 4.26) could be used to connect the Ahp with the Lys residue, giving rise to the cyclic structure shown for 4. ROESY data were also in agreement w ith all other proposed linkages (T able 2-3). The most unusual structural feature of 4 is arguably the brominated tyrosine residue which was recently also found in largamides D, F and G,30 symplocamide A20and pompanopeptin A,31 while other compounds such as scyptolin A32 and cyanopeptolin 95433 are chlorinated at this position. 2.4.2 Absolute Configuration Am ino acid analysis of the acid hydrolyzate of 4 by Marfeys analysis29 using L-FDLA as the derivatization agent revealed that Lys, Thr, Ile and both Val units have L-configuration. Since we were unable to distinguish Land Lallo -Ile by this method, we employed chiral HPLC analysis to verify the presence of L-Ile and thus 2 S,3S configuration of this residue. Since PDAbased Marfeys analysis could not unam biguously identify the FDLA adduct of N,O -diMe-BrTyr due to overlap and low-intensity peaks in the HPLC profile, we establ ished the configuration of this residue by LC-MS based advanced Marfeys analysis.27 Selective ion monitoring for the adduct peaks and their MS/MS base peak daught er ions and comparison with standards proved L-configuration of the aromatic amino acid unit. Lastly, CrO3 oxidation generated L-glutamic acid, so that in combination with ROESY data for the Ahp unit in 4 (which were identical to the ones for 3), the absolute configurat ion of Ahp had to be 3 S,6 R

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28Table 2-3. NMR data for Kempopeptin B ( 4) in DMSOd6 at 600 MHz (1H) and 150 MHz (13C) Unit C/H no. H (J in Hz) C, mult. COSYa HMBCb,c Key ROESYa,c Val-1 1 172.3, qC 2 4.63, dd (9.4, 5.5) 54.8, CH H-3, NH 1, 1 ( N,O -diMe-Br-Tyr) 3 2.04, m 30.5, CH H3-4, H3-5 1, 2, 4, 5 4 0.85, d (6.8) 19.3, CH3 H-3 2, 3, 5 N -Me ( N,O -diMe-Br-Tyr) 5 0.73, d (6.8) 17.6, CH3 H-3 2, 3, 4 N -Me ( N,O -diMe-Br-Tyr) NH 7.68, d (9.4) H-2 1 ( N,O -diMe-Br-Tyr) H-2 (N,O -diMe-Br-Tyr), N -Me ( N,O -diMe-BrTyr), 6-OH (Ahp) N,O -diMe-Br-Tyr 1 169.4, qC 2 5.03, dd (11.3, 2.6) 60.6, CH H-3a, H-3b 3 H-3a, H-5, H-9, N -Me, H-2 (Ile), NH (Val-1) 3a 3.20, dd (.0, 2.6) 32.9, CH2 H-2, H-3b H-2, H-5 3b 2.78, dd (.0, 11.3) H-2, H-3a 4 131.3, qC 5 7.39, d (1.8) 133.5, CH H-9 3, 6, 7, 9 H-2, H-3a, NMe, H-2 (Ile) 6 111.0, CH 7 154.6, qC 8 7.01, d (8.4) 113.0, CH H-9 4, 6, 7 9 7.17, dd (8.4, 1.8) 130.2, CH H-5, H-8 3, 5, 7 H-2 OMe 3.74, s 56.1, CH3 7 N -Me 2.72, s 30.2, CH3 2, 1 (Ile) H-2, H-5, H3-4 (Val-1), H3-5 (Val-1), NH (Val-1) Ile 1 169.7, qC 2 4.35, br d (10.7) 54.2, CH H-3 1, 3, 6 (Ahp) H-3, H-5, H3-6, H-2 ( N -Me-Br-Tyr), H-5 ( N,O diMe-Br-Tyr) 3 1.79, m 32.8, CH H-2, H-4a, H-4b, H3-6 H-2 4a 1.0, m 23.7, CH2 H-3, H-4b, H3-5 4b 0.629, m H-3, H-4a, H3-5 5 0.630, br t (6.9) 10.3, CH3 H-4a, H-4b 3, 4 H-2 6 .15, d (6.4) 13.8, CH3 H-3 2, 3, 4 H-2 Ahp 2 170.4,d qC 3 4.42, m 48.8, CH H-4a, H-4b, NH H-4b, H-5, NH 4a 2.55, m 21.7, CH2 H-3, H-4b, H-5 H-4b, 6-OH, NH 4b 1.71, m H-3, H-4a, H-5 H-3, H-4a 5 1.73, m (2H) 29.7, CH2 H-4a, H-4b, H-6 H-3, H-6 6 4.92, d (2.9) 74.1, CH H-5, 6-OH H-5, 6-OH 6-OH 6.15, d (2.9) H-6 H-4a, H-6, NH (Val-1) NH 7.34, d (9.3) H-3 H-3, H-4a, H-2 (Lys)

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29Table 2-3. Continued. Unit C/H no. H (J in Hz) C, mult. COSYa HMBCb,c Key ROESYa,c Lys 1 169.3,d qC 2 4.26, br 52.1, qC H-3a, H-3b, NH H-3a, H-4, NH, NH (Ahp) 3a 2.00, m 29.0, CH2 H-2, H-3b, H2-4 H-2 3b 1.41, m H-2, H-3a, H2-4 NH 4 1.23, m (2H) 22.1, CH2 H-3a, H-3b, H2-5 H-2 5 1.47, m (2H) 26.3, CH2 H2-4, H2-6 6 2.71, m (2H) 38.6, CH2 H2-5 NH 8.44, d (8.4) H-2 1 (Thr) H-2, H-3b, H-2 (Thr), H-3 (Thr), NH (Thr) NH2 7.60, br s (2H) Thr 1 169.3, qC 2 4.59, dd (10.2, 6.5) 56.3, CH H-3, NH 1 (Val-2) H-3, H-4, NH (Lys), NH (Val-2) 3 5.49, br q (6.5) 71.7, CH H-2, H3-4 4, 1 (Val-1) H-2, NH (Lys) 4 1.20, d (6.5) 17.7, CH3 H-3 2, 3 H-2 NH 7.80, d (10.2) H-2 1 NH (Lys) Val-2 1 172.4, qC 2 4.31, dd (9, 7.1) 57.6, CH H-3, NH 1 H-3, NH, H3-4 (Ba) 3 2.01, m 30.0, CH H3-4, H3-5 H-2 4 0.83, d (7.6) 19.3, CH3 H-3 2 5 0.82, d (7.6) 18.1, CH3 H-3 2 NH 7.82, d (9) H-2 1 (Ba) H-2, H-2 (Thr), H2-2 (Ba) Ba 1 172.5, qC 2 2.16, m (2H) 37.1, CH2 H2-3 1, 3, 4 H3-4, NH (Val-2) 3 1.49, m (2H) 18.9, CH2 H2-2, H3-4 1, 2, 4 4 0.84, t (7.5) 13.6, CH3 H2-3 2, 3 H2-2, H-2 (Val-2) a Recorded at 500 MHz. b Protons showing HMBC correlations to the indicated carbon (600 MHz). c Refers to nuclei within the same unit unless indicated otherwise. d Interchangeable. No HMBC correlations observed. Carbons assigned ba sed on remaining unassigned signals in the 13C NMR.

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30 2.5 Biological activity Protease-Inhibitory Activity. There have been numerous publications describing the isolation of related Ahp-containi ng protease inhibitors from cyanobacteria, which are assumed to be enzyme substrate mimics.34,35,36 Many Ahp-containing cyclodeps ipeptides isolated from cyanobacteria are known inhibitors of serine pr oteases such as elastase, chymotrypsin and trypsin.33,36 Lyngbyastatin 7 (1) and somamide B ( 2) inhibited elastase with IC50 values of 8.3 5.4 nM and 9.5 5.2 nM. Compared with elastase activity, chymotrypsin activity was less compromised upon enzyme incubation with compounds 1 and 2, IC50 values being 2.5 0.2 M and 4.2 0.5 M. Expectedly trypsin activity was unaffected by treatment with compounds 1 and 2 (up to 30 M tested). Kempopeptin A ( 3) inhibited elastase activity with an IC50 of 0.32 0.07 M and slight selectivity over chymotrypsin with IC50 of 2.6 0.1 M, while 3 was unable to inhibit trypsin at the highest concentrated tested (67 M). Conversely, Kempopeptin B ( 4) inhibited only tryps in activity at the IC50 of 8.4 0.2 M, but not elastase and chymotrypsin. Table 2-4. Protease Inhibitory Activity (IC50) from Metabolites Isolated from the Lyngbya sp. from Kemp Channel Elastase Chymotrypsin Trypsin Kempopeptin A ( 3 ) 320 70 nM 2,600 100 nM >67,000 nM Kempopeptin B (4 ) >67,000 nM >67,000 nM 8,400 200 nM Lyngbyastatin 7 ( 1 ) 8.3 5.4 nM 2,500 200 nM >30,000 nM Somamide B (2 ) 9.5 5.2 nM 4,200 500 nM >30,000 nM Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

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31 2.6 Structure-Activity Relationship (SAR) It is known that the am ino acid residue be tween Ahp and Thr binds to the enzymes specificity pocket and thus plays an important role in determining the selectivity towards serine proteases.36,38-40A hydrophobic amino acid at this positi on commonly confers preference for elastase and chymotrypsin while a basic amino aci d such as lysine or arginine is necessary for trypsin inhibition. Figure 2-4. SAR comparison of protease inhibitors from Lyngbya sp. In lyngbyastatin 7 ( 1) and somamide B ( 2), a 2-amino-2-butenoic acid (Abu) unit presumably occupies the specificity pocket, while all other core residues are the same as in 3. This allows direct comparison of their activ ities. Since the side chain co mposition is less important for activity as long as at least two residues flank the cyclic core,24 the Leu (in 3) to Abu (in 1 and 2) change within the core structure seems to increa se elastase-inhibitory activity but not enhance chymotrypsin-inhibitory activit y. A postulated stabilization of the ethylidene moiety by CH/ Confers more selectivity for elastase due to CH/ interaction 1 Confers selectivity for elastase and chymotrypsin 3 4 2 Confers selectivity for trypsin Confers selectivity for elastase

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32 interaction may be responsible fo r the potent elastase activity,41 leading to more pronounced selectivity of 1 and 2 for both proteases than 3. In compound 4, lysine residue presumably occupies the specificity pocket. Since Lys is a basic amino acid it expectedly confers for trypsin-inhibitory activity. 2.7 Summary* We have described four new prot ease inhibitors, lyngbyastatin 7 (1), somamide B ( 2), kempopeptins A ( 3) and B ( 4), from a marine cyanobacterial collection. The discovery of four different protease inhibi tors with three distin ct selectivity profiles from a single homogenous cyanobacterial collection illustrates the potenti al of cyanobacteria to execute their own combinatorial biosynthesis and structure optimization. Protease inhibitors are known to be co-bio synthesized with the cyanobacterial toxins microcystins;42 in fact, metabolites other than micr ocystins have been shown to enhance microcystin activity.43 The biosynthesis of protease inhib itors in nontoxic cya nobacterial strains has precedence as well.44 Proteases are known to perform ma ny beneficial functions that are essential to life. In humans, serine proteas es, particularly trypsi n and chymotrypsin, are important pancreatic digestive enzymes.45,46 Elastase breaks down elastin, an elastic fiber which, along with collagen, is important for determinin g mechanical strength of connective tissues. It is known that overactivity of elasta se is linked to many diseases like emphysema, adult respiratory distress syndrome, and ischemic reperfusion injury, as well as cutaneous wrinkling.15 While the role of these protease inhibitors in nature is not fully understood, a de fensive function against other microorganisms or consumers is probable. Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

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33 Among three important serine prot eases tested, kempopeptin A ( 3) preferentially inhibited elastase and chymotrypsin over trypsin, whereas kempopeptin B ( 4) only inhibited trypsin. Lyngbyastatin 7 (1) and somamide B ( 2) inhibited chymotrypsin to the same extent as kempopeptin A ( 3) did, however, elastase activity was even more affected. The potency of compounds 1 and 2 and their selectivity for inhibiting elas tase over chymotrypsin was attributed to the Abu residue, which replaced the Leu moiety in the cyclic core of 3.

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34 CHAPTER 3 SECONDARY METABOLITES FROM Symploca sp 3.1 Overview Marine cyan obacteria especially Symploca spp., have been scarcely investigated as compared to more prevalent Lyngbya spp., but recently have been proven to be source of an array of biologically active and structurally diverse series of peptides and depsipeptides as described in the Introduction. Dolast atin 10, originally isolated from a sea hare, has been isolated from a Palauan Symploca sp. and been evaluated in phase I an d II anticancer clinical trials, which inspired us to target this genus. Thus in the research of identifying new drug leads from cyanobacteria in Florida, extracts of cyanobacteria of the genus Symploca collected at Pillars, Key Largo, Florida Keys were investigated and yielded one potent cytot oxin, largazole, which showed nanomolar antiproliferative activity and differential growth-inhibitory activity against transformed versus non-transformed cells comp ared to other validated antitumor natural products. Its isolation, structure determination, biological activ ity and molecular target are described below in detail. Scheme 3-1. Isolation Strategy for Symploca sp. (Non-Polar Extract)

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35 3.2 Largazole Largazole, a novel cytotoxin, was isolat ed from the marine cyanobacterium Symploca s p. Figure 3-1. Structure of largazole ( 5) 3.2.1 Isolation and Structure Determination of Largazole* A sample of Symploca sp. was collected from Key Largo, Florida Keys, and extracted with organic solvents. The resulting cytotoxic crude extract was subjected to bioassay-guided fractionation by solvent partition, silica gel chromatography, and re versed-phase HPLC to yield largazole (5 ) as a colorless, amorphous solid. 1H NMR and 13C NMR data coupled with a [M + H]+ peak at m/z 623.2397 in the HRESI/APCIMS of 5 suggested a molecular formula of C29H42N4O5S3. The 1H NMR spectrum exhibited two signals characteri stic for secondary amides ( 2-NH 7.15, 14-NH 6.45). Further twodimensional NMR analysis in CDCl3 using COSY, HSQC, and HMBC data indicated that these exchangeable protons belong to valine and modifi ed glycine residues, re spectively (Table 3-1). The putative glycine carbonyl ( C-13 167.9) was part of a 2,4-disubs tituted thiazole unit as evidenced by HMBCs from the only aromatic methine ( H-12 7.76, C-12 124.2) to C-13 and to another quaternary sp2 carbon, C-11 ( C 147.4). Furthermore, HMBCs from a methyl singlet ( H-9 1.87) to carbonyl C-6 ( C 173.5), quaternary carbon C-7 ( C 84.4), and methylene carbon C8 ( C 43.3), combined with an HMBC from H-8a ( H 4.04) to C-10 ( C 164.6) suggested the Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008 130, 1806 1807. Copyright (2008) American Chemical Society.

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36 presence of a 2-substituted th iazoline-4-methyl-4-carboxylic aci d unit (C-6 to C-10). The only other HMBC to C-10 was from the thiazole prot on H-12, indicating that C-10 bore the thiazole substituent. The methyl thiazoline carboxylate an d the amino terminus of the valine residue were unambiguously connected via an amide linka ge based on HMBC data (Table 3-1). The remaining signals in the 1H NMR spectrum belonged to two spin systems, as concluded from COSY analysis. One of the units was a 7-subst itued 3-hydroxyhept-4-enoic acid moiety (C-15 to C-21) with E -geometry of the double bond based on a large coupling constant for 3JH-18, H-19 of 15.6 Hz, consistent with NOESY cross-peaks between H-18 to H2-20. This unit was attached to the amino terminus of the glycine-derived un it as shown by HMBCs from 14-NH and H-14a/b to C-15 as well as ROESY cross-peaks between 14-NH and H-16a and H-16b. The last unit was an n-octanoyl group (C-22 to C-29) which was c onnected with C-21 based on HMBC from H2-21 to C-22. The low field chemical shift for C-22 ( C 199.4) coupled with the fact that one sulfur atom remained yet to be assigned was strong evidence for a thioester functiona lity. Finally, to account for the molecular formula requirements and fo r the low-field chemical shift of H-17 ( H 5.66) suggestive of an acyloxy substituent, C-17 had to be ester-linked to the carboxyl terminus of valine. This was further supported by a weak NOE between H-17 and H3-5 ( H 0.50), leading to the cyclic planar structure shown for 5. MSn (Figure 3-2) analysis is consistent with the proposed structure. 3.2.2 Absolute Configuration* To assign the absolute configuration of the three chiral centers, our strategy was to generate optically active fragme nts, for which enantiomeric standards are readily available. Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008 130, 1806 1807. Copyright (2008) Am erican Chemical Society

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37 Table 3-1. NMR Spectral Data for Largazole ( 5) in CDCl3 (600 MHz)* C/H no. H ( J in Hz) C, mult. HMBC 1 168.9, qC 2 4.61, dd (9.2, 3.3) 57.7, CH 1, 3, 4, 5, 6 3 2.10, m 34.2, CH 1,c 2c 4 0.68, d (7.2) 18.9, CH3 2, 3, 5 5 0.50, d (7.2) 16.6, CH3 2, 3, 4 2-NH 7.15, d (9.2) 1, 6c 6 173.5, qC 7 84.4, qC 8a 4.04, d (11.4) 43.3, CH2 6, 7, 10 8b 3.27, d (11.4) 6, 7, 9 9 1.87, br s 24.2, CH3 6, 7, 8 10 164.6, qC 11 147.4, qC 12 7.76, s 124.2, CH 10,c 11, 13 13 167.9, qC 14a 5.29, dd (.4, 9.6) 41.1, CH 13, 15 14b 4.27, dd (.4, 2.5) 13, 15 14-NH 6.45, dd (9.6, 2.5) 15c 15 169.4, qC 16a 2.86, dd (.5, 10.5) 40.5, CH 15, 17, 18 16b 2.68, dd (.5, 1.8) 15 17 5.66, ddd (10.5, 7.2, 1.8) 72.0, CH 18 5.51, dd (15.6, 7.2) 128.4, CH 17, 20 19 5.82, dt (15.6, 7.2) 132.7, CH 17, 20 20 2.31 br q (7.2) (2H) 32.3, CH2 18, 19, 21 21 2.90 t (7.2) (2H) 27.9, CH2 19, 20, 22 22 199.4, qC 23 2.52, t (7.5) (2H) 44.1, CH2 22, 24, 25 24 1.64, m (2H) 25.6, CH2 22, 23, 25/26 25 1.29, m (2H) 28.9, CH2 26 26 1.25, m (2H) 28.9, CH2 25, 27 27 1.26, m (2H) 31.6, CH2 28 1.28, m (2H) 22.6, CH2 29 0.87, br t (6.9) 14.0, CH3 27, 28 a Protons showing HMBC correlations to the indicated carbon. b Optimized for nJ = 7 Hz if not indicated otherwise. c Optimized for nJ = 3.5 Hz. Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008 130, 1806 1807. Copyright (2008) Am erican Chemical Society

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38 m/z 497 m/z 497 m/z 311 m/z 212 m/z 623[M+H]+ESI-MS 5 5a Rearrangement m/z 451 5b 5c m/z 451 5c ESI-MS/MS ESI-MS/MS/MS m/z 357 5d 5e 5f O N H O S N S N S H O NH O O O N H O S N S N H2S O NH O H H N H O S N S N H2S N O OH N H O S N S N H2S O NH O N H O S N S N H2S N O N H O S N S N H2S NH O O OH H3N S N S N H3N S N S N N O Figure 3-2. ESI-MSn fragmentation pattern for largazole ( 5 ) Specifically, ozonolysis followed by oxidative workup and acid hydrolysis generated 2methylcysteic acid, valine, and malic acid. The pr oduct mixture was subjected to chiral HPLC analysis, comparing retention times with those of authentic standards. This analysis identified Lvaline, (R )-2-methylcysteic acid, and L-malic acid, establishing the absolute configuration of 5 as 2S,7R ,17S.

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39 Scheme 3-2. Degradation Strategy to Liberate Chiral Subunits 3.3 Biological Evaluation* 3.3.1 Cell Viability Assays Largazole ( 5 ) potently inhibited the growth of highly invasive transform ed human mammary epithelial cells (MDA-MB-231) in a dose-dependent manner (GI50 7.7 nM) and induced cytotoxicity at higher concentrations (LC50 117 nM). In contrast, nontransformed murine mammary epithelial cell s (NMuMG) were less susceptible to compound 5 (GI50 122 nM, LC50 272 nM). We have not observed this remarkable selectivity for other validated antitumor natural products tested in parallel (Table 3-2). Similarly, the selectivity of 5 for transformed fibroblastic osteosarcoma U2OS cells (GI50 55 nM, LC50 94 nM) over nontransformed fibroblasts NIH3T3 (GI50 480 nM, LC50 > 8 M) was unmatched by other natural product drugs tested (Table 3-2). The differential growth-inhibitory activity between transformed and nontransformed cells suggests that cancer cells ar e preferentially targeted by 5. The growth of cancer cell lines derived from colon (HT29) and neuroblastoma (IMR-32) was also strongly inhibited by 5 (GI50 / LC50 12 nM/22 nM; 16 nM/22 nM). Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc 2008 130, 1806 1807. Copyright (2008) American Chemical Society Reproduced in part with permission from Ying Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. web release date: 29-May-2008; DOI: 10.1021/ja8013727 press. Copyright (2008) Am erican Chemical Society

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40 Table 3-2. Growth-Inhibitory Activity (GI50) of Natural Product Drugs MDA-MB-231 NMuMG U2OS NIH3T3 Largazole ( 5 ) 7.7 nM 122 nM 55 nM 480 nM Paclitaxel 7.0 nM 5.9 nM 12 nM 6.4 nM Actinomycin D 0.5 nM 0.3 nM 0.8 nM 0.4 nM Doxorubicin 310 nM 63 nM 220 nM 47 nM 3.3.2 HDACs as Molecular Targets Histone deacetylases have been evolving as in teresting targets in anticancer drug development.47 The deacetylation of the -amino group of histone lysine residues by histone deacetylases is considered as important regulatory mechanism of gene expression.48 One HDAC inhibitor suberoylanilide hydroxamic acid (SAHA),49 has been FDA approved for treatment of cutaneous T-cell lymphoma and others are in clinical trials such as CRA-026440.50 We expected largazole51 to be a prodrug that upon hydrolysis gene rates an HDAC inhibitor similar to FK228 (Figure 3-3),52 where reduction of disulfide bond gene rates a bioactive mercapto unit. HDAC inhibitors have been known to display selec tive growth inhibition against transformed cells,53-56 similar to that observed for largazole ( 5). To test the hypothesis that la rgazole acts as HDAC inhibitor, due to low amounts of natural largazole, we used synthetic largazole whic h had similar physical pr operties, NMR and mass data for all subsequent assays (provided by Dr. Jiyong Hong, Department of Chemistry, Duke University). We measured the cellular HDAC activity in HCT-116 cells upon treatment with largazole. It was observed th at after 8 h of treatment HDAC activity in HCT-116 cells decreased in a dose-dependent manner (Figure 3-4a). Importantly, the IC50 for HDAC inhibition closely resembles the GI50 of largazole in this cell line (Figure 3-4a, Table 3-3). This

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41 resemblance may indicate that HDAC might be the relevant target responsible for largazoles antiproliferative effect. In order to confirm this we further performed immunoblot analysis of an endogenous HDAC substrate, acetyla ted histone H3, which also re vealed the same results as seen for cellular HDAC activity (Figure 3-4b). Figure 3-3. Structural similarity between FK228 and largazole (5) and modes of activation* Reproduced in part with permission from Ying Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. web release date: 29-May-2008; DOI: 10.1021/ja8013727 press. Copyright (2008) Am erican Chemical Society

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42 Figure 3-4. Target identi fication. (a) Largazole ( 5) inhibits cellular HDAC activity in HCT-116 cells (8 h) based on deacetylation of a fluorogenic substrate (BIOMOL). Growthinhibitory activity (48 h) is similar. (b ) Immunoblot analysis showing inhibiton of endogenous histone H3 deacetylation in a dos e-response fashion (8 h). Trichostatin A (TSA) was used as a positive control.* We further confirmed the hypothesis that the thiol 6 is the reactive species using HeLa cell nuclear protein extract rich in class 1 HDACs 1, 2, and 3 (BIOMOL). For carrying out this experiment we teamed up with Dr. Jiyong Hong, Department of Chemistry, Duke University who provided us with synthetic largazole and a ll other synthetic analogs tested here (compounds 6 to 9). We determined enzymatic activity directly and found that thiol analog 6 inhibited the HDACs in the nuclear extract of HeLa cells with a similar IC50 value (Table 3-3). Largazole ( 5 ) and the thiol analog 6 exhibited similar cellular activity against HDACs derived from nuclear Reproduced in part with permission from Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. web release date: 29-May-2008; DOI: 10.1021/ja8013727 press. Copyright (2008) Am erican Chemical Society

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43 HeLa extracts as well as antiproliferative activ ity. However it is likely that largazole underwent hydrolysis under assay conditions. Acetylanalog( 7 ) Thiolanalog( 6 ) Hydroxylanalog( 9 ) Macrocycle( 8 ) O N H O S N S N HS O NH O O N H O S N S N HO O NH O O N H O S N S N O NH O O N H O S N S N O NH O S O Figure 3-5. Structures of s ynthetic largazole analogs* (* provided by Dr. Jiyong Hong, Department of Chemistry, Duke University) Table 3-3. IC50 and GI50 Values for HDACs and Growth Inhibition (nM) HCT-116 growth inhibition HCT-116 HDAC cellular assay HeLa Nuclear Extract HDACs 5 44 10 51 3 37 11 6 38 5 209 15 42 29 7 33 2 50 18 52 27 8 >10,000 >10,000 >10,000 9 >10,000 >10,000 >10,000

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44 3.3.3 Structure-Activity Relationship (SAR) Initial SAR studies were pe rform ed which allowed us to define the pharmacophore of largazole. The acetyl analog 7 showed the same activity as largazole ( 5) in the cellular HDAC assay and the same antiprolifera tive activity, indicating that the carboxylic acid po rtion of the thioester merely contributes to HDAC activity or antiproliferative activity as long as free thiol is formed. To further define the pharmacophore other key analogs of 5 were tested. One with hydroxyl group 9 instead of sulfhydryl group, which is important for chelating with Zn 2+ in the active site of HDACs. It was found that hydroxyl analog 9 was inactive as expected since we know that the sulfhydryl group is needed for chel ation (Table 3-3). The macrocyclic ring lacking the sulphur containing a liphatic chain, compound 8, was inactive against HDACs and lacked antiproliferative activ ity (Table 3-3). This suggested that the thiol group is essential for both HDAC-inhibitory as well as antiproliferative activity. 3.3.4 HDAC Isoform Selectivity HDAC enzym es are usually divided into f our major groups. Class I HDACs are widely expressed in tissues and are constitutively nuclear proteins with HDAC1 and HDAC2 mainly forming transcription repressi on complex. Class II HDACs show various degrees of tissue specificity and shuttle between nucleus and cytoplasm. Class III enzymes mainly consist of sirtuins and their mechanism depends on NAD+ as a cofactor, while the more recently discovered class IV enzyme (HDAC11) is Zn2+ dependent.49 To determine a preliminary HDAC isoform selectivity profile, we tested largazole ( 5) and 6 against recombinant HDAC1 (class I) and HDAC6 (class II) (Table 3-4). Compound 6 inhibited HDAC1 activity at low nanomolar concentrations and was 150-fold less active against HDAC6. This selectivity for HDAC1 was also observed for FK228.52 Furthermore, largazole (5) was found to be less active against both

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45 HDAC1 and HDAC6 enzymes than 6, which may suggest incomplete hydrolysis under assay conditions. Table 3-4. IC50 Values for HDAC1 and HDAC6 Inhibition (nM) HDAC1 (class I) HDAC6 (class II) Largazole ( 5 ) 25 11 5,700 3,600 Thiol ( 6 ) 2.5 1.4 380 76 Trichostatin A 4.9 0.8 18 12 3.4 Summary We described the isolation of a novel and pot ent antiprolifer ative agent from the marine cyanobacterium Symploca sp. It displayed remarkable se lectivity for transformed versus nontransformed cells when compared to other antitumor natural products, indicating that largazole 5 preferentially target cancer cells. Further mechanistic studies were done and it was found that largazole acts as HDAC inhibitor and that its antiproliferative activity correlates with its HDAC inhibitory activity. A preliminary HD AC isoform selectivity profile for largazole suggested that largazole poten tly inhibits class I HDACs.

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46 CHAPTER 4 EXPERIMENTAL SECTION 4.1 General 4.1.1 Spectral Analysis NMR Anal ysis. 1H and 13C NMR data were acquired on a Bruker Avance 500 MHz /Bruker Avance 600 MHz spectrometer with a 5-mm probe operating at 600 and 150 MHz for lyngbyastatin 7 ( 1), somamide B ( 2), and kempopeptin A ( 3), whereas Bruker Avance II 600 MHz equipped with a 1-mm triple resonance high -temperature superconduc ting cryogenic probe. was used for kempopeptin B ( 4) and largazole ( 5 ). 2D NMR experiments were performed on Bruker Avance II 600 MHz equipped with a 1mm triple resonance high-temperature superconducting cryogenic probe for kempopeptin A ( 3), kempopeptin B ( 4), and largazole (5 ), and Bruker Avance 500 MHz spectrometer with a 5-mm probe was used for lyngbyastatin 7 ( 1) and somamide B ( 2). NMR spectra were recorded in CDCl3 and DMSO using residual solvent signals as internal standards. Chemical shifts are referenced to chloroform ( H 7.26, C 77.0) and for DMSO ( H 2.49, C 39.5). Multiplicities are indica ted as : br (broad), s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). The HSQC experiments were optimized for 1JCH = 145 Hz, and the HMBC experiments for nJCH = 7 or 3.5 Hz. Mixing times for the ROESY and NOESY experiments were 400 ms and 800 ms. IR, UV, Optical Rotations. IR spectra were recorded on a Bruker Vector 22 instrument. UV spectra were measured on a SpectraMax M5 (Molecular Devices). Op tical rotations were obtained on a Perkin-Elmer 341 polarimeter. Mass Spectrometric Analyses. HRMS data were obtained using an Agilent LC-TOF mass spectrometer equipped with ESI/APCI multimode ion source detector at the UCR Mass Spectrometry Facility, Department of Chemistry, a nd University of California at Riverside. LC-

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47 MS data were obtained using an Agilent 1100 equipped with a ThermoFinnigan LCQ by ESI (positive mode) at the Department of Chemistry, University of Florida. 4.1.2 Amino Acid Standards Comm ercially available amino acids for the chiral HPLC and Marfeys analysis were purchased from SigmaAldrich. ( R )and ( S)-2-methylcysteines were provided by ResCom (DSM Pharma Chemicals). N,O -diMe-L-Br-Tyr was provided by Dr. Susan Matthew. Synthesis of ( R )and ( S )-2-methylcysteic Acid. : A sample of ( R )-2-methylcysteine hydrochloride (5.0 mg) was treated with 2 mL of a mixture of H2O2HCO2H (1:9) at 0 oC for 2 h. The product mixture was concentrated to dryness by evaporation to give (R )-2-methylcysteic acid. The residue was then r econstituted in 250 L of H2O for amino acid analysis by chiral HPLC. Similarly, ( S)-2-methylcysteine hydrochlor ide was reacted to yield ( S)-2-methylcysteic acid. 4.2 Biological Material 4.2.1 Lyngbya Collec tion* A sample of Lyngbya sp. was collected from a mangrove channel, Kemp Channel, at the northern end of Summerla nd Key, Florida Keys (24o39.730 N, 81o27.791 W) in May 2006. It was suspected to be L. majuscula of gray-black color, though it appears thinner than generally described (cell width: 17.3 m; sheath: 0.9 m; le ngth: 3.8 m). A voucher sp ecimen is retained at the Smithsonian Marine Station.24 Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy

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48 4.2.2 Symploca Collec tion* A sample of Symploca sp. was collected from Pillars, Key Largo (Florida Keys, USA) in August 2003. The specimen had upright, golden-brow n, feather-like filaments consistent with this genus. Filaments measured 5-6 m in widt h including a fine sheath and 8-9 m in length.51 4.3 Extraction and Isolation 4.3.1 Isolation of Lyngbya Metabolites The freeze-dried sample of Lyngbya sp. was extracted with CH2Cl2MeOH (1:1). The resulting lipophilic extract (24.1 g) was partitioned between he xanes and 20% aqueous MeOH, the methanolic phase was evaporated to dryness and the residue was further partitioned between n-BuOH and H2O. The n-BuOH layer was concentrated and subjected to chromatography over silica gel using CH2Cl2 and increasing gradients of i -PrOH. Consecutive fractions that eluted with 50 and 75% i -PrOH were individually applied to C18 SPE cartridges and elution was initiated with H2O followed by aqueous solutions cont aining 25, 50, 75, and 100% MeOH. Both times, the fractions eluting with 75% aqueous MeOH were then purified by semipreparative reversed-phase HPLC (YMC-Pack ODS-AQ, 250 mm, 2.0 mL/min; UV detection at 220 and 254 nm ) using a MeOHH2O linear gradient (50% for 60 min and then 100% MeOH for 10 min). The fraction that had eluted with 50% i -PrOH from silica gel yielded kempopeptin A ( 3) at tR 30.2 min (1.0 mg) a nd lyngbyastatin 7 ( 1) at tR 35.2 min (7.4 mg) while the 75% i PrOH fraction furnished additiona l amounts of lyngbyastatin 7 ( 1) (3.1 mg) and somamide B ( 2) eluted at tR 26.2 min (1.2 mg). Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc 2008, 130, 1806 1807. Copyright (2008) American Chemical Society Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy

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49 The fraction that eluted w ith 100% MeOH from silica gel was subjected to Si SPE cartridge and eluti on started with CH2Cl2 followed by increasing gradients containing 20, 40, 60, 80, and 100% MeOH. The fraction eluting with 20% methanolic CH2Cl2 was then purified by semipreparative reversed-phase HPLC (Y MC-Pack ODS-AQ, 250 10 mm, 2.0 mL/min; UV detection at 220 and 254 nm ) using a MeOHH2O (0.05% TFA) linear gradient (60% for 40 min and then 100% MeOH for 15 min). The fr action that had eluted with 100% MeOH from silica gel yielded kempopeptin B (4 ) at tR 25.2 min (1.6 mg). 4.3.2 Isolation of Symploca Metabo lites* Symploca sp. was freeze-dried and extracted with MeOH-EtOAc (1:1). The resulting lipophilic extract (0.29 g) was partitioned between hexanes and 80% aqueous MeOH. The aqueous MeOH layer was concentrated and fr actionated by silica gel chromatography using CH2Cl2 containing increasing amounts of i -PrOH followed by methanol. The fraction that eluted with 5% i -PrOH was then purified by semipreparat ive reversed-phase HPLC (YMC-pack ODSAQ, 250 10 mm, 2.0 mL/min; UV detecti on at 220 and 254 nm ) using a MeOHH2O linear gradient (40% for 75 min and then 100% MeOH for 10 min). Largazole ( 5 ) eluted at tR 61.5 min (1.2 mg) and along with other less toxic minor components which are still under investigation. Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc 2008 130, 1806 1807. Copyright (2008) Am erican Chemical Society

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50 4.4 Physical Data 4.4.1 Lyngbya Metabolites* Lyngbyastatin7 (1): colorless, amorphous powder; [ ]20 D .4 ( c 0.27, MeOH); UV (MeOH) max (log ) 230 (3.80), 280 (sh) (3.12); IR (film) 3373 (br), 2961, 1733, 1645 (br), 1539, 1446, 1203, 1026 cm-1; 1H NMR, 13C NMR, COSY, HMBC, and ROESY data, see Table 2-1; HRESI/APCIMS m/z [M + Na]+ 969.4710 (calcd for C48H66N8O12Na 969.4698). Somamide B (2): colorless, amorphous powder, UV (MeOH) max (log ) 230 (3.74), 280 (sh) (3.10); NMR data, see ref 25 ; HRESI/APCIMS m/z [M + Na] + 941.4407 (calcd for C46H62N8O12Na 941.4385). Kempopeptin A (3): colorless, amorphous powder; [ ]20 D 45 ( c 0.05, MeOH); UV (MeOH) max (log ) 210 (3.66), 280 (sh) (2.67); IR (film) 3374 (br),2958, 2924, 1735, 1655 (br), 1541, 1449, 1257, 1203, 1139 cm-1; 1H NMR, 13C NMR, COSY, HMBC, and ROESY data, see Table 2-2; HRESIMS m/z [M + Na]+ 1013.4965 (calcd for C50H70N8O13 Na 1013.4960). Kempopeptin B (4) : colorless, amorphous powder; [ ]20 D ( c 0.16, MeOH); UV (MeOH) max (log ) 210 (3.80), 280 (sh) (3.12); IR (film) 3356 (br), 2926, 1738, 1736, 1658 (br), 1530, 1442, 1257, 1205, 1139 cm-1 ; 1H NMR, 13C NMR, COSY, HMBC, and ROESY data, see Table 2-3; HRESIMS m/z [M + H]+ 993.4663 (calcd for C46H74 79BrN8O11 993.4660), 995.4656 (calcd for C46H74 81BrN8O11 995.4640), 1:1 ion cluster. Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600 Copyright (2007) American Chemical Societ y and American Society of Pharmacognosy Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

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51 4.4.2 Symploca Metabo lites* Largazole (5): colorless, amorphous solid; [ ]20 D + 22 ( c 0.1, MeOH); UV (MeOH) (log ) 210 (4.07 ), 260 (sh) (3.61); IR (film) 2924, 2853, 1725, 1694, 1611, 1659, 1641, 1630, 1596, 1512, 1249, 1117, 1067, 1034, 894 cm-1; 1H NMR, 13C NMR, and HMBC data, see Table 3-1; HRESI/APCIMS m/z [M + H]+ 623.2397 (calcd for C29H43N4O5 S3 623.2396). 4.5 Determination of Absolute Configuration The absolute configuration of -am ino acid and -hydroxy acids was determined either by using chiral HPLC analysis of the acid hydrol yzates or by advanced Marfey analysis. The absolute configuration of the amino acids in the hydrolyzate was determined by direct comparison with the retention times of authentic standards. In order to determine absolute configuration of those amino acids where the carboxyl group was incorporat ed into a thiazole ring or to liberate cysteic aci d from cysteine-derived substituted thiazolines, ozonolysis was performed prior to acid hydrolys is followed by oxidative workup. 4.5.1 Lyngbyastatin 7 and Somamide B A sample (~50 g) of compounds 1 and 2 were subjected to acid hydrolysis (6 N HCl) at 110 oC for 24 h. The hydrolyzates were evap orated to dryness, dissolved in H2O (100 L), and divided into two equal portions. To one portion were added 1 M NaHCO3 (50 L) and a 1% v/v solution of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) in acetone, and the mixture was heated at 80 oC for 3 min. The reaction mixture was th en cooled, acidified with 2 N HCl (100 L), dried and dissolved in H2OMeCN (1:1). Aliquots were subjected to RP HPLC (Alltech Alltima HP C18 HL 5 m, 250 4.6 mm, UV detection at 340 nm) using a linear Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc 2008 130, 1806 1807. Copyright (2008) Am erican Chemical Society Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy

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52 gradient of MeCN in 0.1% (v/v) aqueous TFA (30% MeCN over 50 min). The retention times ( tR, min) of the derivatized amino acids in the corresponding hydrolyzates of compound 1 and 2 matched with those of L-Thr (13.8), L-Val (23.6), L-Phe (28.5), and N -Me-L-Tyr (40.6) and also peaks for L-Glu (16.2). Here glutamic acid must ha ve derived from glutamine present in 1 and 2. For comparison, the L-FDLA derivatives of the other sta ndard amino acids not detected in the hydrolyzates had the fo llowing retention times ( tR in min): Lallo -Thr (14.8), Dallo -Thr (16.9), D-Thr (19.1), D-Val (32.5), D-Phe (35.5), N -Me-D-Tyr (42.6), and D-Glu (17.6). CrO3 oxidations of 1 and 2 followed by acid hydrolysis were carried out as described.26 The resulting hydrolyzates were derivatized with L-FDLA and aliquots subjected to reversedphase HPLC as above. When comp ared to the Marfey profiles wi thout prior oxidation, the HPLC profiles for derivatives re sulting from compounds 1 and 2 showed an increased intensity for LGlu and L-Phe peaks, both peaks were already pres ent in the original profile; however, they appeared to be larger after oxidation, while the corresponding D-amino acid derivatives were not detected. 4.5.2 Kempopeptin A* A sample (~100 g) of compound 3 was subjected to acid hydrolysis at 110 oC for 24 h. The hydrolyzate was evaporated to dryness, dissolved in H2O (100 L), and divided into two equal portions. To one portion, 1 M NaHCO3 (50 L) and 1% v/v solution of 1-fluoro-2,4dinitrophenyl-5-L-leucinamide (L-FDLA) in acetone were added and heated at 80 o C for 3 min. The reaction mixture was then cool ed, acidified with 2 N HCl (100 L), dried and dissolved in H2OMeCN (1:1). Aliquots were subjected to reversed-phase HPLC (Alltech Altima HP C18 HL 54, 250 4.6 mm, PDA detection from 200 to 500 nm) using a linear gradient of MeCN in Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

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53 0.1% (v/v) aqueous TFA (30% MeCN over 50 min). The retention times ( tR, min) of the derivatized amino acids in the corresponding hydrolyzate of compound 3 matched with those of L-Thr (14.4), L-Val (24.7), L-Phe (29.6), L-Pro (19.7), L-Leu (28.8), N -Me-L-Tyr (42.1). Conversely, the L-FDLA derivatives ( tR, min) of LalloThr (15.6), D-Thr (20.5), Dallo -Thr (17.1), D-Val (33.9), D-Phe (36.7), D-Pro (23.1), D-Leu (39.5), and N -Me-D-Tyr (43.8) were not detected in the hydrolyzate. CrO3 oxidation of 3 followed by acid hydrolysis wa s carried out as described.26 The resulting hydrolyzate was derivatized with L-FDLA and aliquots subjected to reversed-phase HPLC as above. When compared to the Marf ey profiles without prior oxidation, the HPLC profiles derived from compound 3 showed one new peak for L-Glu ( tR 16.8 min), but not D-Glu ( tR 17.8 min). 4.5.3 Kempopeptin B* A sample (~100 g) of compound 4 was subjected to acid hydrolysis at 110 oC for 24 h. The hydrolyzate was evaporated to dryness, dissolved in H2O (100 L), and divided into two equal portions. To one portion, 1 M NaHCO3 (50 L) and 1% v/v solution of 1-fluoro-2,4dinitrophenyl-5-L-leucinamide (L-FDLA) in acetone were added and heated at 80 o C for 3 min. The reaction mixture was then cool ed, acidified with 2 N HCl (100 L), dried and dissolved in H2OMeCN (1:1). Aliquots were subjected to reversed-phase HPLC (Alltech Altima HP C18 HL 54, 250 4.6 mm, PDA detection from 200 to 500 nm) using a linear gradient of MeCN in 0.1% (v/v) aqueous TFA (30% MeCN over 50 min). The retention times ( tR, min) of the derivatized amino acids in the corresponding hydrolyzate of compound 4 corresponded to those of L-Thr (14.4), L-Val (24.7), and L-Ile/Lallo -Ile (27.0); the latter had the same retention times Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. submitted to J. Nat. Prod. Copyright (2008) American Chemical Society an d American Society of Pharmacognosy

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54 requiring chiral HPLC analysis of the acid hydrolyzate. Peaks for L-FDLA derivatives of the corresponding isomers were not detected ( tR, min): LalloThr (15.6), D-Thr (20.5), Dallo -Thr (17.1), D-Val (33.9), D-Lys (42.5), D-Ile/Dallo -Ile (37.2). N,O -diMe-Br-Tyr adducts could not be reliably detected using this UV-base d method, so that LC-MS was used instead. CrO3 oxidation of 4 followed by acid hydrolysis wa s carried out as described.26 The resulting hydrolyzate was derivatized with L-FDLA and aliquots subjected to reversed-phase HPLC as above. When compared to the Marf ey profiles without prior oxidation, the HPLC profiles derived from compound 4 showed one new peak for L-Glu ( tR 16.8 min), but not D-Glu ( tR 17.8 min). Chiral HPLC analysis for compound 4 : Due to overlap of L-FDLA adducts of L-Ile and Lallo -Ile during Marfeys analysis, the acid hydrolyzate derived from 4 was subjected to chiral HPLC analysis (column, Phenomenex Chirex phase 3126 N,S-dioctyl-(D)-penicillamine, 4.60 250 mm, 5 m; solvents, 2 mM CuSO4 in H2O/MeCN (95:5) or 2 mM CuSO4; flow rate 1.0 mL/min; detection at 254 nm). The absolute configuration of Ile in the hydrolyzate of 4 was determined to be L-Ile by direct comparison with the re tention times of authentic standards, while the configurations of th e other amino acids obtained fr om Marfeys analysis were confirmed. The retention times (tR, min) for standard amino acids were as follows: L-Val (16.6), D-Val (21.8), L-Ile (40.8), D-Ile (52.0), Lallo -Ile (34.6), DalloIle (43.1) (solvent mixture 95:5); L-Lys (5.2), D-Lys (6.4), L-Thr (10.8), D-Thr (13.6), Lallo -Thr (15.1), and Dallo -Thr (17.8) (solvent 2 mM CuSO4). Advanced Marfeys analysis of 4 :The hydrolyzate of compound 4 was derivatized with L-FDLA and analyzed by LC-MS accordi ng to the advanced Marfeys method28 [column: Phenomenex, Synergi 4u Hydro-RP 80A, 2 150 mm, 4 m; mobile phase: 0.1% HCOOH in

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55 MeCN (A) in 0.1% HCOOH in H2O (B)] using a step gradient (10% for 15 min, 50-70% for 55 min, then 70-95% for 60 min).T he synthesis of standard N,O -diMe-L-Br-Tyr will be described elsewhere.31 The L-FDLA mono derivative of standard N,OdiMe-Br-Tyr was detected at a retention time of 28.3 min, while the reaction with racemic DL-FDLA gave rise to two peaks at 28.3 and 29.4 min; the latter on e can be attributed to the D-FDLA derivative of N,OdiMe-BrTyr Hence, the retention times of the enantiomer (L-FDLA derivative of N,OdiMe-Br-Tyr) could be inferred (29.4 min). Select ive ion monitoring for CID ESI-MS2 product ions at m/z .522/524 (putative decar boxylation product) from the parent ion at m/z 568/570 [M+H] + for L-FDLA adducts from the hydrolyzate of 4 detected a compound with retention time at 28.3 min, corresponding to the derivative for N,OdiMe-Br-Tyr. During acid hydrolysis N,OdiMe-Br-Tyr underwent partial O -demethylation so that both ami no and phenolic hydroxyl group were available for reaction with L-FDLA. Thus an additional peak at m/z 862/864 [M+H] + corresponding to the bis-L-FDLA derivative of N,OdiMe-Br-Tyr was detected (tR 52.6 min), matching with the additional peak when standard N,OdiMe-Br-Tyr was subjected to acid hydrolysis prior to derivatization with L-FDLA. The retention time for the bis-L-FDLA adduct of N,OdiMe-Br-Tyr was inferred as above from the retention time obtained for the bis-D-FDLA adduct of N,OdiMe-Br-Tyr ( tR 54.8 min). Consequently, the ab solute configuration of the N,OdiMe-Br-Tyr residue in 4 had to be L. 4.5.4 Largazole* A sample of compound 5 (~100 g) was dissolved in 4 mL CH2Cl2 and subjected to ozonolysis at room temperature for 30 min. Th e solvent was evaporated and the residue was treated with 0.6 mL of H2O2HCO2H (1:2) at 70 oC for 20 min. The solvent was evaporated and Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc 2008 130, 1806 1807. Copyright (2008) Am erican Chemical Society

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56 the resulting oxidation product was hydrol yzed with 0.5 mL of 6 N HCl at 110 oC for 24 h. The hydrolyzed product was dried and analyzed by chiral HPLC (column, Phenomenex Chirex phase 3126 N,Sdioctyl-(D)-penicillamine,4.60 250 mm, 5 m; solvent 1, 2 mM CuSO4 in 95:5 H2O/MeCN, pH 4.50; solvent 2, 0.5 mM Cu(OAc)2 /0.1 M NH4OAc in 85:15 H2O/MeCN, pH 4.60; flow rate 1.0 mL/min; detection at 254 nm). The absolute configuration of the amino acids in the hydrolyzate was determined by direct comp arison with the retention times of authentic standards. The retention times (tR, min) for solvent 1 were as follows: Gly (5.3), L-Val (12.6), DVal (16.4), ( S)-2-Me-Cysteic aci d (20.0), and ( R )-2-Me-Cysteic acid (23.9 ). The retention times ( tR, min) of the hydrolyzate components were 5.3, 12.6, 23.9, indicating the presence of Gly, LVal and (R )-2-Me-Cysteic acid in the product mixture. So lvent 2 was used to detect malic acid. Standard L-malic acid eluted at tR 7.6 min and D-malic acid at tR 20.4 min. Malic acid in the hydrolyzate eluted after 7.6 min, indicating the presence of the L isomer. Gly, L-Val, and ( R )-2Me-Cysteic acid eluted after 4.0, 5.8, 6.5 min, respectively. 4.6 Testing for Biological Activity* 4.6.1 Protease Activity Assay The test samples for 14 were prepared in DMSO by (log/ 2)-fold dilutions ranging from 1 mM to 100 pM. All the assays were performed in triplicate. Phenyl methylsulfonyl fluoride (PMSF) was used as a positive control in the enzyme assays. To test the inhibition of porci ne pancreatic elastase (Elast ase-high purity; EPC, EC134), 75 g/mL solution of elastase was prep ared using Tris-HCl (pH 8.0). The Km for elastase was Reproduced in part with permission from Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem.. Soc 2008 130, 1806 1807 (4.6.3). Copyright (2008) American Chemical Society Reproduced in part with permission from Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. web release date: 29-May-2008; DOI: 10.1021/ja8013727 press. Copyright (2008) Am erican Chemical Society Reproduced in part with permission from Taor i, K.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007 70 15931600. Copyright (2007) American Chemical Society and American So ciety of Pharmacognosy

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57 determined to be 1.5 mM for N -succinyl-Ala-Ala-Alap-nitroanilide, a concentration that was used subsequently for the inhib itor dose-response experiments. Af ter pre-incubation of 165 L of Tris-HCl (pH 8.0), 10 L of elas tase solution, and 10 L of test samples in DMSO (5% final concentration) in a microtiter plate at 30 oC for 20 min, 15 L of substrate solution (1.5 mM final concentration) was added to the mixture. The in crease in absorbance was measured by plotting enzyme activity against substrate concentrations in the presence of different inhibitor concentrations (Lin eweaver-Burk plot). Inhibitory activity against -chymotrypsin (bovine pancreas; Sigma, C4129) was determined as follows. A 1 mg/mL solution of ch ymotrypsin was prepared in assay buffer (50 mM Tris-HCl/100 mM NaCl/1 mM CaCl2, pH 7.8). After preincubation of 80 L of assay buffer solution, 10 L of enzyme solution, and 10 L of te st solution in DMSO in a microtiter plate at 37 oC for 10 min, 50 L of substrate solution (N -succinyl-Gly-Gly-Phe-p -nitroanilide, 0.75 mM final concentration corresponding to Km) was added to the mixture. The increase in absorbance was measured for 30 min at in tervals of 5 min at 405 nm. Inhibitory activity against trypsin was assaye d as described above fo r chymotrypsin, using trypsin from porcine panc reas (Sigma, T0303) and N -benzoyl-DL-arginine-4-nitroanilide hydrochloride as the substrate solution. 4.6.2 Cell Culture All cells wer e maintained at 37 oC humidified air (5% CO2) and assayed in high-glucose DMEM (Invitrogen) supplemented with 10% FBS (Hyclone). 4.6.3 General Cytotoxicity Assay HT29 colon adenocarcinoma cells were used for the bioassay-guided fractionation. To determine cell type selectivit y, cells from six cancer cell lin es (U2OS, HeLa, HT29, HCT-116, MDA-MB-231, IMR-32) and two nontransformed ce ll lines (NMuMG and NIH3T3) were plated

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58 in 96-well plate format and incubated at 37 oC (5% CO2) for 24 h and then treated with various concentrations of compounds or solvent control. After another 48 h of in cubation, cell viability was measured using MTT according to manuf acturers instructions (Promega). GI50 and LC50 values were calculated as GI50: concentration where 100 x (T-To) / (C-To) =50 LC50: concentration where 100 x (TTo) / To = [ T = absorbance in treated wells (48 h); To = absorbance in control wells (48 h)] 4.6.4 Cell-free HDAC Enzymatic Assay with HeLa Nuclear Extract Assays were carried out accord ing to the manufacturers protocol (BIOMOL). Briefly, assay buffer (25 L in blank and 10 L in cont rol) and inhibitor (ranging from 10 M to 320 pM, in EtOH) were added to test sample wells of the microtiter plate. HDAC-enriched nuclear protein extract from HeLa cells (BIOMOL, 4 g in 15 L) was added to all wells except in noenzyme control. The assay pl ate was equilibrated at 37 oC, and then 25 L of substrate fluor de Lys TM substrate was added to a final concentrat ion of 50 M. HDAC reaction was allowed to proceed for 15 min and then stopped by a ddition of 50 L per well of 1X fluor de Lys TM Developer containing trichostatin A at 2 M. After developer addi tion, plates were incubated for 15 min at 37 oC and fluorescence was read (Ex 360 nm, Em 460 nm). 4.6.5 Cellular HDAC Activity Assay HCT-116 cells were seeded in 100 L m edium per well at a density of 35,000 cells/well and grown for 24 h in a sterile 96 -well solid bottom plate. The a ssay was carried out according to the manufacturers instructions (BIOMOL). Briefly, after 24 h the medium was replaced with 50 L/well of medium containing 200 M fluor de Lys TM substrate and 1 M of trichostatin A (TSA, positive control, in DMSO) and test comp ounds (largazole, acetyl derivative, in EtOH) ranging from 10 M to 3.2 nM (lg/2-fold dilu tions) and thiol compound ranging from 10 M to

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59 320 pM. Plates were incubated at 37 oC for 8 h. after the treatment time, 50 L per well of 1X fluor de Lys TM Developer containing tr ichostatin A at 2 M was added. After developer addition, plates were inc ubated for 15 min at 37 oC and fluorescence was read (Ex 360 nm, Em 460 nm). 4.6.6 Enzymatic Assay with Recomb inant Human HDAC1 or HDAC6 Assays were carried out accord ing to the manufacturers protocol (BIOMOL). Assay buffer (25 L in blank and 20 L in control) and inhibitor (ranging from 10 M to 320 pM, in DMSO) were added to the test sample wells of the microtiter plate. The assay plate was allowed to equilibrate at 37 oC. 5 L (100 ng/L) of HDAC1 (>1 Unit/g) or HDAC6 (>2 Units/g) enzyme was added to all the wells except in th e no-enzyme control followed by addition of 25 L of substrate fluor de Lys -SIRT1 substrate to a final conc entration of 10 M. HDAC reaction was allowed to run for 30 min at 37 oC and then stopped by addition of 50 L per well of 1X fluor de LysTM Developer II containing trichostatin A at 2 M. After developer addition, plates were incubated at assay temperature for 45 min for signal to develop, and fluorescence was read. (Ex 360 nm and Em 460 nm). 4.6.7 Immunoblot Analysis HCT-116 cells (650,000 cells/dish) w ere seeded in 10-cm dishes and 24 h later treated with various concentrations of largazole, trichostatin A or corresponding solvent controls (EtOH for largazole and DMSO for trichostatin A). Followi ng incubation for 8 h, whole-cell lysates were prepared using Phosphosafe lysis buffer (Novagen), proteins extracted, and protein concentration measured using the BCA method (Pierce). Cell ly sates containing equal am ounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, probed with antibodies and detected with the Supersignal Femto Western blotting ki t (Pierce). Anti-acetyl-histone H3 (Lys9/18)

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60 antibody was obtained from Millipore and anti-actin and anti-rabbit antibody were purchased from Cell Signaling.

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61 APPENDIX NMR SPECTRA

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62 Figure A-1. 1H Spectrum of Lyngbyastatin 7 ( 1 ) in DMSO-d6

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63 Figure A-2. 13C Spectrum of Lyngbyastatin 7 ( 1 ) in DMSO-d6

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64 Figure A-3. COSY Spectru m of Lyngbyastatin 7 ( 1) in DMSOd6

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65 Figure A-4. ROESY Spectrum of Lyngbyastatin 7 ( 1) in DMSOd6

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66 Figure A-5. TOCSY Spectrum of Lyngbyastatin 7 (1) in DMSOd6

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67 Figure A-6. HMQC Spectr um of Lyngbyastatin 7 ( 1) in DMSOd6

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68 Figure A-7. HMBC Spectrum of Lyngbyastatin 7 ( 1) in DMSOd6

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69 Figure A-8. 1H Spectrum of Somamide B ( 2) in DMSO-d6

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70 Figure A-9. 1H Spectrum of Kempopeptin A ( 3) in DMSOd6

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71 Figure A-10. 13C Spectrum of Kempopeptin A ( 3 ) in DMSO-d6

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72 Figure A-11. COSY Spectrum of Kempopeptin A ( 3) in DMSOd6

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73 Figure A-12. ROESY Spectrum of Kempopeptin A ( 3) in DMSO-d6

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74 Figure A-13. TOCSY Spectrum of Kempopeptin A ( 3) in DMSO-d6

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75 Figure A-14. HSQC Spectrum of Kempopeptin A ( 3) in DMSOd6

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76 Figure A-15. HMBC Spectrum of Kempopeptin A ( 3) in DMSOd6

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77 Figure A-16. 1H Spectrum of Kempopeptin B ( 4 ) in DMSO-d6

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78 Figure A-17. 13C Spectrum of Kempopeptin B ( 4 ) in DMSO-d6

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79 Figure A-18. COSY Spectrum of Kempopeptin B ( 4) in DMSOd6

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80 Figure A-19. ROESY Spectrum of Kempopeptin B ( 4) in DMSOd6

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81 Figure A-20. TOCSY Spectrum of Kempopeptin B ( 4) in DMSO-d6

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82 Figure A-21. HSQC Spectrum of Kempopeptin B ( 4) in DMSOd6

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83 Figure A-22. HMBC Spectrum of Kempopeptin B ( 4) in DMSOd6

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84 Figure A-23. 1H Spectrum of Largazole ( 5) in CDCl3

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85 Figure A-24. 13C Spectrum of Largazole ( 5 ) in CDCl3

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86 Figure A-25. COSY Spectrum of Largazole ( 5 ) in CDCl3

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87 Figure A-26. HSQC Spectrum of Largazole ( 5 ) in CDCl3

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88 Figure A-27. HMBC Spectrum of Largazole ( 5 ) in CDCl3 (optimized for nJCH = 7 Hz)

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89 Figure A-28. HMBC Spectrum of Largazole ( 5 ) in CDCl3 (optimized for nJCH = 3.5 Hz)

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90 Figure A-29. NOESY Spectrum of Largazole ( 5 ) in CDCl3

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95 BIOGRAPHICAL SKETCH Kanchan Taori was born in Maharashtra, India. She completed her BS in pharmaceutical chemistry at Indore University in May 1999. After her BS she completed her masters in organic chemistry at Institute of Chemical Sciences, Indo re University, where she was ranked first in her class. Kanchan then joined Anchrom Analytical Lt d. in Mumbai, India, wh ich is a subsidiary of CAMAG Switzerland. Subsequently, she worked for Indofil Chemicals Company and then Hindustan Lever Research Center (HLRC), which is a subsidiary of Unilever. At HLRC, she became interested in natural product chemistry. Af ter four years of industrial experience, this research interest continued on, wh en she joined the University of Florida as a graduate student under the mentorship of Dr. Hendrik Luesch in January 2006. Here, she worked on the isolation and characterization of novel co mpounds from marine cyanobacter ia. She received a Masters of Science in Pharmacy with specialization in medicinal chemistry in August of 2008.