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New antibiotics from a marine isolate of Bacillus laterosporus Barsby, Todd 2002

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N E W A N T I B I O T I C S F R O M A M A R I N E I S O L A T E O F BACILLUS LATEROSPORUS by T O D D B A R S B Y B . S c , University of British Columbia, 1996 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Chemistry We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A July 2002 © Todd Barsby, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT The continued chemical investigation of a marine isolate of Bacillus laterosporus has resulted in the discovery of nine new metabolites, seven of which displayed antibiotic activity. The bogorols A - E (26-30) were found to comprise a novel structural template of the cationic peptide class of antibiotics, while the basiliskamides A and B (50 and 51) were discovered to possess potent antifungal activity versus both Candida albicans and Aspergillus fumigatus. Two structurally novel acyldipeptides, tupuseleiamides A and B (55 and 56), were the result of a serendipitous discovery. N H 2 51 56 Ill The structures in this thesis were elucidated using a combination of N M R spectroscopy, mass spectrometry, and chemical degradation coupled with chiral G C analyses. The fragmentation pattern observed in the mass spectrum of 26 allowed for the rapid structure elucidation of 27-30. A n empirical method based on steric and electronic arguments was used in conjunction with a series of partial acid hydrolyses to rationalize the relative placement of the enantiomeric pairs of amino acids within the constitution of 26. A series of common structural features defined the novel bogorol cationic peptide template. The C and N termini of the linear peptides were found to be capped by an amino alcohol and an a-hydroxy acid, respectively, leaving the cationic residues to reside solely in the interior of the peptide. In addition, the bogorols contained the uncommon amino acid, £'-2-amino-2-butenoic acid. A preliminary investigation of the secondary structure of the bogorols revealed their propensity to exist as a-helices, thus allowing 26-30 to adopt the amphipathic structure characteristic of cationic peptide antibiotics. The bogorols were found to exhibit in vitro antimicrobial activity versus methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus spp. comparable to that found with other cationic peptide antibiotics. Finally, progress was made towards the total synthesis of 26. The de novo biosynthesis of 2,6-dimethyl-5-heptenal (60) by the dendronotid nudibranch, Melibe leonina, was investigated using a stable isotope incorporation experiment with [l,2-13C2]acetate. This study represents the first demonstration of de novo terpene biosynthesis by a dendronotid nudibranch. A s well , it is the first example to show that nudibranchs are capable of de novo monoterpene biosynthesis. The work reported with M. leonina supports recent evolutionary theory with regards to nudibranch de novo biosynthesis. o 60 T A B L E O F C O N T E N T S Abstract Table of Contents List of Tables List of Figures List of Schemes List of Abbreviations Acknowledgements C H A P T E R 1: General Introduction 1.1. The Antibiotic Armory 1.2. Antibiotic Resistance 1.3. Marine Bacteria as a Source of New Drugs 1.4. The Marine Bacillus as a Source of New Antibiotics 1.5. The Research Program C H A P T E R 2: Cationic Peptide Antibiotics from Bacillus laterosporus 2.1. A Br ief Review of Cationic Peptide Antibiotics 2.2. Introduction 2.3. Isolation 2.4. Structure Elucidation of Bogorol A (26) 2.5. Stereochemistry of Bogorol A (26) 2.6. Secondary Structure 2.7. Structure Elucidation of Bogorol B (27) V 2.8. Structure Elucidation of Bogorol C (28) 65 2.9. Structure Elucidation of Bogorol D (29) 69 2.10. Structure Elucidation of Bogorol E (30) 73 2.11. Biological Activity 77 2.12. A Discussion of the Progress Towards the Total Synthesis of Bogorol A (26) 78 2.13. Conclusion 84 C H A P T E R 3: Antifungal Metabolites from Bacillus laterosporus 87 3.1. Introduction 87 3.2. Isolation 87 3.3. Structure Elucidation of Basiliskamide A (50) 88 3.4. Stereochemistry of Basiliskamide B (50) 97 3.5. Structure Elucidation of Basiliskamide B (51) 102 3.6. Biological Activi ty 103 3.7. Conclusion 105 C H A P T E R 4: Other Metabolites from Bacillus laterosporus 106 4.1. Introduction 106 4.2. Isolation 106 4.3. Structure Elucidation of Tupuseleiamide A (55) 107 4.4. Structure Elucidation of Tupuseleiamide B (56) 117 4.5. Fatty A c i d Biogenesis 120 4.6. Conclusion 125 VI C H A P T E R 5: General Conclusion for Chapters 2,3, and 4 126 C H A P T E R 6: De Novo Terpene Biosynthesis by Melibe leonina 128 6.1. General Introduction 128 6.2. Rationale 130 6.3. [ l ,2 - 1 3 C 2 ] -NaOAcBiosynthe t ic Experiment 132 6.4. Results 132 6.5. Conclusion 136 E X P E R I M E N T A L S E C T I O N 138 Materials and methods 138 Chapter 2 140 Chapter 3 149 Chapter 4 152 Chapter 6 153 A P P E N D I X 154 A . 1. Appendix for Chapter 2 154 A.2 . Appendix for Chapter 3 186 A . 3 . Appendix for Chapter 4 195 A.4 . Appendix for Chapter 6 200 Bibliography 202 vii L I S T O F T A B L E S Page Table 1.1.1. Examples of clinically used antibiotics. 2 Table 2.4.1. N M R data for bogorol A hexaacetate (31). 29 Table 2.5.1. Chiral G C retention times of bogorol A hexaacetate (31) hydrolyzate. 41 Table 2.5.2. Contribution to peptide six numbers. 47 Table 2.7.1. Chiral G C retention times of bogorol B - E (27-30) hydrolyzates. 63 Table 2.7.2. Chemical shifts of H B - 3 within the bogorol family (26-30). 64 Table 2.11.1. Min imum inhibitory concentration (|lg/mL) of bogorols A - E (26-30). 77 Table 3.3.1. J H and 1 3 C N M R data for basiliskamide A (50) recorded in D M S O - d 6 . 89 Table 3.5.1. ' H and 1 3 C N M R data for basiliskamide B (51) recorded in D M S O - d 6 . 103 Table 3.6.1. Activi ty of basiliskamide A (50) and amphotericin B against clinical 104 isolates of Candida albicans as determined by macrobroth dilution. Table 3.6.2. Comparative activity of basiliskamides A (50), B (51), and YM-47522 104 (54) as determined by agar dilution. Table 3.6.3. Antimycobacterial activity of the basiliskamides as determined by agar 104 dilution. Table 4.3.1. ! H and 1 3 C N M R data for tupuseleiamide A (55) recorded in D M S O - d 6 . 108 Table 4.4.1. ] H and 1 3 C N M R data for tupuseleiamide B (56) recorded in DMSO-d6. 119 Table 6.4.1. Identification of 2,6-dimethyl-5-heptenal (60). 136 Table A.2 .1 . ' H N M R chemical shifts for Mosher ester derivatives of basiliskamide A 187 (50), 50a, and 50b, recorded in D M S O - d 6 -vi i i L I S T O F F I G U R E S Page Figure 2.1.1. Four classes of peptide antibiotics. 14 Figure 2.1.2. A general depiction of membrane destabilization. 15 Figure 2.1.3. Cationic peptide antibiotics undergoing clinical trials. 16 Figure 2.4.1. 500 M H z *H N M R Spectrum of bogorol A (26) in D M S O - d 6 . 20 Figure 2.4.2. 800 M H z [ H N M R Spectrum of bogorol A hexaacetate (31) in 22 D M S C M 6 . Figure 2.4.3. 100 M H z 1 3 C N M R Spectrum of bogorol A hexaacetate (31) in 23 D M S O - c i 6 . Figure 2.4.4. 800 M H z C O S Y Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 . 24 Figure 2.4.5. 800 M H z T O C S Y Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 - 25 Figure 2.4.6. 800 M H z H S Q C Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 . 26 Figure 2.4.7. 800 M H z H M B C Spectrum of bogorol A hexaacetate (31) in D M S O - J ^ . 27 Figure 2.4.8. 800 M H z N O E S Y Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 . 28 Figure 2.4.9. 800 M H z N O E S Y Spectrum; N H region expansion of bogorol A 36 hexaacetate (31) in D M S O - ^ . Figure 2.4.10. Universal nomenclature of peptide fragment. 37 Figure 2.4.11. ESI -QIT-MS of bogorol A (26). 38 Figure 2.4.12. E S I - Q I T - M S / M S of bogorol A ' s (26) parent ion. 39 Figure 2.5.1. 800 M H z N O E S Y Spectrum with selected correlations; a-methine region 43 expansion A of bogorol A hexaacetate (31). Figure 2.5.2. 800 M H z N O E S Y Spectrum with selected correlations; a-methine region 44 expansion B of bogorol A hexaacetate (31). Figure 2.5.3. Selected N O E S Y correlations of bogorol A hexaacetate (31). 45 Figure 2.5.4. Coiled structure for acids, numbering shown. 46 Figure 2.5.5. Rule of six numbering system of a peptide bond. 46 IX Figure 2.5.6. A dipeptide model for the early stages of the hydrolysis of 26 and the 48 assignment of residue average six numbers. Figure 2.5.7. Early times of the amino acid analysis of bogorol A (26) hydrolyzate and 50 26 with residue average six numbers. o Figure 2.5.8. Late times of the amino acid analysis of bogorol A (26) hydrolyzate and 51 26 with residue average six numbers. Figure 2.6.1. Helical secondary structures. 54 Figure 2.6.2. C D Spectrum of 300 | l M bogorol A hexaacetate (31). 56 Figure 2.7.1. 500 M H z ' H N M R Spectrum of bogorol B (27) in D M S O - d 6 . 59 Figure 2.7.2. ESI -QIT-MS of bogorol B (27). 60 Figure 2.7.3. E S I - Q I T - M S / M S of bogorol B ' s (27) parent ion. 61 Figure 2.7.4. Nonribosomal peptide synthetase. 62 Figure 2.8.1. 500 M H z ] H N M R Spectrum of bogorol C (28) in D M S O - d 6 . 66 Figure 2.8.2. ESI -QIT-MS of bogorol C (28). 67 Figure 2.8.3. E S I - Q I T - M S / M S of bogorol C 's (28) parent ion. 68 Figure 2.9.1. 500 M H z ' H N M R Spectrum of bogorol D (29) in D M S O - J 6 . 70 Figure 2.9.2. ESI -QIT-MS of bogorol D (29). 71 Figure 2.9.3. E S I - Q I T - M S / M S of bogorol D 's (29) parent ion. 72 Figure 2.10.1. 500 M H z ! H N M R Spectrum of bogorol E (30) in DMSO-ck . 74 Figure 2.10.2. ESI -QIT-MS of bogorol E (30). 75 Figure 2.10.3. E S I - Q I T - M S / M S of bogorol E 's (30) parent ion. 76 Figure 2.13.1. Depiction of 26 as an a-helix. 85 Figure 3.3.1. 100 M H z 1 3 C N M R Spectrum of basiliskamide A (50) in D M S O - d 6 . 90 Figure 3.3.2. 400 M H z ' H N M R Spectrum of basiliskamide A (50) in D M S O - ^ . 91 Figure 3.3.3. 500 M H z C O S Y Spectrum of basiliskamide A (50) in D M S O - d 6 . 92 Figure 3.3.4. 500 M H z H M Q C Spectrum of basiliskamide A (50) in D M S O - J 6 . 93 X Figure 3 . 3 . 5 . 5 0 0 M H z H M B C Spectrum of basiliskamide A (50) in DMSO-d6. 9 4 Figure 3 . 4 . 1 . Results from selected nOe difference experiments on basiliskamide A 9 7 (50). Figure 3 . 4 . 2 . 5 0 0 M H z H M Q C Spectrum of acetontide (53) in D M S O - d 6 . 9 9 Figure 3 . 4 . 3 . Decoupling experiment ( 4 0 0 M H z ) with acetonide 53. 100 Figure 3 . 5 . 3 . A8 (ppm) Values for the Mosher ester derivates 50a and 50b. 1 0 1 Figure 4 . 3 . 1 . 4 0 0 M H z *H N M R Spectrum of tupuseleiamide A (55) in D M S O - c 7 6 . 1 0 9 Figure 4 . 3 . 2 . 1 0 0 M H z l 3 C N M R Spectrum of tupuseleiamide A (55) in D M S O - d 6 . 1 1 0 Figure 4 . 3 . 3 . 5 0 0 M H z H M Q C Spectrum of tupuseleiamide A (55) in D M S O - d 6 . 1 1 1 Figure 4 . 3 . 4 . 5 0 0 M H z C O S Y Spectrum of tupuseleiamide A (55) in DMSO-<i 6 . 1 1 2 Figure 4 . 3 . 5 . 5 0 0 M H z H M B C Spectrum of tupuseleiamide A (55) in D M S O - c i 6 . 1 1 3 Figure 4 . 3 . 6 . Select H M B C correlations for 55. 116 Figure 4 . 4 . 1 . 4 0 0 M H z ' H N M R Spectrum of tupuseleiamide B (56) in DMSO-rf 6 . 118 Figure 4 . 5 . 1 . 100 M H z 1 3 C N M R Spectrum of tupuseleiamide A (55) in D M S O - J e - 1 2 3 [ l , 2 - 1 3 C 2 ] - N a O A c experiment. Figure 4 . 5 . 2 . Normalized l 3 C signals from N a O A c labelling experiment. 1 2 4 Figure 6 . 1 . 1 . Nudibranch terpenes produced by de novo biosynthesis. 1 2 9 Figure 6 . 2 . 1 . Photograph of M. leonina. 1 3 0 Figure 6.4.1. 4 0 0 M H z ' H and 100 M H z l 3 C N M R Spectra of 2,6-dimethy-5-heptenal 1 3 3 (60) in C D C 1 3 . Figure 6 . 4 . 2 . 1 0 0 M H z l 3 C N M R Spectrum of 2,6-dimethyl-5-heptenal (60) - 1 3 4 [ l , 2 - 1 3 C 2 ] - N a O A c experiment. Figure 6 . 4 . 3 . I 3 C Signals from N a O A c labelling experiment. 1 3 5 Figure A . 1 .1 . 8 0 0 M H z ' H N M R Spectrum; NH-region expansion of bogorol A 1 5 5 hexaacetate (31). Figure A . 1 .2 . 800 M H z *H N M R Spectrum; a-methine region expansion of bogorol A 1 5 6 hexaacetate (31). XI Figure A . 1.3. 800 M H z *H N M R Spectrum; aliphatic region expansion of bogorol A 157 hexaacetate (31). Figure A . 1.3a. 800 M H z ' H N M R Spectrum; methyl region expansion of bogorol A 158 hexaacetate (31). Figure A.1.4. 100 M H z 1 3 C N M R Spectrum; downfield region expansion of bogorol A 159 hexaacetate (31). Figure A.1.5 . 100 M H z I 3 C N M R Spectrum; upfield region expansion of bogorol A 160 hexaacetate (31). Figure A . 1.6. 800 M H z C O S Y Spectrum with selected correlations; methyl region 161 expansion of bogorol A hexaacetate (31). Figure A.1.7. 800 M H z C O S Y Spectrum with selected correlations; aliphatic region 162 expansion of bogorol A hexaacetate (31). Figure A . 1.8. 800 M H z C O S Y Spectrum with selected correlations; a-methine region 163 expansion of bogorol A hexaacetate (31). Figure A . 1.9. 800 M H z C O S Y Spectrum with selected correlations; a-methine region 164 expansion B of bogorol A hexaacetate (31). Figure A . 1.10. 800 M H z C O S Y Spectrum with selected correlations; N H region 165 expansion of bogorol A hexaacetate (31). Figure A . l . 1 1 . 800 M H z T O C S Y Spectrum with selected correlations; N H region 166 expansion A of bogorol A hexaacetate (31). Figure A . 1.12. 800 M H z T O C S Y Spectrum with selected correlations; N H region 167 expansion B of bogorol A hexaacetate (31). Figure A . l . 1 3 . 800 M H z T O C S Y Spectrum with selected correlations; N H region 168 expansion C of bogorol A hexaacetate (31). Figure A . 1.14. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region 169 expansion A of bogorol A hexaacetate (31). Figure A . 1.15. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region 170 expansion B of bogorol A hexaacetate (31). Figure A . 1.16. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region 171 expansion C of bogorol A hexaacetate (31). Figure A . 1.17. 800 M H z H S Q C Spectrum with selected correlations; aliphatic region 172 expansion A of bogorol A hexaacetate (31). Xll Figure A . 1.18. 800 M H z H S Q C Spectrum with selected correlations; methyl region 173 expansion A of bogorol A hexaacetate (31). Figure A . 1.19. 800 M H z H S Q C Spectrum with selected correlations; aliphatic region 174 expansion B of bogorol A hexaacetate (31). Figure A . 1.20. 800 M H z H S Q C Spectrum with selected correlations; aliphatic region 175 expansion C of bogorol A hexaacetate (31). Figure A . 1.21. 800 M H z H S Q C Spectrum with selected correlations; a-methine region 176 expansion of bogorol A hexaacetate (31). Figure A . 1.22. 800 M H z H S Q C Spectrum with selected correlations; downfield region 177 expansion of bogorol A hexaacetate (31). Figure A . 1.23. 800 M H z H M B C Spectrum with selected correlations; methyl region 178 expansion of bogorol A hexaacetate (31). Figure A . 1.24. 800 M H z H M B C Spectrum with selected correlations; aliphatic region 179 expansion A of bogorol A hexaacetate (31). Figure A . 1.25. 800 M H z H M B C Spectrum with selected correlations; aliphatic region 180 expansion B of bogorol A hexaacetate (31). Figure A . 1.26 800 M H z H M B C Spectrum with selected correlations; aliphatic region 181 expansion C of bogorol A hexaacetate (31). Figure A . 1.27. 800 M H z H M B C Spectrum with selected correlations; a-methine region 182 expansion of bogorol A hexaacetate (31). Figure A . 1.28. 800 M H z H M B C Spectrum with selected correlations; N H region 183 expansion of bogorol A hexaacetate (31). Figure A . 1.30. 800 M H z N O E S Y Spectrum with selected correlations; NH/aliphatic 184 region expansion of bogorol A hexaacetate (31). Figure A . 1.31. 800 M H z N O E S Y Spectrum with selected correlations; NH/a-methine 185 region expansion of bogorol A hexaacetate (31). Figure A.2 .1 . nOe Experiments on basiliskamide A (50) in DMSO-<i<5. 188 Figure A.2.2. 400 M H z ' H N M R Spectra of Mosher ester (50a and 50b) derivative of 189 basiliskamide A (50) in D M S O - c i 6 . Figure A.2.3. 400 M H z ' H N M R Spectrum of basiliskamide B (51) in D M S O - d 6 . 190 Figure A.2.4. 100 M H z 1 3 C N M R Spectrum of basiliskamide B (51) in D M S O - J 6 . 191 Xlll Figure A.2.5. 500 M H z C O S Y Spectrum of basiliskamide B (51) in D M S O - d 6 . 192 Figure A.2.6. 500 M H z H M Q C Spectrum of basiliskamide B (51) in D M S O - d 6 . 193 Figure A.2.7. 500 M H z H M B C Spectrum of basiliskamide B (51) in D M S O - d 6 . 194 Figure A.3 .1 . 100 M H z 1 3 C N M R Spectrum of tupuseleiamide B (56) in D M S O - d 6 . 196 Figure A.3.2. 500 M H z C O S Y Spectrum of tupuseleiamide B (56) in D M S O - d 6 . 197 Figure A.3.3. 500 M H z H M Q C Spectrum of tupuseleiamide B (56) in D M S O - d 6 . 198 Figure A.3.4. 500 M H z H M B C Spectrum of tupuseleiamide B (56) in D M S O - c i 6 . 199 Figure A.4 .1 . 500 M H z H M Q C Spectrum of 2,6-dimethyl-5-heptenal (60) in C D C 1 3 . 201 LIST OF SCHEMES Scheme 2.5.1. Production of Hmp stereoisomers. Page 42 Scheme 2.12.1. Phomopsin A (35) and the formation of an £-dehydroamino acid. 79 Scheme 2.12.2. Retrosynthetic analysis of bogorol A (26). 80 Scheme 2.12.3. Synthetic plan for bogorol A (26). 82 Scheme 3.4.1. Conversion of 50 to acetonide 53. 98 Scheme 4.5.1. Linear and branched-chain fatty acid biosynthesis. 121 Scheme 4.5.2. Branched-chain fatty acid primers. 121 Scheme 4.5.3. Branch-chained labelling pattern for 55. 122 Scheme 6.4.1. The mevalonate pathway. 130 xiv L I S T O F A B B R E V I A T I O N S [a]25D A , A l a Aba, B A c A l a , A A r B , Aba B O C br C C D CDCI3 C H 2 C 1 2 C O C o A cone C O S Y d dd 5 D B U angstrom specific rotation at wavelength of sodium D line and 25°C alanine 2-amino-2-butenoic acid acetate alanine argon 2-amino-2-butenoic acid ferf-butoxycarbonyl broad cysteine circular dichroism deuterochloroform dichloromethane carbonyl coenzyme A concentrated homonuclear correlation spectroscopy doublet doublet of doublets chemical shift in parts per mill ion l,8-diazobicyclo[5.4.0]undec-7-ene XV D E A D diethyl azodicarboxylate D H P dihydropyran D D 3 A L diisobutylaluminum hydride D I P E A Ar,/V-diisopropylethylamine D M A P 4-A^N-dimethylaminopyridine D M F dimethylformamide D M S O dimethyl sulfoxide E glutamic acid e extinction coefficient equiv equivalent ESI electrospray ionization E t O A c ethyl acetate F phenylalanine Fmoc 9-fluorenylmethoxycarbonyl FTIR fourier transformation infra-red spectroscopy G , G ly glycine G C gas chromatography Gly , G glycine H + acid HC1 hydrochloric acid H M B C heteronuclear multiple bond multiple quantum correlation spectroscopy H m p , X 2-hydroxy-3-methylpentanoic acid H M Q C heteronuclear multiple quantum correlation spectroscopy H 2 0 water XVI H P L C H R D C T M S H R E M S H R E S I - T O F H R F A B M S i He, I I, He IR J, Met(O) J L , Leu -^max Leu, L Lys, K m M , Met M + m/z Me M e C N M e O H Met, M Met(O), J high performance liquid chromatography high resolution desorption chemical ionization mass spectrometry high resolution electron impact ionization mass spectrometry high resolution electrospray ionization time of flight high resolution fast atom bombardment mass spectrometry N M R resonance due to impurity in sample isoleucine isoleucine infra-red methionine sulfoxide scalar coupling constant in H z leucine wavelength at absorbance maximum leucine lysine multiplet methionine molecular ion mass-to-charge ratio methyl acetonitrile methanol methionine methionine sulfoxide M I C minimum inhibitory concentration M R S A methicillin resistant Staphylococcus aureus M S mass spectrum M T B Mycobacterium tuberculosis M T P A (a-methoxy)(a-trifluoromethyl)phenylacetic acid -Mosher 's acid N asparganine N / A not applicable N a B H 4 sodium borohydride N a 2 C 0 3 sodium carbonate N a N 3 sodium azide N a O A c sodium acetate N a 2 S 0 4 sodium sulfate N E t 3 triethylamine N H 4 C 1 ammonium chloride N M R nuclear magnetic resonance nOe nuclear Overhauser effect N O E S Y nuclear overhauser and exchange spectroscopy N P normal phase 0 , Orn ornithine ODS octadecyl silyl Orn, 0 ornithine Pd-C paladium on carbon pet. ether petroleum ether P F P A - I P E pentafluoropropionyl amide isopropyl ester XV111 Ph P P h 3 ppm PPTS pTsOH P y B O P q QIT R Rf R N A R P rt S S A R S C U B A S i 0 2 S O C l 2 sp. spp. t t phenyl triphenylphosphine parts per million pyridinium p-toluenesulfonate p-toluenesulfonic acid monohydrate benzotriazol-1 -yl-oxytripyrrolidinophophonium hexafluorophosphate quartet quadrupole ion trap arganine retardation factor ribonucleic acid reversed-phase room temperature solvent singlet serine structure-activity relationship self-contained underwater breathing apparatus normal phase silica gel thionyl chloride species (singular) species (plural) triplet time T, Thr threonine T B tuberculosis tBu tert-butyl T F A ' trifluoroacetic acid T H F tetrahydrofuran T H P tetrahydropyran T h r , T threonine T L C thin layer chromatography Tip, W tryptophan Tyr, Y tyrosine U V ultra-violet V , V a l valine V a l , V valine V o l , Z valinol w signal due to water W , T r p tryptophan wt% percent by weight V R E vancomycin resistant enterococci Y , Tyr tyrosine X , Hmp 2-hydroxy-3-methylpentanoic acid Z, V o l valinol XX A C K N O W L E D G E M E N T S This thesis stands on the words of encouragement and support of many a friend and family member. Foremost, I would like to acknowledge Professor Raymond J. Andersen, for both approaching science with a contagious enthusiasm and for the diversity of his interests, which allowed me to attempt a myriad of projects. The many members of Prof. Andersen's lab, past and present, have made the undertaking of this thesis a thoroughly enjoyable task. Thanks especially to the brilliant Dave Wil l iams; I couldn't have asked for a better labmate. As well, Roger Linington and Urmi la Deo Jangra, deserve a special mention for making a day at the office so pleasurable, and for all their hard work at keeping this thesis going. Finally, my friendship with Rob Britton should also be duly noted, as there is no one I trust more at -60 ft below the ocean's surface. To my friends and fellow chemists, Rob, Roger, Michael, Carl and Art for their captivating conversations at coffee-time, and for making kayaking with kegs seem so everyday, thank you all. Thanks, especially, to my parents, Lyle and LeeAnn, for their constant support that has allowed me to work at what I enjoy, even if nobody else understands it. To my in-laws, Bob and Jane, for sharing with me their enthusiasm for the marine environment and for simply remarking that they were looking forward to reading my thesis, thank you. A n d of course, I thank Ginger Warden with all my heart, for always being there when the chemistry wasn't, but mostly for making the last five years the most rewarding of my life so far. Lastly, I would also like to acknowledge the fine folks at the Department of Chemistry's N M R facility, M S facility, and Biological Services. A s well , a thank you to all the members of SeaTek Marine Biotechnology, the National High Field N M R Center, U B C ' s Biotechnology Laboratory, and the Department of Fisheries and Oceans West Vancouver Laboratory, who contributed to the work presented herein. 1 C h a p t e r 1. G e n e r a l I n t r o d u c t i o n 1.1. The Antibiotic Armory The search for new antibiotics started in earnest after the successful implementation of large-scale penicillin G (1) production during Wor ld War II, the first major conflict where more soldiers died in battle than from infection. 1 Alexander Flemming had discovered the (3-lactam antibiotic penicillin G over a decade earlier as a natural product of the fungus Penicillium notatum2 With Flemming's discovery, came insight into the degree of chemical warfare being waged at the microbial level; chemical warfare which, as the penicillin experience showed, could be exploited for humankind's benefit. The triumph of penicillin was followed quickly by the discovery of the second major group of fungal (3-lactam antibiotics, the cephalosporins (cephalosporin C , 2).1 1 2 The social ramifications of the discovery of antibiotics have been enormous; within twenty years of the creation of a therapeutic armory to combat infection, Americans saw a ten year increase in their life expectancy.3 However, by the end of the 1950s, most of the classes of today's clinically used antibiotics had been discovered (Table 1.1.1).4 The fluoroquinolines (1970s) and the oxazolidinones (2000), have been the only new antibiotics, which act via novel modes of action, added to the therapeutic armory in the last forty years. 4 , 5 A s a consequence of the enormous cost and time required to develop a new class of antibiotics, 6 the principal advances over the years have come from second and third generation semi-synthetic modifications to existing antibiotics. 4 The result of which has been the chronic use of a select number of antibiotics that act via a few modes of action; thereby setting the stage for the rapid rise of antibiotic resistance. Table 1.1.1. Examples of clinically used antibiotics (information from D a x 4 and Walsh 5 ) . Antibiotic Class (structure shown) Source Bacterial Target Representative Structure (3-lactam (Penicillin G) Fungus Penicillium sp. Cephalosporium sp. Cell-wall Biosynthesis H H C0 2 H Tetracycline (Tetracycline) Bacteria Protein Streptomyces sp. Biosynthesis OH O OH O Aminoglycoside Bacteria Protein (Kanamycin A) Streptomyces sp. Biosynthesis NH 2 HO—i-L-0 HO O H i HO 0 h b - T - l - T ~ 0 H 2 N — * ' — - 1 — I p~-J—j—OH f - | - J - N H 2 OH NH 2 Macrolide Bacteria Protein (Erythromycin) Streptomyces sp. Biosynthesis NMe2 Glycopeptide (Vancomycin) Bacteria Streptomyces sp. Cell-wall Biosynthesis Fluoroquinolone Synthetic (Ciprofloxacin) D N A Repair and Replication HN F - ^ ^ A ^ C O O H ^ A o. f~9 H Oxazolidinone Synthetic Protein °vZ/N _ <C^ _ N^ )^/N^ (Linezolid) Biosynthesis / X 3 1.2. Antibiotic Resistance The emergence of antibiotic resistance has been a natural consequence of the selective pressure generated by antibiotic usage. This disheartening hypothesis was first supported by the discovery of |3-lactam resistance in Staphylococcus aureus, two years after the introduction of penicillin. 7 The reader is directed to an excellent review by Walsh for a discussion of the main mechanisms of resistance, including the formation of drug efflux pumps, the creation of hydrolytic (3-lactamases, and substrate reprogramming. 5 Ten years ago, a special issue of Science highlighted the problems and potential solutions • ' 8 9 to the phenomenon of antibiotic resistance. ' In general, the responsible use of existing antibiotics, the search for new structural classes, as well as the use of synergistic drugs to bolster old ones were the parallel courses of action emphasized. Today the crisis of antibiotic resistance continues, fuelled by the volume of antibiotics used in hospital, 1 0 agricultural 1 1 and other commercial settings, 1 2 which results in the release of copious quantities of antibiotics into the environment each year. 1 3 The combination of the selective pressure of the antibiotics and the ability of bacteria to exchange genetic information between differing genera and species has facilitated the spread of antibiotic resistance. Although previously relegated to hospital settings, some pathogens, like methicillin resistant S. aureus ( M R S A ) and vancomycin resistant Enterococcus spp. ( V R E ) are now emerging in the general community. 1 4 ' 1 5 Tuberculosis (TB), which for years had been on the decrease in the United States, is on the rise, accompanied by multi-drug resistant species. 1 6 ' 1 7 A s well , the eventual emergence of antifungal drug resistance is seen as inevitable. 1 8 This disturbing trend is exacerbating an already tenuous health-care situation. The reports, from Europe, the United States, Japan and the Far East, of the isolation of M R S A strains with reduced susceptibilities to vancomycin, the antibiotic of last-resort, has confirmed fears that 4 a new era of untreatable super-bugs is approaching. 1 9 Currently, fi-lactam resistance has increased the attributable mortality from a S. aureus infection from 8% to 17%, and given the recent trend towards vanomycin resistance, a greater increase in mortality can be expected in the future. Once established in the natural population, and in the absence of a selective pressure, antibiotic resistant organisms wi l l not easily disappear.2 1 As a consequence of our previous inaction in controlling the trend of antibiotic resistance, 22 an emerging problem has developed into a crisis. While a concerted effort in limiting the environmental inundation by antibiotics is critical for controlling the future emergence of antibiotic resistance, those pathogens currently resistant are now endemic, particularly in our hospitals. If the medical establishment is to regain control of common but potentially deadly infections, then it is imperative that we look again to "new drug discovery to improve our therapeutic armory." 2 3 1.3. Marine Bacteria as a Source of New Drugs A recent review by Newman et al. documents the important role that natural products have played in drug discovery, particularly with regards to antimicrobials. 2 4 Historically, terrestrial soil bacteria have been a prolific source of antibiotics. However, in the last decade there has been a concerted and successful effort to exploit the biomedical potential of the previously ignored marine microorganisms, particularly marine bacteria. 2 5" 3 1 Aside from their isolation source, the characteristics and properties that differentiate a marine bacterium from a terrestrial bacterium are not rigorously defined. 3 1 Generally marine bacteria are viewed as possessing a requirement for sodium (seawater) for growth. 3 2 However, examples of marine bacteria do exist where they have been shown to subsist on extremely low sodium levels, to the extent that the contaminant levels found within various media were adequate for growth. 3 3 In this thesis, the term 'marine bacteria' refers to salt-tolerant, not salt-5 obligate, bacteria isolated from the marine environment. Given that the marine and terrestrial environments are not closed systems, it is expected that there wi l l be an overlap of metabolically active bacteria between the two systems. The development of molecular biological techniques, which have allowed for the examination of the ocean's bacterial community, 3 4 has revealed small scale variability within the water column. 3 5 In their examination of the bacterium-bacterium antagonistic interactions that may account for the millimeter-scale variation, Long and Azam found that 5 3 . 5 % of their bacterial isolates produced antibiotic activity against other marine bacteria. 3 6 Thus, antibiotics are hypothesized to play an important role in bacterial community structuring. This hypothesis has been supported by competition studies that observed the induction of antibiotic production by a marine Streptomyces sp. when grown in the presence of other marine bacteria. 3 7 Importantly, the induced antibiotic levels inhibited the growth of the potential competitors. Marine bacteria are not confined solely to the water column. In fact, they have colonized practically every microenvironment of the oceans, including the sediments, the surfaces of all animate and inanimate objects, and the insides of marine organisms, as potential symbionts. 2 8 Indeed, bacteria have been hypothesized as the true producers of many of the complex, and uniquely marine, natural products which were originally isolated from marine invertebrates.3 8 For example, it has been suggested that the cytotoxic bryozoan metabolites, the bryostatins (bryostatin A , 3) are actually produced by a recently described symbiont, Candidatus Endobugula sertula. 4 0 6 OOQ. 3 4 As well, as chronicled by Faulkner et a l . , 4 1 the complex peptide theopalauamide (4) 4 2 isolated from a sponge in Palau has been shown by cell separation experiments to be associated, not with the sponge cells, but with the filamentous bacterial cells. In both these instances, the bacterial symbionts have resisted culturing. The story of the bryostatins, which are undergoing anti-cancer clinical trials, highlights a key issue that marine microbial natural product drug discovers are faced with, namely the current limitations of modern microbiological methods. The bryostatins and the theopalauamides, with their complex and unique structures, provides tantalizing evidence of the possible rewards of working to further microbial cloning and culturing techniques to fully exploit the microbial biodiversity of the oceans. Nevertheless, antibiotics have been found by successfully culturing marine bacteria isolated from seawater, marine sediments, and the surfaces of marine organisms. Some of the first compounds isolated from marine bacterium were the antibiotic brominated pyrroles pentabromopsuedilin (5), tetrabromopyrrole (6), and hexabromo-2,2'-bipyrrole (7), produced by seawater isolates of Altermonas spp 4 3 , 4 4 7 The high levels of bromine in 5, 6, and 7, which is abundant in seawater but not terrestrially, provided some of the first experimental evidence in support of the hypothesis that marine bacteria are a source of novel chemistry. Currently, some of the strongest insight into the chemical potential of marine bacteria has resulted from the collaboration of Fenical and Jensen. The salinamides A - B 4 5 (salinamide A , 8) and the cyclomarins A - C 4 6 (cyclomarin A , 9) were isolated from Streptomyces spp. discovered associated with the surface of a jellyfish and within marine sediments, respectively. Both 8 and 9 displayed antiinflammatory activity, while 8 also showed moderate antibiotic activity. These molecules showcase the unusual metabolic capabilities of marine bacteria. In the case of 8, which has subsequently been found from a terrestrial source, nature is revealed as an unrivaled source of chemical diversity. 9 8 Drawing parallels to terrestrial microbiology, marine bacteria from sediments have been the most intensely studied, and deservedly so, as evidenced by the structure of 9, as well as by the discovery of both marinone 4 7 (10) and the wailupemycins A - C 4 8 (wailupemycin A , 11). Marinone (10) was isolated, alongside its debromo analogue, from cultures of a marine sediment derived, taxonomically-novel, actinomycete that was cultured with 7 5 % seawater. The antibacterial mixed terpene/polyketide 10 possess an unprecedented carbon, skeleton, becoming the first of the marinone class of naphthoquinone antibiotics. The wailupemycins (A, 11) were isolated from a marine sediment derived Streptomyces sp. Octaketide 11 was found along with a series of related enterocin octaketides which had previously been discovered from a terrestrial source. Further work by Piel et al 4 9 showed that the marine derived 11 and its analogues were generated by a series of competitive cyclization pathways encoded within the enterocin gene cluster of the marine Streptomyces sp. Most significantly, 11 and its analogues were only produced when the marine Streptomyces sp. was cultured in a saltwater-based medium. These few examples serve to highlight marine bacteria as a source of potential new drugs and novel chemistry. The genus Bacillus, well known for its production of peptide antibiotics in terrestrial species, 5 0 is comparatively unstudied with respect to marine isolates. As such, they represent a potentially rewarding start for the search for new antibiotics from marine bacteria. 9 1.4. The M a r i n e Bacillus as a Source of New Antibiot ics In order to appreciate the relative paucity of chemical structures isolated from marine Bacillus spp., it is important to examine a few of the main peptide antibiotics that have been isolated from terrestrial Bacillus spp. These include the tyrocidines (tryocidine A , 12), gramicidins (gramicidin C, 13), polymixins (polymixin B , 14) and the bacitracins (bacitracin A , 15). 4 The degree of chemical diversity that terrestrial Bacillus spp. are capable of employing is revealed within the cyclic structures of 12,14, and 15 and the linear structure of 13. The peptide 10 antibiotics of the terrestrial Bacillus sp. are typically comprised of both common amino acids and unusual amino acids, as is seen with the a-y-diaminobutyric acid residues of 14 and the thiozole ring containing residue of 15. These peptides have found various levels of commercial success as topical antibiotics. In general though, they have not been significant additions to the antibiotic armory due to issues of solubility, toxicity, and absorption.4 As well, peptide antibiotics face the inherent problem of gastrointestinal instability, which is associated with most peptides in acidic environments, limiting their systemic use. In their 1977 review, Katz and Demain reported a list of forty-four groups of peptide antibiotics from ten different species of terrestrial Bacillus, of which the four previous peptides represent four of the groups detailed. 5 0 In contrast, there has been approximately seven groups of molecules reported from various marine isolates of Bacillus spp. over the years, only three of which were peptides. The nonpeptidic molecules include the amino sugar antibiotic 16, isolated from cultures of a Bacillus sp. obtained from sediments collected at -4310m in the Pacific basin, 5 1 the aromatic diol guaymasol (17),52 the cytotoxic isocoumarin PM-94125 (18),53 and the antibiotic macrolactin F (19).54 The three examples of peptides isolated from marine isolates of Bacillus spp. include the surfactin acyldepsipeptide analogue, halobacillin (20),55 the Sri K1 depsipeptide homocereulide (21), and the cyclic antibacterial loloatins A - D (loloatin B , 22). 16 17 18 11 H 2 N 19 20 21 22 There are some significant structural similarities between the chemistry of the marine and terrestrial Bacillus spp. The cyclic decapeptide structures of the loloatins (21) are related to that of the tyrocidines (12); however, the loloatins are zwiterionic peptides, while the tyrocidines are cationic, affording the two families vastly different electronic character. A s well , halobacillin (20) belongs to the same iturin class of molecules as surfactin, the powerful biosurfactant. 5 2 The surfactins originate from terrestrial Bacillus spp. Iturin 20, is the first surfactin of the iturin family to be isolated from a marine source, it is produced only in a saltwater based media, and is differentiated from surfactin by the replacement of a glutamic acid with a glutamine, which affords 20 a major difference in electronic character. The acyldepsipeptide 20 also showed pronounced difference in biological activity compared to other iturins. 12 These two examples, when compared to the diversity of peptide antibiotics produced by the terrestrial Bacillus, reveal the marine Bacillus as containing an undeveloped potential for the production of peptides that may possess antibacterial properties. 1.5. The Research Program The battle against infectious microorganisms continues to drive scientific research. In the late 1980s, in collaboration with microbiologist Dr. M . T. Ke l ly , an antibiotic discovery research program was established in our laboratory. The program's mandate was twofold. Firstly, we sought to isolate and identify marine bacteria, from both temperate and tropical oceans, that produced antibiotics. Secondly, we intended to isolate and elucidate the structures of the antibiotics so that we could explore their potential as therapeutic agents that would aid in the fight against infection. Specifically, the ongoing research program was established to address the increasing prevalence of antibiotic resistance amongst human pathogens, against which the current arsenal of therapeutic treatments was proving ineffective. It was founded on the hypothesis that marine bacteria, given their unique medium, are capable of producing secondary metabolites hitherto unseen from terrestrial species. Chemical extracts of the isolates were tested for the presence of antibiotics by their ability to inhibit the growth of a panel of human pathogens, which included M R S A , V R E , Eshcerichia coli, Mycobacterium tuberculosis ( M T B ) , and the yeast, Candida albicans. B y 1993, Dr. Ke l ly and coworkers had screened 500 organisms isolated from the water column, sediments, and the surfaces of plants and animals of the Northeastern Pacific Ocean. 5 8 Eleven of those isolates were found to produce antibiotics. The chemical investigation of two of these bacteria by Dr. J. Needham resulted in the discovery of the structurally novel oncorhycolide 5 9 13 (23), and moriamides A - C 6 0 (moriamide B , 24) of which 24 was found to be a potent antibiotic versus M R S A . In 1997, the microorganism collection had been extended to include those from the tropics, increasing in number to approximately 6000 isolates. Approximately 3.5% of the isolates showed antimicrobial activity. 6 1 The chemical investigations of three of these isolates by Dr. J. Gerard resulted in the discovery of the anti-mycobacterial massetolides A - H ~ (massetolide A , 25) and the loloatins A - D 5 7 ' 6 3 (loloatin B , 22), which were found to be broad-spectrum antibiotics. Dr. Ke l ly at SeaTek Marine Biotechnology, Inc is currently developing the loloatins as potential topical antibiotics. 25 This thesis represents the third installment of work that has resulted from the ongoing collaboration with Dr. Kel ly . Chapters 2, 3, and 4 chronicle the discovery of new antibiotics and novel metabolites that arose from the continued investigation of the marine isolate Bacillus laterosporus M K - P N G - 2 7 6 A , the producing organism of the loloatins (22). Chapter 6 reflects my growing interest in marine chemical ecology. It stands separate from the principal work in this thesis, and is presented as such. 14 Chapter 2. Cationic Peptide Antibiotics from Bacillus laterosporus 2.1. A Brief Review of Cationic Peptide Antibiotics In response to the antibiotic resistance crisis discussed in chapter 1, there has recently been a renewed interest in discovering novel classes of antibiotics that have different mechanisms of act ion. 6 4 ' 6 5 One broad class of compounds attracting a lot of attention is the "cationic peptide antibiotics" 6 6 that are widespread in the innate immune systems protecting living organisms from microbial infections. 6 7" 6 9 These peptide antibiotics are particularly appealing because they k i l l bacteria quickly, in part by physically disrupting cell membranes, and as a consequence they appear to avoid the rapid emergence of resistance. 6 6 Figure 2.1.1. Four classes of peptide antibiotics (adapted from Hancock 6 6 ) , (a) (3-stranded (3 p-strands with disulphide bridges), (b) oc-helical, (c) extended helix, (d) loop (with disulphide bridge). (+) positive charge, (N) amino termini. Hancock 6 6 ' 7 0 ' 7 1 has covered the chemical and biological properties of cationic peptides in a number of excellent reviews. Antibacterial cationic peptides have two distinguishing features. Firstly, their structures usually contain no more than a single negatively charged amino acid and an excess of basic amino acids (lysine, arginine, ornithine, etc.), resulting in a net positive charge 15 of a least +2 (often +4, +5, or +6) even at neutral pH. Secondly, these peptides can fold in three dimensions so that the positively charged basic amino acid residues form a polar hydrophilic face while the lipophilic amino acid residues form a non-polar hydrophobic face (i.e. they are amphipathic). Four distinct folding types that encompass these characteristics have been identified (Figure 2.1.1). They are the [3-sheet structures stabilized by disulphide bridges, a-helices, extended helices (polyprohelices) with a predominance of one or more amino acids, and loop structures. There are a number of proposed models attempting to explain the steps leading to bacterial cell death by cationic peptide antibiotics. A common feature of these models is the interaction of the cationic peptide with the bacterial cell membrane. Early models predicted that the destabilization/permeabilization of the cell membrane by surface associated cationic peptides was the cause of bacterial cell death; however, there is mounting evidence that cell death is actually the result of antibiotic interactions with multiple, currently undefined, intracellular 73-75 targets. The following scenario (Figure 2.1.2) is a simplified depiction of a complicated and unresolved process, and the reader is directed to the recent works of Hancock et a l . 7 3 " 7 5 for more detailed discussions. Figure 2.1.2. A general depiction of membrane destabilization (adapted from Hancock ). (1) Peptide in solution with random conformation. (2) Negatively charged cytoplasmic membrane induces the secondary structure and leads to binding with membrane. (3) Peptide aggregation leads to transient pore/crack formation. 16 Prokaryotic cell membranes have a net negatively charged surface, which consequently has a high affinity for cationic peptides. The interaction with the cell membrane is believed to induce the peptide's amphipathic secondary structure, leading to the aggregation of antibiotics on the cell surface. The antibiotic aggregates cause short-lived cracks/pores of various sizes to form in the cell membrane, which allow the cationic antibiotics to enter the cytoplasm (termed self-promoted uptake), where they subsequently interact with their intracellular targets to cause cell death. Cationic peptide antibiotics typically show broad spectrum activity against Gram-positive bacteria, Gram-negative bacteria, and even fungi with M I C ' s in the range of 1-8 |Ltg/mL against both susceptible and antibiotic resistant pathogens. Intriguingly, cationic antimicrobials bind to extracellular bacterial products (e.g. lipopolysaccharides) that are produced during an infection and which lead to septic shock; as a consequence, these peptides block the onset of sepsis. This stands in contrast to traditional antibiotics, like (3-lactams, which tend to promote the production of bacterial toxins. 7 6 Protegrin-1 and magainin II, cationic peptide antibiotics isolated from pigs 7 7 and frogs, 7 8 respectively, have served as lead structures for the development of analogues that are in various stages of preclinical and clinical evaluation as topical antibiotics (Figure 2.1.3). Figure 2.1.3. Cationic peptide antibiotics undergoing clinical trials. Helix wheel projection of magainin II (from Ding et al . 7 9 ) . (3-sheet protegrin-1 with two disulphide linkages. hydrophobic residue magainin II portegrin 1 17 2.2. Introduction The marine bacterial isolate M K - P N G - 2 7 6 A , tentatively identified by Dr. Kel ly as B. laterosporus by the analysis of cellular fatty acids and 16S R N A , was obtained from the tissues of an unidentified tube worm collected at -15 m off the coast of Loloata Island, Papua New Guinea. The crude extract of the B. laterosporus solid culture showed broad-spectrum antibiotic activity against the- following human pathogens: M R S A , V R E , M T B , Candida albicans, and Eshcerichia coli. A s mentioned in section 1.4, the original bioassay-guided fractionation by Dr. J. Gerard resulted in the isolation of the loloatins A - D . Although potent antibiotics, the presence of the loloatins alone did not explain the breadth of activity displayed by the crude extract; in particular, the presence of the Gram-negative (E. coli) activity was left unaccounted for. A bioassay-guided fractionation using E. coli as the test organism resulted in the isolation and structure determination of the linear cationic peptides, bogorols 1 A - E (26-30). OH H o Ri H 0 R2 H 0 NH 2 Bogorol A (26) R , = Bogorol B (27) R , = Bogorol C (28) Rv = Bogorol D (29) R, = Bogorol E (30) R , = r i (Val) I s (Met) R 2 = R , = (Met(O)) R 2 = (Val) (He) (He) 1 The names assigned to the novel compounds presented in this thesis are taken from geographic place names around the islands of Motupore and Loloata, Papua New Guinea. 18 2.3. Isolation The lyophilized B. laterosporus M K - P N G - 2 7 6 A cells (21.5 g) were immersed in M e O H and extracted three times over a period of six days. The methanolic extracts were combined, filtered, and concentrated in vacuo to give a brown/gray tar. The tar was taken up in 200 m L H 2 0 / M e O H (10:1) and partitioned with 100% E t O A c (3 x 100 mL) . The combined E tOAc extracts were reduced in vacuo to give a crystalline solid (6.5g). In two portions, the residue was subjected to size-exclusion chromatography on a Sephadex LH-20® (100% M e O H ) column to give 500 mg of a fast eluting, ninhydrin positive fraction (Rf = 0 on reversed-phase T L C (100% MeOH)) . This fraction was loaded onto a reversed-phase lOg Sep-Pak® previously equilibrated with 100% H 2 0 , by dissolving the sample in a minimum of M e O H . Upon addition of the M e O H solution to the top of the column an equivalent volume of water was added, precipitating the active component. The column was flushed first with 50 m L of 100% H 2 0 , which was subsequently discarded, followed by 100 m L of H 2 0 / M e C N (6:4) with 0.2% T F A . The 6:4 H 2 0 / M e C N eluant gave 90 mg of a mixture of large molecular weight peptides (1556-1618 daltons) which were responsible for the observed anti-E. coli biological activity. The crude peptides were further separated into nine fractions by reversed-phase H P L C using a Dynamax-60™ C18 column, eluting with H 2 0 / M e C N (6:4) and 0.2% T F A . Repeated H P L C recycling using an Inertsil® C18 column resulted in the isolation of bogorols A (26, 3.2 mg), B (27, 4.1 mg), C (28, 1.5 mg), D (29, 4.4 mg), and E (30, 2.5 mg). 19 2.4. Structure Elucidation of Bogorol A (26)" 26 The structure of bogorol A (26) was elucidated by.a combination of chiral G C analysis of 26's acid hydrolyzate, ESI -QIT-MS and M S / M S analysis, and High Field (800 M H z ) one and two dimensional N M R spectroscopy of its hexaacetate (31) derivative. A s limited resolution in the 500 M H z ' H N M R spectrum (Figure 2.4.1) of 26 precluded a complete structure elucidation, 26 was converted to its hexaacetate derivative 31 and N M R data was acquired at 800 M H z . 1 1 1 The peptide 26 was isolated as an optically active white solid that gave a [M+H] + ion at m/z 1584.0875 in the H R E S I - T O F mass spectrum, appropriate for the molecular formula C80H142N16O16, which indicated that 26 had nineteen degrees of unsaturation. Inspection of the 500 M H z *H N M R spectrum (Figure 2.4.1) revealed signal groupings characteristic of a peptide, with an abundance of amide N H (5 8.3 - 7.6) and a-methine (8 4.6 - 4.0) resonances. A large group of methyl resonances (5 0.9 - 0.7) indicated the highly aliphatic nature of the peptide, which stood in contrast to the polar conditions required for its isolation. "Traditionally, common amino acids have been afforded both a standard three and one letter code; where appropriate, these have been used. In the case of novel amino acids and their derivatives found in this chapter, they too have been assigned three and one letter codes as stated in the text. By mirroring the standard convention, the aim is for these codes to facilitate a more fluid description of the analyses performed in this thesis. '"800 MHz NMR data was acquired and processed by the staff at the National High Field N M R Center, Department of Chemistry, University of Alberta, Edmonton. 20 21 The treatment of 26 with a 3:1 mixture of pyridine and acetic anhydride at room temperature for 24 h, gave bogorol A hexaacetate (31). Hydrolysis of the hexaacetate 31 at 105 °C for 72 h with 6 N HCI and examination of the pentafluoropropionamide iospropyl ester derivatives of the liberated amino acids via chiral G C confirmed the presence of L-valine (Val , V ) , L-isoleucine (lie, I), L-leucine (Leu, L ) , D-leucine (Leu, L ) , D-tyrosine (Tyr, Y ) , D-ornithine (Orn, O), L-lysine (Lys, K ) and D-lysine (Lys, K ) (see section 2.5, table 2.5.1). The hexaacetate 31 gave a [M+1( 1 3 C) I V + H ] + ion in the H R F A B M S at m/z 1837.15212, appropriate for the molecular formula of C92H154N16O22; indicating the formation of a hexaacetate. Six new methyl resonances that were not in the ' H N M R spectrum of the natural product 26 appeared in the 800 M H z [ H N M R (Figure 2.4.2) spectrum of 31. Three of them had chemical shifts (5 1.75, 1.76 and 1.77) appropriate for acetamides presumed to be on the side chains of lysine and ornithine; one had a chemical shift (5 2.23) appropriate for a phenol acetate which had to be on the tyrosine residue; and two of them had chemical shifts (5 1.95 and 2.07) typical of aliphatic acetate esters that had to be associated with fragments other than the amino acids identified by hydrolysis. Inspection of the ! H (Figure 2.4.2), 1 3 C (Figure 2.4.3), C O S Y (Figure 2.4.4), T O C S Y (Figure 2.4.5), H S Q C (Figure 2.4.6), H M B C (Figure 2.4.7) and N O E S Y (Figure 2.4.8) N M R spectra allowed for the complete assignment of the *H and 1 3 C N M R resonances observed for 31 (Table 2.4.1). The hexaacetate 31 was found to be composed of three valines (VI - V 3 ) , three leucines (LI - L3) , two lysines ( K l and K2) , one ornithine (O), one tyrosine (Y) and one isoleucine (I). The remaining residues, denoted X , B, and Z , were characterized by interpretation of the N M R data (Appendix Figures A . 1.1 - 30), and where appropriate, confirmed by chiral G C analysis (see section 2.5). I V Natural abundance 1 3 C accounts for 1.1 % of all C atoms; with 92 C atoms per molecule, approximately one C atom per molecule of 31 will be a 1 3 C atom. 22 23 24 Appendix Figure A. 1.6 11 A i •i Y Figure A. 1.7 • Figure A. 1.8 • 1 I Figure A. 1.9 Figure A. 1.10 CO ft h in oo ' 1 ^ ' 1 1 1 I 1 1 1 1 I ft H ft -\—i—i—|—i—i—i—i—j—i—i—i—i | r 4 in vo i—i—i—i—i—i—i—i—I—i r co Figure 2.4.4. 800 M H z C O S Y Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 . 25 Figure A. 1.16 II • \ I _* I 1 Figure A. 1.15 ~1-T~3T1 p« ft gure A.1.14 P I T 1 Appendix Figure A. 1.11 Figure A. 1.12 Figure A. 1.13 > + 00 I 1 0\ m g ft ft I o Figure 2.4.5. 800 M H z T O C S Y Spectrum of bogorol A hexaacetate (31) in D M S O - d 6 i 26 Appendix Figure A. 1.17 Figure A. 1.18 Figure A. 1.19 •Figure A. 1.20 o CN m o, C N Figure A. 1.21 in CO) a —' o ro o in 1 1 ' I o l O ' I ' 1 o LO Figure 2.4.6. 800 M H z H S Q C ; aliphatic region of bogorol A hexaacetate (31) (for expansion of aromatic region see Appendix Figure A . 1.22). Appendix Figure A. 1.23 i • * • ..pFigure A. 1.25 Figure A. 1.26 Figure A . 1.27 Figure A. 1.28 i— f-• i i i n I i i i i I i i i i I i i i i I i i i i I i i i i | i i i i | i i i i | M , i , i i . I | 1 *S © © © o © o o " <=> c LO o oo at o rH Figure 2.4.7. 800 M H z H M B C Spectrum of bogorol A hexaacetate (31) in D M S 0 - d 6 (Downfield aromatic and carbonyl signals are folded into spectra; in these instances, the addition of 120 ppm gives the corrected chemical shifts). 28 Figure 2.4.8. 800 M H z N O E S Y Spectrum of bogorol A hexaacetate (31) in D M S O - J 6 . 29 c o in CN 0 0 W O z U tN N i i i X m m z of —1 PQ X cn »n CN —" m x x z r PQ Z -I l Z CN i pq X Z I o cn i pq oo CO 00 SO 00 0 0 u o H oo 00 o in >n 00 r--r--cn so o ro U PQ 0 0 O U IX 6 o O '5 c •4—< c CO OH CO 6 >^  X o PQ — r ha d 00 so cn 00 < tN CN oo" cn tN os" oo" oo vo m 00 ~" ' o c3 cn vo d cn d cn cn VO O tN cn oo Os r--d 00 00 d i> cn d cn vd vb so vo tN '—1 cn * " 1 *—' •-1 m oo o in — -oo" oo cn oo in d d — ; m" r - m m" o" in in tN i o c < I tN S co H - l o r-" o 'o 3 CO VD VO in Ov cn vo o cn CN p —'< cn tN CN as as cn cn tN CN m" >n" d d CO o m c o <—1 cn -rf m vo oo ^ d o \ o " vo" cn cn oo cn oo vo vo S d ~ — SO >n K T3 CN K CT °d T J I co m m oo O in m oo h in ' — i oo oo o ~ —< d d CN in VO VO in T I OS 00 O —1 cn so -sr cn m so oo oo ^ d d U cn lib SO SO so cn cn CN os i n i n d ^ d ? n -« CN - i —' OS cn so oo r-~ d d cn CN oo CN «n os p CN —< d cni cn <-< m CN CN CN CN c/3 o u i-i CN c n c n r j - m U U U Z — CN cn •u z —i CN cn m O u 30 CO CO z Z ffi - z u I o Z 6 > Z •z I CN > z CO m CO NO m d CN O CN r-o o o oo o ON OO o m ON ON CN CO CN o o o q o ON CN CN ON CN CN ON ON , \ 00 o o r- NO NO CO u o CO ON <-< CN co " ON" T T " c o NO c o i n CO ~- —< —-ON oo" ON co" . ON r -CN ON CN NO c o ON 1> <—< CN C 3 U C/3 CN r-CN NO CO oo" O r-c o ed O O > •sr' w CN CN '—' CO CO NO NO CO CO "S3 > o CN o o ON" ON' CN oo" t--o o CN ^ r-^  o d <N — > ON ON CN" ON ON 15 CN CN J> . oo" oo" ^ o o o CN > ON m d CO o o r-~ d C N r-- r-- C N T—1 in I> r-- r-- q ON 00 ON * - < d d —'- d c o l>" co " CN" r-" C N o" r-" o o NO" C N NO C N c o r-; ON ON ON —< CN K T3 CN K o o o o o o ON ON CN CO NO in ON CO c o c o ON NO NO t--NO c o CN CN c o CN q m o ON r- ON o o o o o NO 00 *-« CN -1 d d T T ' 1—H d d •ST ON ON 00 OO d wo CO o o o o q ON o q CN d o q CN m ON CN in CN c o NO CN CN NO in NO c o CN d >n r - i> m ON CN o o m X K O ffi O ffi 2 ; ^ CN CN cn cn Z U U U Z - N n m in U Z CN cn U Z 31 X X Z Z i i CN cn > > X U I X Z l z. i 5 Z C N i i C N C N i—) H J 2 Z cn C N C N W ~ ~ cn C N ^ cn cn O < N CN o oo d in oo 00 ON r- cn cn so so Os cn CN so Os C N O OS OS m" in £ 8 S • „ cS in in 0 3 C/3 00 d oo" m m - H o s Os C N C N r-" C N so" od C N in 00 d — : 00 od r- so so 00 00 cn m XT. cn Os *-< —'- —< C N cn" cn" oo" o " cn s q s q •<* C N ~* ~* 00 r-" r-" cn" cn" in C N OS C N s q C N cn OS C N cn > cn" 13 > CD 13 > in t— so ' N t ' OO o" OS cn" d cn o oo o OS cn cn d d cn cn C N C N 3 00 00 CN t- r-«n m oo SO Os os r-• —; d 3 m so so so r~- <—1 os os t~~: ^ ^ ^ d OS C N O SO" cn CN C N O o oo cn os in C N oo - ^ d in" cn" cn" cn p in T t ; in ( J Q —3 C N K X 3 C N K X 3 m i - H r-- cn 00 OS 00 00 OS CN s q CN -1 d d in m *—1 o , | so r-d Os OS r-- CN —: CN m m cn CN O X CN cn U 2 CN CN cn O cn m in cn r- in CN r- -i s q C N od oo C N cn oo od SO C N —( ( N X O X' 2 u u m o O X U Z ' so SO OS 00 ^ os o - d d oq cn o oq r~~- o os t~> m cn —> T - H C N cn TI-in p od so u z Os cn cn in C N TJ - in oo T t ^ ^ o (N h -~ d ^r' oo C N -CT CN CN - H CN cn ^ 32 X Z cn" i CN r-{ r-" ffi in ^ w-T Z TT" V i i cn 1 ^ ^ i j ^ oo 0 0 ^ " m i TT" C N X U i CN I I ^-l Z i CN CN X z ffi cn Z CN <N cn C N w J J in in ^ t " „ m T t m c n -^ f" cn - r f <~i cn 3 C N r n i C N cn i C N cn ^ ^ ^ ^ h J ON ON oo CN ON m O CN m r-CN oo" ON CN r-" m oo CN CN •4—» (0 o 03 _ _ - C N C N ( N r- >, © d C N <D _c m in NO o CN cn CN" oo CN ON cn CN ON cn C N C N C N 00 C N cn r-" r-f 00 00 NO NO O CN 00 NO cn ON cn O CN ON CN ON d ^ r i D -t-» <D O cd P 1— H a>" c CN m r-cn O m CN oo" ON CN o r-C N in 00 Tf" p CN o" d m o" ON ^ ^-i cn ON NO O CN cn O ON C N 00 NO cn cn" 3 <U cn u _c ' o 3 <U OO -a T 3 •E E <*n T 3 C5 T3 oo T 3 o oo cn CN CN ON CN O CN CN CN in 00 ON ON cn O 00 C N cn ON C N ON NO «' « d M V cn cn C N C N OO 00 C N C N cn NO CN —< CN o CN 00 cn d oo >n cn in ON CN ON CN CN CN NO CN oo o O O o d ON oo cn cn CN NO d m cn >-1 •—' 1—' CN O X u z —< C N C N cn X o X m Z U U O X u z — C N C N cn t^" in O X oo U U 33 X CN X N CO oo oo^ oo C-{ r-~ r»" in >n in •<fr TT" <<t i i ^ ^ i cn r-c--d C N oo d NO" CO r-" C N m oq co o" oo" CO CO d p 5-o — ' C N C N C N ON ON o co —< od O N co co IN in oo oo C N C N O " O " 5? ^ d CN oo oo m C N C N C N C N i n i n C N d C N d d N O CN" cn 00 — d PH" N O N O C N C N •sr. C<0 C O oo —< p^ o c > m oq cn O" NO — 00 n-' d oo r-oo r~-r- r - r- T t O N PH PH m h ff) r H PH 6o ^ C N C N OO oo T 3 T3 00 t— ' — N O N O C N r--C N T f cn cn oo r— —'• —'• ~ d d r- 00 d O X U Z —' o ON cn C N -H* C N C N C N C N C N cn m r- co C N m N O m o m oo oo oo ^ d d O N oq — H co oo 00 O N 00 CO C N —i -H N O cn C N d * r- o PH C N C N cn . o X in m U U 6 6 00 00 s s Q Q c N N T3 E CD S S iod o o D o o u oo i . C a ta O ed ed •—> to '5 '3 ;_ CT o - o O o o ca C3 C3 03 T3 3 o C D . OJ 00 00 oo 34 20.5 / C H 3 2 0 7 H M B C Correlation ^5 C H 3 O The first unit, X, was determined to be the acetyl derivative of a 2-hydroxy-3-methyl-pentanoyl (Hmp) group. H M B C correlations observed between a carbonyl resonance at 8 170.1 (Ac-CO) and both a methine proton (HI) at 8 4.75 (d, J = 5.6 Hz) and an acetate methyl resonance at 8 2.07 identified the secondary acetate functionality in this fragment. The H I resonance showed a single C O S Y correlation to the H2 methine at 8 1.85 that was further correlated to a methyl resonance at 8 0.88 (H5) and to a pair of geminal methylene proton resonances (H3, 3') at 8 1.15 and 1.50, which in turn were both correlated to a methyl resonance at 8 0.83 (H4). This set of C O S Y correlations demonstrated that the acetoxy methine (HI) carbon was attached to a sec-butyl group and a H M B C correlation observed between H I and a 1 3 C resonance at 6 167.8 provided evidence that it was also attached to a carbonyl, establishing the substructure X. A n olefinic methine resonance (H2) at 8 5.66 (q, J = 7.2 Hz) and an olefinic methyl resonance (H3) at 8 1.77 (d, J = 7.2 Hz) in the ' H N M R spectrum of 31 could be assigned to a 2-H M B C Correlation B 35 amino-2-butenoic acid residue (Aba) B. H M B C correlations were observed between the olefinic methine H2 (8 5.66) and the a-carbon C I (8 130.8) and the carbonyl resonance (8 168.9) in this dehydroamino acid. A N H resonance at 8 9.54 and the olefinic methyl H3 showed H M B C correlations to the (3 carbon C2 (8 120.7), and thus established the substructure B. H M B C Correlation (1.86 3 H 4 0.85 1.74 z A pair of geminal methylene proton resonances (H5,5') at 8 3.85 and 4.10 and a methyl resonance (Ac-Me) at 8 1.95 all showed H M B C correlations to a carbonyl resonance at 8 170.2, demonstrating that the second aliphatic acetate ester involved a primary alcohol. C O S Y correlations were observed from H5,5 ' to the H I methine at 8 3.78 that was correlated to another methine (H2) at 8 1.74, which was in turn correlated to two methyl resonances at 8 0.86 (H3) and 0.85 (H4), indicating that the acetylated primary alcohol was part of a valinol (Vol) substructure Z . H M B C Correlation The chiral G C analysis (Section 2.5) indicated the presence of an isoleucine residue; however, the low intensity of the isoleucine (I) signal in the hydrolyzate warranted a complete account of its assignment by the N M R data. A n inspection of the *H N M R spectrum of 31's 36 methyl region revealed a resolved triplet (H5) at 5 0.75 (t, J = 7.2 Hz) that correlated in the C O S Y spectrum with geminal methylene proton resonances at 8 1.37 and 1.02 (H3,3'). In turn, C O S Y correlations were observed from H3,3 ' to a methine (H2) at 8 1.72 which was further correlated to a methyl (H4) at 8 0.77 and an a-methine (HI) at 8 4.23. A C O S Y correlation from the H I methine to a N H signal at 8 7.67 and a H M B C correlation from H I to the carbonyl (8 170.8), established the substructure I. Together, the eleven amino acids and the three other substructures (X, B , and Z) accounted for all nineteen degrees of unsaturation in 26, which indicated a linear amino acid arrangement for the peptide. The sequence of peptide bonds, and therefore the constitution of 26, were established by the inspection of the H M B C correlations from the N H signals to their adjacent carbonyls, and by the N O E S Y spectrum of 31, which contained an uninterrupted ladder of NN (i,i+i) correlations (Figure 2.4.9) that linked the adjacent amino acids. Eleven correlations can be observed, starting with Leu 1/Orn and extending through the peptide to Leu3/Vol . Figure 2.4.9. 800 M H z N O E S Y Spectrum; N H region expansion of bogorol A hexaacetate (31). 37 Thus, by analysis of the N M R data, the constitution of bogorol A (26) was completed. However, given the laborious isolation procedure that was required to obtain sufficient quantities of pure 26 for the N M R experiments, it was felt that a mass spectrometry protocol using 26 as a structural template would facilitate the rapid structure elucidation of the remaining bogorols B - E (27-30). The widespread use of mass spectrometry in the structure elucidation of peptides and proteins has resulted in the creation for a universal nomenclature of the fragments. (Figure 2 .4 .10) . 8 0 R 1 O i II - - N - C 4 C H a --x — • y -z ? 2 n " I © © R 2 O • C H - C - - - - N - C - C © H 3 N - C H - C - -H b Figure 2.4.10. Universal nomenclature of peptide fragmentation. According to the nomenclature, the commonly observed acylium/ammonium fragmentation observed with peptides is denoted as a y/b type. The y/b fragments result by the eventual protonation of the amide nitrogen. 8 1 However, with peptides that contain basic residues like lysine and ornithine, the basic residues undergo protonation and the peptide bond fragmentation is believed to proceed slightly removed from the charge site. 8 2 The utilization of an electrospray ionization (ESI)/quadrupole ion trap (QFT) instrument allowed for the rapid acquisition of both M S (Figure 2.4.11) and M S / M S (Figure 2.4.12) data using low amounts of bogorols ( - 10 jLtg). A n inspection of the mass spectrometry data supported the structure of 26, and provided a fragmentation template for rapidly determining the sequence variations that produced the remaining bogorols (27-30). The ESI -QIT-MS of 26 showed a strong mJz parent ion ([M+H] + ) at 1585.3, as well as its sodium adduct at m/z 1607.1, and their corresponding doubly charged species, mflz 814.7 and 792.5 , respectively. 40 Fragmentation proceeded from the Hmp-capped N-terminus, resulting in the loss of m/z 198 (b 2, Hmp-Aba), followed by the consecutive loss of three amino acids, L e u l (Ab3-b2 113); Orn (Ab4-b3 114); and lie (Ab5-b4 113). The fragments, ys and y6, allowed for the assignment of a fourth contiguous amino acid, V a i l (Ay5.y6 99). Fortuitously, the M S / M S fragmentation of 26's parent ion, afforded the complementary fragmentation pattern that started with the loss of V o l to give b ^ . The fragmentation continued with the loss of the next five consecutive amino acids: Leu3 (Abi3-bi2 113), Tyr (Abi2-bii 163), Lys2 (Abu-bio 128), Leu2 (Abio-b9 113), and Val3 (Ab9-b8 99). Together, y 7 and bg, positioned L y s l (A(y7+b8)-i585) 128); as well, y-j and y5 confirmed the positioning of the two consecutive valines, V a l l - V a l 2 ( A y 6 . y 7 198). Thus, the M S analysis confirmed the constitution of bogorol A (26) and provided a fragmentation template that could be used to rapidly elucidate the structure of the remaining bogorols (27-30). Further work was performed with 26 and its hexaacetate 31 to determine the absolute stereochemistry of bogorol A (26). 2.5. Stereochemistry of Bogorol A (26) The fourteen residues that comprise 26 contain a total of sixteen stereocenters that required assignment. Fortunately, there exists a variety of standard techniques for the determination of the stereochemistry of common amino acids. B y using a combination of acid hydrolysis and G C analysis, the stereochemistry of thirteen centers in twelve residues were routinely determined (see below). The stereochemical analyses of the Ffmp (X) and Aba (B) units, which contained the remaining three stereocenters, were undertaken by minor modifications to the acid hydrolysis/GC protocol mentioned previously, and by N M R spectroscopy, respectively. The remaining critical question regarding the structure of 26 involved rationalizing the relative sequence positioning of the enantiomeric (D/L Leu and D / L Lys) pairs of amino acids. The configurations of the component amino acids were established by acid hydrolysis of 31 followed by the chiral G C analysis of the P F P A - I P E amino acid derivatives (Table 2.5.1). Table 2.5.1. Chiral G C retention times of bogorol A hexaacetate (31) hydrolyzate. 3 Standard Time Bogorol A (min) (26) S, R-Hmpb 16.95 -R, S-Hmp b 17.18 -S, S-Hmp b 17.40 17.41 R, i?-Hmp b 17.61 -D-Val 19.02 -L - V a l 19.46 19.50 D-Vo l 19.64 19.67 L - V o l 19.82 -D-Ile 22.34 -L-Ile 22.77 22.74 D-Leu 23.89 23.90 L-Leu 24.74 24.74 D-Tyr 37.07 37.08 L-Tyr 37.21 • -D-Orn 37.88 37.91 L-Orn 38.06 -D-Lys 39.96 40.01 L-Lys 40.12 40.16 a A l l identifications were made by co-injections with standards. bAnalyzed as isopropyl esters, with a different temperature gradient (see Experimental). 42 Thus, the three valine residues ( V I - V 3 ) contained the L-amino acid configuration, while the multiple leucine (L1-L3) and lysine ( K l and K2) residues were comprised of both the L and D stereoisomers. The valinol (Vol) residue and all other amino acids were compared with derivatized, commercially available standards, while the four possible stereoisomers of 2-hydroxy-3-methylpentanoic (Hmp) acid were synthesized as follows (Scheme 2.5.1). ^ f ? 0 N a 1. N a B H 4 I II i O 32 2. H + 3. H P L C Separation., ° I 0 A 32a : R,R/S,S OH OH ° I O A 32b : R,S/S,R Chiral G C Chiral G C Scheme 2.5.1. Production of Hmp stereoisomers. The sodium salt of racemic 3-methyl-2-oxopentanoic acid (32) was reduced with sodium borohydride. Normal phase H P L C was used to separate the product into the diastereomeric pairs 32a and 32b (R,R/S,S and R,S/S,R). Comparison with commercially available samples of D (2R,3R) and D-allo (2R,3S) Hmp acid via chiral G C analysis of their isopropyl esters allowed for the identification of the configurations of the individual enantiomers. Hmp acid was obtained from 26 by ether extraction of the 6N HC1 hydrolyzate. Chiral G C comparison of the isolated Hmp's isopropyl ester with the synthetic samples showed that 26 contained the (2S,3S)-Hmp acid. p i T The configuration of the 2-amino-2-butenoic acid (Aba, B) olefin, A ' , was established as E by the analysis of the N O E S Y spectrum (Figures 2.5.1 and 2.5.2). 43 Figure 2.5.1. 800 M H z N O E S Y Spectrum with selected correlations; a-methine region expansion A of bogorol A hexaacetate (31). 44 Figure 2.5.2. 800 M H z N O E S Y Spectrum with selected correlations; a-methine expansion B of bogorol A hexaacetate (31). 45 N O E S Y correlations were observed between the olefinic methyl H B - 3 (5 1.77) and the a-methine of the adjacent leucine, H L i - l (5 4.30). A s well , the olefinic proton H B - 2 (5 5.66) correlated to several of the Hmp (X) signals at 5 4.75, 1.85 and 0.88 (Figure 2.5.3). N O E S Y Correlation Figure 2.5.3. Selected N O E S Y correlations of bogorol A hexaacetate (31). The remaining stereochemical question involved the sequence positioning of the D / L Leu and D / L Lys . During the G C analysis/acid hydrolysis mentioned previously, the acid hydrolysis was performed for 72 h. However, it was discovered during an earlier attempt at the acid hydrolysis for t = 24 h that only partial hydrolysis was obtained, no isoleucine residue was observed, while the valine level was very low. Intriguingly, it was noted that the relative abundance of the individual enantiomers of the D / L lysines and D / L leucines pairs were not stoicheometric, which suggested a possible means for enantiomeric sequence discrimination. A n investigation into the literature was undertaken to further assess whether the observed selectivity of acid hydrolysis could be exploited with regards to elucidating the sequence positioning of bogorol A ' s (26) enantiomeric amino acids. According to R . L . H i l l ' s 1966 review of the hydrolysis of proteins, 8 3 partial acid hydrolysis occurs with a degree of selectivity dictated by steric and electronic factors. In particular, amide linkages to valine and isoleucine are found to be the most resistant to hydrolysis. The studies cited by H i l l showed that in the initial stages of an acid hydrolysis with strong acid and high temperatures the hydrolyzate is comprised principally of dipeptides, which are found to be particularly resistant to further hydrolysis due to the proximity of their positively 46 charged ammonium groups. Eventually the dipeptides, according to their constitution, hydrolyze at different rates. In 1950, M . S . Newman, while discussing the steric factors involved in the acid catalyzed esterification of carboxylic acids, proposed an empirical rule entitled the rule of six (Figure 2.5.4). 8 4 H C Vb 2 C 3C - C - C - C 3 4 H 6 6 Figure 2.5.4. Coiled structure for acids, numbering shown (adapted from Newman 8 4 ) . The rule stated that "in reactions involving addition to an unsaturated function, the greater number of atoms in the six position, the greater wi l l be the steric effect." 8 4 The total number of atoms in the six position was referred to as the molecule's six number. Newman's rule of six was an empirical rule that was meant to be a simple substitute for molecular models. 8 5 It found widespread use in the 1950s and was adapted for studies with amides, esters, ketones, and olefins. 8 6 87 In 1963, R . E . Whitfield reinterpreted the rule of six for the hydrolysis of dipeptides. Whitfield's modification assigned empirical values to dipeptides that correlated steric factors to the relative rates of dipeptide hydrolysis. Using the generic peptide bond seen in figure 2.5.5, the number of atoms that are at positions six can be added to give a six number for that particular dipeptide. 5 H 5 H 4 c o i H y H H N - C - C - N - C - C 3 2 H 4 & 3 — A B — Figure 2.5.5. Rule of six numbering system of a peptide bond (from Whitfield ). 47 Whitfield showed that the assignment of a six number to a dipeptide correlated well with its hydrolysis rates, to the extent that the hydrolysis of a dipeptide could be grouped as rapid, moderate or slow (a spread which covers approximately a factor of sixty). The higher the six number, the slower the hydrolysis of that dipeptide. Dipeptides with valine or isoleucine tended to have higher six numbers and thus be more resistant to hydrolysis. The contribution of individual amino acids to the dipeptide's six number depends on its positioning at either site A or B of figure 2.5.5. The number of atoms in position six for the amino acids found in bogorol A (26) is presented in table 2.5.2. Table 2.5.2. Contribution to peptide six numbers. Number of atoms in position Number of atoms in position six six Amino A c i d A B Amino A c i d A B Hmp a 6 3 V a l 6 3 Aba 1 3 0 2 Lys 3 3 Leu Orn 3 3 3 3 Tyr V o l a 2 6 3 3 He 6 3 "The modified terminal amino acids were assigned the equivalent six number for Val (Vol) and He (Hmp); however, Whitefield cites that hydroxyl groups are known to accelerate the rate of cleavage, and therefore these values are probably an over estimate.bAba exists as an acid sensitive enamine while in site A, which under acidic conditions will convert to its ct-keto-amide that contains a geometry that would severely restricting its steric hindrance and, thus is assigned a six number of 0. For example, a dipeptide with Tyr at site A and Leu at site B would have a six number of 5, (2 +3). 48 Using this information, a model of the dipeptide formation that would occur during the hydrolysis of 26 can be constructed as in figure 2.5.6. Six numbers can then be assigned to the dipeptides in order to predict their relative rates of hydrolysis. 1 2 3 4 5 6 7 8 9 10 11 12 13 Hmp—Aba—Leu 1 — O r n - I l e — V a l 1 — V a l 2 — L y s 1 — V a l 3 — L e u 2 — L y s 2 — T y r - . L e u 3 — V o l 26 v Dipeptide Formation Hydrolysis at Even Bond Numbers Hydrolysis at Odd Bond Numbers Six Number Six Number Six Number Six Number Residue Average Six Number: 4.5 7.5 4.5 6 7.5 9 9 7.5 7.5 7.5 6 5.5 5.5 3 Hmp—Aba—Leu 1 — O r n - I l e — V a l 1 — V a l 2 — L y s 1 — V a l 3 — L e u 2 — L y s2—Tyr—Leu3—Vol Figure 2.5.6. A dipeptide model for the early stages of the hydrolysis of 26 and the assignment of residue average six numbers. From the literature the following can be hypothesized: during the acid hydrolysis of 26, a series of dipeptides are produced and their constitution depends on whether hydrolysis occurs initially at the even or odd bond numbers. If one assumes no overriding preference, both can be viewed as occurring with an equal probability. Six numbers are inherently linked to the discussion of dipeptides. In order to differentiate the sequence positioning of enantiomeric amino acids in a logical manner, an empirical value that is linked to an individual residue is required. A n inspection of the six 49 numbers of the various dipeptides in figure 2.5.6 reveals that the majority of residues (all but V a i l and Val2) exist in at least one dipeptide with six number of 6. In particular this was always the case with the lysines and leucines, the residues of acute interest. Thus, an individual residue's predilection for liberation by acid hydrolysis is linked to the rate of the other dipeptide, which does not have a six number of 6. Given the commonality of each residue belonging to at least one dipeptide with a six number of 6, it was decided that the averaging of the six numbers to give a residue average six number would be more appropriate for a direct discussion of specific residues. For example, L y s l is found in the dipeptides L y s l - V a l 3 with a six number of 6 and Val2-Lys2, with a six number of 9, which gives L y s l a residue average six number of 7.5 (Figure 2.5.6), while a similar calculation assigns Lys2 a value of 6. A s this new empirical value reflects a particular amino acid residue's predilection for liberation during the acid hydrolysis of the parent peptide, Lys2 is predicted to be more prevalent initially than L y s l in the hydrolyzate. Following dipeptide formation, the free amino acid residues wi l l appear in the hydrolyzate at differing rates, depending on the hierarchy of the residue average six numbers. The larger the residue average six number, the slower the residue's release into the hydrolyzate; or, at any give time (t) during the hydrolysis, the residue with the largest residue average six number w i l l have the smallest relative abundance. The residue average six numbers of bogorol A's (26) components predicts that the first amino acid residues to appear should belong to Hmp, V o l , and L e u l , followed by Leu3, Tyr, Orn, and Lys2. The slowest residues to appear wi l l be V a i l and Val2 , which wi l l be preceded slightly by De, L y s l , Leu2, and Val3 . Fortuitously, the hierarchy of the residue average six numbers predicts that Lys2 (6) wi l l be liberated more rapidly than L y s l (7.5). As wel l , the three leucines are assigned a range (4.5, 5.5 and 7.5) of residue average six numbers, suggesting the possibility for differentiating the enantiomers through acid hydrolysis. 50 Therefore, a more detailed acid hydrolysis experiment was undertaken to see if the predicted hydrolysis hierarchy of the residue average six numbers could be observed, allowing for the rationalization of the sequence positioning of the enantiomeric pairs of amino acids. A 6 N HCI (aq) solution of 26 containing an internal standard, L-alanine, was divided into six samples that were heated at 110°C for differing times (t). The hydrolysis times were as t = 1.5, 3, 6, 12, 14 and 48 h; after which, the samples were each converted to their P F P A - I P E derivatives and analyzed by chiral G C . The signal peak areas were expressed as relative to the peak area of the internal standard, L-alanine. For ease of interpretation, the results are presented in two parts, with the early stages of hydrolysis, t = 1.5 and 3 h (Figure 2.5.7), presented separately from the later hours (Figure 2.5.8). With figure 2.5.8, for ease of interpretation, only the amino acids, valine, isoleucine, leucine and lysine, which are critical for the evaluation of the predictive model, are presented. 4.5 7.5 4.5 6 7.5 9 9 7.5 7.5 7.5 6 5.5 5.5 3 Hmp—Aba—Leu 1 — O r n - I l e — V a l 1 — V a l 2 — L y s 1 — V a l 3 — L e u 2 — L y s 2 — T y r — L e u 3 — V o l Bogorol A (26) with residue average six numbers Hydrolysis Time (h) Figure 2.5.7. Early times of the amino acid analysis of bogorol A (26) hydrolyzate and 26 with residue average six numbers. During the initial stages of the hydrolysis, the hydrolyzate is predicted, and observed, to be comprised principally of the residues with the smallest residue average six numbers, Hmp (4.5), V o l (3) and L e u l (4.5). Thus, from t = 1.5 h, L e u l can be assigned as L-Leu. This 51 assignment is supported by biosynthetic arguments made in section 2.7. A s well , L -Lys is found to precede the presence of D -Lys , which suggests that D -Lys is at position L y s l (7.5), while L -Lys occupies Lys2 (6). This conclusion is supported by the following more detailed analysis of the later hours. 4.5 7.5 4.5 6 7.5 9 9 7.5 7.5 7.5 6 5.5 5.5 3 Hmp—Aba—Leu 1 — O r n - I l e — V a l 1 — V a l 2 — L y s 1 — V a l 3 — L e u 2 — L y s 2 — T y r — L e u 3 — V o l Bogorol A (26) with residue average six numbers r O-6-i i i i p | Hydrolysis Time(h) Figure 2.5.8. Late times of the amino acid analysis of bogorol A (26) hydrolyzate and 26 with residue average six numbers. The low levels of V a l and He at the different times (t) throughout the hydrolysis, as well as the initial appearance of Hmp, V o l and L e u l , validates the use of residue average six numbers as a proxy for a residue's predilection for liberation during acid hydrolysis. Comparison of the relative ratios of D -Lys and L - L y s at times t = 6 and 12 h, reveals that L - L y s is approximately twice as abundant, supporting the assignment of D -Lys at L y s l and L - L y s at Lys2. 52 As previously determined, the L e u l position was assigned as L -Leu . Given that L -Leu is approximately twice as abundant as the other amino acids at t = 48 h, it can be concluded that two of the three leucines in 26 are L-amino acids. The presence of two L-leucines residues could potentially confound the analysis, however, the highly truncated levels of the D -Leu throughout the time points of the hydrolysis, allows for the assignment of D-Leu at position Leu2 (7.5), which has the highest residue average six number of the three leucines. To summarize, in the initial stages of the hydrolysis, bogorol A (26) is converted to a variety of dipeptides, which can be assigned six numbers that qualitatively correlate steric factors with hydrolysis rates. From these dipeptides, the component amino acid residues can be assigned residue average six numbers, which in the case of bogorol A's residues, reflects a residue's predilection for liberation during the parent peptide's acid hydrolysis. The slowest to be hydrolyzed, or those that wi l l appear at the lowest levels at any given time over the course of the hydrolysis, wi l l be the residues that have the largest residue average six numbers. In general, those amino acids that while in the parent peptide have a N-valine bond tend to have the larger residue average six number, which is in agreement with the observations made in H i l l ' s review. From the constitution of 26, the amino acids that are bonded to a N-valine, and thus should appear at slower rates, are L y s l (7.5) and Leu2 (7.5), both of which have been assigned the D-amino acids. As was shown with the analysis of the acid hydrolyzate of bogorol A (26), the qualitative residue average six number effectively predicted the experimental results. However, as mentioned previously, residue average six numbers are based on an empirical model that attempts to correlate steric factors to hydrolysis rates. B y not incorporating electronic influences, the comparison of residue average six numbers between residues that do not share similar electronic characteristics is potentially confounded. For example, De and L y s l share the same residue average six number (7.5) yet L y s l is present early during the hydrolysis (t = 3 h), 53 while He does not appear until the end of the experiment (t = 48 h). As well , not all amino acid residues are stable under acid hydrolysis conditions, a factor that must be taken into account when comparing different residues. Nevertheless, for elucidating the sequence positions of enantiomeric pairs of amino acids, when the enantiomers have different residue average six numbers, a series of G C analyses can be particularly powerful. From the relative ratios of the liberated amino acids from bogorol A (26) over time, it was proposed that D-Lys and D-Leu, constitute the positions L y s l and Leu2, while the positions Lys2, L e u l , and Leu3 contain L-amino acids, thus completing the primary structure of bogorol A (26). A s with all structure elucidation by spectroscopy, the defined structure is a best fit of the data at hand. To conclusively assign a structure requires structural confirmation by total synthesis. The progress towards the total synthesis of 26 is discussed in section 2.12, 54 2.6. Secondary Structure The propensity of cationic peptide antibiotics to adopt a secondary structure plays an important role in their ability to transverse cellular membranes. Indeed, the variation in secondary structure caused by substitutions in the amino acid constitution can have a great 88 influence on an a-helical peptide's antibacterial effectiveness. Thus, a preliminary analysis using the hexaacetate 31 was undertaken in order to determine i f the bogorol template is capable of adopting a secondary structure consistent with other cationic peptide antibiotics. y -47.0 -4.0 -69.7 89 Figure 2.6.1. Helical secondary structures with model polyalanine helices. Torsion angles, dotted lines represent one amino acid residue. Linear peptides, with their unrestrained termini, have an inherent possibility for greater conformational flexibility over their cyclic counterparts.9 0 Despite this potential, linear peptides are found to have preferred conformations, one of which is the helix (Figure 2.6.1). There are several types of helical structures, a, 3 in, n, etc, which differ slightly in the torsion angles (<|>, \|/) of the amino acid residues. The variations in torsion angles changes both the number of amino acid residues per helical turn, and the number of atoms in the ring that results from the intramolecular NH—0=C hydrogen bonding. 9 1 Thus, a 3 i 0 helix has three amino acids per helical turn, as can be seen by 55 the top-down projection of the model in figure 2.6.1. As well, the loop that results from the intramolecular hydrogen bonding in a 3 ] 0 helix is comprised of ten atoms. Following these definitions, the a-helix was historically referred to as a 3 .6n helix, while the 7t-helix was a 4 . 4 i 6 . Of the helical possibilities, only the a and 3io are common, while the K- helix and a few others i 91,92 are extremely rare. A routine method for determining the secondary structure of peptides in solution is via circular dichroism (CD) spectroscopy. 9 3 The secondary structure of the peptide, particularly the helix, imposes a chiral environment on the chromophores of its amino acid carbonyls; which results in the differential absorption of the circular components of plane polarized light. The effect of the different conformations of the molecule, and within the molecule, on a C D spectrum are additive. 9 4 To get a localized, or residue specific, picture of secondary structure, N M R spectroscopy must be used. 9 5 ' 9 6 With bogorol A hexaacetate (31), a preliminary analysis of its secondary structure was performed using a combination of C D (Figure 2.6.2) and N M R spectroscopy. It must first be noted that solvent effects play a large role in determining the secondary structure of peptides. For example, alcohol solvents, like trifluoroethanol, tend to promote helices; hydrophobic solvents, like nonmicellar sodium dodecyl sulfate (SDS) and detergents, promote P-strand formation; while random coils predominate in aqueous buffers. 9 7 ' 9 8 This influence of solvent on secondary structure can potentially confound comparisons using different techniques with different solvents. Wi th 31, polar aprotic solvents were used for both the C D and N M R spectroscopy ( M e C N and D M S O , respectively) and it is believed that the similar nature of the solvents used should minimize the effects due to solvent. 56 Wavelength (nm) Figure 2.6.2. C D Spectrum of 300 p M bogorol A hexaacetate (31) in M e C N . The C D spectrum of 31 (Figure 2.6.2) is diagnostic of a right-handed helical peptide, containing a positive band at 190 nm and a double minimum at approximately 208 and 222 nm. 9 6 ' 9 9 Although difficult to discern by C D , it has been suggested that the 3io helix can be differentiated from an oc-helix by a truncated 222 nm band relative to its 208 nm band. 9 2 Accordingly, this would suggest that 31 does not possess a 3in helix, and most likely contains the more common a-helix. Rigorous modeling and calculations of intramolecular distances has allowed for a prediction of the through-space ' H - ' r l (e.g. NN ( , , , + i ) ; C a N ( , i ( + i), and C aC' 3(,- , + 3 )) correlations that should be prevalent, and hence diagnostic, of the differing peptide secondary structures.9 5 These N M R analyses have been traditionally done with peptides that are models of specific protein sequences (containing L-proteinaceous amino acids) 9 4 or with poly(L-amino ac id) 1 0 0 helices, and consequently are not particularly relevant when dealing with a peptide comprised of both D and unusual amino acids. However, of the N M R correlation criterion, the N O E S Y spectrum of 31 57 clearly showed the N H - N H (NN ( , , I + i>) correlations (Figure 2.4.9) through-out 31, supporting the presence of an oc-helical secondary structure within 31. As well as through the diagnostic N O E correlations, N M R techniques also offer a simple test of helical structure by the inspection of the ! H N M R spectrum. The Karplus dependent ^JNHO. coupling constants reflect the peptide's dihedral angles. The value of VNHO: <6.0 H Z is used as a standard criterion for he l i c i t y . 1 0 0 , 1 0 1 With 31, the N H signals that were sufficiently resolved to calculate 37NHO with confidence are those for Leu2, Tyr, and Leu3, which give 7.2, 8.0 and 8.8 Hz , respectively. These three amino acids reside close to the pseudo-C-terminus, suggesting that as progress is made towards the C-terminus helicity is lost and, therefore, the helical structure is localized nearer the N-capped terminus. However, given the significance that intramolecular H-bonding plays in a helical structure, it can be assumed that the modifications seen in 31 and 26, at both termini, wi l l be disruptive to a helical secondary structure. Thus, the a-helix is hypothesized as being localized within the interior of 31. Taken together, this preliminary analysis suggests that 31 is capable of adopting a right-handed oc-helical secondary structure that is localized in its interior, which becomes more random in nature as the helix-breaking termini are approached. Given the structural similarities, and the role that secondary structures play in the biological activity of cationic peptide antibiotics, it is probable that bogorols A - E (26-30) are also capable of adopting a similar a-helix while interacting with bacterial cell membranes. 2.7. Structure Elucidation of Bogorol B (27) 58 27 Bogorol B (27) was isolated as an optically active white solid that gave a [M+H] + ion at m/z 1570.0710 in the H R E S I - T O F mass spectrum, appropriate for the molecular formula C 7 9Hi4oNi 6 Oi 6 , which indicated that 27 was a homologue of bogorol A (26). The 500 M H z *H N M R (Figure 2.7.1) of 27, like the ' H N M R spectrum of 26, showed the same broad signals characteristic of a cationic peptide (Figure 2.4.1). The notable difference between the ] H N M R spectra of 26 and 27 was the presence of a new methine multiplet at 8 2.10, slightly deshielded from the valine P-methine multiplets at 8 1.95 in the spectrum of 26. Inspection of the ESI -QIT-MS and M S / M S spectra (Figures 2.7.2 and 2.7.3) coupled with chiral G C analysis (Table 2.7.1) of the individual amino acids, attributed the mass difference to a L-valine (27, Ab3-b2 99) for L-leucine (26, Ab3-b2 113) substitution at residue position three. v A l l other structural aspects of 26 were found to be retained within 27. v The residue positions are numbered from left to right, with Hmp as one; Aba as two; through to Vol as fourteen. 59 6 0 61 62 Due to the nonribosomal v l peptide nature of the bogorols (26-30) it was assumed that the sequence of the enantiomeric amino acids seen within bogorol A (26) is conserved throughout the family. Marahiel et al. have extensively reviewed the modular nature of nonribosomal peptide biosynthesis (Figure 2 .7 .4) . 1 0 2 ' 1 0 3 Modules Domains /A-The Domains ©Condensation domain -peptide bond formation ©Adenylation domain -selects amino acid ©Peptidyl carrier protein domain the module's transport domain ©Epimerization domain -converts L to D amino acids ©Termination domain -essential for product release Figure 2.7.4. Nonribosomal peptide synthetase, (adapted from Scwarzer et a l . 1 0 2 ) Nonribosomal peptide synthetases are megaenzymes which are organized as modules that contain a sequence of linked domains (enzymes) that are required for the incorporation of one amino acid residue in the growing peptide chain. The adenylation (A) domain that is responsible for the selection and attachment of the amino acids to the megaenzyme synthetase can show relaxed substrate specificity; resulting in families of peptides with defined positions where the sequence variations occur. The A-domain's relaxed substrate specificity can be exploited to increase metabolite diversity beyond that naturally occurring. 6 2 Typically, the epimerization of an amino acid occurs further along the module, after its attachment as a thioester on the peptidyl carrier protein (PCP) domain. If epimerization is required within a particular module, then the module wi l l contain an epimerization (E) domain (see module 2, Figure 2.7.4). Thus, as is observed with the substitution of L-valine for L-leucine The term 'nonribosomal' is attributed to the presence of unusual (non-proteinaceous, D) amino acids, which is indicative of a peptide not originating from a ribosome, which is the site of protein biosynthesis. 63 with 2 6 and 27 , the stereochemistry within a family of nonribosomal peptides can be viewed as defined, despite the inherent possibility for sequence variations. Table 2.7.1. Chiral G C retention times of bogorol B - E (27-30) hydrolyzates 3. Standard Time Bogorol B Bogorol C Bogorol D Bogorol E (min) (27) (28) (29) (30) R, 5-Hmp/ 19.08 - - - -S, R-Hmpb S, 5-Hmp/ 19.25 19.33 19.28 19.25 19.25 R, R-Hmpb D - V a l b 24.80 - - - -L - V a l b 25.15 25.10 25.10 25.09 25.08 5 - V o l b 25.22 25.22 25.27 25.21 25.19 R-Wo\b 25.32 - - - -D-Ile 22.20 - - - -L-Ile 22.62 22.61 - 22.52 22.52 D-Leu 23.80 23.72 23.68 23.72 23.69 L -Leu 24.61 24.52 24.50 24.55 24.51 D -Met c 33.10 - - - -L - M e t c 33.35 - - 33.30 -L - M e t ( 0 ) c 33.33 - - - 33.27 D -Tyr 37.00 36.97 36.92 36.97 36.92 L -Tyr 37.15 - - - -D-Orn 37.82 37.77 37.71 37.79 37.74 L -Orn 37.99 - - - -D -Lys 39.88 39.89 39.79 39.87 39.81 L -Lys 40.04 40.05 39.93 40.02 39.95 a Al l identifications were made by co-injections with standards. b A different temperature gradient was used then with the other residues (see Experimental). cUnder the hydrolysis conditions, Met is oxidized to Met(0). , M During the G C analysis, the Hmp acid was analyzed as its O-pentafluoropropionyl isopropyl ester, for which chiral resolution was not possible. It was shown that 27 ' s Hmp acid belonged to the same pair of diastereomers as bogorol A's (26) Hmp acid, and by the previous nonribosomal peptide arguments, they were assumed to be equivalent. The presence of the diagnostic olefinic methine and methyl (HB-2 and HB -3) signals of the Aba residue within the ' H N M R spectrum of 2 7 confirmed its existence, which had been suggested by the b 2 fragment in the ESI -QIT-MS. 64 Through their work on dehydroamino acid synthesis, Olsen et al. showed that the ' H chemical shift of the A b a residue's olefinic methyl group ( H B - 3 ) is particularly sensitive to the olefin's configuration. 1 0 5 Given that the chemical shift of H B - 3 is conserved throughout the bogorol family ( 26 -30 ) (Table 2.7.2), the olefin in bogorol B (27) was determined to be E, as in 2 6 . m M i n i s UJL I I B " Bogorol Vinyl ic 8 Methyl ( H B - 3 ) M u l t i , / • A (26) 1.74 d, 7.5 B (27) 1.79 d, 7.5 C ( 2 8 ) 1.78 d, 7.5 D ( 2 9 ) 1.75 d, 7.5 E ( 3 0 ) 1.78 d, 7.5 Thus, by working with the structural and stereochemical template established for bogorol A (26) , the structure elucidation of bogorol B (27) was completed. 28 Bogorol C (28) was isolated as an optically active white solid that gave a [M+H] + ion at m/z 1556.0565 in the H R E S I - T O F mass spectrum, appropriate for the molecular formula C78H138N16O16, which indicated that 28 was a homologue of bogorol A (26) and B (27). The peptide 28's ' H N M R spectrum (Figure 2.8.1) revealed no notable differences between 28 and 27. However, working from the structural and stereochemical template established with 26 for the bogorol family, the ESI -QIT-MS and M S / M S spectra (Figures 2.8.2 and 2.8.3) isolated the mass difference as occurring from the substitution of L-valine (28, Ab3-b2 99; Ab5-b4 99) for both L-leucine (26, A b 3 -b2 113) and L-isoleucine (26, Abs-b4 113) at positions three and five, respectively. The presence and stereochemistry of bogorol C's (28) Aba was determined as in section 2.7 (Table 2.7.2). Chiral G C analysis (Table 2.7.1) of the acid hydrolyzate confirmed the lack of an isoleucine residue and the remaining amino acid constitution of 28. The nonribosomal peptide stereochemical argument established in section 2.7, completed the structural elucidation of 28. 66 67 69 2.9. Structure Elucida t ion of Bogorol D (29) 29 Bogorol D (29) was isolated as an optically active white solid that gave a [M+H] + ion at m/z 1602.0437 in the H R E S I - T O F mass spectrum appropriate for the molecular formula C79H140N16O16S. The molecular formula indicated that the loss of C H 2 , coupled with the addition of a sulphur atom, led to the increase of eighteen daltons over the mass of bogorol A (26). Inspection of bogorol D's (29) ! H N M R . spectrum (Figure 2.9.1) revealed the presence of a sharp methyl singlet at 8 2.05, an appropriate chemical shift for a methyl sulfide. The ESI-QIT-MS and M S / M S (Figures 2.9.2 and 2.9.3) spectra and the chiral G C analysis (Table 2.7.1) of the acid hydrolyzate localized the mass difference with respect to 26 as the result of a L -methionine (29, Ab3-b2 131) substitution for L-leucine (26, Ab3-b2 113) at position three. As with bogorol B (27) and C (28), the G C and M S analyses revealed the remaining structural elements of 29 to be equivalent to 26, and thus the structure elucidation of bogorol D (29) was completed. 70 73 2.10. Structure Elucidation of Bogorol E (30) o ^ H o I H 0 ^ \ H 6 3 *1 3 4 B N H 2 V I Aba O V A L L K N H 2 Orn T \ Leu2 L y s l 30 Bogorol E (30) was isolated as an optically active white solid that gave a [M+H] + ion at m/z 1618.0405 in the H R E S I - T O F mass spectrum, appropriate for the molecular formula C79H140N16O17S, which suggested that 30 was the oxidation product of bogorol D (29). Indeed, the ! H N M R spectrum of 30 (Figure 2.10.1) displayed a methyl singlet at 5 2.50, appropriate for a methyl sulfoxide. Further, the inspection of the ESI -QIT-MS and M S / M S spectra (Figures 2.10.2 and 2.10.3), coupled with chiral G C analysis of the component amino acids (Table 2.7.1) revealed the mass difference, relative to bogorol A (26), as the result of a L-methionine sulfoxide (29, Ab3-b2 147) substitution for L-leucine (26, Ab3-b2 113) at position three. As with bogorol B (27), C (28), and D (29), all other structural and stereochemical aspects were determined to be equivalent to the template established with 26. Typically, methionine sulfoxide containing peptides are isolated as a mixture of diasteromers as a result of the presence of both R and 5 methyl sulfoxides, which is indicated by a doubling of the methyl sulfoxide signal in both the ' H and 1 3 C N M R spectra. 1 0 6 ' 1 0 7 Wi th bogorol E (30), no doubling of signals was observed in the ' H N M R spectrum, and limited material precluded acquiring a 1 3 C N M R spectrum. During the H P L C purification a closely 74 76 77 eluting fraction, of equivalent molecular mass, was found that could not be sufficiently purified for rigorous identification. Together with the lack of the doubling of the ' H N M R signal, this observation suggests that the R and S sulfoxides were resolved. However, the identity of which enantiomer is present in bogorl E (30) was left undetermined. 2.11. Biological Act iv i ty v " With the structure elucidation of the principle members of the bogorol family completed, the bogorols were submitted to SeaTek for preliminary biological testing. Bogorols A - E (26-30) were tested for antibacterial activity against a panel of human pathogens, which included M R S A , V R E , and Escherichia coli (Table 2.11.1). Table 2 .11.1 . M i n i m u m inhibitory concentration (ftg/mL) of bogorols A - E (26-30) as determined by agar dilution using Mueller-Hinton media. Test Bogorol A Bogorol B Bogorol C Bogorol D Bogorol E Organism (26) (27) (28) (29) (30) M R S A 3.125 - 2 3.125 - 2 3 . 1 2 4 - 2 3.125 - 2 6.25 - 3.125 V R E 1 2 . 5 - 6 . 2 5 1 2 . 5 - 6 . 2 5 1 0 0 - 5 0 2 5 - 12.5 2 5 - 12.5 E.coli 5 0 - 2 5 5 0 - 2 5 1 0 0 - 5 0 1 0 0 - 5 0 >200 The bogorols were all inactive (MICs >200 M-g/mL) against Pseudomonas aeruginosa, Pseudomonas cepacia, and Candida albicans. The bogorols possess good activity versus M R S A , which falls within the 1-8 j ig/mL range found for cationic peptide antibiotics. Intriguingly, simple amino acid substitutions to the bogorol template resulted in some pronounced differences in biological data. In particular, the change from bogorol B (27) to C (28) is a simple valine for an isoleucine substitution, yet 27 is v " All the biological testing in this thesis was performed by Mr. Paul Haden, Ms Helen Wright, and Dr. M . T. Kelly of SeaTek Marine Biotechnology, Inc. 78 eight times more active against V R E , which suggests that a structure-activity study might be very rewarding. 2.12. A Discussion of the Progress Towards the Total Synthesis of Bogorol A (26) To fully explore the bogorol's novel antibiotic template, a rapid and versatile synthesis is required. By designing and implementing a synthetic protocol, it would then be possible to create a variety of bogorol analogues that further explored issues of secondary structure, and the role that the component amino acids play in manifesting antibacterial activity. Particularly interesting, would be to assess the importance of the novel structural elements of the bogorol template, those being the dehydroamino acid, and the neutral C and N-termini. Therefore, steps were undertaken to initiate the total synthesis of bogorol A (26). A survey of the literature revealed numerous books 1 0 8" 1 1 0 and reviews 1 1 1 ' 1 1 2 that detailed the principles and methods of solid and solution phase peptide synthesis, including a comprehensive review by Humphrey and Chamberlin that discussed specifically the coupling methods for nonproteinaceous amino acids. As well, two syntheses of dehydroamino acid containing peptides, kahalalide F 1 1 4 (33) and phomalide1 1 5 (34), were recently published that presented many synthetic methods that could benefit a synthetic approach to the bogorols. H 2 N x v = o N ^ H NH O i H o y)^~-NH / N 0 O HN . . . H N ^ ° \ .0 V - N H H N ^ Q \ — M M NH o 3 3 34 79 The synthesis of 33 was attempted via a variety of solution and solid phase combinations. The dehydration of threonine to produce the Z-Aba residue was performed both on and off the resin. The synthesis highlighted the benefits of using a combination of solution and solid phase techniques to rapidly synthesize a complex peptide along with stereochemical analogues. The solution phase synthesis of 34, which contains the uncommon, E - A b a residue, was attempted by Ward et al.; however, formation of the thermodynamically less favorable E configuration proved difficult. Eventually 34 was obtained by H P L C chromatography as a minor product in the synthesis of its isomer, Z-34. The authors detailed numerous attempts at the formation of the E configuration of the A b a residue, which proved insightful in the planning of the synthesis of 26, which also contains an is-Aba residue. Shortly after the publication of the synthesis of 34, Stoehlmeyer et al. disclosed a stereospecific method for the formation of dehydroamino acids in their attempt to synthesize the highly unsaturated peptide, phomopsin A (35) (Scheme 2 . 12 .1 ) . 1 1 6 E 35 Scheme 2.12.1. Phomopsin A (35) and the formation of an E-dehydroamino acid. The authors found that by adding thionyl chloride to a P-hydroxyamino acid, like threonine, a cyclic sulfamidite is produced, that upon addition of base, undergoes an antiperiplanar elimination to yield the corresponding dehydroamino acid. 80 With the pertinent information at hand, a retrosynthetic analysis (Scheme 2.12.2) of bogorol A (26) that encompassed both solution and solid phase techniques, was envisioned. NH2 38 39 40 41 Scheme 2.12.2. Retrosynthetic analysis of bogorol A (26). A n inspection of the structure of 26 revealed the most synthetically challenging structural aspect to be the presence of an E-dehydroamino acid. T w o accounts in the literature have mentioned difficulty in activating the carboxy group of a dehydroamino acid for subsequent coupl ing . 1 1 4 ' 1 1 7 This lead to Humprey and Chamberlain to suggest that the "smallest synthetically sensible dehydropeptide containing fragment is a trimer wherein the dehydro residue occupies the internal posi t ion." 1 1 3 Thus, the leucine/ornithine amide bond of 26 was chosen as the site of disconnection to afford 36 and 37. The synthesis of the major peptide component, 37, was envisioned by standard Fmoc/Boc solid phase methods on an automated peptide synthesizer, which would easily allow for future 81 changes in the peptide sequence. For the tripeptide 36, Stohlmeyer's stereospecific dehydration of the threonine containing tripeptide 38 was proposed. The protected tripeptide 38 could routinely be made by standard phosphonium couplings of 39 to 40, followed by a coupling to 41. The synthesis of 37 was undertaken by Dr Krystyna Piotrowska of the U B C Biotechnology Laboratory, starting with the commercially available D-valinol loaded trityl chloride (CITrt) resin. Unfortunately, the required (25, 35) stereochemistry of the 2-hydroxy-3-methylpentanoic (Hmp) acid was not commercially available. However,, the sodium salt of its diastereomer ( 2 R , 35) (42) was; thus the inversion of 42's secondary alcohol via a Mitsunubo conversion was deemed a valid approach to the production of 41. The total synthesis of 26 was attempted according to scheme 2.12.3. The THP-hydroxy protected acid 41 was synthesized in five steps from the sodium salt of ( 2 R , 25)-2-hydroxy-3-methylpentanoic acid (42). Firstly, 42 was converted to its benzyl ester 118 1 with benzyl bromide in D M F , as evidenced by the addition of five aromatic signals in the H N M R spectrum. Subsequently, the 2/?-hydroxy group of the benzyl ester was inverted via a Mitsunubo conversion to the p-nitro-benzoate (43),1 1 9 which added a pair of deshielded doublets to the ' H N M R spectrum that are indicative of a ^-substituted aromatic ring. The selective removal of the benzoate group with sodium azide afforded the (25, 35)-2-hydroxy-3-methyl pentanoic acid benzyl ester, 1 2 0 as shown by the loss of the aforementioned aromatic doublets in the ! H N M R spectrum. Importantly, the ' H N M R spectrum's chemical shift differences clearly established the deprotected product as a diastereomer of 42's benzy ester. Lastly, the addition of the T H P protecting group, 1 2 1 followed by the hydrogenolysis of the benzyl group, 1 1 5 gave diastereomeric 41 in a 71% overall yield. The latter conversion was established by both the loss of aromatic signals in the ' H N M R spectrum, and the doubling of signals in the ' H and 1 3 C N M R spectrum. a: benzyl bromide (1.2 equiv), D M F , Ar (g), r.t., 5 h. b: D E A D (5 equiv), PPh 3 (5 equiv), p-nitro benzoic acid (5 equiv), benzene, Ar (g), r.t., 3 h. c: NaN 3 (3 equiv), MeOH, Ar (g), 40°C, 30 h. d: DHP (2 equiv), / /TsOH (cat.), CH 2 C1 2 , Ar (g), r.t., 30 min. e: H 2 (g), Pd-C, EtOAc, r.t., 1 h. f: Fmoc-chloroformate (1 equiv), dioxane, 10% N a 2 C 0 3 (aq), r.t., 4 h. g: DIEA (4 equiv), PyBOP (2 equiv), D M F , Ar (g), r.t., o/n. h: Piperidine (1.2 equiv), D M F , r.t., 1 h. i: as in g. j: S O C l 2 (1.5 equiv), NEt 3 (3 equiv), CH 2 C1 2 , Ar (g), -78°C, 1 h; D B U (2 equiv), C H 2 C 1 2 ) Ar (g), 0°C, 1 h. k: T F A , T H F - H 2 0 , r.t., 1 h. 1: as in g. m: T F A - H 2 0 , p-cresol, r.t., 3 h. Scheme 2.12.3. Synthet ic p lan for bogoro l A (26). 83 L-allo-Threonine (44) was routinely N-Fmoc protected with Fmoc-chloroformate to give 45, 1 2 2 as evidenced by the addition of eight aromatic signals in the ' H N M R spectrum. The protected amino acid 45 was subsequently p y B O P coupled with the L-leucine t-butyl ester hydrochloride (46) to give the dipeptide 47. 1 1 4 The coupling was established by the presence of both the Fmoc aromatic and t-butyl methyl signals in the ' H N M R spectrum. Fmoc deprotection of 47 with piperidine gave the free amine 48, 1 2 3 which was confirmed by the loss of the aromatic signals in the ' H N M R spectrum. The subsequent p y B O P coupling of 48 and 41 gave the elimination substrate tripeptide 38 in 63% overall yield. The latter transformation was established by the F fRDCIMS which gave a [M+H] + ion at m/z 487.33821 appropriate for the molecular formula C25H46N2O7. The increased complexity of the ' H N M R spectrum clearly indicated the addition of the diasteromeric 41 residue. One of the telling changes in the ' H N M R spectrum of tripeptide 38 being the presence of two sets of diasteromeric amide ' H resonances. O f the methods available for the creation of dehydroamino acids, those that give the thermodynamically more favorable Z , 1 2 4 ' 1 2 5 or a mixture of the E and Z isomers , 1 0 5 ' " 5 predominate over those that stereospecifically gives the E isomer. 1 1 6 A s mentioned previously, Stohlmeyer et al. observed that the formation of a cyclic sulfamidite of a (3-hydroxyamino acid wil l lead upon the addition of base, to an antiperiplanar elimination, to give the E or Z isomer, dependent upon the configuration of the initial (3-hydroxyamino acid. A n initial attempt at the two step elimination of 38 with thionyl chloride and D B U failed to give any elimination product. A n examination of the individual steps, showed the rapid formation of the cyclic sulfamidite, as evidenced by T L C and by the loss of half the amide signals in the ' H N M R spectrum. The cyclic sulfamidite failed to eliminate upon addition of D B U . Due to time constraints, Dr. Urmi la Deo Jangra has currently undertaken this project, with a focus on using a less sterically hindered base and/or a less sterically hindered substrate for which to perform the elimination. 84 Once completed, the routine coupling of the tripeptide 36 to 37, followed by both the deprotection and the cleavage from the resin, should yield bogorol A (26). A total synthesis of 26 wi l l confirm the proposed structure of the natural product 26, as well as enable the synthesis of a variety of bogorol analogues. 2.13. Conclusion Re-examination of the crude cellular extract of B. laterosporus M K - P N G - 2 7 6 A has resulted in the discovery of the novel cationic peptides antibiotics bogorols A - E (26-30). The bogorols contain a number of structural features that are typical of nonribosomal peptides. These include the reduction of the C-terminal residue to give valinol (Vol), transformation of the N -terminal isoleucine to 2-hydroxy-3-methylpentanoic (Hmp) acid, incorporation of four D amino acids, and the presence of a dehydroamino acid (Aba). As a consequence of the C - and N -terminal modifications, all of the potentially charged residues in the bogorols reside in the interior of the linear peptide chain. The bogorols appear to be the first known linear cationic peptide antibiotics with both C-terminal aminol and N-terminal oc-hydroxy acid modifications. The structural variations within the bogorol family were found to be the result of repetitive amino acid substitutions at the same two amino acid positions (positions 3 and 5), while the structures of the remaining twelve residues were conserved. From a biosynthetic perspective, this allows for the conclusion that the nonribosomal peptide synthetase's modules that are responsible for positions 3 and 5 (Leul and lie in 26) have a reduced substrate specificity in the adenylation (A) domain. Consequently, amino acid substitutions occur, which results in the creation of the structural diversity seen within the bogorol family. A s argued in section 2.7, and as observed with the bogorol family, the nonribosomal synthetase results in substitutions that proceed with the retention of the stereochemistry inherent to that particular module. 8 5 . While completing the structure elucidation of bogorol A (26), a qualitative method using residue average six numbers was used to rationalize the sequence positioning of the enantiomeric pairs of leucine and lysine that occur in 26. The rule of six empirical analysis was adapted to allow for the qualitative correlation of bogorol A's (26) residues predilection for liberation by acid hydrolysis to their relative abundance in the acid hydrolyzate over time. A preliminary investigation into the secondary structure of 31 revealed its propensity to adopt a helical structure in polar aprotic solvents. A s discussed previously, given the role that secondary structure plays in the biological activity of cationic peptide antibiotics, it is reasonable to assume that the bogorols are also capable of adopting an amphipathic secondary structure when interacting with the negatively charged bacterial cell membranes. Indeed, a depiction of bogorol A (26) in a right-handed oc-helix template using HyperChem® v4.0 software depicts the alignment of the two lysines and the ornithine, resulting in a polar hydrophilic face (Figure Figure 2.13.1. Depiction of 26 as an a-helix. The bogorols showed selective and relatively potent activity against M R S A and V R E , as well as moderate activity versus E. coli. Since they represent a new cationic peptide antibiotic template, they are attractive leads for a structure-activity relationship (SAR) study aimed at optimizing their potential as additions to the therapeutic armory. In an attempt to instigate a 2.13.1). \ 86 S A R study, the development of a versatile synthetic route of 26 was undertaken. When completed, questions regarding the importance of the Michael-acceptor dehydroamino acid, the C- and N-terminal caps, and a variety other structural features, to the bogorols' biological activity can be addressed. The novelty of the bogorol (26-30) structures supports the hypothesis that marine Bacillus spp. represent an underdeveloped source of potential antibiotics, particularly peptide antibiotics. 87 Chapter 3. Antifungal Metabolites from Bacillus laterosporus 3.1. Introduction Concurrent to the original isolation of the bogorols from liquid culture, experiments were undertaken to isolate new members of the loloatin family from the solid agar culture cells of Bacillus laterosporus M K - P N G - 2 7 6 A . During these experiments it was observed that the bacteria] cells were also a source of the bogorols. A late eluting fraction from the gel permeation column also displayed activity against the opportunistic fungal pathogen, Candida albicans. Further bioassay-guided purification of the active fraction using C. albicans as the test organism resulted in the isolation and structure determination of basiliskamides A (50) and B (51). Basiliskamide A (50) Basiliskamide B (51) 3.2. Isolation B. laterosporus M K - P N G - 2 7 6 A cells (21.5 g, dry weight) were immersed in M e O H and extracted over a period of six days. The methanolic extracts were concentrated in vacuo and partitioned between E t O A c (3 x 100 mL) and H 2 0 / M e O H (10:1 200 mL). The E t O A c extract was dried over anhydrous N a 2 S 0 4 , filtered, and reduced to dryness in vacuo (6.5 g). The residue was chromatographed on Sephadex LH-20® (100% M e O H ) in two parts to give 226 mg of a fraction containing a U V absorbing compound. This fraction was subsequently subjected to step gradient (1:1 M e O H / H 2 0 to 100% M e O H ) chromatography on a reversed-phase Waters lOg 88 Sep-Pak®. The U V absorbing fraction (82 mg) was further separated into crude 50 and 51 (28 mg total) by normal phase silica column chromatography (4:1 E t O A c / C H 2 C l 2 ) . Final purification by reversed-phase H P L C (7:3 M e O H / H 2 0 ) yielded pure basiliskamides A (50, 14 mg) and B (51, 9 mg) as clear solids. The basiliskamides exhibited antimicrobial activity against C. albicans, Aspergillus fumigatus, and Mycobacterium tuberculosis. 3.3. Structure Elucidat ion of Basil iskamide A (50) 50 Basiliskamide A (50) was isolated as an optically active, clear solid that gave a [M+H] + ion at m/z 386.23358 in the H R F A B M S appropriate for the molecular formula of C23H31NO4. The structure of 50 was determined by a detailed analysis of the one and two-dimensional N M R spectroscopic data (Table 3.3.1). The 1 3 C N M R spectrum (Figure 3.3.1) of 50 showed only 21 well resolved resonances, indicating that there was an element of symmetry in the molecule. Resonances in the ' H N M R spectrum (Figure 3.3.2) of 50 were all well dispersed, which facilitated the inspection of the C O S Y (Figure 3.3.3), H M Q C (Figure 3.3.4), and H M B C (Figure 3.3.5) spectra, and the subsequent identification of the two major substructures, A and B . 89 Table 3.3.1. *H and l 3 C N M R data for basiliskamide A (50) recorded in D M S O - C J 6 -c# 'H(500 M H z ) (Int., m, /(Hz)) l 3 C(100 M H z ) C O S Y a H M B C " 1 167.4 H2 2 5.55 ( l H , d , 11) 119.3 H3 N H (6.83) 3 6.31 140.5 H2, H4 H5, H6/6 ' (1H, dd, 11, 11) 4 7.40 (1H, m) 128.2 H 3 , H 5 5 5.91 (1H, m) 140.5 H4, H6/6 ' H3 , H6/6 ' 6 2.28 (1H, m) 34.7 H 5 , H 6 ' , H 7 O H 6' 1.99 (m) H5, H6, H7 7 3.49 (1H, m) 69.6 H 6 / 6 ' , H 8 , O H H 8 , H 1 3 8 2.06 (m) 40.7 H 7 , H 9 , H 1 3 H13 9 4.92 (1H, dd, 76.3 H8 H l l / 1 1 ' , H 1 3 , H 1 4 9.5,2) 10 1.67 (1H, m) 35.5 H11/11 ' ,H14 H 1 1 / 1 1 ' , H 1 2 , H 1 4 11 1.25 (1H, m) 26.4 H 1 0 , H 1 1 ' , H 1 2 H.10, H I 2 , H14 11' 1.11 (1H, m) H 1 0 , H 1 1 , H 1 2 12 0.87 (t, 7.5) 10.1 H l l / 1 1 ' 13 0.84 (d, 7.) 11.6 H8 14 0.90 (d, 7) 12.8 H10 H9, H l l / 1 1 ' 15 166.0 H 9 , H 1 6 , H17 16 6.61 (1H, d, 16) 118.0 H17 H17 17 7.65 ( l H , d , 16) 144.6 H16 18 134.0 H16,H19/23 19/23 7.71 (m) 128.4 H20/22 H 1 6 , H 1 7 , H 2 0 / 2 2 20/22 7.41 (m) 128.9 H19/23, H21 H17,H19/23 21 7.40 (m) 130.4 H20/22 H20/22 O H 4.57 (1H, d, 5) H7 N H 2 7.31,6.83 . N H - N H • (2H, S) • • . • . : -"Protons correlated to proton resonance in 1H column. "Protons correlated to carbons in C# column. 90 91 13 14 (PPm) 8.0 6.0 4.0 2.0 Figure 3.3.3. 500 M H z C O S Y Spectrum of basiliskamide A (50) in D M S O - J 6 . 93 94 95 r~^i H M B C Correlation A A broad three proton *H N M R resonance at 8 7.41-7.40 (H20/22, H21), that showed H M Q C correlations to carbon resonances at 8 130.4 (C21) and 128.9 (C20/22), along with a broad two proton ' H N M R resonance at 8 7.71 -(HI9/23), that showed H M Q C correlations to a carbon resonance at 8 128.4 (C19/23), were all assigned to a monosubstituted phenyl ring. The 13 phenyl ring accounted for the element of symmetry required by the C N M R data. A one proton doublet at 8 7.65 (H17) in the ] H N M R spectrum showed C O S Y correlations to another one proton doublet at 8 6.61 (HI6). The two doublets were assigned to a vinyl group that was the only substituent on the phenyl ring, which was confirmed by H M B C correlations observed between the vinyl doublet resonance at 8 7.65 (H17) and the phenyl carbon resonance at 8 128.4 (C19/23). H M B C correlations observed between both the vinyl proton resonances at 8 7.65 (HI7) and 6.61 (HI6) and a carbon resonance at 8 166.0 (C15) showed that the phenyl and vinyl fragments were part of a cinnamoyl residue A . The vinyl protons had a vicinal scalar coupling of 16 H z demonstrating the cinnamoyl residue had the E configuration. 96 7.4(1 13 14 H6CH3 128 C H 3 0.87 H f~~\ H M B C Correlation 7.31 6.83 B Analysis of the C O S Y , H M Q C , and H M B C data collected for 5 0 routinely identified substructure B , including the positions of the A 2 ' 3 and A 4 , 5 olefins, the methyl branches at C8 and CIO, and the presence o f - O R substituents at C7 and C9. H M B C correlations observed between both 8 5.55 (H2) and 6.31 (H3) and a carbon resonance at 8 167.4 (CI) showed that C 2 was attached to a carbonyl carbon. Only one nitrogen and two hydrogen atoms remained unaccounted for by the cinnamoyl A and the linear B fragments, suggesting that the C I carbonyl was a primary amide. A pair of broad one proton resonances at 8 7.31 and 6.83, that showed C O S Y correlations to each other but did not show H M Q C correlations to carbon resonances, were assigned to the primary amide N H protons. The N H resonance at 8 6.83 showed an H M B C correlation to 8 119.3 (C2), confirming the presence of the primary amide at the terminus of the linear fragment B . A C O S Y correlation observed between an O H proton resonance at 8 4.57 and a resonance at 8 3.49 (H7) showed that there was an alcohol functionality at H7 and, therefore, the cinnamoyl fragment had to be attached to the linear carbon chain via an ester linkage at C9. A H M B C correlation observed between the methine resonance at 8 4.92 (H9) and the cinnamoyl carbonyl resonance at 8 166.0 (CI5) confirmed the presence of the ester linkage and completed the structure of 5 0 . 97 3.4. S t e r e o c h e m i s t r y o f B a s i l i s k a m i d e A (50) The configurations of the diene olefins, established by the magnitude of their coupling constants, were confirmed by a series of nOe experiments (Appendix A.2 . Figure A.2.1 , summarized in Figure 3.4.1). nOe 5.55 _y 6.31 ' 4% 2% Figure 3.4.1. Results from selected nOe difference experiments on basiliskamide A (50). The protons H2 and H3 had a vicinal scalar coupling constant of 11 H z typical of Z olefins, while H4 and H5 showed a 15 H z vicinal coupling typical of E olefins. Irradiation of the H3 resonance at 5 6.31 induced a nOe in the H 2 resonance at 8 5.55 in agreement with the Z configuration for A 2 ' 3 olefin. Similarly, irradiation of the H5 resonance at 8 5.91 induced a nOe in the H3 resonance at 8 6.31 supporting the E configuration for the A 4 ' 5 olefin. A study by Rychnovsky et al. surveyed 221 1,3-diol acetonides and concluded that the acetal methyl 1 3 C chemical shifts reliably indicate the relative configurations of positions 1 and 3 . 1 2 6 The authors found that the stereochemistry of the 1,3-diol dictates the conformation of the dioxane ring of their respective 1,3-diol acetonides as being either chair, from a syra-diol, or twist-boat, from an ann'-diol. The dioxane ring conformation is then reflected in the C chemical shifts of the acetal methyls; chair dioxanes have an equatorial and an axial methyl group resonance at ca. 19 ppm and 30 ppm, respectively, while twist-boat methyl group resonances are both found at ca. 25 ppm. Experiments were undertaken to exploit Rychnbvsky's empirical relationship for 1,3-diol acetonides with 50 . A diisobutylaluminum hydride reduction of the ester bond of 5 0 yielded the 98 corn esponding 1,3-diol 52. The treatment of 52 with 2,2-dimethoxypropane and a weak acid resulted in the acetonide derivative 53 (Scheme 3.4.1), which was used to determined the relative stereochemistry at C7 and C 9 . H?N D I B A L T H F , - 7 8 ° C H 2N Ar , 18 h 64% H 2 N 52 O H OH M e O ^ J D M e / \ PPTS THF, .60°C A r , 1 h 58% Scheme 3.4.1. Conversion of 50 to acetonide 53. Analysis of the H M Q C data for 53 (Figure 3.4.2), showed that the acetonide methyl carbon resonances had chemical shifts of 19.8 and 30.4 ppm, typical of acetonides formed from yyn-1.3-diols. Further analysis of the ' H N M R data for the acetonide 53 revealed that the dioxane ring existed in a chair conformation with the C6 and CIO carbons equatorial (Figure 3.4.3). Irradiation of H10 (8 1.56) simplified the H9 doublet of doublets to a doublet with 7 = 1 0 Hz , which indicated that H8 and H 9 were both axial and, therefore, the C I 3 methyl was equatorial. Thus, the relative stereochemistries at C7 , C8 , and C9 are as shown in 53. 99 100 dd = 2 Hz, 10 Hz w 5 3 Decoupler 1.56 ppm \ 10 U  3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 (ppm) d= 10 Hz 6 6' 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 (ppm) Figure 3.4.3. Decoupling experiment (400 M H z ) with acetonide 53 . The absolute configuration at C7 of basiliskamide A (50) was established using Ohtani's empirical rule that utilizes the chemical shift difference (A5) of the corresponding R and S Mosher ester ( M T P A ) derivatives (Figure 3.4.4) . 1 2 7 ' 1 2 8 The A5 values arise from a differential deshielding of the parent molecule by the R and the S Mosher ester's benzene ring, which are reflected in the M T P A plane (A5 > 0, A5 < 0) and provide the basis for Ohtani's analysis. 101 o +0.04 -0.02 +0.06 +0.07 H 2 N i -0.09 i -0.06 +0.04 +Q.16 +0.21 O o. I MTPA +0.01 o M T P A = F 3 C OMe *R = 50a 5 = 50b Figure 3.4.4: A8 (ppm) Values for the Mosher ester derivatives 50a and 50b. The natural product 50 was converted into both its R and S Mosher ester (50a and 50b, well resolved ' H N M R spectrum of 50a and 50b (Appendix A .2 , Figure A.2.2) facilitated the assignment of most of the ' H chemical shifts. The A5 values (A5 = 8s - SR) (Appendix A .2 , Table A.2.1) are clearly reflective of the M T P A plane in 50a and 50b (Figure 3.4.4), and indicate that C7 in 50 has the S configuration. The configuration of C10 was not determined experimentally. However, 50 was found to be a homologue of the known compound YM-47522 (54) and the absolute configuration of C10 in 54 has been determined by synthesis to be R n 9 ' m respectively) by treatment with the corresponding Mosher ester acid chloride. Inspection of the O O 54 As the other chiral centers in 50 and 54 have identical configurations (IS, 85, 9R), it is assumed that 50 also has the R configuration at C10. 102 3.5. Structure Elucidation of Basiliskamide B (51) 3 14 51 Basiliskamide B (51) was also isolated as a clear solid that gave a [M+H] + ion at m/z 386.23358 in the H R F A B M S appropriate for the molecular formula of C23H31NO4, identical to the formula of basiliskamide A (50). Analysis of the one and two-dimensional N M R data (Table 3.5.1; Appendix A . 2 , Figures A.2.3 - A.2.7) obtained for 51 showed that it was simply an isomer of 50, in which the cinnamoyl ester was at C7 instead of C9 . The changes in the  lU N M R chemical shifts of both H 7 (A5 = +2.01) and H 9 (A5 = -1.66) when comparing the *H N M R spectra of 51 and 50 were appropriate for an acyl shift between C9 and CI. A s well , a C O S Y correlation from the O H (5 4.48) to H 9 (5 3.26), and a H M B C correlation from H7 (5 5.40) to C15 (8 165.5) were observed. Finally, the natural products 51 and 50 were both converted to the same diol 52 by D I B A L reduction, demonstrating that both molecules had identical configurations. 103 Table 3.5.1. ' H and 1 3 C N M R data for basiliskamide B (51) recorded in DMSO-cfr. C# 'H(500 M H z ) " C ( l O O M H z ) C O S Y 3 H M B C 5 (Int.. m. /(Hz)) 1 167.4 H2, H3 2 5.57 (1H, d, 11) 119.9 H3 N H (6.86) 3 6.33 (1H, dd, 11, 11) 140.0 H2, H4 H5 4 7.51 (1H, dd, 15, 11) 128.8 H 3 , H 5 H 6 ' , H 2 5 5.87 (1H, dt, 15,7) 138.0 H4, H6/6' H 6 ' , H 3 6 2.53 (m) 31.8 H 5 , H 6 ' , H 7 6' 2.36 (1H, m) H5 , H6, H7 7 5.40 (1H, dt, 10.5, 3) 74.0 H6/6 ' H 6 ' , H 1 3 8 1.92 (m) 39.4 H 9 , H 1 3 H13 9 3.26 (m) 73.0 H8, O H H 1 1 ' , H 1 4 10 1.40 (m) 36.3 H11/11 ' ,H14 H 1 1 / 1 1 ' , H 1 2 , H 1 4 11 1.38 (1H, m) 26.5 H 1 1 ' , H 1 2 H12, H14 11' 1.21 (1H, m) H1-1.H12 12 0.85 (t, 7) 11.8 H I 1/11, 13 0.83 (d, 7) 10.7 H8 14 0.74 (3H, d, 7) 12.1 H10 15 165.5 H 1 6 , H 1 7 , H 7 16 6.59 (1H, d, 16) 118.5 H17 H17 17 7.60 (1H, d, 16) 144.1 H16 H19, H23 18 134.0 H 1 6 , H 1 7 19/23 7.70 (m) 128.2 H20/22 H17, H20/22 20/22 7.40 (m) 129.0 H I 9/23 H I 9/23 21 7.40 (m) 130.2 H I 9/23 O H 4.48 ( l H , m ) H9 N H 2 7.34, 6.86 (2H, s) . i— . . 'Protons correlated to proton resonance in 1H column. Protons correlated to carbons in C# column. 3.6. Biological Activity For perspective, the basiliskamides were tested alongside the clinically used antifungal agent amphotericin B (Table 3.6.1); as well , the results were compared to those of the known homologue 54 (Table 3.6.2). 104 Table 3.6.1. Activi ty of basiliskamide A (50) and amphotericin B against clinical isolates of Candida albicans as determined by macrobroth dilution. M I C (ng .mL) Isolate Number Basiliskamide A (50) Amphotericin B 8167 0.5 0.5 8362 0.5 0.5 8363 0.5 0.5 8364 0.5 0.5 8365 0.5 0.5 8366 0.5 0.5 8367 0.5 05 Table 3.6.2. Comparative activity of basiliskamides A (50), B (51), and YM-47522 (54) as determined by agar dilution (the biological data for 54 was obtained from the literature 1 3 2). M I C (M-g/rnL) Test Organism Basiliskamide A (50) Basiliskamide B (51) Y M - 4 5 7 2 2 1 3 1 Candida albicans 1.0 3.1 25 Aspergillus fumigatus 2_L5_ 5.0 > 50 The basiliskamides were also tested against Mycobacterium tuberculosis, the cause of tuberculosis, and Mycobacterium avium-intracellulare, an important cause of mycobacterial infections in immunocompromized patients such as those with A I D S . The results are shown in table 3.6.3. Table 3.6.3. Antimycobacterial activity of the basiliskamides as determined by agar dilution. M I C (ug/mL) Compound M. tuberculosis M. avium-intracellulare Basiliskamide A (50) 25 100 Basiliskamide B (51) 50 > 100 The basiliskamides were found not to be cytotoxic against normal human fibroblast cells at concentrations less than 100 | ig /mL, compared to amphotericin B which is toxic at concentrations as low as 25 u.g/mL. 105 3.7. Conclusion The antifungal polyketide amides, the basiliskamides, were isolated from a marine isolate of Bacillus laterosporus. Basiliskamide A (50) and B (51) differ in the positioning of a cinnamic ester and an alcohol functionality along their common polyketide core. A s well , basiliskamide A (50) was found to be a homologue of the known compound YM-47522 (54), which is produced by a soil strain Bacillus sp. that remains unidentified at the species level. The polyketide chain of 50 and 54 apparently differ in their biosynthetic starter unit. The structure of YM-47522 (54) is extended by a methylene, which would arise from the initial incorporation of a propionate (C3) unit, as opposed to an acetate (C2) unit. The basiliskamides and YM-47522 (54) are unusual Bacillus metabolites, which are more commonly found to be peptide antibiotics. 5 0 Macrolactin F (19) and analogues, produced by a marine Bacillus sp., 5 4 are apparently the only other known polyketide-derived metabolites isolated from a Bacillus sp. The results of several biological assays indicate that the basiliskamides show promise as antimicrobial agents. Basiliskamide A (50) was found to have excellent activity against several clinical isolates of the yeast Candida albicans. This activity was comparable to that of the clinically used amphotericin B . As well , basiliskamide A (50) was approximately twenty-five times more active against C. albicans and the filamentous fungus Aspergillus fumigatus than its homologue YM-47522 (54). The regioisomer basiliskamide B (51) was also found to be more active than YM-47522 (54). The basiliskamides were both found to have activity against M T B , while 50 also displayed activity against M. avium-intracellulare. Most significantly, alongside the comparable antimicrobial activity to amphotericin B , the basiliskamides showed less cytotoxicity to human cells than the amphotericin B . Taken together, the basiliskamides represent excellent lead structures for the development of effective antifungal agents. 106 Chapter 4 . Other Metabolites from Bacillus laterosporus 4.1. Introduction After the discovery of the loloatins by Dr. J. Gerard, the impetus for the re-examination of the chemistry of B. laterosporus M K - P N G - 2 7 6 A was the persistence of strong biological activity versus Gram-negative bacteria in a liquid culture broth that could not be explained by the intracellular loloatins. Bioassay guided fractionation of the liquid culture organic extract lead to the eventual discovery of the bogorols as described in chapter 1. Prior to the elucidation of the bogorol template, experiments were performed with the goal of minimizing the number of chromatographic steps involved in the isolation of the bogorol family. The removal of a gel permeation step, that separates molecules by their molecular weight, resulted in both an expedient isolation of the bogorol family and the serendipitous discovery of the novel lipopeptides, tupuseleiamides A (55) and B (56). Tupuseleiamide A (55) Tupuseleiamide B (56) 4.2. Isolation A liquid culture broth (800 mL) of M K - P N G - 2 7 6 A was lyophilized to half its volume, neutralized with strong acid, and extracted with E t O A c . The organic extract (1.3 g) was loaded onto a reversed phase Sep-Pak® (lOg) and subjected to a five step gradient elution from 1:1 MeOH/Water to 100% M e O H . The 4:1 MeOHAVater fraction was concentrated to dryness in 107 vacuo (88 mg) and further purified by reversed-phase H P L C , eluting with 2:3 MeCN/Water (0.2% T F A ) to give tupuseleiamides A (55, 2.0 mg) and B (56, 0.3 mg). Neither 55 nor 56 showed any biological activity in our antibiotic assays. 4 .3 . Structure Eluc ida t ion of Tupuseleiamide A (55) The gross structure of tupuseleiamide A (55) was determined by the interpretation of both the one and two-dimensional N M R spectroscopic data and the mass spectrometry data obtained for the natural product 55. The structures of the component amino acids and the fatty acid, as well as their sequence connectivity, were determined by detailed analysis of the H , C , C O S Y , H M Q C , and H M B C data (Table 4.3.1). Finally, the configurations of the amino acids were determined by total acid hydrolysis followed by chiral G C analysis of the P F P A - I P E derivatized amino acids (Table 4.3.2). Tupuseleiamide A (55) was isolated as an optically active colourless solid that gave a H R F A B M S [ M + H ] + peak at m/z 409.23417, corresponding to a molecular formula of C21H32N2O6. The molecular formula indicated that 55 had seven degrees of unsaturation. Inspection of the *H (Figure 4.3.1), 1 3 C (Figure 4.3.2), and H M Q C (Figure 4.3.3) N M R spectra revealed signals that were suggestive of a tyrosine containing peptide. 108 Table 4.3.1. ' H and 1 3 C N M R data for tupuseleiamide A (55) recorded in D M S O - J 6 . C# 'H(500 M H z ) l 3 C ( 1 0 0 M H z ) C O S Y 3 H M B C 5 " (Int., m, /(Hz)) Tyr C O O H 12.61 (1H, br s) 1 172.6 H2, H3 , H 3 ' 2 4.34 ( l H , m ) 53.6 H3 , H 3 \ NH(7.77) H3 , H 3 ' 3 2.89 (1H, m) 35.9 H 3 ' , H 2 H5, H9 3' 2.80 (1H, m) H 3 , H 2 4 127.1 H2, H 3 , H 3 ' , H 6 , H8 5,9 6.96 (2H, d, 8.0) 130.1 H6 H3, H 3 ' 6,8 6.62 (2H, d, 8.0) 114.9 H5 OH(9.14) 7 155.9 , H6, H8, OH(9.14) O H 9.14 (1H, s) -N H 7.77 (1H, d, 6.5) H2 Ser 10 170.0 H11,NH(7.77) 11 4.30 (1H, m) 54.8 H12 ,H12 ' ,NH(7 .75 ) 12 3.52 (1H, m) 61.6 H l l , H 1 2 ' , O H ( 4 . 7 5 ) H l l 12' 3.49 (1H, m) H11 .H12 , OH(4.75) O H 4.75 (1H, m) H 1 2 . H 1 2 ' N H 7.75 (1H, d,7.4) H l l F A 13 172.3 H14 ,H15 ,NH(7 .75) 14 2.10 (2H, t, 8.0) 35.1 H15 H15 15 1.46 (m) 25.2 H 1 4 . H 1 6 H14 16 1.20 (m) 28.9 H 1 5 . H 1 7 H 1 4 . H 1 5 . H 1 7 17 1.23 (m) 26.1 H16 .H18 H 1 5 , H 1 6 , H 1 8 18 1.13 (2H, m) 38.3 H 1 7 . H 1 9 H17, H20, H21 19 1.48 (m) 27.3 H 1 8 , H 2 0 , H21 H20, H21 20 0.83 (d, 6.6) 2.2.4 H19 . H 1 8 . H 1 9 21 0.83 (d, 6.6) 22.4 H19 H 1 8 . H 1 9 'Protons correlated to proton resonance in H column. Protons correlated to carbons in C# column. A n apparent triplet in the ' H N M R spectrum at 8 7.76 both integrated for two protons and showed no carbon correlations in the H M Q C and was therefore assigned as two overlapping amide (NH) resonances. In the *H N M R spectrum, a pair of doublets that integrated for two protons each at 8 6.96 and 6.62, a sharp singlet at 8 9.14 not attached to a carbon, and a 109 I l l 20 21 15 14 _ 18 3 " 2 N 10JL A i . V ^ 6 A 8 1 1 9 H O ' l ^ N 13^ 15^  17 3 O H Tyr COOH Tyr TyrSer 5 6 OH NH NH 9 8 1 A LL 13-- i 1 1 1 (PPm) 12.0 10.0 8.0 6.0 Figure 4.3.3. 500 M H z H M Q C Spectrum of tupuseleiamide A (55) in D M S O - d 6 . 20 21 Ser OH 2 11 12 12' 16 17 3 (ppm) 40 80 120 4.0 - l 1 2.0 160 112 O HHV ° 14 _ 16 _ 18 I 19 10 1^ / V / \ > ^ H 3 O Tyr COOH 20 21' 17 18. 16 19 15-14" 3 3' 12 12' ? 11 Ser-OH < 68 _ 59 ~ Ser-NH_ Tyr-NH Tyr-OH Tyr-COOH SI ^ 5 6 (ppm) NH NH 98 JUL - i 1 1 1 1 1 1 1 ' i i 12.0 10.0 8.0 Ser OH 2 11 12 12' LU 20 21 17 16 15, 14 19 18i * 0 (ppm) 2.0 4.0 6.0 8.0 10.0 6.0 4.0 i — i — i — 2.0 12.0 Figure 4.3.4. 500 M H z C O S Y Spectrum of tupuseleiamide A (55) in D M S O - ^ . 113 9 H H V ° HO 1 9 = 3 £ H io i J<r^ Y l l N 1 3 ^ 1 5 14 16 _ 18 I 19 HO Tyr COOH 20 21 17 19, 15-16" 18 _ r 11 12 68 59 10 . 13,, (ppm) Tyr OH TyrSer > g NH NH i I rl J I 9 o Ser OH 2 11 12 12' IjJu 20 21 17 16 (ppm) 0 no 0 o i ' 40 80 120 160 — i — i — i — i — i — i — i — i — i — i — i -8.0 6.0 4.0 -I n - i r-12.0 10.0 8.0 6.0 4.0 2.0 Figure 4.3.5. 500 M H z H M B C Spectrum of tupuseleiamide A (55) in D M S O - J 6 . 114 methylene signal at 8 2.89/2.80 suggested the side chain of tyrosine. In the downfield region of the 1 3 C N M R spectrum, six aromatic signals, 8 155.9 (CH), 8 130.5 (2 x C H ) , 8 127.1 (CH), 8 114.9 (2 x C H ) supported the initial hypothesis of a tyrosine containing peptide. Chiral G C analysis of the acid hydrolyzate of 55 revealed the presence of the amino acids D-tyrosine and D-serine. Tyrosine and serine accounted for six degrees of unsaturation and twelve carbons. One degree of unsaturation (a third carbonyl resonance) and nine aliphatic carbons were left to complete the structure. A broad singlet at 8 12.61 in the ' H N M R spectrum, attached to no carbon, refined the working structure for 55 to a linear dipeptide (tyrosine and serine) with a free carboxylic acid and an aliphatic side chain. Three substructures, a tyrosine (Tyr), serine (Ser) and a fatty acyl (FA) group, were assigned by inspecting the H M Q C (Figure 4.3.3), C O S Y (Figure 4.3.4) and H M B C (Figure 4.3.5) N M R spectra. H M B C Correlation '7i r 9.14 Tyr With Tyr, C O S Y correlations connected the multiplet a-methine at 8 4.34 (H2) to an amide doublet at 8 7.77 (NH) and the methylene multiplet at 8 2.89 and 2.80 (H3/3') of the tyrosine side chain. Further inspection of the C O S Y and H M B C data routinely established the phenyl ring and completed the substructure Tyr. 115 K1 N' H ) 4.30 7.75 Ser H M B C Correlation B y elimination, the multiplet methine at 8 4.30 (HI 1) was assigned as the Ser a-methine. Inspection of the C O S Y and H M Q C completed the assignment of Ser. A n observed H M B C correlation from H I 1 (8 4.30) to a carbonyl at 8 170.0 was tentatively assigned as a two-bond correlation to C10, however, the lack of an observed H M B C correlation from H12 to C10 initially precluded a definitive assignment. The eventual 1 3 C chemical shift assignment of C10 (8 170.0) resulted from the establishment of the 1 3 C chemical shift of the carbonyl C13 (8 172.3) in substructure F A , which by elimination led to the final assignment of the remaining carbonyl C10. A n inspection of the H M Q C and 1 3 C N M R spectra established the remaining nine carbons of the fatty acid substructure F A as follows: one carbonyl, one methine, five methylenes, and two methyls. The methyls were identified as terminal geminal methyls. A methyl doublet at 8 0.83 (H20/21) in the ' H N M R spectrum that integrated for six protons, showed a H M Q C correlation to a single carbon signal at 8 22.4 (C/20/21). As well, both methyls coupled to the same methine at 8 1.48 (H19) in the C O S Y , indicating they existed in an wo-propyl residue. Hy»H H120H H 1 1 3 F A H M B C Correlation 116 Therefore, the only possible connection between the remaining carbonyl and the wo-propyl group was in a linear arrangement of the remaining five methylenes. Analysis of the C O S Y , H M Q C , and H M B C spectra confirmed the presence of the iso-fatty acid F A . The three substructures, Tyr, Ser, and F A were connected by H M B C correlations detailed in figure 4.3.6. A n observed H M B C correlation from tyrosine's amide N H at 5 7.77 to the carbonyl of serine at 8 170.0 (CIO) established the first peptide bond. A second H M B C correlation from serine's amide N H at 8 7.75 to the F A carbonyl at 8 172.3 (C13) linked the final two substructures. The established connectivity accounted for two amide carbonyls and resulted in the remaining carbonyl being assigned as a free carboxylic acid of tyrosine. ,OH O (i 72.3 H O ^ 7 2 . 6 S r ^ | 7 0 ^ ' " N ' l3V o r^i H M B C Correlation HO Figure 4.3.6. Select H M B C correlations for tupuseleiamide A (55). As mentioned previously, the stereochemistry of the component amino acids were established by total acid hydrolysis of tupuseleiamide A (55) followed by chiral G C analysis of the PFPA-DPE derivatized amino acids (Table 4.3.2). Tyrosine and serine were both found to have the D configuration. Standard Tupuseleiamide A (55) Tupuseleiamide B (56) D-Ser 1.4.04. 13.98 14.00 L-Ser ' 14.28 - -D -Tyr 29.44 29.43 29.48 L -Tyr 29.65 - -a Al l indentifications were made by coinjections with standards. In summary, from the available data, the structure of tupuseleiamide A (55) was determined to be a linear lipopeptide. 117 4.4. Structure Elucidation of Tupuseleiamide B (56) 56 Tupuseleiamide B (56) was isolated as an optically active colourless solid that gave a H R F A B M S [M+Na] + peak at m/z 431.21572, corresponding with an elemental composition of C2iH3 2N206Na. Analysis of the one and two-dimensional N M R data (Table 4.4.1) established 56 as an isomer of tupuseleiamide A (55). Total acid hydrolysis of 56 followed by chiral G C analysis of the P F P A - I P E derivatized amino acids (Table 4.3.2) established the presence of both D-tyrosine and D-serine as in tupuseleiamide A . A n examination of the ' H N M R spectrum (Figure 4.4.1) of 56 revealed the structural change from tupuseleiamide A occurred in the methyl region of the fatty acid, with the methyl doublets of 55 now appearing as an overlapping triplet and doublet. Analysis of the ' H and l 3 C N M R spectra and the C O S Y , H M Q C , and H M B C N M R data (Appendix A . 3 , Figures A.3.1 - A.3.2) characterized the structural difference between 56 and 55 as a methyl shift from C19 to C18, giving an anteiso-fatty acid. The stereochemistry at C18 was not determined. 118 119 Table 4.4.1. ' H and l 3 C N M R data for tupuseleiamide B (56) recorded in D M S O - ^ . C# 'H(500 M H z ) (Int., m, 7(Hz)) n C ( 1 0 0 M H z ) C O S Y 3 H M B C Tyr C O O H 1 12.68 (1H, br s) 172.5 (br) 2 4.32 (m) 53.5 H3 , NH(7.84) 3 2.90 (1H, m) 35.6 H 3 ' , H 2 H5, H9 3' 2.80 (1H, m) H3 4 127.0 H6, H8 5,9 6.96 (2H, d, 8.0) 130.0 H6, H8 H 5 , H 9 6,8 6.62 (2H, d, 8.0) 114.8 H5 , H9 OH(9.14), H6, H8 7 155.4 H5, H9 , H6, H8 O H 9.20 (1H, s) N H 7.84 (1H, d,7.5) H2 Ser 10 179.9 11 4.30 (m) 54.7 H12, H12 ' ,NH(7 .81) 12 3.53 (1H, m) 61.5 H l l , H 1 2 ' , O H ( 4 . 7 5 ) 12' 3.48 (1H, m) H 1 1 , H 1 2 , OH(4.75) O H 4.80 (1H, m) H12, H12 ' N H 7.81 (1H, d, 8.0) H l l F A 13 172.2 H14, NH(7.81) 14 2.09 (2H, t, 14) 35.1 H15 15 1.44 (m) 25.4 H14, H I 6 H14 16 1.26 (m) 26.0 H15, H16 ' 16' 1.19 (m) H 1 5 , H 1 6 , H17 17 1.27 (m) . 28.7 H16, H18 H21 18 1.03 (1H, m) 35.9 H17, H19. H21 H21 19 1.26 (m) 33.4 H I 9 , H20 H20 20 0.81 (t,7.0) 11.0 H19 21 0.80 (d, 6.2)' 18.9 H18 'Protons correlated to proton resonance in H column. Protons correlated to carbons in C# column. 120 4.5. F a t t y A c i d B iogenes is Scientists studying the biosynthesis of bacterial fatty acids have traditionally examined those used in cell membranes, which are typically C i 2 to C ] 7 in length. Recently, the discovery of a novel isoprenoid biosynthetic pathway acting in bacteria and plants , 1 3 3 ' 1 3 4 has brought renewed vigor to natural product biosynthetic studies. Given that the tupuseleiamides are both extracellular metabolites and incorporate small chain fatty acids (C9) into their structure, we were lead to experimentally confirm if the same biosynthetic processes acting in cell membrane fatty acid biosynthesis were occurring with the tupuseleiamides. Bacteria of the genera Streptomyces and Bacillus commonly produce branched chain fatty acids, to the extent that in one species, B. subtillis, over 90% of its fatty acids contain branched-chains. 1 3 5 Branched-chain biosynthesis mirrors that known for linear fatty acids as seen in scheme 4.5.1, with an initial condensation catalyzed by an acyl carrier protein (ACP) and the achievement of chain growth via repeated condensations with malonyl-CoA. However, in the biosynthesis of branched-chain fatty acids, the initial A C P has a substrate specificity for the branched primers: isobutyrl-CoA, isovaleryl-CoA, and 2-methylbutyrl-CoA as opposed to the linear precursor ace ty l -CoA. 1 3 2 ' 1 3 6 These branched precursors are derived from the amino acids, valine, leucine, and isoleucine, respectively (Scheme 4.5.2). 1 137 121 O s R SH ACP ^ C y s ^ E r ^ 0 SH • A SH A C P c ^ C y ^ E r ^ R = C Fatty acid synthase R = I Transfer S R _ A C P / C y s V f n z X O S SH ACP / C y ^ y E n z N Reduction p Dehydration Reduction Claisen reaction O O S SH A C P ^ C y ^ E n z ^ R Scheme 4.5.1. Linear and branched-chain fatty acid biosynthesis. 138 ,COOH (7) H 2N"H L-Leucine . C O O H H 2 N H L-Isoleucine C O O H © O 2-Oxoisocaproic acid 0 C O O H © o CoA O Isovaleryl C o A (Iso branch) CoA O 2-Oxo-3-methyl-valeric acid 2-Methyl-butyryl C o A (Anteiso branch) 1: Branched-chain amino acid aminotransferase; 2: branched-chain oxo acid dehydrogenase complex Scheme 4.5.2. Branched-chain fatty acid primers 137 122 Doubly labelled I 3 C sodium acetate ( [ l ,2 - 1 3 C]-NaOAc) has been successfully employed with biosynthetic studies of bacterial metabolites in the past, 5 8 and was seen as suitable precursor for our study. The incorporation of [ l , 2 - 1 3 C ] - N a O A c into a molecule results in the presence of flanking doublets around the natural abundance signal in the 1 3 C N M R spectrum, providing the means for a rapid, visual, and qualitative assessment of results. Biosynthesis via the branched-chain precursor method would require two equivalents of acetate (via malony-CoA) and result in the labelling pattern seen in scheme 4.5.3 with the fatty acid of 55. O O — = 1 3 C — 1 3 C 2 x C o A - ^ ^ ^ O H H 3 C X ^ \ / C o A malonyl-CoA C H O ^ 3 Type II Synthase Isovaleryl-CoA Scheme 4.5.3. Branch-chained labelling pattern for 55. A biosynthetic experiment was undertaken as follows. [ l , 2 - 1 3 C 2 ] - N a O A c (0.25 g) was added, in filter sterilized water, in two portions at 32 and 56 h to a 1 L fermentation culture of M K - P N G - 2 7 6 A . After 144 h, the culture was harvested. The liquid culture was neutralized with the addition of 6 N HCI and tupuseleiamides A (55) and B (56) were isolated as described in section 4.2. Due to the limited quantity obtained, data analysis could only be performed on 55. The 1 3 C N M R spectrum of 55 (Figure 4.5.1) clearly showed the presence of four sites of l 3 C enrichment, the result of the incorporation of two equivalents of [ l , 2 - l 3 C 2 ] - N a O A c (Figure 4.5.2). Acetate incorporation occurred at carbons 13, 14 and 15, 16 as predicted by a branched-chain biosynthesis with an isovaleryl-CoA starting unit (Scheme 4.5.3). 123 C13* C13 C14* C14 172.3 172.3 (ppm) 35.1 35.1 (ppm) C15* C15 25.2 25.2 (ppm) C16* C16 28.9 28.9 (ppm) Figure 4.5.2. Normalized 1 3 C signals from N a O A c labelling experiment (*) and control spectra of tupuseleiamide A (55) in D M S O - d 6 - 1 ppm width. 125 4.6. Conclusion A re-examination of the chemistry of B. laterosporus M K - P N G - 2 7 6 A has resulted in the isolation of the novel lipopeptides, tupuseleiamides A (55) and B (56). Both tupuseleiamides are comprised of nonproteinaceous D amino acids, which implicates a nonribosomal peptide synthase in their biogenesis. The variation in their structures was the result of a change from an iso- to an anfmo-branched chain fatty acid. The biosynthesis of the branched chain C 9 fatty acids of the tupuseleiamides was explored and found to be occurring via the common branched-chain pathway. Lipopeptides are common bacterial secondary metabolites, and many have been isolated from Bacillus species. 1 3 9 They are defined by their mixed biogenic structures and contain both peptide and fatty acid structural motifs. A n example of a lipopeptide previously isolated from B. laterosporus is the novel protease inhibitor bacithrocin A (57) isolated by a Japanese group. 1 4 0 55. 57 The aldehyde lipopeptide 57 isolated from a terrestrial isolate of the same organism as that studied here, contains several structural similarities to the tupuseleiamides, particularly tupuseleiamide A (55) with its /so-fatty acid. However, despite structural similarities, the reduction of the C-terminus of 57 results in net positive charge compared to the net negative charge of 55. The tupuseleiamides showed no antibiotic activity in our assays, but as the bacithrocin A (57) example shows, acyldipeptides can exhibit biological activity. 126 C h a p t e r 5. G e n e r a l C o n c l u s i o n f o r C h a p t e r s 2 , 3 , a n d 4 While re-investigating the broad-spectrum antibiotic activity of the marine bacterium Bacillus laterosporus many new discoveries were made. Bogorols A - E (26-30), five new peptides which represent a novel class of cationic peptide antibiotics, were isolated while exploring the mi\-E. coli activity of B. laterosporus. The bogorols exhibited only moderate antibiotic activity against E. coli, however, they were found to be potent antibiotics versus M R S A . As well , in the process of isolating the bogorols, the novel acyldipeptides, tupuseleiamides A (55) and B (56) were discovered. Lastly, in the search for new peptide antibiotics, the observation that a chromatography fraction displayed antifungal activity led to the structure elucidation of basiliskamides A (50) and B (51). These polyketide amides, when compared to the clinically used amphotericin B , were found to have excellent potential as clinically useful antifungals. One common characteristic of cationic peptide antibiotics is their synergistic activity when used in conjunction with traditional antibiotics. 6 6 Studies to examine the synergism of the bogorols with both clinically-used antibiotics and the antibiotics (the loloatins (22) and the basiliskamides (50 and 51)) produced by B. laterosporus are in progress. The idea that a bacterium produces multiple antibiotics that can act in synergy is a particularly intriguing one. O f course, this assumes a defensive function for the antibiotics discovered. In the case of the bogorols, given the wealth of material on cationic peptide antibiotics as being the products of a common immune response in nature, this may be a safe assumption. However, with the loloatins, it has been hypothesized that they play a role in sporulation, as is seen with their structural cousins the tryocidines (12). 5 8 As for the basiliskamides and the tupuseleiamides, there is no evidence to suggests any one of the various possible ecological roles over the other, and thus their ecological role remains unknown. Bacteria use chemical signals to mediate a 127 diverse array of metabolic process, as well as to interact with other bacterial, plant, and animal cells. Thus, the possible functions of the compounds discovered in this thesis are immense. 1 4 1 To date, four classes of secondary metabolites, three of which display potent antibiotic activity, have been isolated from the cultures of a single marine bacterium. The range in biological activities, encompasses in vitro inhibition of fungi (both yeasts and filamentous), M R S A , V R E , E. coli, and M T B . The work reported in this thesis clearly supports the hypothesis that marine bacteria are an undeveloped source of new antibiotics. Terrestrial isolates of Bacillus spp. have been widely studied and have been found to produce approximately forty-four different classes of peptide antibiotics. 5 0 The discovery of both the loloatins and the bogorols within the same Bacillus sp. suggest that there exists an overlooked potential for the production of peptide antibiotics within the marine Bacillus genus. 128 C h a p t e r 6. De Novo T e r p e n e Biosynthesis by Melibe leonina 6.1. General Introduction Nudibranchs are marine slugs that are notable for their shell-less bodies and wide variety of colouration. 1 4 2 Remarkably adaptive, these carnivorous animals are distributed throughout the world's coastal ecosystems, where they can commonly be found feeding on sessile invertebrates. 1 4 3 Nudibranch skin extracts have been a rich source of secondary metabolites, particularly terpenes, which are believed to act as chemical defences against potential predators such as f i s h 1 4 4 and sea stars. 1 4 5 Much, but not all of nudibranch terpene chemistry has been linked to a dietary origin through a process known as sequestration. 1 4 6 The exceptions have prompted investigations into whether or not nudibranchs are capable of producing their own terpenes (defences) de novo.141 In 1990, Faulkner et a l . 1 4 8 undertook a survey of the chemical constituents of six Northeastern Pacific dorid nudibranchs of varying geographic distributions and developed a hypothesis that relates geographic variation and de novo biosynthesis. The authors found that nudibranchs that contain the same chemicals over a large geographic area, where their food source (the source of sequestered terpenes) can be assumed to vary, were nudibranchs that had been found to be capable of producing their own defences by de novo biosynthesis. This led the authors to conclude that the consistency of secondary metabolites over a geographic area can be used as an indicator for de novo biosynthesis. B y establishing the de novo biosynthesis of terpenes which are conserved throughout their entire geographic range, the study of Northeastern Pacific dorid nudibranchs has been instrumental in supporting the geographic variation hypothesis . 1 4 9 ' 1 5 0 Dor id nudibranchs have been shown to be capable of biosynthesising a variety of sesquiterpenes ( C 1 5 ) , 1 4 7 ' 1 4 9 " 1 5 2 diterpenes ( C 2 0 ) , 1 5 1 and sesterterpenes ( C 2 5 ) (Figure.6.1.1). 1 5 0 129 Sesquiterpenes . O A c albicanyl acetate Cadlina luteomarginata C H O C H O polygodial Dendrodoris limbata Diterpenes O ^ H nanaimoal isoacanthodoral Acanthodoris nanaimoensis A. nanaimoensis A. hudsoni A c Q A. hudsoni olepupane D. limbata farnesic acid glyceride Archidoris odhneri acanthodoral A. nanaimoensis A. hudsoni diterpenoic glyceride A. montereyensis cadlinaldehyde C. luteomarginata luteone C. luteomarginata Figure 6.1.1. Nudibranch terpenes produced by de novo biosynthesis. Terpenes are biosynthesised by the polymerisation of the C 5 building block, isopentenyl pyrophosphate (59), such that, two C 5 units comprise the monoterpenes, three C5 units make the sesquiterpenes, etc. In animals, 59 is made from the condensation of three acetate units, which proceeds through the intermediate mevalonic acid (58). The entire process is known as the mevalonate pathway of terpene biosynthesis (Scheme 6.1.1). 130 O P P 59 PP = pyrophosphate = Intact Acetate Bond = Atom from Cleaved Acetate Bond Scheme 6.4.1. The mevalonate pathway. Initial precursor labelled biosynthetic research used the highly sensitive 1 4 C radioisotopes. 1 5 4 These studies led to methodological improvements that eventually allowed for the use of the less sensitive but more informative 1 3 C stable isotopes. 1 5 1 Typically these experiments used doubly labelled 1 3 C sodium acetate ( [ l , 2 - 1 3 C2 ] -NaOAc) , which as established in chapter 4, enables a rapid and qualitative assessment of results. 6.2. Rationale Figure 6.2.1. Photograph of M. leonina.155 Melibe leonina is a dendronotid nudibranch. It is found seasonally, often in reproductive congregations, in kelp beds along the entire coast of North A m e r i c a . 1 5 5 M. leonina displays one 131 of the most unusual nudibranch feeding patterns. It uses its oral hood to sweep the water for small planktonic crustaceans (zooplankton). When disturbed, M. leonina is capable of swimming and crabs appear to be its sole predator. 1 4 5 ' 1 5 6 M. leonina produces a characteristic fruity odour that originates in pressure sensitive repugnatorial glands. Experiments have shown that the constituents of the glands are repellent to sea stars and some f i sh . 1 4 5 A n examination of the skin chemistry of M. leonina resulted in the isolation of two truncated monoterpenes, the aldehyde 2,6-dimethyl-5-heptenal (60) which is responsible for the nudibranch's odour and its corresponding acid, 2,6-dimethyl-5-heptenoic acid (61). 1 5 7 It has been proposed that 60 and 61 are responsible for the nudibranch's repugnance. 158 There are approximately thirteen species in the genus Melibe, and of these, M. fimbriata is reported to have a similar characteristic odour, 1 5 9 while M. pilosa has been found by G C - M S to contain 60 and 6 1 . 1 6 0 Thus, the following facts led us to experimentally determine i f 60, and therefore 61, are produced by de novo biosynthesis: 1. M. leonina has a geographic range equivalent to many of the Northeastern Pacific dorids. 2. The terpenes 60 and 61 have been found in other Melibe species throughout the world. 3. Unl ike the dorid nudibranchs, M. lonina does not consume a terpene rich diet. 4. Dendronotid nudibranchs have never been shown to be capable of de novo terpene biosynthesis. 5. Nudibranchs have never been shown to be capable of de novo monoterpene biosynthesis. 132 6.3. [ l , 2 - 1 3 C 2 ] - N a O A c Biosynthetic Exper iment In February 2001, a large congregation of M. leonina was discovered while diving in the kelp bed (-2 to -5 m) at Mi l le r ' s Landing, Bowen Island, B . C . Together with M r . Roger Linington, fourteen individuals were collected by S C U B A and immediately immersed in chloroform (250 mL) . A n additional twenty individuals were transported live in a cooler to the Department of Fisheries and Oceans' West Vancouver Laboratory, where they were kept in a 250 L free flow tank for the duration of the biosynthetic experiment. Given the volatile nature of 60, and the pressure sensitive nature of M. leonina's, glands, an injection protocol similar to that used with the dorid Triopha catalinae,161 which requires minimal handling of the animals, was employed. Nudibranchs were injected with 100 pi of a 0.55 M [ l , 2 - 1 3 C2 ] - N a O A c solution upon arrival at the West Vancouver Laboratory and again after 24 h. The injections were made below the mid-body, on the left, hand side of the posterior cerata, in the general vicinity of the digestive gland. 1 5 8 After the last injection, the animals were left undisturbed for 7 days, after which they were immediately immersed in chloroform (250 mL) . Isolation of 60 from both the control and the experimental animals was performed as previously reported. 1 5 7 The chloroform was filtered away from the animals, partitioned with H 2 0 (100 mL) , dried over Na 2 S04, filtered, and concentrated under reduced pressure to give a fragrant oil that was chromatographed on silica gel with hexane - chloroform (9:1) to give 60. 6.4. Results The aldehyde 60 was isolated as a fragrant o i l , which gave a H R E I M S of 140.12012 suitable for the molecular formula C9H16O. Comparison of its C N M R chemical shifts with the literature values (Table 6.4.1) confirmed its structure. The ' f l N M R spectrum (Figure 6.4.1) was assigned by the analysis of the H M Q C N M R data (Appendix A . 4 , Figure A.4.2). 133 134 135 C8* C8 •".70 (ppm) 1 7 7 0 C6* C6 132 70 , \ 132.70 1 J i - ' u (ppm) C5* C5 123.50 , x 123.50 (ppm) C4* C4 4 25.30 / N 25.30 (ppm) C2* C2 45-90 ( p p m ) 45.90 C9* C9 13 30 x 13.30 1 J J U (ppm) Figure 6.4.3. 1 3 C Signals from N a O A c labelling experiment (*) and control spectra of 2,6-dimethyl-5-heptenal (60) in C D C 1 3 -1 ppm width. 136 Carbon # 5C Literature 8C Control 1 205.0 205.2 2 45.9 45.8 3 30.7 30.7 4 25.4 25.3 5 123.5 123.4 6 132.7 132.7 7 25.7 25.7 8 17.7 17.7 9 13.3 13.3 The aldehyde, 60, isolated from the experimental animals gave a 1 3 C N M R spectrum (Figure 6.4.2) which showed evidence for 1 3 C - 1 3 C coupling between C6 and C8 (7C-c 42), C5 and C4 (JC-c 44), as well as C2 and C9 (7C-c 35) (Figure 6.4.3). 6.5. Conclusion In accordance with the geographic variation hypothesis, the results of this experiment confirm that M. leonina produces 60 by de novo biosynthesis. This study is the first to show that a dendronotid nudibranch is capable of de novo terpene biosynthesis. Previously, the dendronotid Tethys fimbria had been shown to biosynthesize de novo a series of ichthyotoxic prostaglandin lactones (fatty acid derivatives). As well, this study represents the first report of de novo monoterpene biosynthesis by nudibranchs. The ability to produce ones own defences (i.e. terpenes) has been hypothesized to be an evolutionary advancement over the sequestration of dietary chemicals. This hypothesis is based on observations made primarily with dorid nudibranchs, which prey on chemically-rich sponges. However, dendronotid nudibranchs, which are believed to be the most primitive of the four nudibranch suborders, do not prey on sponges. 1 6 4 Indeed, M. leonina consumes zooplankton, which are organisms not known to produce secondary metabolites. Cimino and Ghise l in 1 6 5 addressed this apparent contradiction while discussing the evolution of de novo 137 biosynthesis in the crustacean-eating dendronotid T. fimbria. B y invoking a common nudibranch ancestor that preyed on chemically-rich sponges and/or cnidarians, the evolution of de novo biosynthesis in T. fimbria is explained as the catalyst for the ancestral nudibranch's necessary emancipation from its reliance on its original food source for defence. It follows then, that the ability for M. leonina to biosynthesize 60 would have arisen early in its evolutionary history as a direct result of a common Melibe ancestor preying on terpene rich sponges. However, unlike the dorid nudibranchs, which continue to specialise on sponges, this emancipation would have been a necessary condition for further evolution to its unique current state. As mentioned previously, only M. leonina, M. fimbriata, and M. pilosa are described in the literature as having an odour typical of terpenoids. However, the literature on Melibe spp. is sparse, especially concerning species other than M. leonina. Nevertheless, the nudibranchs of the genus Melibe offer a unique opportunity for which to test Cimino and Ghiselin's theory regarding the evolution of de novo biosynthesis in dendronotid nudibranchs. The main tenets of this theory applied to the Melibe example are twofold, firstly, that Melibe spp. have all arisen from a common ancestor that was required to evolve the capability of de novo biosynthesis in order to develop into the various species of today. Secondly, that de novo biosynthesis is an evolutionary advancement over sequestration. From these tenets, it follows that all of the Melibe spp. should then be capable of de novo terpene biosynthesis. This is in contrast to the dorid nudibranchs, which continue to specialise on terpene rich sponges and, consequently, the presence of de novo biosynthesis in some dorids and not others, can not be rationalised on the basis of a common evolutionary ancestor. Thus, the examination of Melibe spp. for the presence of 60 offers the unique opportunity of a testable hypothesis that would provide insight into evolutionary theory regarding the de novo biosynthesis of secondary metabolites by nudibranchs. 138 E x p e r i m e n t a l Sect ion Mater ia l s and Methods A l l solvents used for flash chromatography, sample extractions, and solvent partitions were of reagent grade quality; those used for H P L C were of H P L C quality, and were filtered and degassed prior to use. Anhydrous solvents (dichloromethane, D M F , T H F , benzene, and methanol) and reagents (DJPEA) required for moisture sensitive reactions were purchased as such. Thionyl chloride and triethyl amine were distilled over calcium hydride. Argon (g) was dried by passage through cone, sulfuric acid, sodium hydroxide, and Drierite®. Size exclusion chromatography was performed using Sephadex LH-20® gel permeation beads. Reversed phase column chromatography was performed using either a 10 or 2 g Waters Sep-Pak®. Normal phase column chromatography was performed using 230-400 mesh silica gel 60. Normal and reversed phase thin layer chromatography was performed on commercially available plates. Visualization occurred by U V absorption at 254 and 280 nm, and/or by staining plates with either ninhydrin in ethanol, vanillin in sulfuric acid and ethanol, or an aqueous solution of potassium permanganate and sodium hydroxide. 1 6 6 'Concentration in vacuo', and 'evaporation under reduced pressure' refers to evaporation of solvents using a Buchi rotary evaporator, except when the solvent was either pyridine, D M F or D M S O , in which case a Hi -Vac equipped with a solvent trap was employed. The following mixtures were used in low temperature baths: 0°C: ice/water and - 7 8 ° C : dry ice/acetone. H P L C separations were performed with the following columns: a Whatman Partisil 10 ODS-3 or N P Magnum; a Rainin Partisil 10-ODS; and a CSC-Inertsil-5p:m. H P L C was performed with the following Waters systems: a 600E H P L C pump with a 486 tunable absorbance detector linked to a chart recorder; a 600E H P L C pump with a 996 photodiode array detector interfaced to a P C with Mi l len ium 2010 chromatography software; and a 501 H P L C pump with a 440 U V detector linked to a chart recorder. 139 G C analyses were performed on a Hewlett Packard 5880A Series G C with a flame ionization detector interfaced with a P C running Chrom Perfect® v5.05 software. IR spectra were recorded with a Perkin-Elmer 16000 series FTIR. U V spectra were recorded with a Carey U V - V i s Spectrophotometer. Specific Rotations were determined using a Perkin-Elmer 241 M C Polarimeter a Jasco P-1010 Polarimeter with a sodium light (589 nm). The C D spectrum was run on a Jasco J-710 spectropolarimeter. N M R spectra were recorded on the following Bruker spectrometers: an A M X 5 0 0 , A M 4 0 0 , WH400, and an A V A 4 0 0 , and processed using WLNNMR®. 800 M H z N M R spectra were collected and processed by the staff at the National High Field N M R Centre ( N A N U C ) . Chemical shifts are quoted in parts per million (ppm), 8, and are referenced to the deuterated solvent used (DMSO-efe 8 H 2.49, 8 C 34.9; C D C 1 3 8 H 7.26, 8 C 77.0). Coupling constants (7) are reported in hertz. Routine Mass spectral analyses were performed by the staff at the U B C Department of Chemistry Mass Spectrometry Centre. ESI -QIT-MS and M S / M S experiments were performed on Bruker-Hewlett Packard 1100 Esquire-LC system; samples were dissolved in methanol. A l l biological testing of compounds, and culturing of B. laterosporus M K - P N G - 2 7 6 A was performed by M r . Paul Haden, M s . Helen Wright and Dr. Michael T. K e l l y of SeaTek Marine Biotechnology, Inc. 140 Chapter 2 Culture Conditions M K - P N G - 2 7 6 A was cultured on trays of solid tryptic soy agar supplemented with NaCl to concentrations of 1 %. Twenty-one 400 m L trays (24 cm x 37 cm x 0.5 cm deep agar) were cultured for five days after which the combined cells and agar were lyophilized. The dry cells were scrapped off the agar. Isolation See section 2.3. Compound Summaries Bogorol A (26) was isolated as a white solid; [oc] 2 5 D = -38.2° (MeOH); . ESI -QIT-MS and M S / M S data see figures 2.3.10 and 2.3.11 ; G C data see table 2.4.1; H R E S I M S [ M + H ] + m/z 1584.0875 ( C 8 0 H i 4 3 N 1 6 O , 6 , calcd 1584.0868). Bogorol B (27) was isolated as a white solid; [ a ] 2 5 D = -50.3° (MeOH); ESI -QIT-MS and M S / M S data see figures 2.6.2 and 2.6.3; G C data see table 2.6.1; H R E S I M S [M+H] + m/z 1570.0710 (C 79Hi4iN 1 60i 6 , calcd 1570.0711). Bogorol C (28) was isolated as a white solid; [ a ] 2 5 D = -64.7° (MeOH); ESI -QIT-MS and M S / M S data see figures 2.7.2 and 2.7.3; G C data see Table 2.6.1; H R E S I M S [ M + H ] + m/z 1556.0565 ( C 7 8 H , 3 9 N i 6 0 , 6 , calcd 1556.0555). Bogorol D (29) was isolated as a white solid; [oc] 2 5 D = -43.5° (MeOH); ESI -QIT-MS and M S / M S data see figures 2.8.2 and 2.8.3; G C data see Table 2.6.1; H R E S I M S [M+H] + m/z 1602.0437 (C79H,4 iNi 6 0 , 6 S, calcd 1602.0432). Bogorol E (30) was isolated as a white solid; [ a ] 2 5 D = -17.2° (MeOH); E S I - Q I T - M S and M S / M S data see figures 2.9.2 and 2.9.3; G C data see Table 2.6.1; H R E S I M S [ M + H ] + m/z 1618.0405 ( C 7 9 H i 4 1 N 1 6 0 i 7 S , calcd 1618.0381). 141 Bogorol A hexaacetate (31). A 3:1 mixture of pyridine and acetic anhydride was added, with stirring, to a round bottom flask charged with 50 mg bogorol A (26). After 16 h at room temperature, the solvent was removed under reduced pressure and the resulting solids were chromatographed on a reversed-phase silica Sep-Pak® (2g), eluting a step gradient with 18 m L of each of the following: 100% H 2 0 , 1:1, 2:3, 3:7, 1:4, 1:9 H 2 0 : M e O H , 100% M e O H . The fractions 1:4, 1:9 and 100 % M e O H were reduced in vacuo and further purified by reversed-phase H P L C (1:1 M e C N : H 2 0 with 0.2% T F A ) to yield bogorol A hexaacetate (31) (16 mg, 27 %) as a white solid. 6H(800 M H z , D M S O - d 6 ) and 8C(100 M H z , D M S O - d 6 ) data see table A . 1.1; H R F A B M S [M+( , 3 C)1+H] + m/z 1837.15212 (C92H156N16O22, calcd 1837.15801). Total Acid Hydrolysis and GC Analysis In a screw-top vial, bogorol (0.5 mg) was dissolved in 6 N HCI (1 mL) and heated at 110°C for 72 h. The HCI was removed under a stream of N 2 gas. A stock solution of i-Propyl acetate was created by slowly adding 1.25 m L acetyl chloride to 5 m L i'-propanol at 0 ° C . /-Propyl acetate (250 uL) was added to the reaction vial and heated to 110°C for a further 45 min followed by solvent removal under a stream of N 2 gas. Dicholoromethane (250 p.L) and pentafluoropropionyl anhydride (100 uL) were added to the reaction vial and heated to 110°C for 15 min. Excess reagent was removed under N 2 gas and the sample was redissolved in dichloromethane (200 | i L ) . Standards were prepared in the same fashion using optically pure L-amino acids and their racemic mixtures. The amino acid standards and the hydrolyzates were analyzed on a 25 m chiralsil-Val Heliflex column with FED detection using the following conditions: He carrier; initial oven temp. 60°C until initial time 4 min; program rate 3°C/min to oven temp. 130°C; program rate 10°C/min to oven temp 190°C (37.5 min.); isothermal until final time 47.5 min. For the analysis of the valine, valinol, and the 2-hydroxy-3-methylpentanoic acid derivatives, the following temperature gradient was used: initial oven temp, 60°C ; initial time, 142 15 min; program rate 4°C/min to oven temp 80°C; program rate 10°C/min to oven temp 190°C; isothermal until final time of 47.5 min. Chiral GC Experiment To 1.5 mg 26 (0.95 |i.mol) was added 1.5 m L of 6 N HC1 containing 3.84 (imol L-alanine. The solution was mixed and then divided into the six 0.25 m L samples in screw top vials. The vials were sealed and placed in a sand bath heating mantle at 105° C (t = 0); one sample was removed at each of t = 1.5, 3, 6, 12, 24 and 48 h and subjected to the procedure outlined in the proceeding "Total A c i d Hydrolysis and G C Analysis" section. The same column conditions were used as in the latter. Reduction of +/- 3-Methyl-2-oxopentanoic Acid To a stirred (0° C) solution of 3-methyl-2-oxopentanoic acid, sodium salt, (32, 50 mg, 0.33 mmol) in 5 m L M e O H was added sodium borohydride (62.2 mg, 1.64 mmol). The solution was stirred for 2 h, after which, 20 m L of 0.2 N HC1 (aq) was added. The acidified solution was extracted with E tOAc (3 x 10 mL) . The combined organic extracts were dried (Na2S04), filtered, and concentrated under reduced pressure to give 32a and 32a (38.3 mg 83%) as a mixture of four stereoisomers. The four stereoisomers were separated into their enantiomeric pairs by normal phase H P L C (95:5, Hexane - Isopropanol) on an analytical column, with U V detection at 209 nm. The enantiomers were converted to their isopropyl esters with tsq-propyl acetate as in the Total Acid Hydrolysis and GC Analysis section and identified by chiral G C analysis using the conditions outlined in the aforementioned section. (R, R; S, S) 8H(400 M H Z ; CDC1 3 ) 4.16 (1H, d , / 4 ) , 1.86 (1H, m), 1.39 (1H, m), 1.28 (1H, m), 1.00(3H, d, 77), 0.90 (3H, t, 7 7.5); 8C(100 M H z ; CDC1 3 ) 179.0, 74.6,38.9, 23.6, 15.3, 11.7. (R, S; S, R) 8 H(400 M H z ; CDC1 3 ) 4.27 (1H, d, 7 3), 1.87 (1H, m), 1.51 (1H, m), 1.33 (1H, m), 0.95 (3H, t, 7 7), 0.85 (3H, d, 7 7); 8 c ( 1 0 0 M H z ; C D C l 3 ) 179.2, 72.8,38.4,25.9, 13.1, 11.8. 143 O OH 62 (2R, 3S)-2-Hydroxy-3-methylpentanoic acid benzyl ester (62). Benzyl bromide (180 1.5 mmol) was added, dropwise, to a stirred solution of the sodium salt of 2-hydroxy-3-methylpentanoic acid (42, 206 mg, 1.3 mmol) in dry D M F (10 mL) under argon (g) at room temperature. After 5 h, the D M F was removed in vacuo. The resulting solids were suspended in 25 m L dichloromethane and washed with 0.1 N sodium hydroxide (aq.) (3 x 10 mL). The combined aqueous washings were extracted with dichloromethane (1 x 20 mL) . The combined organics were dried (Na 2 S0 4 ) , filtered, and evaporated under reduced pressure to give 62 (275 mg, 93%) as an oi l . 8H(400 M H z ; CDC1 3 ) 7.35 (5H, br m), 5.20 (2H, dd, 7 18 and 12), 4.21 (1H, dd ,7 6and3) , 2.68 (1H, d ,76 ) , 1.81 (1H, m), 1.51 (1H, m), 1.27 (1H, m), 0.92 (3H, t, 7 8), 0.76 (3H, d, 7 7); 8C(100 M H z ; CDC1 3 ) 175.3, 135.2, 128.6, 128.5, 128.4, 73.0, 67.3, 38.5, 25.9, 13.1, 11.8; [ a ] 2 5 D = + 3 . 1 ° (CHC1 3 ); HREDVIS M + m/z 222.12580 ( C i 3 H , 8 0 3 , calcd 222.12559). 43 (25, 3S)-2-p-Nitro-benzoate-3-methylpentanoic acid benzyl ester (43). Diethyl azodicarboxylate (0.98 mL, 6.2 mmol) was added dropwise to a stirred solution of ester 62 (275 mg, 1.24 mmol), triphenylphosphine (1.63 g, 6.2 mmol), and p-nitro-benzoic acid in dry benzene 144 (20 mL) under argon (g) at room temperature. After 3 h, the reaction solution was chromatographed ( S i 0 2 , benzene) to remove triphenylphosphine oxide. Further flash chromatography ( S i 0 2 , 95 : 5 pet. ether - diethyl ether) afforded 43 (441 mg, 96 %) as a white solid. 6 H(400 M H z ; CDC1 3 ) 8.22 (2H, d, J 2), 8.20 (2H, d, J 2), 7.32 (5H, br m), 5.20 (3H, m), 2.15 (1H, m), 1.52 (1H, m), 1.38 (1H, m), 1.03 (3H, d, J 7), 0.92 (3H, t, J 7); 5C(100 M H z ; CDC1 3 ) 169.0, 164.3, 150.9, 135.2, 135.0, 130.9, 128.6, 128.5, 125.3, 123.6, 77.5, 67.1, 36.8, 24.6, 15.5, 11.5; [cc] 2 5 D= -0.69° (CHCI3) ; H R E I M S M + m/z 371.13596 ( C 2 0 H 2 i N O 6 , calcd 371.13689). 63 (25, 3S)-2-Hydroxy-3-methylpentanoic acid benzyl ester (63). A stirred solution of ester 43 (196 mg, 0.53 mmol) and sodium azide (103.1 mg, 1.6 mmol) in dry methanol under argon (g) was heated at 40° for 30 h. The solution was reduced in vacuo, and the resulting solids were suspended in diethyl ether (50 mL) and washed with water (3 x 20 mL) . The combined aqueous washings were extracted with diethyl ether (1 x 20 mL) . The combined diethyl ether solution was dried ( N a 2 S 0 4 ) , filtered, and evaporated under reduced pressure. The residue was chromatographed ( S i 0 2 , 9:1 pet. ether - diethyl ether) to give 63 (114 mg, 97%) as an oi l . 8H(400 M H z ; CDC1 3 ) 7.35 (5H, br m), 5.19 (2H, dd, J 22 and 12), 4.10 (1H, br s), 2.94 (1H, d, J 6), 1.81 (1H, m), 1.31 (1H, m), 1.23 (1H, m), 0.95 (3H, d, J 7), 0.84 (3H, t, J 7); 8C(100 M H z ; CDCI3) 174.7, 135.1, 128.5, 128.4, 128.3,74.7, 67.0, 39.0, 23.6, 15.3, 11.6; [ a ] 2 5 D = -3.0° (CHCI3) ; H R E I M S M+m/z 222.12510 ( C i 3 H i 8 0 3 , calcd 222.12559). 145 64 (2S, 3S)-3-Methyl-2-(tetrahydro-2H-pyran-2-yl)oxy-pentanoic acid benzyl ester (64). p-Toluene sulfonic acid (2 mg, 0.01 mmol) was added to a stirred solution of ester 63 (25 mg, 0.11 mmol) and dihydropyran (18.6 U .L, 0.22 mmol) in dry dichloromethane (0.5 mL) under argon (g) at room temperature. After 0.5 h, the reaction solution was diluted with diethyl ether (100 mL) and washed with a 1% sodium bicarbonate solution (3 x 20 mL) , dried (Na2S04), and evaporated under reduced pressure. The residue was chromatographed (SiO"2, 92.5:7.5 hexane -ethyl acetate) to give diastereomeric 64 (28.3 mg, 84%) as an o i l . 5 H(400 M H z ; CDC1 3 ) 7.33 (br m), 5.15 (m), 4.59 (m), 4.19 (d, J 5.5), 3.81 (br m), 3.73 (d, J 7.6), 3.47 (br m), 3.31 (br m), 1.95-1.75 (br m), 1.73-1.63 (br m), 1.62-1.40 (br m), 1.22 (m), 0.93 (d, J 6.9), 0.87-0.82 (br m); 8C(100 M H z ; CDC1 3 ) 172.5, 172.4, 136.0, 135.7, 128.5, 128.4, 128.38, 128.3, 128.2, 128.1, 100.8, 96.9, 82.5, 78.2, 66.2, 66.1, 62.4, 62.2, 38.0, 37.7, 30.33, 30.32, 25.4, 25.2, 24.8, 24.7, 19.2, 19.0, 15.5, 14.5, 11.4, 11.0; H R D C T M S [M+H] + m/z, 307.19107 (CgHseCU, calcd 307.19094). 146 41 (25, 3S)-3-Methyl-2-(tetrahydro-2H-pyran-2-yl)oxy-pentanoic (41). Ester 64 (21.5 mg, 0.07 mmol), in ethyl acetate (1 mL) with 20% w/w 10% Pd-C, was stirred under an atmosphere (balloon) of H 2 (g) at room temperature. After l h , the reaction mixture was filtered through celite and the filtrate was evaporated under reduced pressure to yield 41 (14.9 mg, 98%) as a mixture of diastereomers. 5H(400 M H z ; CDC1 3 ) 4.67 (br m), 4.51 (m), 4.20 (d, J 4.8), 3.96 (br m), 3.91 (d, J 4.6), 3.82 (br m), 3.52-3.45 (br m), 1.97-1.86 (br m), 1.85-1.74 (br m), 1.73-1.64 (br m), 1.63-1.45 (br m) 1.27 (m), 1.00 (d, J 6.8), 0.95-0.85 (m); 5C(100 M H z ; CDC1 3 ) 177.2, 174.6, 102.7, 97.4, 83.2, 78.1, 64.8, 62.4, 38.0, 37.6, 30.7, 30.3, 25.3, 24.9, 24.6, 24.1, 20.5, 19.0, 15.5, 15.2, 11.6, 1 1 . 5 ; H R D C I M S [M+H] + m/z 217.14336 ( C , , H 2 o 0 4 , calcd 217.14398). 45 N-(9H-Fluoren-9-ylmethoxycarbonyl)-L-allo-threonine (45). 9/7-Fluoren-9-ylmethyl chloroformate (819 mg, 3.2 mmol) in dioxane (20 mL) was added dropwise to a stirred solution of L-a//o-threonine (377mg, 3.2 mmol) in dioxane (20 mL) and 10% sodium carbonate (aq.) (20 mL) at 0°. The solution was stirred at room temperature for 4 h, diluted with water (100 mL) 147 and washed with diethyl ether (3 x 50 mL) . The aqueous solution was acidified with 6 N HCI (aq), and extracted with ethyl acetate (3 x 100 mL) , dried ( N a 2 S 0 4 ) , and evaporated under reduced pressure. The crude product was recrystallized from chloroform/hexane to give 64 (700 mg, 65%) as a white solid. 8 H(400 M H z ; DMSO-cfc) 12.5 (br OH) , 7.87 (2H, d, J 7.6), 7.73 (2H, d, J 7.6), 7.49 (1H, d, J 8), 7.40 (2H, m), 7.32 (2H, m), 4.90 (br s), 4.27-4.22 (3H, br m), 3.93 (2H, brm), 1.10 (3H, d, J 5.8); 5C( 100 M H z ; DMSO-d 6 ) 172.2, 156.1, 143.8, 140.7, 127.6, 127.0, 125.3, 120.1, 66.4, 65.7, 60.3, 46.6, 19.6; [oc]2 5 D= +6.4° (CHC1 3 ) ; HRDCEVIS [M+H] + m/z 342.13422 (Q9H19NO5, calcd 342.13415). I 47 N-(9H-Fluoren-9-ylmethoxycarbonyl)-L-allo-threonyl-L-Leucine t-butyl ester (47). D I P E A (90 p i , 0.52 mmol) was added to a stirred solution of P y B O P (136 mg, 0.26 mmol), L -leucine t-butyl ester hydrochloride (58 mg, 0.26 mmol), and acid 45 (46 mg, 0.135 mmol) in dry D M F (2 mL) at room temperature under an atmosphere of argon (g). The reaction solution was stirred overnight. The D M F was removed under reduced pressure and the resulting dark orange oil was chromatographed ( S i 0 2 , 6:4 hexane - ethyl acetate) to afford 47 (69 mg, 96%) as a colorless oi l . 5 H(400 M H z ; CDC1 3 ) 7.73 (2H, d, J 7.4), 7.55 (2H, d, J 7.2), 7.37 (2H, t, J 7.0), 7.27 (2H, m), 6.66 (1H, d, J 7.4) 5.85 (,1-H, d, J 8.0), 4.45-4.30 (3H, br m), 4.18 (1H, t, J 6.8) 4.11 (1H, m ) 3 . 8 9 ( l H , br m), 3.75 (1H, br s), 1.65-1.51 (2H, b rm) , 1.43 (9H, s), 1.27-1.22 (4H, m), 0.88 (6H, t, J 4.8); 6C(100 M H z ; CDC1 3 ) 172.3, 171.0, 156.5, 143.7, 143.6, 141.3, 127.7, 127.0, 125.1, 125.0, 120.0, 82.4, 69.5, 67.3, 59.2, 52.0, 47.0, 40.7, 27.9, 24.9, 22.7, 21.7, 19.6; [ot] 2 5 D= -19.5° (CHCI3) ; H R E I M S M + m/z 510.27333 ( C 2 9 H 3 8 N 2 0 6 , calcd 510.27299). 148 48 L-allo-threonyl-L-leucine t-butyl ester (48). Piperidine (14 u l , 0.14 mmol) was added to a stirred solution of 47 (64 mg, 0.12 mmol) in D M F (2 mL) at room temperature. After 1 h, the D M F was removed under reduced pressure and the crude reaction mixture was chromatographed on a N P lOg Sep-Pak® (100% EtOAc) to yield 48 (25.8 mg, 75%) as an o i l . 8H(400 M H z ; CDC1 3 ) 7.51 (2H, d, J 7.6), 4.42 (1H, m), 3.92 ( 1H , br t, J 6.0), 3.27 (1H, br s), 2.55 (3H, br s), 1.59 (2H, m), 1.50 (1H, m), 1.42 (9H, s), 1.18 (3H, d, J 6.5), 0.90 (6H, dd, J 6.0, 5.7); 5C(100 M H z ; CDC1 3 ) 173.8, 172.2, 82.0, 69.5, 59.8, 51.2, 41.4, 27.9, 25.0, 22.8, 21.9, 18.8; [oc] 2 5 D= - 20.7° (CHCI3) ; H R D C T M S [M+H] + m/z 289.21260 ( C , 4 H 2 9 N 2 0 4 , calcd 289.21273). 38 N-[(2S, 3S)-2-(tetrahydro-2H-pyran-2-yl)oxy-3-methylpentanoate]-L-allo-threonyl-L-leucine t-butyl ester (38). D I P E A (45 pj, 0.26 mmol) was added to a stirred solution of P y B O P (67 mg, 0.13 mmol), amide 48 (24 mg, 0.08 mmol), and acid 41 (14 mg, 0.065 mmol) in dry D M F (1 mL) at room temperature under an atmosphere of argon (g). The reaction solution was stirred overnight. The D M F was removed under reduced pressure and the resulting dark 149 orange oi l was chromatographed ( S i 0 2 , 6.5:3.5 hexane - ethyl acetate) to afford diasteromeric 38 (27.8 mg, 88%) as a colorless oi l . Partially characterized 38 was brought through to the S O C l 2 / N E t 3 dehydration reaction as a mixture of diastereomers. 8H(400 M H z ; CDC1 3 ) 7.50 (d, J 8) 7.30 (d, J 8) 6.71 (d, J 8), 6.43 (d, J 8), 4.50 - 4.31 (m), 4.12 - 4.04 (m), 3.97 - 3.84 (m), 3.49 -3.40 (m), 2.69 (br s), 1.91 -1.65 (m), 1.62- 1.45 (m), 1.43 (s C H 3 ) , 1.28- 1.20 (m C H 3 ) , 0 . 9 8 -0.84 (m C H 3 ) H R D C I M S [M+H] + m/z 487.33821 ( C 2 5 H 4 7 N 2 0 7 , calcd 487.33797). Chapter 3 Culture Conditions A s in the chapter 2 section. Isolation See section 3.2. Compound Summaries Basiliskamide A (50) was isolated as a clear solid; 5H(400 M H z ; D M S O - d 6 ) and 8C(100 M H z ; D M S O - 40 see table 3.3.1; IR (film) D m a x : 3348, 3205, 2966, 2934, 1705, 1697, 1635, 1595, 1450 cm" 1; U V (MeOH) Xmax: 262 nm (8 41 000); [oc] 2 5 D = -78°(MeOH); H R F A B M S [M+H] + m/z 386.23358 ( C 2 3 H 3 2 N 0 4 , calcd 386.23313). Basiliskamide B (51) was isolated as a clear solid; 8H(400 M H z ; DMSO-cfe) and 8C(100 M H z ; D M S O - d 6 ) see table 4.5.1; IR (film) u m a x : 3348, 3205, 2962, 2926, 1702, 1664, 1637, 1595, 1450 cm- 1; U V (MeOH) X m a x : 262 nm (e 43 000); [ a ] 2 5 D (MeOH) = -12°; H R E I M S M + m/z 385.22531 ( C 2 3 H 3 1 N 0 4 , calcd 385.22531) 150 Reduction of Basiliskamide A (26) to 1,3-Diol (CI) To a stirring and cooling flask (-78°, A r (g)) of basiliskamide A (50, 4.7 mg, 12 |u,mol) in 1 m L T H F , were added 4 equivalents of diisobutylaluminum hydride ( D I B A L - H ) (1 M in hexane). The reaction was stirred for a further 18 h, then diluted with E t O A c (3 mL) and quenched by the addition of 2 m L NH 4 C1 (aq), stirring until the reaction mixture turned cloudy (10 min). The mixture was extracted with E t O A c (3 x 5' mL), and the combined organics were reduced to dryness in vacuo. Preparative normal phase T L C (100% EtOAc) followed by reversed phase H P L C (7 : 3 M e O H : H 2 0 ) gave 52 (2 mg, 64%). 5H(500 M H z ; D M S O - d 6 ) 7.44 (1H, dd, 7 15, 11), 7.35 (1H, br s, N H ) , 6.86 (1H, br s, N H ) , 6.36 (1H, dd, 7 11), 6.02 (1H, dt, 7 15, 7), 5.57 (1H, d, 7 11), 4.48 (1H, d, 7 5.4, OH). 4.69 (1H, d, 7 4.4, OH) , 3.80 (1H, m), 3.23 (1H, m), 2.26 (1H, m), 2.07 (1H, m), 1.61 (1H, m), 1.36 (1H, m), 1.35 (1H, m), 1.18 (1H, m), 0.84 ( 3 H , t , 7 7), 0,72 (3H, d, 7 7 ), 0.66 (3H, d, 7 7); H R F A B M S [M+ H]+ m/z 256.19211 ( C 1 4 H 2 6 N 0 3 , calcd 256.19127). Formation of Acetonide (53) To a stirring flask (Ar (g)) of 1.5 mg of 52 (5.9 | imol) in 0.5 m L 2,2-dimethoxypropane was added pyridinium p-toluenesulfonate (5 wt% diolbasiliskamide). The reaction mixture was stirred and heated at 60°C for 1 h. The reaction mixture was filtered through silica (100% EtOAc) and the solvents removed in vacuo. Reversed-phase H P L C (4:1 M e O H : H 2 0 ) gave 53 (1 mg, 58%). 5H(400 M H z ; D M S O - d 6 ) 7.46 (1H, dd, 7 15, H-4), 7.35 (1H, br s, N H ) , 6.85 (1H, br s, N H ) , 6.37 (1H, dd, 7 11, 11, H-3), 5.94 (1H, dt, 7 15, 7, H-5), 5.58 (1H, d, 7 11, H-2), 3.57 (1H, m, H-7), 3.48 (1H, dd, 7 10, 2, H-9), 2.44 (1H, m, H-6), 2.19 (1H, m, H-6'), 1.54 (1H, m, H-10), 1.36 (3H, s, Me-17), 1.33 (1H, m, H-8), 1.30 (1H, m, H - l l ) , 1.25 (1H, m, H - l l ' ) , 1.23 (3H, s, Me-16), 0.83 (3H, t, J 7, Me-12), 0.75 (3H, d, 7 7, Me-13), 0.71 (3H, d, 7 7, Me-14); 8 c ( 1 0 0 M H z ; D M S O - ^ 6 ) 167.8 ( C - l ) , 140.6 (C-3), 138.5 (C-5), 128.9 (C-4), 120.2 (C-2), 97.6 151 (C-15), 74.9 (C-9), 74.0 (C-7), 36.5 (C-6), 35.1 (C-8), 34.6 (C-10), 30.4 (C-16), 26.7 ( C - l l ) , 19.8 (C-17), 12.7 (C-13), 12.1 (C-12), 11.6 (C-14); H R F A B M S [M+H]+ m/z 296.22198 ( C 1 7 H 3 0 N O 3 , calcd 296.2257). Reaction of 50 with (R)-MTPA Acid Chloride To a stirred solution of 50 (1.5 mg, 4.0 jimol) in 0.5 m L dry C H 2 C 1 2 were added a few crystals of D M A P , a drop of triethylamine, and ( i?)-MTPA acid chloride (4 mg, 16.0 u,mol). The solution was stirred for 16 h. The removal of solvent in vacuo, preparative reversed phase T L C (100% M e O H ) , followed by reversed phase H P L C (4:1 M e O H : H 2 0 ) gave the ( t f ) -MTPA ester 50a (0.8 mg, 33%). 8H(500 M H z ; D M S O - d 6 ) 7.72 (3H, br envelope), 7.43 (9H, br envelope), 7.35 (1H, br s), 6.84 (1H, br s), 6.70 (1H, d, J 16), 6.20 (1H, dd, J 11, 11), 5.58 (2H, m), 5.17 (1H, m), 4.98 (1H, m), 3.43 (3H, s), 2.60 (1H, m), 2.26 (2H, br m), 1.72 (1H, m), 1.29 (2H, br m), 1.17 (1H, m), 0.95 (3H, d, J 7), 0.93 (3H, d, J 7), 0.88 (3H, t, J 7); H R F A B M S [M+H]+ m/z 602.27148 ( C 3 3 H 3 9 N 0 6 F 3 , ca lcd 602.272950). Reaction of 50 with (S)-MTPA Acid Chloride A solution of 50 (1.5 mg) in 0.5 m L dry C H 2 C 1 2 was treated as above, but with the (S) -MTPA acid chloride (4 mg, 16.0 umol) to give the (S ) -MTPA ester 50b (0.4 mg, 17%). 8H(500 M H z ; D M S O - J 6 ) 7.72 (3H, br envelope), 7.54 (1H, m), 7.43 (8H, br envelope), 7.38 (1H, br s), 6.91 (1H, br s), 6.72 (1H, d, J 16), 6.36 (1H, dd, J 11, 11), 5.78 (1H, m), 5.64 (1H, d, J 11), 5.13 (1H, m), 4.94 (1H, m), 3.42 (3H, s), 2.63 (1H, br m), 2.35 (1H, m), 2.17 (1H, m), 1.65 (1H, m), 1.27 (2H, br m), 1.15 (1H, m), 0.89 (3H, d, J 7), 0.87 (3H, t, J 7), 0.70 (3H, d, J 7); H R F A B M S [M+H]+ m/z 602.27352 ( C 3 3 H 3 9 N 0 6 F 3 calcd 602.272950). 152 C h a p t e r 4 Culture Conditions M K - P N G - 2 7 6 A was grown on tryptic soy Agar with 1 % N a C l at room temperature for 2-3 days. 800 m L of tryptic soy broth with 1% N a C l was innoculated with a 1.0 McFarland standardized bacterial suspension in 0.85 % saline. The cultures were then grown for an additional 6-7 days at 22°C on a shaker. Isolation See section 4.2 Total Acid Hydrolysis and GC Analysis The general procedure provided in section 6.2 was followed for the hydrolysis and G C analysis. Thus, in a screw-top vial, tupuseleiamide (0.5 mg) was dissolved in 6 N HC1 (1 mL) and heated at 110°C for 16 h. The amino acid standards and the hydrolyzates were analyzed on a 25 m chiralsil-Val Heliflex column with FED detection using the following conditions: He carrier, detector temp 90°C, initial time 4 min, program rate 4°C/min, final oven temp 200°C, final time 27.5 min. Stable Isotope Feeding Experiment A 1 L fermentation culture of M K - P N G - 2 7 6 A was fed, in two portions, a total of 0.25 g [1,2- 1 3 C 2 ] sodium acetate (in 2 x 5 m L portions of filter sterilized water) at 32 and 56 h. The culture was then harvested after 144 h. The liquid culture was neutralized with the addition of 6 N HC1 and tupuseleiamide A (55) was isolated as described in section 4.2. Compound Summaries Tupuseleiamide A (55), was isolated as a clear solid; IR (film) u m a x : 3449, 1728, 1660, 1614, 1516, 1222 c m - 1 ; U V (MeOH) y m a x (e): 262 nm (41 000); [cc] 2 5 D -78° ( M e O H ; 8H(400 153 M H z ; DMSO-4) and 5 C(100 M H z ; DMSO-4) data see table 4.3.1; H R F A B M S [M+H] + m/z 409.23417 (C 2 1 H33N 2 06, calcd 409.23386). Tupuseleiamide B (56), was isolated as a clear solid; IR (film) Umax: 3449, 1717, 1660, 1620, 1517, 1224 cm" 1; U V (MeOH) Ymax (e): 262 nm (41 000); [ a ] 2 5 D -78° (MeOH); ; 6H(400 M H z ; D M S O - d 6 ) and 5 C(100 M H z ; DMS0-4) data see table 4.4.1; H R F A B M S [M+Na] + m/z 431.21572 ( C 2 I H 3 2 N 2 0 6 N a , calcd 431.21580). Chapter 6 Isolation See section 6.3 Compound Summary 2,6-Dimethyl-5-heptenal (60) was isolated as an oil (8.9 mg, Control Experiment); 8H(400 M H z ; CDC1 3 ) 9.59 (1H, d, J 2.0, Aldehyde), 5.06 (1H, t, J 7.2, H-5), 2.34 (1H, m, H-2), 2.01 (2H, m, H-4), 1.75 (1H, m, H-3) 1.57 (3H, s, H-7) 1.54 (3H, s, H-8) 1.38 (1H, m, H-3) 1.07 (3H, d, J 7.2, H-9); 5 C(100 M H z ; CDC1 3 ) see table 5.4.1; H R E I M S M + m/z 140.12012 ( C 9 H i 6 0 , calcd 140.11991). A p p e n d i x A . l . Appendix for Chapter 156 157 159 161 Figure A . 1.6. 800 M H z C O S Y Spectrum with selected correlations; methyl region expansion of bogorol A hexaacetate (31) . 162 t\ • • • • t • • H f t H H H H rt H H H H Figure A . 1.7. 800 M H z C O S Y Spectrum with selected correlations; aliphatic region expansion of bogorol A hexaaceate (31). 163 b — Figure A . 1.8. 800 M H z C O S Y Spectrum with selected correlations; a-methine region expansion A of bogorol A hexaacetate (31). 164 Figure A . 1.9. 800 M H z C O S Y Spectrum with selected correlations; a-methine expansion B of bogorol A hexaacetate (31). 165 I , I , I I M j I I I I | I I I , j I | I I I I | i I I I | I I I I | I I I I | I I I I | | I 1 I I | I I I I | M i I i ! I | I M I | I I I I | : . I | I I I I | I . i I | I I I I | II I I g O N * l 0 C O O ( < < < f < 0 C O H a cn en ro rn CO ^ »» «* ^ ^ h — Figure A . 1.10. 800 M H z C O S Y Spectrum with selected correlations; N H region expansion of bogorol A hexaacetate (31). 166 Figure A . 1.11. 800 M H z T O C S Y Spectrum with selected correlations; N H region expansion A of bogorol A hexaacetate (31). I 167 Figure A . 1.12. 800 M H z T O C S Y Spectrum; N H region expansion B of bogorol A hexaacetate (31). 168 Figure A . 1.13. 800 M H z T O C S Y Spectrum with selected correlations; N H region expansion C of bogorol A hexaacetate (31). 169 Figure A . 1.14. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region expansion A of bogorol A hexaacetate (31). 170 Figure A . 1.15. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region expansion B of bogorol A hexaacetate (31). Figure A . 1.16. 800 M H z T O C S Y Spectrum with selected correlations; a-methine region expansion C of bogorol A hexaacetate (31). 172 Figure A . 1.17. 800 M H z H S Q C Spectrum with selected correlations; aliphatic region expansion A of bogorol A hexaacetate (31). Figure A . 1.18. 800 M H z H S Q C Spectrum with selected correlations; methyl region of bogorol A hexaacetate (31). 174 Figure A . l . 1 9 . 800 M H z H S Q C Spectrum; aliphatic region expansion B of bogorol A hexaacetate (31). 175 Figure A . 1.20. 800 M H z H S Q C Spectrum with selected correlations; aliphatic region expansion C of bogorol A hexaacetate (31). 176 1 M J I f l l l l l l l l U l l l l ,111111111 | I|l 1 | l l l l | i m | M I I | I M I | l l l l | < •£? L L <*> ex <>i •!» VO co o <N • » FigureA. 1.21. 800 M H z H S Q C Spectrum with selected correlations; a-methine region expansion of bogorol A hexaacetate (31). 177 Figure A . 1.22. 800 M H z H S Q C Spectrum with selected correlations; aromatic region expansion of bogorol A hexaacetate (31). 178 Figure A . 1.23. 800 M H z H M B C Spectrum with selected correlations; methyl region expansion of bogorol A hexaacetate (31 ) . 179 Figure A . 1.24. 800 M H z H M B C Spectrum; aliphatic region expansion A of bogorol A hexaacetate (31). 180 B-2 (120.7), 1 (130.8)/B-3 Z-Ac-CO (170.2)/ Z-Ac-Me 1 X - A c - C O (170.1)/ X-Ac-Me in I- « i 1 — r - * - i — i — ' — r -* i CO o in Figure A . 1.25. 800 M H z H M B C Spectrum with selected correlations; aliphatic region expansion B of bogorol A hexaacetate (31). 1 8 1 oo oo Figure A . 1.26. 800 M H z H M B C Spectrum; aliphatic region expansion C of bogorol A hexaacetate (31). 182 LI OAc |_| Q H O V2 3 4 K2 NHAC L3 V3 3 4 NHAc y j O H if I H U f H ° SiH V -^NHAc L2 OAc A c - C O (170.2), l/Z-5'g A c - C O (170.2)/ Z-5 1-3,2, C O (170.8)/1-X-5, 3, 2, C O (167.8), A c - C O (170.1)/ X-l[ 0 ^ Figure A . 1.27. 800 M H z H M B C Spectrum with selected correlations; a-methine region expansion of bogorol A hexaacetate (31). B-CO (163.9), X-CO (167.8)/ B-NH I I I I 1 ^ 1 I I I | I I I I | I I I ' | " I I | I I I I | ' I I I | ' a oo o a «•> -» •* I I I I I I I I I I I j I I I I I I I 1 1 1 1 1 1 1 " 11 11111 1111 1 1 1 1 1 1 1 11 1 1 1 1 Cfl «• «o m in in r 1 A Figure A . 1.28. 800 M H z H M B C Spectrum with selected correlations; N H region expansion of bogorol A hexaacetate (31). 184 I Figure A . 1.29. 800 M H z N O E S Y Spectrum; NH/aliphatic region expansion of bogorol A hexaacetate (31). 185 00 I j _ i r i i i i i i — | ' i i "i—i | i i i i | i—i i i | i i—i i | i i i i — | i i i i | i r i i | — i i i i | r T i i | i r T i | i T i i | ~~ m ir» \o r— oo o\ o w-i r> Figure A . 1.30. 800 M H z N O E S Y Spectrum; NH/a-methine region expansion of bogorol A hexaacetate (31). A .2 . Appendix for Chapter 187 Table A . 2.1. ' H N M R chemical shifts for Mosher ester derivatives of basiliskamide A (50), 50a and 50b. recorded in D M S O - d 6 . . C# S - M T P A ester (50b) R - M T P A ester (50a) 6S-8R ' H (500 M H z ) ' H (500 M H z ) AS (ppm) (ppm) (ppm) 1 2 5.643 5.600 +0.04 3 6.356 6.201 +0.16 4 7.544 * * 5 5.754 5.547 +0.21 6 2.603 2.567 +0.04 6' 2.351 2.320 +0.03 7 5.133 5.167 -0.03 8 2.167 2.260 -0.09 9 4.938 4.980 -0.04 10 1.654 1.718 -0.06 11 1.275 1.291 -0.02 11' 1.154 1.167 -0.01 12 0.868 0.884 -0.02 13 * * * 14 * * * 15 - - -16 6.722 6.704 +.02 17 7.722 7.717 +0.01 18 - - -19/23 * * * 20/22 * * * 21 * * * MTPA-Pheny l * * M T P A - O M e 3.420 3.431 -0.01 N H 2 7.384, 7.325, +0.06, 6.907 6.840 +0.07 *Unable to accurately determine these chemical shifts. 188 189 190 1 9 1 192 13 14 20 21 22 17 12 13 14 11' 10 11 w 9 O H << 16 20 21 22-N H N H 17 19 23 H 2 N , 16 21 V ^ 1 9 23 17 O O H 12 13 14 N H N H 3 2 5 J U J ™ 9 8 w s 9 10 8 " " (ppm) 2.0 4.0 6.0 - i 1— 4.0 8.0 (ppm) 8.0 6.0 .  2.0 Figure A.2.5 . 500 M H z C O S Y Spectrum of basiliskamide B (51) in D M S O - d 6 . 193 13 14 12 13 14 10 2 16 19 23 20 22 4 21 3 5 17 20 21 22 19 17 23 18 15 HpN' 16 N H N H W U L 3 - \ 7 0 23 17 2 11 12 13 14 •5 I  7 J L * O H _A_ w s 9 10 11 11* 8 1 1 1 1 (ppm) 40 80 120 160 -I 1 1 r-(ppm) 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 Figure A.2.6. 500 M H z H M Q C Spectrum of basiliskamide B (51) in D M S O - d 6 . 194 Figure A.2.7. 500 M H z H M B C Spectrum of basiliskamide B (51) in D M S O - d 6 . A.3. Appendix for Chapter 196 197 II rl I || HO 1 9 = 3 ^ H 1 0 J k A ! 4 / -Y l l N 1 3 ^ 1 5 11 i_i 14 16 _ 18 19 21 20 17 HO Tyr COOH Tyr OH 14-3 3' 12 12'^J 11 Ser-OH < 68 59 Ser-NH Tyr-NH Tyr-OH Tyr-COOH <^  - i r — i 1 ' r~ TyrSer 9 NH NH ft d 0 9 S e r 2 11 p, | OH AJLJJLAJLJ 14 19 17 16 16 15 18 (ppm) m 0 0« 0 O 0 t 0 2.0 4.0 6.0 8.0 10.0 - l 1 6.0 1 1 1 1 1 4.0 2.0 12.0 (ppm) 12.0 10.0 8.0 Figure A.3.2. 500 M H z C O S Y Spectrum of tupuseleiamide B (56) in D M S O - d 6 . 198 HO 9 HO 7 Tyr COOH O H H < V O ii " ' II 14 16 18 19 N'l3 ^ ^ ^ ^ T ^ 20 H 21 u H I1 7 ^ Y " f 20 21 3 O 20 21 15 16 17-14, 19-11 12 68-59-10 13 Tyr OH Tyr Ser 9 NH NH 12 Ser 2 11 i2' OH 14 19 17 16 16 15 18 9 o — i 1 1 1 i 1 1 1 ; r-10.0 8.0 6.0 (ppm) 40 80 120 160 (ppm) 12.0 .  4.0 2.0 Figure A . 3 . 3 . 500 M H z H M Q C Spect rum o f tupusele iamide B (56) in D M S O - d 6 . 199 rso 68. 59-120 160 10 13 (ppm) 12.0 10.0 8.0 6.0 4.0 2.0 Figure A.3.4. 500 M H z H M B C Spectrum of tupuseleiamide B (56) in D M S O - d 6 . 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