Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Morgan, Kalindi Dasi
Doctor of Philosophy - PhD
University of British Columbia
The discovery of novel natural products continues to be critical for the development of new pharmaceuticals. Innovative methods to discover novel natural products can reveal previously overlooked chemical diversity. One such method is genome mining, where sequenced bacterial genomes are assessed for the presence of biosynthetic gene clusters. Another such method is bioassay-guided fractionation. Following either of these approaches, the bacteria must be grown and harvested, and novel natural products must be isolated and characterized. In the first part of this thesis, a nitrogen-NMR guided approach was developed to retrieve genetically predicted natural products from bacterial cultures. Piperazic acid (Piz)-containing natural products were targeted because this unique amino acid is often found in peptidic natural products with biological activity and impressive chemical structures. Piz contains a unique nitrogen-proton NMR correlation targeted through ¹H-¹⁵N HSQC. The unique N-H correlation also gave access to Piz’s diagnostic spin system through ¹H-¹⁵N HSQC-TOCSY NMR experiments. These two ¹⁵N NMR experiments were used to monitor for the presence of peptides containing Piz in culture extracts of genome-mined bacteria. Through the application of these ¹⁵N NMR experiments to guide isolation of Piz natural products, four novel compounds were discovered from Streptomyces incarnatus NRRL 8089. Three of these were isolated and structure’s elucidated as part of this thesis work: dentigerumycin F (4.2), dentigerumycin G (4.1) and incarnatapeptin A (4.3). 4.3 demonstrated a unique bicyclic moiety not previously seen in chemical structures, and a fourth compound, incarnatapeptin B (4.4), has in vitro cytotoxicity. In the second part of the thesis, bioassay-guided fractionation is used to screen a small library of marine bacteria in various assays. Using this method, known natural product molecules were uncovered, along with the discovery of two novel natural products from the marine bacterium Salinispora arenicola RJA3005. These two compounds, 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4), were isolated from extracts of wild-type bacteria for the first time. Feeding studies and analysis of ¹³C splitting patterns suggest that these compounds were biosynthesized from bacterium through phosphoenolpyruvate and erythrose precursors. Altogether, this thesis's work develops and harnesses various natural product discovery methods to uncover diverse natural products.
Chemical and Genetic Investigations of Marine and Terrestrial Bacteria towards Bioactive Natural Product Discovery by Kalindi Dasi Morgan B.Sc., The University of British Columbia, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2021 © Kalindi Dasi Morgan, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Chemical and Genetic Investigations of Marine and Terrestrial Bacteria towards Bioactive Natural Product Discovery submitted by Kalindi Dasi Morgan in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Raymond Andersen, Professor, Joint appointment with Chemistry & Earth, Ocean and Atmospheric Sciences, UBC Co-supervisor Katherine Ryan, Associate Professor, Chemistry, UBC Co-supervisor Martin Tanner, Professor, Chemistry, UBC Supervisory Committee Member Andrew MacFarlane, Associate Professor, Chemistry, UBC University Examiner Reinhard Jetter, Professor, Joint appointment with Botany & Chemistry, UBC University Examiner Additional Supervisory Committee Members: Suzana Straus, Professor, Chemistry, UBC Supervisory Committee Member Harry Brumer, Professor, Joint appointment with Chemistry & Michael Smith Laboratories, UBC Supervisory Committee Member iii Abstract The discovery of novel natural products continues to be critical for the development of new pharmaceuticals. Innovative methods to discover novel natural products can reveal previously overlooked chemical diversity. One such method is genome mining, where sequenced bacterial genomes are assessed for the presence of biosynthetic gene clusters. Another such method is bioassay-guided fractionation. Following either of these approaches, the bacteria must be grown and harvested, and novel natural products must be isolated and characterized. In the first part of this thesis, a nitrogen-NMR guided approach was developed to retrieve genetically predicted natural products from bacterial cultures. Piperazic acid (Piz)-containing natural products were targeted because this unique amino acid is often found in peptidic natural products with biological activity and impressive chemical structures. Piz contains a unique nitrogen-proton NMR correlation targeted through 1H-15N HSQC. The unique N-H correlation also gave access to Piz’s diagnostic spin system through 1H-15N HSQC-TOCSY NMR experiments. These two 15N NMR experiments were used to monitor for the presence of peptides containing Piz in culture extracts of genome-mined bacteria. Through the application of these 15N NMR experiments to guide isolation of Piz natural products, four novel compounds were discovered from Streptomyces incarnatus NRRL 8089. Three of these were isolated and structure’s elucidated as part of this thesis work: dentigerumycin F (4.2), dentigerumycin G (4.1) and incarnatapeptin A (4.3). 4.3 demonstrated a unique bicyclic moiety not previously seen in chemical structures, and a fourth compound, incarnatapeptin B (4.4), has in vitro cytotoxicity. In the second part of the thesis, bioassay-guided fractionation is used to screen a small library of marine bacteria in various assays. Using this method, known natural product molecules were uncovered, along with the discovery of two novel natural products from the marine iv bacterium Salinispora arenicola RJA3005. These two compounds, 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4), were isolated from extracts of wild-type bacteria for the first time. Feeding studies and analysis of 13C splitting patterns suggest that these compounds were biosynthesized from bacterium through phosphoenolpyruvate and erythrose precursors. Altogether, this thesis's work develops and harnesses various natural product discovery methods to uncover diverse natural products. v Lay Summary Some living organisms produce specialized organic compounds termed natural products. Natural products are an important resource for the development of new pharmaceuticals. Common medicines derived from natural products include penicillin for bacterial infections, statins for lowering blood cholesterol levels, and cyclosporin A to prevent rejection of organ transplants. As we face antibiotic resistance and increased rates of cancers, the search for new medicines from natural sources continues. In many cases, we now have a detailed understanding of how these organisms construct natural products. This knowledge, in turn, enables the discovery of undetected natural products. In this thesis, I isolated natural products from crude extracts of marine and terrestrial bacteria to search for new potential lead compounds for drug development. Towards that end, I also developed a new method for discovering undetected natural products that combines bacterial genomic information and spectroscopy experiments. vi Preface Chapter 2: A large portion of this material has been modified from Morgan, K. D.; Andersen, R. J.; Ryan, K. S. Piperazic Acid-Containing Natural Products: Structures and Biosynthesis. Nat. Prod. Rep. 2019, 36 (12), 1628–1653. I modified the figures from the published paper to be more compact. Dr. Katherine Ryan and I co-wrote the manuscript for the paper. Initial BLASTP searches were completed in partnership with Dr. Pamoda Ratoneweera. All other bioinformatic analysis was completed on my own. Chapter 3: A portion of this chapter has been modified from Morgan, K. D.; Williams, D. E.; Patrick, B. O.; Remigy, M.; Banuelos, C. A.; Sadar, M. D.; Ryan, K. S.; Andersen, R. J. Incarnatapeptins A and B, Nonribosomal Peptides Discovered Using Genome Mining and 1H/15N HSQC-TOCSY. Org. Lett. 2020, 22 (11), 4053–4057. I completed all the work involved in developing the method including growing the bacteria and running the NMR experiments. My contributions to the writing of the manuscript include completion of the figures and the supporting information reflected in Chapter 3 method development, as well as small aspects of the manuscript. I am responsible for the text presented in this thesis. Chapter 4: A portion of this chapter has been modified from Morgan, K. D.; Williams, D. E.; Patrick, B. O.; Remigy, M.; Banuelos, C. A.; Sadar, M. D.; Ryan, K. S.; Andersen, R. J. Incarnatapeptins A and B, Nonribosomal Peptides Discovered Using Genome Mining and 1H/15N HSQC-TOCSY. Org. Lett. 2020, 22 (11), 4053–4057. Michael Leblanc and I worked on the scale-up solid culturing of Streptomyces incarnatus NRRL 8089. I isolated and elucidated the constitution of dentigerumycin F (4.2) and dentigerumycin G (4.1). I isolated and elucidated the constitution of incarnatapeptin A (4.3) with incomplete evidence for the bicyclic moiety. Dr. vii David Williams methylated incarnatapeptin A and grew the crystal of the methyl ester of incarnatapeptin A. Dr Brian O. Patrick ran and analyzed the x-ray crystallography data. Dr. David Williams re-isolated 4.1, 4.2, and 4.3 to obtain better data for publication and isolated and elucidated the structure of incarnatapeptin B (4.4). The anticancer biological assay was conducted by Carmen A. Banuelos from the Sadar laboratory at the BC Cancer Agency. The disc diffusion bacterial assay was completed by Michael Leblanc, and the MIC assay was completed by Jessie Chen of UBC Bioservices. I co-wrote, with Dr. David Williams, the experimental section and the supporting information for the portions of the publication concerning the isolation and structure elucidation of 4.3 and 4.4. I co-authored the figures pertaining to the structures of 4.3 and 4.4. My supervisors contributed to the body of the manuscript. Although some of the material presented is based on a published article, I am the author of chapter 4. Chapter 5: My contributions for chapter 5 include the work of growing the bacteria, isolating metabolites, dereplicating the known compounds and running the TLC-bioautography assay. Chapter 6: My contributions include the isolation and structure elucidation of (6.1) and (6.2). I carried out some of the scale-up bacterial culturing, with assistance for some of the scale-up bacterial culturing from Michael Leblanc. I completed the bacterial culturing for feeding labelled precursor, followed by isolation and interpreted the resulting carbon-13 spectra. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures ............................................................................................................................. xvi List of Schemes ......................................................................................................................... xxvi List of Symbols ....................................................................................................................... xxvii List of Abbreviations ............................................................................................................. xxviii Acknowledgements .................................................................................................................. xxxi Dedication ................................................................................................................................ xxxii Chapter 1: General Introduction .................................................................................................1 1.1 Natural Products as Drug Leads ..................................................................................... 1 1.1.1 Natural Products in History ........................................................................................ 1 1.1.2 Microbial Natural Products ......................................................................................... 2 1.1.2.1 Marine Bacteria ................................................................................................... 5 1.1.3 Structure Elucidation of Novel Natural Products ....................................................... 6 1.1.4 Natural Product Discovery .......................................................................................... 9 1.1.4.1 The Biological Basis of Natural Product Discovery ......................................... 11 1.1.4.2 Bioassay-Guided Fractionation ......................................................................... 12 1.1.4.3 Mass Spectrometry as a Natural Product Discovery Methodology .................. 15 ix 1.1.4.4 NMR as a Natural Product Discovery Methodology ........................................ 15 1.2 Natural Product Biosynthesis ........................................................................................ 19 1.2.1 Natural Product Classes ............................................................................................ 20 1.2.1.1 Modular Natural Products: Polyketides and Nonribosomal Peptides ............... 21 1.2.1.1.1 Polyketides .................................................................................................. 22 1.2.1.1.2 Nonribosomal Peptides ............................................................................... 25 1.2.1.1.3 NRPS-PKS and PKS-NRPS Hybrids .......................................................... 26 1.2.1.1.4 Tailoring Reactions ..................................................................................... 27 1.2.1.2 Shikimic Acid Pathway..................................................................................... 27 1.2.1.3 Alkaloids ........................................................................................................... 28 1.2.2 Genetic Foundation of Bacterial Natural Product Biosynthesis ............................... 30 1.3 Research Goals.............................................................................................................. 31 Chapter 2: Mining Bacterial Genomes for Piz-containing Natural Products ........................33 2.1 Bioinformatic Processes................................................................................................ 33 2.2 Bioinformatics for Strain Selection Through Genome Mining .................................... 35 2.3 Piperazic Acid-Containing Natural Products ................................................................ 38 2.3.1 Linear Peptides.......................................................................................................... 39 2.3.2 Cyclic Peptides.......................................................................................................... 40 2.3.3 Depsipeptides ............................................................................................................ 41 2.3.3.1 13- and 18-membered Depsipeptides ................................................................ 41 2.3.3.2 Azinothricins: 19-Membered Cyclodepsipeptides ............................................ 42 2.3.3.2.1 19-Membered Cyclodepsipeptides With Long Acyl Tails .......................... 43 2.3.3.2.2 19-Membered Cyclodepsipeptides With Short Acyl Tails ......................... 44 x 2.3.3.2.3 19-Membered Cyclodepsipeptides With NRPS Derived Acyl Tails .......... 45 2.3.4 Sanglifehrin, Mollemycin, Dimeric Compounds and the Bisintercalators ............... 45 2.4 Biosynthesis of Piperazic Acid-Containing Molecules ................................................ 46 2.5 Bioinformatic Analysis Using the Piperazate Cyclizing Gene ktzT ............................. 57 2.6 Bioinformatic Prediction of Selected Strains ................................................................ 60 2.7 Conclusion .................................................................................................................... 63 2.8 Experimental Section .................................................................................................... 64 2.8.1 General Experimental Procedures ............................................................................. 64 Chapter 3: Nitrogen NMR-guided Isolation of Piz-containing NRPs .....................................65 3.1 Chapter Overview ......................................................................................................... 65 3.2 Nitrogen NMR .............................................................................................................. 67 3.3 Method Development: 15N NMR-Guided Fractionation of Piz-containing NRPs ....... 70 3.3.1 Piperazic NMR for Targeted Isolation of Piz Natural Products ............................... 70 3.3.2 Analysis of the Piperazic Acid NMR Signature in a Known Producer .................... 74 3.3.3 Strain and Media Selection Through Diagnostic NMR Resonances ........................ 77 3.3.4 Gene Cluster Analysis of Streptomyces incarnatus NRRL 8089 ............................. 90 3.3.5 15N NMR Guided Fractionation of Piperazic Acid-Containing Metabolites in Streptomyces incarnatus NRRL 8089 ................................................................................ 100 3.4 Conclusion .................................................................................................................. 110 3.5 Experimental Section .................................................................................................. 111 3.5.1 General Experimental Procedures ........................................................................... 111 3.5.2 Bacterial Cultivation ............................................................................................... 112 3.5.2.1 Streptomyces sp. RJA2928, A Known Producer ............................................ 112 xi 3.5.2.2 Growing Genome-Mined Strains .................................................................... 114 3.5.3 15N NMR Guided Fractionation .............................................................................. 118 Chapter 4: Piperazic acid-containing Metabolites from Streptomyces Incarnatus NRRL 8089..............................................................................................................................................122 4.1 Chapter Overview ....................................................................................................... 122 4.2 19-Membered Depsipeptides with Three Piperazic Acids .......................................... 122 4.3 Structure Elucidation of Piperazic Acid-Containing Metabolites ............................. 124 4.3.1 Structure Elucidation of Dentigerumycin G (4.1)................................................... 124 4.3.2 Structure Elucidation of Dentigerumycin F (4.2) ................................................... 145 4.3.3 Investigations into the Equilibria Observed in Dentigerumycins F And G ............ 165 4.3.4 Structure Elucidation of Incarnatapeptin A (4.3) .................................................... 173 4.3.5 Incarnatapeptin B .................................................................................................... 192 4.4 Biosynthetic Proposal for the Incarnatapeptins and Dentigerumycins F and G ......... 193 4.5 Bioactivity ................................................................................................................... 198 4.5.1 Antimicrobial Assays .............................................................................................. 198 4.5.2 Bioactivity of Incarnatapeptins in the Prostate Cancer Assay ................................ 199 4.6 Summary and Conclusion ........................................................................................... 204 4.7 Experimental Section .................................................................................................. 206 4.7.1 General Experimental Procedures ........................................................................... 206 4.7.2 Bioassay Experimental Methods............................................................................. 207 4.7.2.1 MIC Experimental Methods ........................................................................... 207 4.7.2.2 Prostate Cancer Assay Experimental Methods ............................................... 208 Chapter 5: Bacterial Library Construction for Bioassay-guided Discovery ........................209 xii 5.1 Chapter Overview ....................................................................................................... 209 5.2 Strain Selection of Marine Bacteria ............................................................................ 209 5.3 Bioassays for Guiding Fractionation .......................................................................... 210 5.3.1 Phosphatase Assay .................................................................................................. 212 5.3.2 Anti-Inflammatory Assays ...................................................................................... 212 5.3.3 Anti-microbial Assays ............................................................................................ 212 5.4 Results from Bioassay-guided fractionation ............................................................... 213 5.4.1 Phosphatase Assay Results ..................................................................................... 213 5.4.2 Anti-Inflammatory Assay Results........................................................................... 215 5.4.3 Anti-microbial Assay Results: Fusant Strains ........................................................ 218 5.5 Summary and Conclusion ........................................................................................... 220 Chapter 6: Salinispora arenicola RJA3005: Chemistry and Feeding Studies ......................221 6.1 Chapter Overview ....................................................................................................... 221 6.2 Salinispora: A Marine Obligate Genus and it’s Natural Products .............................. 222 6.2.1 Biosynthesis of Rifamycin and Saliniketal ............................................................. 223 6.3 Isolation and Structure Elucidation of Metabolites from Salinispora arenicola RJA3005 ................................................................................................................................. 227 6.3.1 Structure Elucidation of 3,5-dihydroxyphenylpyrone (6.1) .................................... 228 6.3.1.1 Derivatization of 3,5-dihydroxyphenylpyrone (6.1) ....................................... 239 6.3.1.2 Assignment of Configuration .......................................................................... 248 6.3.2 Structure elucidation of 3-acetamide-5-hydroxyphenylpyrone (6.4)...................... 256 6.4 Biosynthesis of Salinispora arenicola RJA3005’s 6.1 ............................................... 264 6.4.1 Labelled Precursor Feeding Studies........................................................................ 266 xiii 6.4.1.1 1,2-13C2-acetate Feeding Experiments ............................................................ 266 6.4.1.2 U-13C6-Glucose Feeding Experiments ............................................................ 270 6.4.2 Bioinformatic Analysis of the Salinspora arenicola RJA3005’s Draft Genome ... 278 6.5 Bioactivity ................................................................................................................... 279 6.6 Summary and Conclusion ........................................................................................... 279 6.7 Experimental Section .................................................................................................. 280 6.7.1 General Experimental Procedures ........................................................................... 280 6.7.2 Isolation of metabolites ........................................................................................... 281 6.7.3 Methylation ............................................................................................................. 283 6.7.4 Mosher’s Ester ........................................................................................................ 283 6.7.5 Feeding studies........................................................................................................ 284 Chapter 7: Conclusion ...............................................................................................................286 References ...................................................................................................................................291 Appendices ..................................................................................................................................314 Appendix A NMR of Unsolved Fractions of Streptomyces incarnatus NRRL 8089 ............ 314 Appendix B NMR Spectra of Dentigerumycin F and G Isolated with Formic Acid taken by Dave Williams, Unpublished .................................................................................................. 318 Appendix C NMR spectra of Incarnatapeptin A as taken by Dave Williams, Published ....... 325 Appendix D Experimental and NMR Spectra of the Methyl Ester* of Incarnatapeptin A taken by Dave Williams, Published. ................................................................................................. 333 Appendix E Crystal Data and Experimental Data provided by Dr Brian O. Patrick .............. 340 Appendix F Experimental Data for MIC of Incarnatapeptins ................................................ 355 Appendix G Other N-N Bond Natural Product Functional Groups ........................................ 357 xiv List of Tables Table 1-1. Overview of the connection between shared precursors ............................................. 20 Table 1-2. Example of Streptomyces codon bias for alanine because of its high G+C content. .. 30 Table 2-1. Selected natural products discovered using genome mining techniques. .................... 38 Table 2-2. List of characterized Piz-containing gene clusters. ..................................................... 48 Table 2-3. Names of selected strains containing KtzT homologs. ............................................... 60 Table 2-4. Selected strains with the encoding organization ......................................................... 62 Table 3-1. NMR properties for relevant nuclei. ............................................................................ 67 Table 3-2. Piperazic acid has unique NMR signatures ................................................................. 71 Table 3-3. Experimental NMR chemical shifts in DMSO-d6 for Piz ........................................... 73 Table 3-4. Analysis of Piz production in a known producer. ....................................................... 76 Table 3-5. Initial genome mined strains with selected media. ...................................................... 78 Table 3-6. Initial genome mined strains with initial nitrogen NMR ............................................. 79 Table 3-7. Additional genome-mined bacterial strains ................................................................. 84 Table 3-8. Remainder of the genome-mined strains ..................................................................... 84 Table 3-9. BLASTP comparison of the NRP and PK domains .................................................... 96 Table 3-10. Streptomyces incarnatus NRRL 8089: list of bioinformatically predicted genes ..... 99 Table 4-1. NMR shifts of dentigerumycin G’s ........................................................................... 144 Table 4-2. NMR shifts of dentigerumycin F in both pyridine-d5 and DMSO-d6. ...................... 164 Table 4-3. NMR assignments for incarnatapeptin A (4.3) in DMSO-d6 .................................... 192 Table 4-4. Antibacterial MIC results for dentigerumycin F ....................................................... 199 Table 5-1. List of strains selected for mini library...................................................................... 211 xv Table 6-1. Gene cluster of required genes for production of AHBA.......................................... 224 Table 6-2. NMR assignments for 6.1, 6.2 and 6.3. Recorded in DMSO-d6 at 600 Mhz. ........... 248 Table 6-3. Results from Mosher’s ester NMR Analysis. ............................................................ 254 Table 6-4. NMR assignments for 6.4. Recorded in DMSO-d6 at 600 MHz. .............................. 263 xvi List of Figures Figure 1-1. Historical natural products. .......................................................................................... 1 Figure 1-2. Select natural products that are in current use as pharmaceutical compounds. ........... 3 Figure 1-3. Daptomycin, an antibiotic isolated from a terrestrial Actinobacteria. ......................... 5 Figure 1-4. Potent bioactive compounds isolated from marine bacteria. ....................................... 6 Figure 1-5. The standard suite of NMR experiments ..................................................................... 9 Figure 1-6. Natural product discovery .......................................................................................... 10 Figure 1-7. General discovery workflow. ..................................................................................... 11 Figure 1-8. The bioassay-guided fractionation process, ............................................................... 14 Figure 1-9. Natural products isolated using bioassay-guided fractionation. ................................ 14 Figure 1-10. Structures solved but not isolated with DANS. ....................................................... 16 Figure 1-11. Natural Products isolated using NMR in various aspects of the discovery process. 17 Figure 1-12. Natural Products isolated using variations of NMR-Genome mining approaches. . 19 Figure 1-13. Representative natural products of the select natural product classes. .................... 21 Figure 1-14. Polyketides are built from basic two-carbon building blocks .................................. 22 Figure 1-15. PKS module and domain organization .................................................................... 23 Figure 1-16. Various types of polyketide natural products ........................................................... 24 Figure 1-17. NRPS module organization and domain functions. ................................................. 26 Figure 1-18. Rapamycin, a mixed natural product utilizing a shikimate derived starting unit..... 29 Figure 1-19. Shikimate pathway and analog natural products. ..................................................... 29 Figure 1-20. The diversity of alkaloid structures. ......................................................................... 30 xvii Figure 2-1. Linear Piz-containing peptides. .................................................................................. 40 Figure 2-2. Cyclic Piz-containing peptides. .................................................................................. 41 Figure 2-3. Structures of 18-membered cyclodepsipeptides and their linear congeners. ............. 42 Figure 2-4. 19-membered cyclodepsipeptides with long acyl tails............................................... 43 Figure 2-5. 19-membered depsipeptides with short acyl tails. ..................................................... 44 Figure 2-6. 19-membered depsipeptides with formamide tails. ................................................... 45 Figure 2-7. Other Piz-containing natural products. ...................................................................... 46 Figure 2-8. Kutzneride gene cluster map with key genes and modules named. ........................... 48 Figure 2-9. Piz-containing natural products produced by nonribosomal peptides synthases……50 Figure 2-10. Piz-containing natural products produced by hybrid gene clusters. ........................ 51 Figure 2-11. Piz-containing natural products produced ................................................................ 53 Figure 2-12. The marformycin gene cluster. ................................................................................ 54 Figure 2-13. Characterized transformations leading to marformycin biosynthesis. ..................... 54 Figure 2-14. Characterized and hypothesized transformations ..................................................... 55 Figure 2-15. Biosynthetic pathway to piperazic acid.................................................................... 57 Figure 3-1. 1H-15N HSQC-TOCSY is an NMR experiment ......................................................... 70 Figure 3-2. Padanamide A’s structure and Piz NMR chemical shifts. ......................................... 74 Figure 3-3. Prepared Petri plates of known Piz producer Streptomyces sp. RJA2928. ................ 75 Figure 3-4. 1H-15N HSQC NMR for Samples L and UL .............................................................. 76 Figure 3-5. NMR spectra of crude metabolite extract of Streptomyces sp. RJA2928 .................. 77 Figure 3-6. 1H-15N HSQC of Streptomyces phaeochromogenes NRRL B-1248.......................... 80 Figure 3-7. 1H-15N HSQC of Streptomyces griseus subsp. griseus NRRL WC-3645 .................. 80 Figure 3-8. 1H-15N HSQC of Streptomyces griseus subsp. griseus NRRL WC-3645 .................. 81 xviii Figure 3-9. 1H-15N HSQC of Streptomyces griseus NRRL F-5144.............................................. 81 Figure 3-10. 1H-15N HSQC of Streptomyces griseus NRRL F-5144............................................ 82 Figure 3-11. 1H-15N HSQC of Streptomyces sp. NRRL F-2664 ................................................... 82 Figure 3-12. 1H-15N HSQC of Streptomyces katrae NRRL F-16271 ........................................... 85 Figure 3-13. 1H-15N HSQC of Streptomyces katrae NRRL F-16271 extract from TSB agar, ..... 85 Figure 3-14. 1H-15N HSQC of Streptomyces sp. NRRL F-2747 extract from TSB agar, ............. 86 Figure 3-15. 1H-15N HSQC of Streptomyces sp. NRRL F-6131 ................................................... 86 Figure 3-16. 1H-15N HSQC of Streptomyces sp. NRRL F-6131 ................................................... 87 Figure 3-17. 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 .......................................... 87 Figure 3-18. 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 .......................................... 88 Figure 3-19. 1H-15N HSQC of Streptomyces sp. NRRL B-1347 extract ...................................... 88 Figure 3-20. 1H-15N HSQC of Streptomyces sp. NRRL B-1347 .................................................. 89 Figure 3-21. 1H-15N HSQC of Streptomyces sp. S920 extract from Mannitol Soy agar .............. 89 Figure 3-22. Comparison of Streptomyces incarnatus NRRL 8089 ............................................. 94 Figure 3-23. Workflow for Piz 15N NMR-guided fractionation. ................................................ 101 Figure 3-24. Full proton spectrum of labelled crude S. incarnatus NRRL 8089........................ 101 Figure 3-25. Full 1H spectrum of labelled/unlabelled mixture ................................................... 102 Figure 3-26. Scale up: Full 1H-15N HSQC spectrum of labelled crude ...................................... 103 Figure 3-27. Scale-up: 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 crude extract .. 103 Figure 3-28. Scale-up: 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 ....................... 104 Figure 3-29. Scale-up: 1H-15N HSQC-TOCSY spectrum of labelled crude ............................... 104 Figure 3-30. 1H-15N HSQC-TOCSY of Streptomyces incarnatus NRRL 8089 ......................... 105 Figure 4-1. Natural products that contain three piperazic acids. ................................................ 124 xix Figure 4-2. 1H spectrum for dentigerumycin G (4.1).................................................................. 125 Figure 4-3. 13C spectrum for dentigerumycin G (4.1) ................................................................ 126 Figure 4-4. 1H-15N HSQC NMR spectrum of dentigerumycin G (4.1) ...................................... 127 Figure 4-5. Piz NMR spin systems for dentigerumycin G (4.1). ................................................ 128 Figure 4-6. 1H-15N HSQC-TOCSY NMR spectrum of dentigerumycin G (4.1) ........................ 128 Figure 4-7. Expansion of 1H-1H TOCSY NMR spectrum of dentigerumycin G (4.1) ............... 129 Figure 4-8. Full TOCSY NMR spectrum of dentigerumycin G (4.1) ........................................ 129 Figure 4-9. COSY60 NMR spectrum of dentigerumycin G (4.1) .............................................. 130 Figure 4-10. 1H-13C HSQC NMR spectrum of dentigerumycin G (4.1) .................................... 130 Figure 4-11. 1H-13C HMBC NMR spectrum of dentigerumycin G (4.1) ................................... 131 Figure 4-12. Connecting the core amino acids for dentigerumycin G. ....................................... 132 Figure 4-13. TOCSY and COSY60 spectra demonstrating the correlations .............................. 132 Figure 4-14. 1H-15N LRHMQC spectrum demonstrating the correlations ................................. 133 Figure 4-15. tROESY correlations .............................................................................................. 133 Figure 4-16. 1H-13C HMBC spectrum demonstrating proton-carbonyl correlations .................. 134 Figure 4-17. 1H-1H TOCSY correlations .................................................................................... 134 Figure 4-18. Building up fragments of dentigerumycin G (4.1) ................................................. 136 Figure 4-19. Correlations confirming the hydroxy-leucine amino acid ..................................... 138 Figure 4-20. 1H-13C HMBC detailing correlations for the PKS linkage portion of 4.1. ............. 140 Figure 4-21. Initial 1H-1H TOCSY spectrum showing multiple signals ..................................... 140 Figure 4-22. 1H-1H TOCSY demonstrating 4.1’s disubstituted oxane ring, ............................... 141 Figure 4-23. 1H-1H TOCSY demonstrating 4.1’s disubstituted oxane ring, ............................... 141 Figure 4-24. 2D J-resolved spectrum.......................................................................................... 142 xx Figure 4-25. 1H spectrum for dentigerumycin F ......................................................................... 146 Figure 4-26. 13C spectrum for dentigerumycin F ........................................................................ 146 Figure 4-27. Expansion of the 1H NMR spectrum for 4.2 .......................................................... 148 Figure 4-28. Chemical shift assignments for the three Piz amino acids of dentigerumycin F. .. 148 Figure 4-29. 1H-15N HSQC NMR spectrum of dentigerumycin F,............................................. 149 Figure 4-30. Expansion of 1H-1H TOCSY NMR spectrum of dentigerumycin F ...................... 149 Figure 4-31. 1H-1H TOCSY NMR spectrum of dentigerumycin F ............................................ 150 Figure 4-32. 1H-13C HSQC NMR spectrum of dentigerumycin F .............................................. 150 Figure 4-33. Connecting the core amino acids for dentigerumycin F. ....................................... 151 Figure 4-34. Expansion of 1H-1H COSY60 NMR spectrum of dentigerumycin F ..................... 152 Figure 4-35. Expansion of 1H-13C HMBC NMR spectrum of dentigerumycin F ...................... 153 Figure 4-36. 1H-15N LRHMQC demonstrating correlations ....................................................... 153 Figure 4-37. Expansion of tROESY NMR spectrum for dentigerumycin F .............................. 154 Figure 4-38. Expansion of the tROESY NMR spectrum for dentigerumycin F ......................... 154 Figure 4-39. Building up fragments of dentigerumycin F .......................................................... 155 Figure 4-40. Expansion of the 1H-13C HMBC spectrum for 4.2 ................................................. 157 Figure 4-41. Correlations confirming the β-OH-Leucine amino acid ........................................ 158 Figure 4-42. 1H-13C HMBC NMR spectrum of dentigerumycin F ............................................. 159 Figure 4-43. Full 1H-13C HMBC NMR spectrum of dentigerumycin F ..................................... 160 Figure 4-44. Expansion of the 1H-1H COSY60 NMR spectrum of dentigerumycin F ............... 160 Figure 4-45. 1H-1H TOCSY NMR spectrum of dentigerumycin F ............................................ 161 Figure 4-46. Expansion of the 1H-13C HSQC NMR spectrum of dentigerumycin F .................. 161 Figure 4-47. Expansion of 1H-13C HMBC NMR spectrum of dentigerumycin F ...................... 162 xxi Figure 4-48. 2D J-resolved spectrum for dentigerumycin F....................................................... 162 Figure 4-49. Comparison of proton spectra of 4.1 ...................................................................... 165 Figure 4-50. Oxane systems of dentigerumycin G (4.1) ............................................................. 166 Figure 4-51. 1H-13C HMBC of room temperature dentigerumycin G (4.1) ................................ 167 Figure 4-52. Full 1H-13C HMBC of room temperature dentigerumycin F (4.2) ......................... 168 Figure 4-53. Expansion of 1H-13C HSQC of dentigerumycin G (4.1) in CD3OD ...................... 169 Figure 4-54. Expansion of 1H-13C HSQC of dentigerumycin G (4.1) in CD3OD ...................... 170 Figure 4-55. Expansion of overlapping 1H-13C HSQC spectra of 4.1 in CD3OD. ..................... 170 Figure 4-56. Overlapping 1H-13C HMBC NMR spectra of dentigerumycin G (4.1) in CD3OD. 171 Figure 4-57. Scheme of equilibrium seen in both (4.1) and (4.2) ............................................... 171 Figure 4-58. The tautomerization in 4.1 between a hemiketal and ketone form. ....................... 173 Figure 4-59. 1H spectrum for author isolated incarnatapeptin A (4.3) ....................................... 174 Figure 4-60. 13C spectrum collected by author for incarnatapeptin A ........................................ 175 Figure 4-61. 1H-15N HSQC spectrum for author isolated incarnatapeptin A in DMSO-d6, ....... 176 Figure 4-62. 1H-15N HSQC spectrum collected by author for incarnatapeptin A ...................... 176 Figure 4-63. Chemical shift assignment for the three Piz amino acids of incarnatapeptin A..... 177 Figure 4-64. 1H-15N HSQC-TOCSY spectrum collected by author for incarnatapeptin A ........ 178 Figure 4-65. Expansion of 1H-1H TOCSY spectrum for author isolated incarnatapeptin A ...... 179 Figure 4-66. 1H-13C HSQC spectrum for author isolated incarnatapeptin A .............................. 179 Figure 4-67. Connecting the core amino acids for incarnatapeptin A. ....................................... 180 Figure 4-68. tROESY correlations between the protons ............................................................ 181 Figure 4-69. 1H-1H COSY spectrum for author isolated incarnatapeptin A ............................... 182 Figure 4-70. 1H-13C HMBC spectrum for author isolated incarnatapeptin A............................. 182 xxii Figure 4-71. 1H-15N LRHMQC spectrum for author isolated incarnatapeptin A ....................... 183 Figure 4-72. 1H-1H COSY spectrum for author isolated incarnatapeptin A ............................... 184 Figure 4-73. 1H-1H TOCSY spectrum for author isolated incarnatapeptin A ............................ 184 Figure 4-74. Full 1H-13C HMBC spectrum for author isolated incarnatapeptin A ..................... 185 Figure 4-75. Building up fragments of incarnatapeptin A .......................................................... 185 Figure 4-76. Expansion of 1H-1H COSY60 spectrum for incarnatapeptin A ............................. 186 Figure 4-77. Left-hand panel: COSY and HMBC correlations .................................................. 187 Figure 4-78. Initial NMR data could not distinguish .................................................................. 188 Figure 4-79. X-ray crystal figure produced for the methyl ester of incarnatapeptin A. ............. 190 Figure 4-80. Biosynthetic proposal for the incarnatapeptins and dentigerumycins F and G. ..... 194 Figure 4-81. Visualizing putative biosynthesis pathways........................................................... 195 Figure 4-82. Proposed biosynthetic pathway for the PKS extender units .................................. 197 Figure 4-83. LNCaP and PC3 cells were exposed for 24 hours ................................................. 201 Figure 4-84. Incarnatapeptin A (4.3) induced cell death ............................................................ 202 Figure 4-85. Viability of LNCaP cells treated with incarnatapeptins A (4.3) or B (4.4) ........... 203 Figure 4-86. Combining genome mining and Piz 15N NMR-guided fractionation..................... 204 Figure 4-87. Structures of incarnatapeptin A (4.3), and dentigerumycins F (4.2) and G (4.1). . 204 Figure 5-1. Compounds isolated from Streptomyces sp. RJA4178. ........................................... 215 Figure 5-2. Compounds isolated from Streptomyces sanglieri RJA4249................................... 217 Figure 5-3. Fusant strains. ........................................................................................................... 218 Figure 5-4. Antimycins responsible for the antifungal activity of the fusant RJA4374. ............ 220 Figure 6-1. AHBA starter unit leading into the polyketide modules. ......................................... 225 Figure 6-2. Key steps during the biosynthesis of AHBA from glucose. .................................... 226 xxiii Figure 6-3. The final step in the shikimate pathway ................................................................... 226 Figure 6-4. 1H spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. ........................................ 229 Figure 6-5. 1H spectrum of 6.1 demonstrating integration, ........................................................ 230 Figure 6-6. 13C spectrum of 6.1, recorded in DMSO-d6, at 150 MHz. ....................................... 230 Figure 6-7. Downfield expansion of 13C spectrum of 6.1 ........................................................... 231 Figure 6-8. Expansion of 13C spectrum of 6.1, recorded in DMSO-d6, at 150 MHz. ................. 231 Figure 6-10. Numbering and structure of 6.1. ............................................................................ 232 Figure 6-11. Selected HMBC correlations .................................................................................. 233 Figure 6-12. 1H-13C HSQC spectrum of 6.1, showing 1J 1H-13C coupling ................................. 234 Figure 6-13. 1H-1H COSY60 spectrum of 6.1, recorded in ........................................................ 234 Figure 6-14. Full 1H-13C HMBC spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. ........... 235 Figure 6-15. Expansion of 1H-13C HMBC resonances for 6.1 .................................................... 235 Figure 6-16. Expansion of 1H-13C HMBC spectrum of 6.1, ....................................................... 236 Figure 6-17. Expansion of 1H-13C HMBC spectrum of 6.1 ........................................................ 236 Figure 6-18. Key 1H-13C HMBC and 1H-1H tROESY correlations of 6.1. ................................ 237 Figure 6-19. Full 1H-1H tROESY spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. .......... 237 Figure 6-20. Expansion of the 1H-1H tROESY spectrum ........................................................... 238 Figure 6-21. Structures of the natural product and methylation derivative ................................ 239 Figure 6-22. 1H spectrum for methylation of the 2-pyrone isomer (6.2) .................................... 240 Figure 6-23. 13C spectrum for methylation of the 2-pyrone isomer (6.2) ................................... 241 Figure 6-24. Chemical shifts for the natural product .................................................................. 241 Figure 6-25. 1H-1HCOSY60 spectrum for methylation of the 2-pyrone isomer (6.2) ................ 242 Figure 6-26. 1H-13C HSQC spectrum for methylation of the 2-pyrone isomer (6.2).................. 242 xxiv Figure 6-27. 1H-13C HMBC spectrum for methylation of the 2-pyrone isomer (6.2) ................ 243 Figure 6-28. 1H-1H tROESY spectrum for methylation of the 2-pyrone isomer (6.2) ............... 243 Figure 6-29. Proton spectrum for methylation of the 4-pyrone form (6.3)................................. 244 Figure 6-30. 13C spectrum for methylation of the 4-pyrone form (6.3) ...................................... 245 Figure 6-31. 1H-1H COSY60 spectrum for methylation of the 4-pyrone form (6.3) .................. 245 Figure 6-32. 1H-13C HSQC spectrum for methylation of the 4-pyrone form (6.3) ..................... 246 Figure 6-33. 1H-13C HMBC spectrum for methylation of the 4-pyrone form (6.3).................... 246 Figure 6-34. 1H-1H tROESY spectrum for methylation of the 4-pyrone form (6.3) .................. 247 Figure 6-35. Overlay of the 1H-13C HMBC’s of the 6.2 and 6.3. ............................................... 247 Figure 6-36. Mosher’s ester reaction with 6.3, a derivative of 6.1. ............................................ 251 Figure 6-37. 1H-13C HSQC spectrum for S-Mosher’s Ester ....................................................... 252 Figure 6-38. 1H-13C Expansion of HSQC spectrum for S-Mosher’s Ester ................................. 252 Figure 6-39. 1H-1H Expansion of COSY60 spectrum for S-Mosher’s Ester .............................. 253 Figure 6-40. 1H-13C HSQC spectrum for R-Mosher’s ester ....................................................... 254 Figure 6-41. Expansion of 1H-13C HSQC spectrum for R-Mosher’s ester ................................. 255 Figure 6-42. Expansion of 1H-1H COSY60 spectrum for R-Mosher’s ester .............................. 255 Figure 6-43. Newman projections of the Mosher’s esters .......................................................... 256 Figure 6-44. Numbering and structure for 3-acetamide-5-hydroxyphenylpyrone (6.4). ............ 257 Figure 6-45. 1H spectrum of 6.4, recorded in DMSO-d6, at 600 MHz. ...................................... 258 Figure 6-46. 13C spectrum of 6.4, recorded in DMSO-d6, at 150 MHz. ..................................... 258 Figure 6-47. 1H-1H COSY60 spectrum of 6.4 ............................................................................ 259 Figure 6-48. 1H-13C HSQC spectrum of 6.4 ............................................................................... 259 Figure 6-49. Full 1H-13C HMBC spectrum of 6.4, ...................................................................... 260 xxv Figure 6-50. Key 1H-13C HMBC correlations for the ring systems of 6.4.................................. 260 Figure 6-51. Expansion of the 1H-13C HMBC spectrum of 6.4 .................................................. 261 Figure 6-52. Expansion of the 1H-13C HMBC spectrum of 6.4 .................................................. 261 Figure 6-53. 1H-15N HSQC spectrum of 6.4 demonstrating the N-H correlation ....................... 262 Figure 6-54. 1H-1H tROESY spectrum of 6.4, recorded in DMSO-d6, at 600 MHz. ................. 262 Figure 6-55. Likely pathway to the biosynthesis of 6.1. ............................................................. 264 Figure 6-56. Vancomycin and kendomycin A ............................................................................ 264 Figure 6-57. Asukamycin and manumycin incorporation .......................................................... 265 Figure 6-58. Biosynthetic strategy leading to resorcinol’s 3,5-dihydroxyphenyl moiety. ......... 265 Figure 6-59. Three possible pathways to 6.1 .............................................................................. 266 Figure 6-60. Doubly labelled acetate leads to varying 13C incorporation patterns ..................... 267 Figure 6-61. 1,2-13C2-acetate feeding ......................................................................................... 268 Figure 6-62. Acetate labelling incorporated into C-8 (δC 45). .................................................... 269 Figure 6-63. Carbon-13 spectrum of 6.1 with added 1,2-13C2-acetate........................................ 270 Figure 6-64. Glucose is a central metabolite .............................................................................. 271 Figure 6-65. When examining the different pathways possible ................................................. 272 Figure 6-66. U-13C6-Glucose feeding experiments demonstrated complex coupling patterns ... 273 Figure 6-67. The resonance at δC 101.5 (C-4) in the U-13C6-glucose feeding experiment ......... 274 Figure 6-68. 13C spectrum from the U-13C6-Glucose feeding experiments ................................ 276 Figure 6-69. 13C spectrum from the U-13C6-Glucose feeding experiments ................................ 277 Figure 6-70. 13C spectrum from U-13C6-Glucose feeding experiments ...................................... 277 Figure 6-71. Partial gene cluster comparison from the contig 17 ............................................... 278 xxvi List of Schemes Scheme 3-1. Gene cluster from Streptomyces incarnatus NRRL 8089 ........................................ 91 Scheme 3-2. 15N NMR-guided isolation scheme of Streptomyces incarnatus NRRL 8089. ..... 109 Scheme 5-1. Isolation scheme of Streptomyces sp. RJA4178 .................................................... 214 Scheme 5-2. Isolation scheme of the Streptomyces sanglieri RJA4249 ..................................... 216 Scheme 5-3. Isolation scheme of the fusant RJA4374 ............................................................... 219 Scheme 6-1 Isolation scheme for Salinispora arenicola RJA3005 culture extract. .................... 228 Scheme 6-2. Methylation of the tertiary alcohol at C-11............................................................ 240 xxvii List of Symbols °C degree Celsius [𝛼]𝐷25 specific rotation at sodium D-line recorded at 25 °C 13C carbon-13 15N nitrogen-15 1H hydrogen/proton C18 linear octadecane groups δ chemical shift in parts per million from tetramethylsilane J coupling constant (in hertz) m/z mass-to-charge ratio xxviii List of Abbreviations 1D one dimensional 2D two dimensional Ac acetyl BC British Columbia BGC biosynthetic gene cluster br broad CCR crotonyl-CoA carboxylase/reductase CD3OD deuterated methanol COSY correlation spectroscopy d doublet # d. days DCM dichloromethane dd doublet of doublets dm doublet of multiplets DMSO dimethyl sulfoxide DMSO-d6 deuterated dimethyl sulfoxide D2O deuterated water dt doublet of triplets EtOAc ethyl acetate g gram HMBC heteronuclear multiple bond coherence xxix HPLC high performance liquid chromatography HRESIMS high resolution electrospray ionization mass spectrometry HSQC heteronuclear single bond coherence Hz hertz IC50 half maximal inhibitory concentration LC-MS liquid chromatography-mass spectrometry hybrid LC-MS/MS liquid chromatography-tandem-mass spectrometry hybrid LRESIMS low resolution electrospray ionization mass spectrometry μg microgram μM micromolar m multiplet Me methyl MeCN/ACN acetonitrile MeOH methanol mg milligram MHz megahertz min minute mL millilitre mRNA messenger ribonucleic acid MRSA methicillin-resistant Staphylococcus aureus MS mass spectrometry NMR nuclear magnetic resonance xxx NOESY nuclear Overhauser effect spectroscopy NRP nonribosomal peptide NRPS nonribosomal peptide syntethase ORF open reading frame ORTEP oak ridge thermal ellipsoid plot Piz piperazic acid PK polyketide PKS polyketide synthase ppm parts per million ppt precipitate pyridine-d5 deuterated pyridine tROESY transverse rotating frame nuclear Overhauser effect spectroscopy s singlet smBGC secondary metabolite biosynthetic gene cluster sp. species t triplet TLC thin layer chromatography TOF time of flight xxxi Acknowledgements I offer my enduring gratitude to the faculty, staff and my fellow students at UBC and in the Andersen and Ryan labs, who have inspired me to continue my work in this field. I owe much to the NMR staff of the UBC Chemistry department; Maria, in particular, with both her NMR expertise and discussions of what it means to be a mother and woman in science. I owe particular thanks to UBC for providing me with a 4Year Fellowship, without this funding it would have been very challenging to navigate the all-encompassing worlds of graduate school and motherhood. I give many thanks to my co-supervisors Dr. Raymond Andersen and Dr. Katherine Ryan for enlarging my vision of science, their patience in general, their willingness to navigate the hurdles of having a joint student, and all the myriads of support in terms of both questions and direction. Special thanks are owed to my husband, Ray Morgan, for supporting and believing in me through all these years. Special thanks are also owed to my mother, whose tender care to my children has been invaluable, and my siblings who have supported me in so many countless ways throughout the years. xxxii Dedication I dedicate this thesis to my sons: Leif and Forest May the love of science and inquiry be a foundation of your lives 1 Chapter 1: General Introduction 1.1 Natural Products as Drug Leads 1.1.1 Natural Products in History Natural products have been used for millennia in human cultures as medicines,1–3 fabric dyes,4 building materials (resins and waxes),5 and fragrances.5,6 Although plant crude extracts have long been used as medicines,2 the active components of those medicines (if present) began to be deciphered less than two hundred years ago. Beginning in 1828, chemists began to isolate pure natural products such as salicin (1.1), quinone, and morphine (1.4).2 Next, they used simple chemistry to transform these isolated natural products into analogues with greater Figure 1-1. Historical natural products. 2 bioavailability and activity. Salicin, isolated from Salix sp., was converted to acetylsalicylic acid (1.3)7 following the understanding that it was transformed into salicylic acid in the blood8 (Figure 1.1a). Morphine, initially isolated as a pain-killing compound along with many other inactive poppy alkaloids, was transformed into codeine (1.5) (Figure 1.1b).2 From these beginnings, natural products have been harnessed to develop world-transforming pharmaceuticals that allow for: successful organ transplants to occur (cyclosporin A9–11), bacterial and fungal infections to be defeated (actinomycin and neomycin12,13), treatment of tuberculosis (streptomycin12), and varying cancers to be battled (taxol (1.9), cytarabine (1.10), trabectedin (1.11), eribulin).14 Since the mid-1980s, natural products and their derivatives have accounted for 58% percent of antibiotics and 32% of anticancer compounds. Currently, 87% of all known human diseases have a treatment that is a natural product or derived from natural products.15 These numbers speak to the continued research potential of natural products as a source of pharmaceutical medicines. 1.1.2 Microbial Natural Products Microbes became an important source for bioactive natural products with Alexander Fleming’s discovery of penicillin in 1929 from Penicillium notatum. By the 1940s, research efforts transformed penicillin into a potent antibiotic drug that changed the face of World War II.16 From this outset, 20,000 secondary metabolites have been discovered from bacteria, and fungi11 with multiple important antibiotics (1.8) discovered from microbes. In addition to antibiotics, microbes have provided antidiabetic medicines, immunosuppressants (1.6), cholesterol-lowering (lovastatin) and antitumor medicines (1.7).16 3 Figure 1-2. Select natural products that are in current use as pharmaceutical compounds. Microbes is an umbrella term that encompasses both fungal and bacterial kingdoms. Given the scope of this thesis, bacterial natural products will be the focus. Bacteria are prokaryotic, lacking internal membrane-bound structures.17 Bacteria reproduce, without recombination, via means of single-cell division into daughter cells. Instead, bacteria rearrange 4 their genes and genomes17 through multiple reproduction-independent18 DNA recombination mechanisms. A significant result of these dynamic DNA processes is that microbial communities can be constructed of multiple strains of the same bacteria species.19 These strains will be unique in their genomic and phenotypic variability in a manner loosely similar to how one might think of individual humans: of the same species but with significant variation.19 Although bacteria are single-celled organisms, they can function ecologically as multicell units when they form colonies from daughter cells that do not separate after division.18 Within the Bacteria Kingdom itself, the Phylum Actinobacteria has dominated bacterial natural product discovery efforts. Actinobacteria is one of the most extensive taxonomic lineages composed mainly of Gram-positive staining bacteria with high G + C DNA content;20 however, diversity marks this lineage more than any common feature. Several orders, such as Micromonosporineae, Streptomycineae, and Actinomycetaceae,20 have been well-studied since they grow well in laboratory conditions and have large genomes showing prodigious capacities for natural product production.21 Many species of this order produce spores that can resist dehydration, mildly hot temperatures and produce the same chemistry when revived. Actinobacteria contain both terrestrial and marine strains,20 and are numerous in most soils, including marine sediments. Actinomycetes are the source of many bioactive natural products,22–24 including daptomycin (1.12, isolated from Streptomyces roseosporus NRRL 11379),25 a powerful antibiotic. 5 Figure 1-3. Daptomycin, an antibiotic isolated from a terrestrial Actinobacteria. 1.1.2.1 Marine Bacteria The heterogeneous marine environment is a treasure house of diverse bacterial strains that can be collected from sediments and the microbiomes of invertebrates.26 Altogether, the marine environment, which covers 70% of this planet’s surface, supports an enormous range of biodiversity.27 In fact, early samples taken on marine sediment found higher microorganism diversity than that found in terrestrial soils.28 Some bacterial genera, such as the Salinispora genus discussed in Chapter 6, require a certain sodium concentration in their environments for the proper functioning of basic metabolic activity and have truly become marine-dependent bacteria.28 Many marine strains, however, are recent adaptations from a terrestrial origin.29 Marine microorganisms experience a complex heterogeneous environment,30 which has been shown to select for microorganisms with a wide range of specialized metabolites.29 This diversity has allowed chemists to isolate and identify numerous bioactive and novel marine natural products.31 Historical examples include the pyrrole antibiotic 2,3,4-tribromo-5(1-hydroxy,2,4-dibromophenyl)pyrrole32 isolated from seawater bacteria and the marine sponge 6 sourced cytarabine (a chemotherapeutic drug)33 and vidarabine (an anti-viral).34 A recent example is salinosporamide A (1.14), a proteasome inhibitor in advanced stages of clinical trials for its anticancer properties.35 Another recent example is the peptide cyclomarin A (1.13),36,37 a potent anti-inflammatory, anti-malarial and anti-viral compound38 isolated from a Streptomyces sp. gathered from marine sediments near San Diego, California. Cyclomarin’s structure and bioactivity have also driven interest in characterizing its biosynthetic pathway and several synthetic efforts.36 Marine organisms continue to yield diverse natural products, with hundreds of pharmaceutically useful novel natural products reported yearly.39 Figure 1-4. Potent bioactive compounds isolated from marine bacteria. 1.1.3 Structure Elucidation of Novel Natural Products An essential aspect of natural product chemistry is characterizing the constitution and configuration of the isolated biological molecules that have utility either as drug leads, insecticides, dye compounds, or other applications. Without knowledge of the natural product structure, there is no capacity for rational synthesis, industrial fermentation, mechanism-of-7 action studies, or biosynthetic pathway characterization. In the early years of natural product chemistry, structure elucidation was a painstaking task involving chemical degradation experiments,40 taking decades of work,40,41 and sometimes producing erroneous results. Even when supplemented with spectroscopic techniques, the chemical degradation experiments necessitated several hundred milligrams (at minimum) of a natural product and were thus biased towards major metabolites.40 Since the 1970s, mass spectrometry42 and nuclear magnetic resonance40,43,44 have been amongst the basic techniques for elucidating the constitution of natural products. Mass spectrometry (MS) relies on an ionization source to ionize molecules (as well as fragment them), a mass analyzer that separates those ions by their mass-to-charge ratio (m/z), and a detector that translates that m/z signals into a mass spectrum.42 A variety of MS instruments are available for analysis, dependent on the target molecules' size and polarity. A reliable method for natural product structure elucidation is Electrospray Ionization (ESI), where a soft ionization source tends not to fragment molecules, generally giving only the parent molecule ion.42 With high-resolution ESI-MS, the parent ion's mass is known to four to five decimal places, which allows calculation of possible molecular formulae with a high degree of certainty. That said, molecular fragmentation in MS follows chemical principles, and, as such, it provides a molecular fingerprint that can, for certain classes of natural products, be useful in structure elucidation and discovery.42 In brief, MS is indispensable in providing the molecular formula for novel natural products, particularly if there are multiple heteroatoms or a high degree of unsaturation. Nuclear magnetic resonance (NMR) is also an indispensable spectroscopic technique that provides predictable information about the chemical environment for each of the atoms in a molecule. In an applied magnetic field, certain isotopes such as 13C, 1H, 14N and 15N have a 8 magnetic moment that causes them to precess about the direction of the magnetic field at a frequency (Larmor) proportional to the strength of the magnetic field and the gyromagnetic ratio unique to the isotope. This property can be manipulated to give us information about the identity and number of neighbours surrounding an NMR active nucleus. Since the 1950s, NMR has been used for organic structure determination and biosynthetic pathway characterization.44 From 1H experiments, chemical shift (δ) data can be collected depending on the slight changes in Larmor frequency influenced by the surrounding electron density, and coupling data can be gathered, which is dependent on the number of proton neighbours each proton has. With instrument evolution, 13C experiments allowed the determination of the carbon chemical shift. Between 1950 and 1990, 2-dimensional (2D) experiments were developed that allowed observation of multi-atom correlations between homonuclear atoms (COSY:1H-1H correlation), slowly into 1-bond correlations between heteronuclear atoms (HETeronuclearCORrelation, HETCOR 1H-13C correlations) and then 2,3-bond correlations between heteronuclear atoms (Heteronuclear Multiple Bond Correlation Spectroscopy, HMBC).44,45 Presently, there is a standard suite of experiments43,44 used as evidence of atom connectivity as outlined in Figure 1-5. Through-space experiments take advantage of nuclear Overhauser enhancements (NOEs) to help identify the relative configuration. As a chemical tool, NMR’s strengths are its almost universal detection, the detailed information it delivers on molecular structure (especially for novel compounds), and its quantitative abilities.46 Additionally, instrumental advances have increased signal/noise proportions at the same concentration while also giving improved resolution, which permits analysis of even microgram amounts of material.44 This capacity to analyze minute material amounts has increased the discovery of minor metabolites, which are natural products produced at low levels. 9 Figure 1-5. The standard suite of NMR experiments utilized to construct the constitution of a novel compound. 1.1.4 Natural Product Discovery The mining of natural products for human use has successfully yielded many life-saving pharmaceuticals, yet continual discovery efforts are needed to improve on that success.12–14,47–49 The ongoing need for new medicines drives the search for novel products with drug lead promise.47,49 In addition to these bioactivities, natural products have utility as dyes, insecticides, herbicides and more. The chemical complexity and bioactivity often found with natural products inspire and inform synthetic organic chemists' work.50,51 The biochemical transformations leading to natural products have motivated chemical biologists to study the associated enzymatic 10 pathways.52–54 Finally, due to the biological interactions mediated by natural products, chemists, biologists and microbiologists study these molecules’ chemical ecology.55,56 As such, the drive to uncover and discover novel natural products is propelled by the need for new medicines and the need to grow a basic scientific understanding of the chemical world around us. Figure 1-6. Natural product discovery mines the chemical diversity of living organisms for useful bioactive molecules. Although there are various discovery strategies, the general workflow is roughly the same (Figure 1-7). Most discovery efforts begin by determining an organism of interest, then extracting metabolites from that organism in some manner, purifying a compound, and elucidating that compound's structure. Despite a recent plateau in the rate of natural product discovery, analysis of discovery patterns still demonstrates the enormous potential for novel chemistry from diverse living creatures.47 The development of new discovery methods is a useful way of driving discovery, and there has also been work to match genotype with chemotype for faster discovery.2,15,46,57,58 Given the centrality of the specific method to optimized natural 11 product discovery, an in-depth examination of some of these methods follows in Sections 1.1.4.2-1.1.4.4, with genome mining59 explored in detail in Chapter 2-1 and 2-2. Figure 1-7. General discovery workflow. 1.1.4.1 The Biological Basis of Natural Product Discovery The process of discovering novel natural products is rooted in the biological diversity of organisms. Given that natural product production has been tied to an organism’s environmental interactions,60,61 and that evolution and ecology are interdependant,62 one could hypothesize that 12 natural product variability may be constrained by slower evolutionary processes.60 However, genomic analysis of bacteria, fungi and plants demonstrates a greater capacity for secondary metabolite production than currently represented in discovered chemistry.63 Although primary metabolic pathways are widely conserved across kingdoms, genera and species, specialized metabolites’ adaptive significance results in distinct metabolic profiles for strains of even the same microbial species.64 As a result, spatially and temporally separated environmental samples of marine sediment, invertebrates, or terrestrial soil (as examples) have the potential to yield distinct chemistry.65–67 Conversely, the effects of horizontal gene transfer (HGT) are such that, for example, rifamycin, a potent antibiotic, can be found in varying bacterial genera from Streptomyces to Salinispora.68 As a result, the challenge of drug discovery from bacteria is the tendency to find known compounds. One solution to this challenge is screening bacterial strain extracts, often seeking minor metabolites with the assistance of their bioactivity, in multiple assays and dereplicating using NMR data, MS data, and refined literature searches. 1.1.4.2 Bioassay-Guided Fractionation Bioassay-guided fractionation is a robust method used in varying capacities since the 1940’s58 for systematic screening of organisms to discover natural products.69–71 Bioassay-guided fractionation continues to yield novel chemistry in natural product discovery.69,70,72–74 Over the last 30 years or so, methods to improve bioassay-guided fractionation include high- throughput screening of hundreds of samples,58,59,75 as well as the refinement of the bioassays towards target-based approaches (such as enzyme assays),58 or faster turn-around time (TLC bioautography method).76 13 Bioassay-guided fractionation focuses on biological activities, usually with a medicinal utility, utilizing a bioassay designed to test for the desired function. This robust method can identify organisms producing bioactive natural products and guide the isolation scheme of a pure natural product. Antimicrobial bioassays test for natural products that will inhibit the growth of specific human pathogens. Cytotoxic bioassays test for natural products with inhibition of specific cancer cell lines. Enzyme assays target natural products that will inhibit enzymes from specific, medically relevant biochemical pathways in the human body. Bioassays can also be used to test for novel insecticides and herbicides.11,77 Utilizing a bioassay that tests for the desired functionality, metabolic extracts from organisms such as bacteria, fungi, marine invertebrate, and plant materials are examined for the desired biological activity. After a positive bioassay result, the “crude” extract can be sequentially and iteratively separated chemically via such tools as solvent-solvent partitioning and chromatographic separation. After each round of separation, the resulting fractions are tested in the selected bioassay. The fraction displaying the most significant bioactivity is chosen for further separation rounds. Ideally, since the active compound concentration should be increasing in ratio to the non-active components of the fractions, an increase in bioactivity metrics should be observable in the bioassay results. Bioassay-guided fractionation points through the complex mix of molecules produced by living things towards the compounds of interest. After this retrieval, data collected from nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) allow structure elucidation. The robust technique of bioactivity guided fractionation has yielded many vital pharmaceuticals such as vancomycin (1.15),78 salinosporamide (1.14),35 and lead compounds such as kutzneride (1.16),72 to name but a few. A drawback is that bioassay-guided fractionation 14 can reveal the same chemistry even from different bacterial species due to the presence of identical specialized metabolites, often because of horizontal gene transfer. Figure 1-8. The bioassay-guided fractionation process, demonstrating sequential separation guided by the results in bioassays of choice. Represented is the expected increase in activity as the active compound is concentrated and isolated from a complex mixture. Figure 1-9. Natural products isolated using bioassay-guided fractionation. 15 1.1.4.3 Mass Spectrometry as a Natural Product Discovery Methodology Mass spectrometry (MS) can be used directly as a discovery tool. The highlight of MS for discovery is that it can be used both for high-throughput identification of known molecules79 and investigations into new compounds. When coupled with metabolomic data analysis methods such as multivariate statistical analyses, self-organizing map analytics (SOM), or molecular networking, strains or extracts with potentially novel components can be prioritized for investigation.80,81 This type of approach has seen the discovery of tambromycin,82 columbamides,83 and other molecules. However, there are still some significant limitations when using mass spectrometry as a discovery tool, such as varying tendencies of molecules to ionize, media interference in crude samples,84 unpredictable fragmentation patterns of some natural product classes,85–87 and the need to use NMR in order to determine the final structure. 1.1.4.4 NMR as a Natural Product Discovery Methodology NMR methods for natural product discovery have been more recently explored. NMR data collected on crude biological extracts shows every correlation in the 2D data essential for structure elucidation; this makes it difficult to determine the structure of the components without extensive isolation efforts. One solution to that problem was the development of the differential analysis of 2D NMR spectra (DANS). DANS was initially developed to focus on identifying phenotypically unique molecules from target organisms with ecological or other differences via NMR without complete isolation. In this way, DANS allowed elucidation of the indole alkaloids TC-705A and TC-705B.88 Indeed, in the chemical ecology field, DANS has proven to be a powerful method for studying signaling molecules of the insect Caenorhabditis elegans,89 with a variant of this strategy used in many metabolomic studies of plant and insect species.90,91 However, as pure compounds are not isolated with this method, it is difficult to test the natural 16 product’s bioactivity. Attempts have been made to target bioactive compounds utilizing variants of the DANS strategy.92,93 These method variants have had limited success, although one attempt identified elaganolone (1.18), previously isolated in traditional bio-activity screening protocols.94 In the search for methods to combine with genome mining, DANS has been used to match orphan gene clusters with the produced molecule by statistical comparison of the NMR spectra from a targeted deletion mutant compared to a wild-type strain. This method identified bacillaene (1.17), an easily degraded molecule that resisted traditional isolation protocols.95 In the end, identification of molecules from within a complex NMR spectrum eases dereplication efforts, although the databases that would strengthen this are fragmented at best.96 Figure 1-10. Structures solved but not isolated with DANS. The van Wezel group reported a strategy closely related to DANS in which they utilized extensive multivariate data analysis of 1H NMR spectra to link bioactive fractions with differences in proton spectra for selection of the desired fraction. For isolation of the simple antibiotic 7-prenylisatin, they utilized a common aromatic proton fingerprint. Assessing differences in proton spectra led to the discovery of iotrochotazine A and the venulosides.97 The common challenge with these proton-based techniques is that proton spectra have a limited 17 chemical shift range, generally 0-15 ppm. This limited range leads to many overlapping signals in crude samples, requiring time-consuming chromatographic steps before confirmation of uniqueness. In 2015, the Wang group employed the larger spectral range of 13C to identify clusters of 13C resonances in fractionation efforts. This strategy led to the discovery of six podoverines (1.19) with considerably more structural diversity than previous NMR-discovered molecules.98 Other methods for NMR-based discovery, heavily reliant on metabolomic methodologies, include hybrid NMR-MS methods combined with MS principal component analysis methods to select a strain or fraction of interest. These hybrid methods have led to the discovery of a new piperamide, ottonoidenamide (1.20)99 and a host of other plant compounds.100 Figure 1-11. Natural Products isolated using NMR in various aspects of the discovery process. Two additional methods have combined targeted NMR methods with genome mining. These are the genomisotopic approach and a phosphate NMR approach. The genomisotopic approach developed by Gerwick et al. uses isotopically labelled precursors as predicted by in silico genetic information to link orphan secondary metabolite gene clusters to the putatively produced natural product.101 In this method, the use of the 1H-15N HMBC experiment to identify a labelled isoleucine (Ile) led to the successful isolation of orfamide A (1.21), representative of a new peptide class. As a drawback, this method required extensive labelling due to dependence on the increase of N-H signal of a labelled proteogenic amino acid with signals in a crowded region 18 of the N-NMR spectrum.101 The complexity of signals in this region biases this method towards isolating major metabolites. In the phosphate NMR method, Metcalf and associates target a specific phosphonate encoding gene, pepM, to select microorganisms for culturing that contained that specific gene.102 They then utilized the diagnostic chemical shift of a C-P in 31P NMR to assess the presence or absence of phosphonates in their crude mixture, which led to the isolation of (2-acetamidoethyl)phosphonic acid, (2-acetamido-1-hydroxyethyl) phosphonic acid (1.22), and (cyano (hydroxy)methyl)phosphonic acid (1.23).102 This approach links a diagnostic 31P NMR signal to a specific type of phosphorous metabolite for novel natural product discovery. It demonstrates the utility of targeted genome mining coupled with specific NMR features for rapid natural product discovery efforts. However, phosphonic acid natural products are a relatively small group of natural products with limited structural diversity. This approach was also not able to complete the structure elucidation of all the isolated products and synthesis was required. Overall, NMR has infrequently been used as a tool for natural product discovery. Given the power of NMR spectroscopy, I wanted to expand the approach of using NMR to match genotype with chemotype, which is the focus of Chapters 2 to 4. 19 Figure 1-12. Natural Products isolated using variations of NMR-Genome mining approaches. 1.2 Natural Product Biosynthesis Natural products are often broadly defined as both the primary and secondary metabolites produced by living organisms.52,103–105 Medicinal and organic chemists are usually referring to secondary metabolites when they discuss natural products.103,106 Secondary metabolites are generally small molecules (smaller than 2000 Daltons) that are often species-specific.107 A large body of evidence supports that organisms produce at least some of their secondary metabolites for defense and inter- and intra-species communication purposes.64,105,108–111 Given their specialized purpose, secondary metabolite production in microbes is tightly regulated.110 In fact, the expression of secondary metabolite enzymes generally occurs when the bacterial growth rate slows upon the heterogenous exhaustion of carbon, nitrogen and phosphorous nutrient sources.52,112 At this point, some primary metabolite building blocks,109–111,113,114 previously stored as pools, are carefully shunted into the biochemical pathways leading to secondary 20 metabolites. Within these pathways, secondary metabolite enzymes in biosynthetic pathways assemble natural products.109 1.2.1 Natural Product Classes Although different sources will vary, there are six classes of natural products generally considered to have a secondary metabolite origin.103,106 These six classes are broadly defined as polyketides, nonribosomal peptides, shikimate derived, alkaloids, purines and pyrimidines, and isoprenoids. Molecules from each of these classes can be glycosylated for additional diversity.103 These six classes all have their varying precursors rerouted from primary metabolite pathways.103 Table 1.1 details a simplistic overview of the connections between primary and secondary metabolites. Of these natural products classes, bacteria tend to produce polyketides, nonribosomal peptides, and shikimic acid metabolites,106 and as such, a more in-depth focus will spotlight each of those classes. Following this will be a brief discussion about alkaloids. See Figure 1-13 for representatives of these classes. Nutrient Source Central Precursor Branch Precursor Primary Metabolites Secondary Metabolites Glucose Acetyl-CoA Malonyl-CoA Fatty acids Polyketides Nonribosomal amino Acids Mevalonic Acid Steroids Terpenoids Krebs Cycle Ribosomal amino acids Nonribosomal amino acids Terpenoids Shikimic acid Ribosomal aromatic amino acids Flavonoids Lignans Alkaloids Nonribosomal amino Acids Table 1-1. Overview of the connection between shared precursors of primary and secondary metabolites. 21 Figure 1-13. Representative natural products of the select natural product classes. 1.2.1.1 Modular Natural Products: Polyketides and Nonribosomal Peptides Although sourced from different building blocks, both polyketides and nonribosomal peptide natural products frequently possess biological activity and are constructed utilizing assembly-line production of the larger molecules from small monomers. To do so, enzymes work together in module units that are selective for monomer variants and build them together, “lego style”, utilizing repetitive chemical logic specific to the natural product class. Additionally, both 22 can have pre- and post-assembly line tailoring. Together, polyketides and nonribosomal peptides make up roughly thirty percent of known natural products.115,116 1.2.1.1.1 Polyketides Polyketides (PK) are produced from acetyl-CoA in two carbon fragments (Figure 1-14a). Owing to the reactive diversity of the carbonyl functional group, those fragments can be arranged into diverse skeletons of natural products (aromatics, pyrones, alkenes, alkanes etc.). Producing organisms use large modular enzymes called polyketide synthases (PKS) constructed of repeating modules of smaller enzyme domains with specific catalytic functions that work together in an Figure 1-14. Polyketides are built from basic two-carbon building blocks derived from either a) acetyl-CoA or b) malonyl-CoA. c) A large diversity of extender units add novelty to polyketides. assembly-line fashion to craft the diverse PK skeletons. The typical module contains a β-ketoacylsynthase (KS) domain, an acyl transferase (AT) domain, and a phosphopantethienylated acyl carrier protein (ACP, or T for thiolation).103,117 The KS domain is the site where malonyl-CoA (Figure 1-14b) undergoes decarboxylative Claisen condensation chemistry (Figure 1-15) to connect the acetyl-CoA monomers.118 The AT domain confers specificity towards a particular acetyl-CoA extender unit (Figure 1-14c shows the diversity of PK extender units). The ACP (T) a b c 23 domain holds new acetyl units as thioesters, whereby the C-2 carbanion nucleophile attacks the electrophilic carbonyl C-1 held as a thioester on the KS domain of the growing chain (Figure 1-15a). These three domains produce chains of ketones or poly-β-ketos.118 However, these polyketides are often reduced either to a hydroxyl, an olefin, or a saturated alkyl, respectively, using first ketoreductase (KS) domains, next dehydratase (DH) domains, and lastly enoyl reductase (ER) domains (Figure 1-15b).117 The termination module contains a final domain called a thioesterase (TE), where the polyketide is offloaded either through hydrolytic cleavage of the thioester bond or a cyclization reaction. Figure 1-15. a) PKS module and domain organization. Two carbon units shown in blue in the diagram b) PKS reducing domains. a b 24 Polyketide modules themselves are often arranged into large genes or subunits containing multiple modules. Within this gene organization, there is usually a starting module (which loads starting units), multiple extending modules (which load extender units), and generally a termination module. Dependent on the type of PKS, the acetyl-CoA building blocks can vary or predictably rearrange malonyl-CoA, the basic extender unit utilized for decarboxylative Claisen condensation. There are four general types of PKS, with many exceptions reported. The first, modular type 1 (non-iterative), can use a variety of PKS extender units and utilize gene clusters composed of multiple modules with unique domain organizations and varying AT specificities. In modular type 1 PKS, most common in bacteria, unusual acetyl-CoA units diversify polyketides with various branching substituents at C-2 (Figure 14-C).118 Next, iterative type 1 and iterative type 2 (II) use only malonyl-CoA extender units and, as the name implies, iteratively re-use the same modules to manufacture polyketides from this simple building block. Type II PKS produce aromatic structures. PKS type III are unique because they are composed only of a dimer of KS domains. Originally thought to be only plant enzymes, it is Figure 1-16. Various types of polyketide natural products produced by variations of enzymatic machinery. 25 now understood that there are bacterial PKS III natural products. PKS III enzymes typically make simple and often aromatic molecules, predominantly using malonyl-CoA as an extender unit, although some bacteria have been shown to use both methylmalonyl-CoA and ethylmalonyl-CoA extender substrates.119 See Figure 1-16 for examples of natural products made by each type of enzymatic machinery. 1.2.1.1.2 Nonribosomal Peptides Nonribosomal peptides (NRP) are constructed from multiple amino acids linked via peptide bonds. They differ from ribosomal peptides in the amino acid building blocks. Twenty to twenty-two amino acids are used to construct ribosomal peptides, whereas over 200 amino acids are known to be utilized to construct NRPs. NRPs are constructed from large modular proteins termed nonribosomal polyketide synthetases (NRPS) in a parallel manner to polyketides. Modules are constructed of three repeated domains: an adenylation (A) domain, a peptidyl carrier protein (PCP, T for thiolation), and a peptide bond condensation domain (C) (Figure 1-17).120,121 The A domain confers specificity towards a particular amino acid. The PCP domain holds new amino acid units as thioesters, whereby the nitrogen nucleophile attacks the electrophilic carbonyl C-1 held as a thioester on the C domain of the growing chain. Modules are organized into initiation modules with only A-CP domains and extender modules with C-A-PCP domains. Other common domains include MT, or methyltransferase, and E or epimerase. Genes will often be composed of multiple modules, with the biosynthetic machinery for large clusters composed of multiple genes with multiple modules. There is also a thioesterase domain (TE) found in termination modules, which determines the mechanism of release from the assembly and determines if the molecule is a cyclic peptide, a depsipeptide, or linear peptide, amongst other possibilities.103,106 26 Figure 1-17. NRPS module organization and domain functions. There are different types of NRPS gene clusters whose gene organization influences the type of nonribosomal peptides that can be biosynthesized. These types are linear, iterative, and non-linear. Module organization of linear NRPS accurately reflects the predicted primary sequence of the peptide, whereas iterative NRPS reuse modules to create peptides of repeated amino acids. Non-linear NRPS, as the name suggests, are non-predictive, with assembly of the natural product often involving a mixture of modules and free-standing domains.120 1.2.1.1.3 NRPS-PKS and PKS-NRPS Hybrids PKS-NRPS and NRPS-PKS are hybrids that connect acetyl-CoA fragments with amino acids.122,123,124 Nonribosomal and polyketide biosynthetic gene clusters use remarkably similar strategies to build a diverse set of molecules through the repetitive formation of C-N bonds and 27 C-C bonds, respectively. Combination of the two biosynthetic classes results in hybrid molecules incorporating both acetyl units and amino acids to craft an incredible array of complex molecules. NRPS/PKS hybrids with the PKS downstream of the NRPS module build C-C bonds through nucleophilic attack of the PK carbanion onto the carbonyl carbon on the growing molecular tethered to an NRPS thiolation domain.122 Conversely, PKS/NRPS hybrids utilize the nitrogen on the downstream NRPS module to attack the carbonyl carbon on the PK T domain, building the molecule with the addition of a C-N bond.122 Examples of these hybrids are abundant in bacteria and will be seen in Chapter 2. 1.2.1.1.4 Tailoring Reactions In addition to the diverse construction strategies mentioned, both NRPS and PKS introduce diversity through tailoring enzymes. These tailoring enzymes can act on substrates tethered to the thiolation carrier proteins or post-assembly on the free substrate.125 The known tailoring reactions that can occur to tethered substrates include cyclization, halogenation, methylation, oxidation, reduction.125 The tailoring reactions that can occur post-assembly include oxidation, reduction, cyclization, methylation and halogenation along with the installation of prenyl groups, acyl groups, carbamoyl groups, and aminotransferases.126 Examples of the results of tailoring reactions are seen in Figure 1-16. 1.2.1.2 Shikimic Acid Pathway While glucose is the nutrient leading to both acetyl-CoA and shikimic acid, the shikimic acid pathway produces six-carbon precursor units directly produced from glucose through a four-carbon precursor, D-erythrose-4-phosphate, and a three-carbon precursor, phosphoenolpyruvate.127,128 Shikimic acid was first isolated in the late 1800s from a fruit of a plant with the Japanese name shikimi-no-ki, and its structure was completely characterized in the 28 1930’s.129 Its role in the biosynthesis of the aromatic amino acids, L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), and L-tryptophan (L-Trp) was elucidated in the 1950s. Additionally, polyketides can incorporate shikimate pathway genes for the production of starting units such as in cahuitamycins,130 ascomycin and rapamycin (1.24).103,106 At times, shikimate acid pathway metabolites will be incorporated in the post-assembly process as seen in ansatrienin (1.25).131 Ansatrienin also incorporates 3-amino-5-hydroxy benzoic acid (AHBA) as its starter unit. AHBA is closely related to shikimic acid being also biosynthesized from D-erthyrose-4-phosphate and phosphoenolpyruvate. The AHBA synthase gene cluster utilizes the same enzymes and building blocks as the shikimate acid pathway, save for one enzyme that incorporates nitrogen and catalyzes aromatization as the final step in constructing AHBA.132 AHBA itself is the starting unit for multiple polyketide natural products such as rifamycin and will be discussed in more detail in Chapter 6. Gallic acid (1.26) is a metabolite that has been completely produced from the shikimate pathway, with feeding studies analyzing 13C splitting patterns demonstrating that it derives from D-erythrose-4-phosphate and phosphoenolpyruvate.133 Shikimate pathway metabolites are present in a large variety of natural products produced by plants and microbes such as phenolics, flavonoids, stilbenes, chalcones, quinones, and the side-chain of the terpene-derived taxol.128 1.2.1.3 Alkaloids Alkaloids are perhaps the most diverse of all the natural products with over 20,000 variations of alkaloids produced by a number of biosynthetic precursors.134,135 Their common feature is a basic nitrogen atom incorporated into the molecule in diverse ways as long as it is not a peptide bond. The nitrogen's may originate from amino acids such as L-ornithine (L-Orn), L- arginine (L-Arg), 29 Figure 1-18. Rapamycin, a mixed natural product utilizing a shikimate derived starting unit. Figure 1-19. Shikimate pathway and analog natural products. L-tryptophan (L-Trp), or others.135–137 Alkaloids are often organized by the name of the heterocycle forming the core of their scaffold and encompass such classes as indolocarbazole alkaloids (1.27) and pyrrole alkaloids (1.28). Additionally, they are often bioactive (1.28 is a powerful antibiotic) with fascinating, complex scaffolds (Figure 1-20).106,138,139 30 Figure 1-20. The diversity of alkaloid structures. 1.2.2 Genetic Foundation of Bacterial Natural Product Biosynthesis Secondary metabolite genes are often clustered into co-regulated genes that contain all required biosynthesis, regulation and resistance genes for the successful production of a secondary metabolite.111,140 These gene clusters often appear in “genomic islands,” discrete regions found on bacterial chromosomes or plasmids. Actinomycetes, the focus of this thesis, tend to have genome sizes in the range of 3 Mb to 12 Mb. An important feature of these genomes is that they are G+C rich bacteria because they tend to use codons with a higher percentage of G+C (Table 1-2).141 In secondary metabolism, open-reading frames (ORFs) consisting of G+C rich codons are often present in operons: multiple genes that are transcribed together onto one long mRNA.142,143 Operons themselves can be clustered together into polyoperons: multiple operons under control of the same regulator for the purpose of a metabolic process.144 Amino Acid Codon Streptomyces Usage (%) Alanine GCC 60 GCG 33 GCA 5 GCT 2 Table 1-2. Example of Streptomyces codon bias for alanine because of its high G+C content. 141 31 On a practical level, this genome structure enables the identification and manipulation of relevant bacterial biosynthetic genes using bioinformatics and molecular biology tools. Another consequence of this co-regulation is that there is often an exact relationship between the order of the genes in a gene cluster and the order in which biosynthetic enzymes transform building blocks into complex secondary metabolites.111 This association is called the colinearity rule, whereby the end product can be predicted by the gene sequence in a gene cluster. Once thought to be the norm, it is now understood that the colinearity rule is often broken.111 These differences in genes house the information for the production of biosynthetic enzymes145 capable of catalyzing the multitude of chemical transformations leading to the diversity of natural products that we see. These biosynthetic enzymes often catalyze stereo- and regioselective chemical reactions. By characterizing these enzymes, they can be harnessed as tools for synthetic chemistry, they can be used in the rational design of synthetic biology and can lead to ever better predictions of gene cluster functions. 1.3 Research Goals The work presented in this thesis was conducted in an effort to discover novel natural products with therapeutic properties. One strategy that was undertaken to accomplish this goal was to isolate molecules with a specific functional group that had previously been shown to be associated with bioactive properties. In Chapters 2, 3 and 4, I targeted the isolation of molecules containing a piperazic acid (Piz) residue. In order to do so, I developed a new method, combining genome mining with 15N NMR. The premise was that nitrogen NMR would allow targeted access to bioinformatically predicted natural products containing Piz. This hypothesis was constructed because Piz has a unique nitrogen-proton correlation that also allows observation of Piz’s spin system in an uncrowded region of the nitrogen NMR spectral window. 32 In Chapter 2, I describe the diversity of Piz-containing natural products and the bioinformatics work I carried out in order to genome mine Piz-containing strains for analysis. In Chapter 3, I describe the development of the 15N NMR-guided method for targeting isolation of Piz-containing compounds from genome-mined strains. In Chapter 4, I describe the structure elucidation and bioactivity of the natural products I isolated using a 15N NMR-guided method for targeting Piz-containing compounds. A second discovery strategy, explored in Chapters 5 and 6, was to use bioassay-guided fractionation to uncover molecules of interest in a small library of marine bacteria. In Chapter 5, I describe the construction of a mini-library and the outcome of bioassay-guided fractionations executed on mini-library strains with best activity. In Chapter 6, I describe how the bioassay results from the mini-library led to the structure elucidation of two natural products not previously found in wild-type bacteria. Based on my findings using this method, the goal was then to utilize stable isotope feeding studies to investigate the biosynthetic transformations leading to the 3,5-dihydroxyphenyl moiety present in one of the novel natural products. Finally, in Chapter 7, I conclude the thesis and detail possible future directions. 33 Chapter 2: Mining Bacterial Genomes for Piz-containing Natural Products This chapter first describes the bioinformatic tools employed to identify biosynthetic gene clusters of interest. Known piperazic acid-containing molecules, the focus of my study, are then described, followed by a description of the biosynthesis of known molecules. Finally, the work done to identify gene clusters of interest is then described. This background sets the stage for the experimental work described in Chapter 3. 2.1 Bioinformatic Processes From the first report of a sequenced bacterial genome in 1995,146 continual advances in DNA sequencing technologies have enabled the sequencing of thousands of microbial genomes, with the current list of sequenced prokaryote genomes in the National Center for Biotechnology Information standing at just about two hundred and fifty thousand (https://www.ncbi.nlm.nih.gov/genome/browse/#!/overview/). Sequence, protein, and genomic databases have been constructed and are maintained by various governments around the world: EBI (Europe and the UK), NIG (Japan), and NCBI (USA). These databases store genomic information for multiple species, including humans, animals, plants, and microbes.147,148 In terms of the microbial data gathered, sequenced genomic information can be used to deduce evolutionary relationships,145,149,150 understand bacterial metabolic community pathways,151 explore the metabolites of uncultivable microbes,152 and has been increasingly used to access secondary metabolites through genome mining.153 The genome sequencing process begins with DNA samples copied into read lengths of contiguous nucleotide sequences dependent on the sequencing technology used. Sanger sequencing was the first developed sequencing technology, it is a slow and expensive procedure, 34 but it produces copies of DNA in fragments or “reads” of about 900 base pairs (bp). Multiple Next Generation Sequencing (NGS) technology platforms were developed to increase the speed of sequencing, but this decrease in time and expense came with the tradeoff of short read length (50-800 bp in length).154,155 This tendency towards short reads is one of the drawbacks of NGS and has led to the “third revolution” in sequencing technology.154,156 These new technologies produce much longer reads, with PacBio capable of sequencing an entire genome into one contig, but they are much slower than NGS and rely on quantified, high-quality DNA.156 Nevertheless, Sanger and NGS sequencing are presently the most common; genomes are generally sequenced in strings of 800-10,000 base pairs. In this process, many repeated fragments are produced. Since genes and gene clusters of co-expressed genes can be larger143 than the reads produced by sequencing, assembly is the method for “stitching” or assembling these fragments together. The fragments are assembled either by aligning to a previously analyzed reference genome or de novo. De novo assemblies use software built on algorithms that can string bigger and bigger reads together based on a statistical likelihood that they are from the same area of the genome.155 Regardless of the method used for assembly (alignment or de novo), when the software can no longer find links between algorithmically stitched fragments, the areas that have now been linked together are termed contigs. With the proliferation of cheap NGS technology, there has also been a proliferation of draft genomes, having up to hundreds of contigs.146 After contigs are assembled, nucleotide information is categorized into the genes through structural annotation,157 and many genes can then be assigned putative functions through functional annotation, which can set the stage for identification of biosynthetic gene clusters. 35 2.2 Bioinformatics for Strain Selection Through Genome Mining Characterization of gene clusters involved in the production of secondary metabolites has enabled the construction of various software programs or web-based interfaces (antiSMASH 4, BLAST, 2nd find, NRPS/PKS analysis https://www.secondarymetabolites.org) that allow for in silico inference of biosynthetic function for genes and biosynthetic gene clusters (BGCs). This inference is based on searching for homologous sequences that possess either nucleotide or amino acid sequence similarity between the characterized enzyme’s gene sequence and an unknown open reading frame (ORF). Homologous sequences share common ancestry – either from gene-duplication events (paralogs) or speciation events (orthologs).158 It has been shown that homologous sequence with a high percentage of sequence similarity likely also share similar functions. BLAST or Basic Local Alignment Search Tool is the foundation of most functional annotation tools as it is linked to the GenBank database. There are various BLAST platforms, including BLASTN that will search nucleotide databases using a nucleotide query, and BLASTP that searches protein databases using a protein query.159 BLAST finds places of local similarity between a query and sequences in its database; termed pairwise alignment. From there, BLAST then displays a percentage of the two sequences that matched (identity %) and how much of the query and the database sequence matched (similarity %). In addition, it gives a statistical probability of how much the identity match was due to random chance – the smaller this statistical probability, the greater the chance that the query and the database sequence are homologous sequences.159 With the above stated, there is a caution that although a certain percentage of sequence similarity generally indicates common ancestry,145 the link between sequence and function is less straight forward.158 This is particularly true of nucleotide sequence homology so the use of BLASTN can be less of informative. Due to this, genome mining efforts 36 which compare the amino acids sequence of a query against amino acid sequences in the database (using the BLASTP algorithm) homology utilizing BLASTP will have a higher likelihood of uncovering homologs with both sequence and function similarity. The other drawback from this sequence similarity approach is that proteins may share a functional similarity, even if evolutionary events have introduced a great deal of sequence changes. In this case then, two sequences may not share a high degree of sequence similarity but may have identical functions.158 PSI-BLAST is a BLAST algorithm that works to overcome this potential limitation – searching for distantly related sequences sharing function.158 PSI-BLAST uses an initial BLASTP search then constructs a multiple sequence alignment of those amino acid sequences to create a scoring matrix to compare sequences on the next iteration.160 During this process it takes into account characterized protein models and conserved protein domains. However, PSI-BLAST is an iterative algorithm, it must be run upwards of 10 to 20 times for the same search query, each time ideally favoring functionally similar proteins. However, if a non-functionally similar protein is introduced due to protein domain similarity, the subsequent iterations may distort the outcome towards non-functionally similar sequences, leading to false positives.158 A way to account for the weaknesses of both pairwise alignment and multiple sequence alignment-based algorithms is to run both for a complete analysis. With the above bioinformatic tools, it can be generally predicted if a BGC encodes for nonribosomal peptide synthetases (NRPS) and/or polyketide synthases (PKS) as these modules and domains have predictable sequence motifs across multiple bacterial species.161,162 The program antiSMASH, which is an acronym for “antibiotics and secondary metabolites analysis shell,” allows users to specifically search finished and draft genomes for gene clusters that code for secondary metabolites like those of NRPS or PKS origin (to name but a few).163 37 antiSMASH will find clusters within sequenced data, provide the functional annotation, list the closest homologous sequences from within the closest characterized secondary metabolite biosynthetic pathways, give a breakdown of A and AT domain specificities in NRPS and PKS modules, and provide a likely linear structure based on colinearity rules. 2ndFind is a web-based tool that will find secondary metabolite gene clusters and list putative functions for the genes within the gene clusters (biosyn.nih.go.jp/2ndfind/). It does not give predictions for A(NRPS) or AT(PK) domain specificity, and as such, must be coupled with a secondary bioinformatic tool such as the web served NRPSpredictor164 or NRPS/PKS Analysis165 for that analysis. These were the main tools used in the genome mining research work discussed in this thesis. Due to the colinear rule, the constitution of bacterial natural product can often be predicted162 by analyzing the gene organization, the A domain (NRPS) specificity, and the AT domains (PKS) specificity of a gene cluster. This is the application of the bioinformatic processes to the genome mining of natural products. This approach has led to an array of discoveries of novel molecules including salinilactam21 (2.1), tambromycin166 (2.2) and avermipeptin B167, thailandamide A168 (2.3), leptolyngbyalide A (2.4), and bacillaene169 (See Table 2.1). Genome mining efforts allow us to explore the genetic machinery of sequenced bacteria for gene clusters of interest. In this thesis, I used genome mining tools to sift through the thousands of sequenced bacteria in public databases to find bacteria predicted to produce Piz-containing natural products. 38 A Small Subset of Natural Products Discovered Through Genome Mining Obtained from canonical gene clusters Salinilactam (2.1) Tambromycin (2.2) Obtained from non-canonical gene clusters Thailandamide A (2.3) Leptolyngbyalide A (2.4) Table 2-1. Selected natural products discovered using genome mining techniques. 2.3 Piperazic Acid-Containing Natural Products As natural products are produced by biosynthetic enzymes in biological systems, novel chemical scaffolds are often linked with enzymes using novel catalytic strategies.170 Pursuing chemical diversity can lead us to useful drug leads and pharmaceuticals while expanding our 39 chemical and biological knowledge; this can inform rational efforts at drug discovery and drug design. Piperazic acid is one such chemically diverse natural product moiety. Piperazic acid (2.5) is a cyclic hydrazine amino acid that has been found in diverse natural products, many of which have potent biological activities. Piperazic acid is a unique non-proteinogenic amino acid in several ways. First, it is a rare amino acid with a nitrogen-nitrogen bond and the only known example of a natural cyclic hydrazine amino acid.171 Second, as an amino acid residue, particularly as the unsaturated derivative, it has a constrained conformation that locks the amino acid in place and can thereby induce β-turns in peptides.172 This unique feature makes piperazic acid and its congener dehydropiperazic acid potentially useful for engineering peptides with β-turns. Third, natural products with piperazic acid residues often have multiple rare structural features and unique biological activities,173 and as such, methods for specifically isolating piperazic acid-containing molecules have the potential to lead to particularly potent natural products. To highlight the utility of finding new piperazic acid-containing peptides, I will catalogue the known piperazic acid-containing natural products to demonstrate the breadth of structural diversity. 2.3.1 Linear Peptides Some of the most exciting and unusual piperazic acid-containing molecules are linear peptides. As a group, the linear nonribosomal piperazic acid peptides are marked by their structural diversity and incorporation of additional unusual amino acids and functional groups. These molecules can be sorted into a few smaller families: antrimycins (2.6), Sch 382582.3 (2.7), matlystatins/YM.24074 (2.8, 2.9), padanamides/actinoramides (2.10, 2.11, 2.12, 2.13), and cahuitamycins (2.14, 2.15). Even within each of these families, of which representatives of each are shown here, structural diversity can be found.173 40 Figure 2-1. Linear Piz-containing peptides. 2.3.2 Cyclic Peptides Composed entirely of amino acids and cyclized by the formation of a peptide bond this piperazic acid-containing family contains one known cyclic tetrapeptide, glomecidin (2.17),174 and multiple hexapeptides (2.18 to 2.22), of which representatives are pictured in Figure 2-2.173 41 Figure 2-2. Cyclic Piz-containing peptides. 2.3.3 Depsipeptides Depsipeptides, peptides where one of the amide linkages is replaced with an ester, are the largest group of nonribosomal peptides containing piperazic acid. The majority of these depsipeptides are cyclodepsipeptides, with several exceptions described below. This group can be further subdivided into 13-membered, 18-membered, and 19-membered cyclodepsipeptides.173 2.3.3.1 13- and 18-membered Depsipeptides The antimycobacterial lydiamycins (2.23) are the sole examples of piperazate-containing 13-member cyclodepsipeptides. The first family of piperazate-containing molecules on record is the 18-membered depsipeptide family of monamycins (2.24) isolated from Streptomyces jamaicensis during bioassay-guided screening efforts against Fusarium oxysporum cubense by Hassel and coworkers in 1959 and whose structures were elucidated in the early 1970’s. Additional 42 18-membered cyclic depsipeptides, of which a subset is shown in Figure 2-3, include the piperazimycins (2.25), gerumycin A (2.26), the svetamycins (2.27), the piperidamycins A (2.28),175 along with the related linear peptides JBIR.39 (2.29) and JBIR.40 (2.30)176. The kutznerides (2.31) also belongs to this class.173 Figure 2-3. Structures of 18-membered cyclodepsipeptides and their linear congeners. 2.3.3.2 Azinothricins: 19-Membered Cyclodepsipeptides The largest group of piperazic acid-containing molecules is the azinothricins named for the azino sub-structure of piperazic acid. The structures in this family are 19-membered cyclodepsipeptides with acyl side chains, isolated from various Streptomyces and related strains. Many of them appear to be hybrid PKS-NRPS derived molecules. The structures of the molecules are remarkably similar 43 across the family, often with similar residues at similar positions, suggesting conserved biosynthetic pathways. Most of these members contain an oxane moiety linking the amino acid sequences of the molecule with the acetyl derived portions of the molecule. Close examination of the substituents of the oxane ring, along with feeding studies in verucopeptin’s and polyoxypeptin’s producers suggest a divergence in biosynthetic strategies with a large portion of the acyl tail containing a strictly polyketide origin vs those whose acyl tail itself has origins from both amino acids and acyl-CoA precursors.173 2.3.3.2.1 19-Membered Cyclodepsipeptides With Long Acyl Tails Azinothricin (2.32), A83586C (2.33), citropeptin (2.34),177 GE3 (2.35),178 kettapeptin (2.36)179, verucopeptin (2.37), and diperamycin (2.38), are all 19-membered cyclodepsipeptides whose long acyl chains are derived from polyketide acetyl units.180 Figure 2-4. 19-membered cyclodepsipeptides with long acyl tails. 44 2.3.3.2.2 19-Membered Cyclodepsipeptides With Short Acyl Tails L-156,602 (2.39) variapeptin (2.40),177 the aurantimycins A.D (2.41 to 2.44), the polyoxypeptins A and B (2.45, 2.46), the oleamycins (2.47, 2.48), IC101 (2.49), the closely related pipalamycin (2.50) all have distinctly shorter substituents on the oxane sub-structure of the azinothricins. A subsection is demonstrated here with the rest discussed in Chapter 4. Feeding studies completed on polyoxypeptin suggest that the branched acyl chain attached to the oxane ring is produced from an intact isoleucine (Ile) unit.181 However, additional biosynthetic characterizations build evidence that many branched PKS extender units are constructed through a crotonyl-CoA pathway.118,182 Figure 2-5. 19-membered depsipeptides with short acyl tails. 45 2.3.3.2.3 19-Membered Cyclodepsipeptides With NRPS Derived Acyl Tails This subset does not have an oxane-based PKS tail. Marformycins A-F (2.51) were isolated from a Streptomyces strain isolated from sediment at 1396 m depth in the South China Sea (Figure 2-6).173 They feature a unique N-formylated side chain while containing only a single piperazic acid and an O-methylated tyrosine. Related natural products are the depsidomycins (2.52), and the piperastatins (2.53).173 Figure 2-6. 19-membered depsipeptides with formamide tails. 2.3.4 Sanglifehrin, Mollemycin, Dimeric Compounds and the Bisintercalators Increasing the structural diversity of Piz-containing compounds, sanglifehrin (2.54), mollemycin (2.55), himastatin (2.56), chlopostatin, luzopeptin (2.57) and quinoxapeptin uniquely add to known natural products. As of now, each stands in a family on its own. Altogether it can be seen that piperazic acid-containing natural products are rich with diversity and, as such, are excellent leads for natural product discovery efforts.173 46 Figure 2-7. Other Piz-containing natural products. 2.4 Biosynthesis of Piperazic Acid-Containing Molecules All known piperazic acid-containing molecules come from actinobacteria, although recent work has predicted their presence in more widely flung bacteria.183 The first published gene cluster of a Piz-containing natural product was for kutznerides (2.31) (ktz), an 18-membered depsipeptide showing it to be produced by a nonribosomal peptide synthase with three major NRPS modules and the enzymes required for pre and post-assembly modifications (Fig 2.8).184 This gene cluster 47 encodes three multidomain nonribosomal peptide synthetase proteins, KtzE, KtzG, and KtzH, collectively encoding six adenylation domains, with one of the A-domains in KtzH split into two pieces by a methyltransferase domain, consistent with the structures of the kutznerides. The KtzE module contains five domains (A,T,E,C,T) and was postulated to load in the unusual amino acid 2-(1-methylcyclopropyl)- D-glycine. The KtzG module, which contains three domains (A, KR, T), with a valine-for-aspartate substitution in the A domain, was shown to have substrate specificity for a ketoacid - specifically α-ketoisovaleric acid. Other standalone adenylation domains KtzB and KtzN, were shown to be involved in the assembly of unusual amino acids or as replacements for inoperable A domains in the larger modules.184 Subsequent studies by the Walsh and Garneau-Tsodikova groups interrogated the biosynthesis of these amino acids. Among the biosynthetic arsenal leading to the kutznerides are enzymes KtzQ and KtzR, flavin-dependent halogenases;185 KtzD and KthP, Fe(II), α-ketoglutarate dependent halogenases;186,187 KtzA, the acyl-CoA dehydrogenase-like flavoprotein that forms a cyclopropyl ring;187 KtzP and KtzO, non-heme iron oxygenases that catalyze opposite stereospecific hydroxylations;188 and a methyltransferase domain in KtzH that catalyzes O-methylation of a thioester-tethered serine.189 Additionally, the enzyme KthP, encoded outside of the immediate boundaries of the cluster, catalyzes stereospecific chlorination of a thioester-tethered L-piperazic acid, giving the (S)-5-chloro-L-piperazic acid congener. Since the publication of the ktz cluster in 2007, thirteen additional gene clusters for piperazic acid-containing molecules have been published covering a majority of the major structural classes for these molecules (Table 2-2).173 48 Figure 2-8. Kutzneride gene cluster map with key genes and modules named. Red genes indicate nonribosomal peptide origins. Purple square indicates ktzT, the piperazate synthase. Compound family Compound Natural product class Year Accession no Linear peptides Padanamides NRP/PK 2013 KC915040 Cahuitamycins NRP/ Shikimic 2016 KU363800 Matlystatin NRP/PK/ terpene 2017 NZ_KB907724 Cyclic peptides NW-G (AFN) NRP 2018 MH497044 18-membered depsipeptides Gerumycin A & B NRP 2015 CP010990 Gerumycin C NRP 2015 CP011868 Kutzneride NRP 2007 EU074211 19-membered depsipeptides Polyoxypeptin NRP/PK 2014 KF386858 Aurantimycin NRP/PK 2016 NZ_AOPZ01000147.1 Dentigerumycin A NRP/PK 2015 CP011862 Dentigerumycin B NRP/PK 2017 NZ_MWFK01000002 Marformycin NRP 2015 KP715145 Dimeric Himastatin NRP 2011 FR823394 Sanglifehrin Sanglifehrin PK/NRP 2011 FJ809786 Table 2-2. List of characterized Piz-containing gene clusters.173 Additional nonribosomal peptide synthase gene clusters were characterized for the gerumycins (2.26, 4.12-4.13), an 18-membered depsipeptides family. Gerumycin A and B were shown to be produced by Pseudonocardia sp. EC080625-04, interestingly by a gene cluster with two loci separated by ~92 kilobase pairs, while gerumycin C was produced by Pseudonocardia sp. ktzD ktzB ktzN ktzR ktzQ 49 HH130629-09 (Fig 2.9). The dimeric depsipeptide himastatin was also shown to be produced by nonribosomal peptide machinery in Streptomyces himastatinicus ATCC 53653 genome (Fig 2.10). The cyclic peptide family, the alboflavusins (formerly the NW-G series), were most recently shown, somewhat expectedly, to be produced in Streptomyces alboflavus strain 313 by a nonribosomal peptide synthetase (Fig 2.10). Biosynthesis is proposed to involve the construction of piperazic acid as a free amino acid from L-ornithine, then loading of amino acids onto a nonribosomal peptide synthetase assembly line.190 Gene clusters characterized for the linear peptides (Figure 2-10) all show distinct construction strategies, perhaps reflective of their structural diversity. Padanamides and actinoramides are linear tetrapeptides marked by unusual and distinct heterocycles at the C-termini of the molecules. Characterization of the padanamide gene cluster demonstrated that biosynthetic production involves a hybrid non-canonical NRPS/PKS gene cluster with the N-carbomylation of an intermediate nonribosomal amino acid deciding the formation of the final heterocycle.191 Cahuitamycins are the only the shikimic acid-NRPS hybrid produced Piz-containing natural product with a shikimic acid pathway-derived starter unit while the rest of the scaffold is produced by NRPS modules. Specifically, cahuitamycins A and B’s aromatic starter unit is produced from the salicylate synthase enzymes, CahI and CahJ while cahuitmycin C, on the other hand, has a salicylate derived starter unit – 6-methylsalicylate starter unit, - whose enzymatic machinery is encoded by genes in a second distinct locus. Matlystatins, yet another fascinating linear peptide family, are produced from a gene cluster that utilizes precursors from the PKS, the NRP, and the terpene pathways. This multi-hybrid biosynthetic strategy may help explain the fascinating structures that make up this family. Not a linear peptide, but also utilizing a unique hybrid 50 biosynthetic strategy, sanglifehrin’s (2.53) gene cluster loads in Piz as its starting unit and then completes the rest of its biosynthesis with polyketide machinery. Figure 2-9. Piz-containing natural products produced by nonribosomal peptides synthases. Red genes indicate non-ribosomal peptide origins. Purple square indicates ktzT homolog - the piperazate synthase. Finally, the gene clusters have been characterized for the 19-membered depsipeptides. Gene clusters for polyoxypeptin (2-45) and aurantimycin (2-41) have been published (Figure 2-11 and Figure 2-14).192,193 Each of these gene clusters is large (~75 kb for polyoxypeptin, and ~60 kb for aurantimycin) and contain both Type I polyketide synthase and nonribosomal peptide 51 synthetase genes, as expected from the structures of the corresponding molecules. For polyoxypeptin, deletion of any of the NRPS genes plyC, plyD, plyQ, plyI, and plyS abolishes the Figure 2-10. Piz-containing natural products produced by hybrid gene clusters. Red genes indicate non-ribosomal peptide origins. Blue genes indicate polyketide origins. Yellow genes indicate shikimate pathway origins. Green genes indicate terpene origins. Purple square indicates ktzT homolog - the piperazate synthase. production of polyoxypeptins.192 Additionally, deletion of the P450 gene plyM abolishes polyoxypeptin A production and accumulates polyoxypeptin B, suggesting that PlyM catalyzes hydroxylation of polyoxypeptin B to give polyoxypeptin A. The structure of the C15-acyl chain of 52 polyoxypeptin suggests that the unusual extender unit 2-(2-methylbutyl)malonyl-CoA is required for its assembly. Prior labelling studies have shown that this 2-(2-methylbutyl)malonyl could derive from isoleucine,181 possibly via a transamination reaction. However, deletion of a candidate aminotransferase gene (plyN) did not abolish production of polyoxypeptins, suggesting that the aminotransferase might be elsewhere in the genome.192 In the case of the related compound aurantimycin, the extender unit should instead be 2-(2-methylpropyl)malonyl, which could possibly arise from a similar transamination reaction, instead from valine. For the aurantimycin gene cluster (art), deletion of the artG, a candidate NRPS gene, abolishes production of aurantimycins, experimentally linking the gene cluster to production of aurantimycins.193 Dentigerumycin A is another example of an actinobacteria-derived azinothricin depsipeptide,194 produced by a NRPS-PKS hybrid gene cluster in a Pseudonocardia strain isolated from an ant (Apterostigma dentigerum). It has four main multidomain NRPS modules whose organization is: (C,A,T,TE); (E,C,A,T); (C,A,T); (T,E,C,A,T) and multiple standalone condensation, adenylation, and thioesterase domains (Figure 2-11). Three structurally similar molecules, named dentigerumycins B-D (4.5-4.7), were isolated from another strain, Streptomyces sp. M41 also from an ~100 kb NRPS-PKS hybrid gene cluster, albeit with five main multi-domain NRPS modules (one of which is immediately adjacent to the PKS modules) and multiple standalone NRP domains (Figure 2-11). The linear structures of dentigerumycins C-D are suggestive of a premature off-loading of a hybrid PK-NRP intermediate prior to incorporation of the final L-threonine residue. In 2018, another congener, dentigerumycin E (4.8), was isolated from the co-culture of a marine-derived Streptomyces sp. JB5 with a Bacillus sp., and a putative gene 53 Figure 2-11. Piz-containing natural products produced by nonribosomal peptide/polyketide hybrids. Red genes indicate nonribosomal peptide origin. Blue genes indicate polyketide origin. Purple square indicates ktzT homolog - the piperazate synthase. cluster for 4.8 was identified from the genome of Streptomyces sp. JB5 however, the gene sequence for this gene cluster has not been deposited with an open-access database.195 Ju and co-workers isolated the marformycin gene cluster (mfn), which encodes three multidomain NRPS modules (MfnC, MfnD, MfnE), three standalone NRP domains, along with a series of tailoring enzymes (Figure 2-12 and Figure 2-13).196 They established that the tailoring 54 enzyme, MfnG, O-methylates free D/L-tyrosine, before loading this amino acid onto the NRPS. Additionally, they provided evidence that MfnA catalyzes N-formylation and that the cytochrome P450 MfnN catalyzes hydroxylation of the piperazic acid residue as the terminal step in the biosynthesis. Finally, they demonstrated that MfnO (a Type IV aminotransferase) and MfnH (a 116-amino acid protein of unknown function) work in concert to catalyze the interconversion of diastereomers L-Ile and L-allo-Ile197, giving a precursor to the marformycins. Figure 2-12. The marformycin gene cluster. Red genes indicate nonribosomal peptide origin. Purple square indicates ktzT homolog - the piperazate synthase. Figure 2-13. Characterized transformations leading to marformycin biosynthesis.173 55 Figure 2-14. Characterized and hypothesized transformations leading to: a) polyoxypeptin biosynthesis b) aurantimycin biosynthesis.17356 All these gene clusters have a common feature in the hydrazine piperazic acid amino acid that has driven much of their interest. The biosynthetic origins of Piz were of curiosity since the first elucidation of monamycins in the early 1970s. Piz’s biosynthetic route was probed using radiolabelled ornithine in the 1970s, research whose result erroneously led to the conclusion that L-ornithine was not a precursor. In the 1990’s, the biosynthetic pathway of valanimycin, a nitroso-containing natural product, was investigated, which suggested that a hydroxylamine was involved in the formation of its nitrogen-nitrogen bond.198 When the Walsh group was exploring kutzneride’s (2.31) biosynthesis in 2012,199 they noticed a L-Orn N-hydroxylase homolog gene (ktzI) in 2.31’s gene cluster. This observation, coupled with the work on the biosynthesis of valanimycin, led them to re-investigate L-Orn as a Piz precursor. Walsh and coworkers identified KtzI, encoded in the gene cluster corresponding for the piperazic acid-containing kutznerides, as a flavin-dependent L-ornithine N-hydroxylase and demonstrated that free N-OH-L-ornithine (2.57) is an intermediate on the pathway to piperazic acid (2.5).199 This crucial result established that the δ-nitrogen of ornithine is activated as a hydroxylamine. The question of how the N-N bond was formed was still a mystery until Yiling Du of the Ryan lab recognized that a small open reading frame -orf4 in the kutzneride gene cluster was misannotated as a regulator. Additionally, homologous open reading frames were found in many characterized Piz-containing gene clusters with either adjacent or nearby ktzI homolog genes. Subsequent work by the Ryan lab identified this enzyme as KtzT, a heme-dependent, but not oxygen-dependent, piperazate synthase (Figure 2-15), with both ktzI and ktzT genes generally present in every gene cluster linked to a piperazic acid-containing molecule.173 57 Figure 2-15. Biosynthetic pathway to piperazic acid involves N-hydroxylation of L-ornithine by KtzI and N-N bond formation by KtzT.173 2.5 Bioinformatic Analysis Using the Piperazate Cyclizing Gene ktzT From the characterized biosynthetic pathways of piperazic acid-containing natural products, it is evident that production of this family involves many non-canonical pathways. For one, cahuitamycins employ two different starting units, the enzyme machinery for one of them usually found at a great distance from the rest of the producing cluster.130 Secondly, the machinery for characterized 19-membered depsipeptide gene clusters show non-colinearity with multiple unattached TE domains and other anomalies.173 Thirdly, the piperazate synthase itself has been shown to be an unusual heme-dependent enzyme that does not rely on oxygen for N-N bond formation.200 In addition to the interesting biochemistry that accompanies the production of piperazic acid-containing natural products, many of these natural products possess a wide variety of potent bioactivities173 and, as such, there is the correlation that the discovery of new piperazic acid-containing natural products will have a high likelihood of revealing novel bioactive drug leads. With this logic in hand, I chose to target Piz-containing natural products through genome-mining. 58 In vitro biochemical characterization had confirmed that the KtzT (Accession no. ABV56600.1) enzyme, found within the kutzneride gene cluster (Figure 2-8), was the piperazate synthase involved in the production of the N-N bond found in piperazic acids.200 This same work by the Ryan lab confirmed that the N-hydroxylated L-Orn (2.57) product of KtzI was the piperazate synthases’ substrate. Finally, as all known characterized gene clusters for Piz-containing natural products contain the NRPS modules necessary for incorporation of a nonribosomal amino acid, it can be expected that any Piz-producing gene cluster contains an NRPS module. KtzH is the NRPS module within the Kutzneria sp. 744 (Figure 2-8) whose A domains have been predicted to specify for piperazic acid.199 Thus, in short, the minimal enzymatic machinery so far understood to be involved in the production of piperazic acid-containing NRP’s, is a piperazate cyclase, a hydroxylase, and an NRPS module or modules, which have A-domains with specificity for Piz amino acids. Both hydroxylases and NRPS modules are common across many types of associated natural products. Thus, to target the discovery of the Piz amino acid, I hypothesized that the piperazate synthase should be unique to Piz-containing natural products. With this in mind, I hypothesized that the KtzT (Accession no ABV56600.1) amino acid sequence could be utilized as a genome mining probe in BLASTP201 searches of sequenced bacterial genome assemblies contained within the NCBI database to find bacterial strains with Piz-producing potential. However, the gene cluster associated with the production of the kutznerides, and thus containing KtzT, which belongs to Kutzneria sp. 744, is distantly related to the more ubiquitous Streptomyces genus within the Actinobacteria phylum.20,157 Due to this taxonomical distance, I accepted 50% sequence homology to KtzT to indicate a putative piperazate synthase homolog. I expected that true piperazate synthases would be found near or adjacent to an ORF homologous to a lysine/ornithine hydroxylase as well as an 59 NRPS module homolog. These requirements, then, added secondary and tertiary pieces of information to build evidence towards a strain’s ability to produce Piz-containing natural products. The sequenced genomes found through BLASTP containing an ORF with high sequence homology to KtzT were visually inspected using the NCBI in-house genome browser to determine if they contained proximal hydroxylases (KtzI homologs) and NRPS machinery (KtzH homologs). On occasion, a draft genome would contain KtzT and KtzI homologs on a short contig that displayed no NRPS machinery. In these instances, BLASTP searches were completed against the entire draft genome sequence of the respective strain using KtzH (Accession no. ABV56588.1) as a query. In this manner, I filtered down the approximately 200 KtzT homologs to a shorter list of about 30 strains of sequenced bacteria that had all aspects of genetic coding for the necessary machinery. The majority of these (a subset is shown in Table 2-3) were from the Streptomyces genus and all of them from the Actinobacteria phylum. 60 Strain KtzT homolog Accession no. Accession no. of contig containing KtzT homolog Streptomyces sp. NRRL F-6131 WP_030310216.1 NZ_JOHN01000049.1 Streptomyces sp. NRRL B-1347 WP_030688266.1 NZ_JOJM01000099.1 Streptomyces tsukubensis NRRL18488 EIF94522.1 AJSZ01000008.1 Streptomyces sp. NRRL F-2747 WP_051805955.1 NZ_JOIS01000036.1 Streptomyces sp. NRRL F-5053 WP_030885178.1 NZ_JOHT01000002.1 Streptomyces sp. NRRL S-920 WP_051819333.1 NZ_JODF01000006.1 Streptomyces katrae NRRL B-16271 WP_030301364.1 NZ_JNZY01000034.1 Streptomyces phaeochromogenes NRRL B-1248 WP_055612424.1 NZ_LIQZ01000085.1 Streptomyces griseus subsp. griseus NRRL WC-3645 WP_037672886.1 NZ_JOJE01000006.1 Streptomyces griseus NRRL F-5144 WP_030722408.1 NZ_JOGA01000032.1 Streptomyces sp. NRRL F-2664 WP_030773938.1 NZ_JOFX01000040.1 Streptomyces sp. NRRL B-1140 WP_053672929.1 NZ_LGEA01000110.1 Streptomyces incarnatus NRRL 8089 AKJ15811.1 CP011498.1 Table 2-3. Names of selected strains containing KtzT homologs. 2.6 Bioinformatic Prediction of Selected Strains From within the smaller subset of strains that contained homologs to the three (presumed) necessary enzymes - KtzT, KtzI, and KtzH - the appropriate contigs were analyzed using both the bioinformatics software pair 2ndFind (http://biosyn.nih.go.jp/2ndfind/) with NRPS/PKS finder165 and the standalone antiSMASH.163,202,203 This effort 1) confirmed that a contig or series of contigs within a (draft) genome contained homologs to KtzT, KtzI and KtzH; 2) allowed analysis of the A domain prediction; and 3) produced a linear sequence prediction. Through this analysis, I was ideally hoping to confirm that the clusters of interest had A domains specific to piperazic acid. Initial searches through NRPS/PKS finder and antiSMASH3 did not find any Piz specific A domains. There were, however, multiple A domains for which the software programs could not predict the loaded amino acid – termed X - and the assumption was made that some of 61 these X’s were, in fact, Piz. With the release of antiSMASH4,163 the included SANDPUMA ensemble algorithm204 proved capable of predicting most of the A-domains with Piz specificity. The gene clusters of some of the chosen strains were run back through antiSMASH4 to confirm initial assumptions. Further analysis of characterized gene clusters, as described in Section 2.4, suggested that the larger NRP multidomain modules appear to be involved in the factory-like assembly of the coded natural product. In contrast, the smaller stand-alone NRP modules or domains appear to be involved in the production of pre-assembly transformations for unusual amino acids. Given the non-linear nature of many of the gene clusters, the A domains of multidomain NRPS modules were compared against characterized gene clusters (Figures 2-9 to 2-12). The putative amino acid sequence predictions and known compounds' structures were also compared as a dereplication process. These comparisons filtered the hundreds of possible hits to a list of 13 strains (Table 2-3 and Table 2-4) that were obtainable from the US government’s Agricultural Research Service Culture Collection (NRRL) collection. 62 Table 2-4. Selected strains with the encoding organization of the multidomain NRPS modules with predicted A-domain specificity.Strain Predicted NP class Predicted NRPS A- domain colinear sequence for the A domains within main NRPS modules. Closest known Cluster/ Streptomyces sp. NRRL F-6131 NRP/PK Sanglifehrin Streptomyces sp. NRRL B-1347 NRP Kutzneride Streptomyces tsukubensis NRRL18488 NRP/PK Polyoxypeptin Streptomyces sp. NRRL F-2747 NRP Marformycins Streptomyces sp. NRRL F-5053 NRP Vazabitide A Streptomyces sp. NRRL S-920 NRP Himastatin Streptomyces katrae NRRL B-16271 NRP/PK Aurantimycins Streptomyces phaeochromogenes NRRL B-1248 NRP Himastatin Streptomyces griseus subsp. griseus NRRL WC-3645 NRP Kutzneride Streptomyces griseus strain NRRL F-5144 NRP/PK Polyoxypeptin Streptomyces sp. NRRL F-2664 NRP/PK Himastatin Streptomyces sp. NRRL B-1140 NRP/PK Polyoxypeptin Streptomyces incarnatus NRRL 8089 NRP/PK Polyoxypeptin 63 2.7 Conclusion In conclusion, the ability to cheaply sequence bacterial genomes has led to large databases containing tens of thousands of sequenced bacterial genomes. The concurrent characterization of secondary metabolite enzymes has led to the ability to predict which classes and structures of natural product a bacterial strain can putatively produce. Bioinformatic software, based on the biological characterization of the strings of nucleotides, can aid in the analysis of the prodigious amounts of data. Within natural products, piperazic acid-containing molecules are an exciting class of natural products due to their structural diversity and often potent biological activities. Using the amino acid sequence for the highly specific KtzT piperazate synthase as a probe to sort through the thousands of sequenced bacteria contained within open-access biological databases, I was able to select a small group of thirteen bacteria. These bacteria possessed genomes that were predicted to code for the enzymatic machinery necessary to produce piperazic acid-containing natural products. After this effort, the next question became how to find suitable culture conditions for Piz-containing metabolite production and isolate the pure metabolites from crude culture extracts in sufficient quantities to allow structure elucidation of their chemical structures. The first step in this complex process was to observe the Piz amino acid from within the complex of bacterial metabolite to determine if the bacteria was even producing the genomically-predicted Piz-containing natural product under specific laboratory conditions. 64 2.8 Experimental Section 2.8.1 General Experimental Procedures Genome mining efforts were completed using browser based BLASTP201 followed by utilization of the NCBI website gene viewer for manual curation of contigs/genomes. Obtained contigs or complete genomes were further analyzed using the web based 2ndFind analytical software (http://biosyn.nih.go.jp/2ndfind/) in conjunction with NRPS/PKS Website analysis165and antiSMASH 3, 4 and 5202,203,205 with antiSMASH 4 using the SANDPUMA204 algorithm generally used for identification of Piz-linked A-domains. 65 Chapter 3: Nitrogen NMR-guided Isolation of Piz-containing NRPs 3.1 Chapter Overview Methods for the focused isolation of low-abundance natural products with specific chemical sub-structures could expand known bioactive chemical diversity for drug discovery. As discussed, Piz-containing molecules have structural novelty and bioactivity that often accompanies the incorporation of this nonribosomal amino acid. To target Piz-containing molecules, I envisaged developing a method for selecting strains by genome mining for piperazic acid biosynthetic genes, as discussed in Chapter 2, followed by NMR-based tracking of fractions for chemical shifts uniquely associated with the piperazic acid residue itself. Such an approach would allow me to target low-abundant metabolites. The first step in using nitrogen NMR experiments to discover Piz-containing compounds was an analysis of the Piz NMR signature. To validate this method, I employed the known Piz-containing natural product producer Streptomyces sp. RJA2928. This strain was previously shown to produce the padanamides206 through a gene cluster191 containing a KtzT homolog.200 It was then necessary to determine if a piperazic acid signal was visible in Streptomyces sp. RJA2928’s crude extract. As a point of comparison, 1H-1H TOCSY and 1H-13C HSQC-TOCSY experiments were conducted on the crude extract, but the spectra obtained proved too complex to assign a Piz spin system. Simultaneously, 1H-15N HSQC NMR experiments were conducted on Streptomyces sp. RJA2928 crude extract and the spectra demonstrated that the Piz N-H correlation (δH 4.52, δN -299.8) was visible if Piz was present. However, given the crude NMR spectra's low signal-to-noise ratio, it was not possible to unequivocally rule out a false positive based on this N-H correlation. Two additional steps were developed: 1) feeding the labelled 15N2-L-Orn precursor to the natural product producers to increase the Piz N-H 15N NMR signal and 2) carrying out a second 66 experiment, the 1H-15N HSQC-TOCSY NMR experiment, to access the diagnostic spin system (Table 3.2) in a less crowded spectral window. Additionally, as the 1H-15N HSQC-TOCSY NMR experiment is less sensitive than the 1H-15N HSQC NMR experiment, incorporation of the 15N-labelled Piz precursor 15N2-L-Orn would allow access to the 1H-15N HSQC-TOCSY NMR experiment in a crude extract and when smaller quantities of the natural product were present. Upon confirming that the 15N NMR Piz signal and the spin system (with labelling) were visible in Streptomyces sp. RJA2928’s crude extract, bacteria previously selected through genome-mining (see Chapter 2.5 and 2.6) were grown on various agar media. The presence of the 15N NMR Piz signal was then used to select a bacterium for large-scale growth in a medium in which the microbe was producing Piz-compounds. The species Streptomyces incarnatus NRRL 8089 showed a clear 15N NMR Piz signal when grown on ISP4 agar medium and had a gene cluster bioinformatically predicted to have an appreciable amount of novelty. Then, Streptomyces incarnatus NRRL 8089 was grown with 15N-labelled Piz precursor 15N2-L-Orn added to ISP4 agar media. The crude extract containing labelled material was assessed for the N-H correlation and then a Piz spin system utilizing the 1H-15N HSQC-TOCSY NMR experiment. These NMR experiments on the labelled crude extract allowed unequivocal observation of the presence of a Piz-containing natural product from the extract. To obtain enough material for final isolation and structure elucidation, a larger amount of this bacterium was grown without a labelled precursor. The labelled and unlabelled extracted material were combined. The 15N NMR Piz signal and spin system were then used to guide isolation of Piz-containing metabolites from the Streptomyces incarnatus NRRL 8089 bacterium resulting in four novel compounds, three of which I isolated and then elucidated the constitutional framework as part of this thesis work. 67 3.2 Nitrogen NMR Nuclear Magnetic Resonance (NMR) is a standard method for structure elucidation of organic molecules. With steady instrument improvements and new techniques, useful NMR data can be increasingly gathered with less material and more complex molecules.44 Of the commonly found elements in natural products – hydrogens (i.e. protons), carbon, oxygen, nitrogen, and phosphorous– experiments can be run to directly probe small variations in structural environments for 1H, 13C,14N, 15N, and 31P nuclei (Table 3.1). Indirectly, chemical shift and carbon multiplicity can help to infer the presence of electronegative oxygen.207,208 The primary nuclei investigated by NMR in routine natural product structure elucidation are 1H and 13C. In particular, 1H, with its large natural abundance and large gyromagnetic ratio, is a nucleus for which it is relatively easy to obtain NMR data with even nanogram-scale material.209 The 13C nucleus is far less abundant with a smaller gyromagnetic ratio; collecting data for a natural product’s carbon skeleton requires more material and longer collection times. However, with indirect detection and high field strength, it can be obtainable at the microgram scale.209,210 Nitrogen has two NMR visible isotopes, 14N and 15N. 14N is the more abundant isotope; however, 14N signals suffer from significant line broadening due to quadrupolar relaxation.211 Thus, 15N is the more commonly observed nitrogen isotope.211 Nuclide Spin I Natural Abundance (%) Relative Sensitivity Gyromagnetic ratio γ NMR frequency [MHz] 1H 1/2 99.985 1.00 26.7519 100 13C 1/2 1.108 1.59 x 10-2 6.7283 25.144 14N 1 99.63 1.01 x 10-3 1.9338 7.224 15N 1/2 0.37 1.04 x 10-3 -2.7126 10.133 Table 3-1. NMR properties for relevant nuclei. However, the 15N isotope suffers from super-low natural abundance and a small gyromagnetic ratio (Table 3-1).212 Methods for overcoming both 15N and 13C signal 68 insensitivities incorporate a suite of two-dimensional NMR experiments where the sensitivity of the proton is transferred to the more insensitive nuclei increasing the signal-to-noise (S/N) ratio and permitting data analysis of lower abundance metabolites.212,213 A good example of this is the difference in sensitivity between the 13C signal in 1H-13C HSQC and 1H-13C HMBC spectra and one-dimensional 13C carbon spectra. In two-dimensional experiments, carbon signals can be collected (given enough protons in the molecule) through inverse detection even when material limitations prevent the collection of high quality one-dimensional 13C spectra. In general, there are two phenomena that are exploited to transfer sensitivity from the proton to insensitive nuclei like 13C and 15N: one is the nuclear Overhauser effect, and the other is polarization transfer for scalar coupled nuclei.214,215 The base experiment for non-selective sensitivity enhancement for scalar coupled nuclei is called the Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) experiment.214 The maximum sensitivity transferable by the nuclear Overhauser effect is determined by the gyromagnetic ratio of a neighboring nuclei as a dividend of twice the gyromagnetic ratio of the observed nuclei. Due to this relationship, the nuclear Overhauser effect is not of value for sensitivity enhancement of the 15N nuclei with its negative gyromagnetic ratio.215 However, polarization transfer in scalar coupled nuclei is independent of the sign of the gyromagnetic ratio and, as such, is very useful for 15N experiments.214 The one caveat is that since this polarization transfer relies on scalar coupling there must be through-bond connectivity for this enhancement to occur. Because of the larger differences in the gyromagnetic ratio between 1H and 15N and thus, a larger difference in populations of the 1H low energy and high energy spin states, the inverse detection of 15N in these 2D experiments, where polarization is transferred from 1H to 15N, results in increased detection sensitivity for 15N.215 With experiments built off the INEPT foundation,214 15N is routinely used in NMR methods for elucidating amino 69 acids for protein structure determination.216,217 The low natural abundance can be circumvented by incorporating stable isotope (or labelled) precursors into natural products to increase the 15N signal.218–221 The use of 15N in small molecule structure determination has had varied use in the literature.212,213 One barrier to its use has been the tendency to report nitrogen referenced to various internal and external references,211,222 which has made it difficult to look to the literature for typical chemical shifts that are widely applicable. Fortunately, over the last several decades, there has been a shift to reference 15N NMR spectra to either nitromethane (IUPAC recommendation) or liquid ammonia set equal to zero.212 Additionally, experimental work has been done to compare the shifts of nitromethane and liquid ammonia, meaning chemical shift values can be easily converted between these two 15N references.208,211,212,223 One advantage of nitrogen NMR is the large spectral range (~900 ppm) in different chemical environments such that different nitrogen-containing functional groups will have widely dispersed 15N chemical shifts.224 Within both proteins and secondary metabolites, there are also usually fewer nitrogen atoms (compared to protons or carbons), which means that the spectra are not as crowded as proton and carbon spectra. Specific experiments have been designed to make use of this larger spectral dispersion to provide a means of separating crowded proton and carbon signals. One such experiment is the 1H-15N-HSQC-TOCSY.224 This experiment allows observations of spin systems, i.e. the set of protons that are all scalar coupled to an adjacent proton (Figure 3.1), connected through a nitrogen’s proton. For clarity, this experiment provides TOCSY transfer from the 1HN spin to the 1Hα spin and on through adjacent J-coupled protons. Because the TOCSY transfer begins with the 1HN spin, the trace of the spin system begins with the 15N resonance at which the 1HN is attached. 1H-15N HSQC experiments can be used to link a primary or secondary nitrogen with its attached proton, which due to the 70 large dispersion of nitrogen signals, allows observation of spin systems outside the crowded proton spectral window.224 The TOCSY portion of the 1H-15N HSQC-TOCSY then allows observation of the magnetization transfer through scalar-coupled protons in order to see a complete amino acid 1H spin systems in peptide natural products (Figure 3.1). Figure 3-1. 1H-15N HSQC-TOCSY is an NMR experiment that allows us to observe the proton spin system of a single amino acid embedded in a peptide, beginning with the N-H correlation. The carbonyl of the peptide bond provides an insulator functionality that constrains observation to single amino acid spin systems in the TOCSY spectrum. 3.3 Method Development: 15N NMR-Guided Fractionation of Piz-containing NRPs 3.3.1 Piperazic NMR for Targeted Isolation of Piz Natural Products I was able to use genomic mining to predict which bacterial strains could be capable of producing novel Piz-containing NRPs; however, a robust method for isolating the metabolites was required. While liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem-mass spectrometry (LC-MS/MS) hybrid systems are commonly coupled with genome mining, depsipeptides (a common class of Piz-containing natural products) 71 usually have unexpected fragmentation patterns. Furthermore, Piz-containing peptides can often have the same mass but different structures (Figure 2-21), and I lacked high-resolution LC-MS/MS capacities in the department when the project first began. Finally, NMR is required for the final structure elucidation of complex peptides. Thus, LC-MS did not seem the best tool for analysis of bacterial extracts for novel Piz-containing natural products. Considering this, I noted that proteinogenic amino acids have characteristic NMR spin systems visible in 1H-1H TOCSY NMR experiments, which are routinely used for protein structure elucidation.224 Although piperazic acid is a nonribosomal amino acid, I hypothesized that it, like proteogenic amino acids, would likely have a characteristic spin system that was distinguishable in such experiments like 1H-1H TOCSY or 1H-13C HSQC-TOCSY. To substantiate the hypothesis that Piz would have a characteristic spin system, I analyzed experimental data on Piz-containing natural products reported in the literature (Table 3-2) and observed that Piz-containing natural products have predictable and unique NMR proton spin systems. Furthermore, in several reported literature values, the shielded secondary nitrogen Table 3-2. Piperazic acid has unique NMR signatures for its nitrogen, nitrogen-attached proton, and complete amino acid spin system. experimentally shown to possess a chemical shift of ~ δN −300 (referenced to CH3NO2) in a unique area of the nitrogen spectral field (Table 3.3). Given the partial negative charge predicted to reside on the N-6 of piperazic acid, this increased shielding due to increased electron density 72 is predictable.225 Importantly, this unique nitrogen shift is coupled to a distinctly shielded proton compared to amide protons in peptide-based natural products. This nitrogen-attached proton has a chemical shift shown experimentally to fall between δH [4.1 to 5.47] in DMSO-d6 (Table 3.2 and 3.3), with even greater shielding in other deuterated solvents such as CDCl3 and ACN-d3. This shift contrasts with the normal NMR chemical shift range of amide protons seen as predictable markers of peptides, which tend to fall between δH [6.7 to 8.7] in DMSO-d6.211 This analysis suggested that the 1H-15N HSQC NMR experiment could potentially identify these compounds from within a crude mixture of bacterial metabolites that commonly also contain amino acids, simple peptides and diketopiperazines. The potential of observing the presence or absence of piperazic acids from within a crude bacterial extract would allow for: 1) selecting laboratory conditions in which the genome-mined bacteria were producing the Piz-containing metabolite, and 2) directing a pathway for the isolation workflow to streamline isolation work. Thus, if the N-H signal was visible in the crude extract, the 15N NMR-guided fractionation workflow could mimic the bioassay-guided fractionation workflow. Bioassay-guided fractionation has been the gold standard for decades because it allows the isolation chemist to focus on both the organisms and fractions of interest, leading (ideally) to bioactive compounds (Figures 1.8 and 1.9).226 Using nitrogen NMR experiments to select for the presence of piperazic-acid compounds would not lead directly to bioactive compounds; however, piperazic acid-containing compounds are overwhelmingly associated with bioactivity173 and often with structural novelty. 73 Table 3-3. Experimental NMR chemical shifts in DMSO-d6 for Piz within natural products as obtained from literature sources.227 Natural Product Type of Piz α β γ δ N-NH N-NH N-NH δH (δC) δH δH δH (δC) δH δN δN dentigerumycin C* L-Piz 4.55 (52.6) 1.8 1.3 2.7 (46.4) 5.47 D-Piz 5.58 (47.7) 1.8 1.42 2.7 (46.8) 5.2 D-Piz 5.44 (47.7) 1.8 1.43 2.7 5.18 dentigerumycin D* L-Piz 4.84 (50) 1.8 1.48 2.7 (46.4) 5 D-Piz 5.58 (47.8) 1.8 1.48 2.7 (46.4) 5.04 D-Piz 5.44 (47.6) 1.8 1.53 2.89 (45.6) 5.11 matlystatin* L-Piz 5.00 (49.7) 1.8 1.36 2.7(46.3) 4.74 Sch 382582* L-Piz 5.12 (49.52) 1.8 1.49 2.8 (46.38) 4.92 hytramycins* D-Piz 4.94(51.91) 1.8 1.5 2.8 (48.48) 4.96 D-Piz 5.14(49.03) 1.8 1.47 2.8 (48.64) 4.99 L-Piz 5.7 (48.58) 1.8 1.45 2.8 5.07 padanamides* L-Piz 4.97 (50.9) 1.8 1.35 2.79 (46.8) 4.5 -299.9 -232 piperidamycin A Ave x 3 5.81 (47.62) 1.8 1.5 3.00/2.58 (46.48) 5.2 γ-OH 4.81 (48.77) 2.3, 1.8 3.6 2.8 (52.38) 4.97 sanglifehrins peptide bond to N5 1.92 (58.0) 1.45 1.65/1.30 4.20/2.65 (40.5) 4.82 -297.5 -239.7 soliseptide A L-Piz 5.06 (50.4) 2.06/1.54 1.54/1,46 2.97/2.59 5.43 γ-OH 4.97 (46.7) 2.03/1.82 3.65 2.9/2.85 (52.6) 5.49 svetamycins γ-chl 5.73(49.8) 2.41/1.90 4.36 2.66/3.38 (54) 5.23 δ-Me 4.88 (49.7) 2.17/1.76 1.56/1.51 2.69 (52.3) 4.14 δ,δ-dMe 4.88 (49.7) 2.07/1.96 1.42/1.34 2.69 (50.9) 4.57 himastatin γ-OH 5.08 2.44/1.92 3.77 3.02/2.79 5.37 *α, β, and γ positions are averages of the diastereotopic δH 74 3.3.2 Analysis of the Piperazic Acid NMR Signature in a Known Producer To assess the viability of a 15N NMR-based method for screening for piperazic acid-containing peptides, I first evaluated the crude metabolite mixture of Streptomyces sp. RJA2928. This species was previously shown to produce the Piz-containing natural product padanamide206 (Figure 3-2) and had a known gene cluster containing a ktzT homolog200. Initially, this species was grown on 2 L of agar medium to provide 600 milligrams (mg) of a crude metabolite mixture (hereby referred to as “crude”) from which an aliquot was evaluated for the Piz NMR N-H correlation using the 1H-15N HSQC experiment as well as the Piz spin system using 1H-1H TOCSY and 1H-13C HSQC-TOCSY NMR experiments. The spectra obtained from 1H-1H TOCSY and 1H-13C HSQC-TOCSY proved sufficiently complex to prevent unequivocal confirmation of the production of padanamides within the crude mixtures, while the 1H-15N HSQC experiment did not show the Piz N-H correlation. As I was uncertain if this lack of signal was simply because the N-H correlation was not visible in the crude mixture, fractionation of the crude was performed. Streptomyces sp. RJA 2928’s crude metabolite mixture was passed through a SephadexTM LH-20 column to create three fractions which were each evaluated using 1H-15N HSQC experiment, 1H-1H TOCSY and 1H-13C HSQC-TOCSY NMR experiments. Analysis of the three spectra revealed no change from the crude mixture, and it seemed apparent Figure 3-2. Padanamide A’s structure and Piz NMR chemical shifts. 75 that the nitrogen signal likely needed to be enhanced with labelled 15N2-L-Orn. I also considered that the frozen Streptomyces sp. RJA2928 used for inoculation and growth was a late-stage generation that might have degenerated into being a non-producer of padanamides. Unpublished work in the Ryan and Andersen labs had demonstrated that padanamides could change through colony generations. To resolve these questions and to investigate the detection limit of the experiment, first-generation Streptomyces sp. RJA2928 was revived from the -80 ℃ cryostorage and grown in small quantities both with labelled 15N2-L-Orn (L) and with unlabelled L-Orn (UL) (Figure 3-3). Streptomyces sp. RJA2928 Media Volume Additives Crude EtOAc Extract MM1 30 mL 20 mg 15N2-L-Orn 11.5 mg MM1 30 mL 20 mg L-Orn 10.5 mg Figure 3-3. Prepared Petri plates of known Piz producer Streptomyces sp. RJA2928. After extraction, the crude’s L and UL were evaluated for Piz NMR signals (Table 3.4, Figure 3.4). A signal in the region of ~ δN -300 ppm and δH 4.52 ppm was visible in both samples as seen in Figure 3-4, but it was difficult (to impossible) to distinguish from noise in the unlabelled sample. These results showed that if the producer made the Piz-containing natural product, the correlation was visible, albeit very weakly, even in small amounts of material and with natural abundance 15N. However, adding labelled 15N2-L-Orn to the medium provided strongly enhanced S/N and diagnostic N-H Piz NMR signature observation. The 1H-15N HSQC-TOCSY NMR experiment was then performed to access the diagnostic Piz amino acid spin system. However, it is an insensitive experiment compared to the 1H-15N HSQC experiment, requiring both increased compound concentration and increased time (up to 7-9 hours vs 48 mins), and attempts to collect this data on samples L and UL failed. L UL 76 Figure 3-4. 1H-15N HSQC NMR for Samples L and UL of known Piz producer Streptomyces sp. RJA2928. In order to obtain more material to verify that the Piz spin system from padanamides was observable in the crude, the quantity of bacteria grown was increased. A tray (37 cm X 20 cm) of solid MM1 agar was prepared with 100 mg 15N2-L-Orn added to 400 mL of MM1 marine media. After growth and extraction, a small aliquot of this sample ScL, (~20 mg) was first subjected to the 1H-15N HSQC experiment followed by the 1H-15N HSQC-TOCSY experiment. The crude obtained from this experiment using medium containing 15N2-L-Orn gave strong enough NMR signals to allow observation of both the N-H correlation and the Piz spin system (Figure 3.5) in the 1H-15N HSQC-TOCSY NMR experiment. The reduction of NMR acquisition time in the 1H-15N HSQC was also a valuable benefit, as shown in Table 3-4. Sample Agar Medium Amount of Media L-Orn (mg) 15N2-L-Orn (mg) 1H-15N HSQC NMR Time for 15N experiment 1H-15N HSQC-TOCSY L MM1 30 mL 0 20 Yes 96 min Not Observable UL MM1 30 mL 20 0 Very weak 96 min Not Observable ScL MM1 400 mL 0 100 Yes 48 min* Yes Table 3-4. Analysis of Piz production in a known producer. *Less intense signal available in ~15-20min. 77 Figure 3-5. NMR spectra of crude metabolite extract of Streptomyces sp. RJA2928 grown with labelled 15N2-L-Orn. a) 1H-15N HSQC spectrum demonstrating a Piz N-H correlation b) 1H-15N HSQC-TOCSY spectrum demonstrating padanamides’ Piz spin system. 3.3.3 Strain and Media Selection Through Diagnostic NMR Resonances Initially, several strains predicted to have the genomic capacity to produce Piz-containing natural products, as seen in Table 3-5, were ordered from the Agricultural Research Service (NRRL) Culture Collection. NRRL collection strains were specifically chosen as they are: 1) characterized Biosafety Level-One non-pathogenic bacteria that create minimal hazards;228 2) readily available to any research program; and 3) free-of-charge if under a certain number were ordered. Next, it was considered that secondary metabolite production varies depending on environmental conditions.229 In the laboratory, this results in a variance of secondary metabolites produced when growth media varies, so there is no certainty that genotype will result in the desired metabolite phenotype. Therefore, several growth media were selected for each NRRL strain (Table 3.5). The four strains listed in Table 3.5 were obtained from the Agricultural Research Service Culture Collection, revived and grown on large Petri plates with added labelled precursor, harvested, and extracted to obtain 15-30 mg of crude material. 1H-15N HSQC spectra Sample ScL: 1H-15N HSQC Sample ScL: 1H-15N HSQC-TOCSY TD=32 TD=128 78 were obtained for each crude sample. Several species were selected based on these results for scale-up. Appearance of Bacteria on Agar Media Strain L-Orn MM1 F MS Streptomyces phaeochromogenes NRRL B-1248 Labelled white spores on brown stain white spores, yellow stain Streptomyces griseus subsp. griseus strain NRRL WC-3645 Labelled white spores, leathery pinkish white, leathery Streptomyces griseus NRRL F-5144 Labelled yellow spores on deep yellow stain yellow/green spores w. lemon yellow stain Streptomyces sp. NRRL F-2664 Labelled pink spores, dark brown stain white spores, golden yellow stain Table 3-5. Initial genome mined strains with selected media. Based on the spectrum seen in Figure 3-6, the strain Streptomyces phaeochromogenes NRRL B-1248 was grown on a tray of MM1 F agar with 100 mg of 15N2-L-Orn added to the media. After growth and extraction, it was fractionated utilizing the Piz N-H correlation at δH 5.55; δN −304 as the guide. At the end of the isolation workflow, the N-H signal was present, but the compound was not present in amounts that allowed collection of a 2-D data set towards structure elucidation. From this result, it was understood that although a strong signal from labelled material could act as a guide in the isolation process, it would not guarantee that there was enough material for isolation and structure elucidation. The decision was made to focus on other strains rather than scale up this strain. Subsequently, Streptomyces griseus subsp. griseus strain NRRL WC-3645 (Figure 3-8) was grown with a small amount of labelled material and a larger amount of unlabelled material. The extracts obtained from both media were combined together and separated using the N-H correlation, δH 5.88; δN −309.2, as a guide without the use of the 1H-15N HSQC-TOCSY experiment. This N-H correlation was slightly outside the boundaries observed from examining literature values in Table 3-2 yet was close enough to these values to investigate if this N-H 79 correlation belonged to a Piz-containing compound. Upon separation through bench-top chromatographic steps and purification on reversed-phase HPLC, the compound possessing this signal was determined to be para-aminobenzoic acid (PABA), a common primary metabolite. This result demonstrated that 1) the secondary piece of data provided by 1H-15N HSQC-TOCSY experiment was invaluable for increasing isolation efficiency; and 2) combining labelled and unlabelled material allowed a nitrogen-NMR guided fractionation to isolate at least a primary metabolite. Nitrogen NMR Strain Media Extract δH. δN Streptomyces phaeochromogenes NRRL B-1248 MM1 F 5.55 ; -304 Figure 3-6 Streptomyces griseus subsp. griseus strain NRRL WC-3645 MM1 F 5.88; -309.2 Figure 3-7 4.17, -299 MS 5.88; -309.2 Figure 3-8 Streptomyces griseus strain NRRL F-5144 ISP4 5.49, -302 (v weak) Figure 3-10 5.12, -300 (v weak) TSB 5.45, -302 Figure 3-9 Streptomyces sp. NRRL F-2664 MS 5.41, -302 Figure 3-11 Table 3-6. Initial genome mined strains with initial nitrogen NMR experiments per media grown. Of these strains, initial attempts to grow Streptomyces griseus strain NRRL F-5144 did not result in observations of the N-H Piz correlation. Streptomyces griseus strain NRRL F-5144 was grown on several more media including ISP4 and TSB (Figure 3-9 and 3-10). The bacterium was cultured in small amounts but this did not provide enough material for isolation of the suspected polyoxypeptin congener. Larger amounts of this bacterium would need to be grown for successful isolation efforts. 80 Figure 3-6. 1H-15N HSQC of Streptomyces phaeochromogenes NRRL B-1248 extract from MM1 F agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-7. 1H-15N HSQC of Streptomyces griseus subsp. griseus NRRL WC-3645 extract from MM1 F agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 81 Figure 3-8. 1H-15N HSQC of Streptomyces griseus subsp. griseus NRRL WC-3645 extract from Mannitol Soy agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-9. 1H-15N HSQC of Streptomyces griseus NRRL F-5144 extract from TSB agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 82 Figure 3-10. 1H-15N HSQC of Streptomyces griseus NRRL F-5144 extract from ISP4 agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-11. 1H-15N HSQC of Streptomyces sp. NRRL F-2664 extract from Mannitol Soy agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 83 Nine additional strains (Table 3-7) were obtained from the Agricultural Research Service Culture Collection and revived in tryptic soy broth to continue the investigation into genome-mined strains. After successful growth, each bacterium was grown on four different agar media prepared as large Petri plates. The EtOAc extracts of each medium’s trial were dried under reduced pressure to yield 20-30 mg of crude material. 1H-15N HSQC spectra were obtained for each crude sample after dissolving in DMSO-d6 with TD=32 scans in the F1 dimension and 48 scans in the F2 dimension (Table 3-8, Figures 3-12 to 3-21). The in silico prediction of KtzT homolog-containing gene clusters were reexamined for extracts in which a putative Piz N-H correlation was observed. Strains were selected for growth with labelled 15N2-L-Orn based on their Piz N-H NMR signal and potential novelty of the cluster. 84 Appearance of Bacteria on Agar Media Strain L-Orn MM1 F MS ISP4 TSB Streptomyces katrae NRRL B-16271 Unlabelled small white pink spores cream, few white spores cream, some white spores, light pink stain Off-white spores Streptomyces tsukubensis NRRL18488 Unlabelled Did not grow Streptomyces sp. NRRL F-2747 Unlabelled large white colonies turn pink with time. Pink/brown stain small white colonies large pink spores small white colonies Streptomyces sp. NRRL F-5053 Unlabelled cream small cream, small cream, white spores lots of white spores Streptomyces sp. NRRL F-6131 Unlabelled mauve stain, white spores light brown stain, no spores pink stain, lots of white spores orange stain, wrinkled Streptomyces sp. NRRL B-1140 Unlabelled Did not grow Streptomyces incarnatus NRRL 8089 Unlabelled brown stain tiny, clear white spores small, clear tiny clear spores Streptomyces sp. NRRL B-1347 Unlabelled Off-white colored, leathery Off-white spores green stain, yellow spores wrinkled, cream Streptomyces sp. S920 Unlabelled thin white film large white wrinkled pinkish colonies very few white wrinkled Table 3-7. Additional genome-mined bacterial strains listed with the various growing media. Nitrogen NMR Strain Media Extract δH. δN Streptomyces katrae NRRL B-16271 MS 5.43, -303 Figure 3-12 TSB 5.43, -303 Figure 3-13 Streptomyces sp. NRRL F-2747 TSB 5.48, -301.0 Figure 3-14 Streptomyces sp. NRRL F-5053 None No signal N/A Streptomyces sp. NRRL F-6131 MM1 F 5.45; -301 Figure 3-15 TSB 5.45; -301 Figure 3-16 Streptomyces incarnatus NRRL 8089 ISP4 5.43, -301 Figure 3-17 5.11, -300 (v.v weak) Figure 3-17 TSB 5.43, -302 Figure 3-18 Streptomyces sp. NRRL B-1347 MM1 F 5.45; -303.6 (v weak) Figure 3-19 MS 5.40, -302.0 Figure 3-20 Streptomyces sp S920 MS 5.58, -302.6 Figure 3-21 Table 3-8. Remainder of the genome-mined strains and their nitrogen NMR N-H correlations. 85 Figure 3-12. 1H-15N HSQC of Streptomyces katrae NRRL F-16271 extract from Mannitol Soy agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-13. 1H-15N HSQC of Streptomyces katrae NRRL F-16271 extract from TSB agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 86 Figure 3-14. 1H-15N HSQC of Streptomyces sp. NRRL F-2747 extract from TSB agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-15. 1H-15N HSQC of Streptomyces sp. NRRL F-6131 extract from MM1 F agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 87 Figure 3-16. 1H-15N HSQC of Streptomyces sp. NRRL F-6131 extract from TSB agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-17. 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 extract from ISP4 agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 88 . Figure 3-18. 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 extract from TSB agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-19. 1H-15N HSQC of Streptomyces sp. NRRL B-1347 extract from MM1 F agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 89 Figure 3-20. 1H-15N HSQC of Streptomyces sp. NRRL B-1347 extract from Mannitol Soy agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. Figure 3-21. 1H-15N HSQC of Streptomyces sp. S920 extract from Mannitol Soy agar, recorded at 600 MHz, in DMSO-d6. Red box indicates presence of Piz-like N-H correlations. 90 3.3.4 Gene Cluster Analysis of Streptomyces incarnatus NRRL 8089 As discussed in Section 2.6, BLASTP searches for KtzT homologs returned a KtzT homolog contained within a contig with Accession no. CP011498.1. This contig was assembled from a draft sequencing of the actinomycetes species Streptomyces incarnatus NRRL 8089, which resulted in 76 contigs containing 8,266 predicted genes for this 8.9 Mb genome.230 Streptomyces incarnatus NRRL 8089 was isolated from Indian soil in 1977 and identified as a novel bacterial species.231 Interestingly, it has been shown to produce analogs of the potent antifungal natural product sinefungin231 as well as contain the biosynthetic machinery capable of producing an analog of the unusual natural product foxicin.232 Streptomyces incarnatus NRRL 8089 appears to be ‘a biosynthetically talented’ species.233 antiSMASH analysis of contig CP011498.1 revealed that the entire 177 kilobase pair (kb) contig was a hybrid NRPS-PKS gene cluster. This is an unusually large size for such a gene cluster. Biosynthetic studies have demonstrated that hybrid NRPS-PKS gene clusters are larger than single origin clusters; with an average of 71kb for hybrid, 51 kb for PKS, and 45 kb for NRPS clusters. The largest predicted hybrid has been bioinformatically shown to be 185 kb.234 Still, gene clusters for characterized Piz-containing 19-membered depsipeptides have ranged in size from 61-100 kb. However, as of now, there has not been a full biosynthetic characterization of all the transformations that lead to any one Piz-containing 19-membered depsipeptide. It is thus possible that there has been some premature shortening of predicted secondary metabolite biosynthetic gene clusters (smBGC). In this case, a size of 177 kb for the Streptomyces incarnatus NRRL 8089 gene cluster could be accurate but with current knowledge this is unlikely. 91 Scheme 3-1. a) Gene cluster from Streptomyces incarnatus NRRL 8089 that contains a homolog to ktzT, represented here from genes AKJ15806.1 to AKJ15860.1 b) A close-up on the gene cluster emphasizing the NRP and PK modules, predicted domains and A/AT domain specificities. After the initial antiSMASH analysis, each gene of the contig was analyzed utilizing the browser-based program FramePlot to confirm that each assigned open reading frame had typical Streptomyces G/C codon usage and contained ribosomal binding sites. After analysis by FramePlot, each gene was then analyzed using BLASTP to confirm the functional annotation assigned by antiSMASH (Table 3-10). Next, in silico analyses were performed to obtain a clearer picture of the beginning and end of the S. incarnatus NRRL 8089 putatively Piz-containing smBGC. A MultiGeneBlast analysis159 was performed to compare against similar gene clusters in anticipation that one of those gene clusters would contain clear cluster boundaries. Such a gene cluster was not observed. However, through BLASTP searches, it was evident that the individual genes routinely returned the highest sequence homology to genes in the strain Streptomyces sp. 75 (Table 3.10). Streptomyces sp. 75’s predicted gene cluster had flanking transposases over an ~90 kb region. The NRRL 8089 contained a transposase on one 92 end (Accession no. AKJ15852.1) with limited sequence homology to one of the Streptomyces sp. 75’s transposase. In between the two Streptomyces sp. 75’s transposases, NRRL 8089 genes demonstrated high sequence homology to the vast majority of Streptomyces sp. 75’s gene. On the other side of the second transposase, the identity and sequence homology between the genes of Streptomyces sp. 75’s and S. incarnatus NRRL 8089 significantly decreased. Thus, the two Streptomyces sp. 75 transposases were used to roughly estimate the size of Streptomyces incarnatus NRRL 8089’s putative Piz-containing NRP-PK producing gene cluster as a highly speculative 90 kb (Scheme 3-1-a). Initial in silico analysis of the Streptomyces incarnatus NRRL 8089 gene cluster predicted that it was producing an azinothricin-type molecule (Scheme 3-1-b). antiSMASH, PKS/NRPS analysis, nor NRPSpredictor2 were able to give a complete A-domain specificity prediction on the number of Piz amino acids present in the produced molecules. Initially, the SANDPUMA algorithm predicted the presence of two Piz’s within the molecule, and the other two algorithms did not predict the presence of Piz-specific A domains. In seeking novel compounds, it was noted that there are numerous azinothricin-type compounds with two Piz’s (Figures 2-4 and 2-5). Further comparison (Figure 3-22) with the module and domain organization of biosynthetically-characterized 19-membered depsipeptide compounds that contain both NRP and PK modules [polyoxypeptin (2.45), aurantimycin (2.41) (seen in Figure 2.5) dentigerumycin A (4.5), and dentigerumycin B (4.6) with 4.7 and 4.8 being linear peptide congeners) (seen in Figure 4.1)] was then completed. This led to the observation that the S. incarnatus NRRL 8089 gene cluster contained a fifth main NRPS module leading to a total of seven A domains. When contrasted with the six A domains found within the main NRPS modules in all known 19-membered depsipeptides (Figure 3-22) this difference led to the 93 speculation that the produced compound could have some novelty. However, there was uncertainty as to why the two adjacent NRPS modules both contained TE domains. Several hypotheses were: one of the TE domains was non-operational although that was not indicated via the in silico analysis; the bacteria was producing a new variant of the Piz-containing NRP-PKS depsipeptides; or that the bacteria was somehow funneling the assembly into one (AKJ15828.1) or the other (AKJ15829.1) of the NRPS TE domain-containing modules. BLASTP analysis of the major NRP and PK domains (Table 3.9) against the all five characterized 19-membered depsipeptide compounds demonstrated clear relation to this type of compound but with lower sequence homology in NRPS modules AKJ15828.1 and AKJ15829.1, suggesting possible divergence. 94 Figure 3-22. Comparison of Streptomyces incarnatus NRRL 8089 predicted gene organization with the gene organization of partially characterized 19-membered azinothricin-like gene clusters. 95 Table 3-9 BLASTP comparison of the genome mined Streptomyces incarnatus NRRL 8089 with characterized 19-membered depsipeptides. Natural Product Genome-mined Strain Polyoxypeptin (2.45) Aurantimycin (2.41) Dentigerumycin A (4.5) Dentigerumycin B/C/D (4.6, 4.7**, 4.8**) Marformycins (2.51) Bacterial Strains Streptomyces incarnatus NRRL 8089 Streptomyces sp. MK498-98F14 Streptomyces aurantiacus JA 4570 Pseudonocardia sp. AL041005-10 Streptomyces sp. M41 Streptomyces drozdowiczii strain SCSIO 10141 taxid: 1414447 taxid: 1286094 taxid: 47760 2 taxid id’s for different sequenced genomes of the same strain taxid : 445576 2 reference genomes deposited under this taxid id taxid 1955065 taxid: 202862 Proposed function Accession no. Identity/Similarity to the Streptomyces incarnatus NRRL 8089 protein sequence for a gene. Accession no. or Enzyme name of gene protein sequence with highest identity. L-lysine 6-monooxygenase AKJ15810.1 45/5 AGZ15458.1 72/95 EPH43660.1 59/95 ALE77168.1 84/99 WP_081222078.1 MfnI 50/94 transcriptional regulator (KtzT homolog) AKJ15811.1 No significant similarity (nss) 75/99 EPH43659.1 old: 53/100 WP_083474028.1 new: 52/97 WP_145983356.1 87/100 WP_081222077.1 MfnJ 54/99 thioesterase AKJ15821.1 34/95 AGZ15477.1 EPH39677.1 62/96 ; EPH43674.1 35/99 33/87 WP_062394358.1 74/94 WP_081222075.1 nss FAD-dependent oxidoreductase AKJ15825.1 87/99 AGZ15457.1 WP_016638468.1 79/99 69/99 WP_062394347.1 87/99 WP_081222067.1 nss peptide synthetase AKJ15826.1 82/99 AGZ15458.1 80/95 WP_158691993.1 65/98 ALE77164.1 72/99 WP_081222066.1 MfnC 45/98 peptide synthetase AKJ15827.1 74/100 AGZ15459.1 69/100 WP_016638470.1 59/96 ALE77166.1 70/99 WP_081222065.1 MfnC 47/99 peptide synthetase AKJ15828.1 57/99 AGZ15460.1 59/100 WP_016638471.1 43/80 ALE77165.1 78/100 WP_081222064.1 MfnE 45/99 peptide synthetase AKJ15829.1 60/99 AGZ15460.1 75/100 WP_016638471.1 44/65 ALE77165.1 57.24/99 WP_081222064.1 MfnE 44/99 thioesterase AKJ15830.1 84/100 AGZ15461.1 WP_106967774.1 82/100 35/95 ALE77171.1 85/98 WP_081222063.1 nss 96 cytochrome P450 AKJ15833.1 81/100 AGZ15470.1 86.54/100 WP_016638746.1 65.06/99 WP_062394378.1 85/100 WP_081222060.1 MfnN 31/91 thioesterase AKJ15834.1 80/99 AGZ15471.1 (thioesterase II) 76/99 WP_016638477.1 31/82 ALE77169.1 76/96 WP_081222059.1 modular polyketide synthase AKJ15895.1 75/100 AGZ15472.1 77/100 WP_078620910.1 53/99 ALE77181.1 70/99 WP_081222058.1 Type I polyketide synthase AKJ15835.1 74/99 AGZ15473.1 71/99 WP_106967775.1 49/93 WP_062394429.1 ; [KR-T-KS-AT-DH-KR-T-KS]* 58/35 ALE77178.1 71/100 WP_143661103 Type I polyketide synthase AKJ15836.1 76/98 AGZ15474.1 75/100 WP_016638480.1 50/84 WP_062394412.1; [KS-AT-DH-ER-KR]* 67/29 ALE77178.1 73/99 WP_081222056.1 acyl transferase AKJ15837.1 82/99 AGZ15475.1 79/99 WP_016638481.1 62/99 ALE77175.1 76/99 WP_081222055.1 non ribosomal peptide synthetase AKJ15896.1 73/100 AGZ15476.1 70/100 WP_078620912.1 47/99 ALE77173.1 69/100 WP_081222054.1 *NRPS/PKS Analysis was used to determine domains of select modules which were highly truncated in the reference genome reported for dentigerumycin A ** 4.7 and 4.8 are linear peptide congeners of the cyclic depsipeptide 4.6; they are all produced from the same gene cluster Table 3-9. BLASTP comparison of the NRP and PK domains of the genome mined Streptomyces incarnatus NRRL 8089 with characterized 19-membered depsipeptides.97 Table 3-10. Streptomyces incarnatus NRRL 8089: list of bioinformatically predicted genes from the contig with accession no. CP011498, pg. 1 of 3 Size (a.a) Accession # Proposed Function Closest Homolog Source Identity/ Similarity 849 AKJ15806.1 amidinotransferase WP_115914648.1 Streptomyces sp. 75 91/100 942 AKJ15807.1 hypothetical protein WP_115914649.1 Streptomyces sp. 75 74/56 909 AKJ15808.1 acetylglutamate kinase WP_115914654.1 Streptomyces sp. 75 70/97 237 AKJ15809.1 MbtH family protein WP_115914744.1 Streptomyces sp. 75 88/85 1353 AKJ15810.1 L-lysine 6-monooxygenase WP_115914650.1 Streptomyces sp. 75 86/100 645 AKJ15811.1 FMN-binding negative transcriptional regulator WP_115914651.1 Streptomyces sp. 75 90/100 777 AKJ15812.1 ABC transporter permease WP 115907869.1 Streptomyces sp. 75 83/99 975 AKJ15813.1 ATP-binding cassette domain-containing protein WP_093931193.1 Streptomyces sp. 75 84/97 693 AKJ15814.1 hypothetical protein WP_115914652.1 Streptomyces sp. 75 86/100 741 AKJ15815.1 MarR family transcriptional regulator WP_115907872.1 Streptomyces sp. 75 68/99 1215 AKJ15816.1 acetylornithine transaminase WP_115914653.1 Streptomyces sp. 75 89/99 906 AKJ15892.1 acetylglutamate kinase WP_115914654.1 Streptomyces sp. 75 87/100 1152 AKJ15817.1 bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase ArgJ WP_115914655.1 Streptomyces sp. 75 91/100 1029 AKJ15818.1 N-acetyl-gamma-glutamyl-phosphate reductase WP_115914656.1 Streptomyces sp. 75 93/100 1182 AKJ15819.1 class A beta-lactamase-related serine hydrolase WP_115914657.1 Streptomyces sp. 75 75/93 984 AKJ15820.1 hypothetical protein WP_115914658.1 Streptomyces sp. 75 85/100 744 AKJ15821.1 thioesterase WP_115914659.1 Streptomyces sp. 75 87/99 216 AKJ15822.1 MbtH family protein WP_115914660.1 Streptomyces sp. 75 93/98 645 AKJ15893.1 regulator WP_115914745.1 Streptomyces sp. 75 92/100 1581 AKJ15823.1 amino acid adenylation domain-containing protein WP_115914661.1 Streptomyces sp. 75 88/100 303 AKJ15824.1 acyl carrier protein WP_115914662.1 Streptomyces sp. 75 97/76 1188 AKJ15825.1 NAD(P)/FAD-dependent oxidoreductase WP_115914663.1 Streptomyces sp. 75 98/100 7794 AKJ15826.1 amino acid adenylation domain-containing protein WP_115914664.1 Streptomyces sp. 75 93/100 7767 AKJ15827.1 amino acid adenylation domain-containing protein WP_115914665.1 Streptomyces sp. 75 89/100 3888 AKJ15828.1 peptide synthase WP_115914666.1 Streptomyces sp. 75 92/100 3789 AKJ15829.1 nonribosomal peptide synthetase [Streptomyces ] WP_115914666.1 Streptomyces sp. 75 60/99 735 AKJ15830.1 thioesterase WP_115914746.1 Streptomyces sp. 75 93/100 98 Table 3-10. continued pg. 2 of 3 972 AKJ15831.1 ATP-binding cassette domain-containing protein WP_115914667.1 Streptomyces sp. 75 94/100 726 AKJ15894.1 ABC transporter permease WP_115914668.1 Streptomyces sp. 75 95/100 261 AKJ15832.1 acyl carrier protein WP_115914669.1 Streptomyces sp. 75 91/100 1251 AKJ15833.1 cytochrome P450 WP_115914670.1 Streptomyces sp. 75 92/100 738 AKJ15834.1 thioesterase WP_115914671.1 Streptomyces sp. 75 90/99 3084 AKJ15895.1 type I polyketide synthase WP_115914672.1 Streptomyces sp. 75 87/99 5577 AKJ15835.1 SDR family NAD(P)-dependent oxidoreductase WP_115914673.1 Streptomyces sp. 75 87/100 6585 AKJ15836.1 SDR family NAD(P)-dependent oxidoreductase WP_115914674.1 Streptomyces sp. 75 87/100 3111 AKJ15837.1 acyltransferase domain-containing protein WP_115914675.1 Streptomyces sp. 75 92/99 3132 AKJ15896.1 nonribosomal peptide synthetase WP_115914676.1 Streptomyces sp. 75 88/100 1359 AKJ15838.1 crotonyl-CoA carboxylase/reductase WP_115914677.1 Streptomyces sp. 75 92/100 990 AKJ15839.1 ketoacyl-ACP synthase III wp 115914678.1 Streptomyces sp. 75 94/100 885 AKJ15840.1 3-hydroxybutyryl-CoAdehydrogenase WP_115914679.1 Streptomyces sp. 75 92/99 759 AKJ15841.1 thioesterase WP_115914680.1 Streptomyces sp. 75 84/100 1500 AKJ15842.1 3-(3-hydroxy-phenyl)propionate hydroxylase REE26142.1 Streptomyces sp. 75 83/99 1578 AKJ15843.1 acyl-CoA carboxylase subunit beta WP 115914681.1 Streptomyces sp. 75 94/100 519 AKJ15844.1 hypothetical protein WP_115914682.1 Streptomyces sp. 75 92/100 843 AKJ15845.1 hypothetical protein WP_115914683.1 Streptomyces sp. 75 93/99 1008 AKJ15846.1 ATP-binding cassette domain-containing protein WP_115907870.1 Streptomyces sp. 75 82/94 777 AKJ15847.1 ABC transporter permease WP_115907869.1 Streptomyces sp. 75 83/99 231 AKJ15848.1 [protein-PII] uridylyltransferase WP_115905648.1 Streptomyces sp. 75 39/36 198 AKJ15849.1 hypothetical protein WP_115911074.1 Streptomyces sp. 75 50/26 396 AKJ15897.1 dipeptidase WP_115906062.1 Streptomyces sp. 75 38/29 2589 AKJ15850.1 FtsX-like permease family protein WP_115907726.1 Streptomyces sp. 75 31/28 723 AKJ15851.1 ABC transporter ATP-binding protein WP_115907746.1 Streptomyces sp. 75 42/99 303 AKJ15852.1 IS630 family transposase WP_115914754.1 Streptomyces sp. 75 31/35 753 AKJ15853.1 Thioesterase WP 115914629.1 Streptomyces sp. 75 51/96 324 AKJ15854.1 Acyl carrier protein WP 115914627 Streptomyces sp. 75 32/69 525 AKJ15855.1 putative flippase GtrA REE26146.1 Streptomyces sp. 75 84/98 189 AKJ15856.1 beta-galactosidase WP_115907185.1 Streptomyces sp. 75 29/83 1227 AKJ15857.1 cytochrome P450 WP_115914684.1 Streptomyces sp. 75 88/100 99 Table 3-10. Streptomyces incarnatus NRRL 8089: list of bioinformatically predicted genes from the contig with accession no. CP011498, pg. 3 of 3. 903 AKJ15898.1 1-deoxy-D-xylulose-5-phosphate synthase WP_115910440.1 Streptomyces sp. 75 32/66 693 AKJ15858.1 Transketolase WP 115910169.1 Streptomyces sp. 75 36/86 1128 no annotation in 8089 MFS transporter WP_115914646.1 Streptomyces sp. 75 46/95 1011 AKJ15859.1 Aldo/keto reductase WP 115910951 Streptomyces sp. 75 40/96 561 AKJ15860.1 ribosomal-protein-alanine N-acetyltransferase REE32307.1 Streptomyces sp. 75 35/63 100 3.3.5 15N NMR Guided Fractionation of Piperazic Acid-Containing Metabolites in Streptomyces incarnatus NRRL 8089 A significant challenge in any natural discovery program is to separate the desired compounds from the complex mixture of primary and secondary metabolites in an organism’s crude metabolite extract. Streptomyces incarnatus NRRL 8089 was chosen as a strain to further investigate due to its clear and stable 15N NMR signature as well as the potential novelty indicated by its putative gene cluster. In the proton spectrum of S. incarnatus NRRL 8089 crude mixture shown in Figure 3-24, a complicated crude extract is observed. From this proton spectrum of the crude extract alone, it would be challenging, if not impossible, to make an informed decision on whether the extract contained any Piz-containing natural products. Even with initial fractionation (Figure 3-25), it would be near impossible to make an informed decision on where to focus isolation efforts based on proton spectra alone. Given this chemical complexity of the extraction of an organism’s metabolic products, bioassays have long been harnessed as a powerful tool to guide natural product isolation (Figures 1-8 and 1-9). In the search for novel Piz-containing natural products, I had hypothesized that the Piz 15N NMR signature N-H correlation and resulting N-H centred spin system could be used as a guide to isolate the in silico predicted molecules (Figure 3-23). This research aimed to isolate Piz-containing natural products from Streptomyces incarnatus NRRL 8089 utilizing the observed nitrogen-proton resonance, complete structure elucidation of those compounds, and then test them for bioactivity across a range of bioassays. 101 Figure 3-24. Full proton spectrum of labelled crude S. incarnatus NRRL 8089 metabolites recorded at 600 MHz in DMSO-d6. Figure 3-23. Workflow for Piz 15N NMR-guided fractionation. 102 Figure 3-25. Full 1H spectrum of labelled/unlabelled mixture of Fraction B of S. incarnatus NRRL 8089 metabolites recorded at 600 MHz in DMSO-d6. In order to confirm the observed Piz N-H signal (Figure 3-17), one tray (37 cm X 20 cm; 400 mL) of Streptomyces incarnatus NRRL 8089 was grown on ISP4 agar medium with the addition of labelled 15N2-L-Orn. The resulting crude extract was then subjected first to 1H-15N HSQC (Figure 3-26 & 3-27) and then 1H-15N HSQC-TOCSY (Figure 3-29) NMR experiments. These experiments confirmed the previously seen Piz N-H correlation at δH 5.43; δN -301, as well as a Piz-like spin system (Tables 3-2 and 3-3). The 1H-15N HSQC spectrum in Figure 3-28 was taken with half the number of increments in the F1 dimension as in the spectrum in Figure 3-27 so it required only half the time. Nevertheless, the information obtained is comparable in both spectra. 103 Figure 3-26. Scale up: Full 1H-15N HSQC spectrum of labelled crude S. incarnatus NRRL 8089 metabolites recorded at 600 MHz in DMSO-d6. Inset: Piz-like N-H correlations. Figure 3-27. Scale-up: 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 crude extract obtained from 400 mL of ISP4 agar with addition of 200 mg of 15N2-L-Orn. 600 MHz, in DMSO-d6; F2:96 scans F1: TD = 64. 104 Figure 3-28. Scale-up: 1H-15N HSQC of Streptomyces incarnatus NRRL 8089 crude extract obtained from 400 mL of ISP4 agar with addition of 200 mg of 15N2-L-Orn. 600 MHz, in DMSO-d6; F2:96 scans F1: TD = 32. Figure 3-29. Scale-up: 1H-15N HSQC-TOCSY spectrum of labelled crude S. incarnatus NRRL 8089, recorded at 600 MHz in in DMSO-d6. Piz-spin system projected on to the F2 axis and seen within the inset. 105 Figure 3-30. 1H-15N HSQC-TOCSY of Streptomyces incarnatus NRRL 8089 crude extract obtained from 400 mL of ISP4 agar with addition of 200 mg of 15N2-L-Orn. 600 MHz, in DMSO-d6; F2:128 scans F1: TD=128. Earlier work on these genome-mined strains had demonstrated that incorporation of a stable isotopically labelled precursor into a bacterial metabolite could enhance the 15N NMR Piz signal such that it was visible in a crude extract yet could also mask a low compound concentration. Thus, signal strength cannot always be correlated with enough material to complete the discovery process. This result can be compared to bioassay-guided fractionation when a potent bioactive minor metabolite demonstrates profound bioassay results. To obtain enough compound for structure elucidation, the strategy (when working with bacteria) is to scale-up or grow larger quantities of the producing bacterium. Therefore, to obtain more material for isolation and structure elucidation, Streptomyces incarnatus NRRL 8089 was grown on 8 L of ISP4 agar medium with the addition of unlabelled L-Orn. The resulting pale yellow waxy crude extract was combined with the 15N labelled crude extract obtained from one tray of bacteria culture as discussed above. The combined crude material was subsequently partitioned between EtOAc and water, with a significant portion of this EtOAc-soluble fraction insoluble in 106 MeOH and soluble in hexanes. Both hexane and MeOH soluble fractions were tested for the Piz N-H signal using the 1H-15N HSQC and 1H-15N HQSC-TOCSY NMR experiments, with the signal found in the MeOH soluble fraction. The MeOH soluble fraction was then subjected to a SephadexTM LH-20 column with each subsequent fraction then tested for the Piz N-H signal using the 1H-15N HSQC and 1H-15N HQSC-TOCSY NMR experiments. The signals were found in an earlier eluting fraction, which was then subjected to a step-gradient normal-phase Sep-Pak with the MeOH wash containing the Piz NMR signal of interest. This MeOH-soluble fraction was then fractionated on a C18 Sep-Pak with the fractions C and D containing the Piz NMR signal of interest. These fractions were moved to final purification through reversed-phase HPLC. During HPLC purification, however, there proved to be too little sample for adequate method development. Increased scale-up was thus necessary and the S. incarnatus NRRL 8089 was grown as 58 L of ISP4 agar lawns supplemented with unlabelled L-Orn. This was combined with the material obtained from growing S. incarnatus NRRL 8089 on 800 mL of ISP4 agar lawns supplemented with 15N2-L-Orn. Given the large amount of hydrophobic material previously seen in the crude metabolite extract, the obtained crude was first partitioned between hexanes and distilled water with the aqueous layer being further partitioned with EtOAc. The hexanes, EtOAc, and water soluble partitions were subjected to nitrogen NMR experiments with the Piz-signal found in the EtOAc soluble partitions. The EtOAc-soluble fraction was then triturated with cold MeOH and gravity filtered to remove the fatty residues. The MeOH soluble fraction was then fractionated on a SephadexTM LH-20 column, combined via TLC spots, and the resulting fractions subjected to a 1H-15N HSQC NMR experiment, and those fractions with the nitrogen correlation of interest were then subjected to the 1H-15N HQSC-TOCSY experiment. Again, the early eluting fraction 107 contained the Piz-signal of interest. To optimize the C18 flash chromatography, a small portion of the EtOAc soluble material was fractionated using a C18 Sep-Pak and the obtained fractions analyzed via nitrogen NMR. This separation was replicated on a larger scale on a C18 flash column (30 cm x 20 cm) with fractions C and D demonstrating the Piz N-H correlation and spin system in the 1H-15N HSQC and 1H-15N HQSC-TOCSY NMR experiments. Fraction D, with less material, was set aside. A portion of the fraction C was then subjected to reversed-phase HPLC where-in the HPLC separation was divided into 10 min time signatures since the eluent contained no distinguishing UV absorption pattern. Subsequent 15N NMR analysis with the 1H-15N HSQC experiment demonstrated loss of the Piz signal until all the fractions were recombined. Thus, the separation was shown to require a low PH buffer to prevent significant tailing and compound drag across the column. With the addition of 0.01%TFA to the water solvent, clear HPLC peaks were obtained with the peaks labelled 8089.Et.B.C.11, 8089.Et.B.C.13, 8089.Et.B.C.14, 8089.Et.B.C.15, 8089.Et.B.C.16 and 8089.Et.B.C.17 demonstrating the Piz signal in the 1H-15N HSQC experiment (Scheme 3.2). Over the time required to separate all of fraction C, the peak labelled 8089.Et.B.C.15 demonstrated shifts in intensity and the appearance of two peaks, with NMR spectra demonstrating a large mix of compounds, and this was set aside. The peak labelled, 8089.Et.B.C.17, took over 20 minutes to fully elute from the column despite the acidic conditions; subsequent NMR analysis demonstrated that it was not a clean compound and this too was set aside. Thus, structure elucidation efforts, to be described in Chapter 4, were focused on the pure compounds initially named via HPLC peak: 8089.Et.B.C.11, 8089.Et.B.C.13, 8089.Et.B.C.14, and 8089.Et.B.C.16. Each of those compounds isolated as HPLC peaks, displayed the Piz N-H correlation in the 1H-15N HSQC experiment. 8089.Et.B.C.14 appeared to be a very interesting compound with the appearance of NMR correlations unique from the rest 108 and a mass of 796.1 as indicated by LRESIMS. However, it was produced in very low titers, was difficult to purify as it was adjacent to the abundant 8089.Et.B.C.13, and the structure was changing over the NMR collection time. With the minimal quantities collected and the changing spectra, structure elucidation proved especially challenging (Data Set in Appendix A) and could not be completed. 8089.Et.B.C.11 (4.3) was also produced in minor titers, however, the NMR spectra collected were not changing over NMR collection time – thus, as described in Chapter 4, structure elucidation was completed. 8089.Et.B.C.13 (4.2) and 8089.Et.B.C.16 (4.1) also demonstrated changing spectra over the NMR collection time. However, these compounds were the relatively abundant members from this family of minor compounds. As such, the structure elucidation, described in Chapter 4, was completed through careful analysis of the spectra along with collection in various deuterated solvents and varying the temperature of the samples. 109 Scheme 3-2. 15N NMR-guided isolation scheme of Streptomyces incarnatus NRRL 8089. Yellow indicates the presence of the Piz N-H signal and connected spin system in nitrogen NMR experiments. The bacterium was producing very minor amounts of 8089.Et.B.C.11, and portions of this small amount was lost as the isolation scheme was developed. As such, there was not enough material for unequivocal structure elucidation of incarnatapeptin A at this stage. The crude bacterial extract obtained from the growth of 87 L of ISP4 with added was given to David E. Williams, a research associate in the Andersen Lab, who isolated more of the compound with the previously isolated compound utilized as an HPLC standard. Additionally, the methyl ester of (4.3) (4.2) (4.1) 110 incarnatapeptin A was produced and gave a crystal whose crystal structure was used for stereochemical assignment (Appendix E). The NMR data sets from this separation and derivatization are in Appendix D. Through these efforts incarnatapeptin B (4.4) was also found from Streptomyces incarnatus NRRL 8089’s extract; this data and work are not reported here.227 David Williams re-isolated 8089.Et.B.C.13 (4.2) and 8089.Et.B.C.16 (4.1) with formic acid modifier instead of trifluoroacetic acid, which prevented the previously observed equilibrium (to be discussed in Chapter 4). The NMR data sets from this separation are also of superior quality, served to support the previous structure assignments of the hemiketal form of the compounds and are in Appendix C. 3.4 Conclusion Here I have shown that piperazic acid is likely to have predictable NMR features and then showed that 15N NMR experiments could be used to observe the diagnostic aza nitrogen chemical shift and attendant proton chemical shifts, even from a crude bacterial metabolite extract. Furthermore, with stable isotopic feeding of the biosynthetic precursor 15N2-L-ornithine, the N-H correlation was visible even in extracts from small amounts of bacterial culture. Furthermore, with feeding 15N2-L-ornithine, I observed the entire Piz spin system from a crude bacterial metabolite extract using a 1H-15N HSQC-TOCSY NMR experiment. The application of these NMR experiments allowed me to select optimal growing conditions for isolation of Piz metabolites from strains of bacteria selected through genome mining. Altogether, this work demonstrated that 15N NMR experiments could be used to guide the isolation of novel Piz compounds from a genome-mined strain predicted to contain Piz-producing machinery. 111 3.5 Experimental Section 3.5.1 General Experimental Procedures All solvents used for partitioning and flash chromatography were Sigma-Aldrich HPLC grade, used without further treatment. For HPLC work, Sigma-Aldrich HPLC grade solvents were filtered through Milli-Q filter paper prior to use. Distilled water was used to prepare all growth media for terrestrial bacteria. Seawater collected by M. Leblanc was used to prepare growth media for all marine bacteria. Milli-Q H2O was used for partitioning, reversed-phase Sep-Pak and HPLC. The 1H, 13C and 15N NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual DMSO-d6 peak (chemical shift  2.50) and 13C chemical shifts are referenced to the DMSO-d6 solvent peak ( 39.51). The 15N NMR spectra are in reference to CH3NO2 (0 ppm) with a variability of 1 ppm. Low resolution electrospray-ionization mass spectrometer (LRESIMS) were recorded on Bruker HCT Ultra PTM Discovery System and high-resolution electrospray-ionization mass spectrometer (HRESIMS) were recorded on a Waters / Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. Merck Type 5554 silica gel plates and Whatman MKC18F plates were used for analytical thin layer chromatography (TLC). Sephadex™ LH-20 purchased from Sigma was used for gel filtration chromatography. para-Anisaldehyde stain with subsequent heat application was used to visualize TLC spots. Sep-Pak SPE Silica and C18 cartridges (2 g, 5 g, and 10 g) were purchased from Waters. C18 Sep-Pak’s with free-flowing solid phase were dismantled and repacked into larger columns for large-scale C18 flash chromatography. Labelled 15N2-L-Orn (99%) was obtained from Cambridge Isotope Laboratories, Inc and unlabelled L-Orn was obtained from either 112 Sigma-Aldrich or Fisher Scientific. All other media components were obtained from Fisher Scientific. Reversed phase HPLC purifications were performed on a Waters 1525 Binary HPLC pump attached to a Waters 2998 Photodiode Array Detector and equipped with Empower Software. 3.5.2 Bacterial Cultivation 3.5.2.1 Streptomyces sp. RJA2928, A Known Producer First growth : Five trays with 400 mL of autoclave-sterilized MM1 agar were prepared by (1) autoclaving the medium combined with seawater; (2) allowing the agar to cool; and (3) spreading each tray with 5 mL of a liquid culture of the ~fifth-generation of the padanamide producer Streptomyces sp. RJA2928.206 The five trays were grown for 14 d (22 ℃), then the combined agar and mycelia were cut into small squares, transferred to a plastic container, and extracted three times with EtOAc. The three EtOAc extractions were combined and dried in vacuo to provide ~600 mg of a crude metabolite mixture. An aliquot of ~20 mg was dissolved in DMSO-d6 and the sample was subjected to 1H-15N HSQC, 1H-13C HSQC-TOCSY and 1H-1H TOCSY with no Piz correlations observed. Next, the crude metabolite mixture was fractionated utilizing Sephadex™ LH-20 (MeOH) leading to three fractions A (brown oily solid, 126 mg), B (brown oily solid, 237 mg), and C (brown solid, 105 mg). An aliquot from each fraction was subjected to 1H-15N HSQC, 1H-13C HSQC-TOCSY and 1H-1H TOCSY, again with no Piz correlations observed. Second growth and limit detection: 25 mL of MM1 broth was inoculated with an aliquot of partially thawed first-generation Streptomyces sp. RJA2928. Small Petri plates were prepared with 30 mL agar media containing 20 mg 15N2-L-Orn (L) and 30 mL agar media/20 mg L-Orn (UL) that had been added via syringe sterilization to the warm liquid agar before pouring. The 113 prepared Petri plates were inoculated with ~1 mL of the padanamide producer RJA2928, grown for 14 days, and then harvested separately. Each sample was extracted with EtOAc (x3), the extractions were combined and dried in vacuo to provide crude mixtures of L (11.5 mg) and UL (10.8 mg) (hereby referred to as “crude”). The 1H-15N HSQC experiment was collected with F2:96 scans; F1: TD = 32 for samples L and UL. Increasing amount of labelled media for bacterial growth: A tray of solid MM1 agar was prepared by first autoclaving 350 mL of MM1 agar with seawater and then adding 100 mg 15N2-L-Orn. The labelled amino acid was incorporated by first dissolving it in previously autoclaved sea water and then filter sterilizing it into the 350 mL of MM1 agar at around 70 ℃. After the agar cooled, this tray was spread with 5 mL of a liquid culture of Streptomyces sp. RJA2928, grown for 14 days and then harvested into EtOAc. The EtOAc extract was gravity filtered to separate solvent from agar and mycelium, then fresh portions of solvent were added to the residual agar and mycelia (2x). The resulting EtOAc portions were combined and dried under reduced pressure to yield 206 mg of a gummy brown crude extract called sample ScL. A small aliquot of ~20 mg was dissolved in DMSO-d6; the 1H-15N HSQC experiment was collected with F2:48 scans; F1: TD = 32 and the 1H-15N HSQC-TOCSY was collected F2:128 scans; F1: TD = 128. As the 1H-15N HSQC-TOCSY is traditionally collected with a higher F1:TD value than the number of scans in the F2 direction, various ratios of F2 scans to F1:TD values were sampled. In all cases, running the 1H-15N HSQC-TOCSY with equivalent or greater F2 scans than F1:TD values yielded observation of the more complete Piz spin system; F2:128 scans; F1: TD = 128 appeared to be ideal with a F1:TD = 96 also yielding decent observations 114 3.5.2.2 Growing Genome-Mined Strains Growth of genome-mined bacteria: The four strains listed in Table 3.4 were obtained from the Agricultural Research Service Culture Collection and revived in tryptic soy broth (TSB, purchased from Fisher Scientific) prepared with distilled water and grown at 28 °C for 3-7 d with shaking at 220 RPM. After 3-7 d, the inoculum was spread on two large Petri plates each containing 50 mL of either MM1 fresh water agar (0.5 g of glucose, 0.5 g of yeast extract, 1.0 g of peptone, 0.01 g of FeSO4•7H2O, 0.02 g of Na2HPO4, 15.0 g of agar, 1 L of distilled water) or Mannitol Soya Flour agar (MS, 20 g of mannitol, 20 g soya flour, 100 mM calcium carbonate, 20.0 g of agar, 1 L of distilled water). Each of these media had 20-30 mg of 15N2-L-Orn diluted in minimal sterilized water and then filter sterilized into the autoclaved agar media. Each Petri plate was grown for 14 d, and then the agar and mycelium were harvested together as small squares of agar lawn and extracted with EtOAc (3 x 150 mL). The EtOAc extracts were combined and dried under reduced pressure to yield 15-30 mg of crude material. 1H-15N HSQC spectra were obtained for each crude sample after dissolving in DMSO-d6 with TD=32 in the F1 dimension and 48 scans in the F2 dimension. If no signal was obtained, more scans were added to the file using the command go. Streptomyces phaeochromogenes NRRL B-1248 small scale-up: The strain Streptomyces phaeochromogenes NRRL B-1248 was grown on 400mL of MM1 F agar which had had 100 mg of 15N2 LOrn added as described above for RJA2928. After 14 days, the agar medium was extracted with EtOAc (3x) with the combined layers dried under reduced pressure to reveal 43 mg of crude material. This crude material was assessed for the Piz N-H correlation at δH 5.55; δN -304 and then separated via SephadexTM LH-20 (MeOH), leading to three fractions A (22mg, yellow oil), B (16.5 mg, yellow oily solid), and C (3.5 mg, brown solid) which were assessed for 115 the Piz N-H correlation. The fraction A (22 mg) containing the Piz signal at δH 5.55; δN -304 was further separated via step gradient C18 flash chromatography (Sep-Pak 2g). The fraction eluting with 3:1 H2O:MeOH (v/v, 7 mg) contained the Piz N-H correlation at δH 5.55; δN -304 and was further separated via reversed-phase HPLC using an InertSustain C18, 5µm, 10 x 250 mm column. Samples obtained were not sufficient for structure elucidation. Streptomyces griseus subsp. griseus strain NRRL WC-3645 small scale-up: Streptomyces griseus subsp. griseus strain NRRL WC-3645 (Figure 3-8) was grown on mannitol soy agar as 16 L of autoclaved agar medium, with 250 mg unlabelled L-Orn/400mL added via filter-sterilization when at ~70 ℃, poured as 44 sterilized trays. After 14 days of growth, mycelia and agar were harvested into EtOAc, soaked for a minimum of 24 hours (x3), then gravity filtered and dried in vacuo to obtain a crude extract. This unlabelled extract was combined with the extract obtained from the growth of 200mL of mannitol soy agar, which had had 40 mg of 15N2 LOrn added as described above. The 1H-15N HSQC experiment was performed on the labelled extract before combining. The combined crude extract (1 g) was separated via SephadexTM LH-20 (MeOH), whereby fraction C (28 mg) contained the N-H correlation of interest via NMR monitoring. This fraction was further separated via a C18 Sep-Pak (5 g) whereby the fraction eluting with 9:1 H2O:ACN (v/v) demonstrated the N-H correlation of interest. Further separation via reversed-phase HPLC using an InertSustain C18, 5µm, 10 x 250 mm column, gradient solvent system (9:1 H2O:ACN to 2:3 H2O:ACN over 60 minutes) led to the isolation of para-aminobenzoic acid (PABA) which demonstrated the N-H correlation of interest. Growth of additional genome-mined bacteria: Continuing the investigation into genome-mined strains, nine strains were obtained from the Agricultural Research Service Culture Collection as listed in Table 3.7 and revived in tryptic soy broth (TSB, purchased from Fisher 116 Scientific) prepared with distilled water and grown at 28 °C for 3-7 d with shaking at 220 RPM. After 3-7 d the inoculum was spread on 4 large Petri plates each containing 60 mL of either MM1 fresh water agar (0.5 g of glucose, 0.5 g of yeast extract, 1.0 g of peptone, 0.01 g of FeSO4•7H2O, 0.02 g of Na2HPO4, 15.0 g of agar, 1 L of distilled water); ISP4 (10 g of soluble starch, 1 g of dipotassium phosphate, 1.0 g of magnesium sulfate USP, 1.0 g of sodium chloride, 2.0 g of ammonium sulfate, 2.0 g of calcium carbonate, 0.001 g of FeSO4•7H2O, 0.001 g of ZnSO4•7H2O, 0.001 g of Mn(II)Cl2, 20.0 g of agar, 1 L of distilled water); tryptic soy agar (TSA, purchased from Fisher Scientific), or Mannitol Soya Flour agar (MS, 20 g of mannitol, 20 g soya flour, 100 mM calcium carbonate, 20.0 g of agar, 1 L of distilled water) with the addition of 60 mg of L-Orn diluted in minimal sterilized water and then filter sterilized into the autoclaved agar media. Each Petri plate was grown for 14 d, and then the agar and mycelium were harvested together as small squares of agar lawn and extracted with EtOAc (3 x 150 mL). The EtOAc extracts were combined and dried under reduced pressure to yield 20-30 mg of crude material. 1H-15N HSQC spectra were obtained for each crude sample after dissolving in DMSO-d6 with TD=32 scans in the F1 dimension and 48 scans in the F2 dimension. If no signal was obtained, more scans were added to the file using the command go on the Bruker TopSpin software. Streptomyces incarnatus NRRL 8089 Media Selection: To identify the optimum lab conditions, Streptomyces incarnatus NRRL 8089 was revived and inoculated on four different agar media each containing unlabelled L-Orn. One large Petri plate prepared from 60 mL of each media was inoculated with the desired bacteria. After 2 weeks of growth the bacteria was harvested into enough ethyl acetate to cover the cut agar medium. The ethyl acetate extracts were filtered through cotton wool to remove the mycelia and the agar and dried in vacuo to give crude extracts; ISP4 (6.6 mg); MM1 F (7.9 mg); TSB (19.8 mg) and Soy Mannitol (10.1 mg). The 117 resulting crude extracts were dissolved in ~ 0.5 mL of deuterated DMSO-d6, placed in standard 5mm NMR tubes, and were then subjected to 15N-HSQC NMR experiments, where a weak 1H-15N (δH 5.01, δN −300) correlation was seen in the crude extract from bacteria grown on ISP4 (Figure 3-5), possibly consistent with a Piz residue(s). Streptomyces incarnatus NRRL 8089 small scale-up with labelled material: Subsequently, Streptomyces incarnatus NRRL 8089 was inoculated on a 400 mL lawn of solid ISP4 agar which had 200 mg of labelled 15N2-L-Orn dissolved in 50 mL of previously autoclaved distilled water that was added via syringe-filter to 350 mL of autoclaved ISP4 agar cooled to ~70oC, with the combined portions poured into an autoclaved aluminum foil-covered stainless steel pan. The decision was made to use 200 mg labelled 15N2-L-Orn per 400 mL media when growing Streptomyces incarnatus NRRL 8089 versus the 100 mg labelled 15N2-L-Orn per 400 mL medium used when growing padanamide producer Streptomyces sp. RJA2928 because it had been shown that NRRL 8089 was a producer of a sinefungin analog231 which utilized L-Orn in its biosynthetic pathway.235 As a precaution before scale-up, the structure and available NMR data for sinefungin236 was analyzed to ensure there were no overlapping N-H signals with Piz. 5 mL of five day liquid growth of Streptomyces incarnatus NRRL 8089 was spread on the cooled and solidified ISP4 agar as inoculum with the pan recovered with foil and then left undisturbed for 14 days. After 14 days of growth, the lawns of agar were cut into small pieces and submerged in EtOAc for a minimum of 24 hours for solid-liquid extraction of the bacterial metabolites. Following the minimum period of 24 hours, the EtOAc portion was gravity filtered through cotton wool to separate the agar and bacterial mycelia from the saturated solvent and a fresh EtOAc portion was applied (2x). The EtOAc (3x) portions were then combined and dried in vacuo and the crude extract was subjected first to 1H-15N HSQC (Figure 3-6) and then to 1H-15N 118 HSQC-TOCSY (Figure 3-7) NMR experiments, giving the proton-nitrogen correlation and Piz-like spin systems seen in, consistent with the Piz system observed in Streptomyces sp. RJA2928. 3.5.3 15N NMR Guided Fractionation Initial large scale-up of Streptomyces incarnatus NRRL 8089: To obtain more material for isolation and structure elucidation, Streptomyces incarnatus NRRL 8089 was inoculated on agar lawns prepared on large stainless steels pans through pouring 8 L of ISP4 agar medium to which 0.9 g/400 mL of unlabelled L-Orn was added. After 14 days of growth, the combined cells and solid agar medium were extracted with EtOAc which was dried under reduced pressure to form a crude metabolite mixture as previously described. This pale yellow waxy crude extract was combined with previously grown labelled extract for a total of 266.1 mg. This crude material was then partitioned between EtOAc and water and then dissolved in a minimal amount of MeOH to place on a Sephadex LH-20 column. A significant portion of this partition precipitated out of the MeOH solvent and the precipitate was removed by gravity filtration and rinsed with cold MeOH. The precipitate proved to be most soluble in hexanes leaving a waxy yellow mass, while the MeOH soluble partition proved to be a brown oily solid. Both hexane and MeOH soluble fractions were tested for the Piz signal using the 1H-15N HSQC NMR experiment and the signal was found in the MeOH soluble fraction. The MeOH soluble fraction was fractionated using on a Sephadex LH-20 column and eluted with MeOH:DCM (4:1 v/v) into multiple small fractions. Fractions were spotted using thin layer chromatography (Alumina backed Silica, DCM:MeOH, 9:1, UV 254 nm and p-anisaldehyde stain) and combined based on common appearance in the UV and stain visualization methods (A, B, C, D). Each fraction obtained from Sephadex LH-20 was subjected to 1H-15N HSQC NMR Experiment, those fractions with the signal were then subjected to the 1H-15N HQSC-TOCSY experiment. The fraction B (53.6 mg, brown oil) was then 119 subjected to Si gel flash chromatography using a step-gradient whereby the piperazic-acid diagnostic NMR signature was seen in the final MeOH wash. This MeOH soluble fraction (18 mg) was then fractionated via C18 Sep-Pak (2 g) with step-gradients of 1:3, 1:1, 3:1, and 1:1 ACN/H2O with the fractions C (1:1 H2O:ACN v/v; 1.2 mg; yellowish oil) and D (1:3 H2O:ACN v/v; 3.1 mg; yellowish-brown oily solid) containing the Piz NMR signal of interest. These fractions were moved to final purification through reversed-phase HPLC using an InertSustain C18, 5 µm, 10 x 250 mm column. After HPLC purification, however, there proved to be too little sample for structure elucidation. Scale-up of Streptomyces incarnatus NRRL 8089 and isolation of dentigerumycins F (4.2) and G (4.1) and incarnatapeptins A (4.3): To obtain more material for isolation of the Piz compounds, Streptomyces incarnatus NRRL 8089 was cultured on 58 L volume of ISP4 medium with added filter sterilized unlabelled L-Orn (4.5 g / 2 L) prepared as 160 pans of solid agar and grown at rt for 14 d. When the majority of spores turned a light grayish-pink, the agar media, bacterial mycelia and spores were cut into small squares and extracted with EtOAc (3x). The resulting crude was subjected first to 1H-15NHSQC and then 1H-15NHSQC-TOCSY experiments to obtain weak nitrogen-proton correlations. Concurrently, S. incarnatus NRRL 8089 was cultured on an additional two pans of ISP4 agar with added filter-sterilized 15N2-L-Orn prepared as above (final volume/tray 400 mL), harvested and extracted with EtOAc (3x). The extracts were combined and concentrated under reduced pressure. The resulting crude extract was analyzed by 1H-15N HSQC and 1H-15N HSQC-TOCSY NMR experiments to obtain the nitrogen-proton correlations. This material contained the nitrogen signal required for NMR observation throughout subsequent isolation steps. To obtain enough material for isolation, this crude material containing labelled piperazic-acid residues was then combined with the crude material 120 of the larger growth of Streptomyces incarnatus NRRL 8089 grown with unlabelled L-Orn. The combined EtOAc extracts were partitioned between H2O (750 mL) and hexanes (250 mL x 3). The resulting aqueous extract was partitioned with EtOAc (4 x 300 mL). No nitrogen-proton correlations of interest were observed in the waxy yellow hexanes soluble material and it was set aside. The EtOAc soluble material was triturated with cold MeOH (~5 mL) to give a white precipitate and a brown liquid which, when concentrated in vacuo gave a brown oil. The white ppt and the brown oil were subjected to the piperazic-acid diagnostic nitrogen experiments with the nitrogen-proton correlations of interest observable only in the brown oil. This MeOH soluble material (900 mg; brown oil) was then chromatographed on SephadexTM LH-20 with 4:1 (v:v) MeOH/CH2Cl2 as eluent. The resulting test tube fractions were spotted using thin layer chromatography (Alumina backed Silica, DCM:MeOH, 9:1, UV 254 nm and p-anisaldehyde stain) and combined based on common appearance in the UV and stain visualization methods to give five fractions (A, B, C, D, E) which were subjected to the piperazic-acid diagnostic nitrogen experiments. The fraction labelled B (246.5 mg ; brown oily solid) contained the nitrogen-proton correlations and spin-system of interest. This fraction was then fractionated via C18 flash chromatography, where the NMR signature was seen in the fractions eluting with 2:3 and 5:6 ACN/H2O. To further optimize the C18 flash chromatography, a small portion of fraction B was then fractionated using a C18 Sep-Pak (2 g) column with step-gradients of 1:4, 21:39, 2:3, 5:6 and 1:1 ACN/H2O where it was determined via 1H-15N HSQC experiments that the Piz-Signal was eluting in H2O/ACN (2:3 and 5:6) with no signal present in H2O/ACN (1:1). The remaining EtOAc portion was then dry-loaded onto a large C18 flash column (30 mm x 20 cm) containing 40 g of C18 gel and slowly eluted using increasing amounts of ACN (1:4, 21:39, 2:3, 5:6 and 1:1 ACN/H2O v/v). The fractions C (42.2 mg; 2:3 H2O:ACN v/v ) and D (22 mg; 5:6 H2O:ACN v/v) 121 demonstrated the Piz signal in 1H-15N HSQC. A portion of Fraction D was subjected to LRESIMS and the rest was set aside. A portion of the fraction C was then subjected to reversed-phase HPLC. Fractions were collected divided by 10 min time signatures as it was known through TLC that these compounds had no significant UV chromophore. Subsequent 15N NMR analysis with the 1H-15N HSQC experiment demonstrated loss of the Piz signal until all the fractions were recombined. The separation was shown to require a low PH buffer to prevent significant tailing and dragging across the column. With the addition of 0.01% TFA to the water solvent with a solvent system of 1:1 ACN/H2O, clear HPLC peaks were obtained with the peaks labelled 11, 13, 14, and 16 demonstrating the Piz signal in the 1H-15N HSQC experiment (Scheme 3.2). Thus, pure samples of dentigerumycin F (4.2) and G (4.1) and incarnatapeptin A (4.3) were obtained from the fraction eluting with 2:3 ACN/H2O via purification with reversed-phase HPLC using an InertSustain C18, 5 µm, 10 x 250 mm column with 1:1 ACN/H2O with 0.05 % (v/v) TFA as eluent. 122 Chapter 4: Piperazic acid-containing Metabolites from Streptomyces Incarnatus NRRL 8089 4.1 Chapter Overview This chapter will describe the structure elucidation of the novel Piz-containing natural products dentigerumycin G (4.1), dentigerumycin F (4.2), and incarnatapeptin A (4.3) isolated from Streptomyces incarnatus NRRL 8089 utilizing the Piz-targeted Nitrogen-NMR guided methodology described in Chapter 3. Incarnatapeptin A is a linear acid that contains a unique bicyclic ring that has not previously been seen in natural products. Dentigerumcyin F and G are linear peptides that are related to dentigerumycin C and D. Since these natural products all contain three piperazic acids, a brief review of other peptides containing three piperazic acid residues will be presented. Additionally, an overview of the unusual equilibrium observed in the oxane rings of dentigerumycin F and G will be presented. A biosynthetic proposal for the pathway that leads to both the incarnatapeptins and dentigerumycin F and G will be introduced. Finally, the antimicrobial assay results for incarnatapeptin A, dentigerumycin F and G will be discussed, while the bioactivity of incarnatapeptin A and incarnatapeptin B (4.4) in prostate cancer cytotoxicity assays will be examined. 4.2 19-Membered Depsipeptides with Three Piperazic Acids Genome mining identified only two Piz residues in the Piz-containing natural products produced by Streptomyces incarnatus NRRL 8089, but 1H-15N-HSQC nitrogen NMR data indicated the presence of three piperazic acids. Several natural products (Figure 4-1) that contain three piperazic acids are known, and a subset of them will be discussed below to provide context for the discovery research described in this chapter. The dentigerumycins are 19-membered cyclic depsipeptides (4.5, 4.6 and 4.9) or their linear peptide congeners (4.7 and 4.8). Dentigerumycin A (4.5), isolated from a Pseudonocardia strain, contains the amino acid 123 sequence L-Ala, 3-OH-L-Piz, D-Piz, N-OH- L-Ala, D-Piz, β-OH-Leu; dentigerumycins B (4.6), C (4.7) and D (4.8), all isolated from a Streptomyces strain, contain the core amino acid sequence, L-Piz, D-Piz, N-OH-Gly, D-Piz, β-OH-Leu. 4.6 has an additional amino acid, N-OH-L-allo-Thr, which is used to form the cyclic molecule. Dentigerumycin E (4.9), isolated from another Streptomyces strain, shares the amino acid sequence of dentigerumycin B save for the cyclizing N-OH-L-Thr, but it has a uniquely oxidized substituent on its oxane ring.173 Other classes of natural products that contain three Piz moieties are peptides (4.10 and 4.11) and 18-membered depsipeptides (2.26, 4.12, 4.13). The hytramycins (4.10 and 4.11), peptides isolated from a Streptomyces species, have an amino acid sequence of L-Piz, D-Val/Ile, L-Leu, D-Piz, N-Me-Ala, and D-Piz. Hytramycin’s gene cluster has not been identified. The gerumycins (2.26, 4.12, 4.13), 18-membered depsipeptides produced by a Streptomyces species, have an amino acid sequence of α-Me-L-Ser, 3-OH L-Piz, 3-Cl-D-Piz, L-Ala, (3-OH) D-Piz. In 18-membered depsipeptides (Figure 2-3), it is presumed that the ring closure occurs through the loading of a final hydroxy acid monomer in place of an amino acid; in the gerumycins, the final loading of lactic acid leads to 2.26 and 4.12, whereas the loading of 2-hydroxybutanoic acid leads to 4.13.173 124 Figure 4-1. Natural products that contain three piperazic acids.173 4.3 Structure Elucidation of Piperazic Acid-Containing Metabolites from Streptomyces Incarnatus NRRL 8089 4.3.1 Structure Elucidation of Dentigerumycin G (4.1) The structure elucidation of dentigerumycin G (4.1) will be described first, as it was the first structure from the isolated piperazic acid-containing nonribosomal peptides to be solved due to its greater abundance. Although its structure elucidation was complicated due to the presence of hemiketal/dehydrated forms, the abundance of the material (~1 mg) that was available for obtaining a dataset gave strong enough NMR signals to assess which resonances belonged to which form. Analyzing the samples in various deuterated solvents aided the characterization, as did assessing both high-res and low-res mass spectra in varying conditions. 125 Figure 4-2. 1H spectrum for dentigerumycin G (4.1) recorded after sample was in the fridge for ~1 week, in DMSO-d6, recorded at 600 MHz. 126 Figure 4-3. 13C spectrum for dentigerumycin G (4.1) demonstrating the large numbers of carbons in the compound, recorded in DMSO-d6 at 150 MHz. Dentigerumycin G (4.1) gave a [M + Na]+ ion in the HRESIMS at m/z 807.4237 appropriate for a molecular formula of C35H60N8O12 that also requires 10 sites of unsaturation. Analysis of the 1H, 1H-1H gCOSY60, 1H-1H gTOCSY/1H-13C gHSQC/1H-15N gHSQC/1H-13C gHMBC/ 1H-15N gHSQC-TOCSY NMR data recorded in DMSO-d6 revealed 4.1 as an acid that contains three Piz residues, an N-hydroxy alanine residue, a β-OH-leucine, along with an acyl side chain containing a tetrahydropyran ring bearing vicinal methyl and ethyl substituents in a trans-configuration. The 1H NMR data showed a broad 1H resonance at δH 13.26 (Figure 4-2) typical of a carboxylic acid, which led to the working hypothesis that this molecule was a linear peptide with a free carboxylic acid at its C terminus. Three exchangeable proton resonances at δH 4.72 (δN -301.1), 5.08 (δN -300.0), and 5.16 (δN -300.5) showed one-bond correlations to nitrogen resonances in the 1H-15N HSQC spectrum, and these were the signals that guided the isolation of 127 the compound (Figure 4-4). These Piz-like correlations were subsequently shown to be saturated piperazic acid spin systems (Figure 4-5) in both the 1H-15N HSQC-TOCSY (Figure 4-6) and 1H-1H TOCSY (Figure 4-7 and 4-8) spectra. In particular, δH 4.72 (NH-1) demonstrated correlations into H-5a/b, H-4a/b, H-3a/b, and H-2a/b ; δH 5.16; (NH-4) showed correlations into H-10a/b, H-9a/b, H-8a/b, and H-7a/b; while δH 5.08 (NH-6) had correlations into the methylene protons H-18a/b, H-17a/b, H-16a/b, and H-15. Further evidence was seen in the HSQC spectrum which showed that all methylene carbons adjacent to the Piz N-H’s possessed the typical deshielded carbon resonances of this position (see Table 3-2) in these moieties: C-5 (δC 46.7), C-10 (δC 46.2) and C-18 (δC 45.3). Figure 4-4. 1H-15N HSQC NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. N-H correlations which identified 4.1 as a Piz-containing molecule. 5.16, -300.5 & 5.08, -300 Piz 2 N-H : 5.16 Piz 3 N-H : 5.08 4.72, -301 Piz 1 N-H : 4.72 128 Figure 4-5. Piz NMR spin systems for dentigerumycin G (4.1). These spin systems, seen in the 1H-15N HSQC-TOCSY NMR experiment were the second data point to confirm 4.1 as a Piz containing molecule while also providing a handle for structure elucidation. Figure 4-6. 1H-15N HSQC-TOCSY NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO- d6. Piz spin systems, which roughly matched experimental values for a typical Piz, outlined in red. 2.2, 1.76, 1.4-1.3 3.00, 2.8, 2.6 4.72, -301 5.16, -300.5 & 5.08, -300 129 Figure 4-7. Expansion of 1H-1H TOCSY NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. Demonstrated is the Piz 1, 2 and 5 spin systems (Figure 4-5), from the N-H proton on one end of the spin system and from the α-methine on the other end of the spin system. Figure 4-8. Full TOCSY NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. Piz 2; Piz 3 N-H Piz 1 N-H Piz 1 α-methine Piz 2 α-methine Piz 3 α-methine H-4 H-3 H-3 H-5 H-5 H-4 H-4 H-3 H-3 H17 H16 H16 H18 H-9 H-8 H-8 H10 H17 H16 H-9 H-8 H-8 130 Figure 4-9. COSY60 NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. Figure 4-10. 1H-13C HSQC NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. This is demonstrating the unusually deshielded methines of Piz 2 and Piz 3. Also seen is the lack of correlation of the N-H protons into a carbon. Piz 2 α-methine Piz 3 α-methine Piz 1 α-methine Piz 2; Piz 3 N-H Piz 1 N-H 131 Figure 4-11. 1H-13C HMBC NMR spectrum of dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. In silico analysis (antiSMASH predictions, Table 2-4) predicted an N-hydroxylated alanine, which was confirmed through 1H-1H gCOSY and 1H-1H TOCSY correlations between an α-methine proton at δH 5.53 (H-12) and a methyl doublet at δH 1.34 (H-13) both of which showed HMBC correlations into a carbon resonance at δC 172.01 (C-11), as seen in NMR spectra expansions in Figure 4-13. The nitrogen-attached hydroxyl was suspected due to a broad proton resonance at δH 9.55, which is characteristic of a hydroxymate proton resonance. This was corroborated via 1H-15N LRHMQC cross peaks between H-13 (δC 1.34) and N-5 (δN -199.9) (Figures 4-14). The amino acid sequence of this free acid was confirmed as Piz, Piz, N-OH-Ala, Piz (Figure 4-12) through a combination of HMBC correlations between protons and the peptide carbonyl resonances and tROESY resonances between nitrogen-attached Piz protons and the adjacent α-methine protons (Figure 4-15 & 4-16). H-23/24, C-21 (75.6) 132 Figure 4-12. Connecting the core amino acids for dentigerumycin G. Figure 4-13. TOCSY and COSY60 spectra demonstrating the correlations between N-OH-Ala’s methyl and α-methine protons leading to the assigned structure of 4.1. Recorded at 600 MHz in DMSO-d6. 133 Figure 4-14. 1H-15N LRHMQC spectrum demonstrating the correlations between N-OH-Ala’s methyl protons and the hydroxymate nitrogen leading to the assigned structure of 4.1. Recorded at 600 MHz in DMSO-d6. Figure 4-15. tROESY correlations between the proton of the nitrogen-attached hydroxyl proton and Piz 3’s α-methine proton leading to the assigned structure of 4.1. Recorded at 600 MHz in DMSO-d6. 1.34, -204.0 5.47, 9.55 134 Figure 4-16. 1H-13C HMBC spectrum demonstrating proton-carbonyl correlations that helped connect the amino acids thus leading to the assigned structure of 4.1. Recorded at 600 MHz in DMSO-d6. Figure 4-17. 1H-1H TOCSY correlations between the γ-methine (H-22), β-methine (H-21), α-methine (H-20) and nitrogen proton (NH-8) of β-OH-Leu which helped lead to the assigned structure for 4.1. The H-21/H-22 signal was very weak, so the spectrum is shown deep into the contours. Recorded at 600 MHz in DMSO-d6. 3.41, 5.42 (H-20) 3.41, 7.23 (NH-8) 5.42, 3.41(H-21) 5.47, 171.6 (C-12)5.54, 171.9 (H-13)1.34, 171.9 1.77, 171.6 (H-32)1.2, 176.5 (H-32)1.27, 172.3 (H-20)5.42, 171.5 7.23, 172.3 N-H Leu 5.42, 7.23(NH-8) 3.41, 1.88(H-22) 3.41, H-21 5.42, H-20 135 Further analysis of the 2-D data revealed a β-OH-Leucine with characteristic methyl doublets at C-23 (δC 14.7, δH 0.82) and C-24 (δC 20.2, δH 0.82) showing HMBC correlations from δH(C-23/24) 0.82 into C-21(δC 75.6) (Figure 4-11). The 1H-1H TOCSY spectrum of a cold sample (Figure 4-17) revealed 3 adjacent methines at H-20 (δH 5.42), H-21(δH 3.41), and H-22(δH 1.69) as well as a cross peak from H-20 to an amide proton at δH 7.29. Although no hydroxyl proton was observed in this data, the deshielded methine carbon resonance at δC 75.6 for C-21 supported this assignment as did later spectra obtained from 4.1 isolated without TFA (Appendix). Such a β-OH-Leucine is conserved across the majority of azinothricin type molecules.173 Thus, the presence of this amino acid, along with the three Piz amino acids discussed earlier, suggests that 4.1 was the product of the Streptomyces incarnatus NRRL 8089 NRPS-PKS hybrid gene cluster found using the KtzT probe. Furthermore, despite key gene organization differences between Streptomyces incarnatus NRRL 8089 ’s gene cluster and dentigerumycin A’s Pseudonocardia producer, (Figure 3-22 and Table 3-9) 4.1 was seen to be structurally related to dentigerumycin A (4.5) except for a missing cyclizing alanine. However, as the 1H NMR data demonstrated evidence that 4.1 was a free acid, this molecule was presumed to be a linear molecule in a similar fashion to dentigerumycin C (4.7)and D (4.8).237 In fact, this molecule proved to be a constitutional isomer of dentigerumycin C237 (Figure 4-1), sharing the molecular formula C35H60N8O12, and the calculated molecular mass of 807.4228. 136 Figure 4-18. Building up fragments of dentigerumycin G (4.1) to help confirm the structure of the PKS portion. Given the close association to the linear dentigerumycins, it was essential to establish the structural differences. After assigning Fragment A via NMR correlations, it became apparent that the first point of difference from dentigerumycin C (4.7) was in the core amino acids for which the molecular formula of the fragment is seen above in Figure 4-18. Fragment A was calculated to have the molecular formula of C18H30N7O6, while the comparable fragment in dentigerumycin C has a molecular formula of C17H28N7O6 (Figure 4.1).237 This left seventeen carbons, thirty protons, one nitrogen, and six oxygens that were yet to be assigned within the structure. Analysis of the acyl side chain was complicated by the hemiketal/dehydro equilibrium (discussed in Subsection 4-3-3), which developed within the data collection time frame. However, the structure of the molecule was built by analysis of the NMR data that described the standard β-OH-Leu and was extended by evidence of a hydroxyl and methyl substituent attached to a quaternary carbon as seen in Figure 4-19. The methyl singlet at H-32 (δC 21.2, δH 1.20) showed clear HMBC correlations into deshielded quaternary carbons at δC 76.9 (C-26) and δC 98.0 (C- 137 27), both of whose resonances indicated the likely attachment to electronegative oxygen217,238 (Figure 4-20). Additionally, H-32 demonstrated HMBC correlations into a carbonyl carbon at δC 172.3 (C-25) (Figure 4-20), which was also seeing 2J correlations from the N-H (δH 7.23) of the β-OH-Leucine thus helping to connect the NRPS and PKS portions of the molecule. In the first set of data recorded, this H-32 proton singlet was captured as two peaks (A: δH 1.20 and B : δH 1.27). For the methyl singlet peak at δH 1.20 there appeared to be very low relative integration, despite strong HMBC resonances into multiple quaternary carbons. By capturing the proton spectra at varying times throughout the data collection and at different initial temperatures, it became clear that the relative integration for the proton at δH 1.20 was changing. This will be discussed in greater detail in Subsection 4-3-3. Furthermore, although the hydroxyl proton was not visible in the data, the deshielded carbon resonances of C-26 and C-32 strongly suggested the presence of a hydroxyl substituent. The singlet coupling pattern of the H-32 proton also provided evidence that there were no coupled protons attached to C-26. Fragment B seen in Figure 4-18 was thus constructed as a partial molecular formula of C9H16NO4. This left eight carbons, fifteen protons, and two oxygens that were yet to be assigned within the structure and as such likely contained within the remainder of the molecule. With this information in hand, it was possible to elucidate the remainder of 4.1. Through analysis of the 2D data, it became evident that the remaining C8H15O2 were forming a disubstituted oxane ring. The oxane ring was revealed through gCOSY60 and 1H-1H TOCSY correlations from the H-31 methine (δH3.58) to the shielded methine proton at H-30 (δH0.97) and extended methylene and methyl protons of H-33 (δH1.39, 0.96) and H-34 (δH 0.82). The methine proton at H-30 was distinctly attached to a deshielded carbon resonance (δC 41.9) 138 anchoring the structure elucidation efforts despite the challenges associated with solving closely related spin systems appearing in the NMR spectra due to an apparent equilibrium between two forms of the molecule. Figure 4-19. Correlations confirming the hydroxy-leucine amino acid and the PKS portion of 4.1. The methyl doublet at H-35 (δH 0.99) was distinctive, demonstrating clear COSY60 correlations into H-31 as well as HMBC correlations into C-31 (δC 69.1). More challenging to confirm was the methyl group of the suspected ethyl substituent chain of H-33 and H-34 because the oxane ring substituent resonances were overlapped with the β-OH-Leu methyl doublets (H24/23 δH 0.82). The overlapping resonances made NMR assignment challenging. Further adding to this challenge was that these upfield resonances were also present for both a hydrated and dehydrated oxane ring, resulting in an exceedingly complex region of the NMR spectra. An initial TOCSY spectrum recorded on the sample demonstrated the complicating equilibrium (Figure 4-21). Subsequently, a TOCSY spectrum recorded immediately after the sample of 4.1 had been stored in the fridge for one week demonstrated that H-34, H-33, H-30, H-31 and H-29 were in a spin system (Figure 4-22). A TOCSY spectrum recorded after the sample sat at room 139 temperature for two weeks (Figure 4-23) demonstrates the re-equilibration of the molecule between a hemiketal and olefin form at C-27/C-28, leading, once again, to a complicated spectrum. HMBC correlations (Figure 4-20) between the deshielded C-30 (δC 41.92) and both the methine of H-31 (δH 3.58) and the methyl at δH 0.99 (H-35), along with the correlations between the deshielded C-31 (δC 69.00) and the methine multiplet of H-30 (δH 0.97) supported an adjacent nature of the oxane substitutions. 2D J-resolved spectra were recorded to confirm the coupling patterns (Figure 4-24) of H-34 and H-35. The H-35 methyl substituent was confirmed as a doublet in both forms, coupling to the methine proton of H-31. The H-34 methyl substituent was confirmed as a triplet coupling to the methylene protons of H-33. Thus, gCOSY60 and HMBC correlations, along with coupling patterns established through a J-resolved spectrum, demonstrated that the oxane ring had vicinal methyl and ethyl substituents. This unique ring pattern differentiated 4.1 as a novel linear piperazic acid-containing natural product. 2D t-ROESY correlations between the H-31 methine proton (δH 3.58) and the methylene protons of H-33a/b (δH 1.39, 0.99) gave evidence for a trans relative configuration of the two sidechains. 2D t-ROESY correlations taken from data showing the hydroxyl chemical shifts (Appendix B) confirmed the hydroxyl at C-27 was on the same face of the six-membered ring as the methine proton at H-31 (δH 3.58). The absolute configuration of the molecule was assigned using the crystal structure of incarnatapeptin A (4.3) given that they are produced from the same biosynthetic pathway and have closely related NMR resonances for the amino acids and PKS-NRPS link, this will be discussed in Section 4.4. 140 Figure 4-20. 1H-13C HMBC detailing correlations for the PKS linkage portion of 4.1. Recorded in DMSO-d6, at 600 MHz. Figure 4-21. Initial 1H-1H TOCSY spectrum showing multiple signals due to an equilibrium in the PKS portion of 4.1,recorded in DMSO-d6 at 600 MHz. C-26, 76.9 C-27, 98.0 C-25, 176.5 H-32, 1.21 H-31 H-35 H-35 141 Figure 4-22. 1H-1H TOCSY demonstrating 4.1’s disubstituted oxane ring, recorded on a sample sitting in the fridge for one week, recorded in DMSO-d6, at 600 MHz. Figure 4-23. 1H-1H TOCSY demonstrating 4.1’s disubstituted oxane ring, recorded on a sample sitting at room temperature for two weeks demonstrating a return to intense duplicated resonances, particularly for H-35. Recorded in DMSO-d6, at 600 MHz. H-31 H-35 H-30 H-33 H-29 H-31 H-35 H-35 142 Figure 4-24. 2D J-resolved spectrum demonstrating the coupling patterns for the various methyl groups in 4.1 recorded from a sample that had been sitting at room temp so demonstrating multiple resonances for H-35. Recorded in DMSO-d6, at 600 MHz.H-35 H-35 H-34 H-34 H-34 143 8089-16: Dentigerumycin G (4.1) DMSO-d6 CD3OD Hemiketal Form Olefinic Form Olefinic Form Hemiketal Form Residue Position δC/δN δH (J = Hz) δC/δN δH (J = Hz) δC/δN δH (J = Hz) δC/δN δH (J = Hz) L-Piz 1 1 C 172.4 172.4 171.9 2 CH 50.6 4.9 m 50.6 4.9 m 52.4 5.05 52.4 5.05 3 a/b CH2 24.7 1.76, 2.12 m 24.7 1.76, 2.12 m 26.7 1.86, 2.27 m 26.7 1.86, 2.27 4 a/b CH2 21.3 1.35, 1.52 m 21.3 1.35, 1.52 m 22.3 1.64, 1.52 m 22.3 1.64, 1.52 5 a/b CH2 46.7 2.61, 2.99 m 46.7 2.61/2.99 m 48.4 2.74, 3.0 m 48.4 2.74, 3.0 NH-1 NH -301.0 4.72 -301.0 4.72 n/a n/a n/a n/a COOH OH 13.09 br.s 13.09 br.s n/a n/a n/a n/a D-Piz 2 6 C 172.0 172.0 174.8 7 CH 48.3 5.53 m 48.3 5.53 m 50.5 5.68 50.5 5.68 8 a/b CH2 24.6 2.0, 1.75 m 24.6 2.0, 1.75 m 26.7 2.13, 1.88 26.7 2.13, 1.88 9 a/b CH2 20.7 1.47, 1.52 m 20.7 1.47, 1.52 m 22.3 1.61 22.3 1.61 10 a/b CH2 46.2 2.59, 2.9 m 46.2 2.59, 2.9 m 48.4 2.76, 3.02 48.4 2.76, 3.02 NH-4 NH -300.5 5.16 -300.5 5.16 n/a n/a n/a n/a N-OH-L-Ala C11 C 172.0 172.0 174.3 173.20 12 CH 51.6 5.53 m 53.6 5.55 54.8 5.63 54.4 5.76 13 CH3 14.6 1.34 d (7.4) 14.8 1.35 14.5 1.41 14.4 1.41 N-5 -204.0 n/a n/a n/a n/a N5-OH 9.55 br.s n/a n/a n/a n/a D-Piz 3 14 C 171.6 171.6 172.9 15 CH 47.8 5.47 m 47.8 5.47 m 50.2 5.5 50.2 5.5 16 a/b CH2 24.9 2.1, 1.77 24.9 2.1, 1.77 26.7 2.24, 1.85 26.7 2.24, 1.85 17 a/b CH2 21.6 1.49, 1.34 21.6 1.49, 1.34 22.3 1.61 22.3 1.61 18 a/b CH2 45.3 2.8 m 45.3 2.8 m 47.4 2.96, 3.0 47.4 2.96, 3.0 NH-6 NH -300.0 5.08 -300.0 5.08 n/a n/a n/a n/a β-S-OH- L-Leu 19 C 171.5 171.5 171.2 172.4 144 20 CH 49.8 5.42 m 49.8 5.42 m 50.5 5.55 50.5 5.56 21 CH 75.6 3.34 br.d 75.6 3.41 m 77.1 3.7 77.1 3.67 22 CH 28.8 1.88 m 28.8 1.88 m 28.4 1.87 28.4 1.87 23 CH3 14.7 0.82 d (6.8) 14.7 0.82 d (6.8) 15.5 0.92 15.5 0.91 24 CH3 20.2 0.82 d (6.8) 20.2 0.82 d (6.8) 20.3 0.97 20.8 0.94 NH-8 NH -267.0 7.66 d (10.0) -267.0 7.23 d(10.0) n/a n/a n/a n/a 21-OH not visible n/a n/a n/a n/a PKS 25 C 176.5 172.3 174.2 177.9 26 C 76.9 75.2 76.7 77.0 27 C 98.0 154.1 154.5 101.0 28 a/b CH2 28.0 2.15, 1.95 93.1 4.81 97.3 4.95 26.7 2.15, 1.60 29 a/b CH2 24.3 1.69 24.1 2.02, 1.68 25.5 2.14, 1.75 23.4 1.74, 1.39 30 CH 41.9 0.97 m 38.6 1.26 m 40.5 1.38 m 43.8 1.08 m 31 CH 69.1 3.58 m 74.1 3.35 m 73.5 3.41m 72.2 3.69 m 32 CH3 21.2 1.2 s 23.7 1.27 s 30 1.29 s 21.2 1.35 s 33 a/b CH2 23.7 1.39, 0.96 m 24.1 1.06, 0.98 m 25.2 1.54, 1.12 m 24.9 1.02, 0.88 m 34 CH3 10.1 0.82 t (7.5) 11.3 0.87 t (7.5) 11.0 0.92 t (7.5) 11.0 0.89 t (7.5) 35 CH3 19.1 0.99 d (6.5) 19.1 1.14 d (6.5) 19.0 1.23 d (6.5) 19.0 1.08 d (6.5) 26-OH not visible not visible n/a n/a 27-OH not visible not visible n/a n/a Table 4-1. NMR shifts of dentigerumycin G’s hemiketal and dehydrated forms recorded in either DMSO-d6 or CD3OD at 600 MHz. 145 4.3.2 Structure Elucidation of Dentigerumycin F (4.2) The structure elucidation of dentigerumycin F (4.2) will be described next, which is the order in which the isolated piperazic acid-containing nonribosomal peptides structures were solved, despite dentigerumycin F eluting from the column first. The data set for 4.2 was obtained in pyridine-d5 due to earlier challenges solving the structure of dentigerumycin G (4.1) using spectra recorded in DMSO-d6. The complexity of the data in DMSO-d6 was later understood to be due to an equilibrium between hemiketal and dehydrated forms of the molecule. Interestingly, when 4.2 was placed in pyridine-d5, the rate of equilibrium appeared significantly reduced with the hemiketal form favoured —this greatly aided structure elucidation of 4.2. After the initial datasets were recorded, pyridine-d5 was removed, and DMSO-d6 was added, leading to more complex spectra but with the ability to assign resonances (Table 4-2) due to work already completed on 4.1. 146 Figure 4-25. 1H spectrum for dentigerumycin F recorded in pyridine-d5 at 600 MHz. Figure 4-26. 13C spectrum for dentigerumycin F recorded in pyridine-d5 at 150 MHz. Dentigerumyin F (4.2) was isolated as a clear oil that gave a [M + Na]+ ion in the HRESIMS at m/z 806.4369 appropriate for a molecular formula of C35H61N9O11 that requires ten 147 sites of unsaturation. Analysis of the 1H, 1H-1H gCOSY60, 1H-1H gTOCSY/1H-13C gHSQC/1H-15N HSQC/1H-13C and gHMBC NMR data recorded in pyridine-d5 for 4.2 revealed that 4.2 contains three Piz residues (one of which contained a terminal amide), an N-hydroxy alanine residue (N-OH-Ala), a 3-OH-leucine, along with an acyl side chain containing an oxane ring bearing vicinal methyl and ethyl substituents in a trans relative configuration. The 1H NMR data showed two amide protons at δH 8.27 and 8.09 (Figure 4-25), demonstrating 1H-15N HSQC correlations into a nitrogen at δN -275.8, which led to describing this molecule as a likely linear free-amide peptide-containing molecule. The three exchangeable proton resonances at δH 5.50 (δN -300.3), 5.75 (δN -300.0), and 5.67 (δN -300.9) showed one-bond correlations to nitrogen resonances in the 1H-15N HSQC spectrum and were the signals followed throughout the isolation of this compound (Figure 4-29). These Piz-like correlations were subsequently shown to be saturated piperazic acid spin systems (Figure 4-28) in the 1H-1H TOCSY (Figure 4-30 and 4-31) spectrum. In particular, δH 5.50 (NH-1) demonstrated correlations into H-5a/b, H-4a/b, H-3a/b, and H-2a/b ; δH 5.67 (NH-4) showed correlations into H-10a/b, H-9a/b, H-8a/b, and H-7a/b; while δH 5.75 (NH-6) had correlations into the methylene protons of H-18a/b, H-17a/b, H-16a/b, and H-15. Further evidence was seen in the HSQC spectrum, which showed that all methylene carbons adjacent to the Piz N-H’s possessed the deshielded carbon resonances typical of these moieties: C-5 (δC 47.7), C-10 (δC 47.4) and C-18(δC 46.9). 148 Figure 4-27. Expansion of the 1H NMR spectrum for 4.2, demonstrating a clear triplet coupling pattern at the upfield resonance δH 0.77. Figure 4-28. Chemical shift assignments for the three Piz amino acids of dentigerumycin F. 149 Figure 4-29. 1H-15N HSQC NMR spectrum of dentigerumycin F, recorded at 600 MHz in pyridine-d5, showing the three Piz N-H correlations along with the primary amide N-H2 correlations. Figure 4-30. Expansion of 1H-1H TOCSY NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 demonstrating the three Piz spin systems with scalar coupling from the Piz nitrogen-attached proton as well as the Piz α-methine protons. (H-13)1.69, 6.19 Piz 2 N-H Piz 1 N-H & α-methine Piz 3 N-H Piz N-H correlations Primary amide N-H correlations H-4 H-3 H-3 H-5 H-5 H17 H16 H16 H18 H18 H-9 H-9 H-8 H-8 H10 H10 H17 H16 H16 H-9 H-9 H-8 H-8 H10 H10 Piz 2 α-methine Piz 3 α-methine 150 Figure 4-31. 1H-1H TOCSY NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 also demonstrating the clear correlations between H-20, H-21 and NH-8. Figure 4-32. 1H-13C HSQC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5. Piz 2 α-methine Piz 3 α-methine Piz 1 α-methine H-20, NH-8 H-20, H-21 H-21, H-20 H-21, NH-8 151 Figure 4-33. Connecting the core amino acids for dentigerumycin F. The presence of the N-hydroxylated alanine was confirmed both via COSY60 and 1H-1H TOCSY correlations between the methyl doublet resonating at δH 1.67 (H-13) and the deshielded α-methine proton resonating at δH 6.19 (H-12) (Figure 4-34), the presence of 1H-15N LRHSQC correlations between the methyl doublet and the shielded nitrogen (δH 1.67, δN -197.6) suggestive of a hydroxamate moiety (Figure 4-36), and a 1H-13C HMBC correlation between the methyl doublet (δH 1.67) and the deshielded α-carbon (δC 54.2, C-12) (Figure 4-35). The hydroxamate proton was not visible due to the basic nature of the pyridine NMR solvent. tROESY correlations between adjacent α-methine’s and the nitrogen-attached Piz protons established the placement of Piz 1, Piz 2 and N-OH-Ala (Figure 4-37). Since the nitrogen-attached hydroxyl was a very broad resonance in pyridine-d5, it could not be used to establish the connection between N-OH-Ala and 152 Piz 3. However, tROESY correlations were visible between the methyl resonances at H-13 and one of the proton resonances at H-16 (Figure 4-38). 3D models demonstrated that the distance between H-13 and H-16 was 2.4A, well within the through-space range for strong NOEs.221 Thus, the amino acid sequence of 4.2 was confirmed as Piz, Piz, N-OH-Ala, Piz (Figure 4-33). Figure 4-34. Expansion of 1H-1H COSY60 NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 which shows the correlation between neighboring H-13 and H-12 protons. (H-13)1.67, (H-12) 6.19 153 Figure 4-35. Expansion of 1H-13C HMBC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 which shows the correlation between the H-13 resonance and C-12 and C-11. Figure 4-36. 1H-15N LRHMQC demonstrating correlations between N-OH-Ala’s methyl protons and the nitrogen-attached hydroxyl group for dentigerumycin F, at 600 MHz in pyridine-d5. (H-13) 1.67 1.67, (C-12) 54.2 1.67, (C-11) 173.5 (H-13)1.67, (N-OH) -197.6 154 Figure 4-37. Expansion of tROESY NMR spectrum for dentigerumycin F, recorded at 600 MHz in pyridine-d5, demonstrating the through-space correlations connecting Piz 1, Piz 2 and the N-OH-Ala amino acids. Figure 4-38. Expansion of the tROESY NMR spectrum for dentigerumycin F, recorded at 600 MHz in pyridine-d5, demonstrating the through-space correlations connecting Piz 3 and the N-OH-Ala amino acids. (H-7) 6.11, (NH-1) 5.5 (H-12) 6.19, (NH-4) 5.67 (H-16) 2.48, (H-13) 1.67 (H-16) 2.48, (H-15) 6.24 155 Figure 4-39. Building up fragments of dentigerumycin F to help confirm the structure of the PKS portion. To differentiate the structures of constitutional isomers dentigerumycin F (4.2) and dentigerumycin D (4.8) fragments of the molecules were assessed for mass and formula differences. The first point of difference was seen in the core amino acid mass (Figure 4-39); Fragment A has a molecular formula of C18H31N8O5 while the comparable fragment in dentigerumycin D237 has a molecular formula of C17H29N8O5 (molecular formula for this fragment of dentigerumycin D was determined using ChemDraw on the published structure). For 4.2, this left, as in 4.1, seventeen carbons, thirty protons, one nitrogen, and six oxygen atoms that were yet to be assigned within the structure. Recorded in pyridine-d5, the hemiketal/dehydro equilibrium (discussed in Subsection 4-3-3) for dentigerumycin F (4.2) was not as pronounced; however, it was still a complicating factor in the structure elucidation. Through NMR analysis, it became evident that the remainder of the molecule was identical to 4.1. Notably, 4.2 also 156 contained a methyl singlet at C-32 (δC 21.7, δH 1.64) which showed clear HMBC correlations into a quaternary carbon at δC 78.2 (C-26), δC 100.2 (C-27) along with a carbonyl carbon at δC 178.5 (C-25) (Figure 4-42). The hydroxyl substituent of the C-26 quaternary carbon was not visible in the data due to the basic nature of the deuterated pyridine utilized as NMR solvent. However, the deshielded carbon resonances at C-26 and the singlet methyl at C-32 also provided evidence that there were no coupled protons attached to C-26. Further analysis of the 2-D NMR data revealed the characteristic β-OH-Leucine in the TOCSY spectrum with a spin system inclusive of a methine doublet of doublets at H-21 (δH 4.12), demonstrating TOCSY correlations to a methine at H-22 (δH 2.45), methyl doublets at H-23 (δH 1.44, δC 16.0) and H-24 ( δH 1.19, δC 21.1) (Figure 4-45), a methine proton at H-20 (δH 6.40) and an amide proton at δH 8.71 (NH-8) (Figure 4-31). COSY data demonstrated cross peaks from the H-22 methine to the two methyl doublets at H-23 and H-24 (Figure 4-44). Strong HMBC correlations from the N-H amide proton at δH 8.71 to the C-25 carbonyl at δC 178.5; from H-20 to the carbonyls at C-19 (δC 174.2) and C-25; and from H-21 to C-19, built the connection between the NRPS and PKS portions of the molecule (Figure 4-40). Fragment B seen in Figure 4-39, was then constructed with a partial molecular formula of C9H16NO4. 157 Figure 4-40. Expansion of the 1H-13C HMBC spectrum for 4.2 demonstrating correlations connecting the NRPS and PKS portions of the molecule through H-20, H-21 and NH-8 into C-19 and C-25. Also seen are the correlations which linked Piz protons to adjacent carbonyls. Recorded at 600 MHz in pyridine-d5. With this information, it was seen that there were eight carbons, fifteen protons and two oxygens that were yet to be assigned within the structure. With this information in hand, it was possible to elucidate the remainder of the molecule. Through analysis of the 2-D data, it became evident that the remaining C8H15O2 formed a disubstituted oxane ring that was identical to the construction of the PKS sidechain seen in the free acid. The oxane ring was again revealed through gCOSY60, 1H-1H TOCSY and HMBC correlations. First, the COSY spectrum allowed clarification of the suspected proton methyl groups (Figure 4-44). The H-34 methyl shift (δH 0.77, δC 10.7), had a shielded chemical shift often associated with aliphatic methyl groups and was well separated from the β-OH-Leucine two methyls in pyridine-d5 such that its triplet coupling pattern was very clear. This triplet splitting pattern was confirmed via the 2D J-resolved NMR experiment (Figure 4-48). In pyridine-d5, the resonance for one of the leucine’s (Piz 1)4.9, 172.4 6.11, 173.9 (Piz 3)6.24, 172.9v (H-13)1.67, 173.5 (H-21)4.12, (C-19) 174.2 (H-32)1.64, (C-25)178.5 174.2 (H-20)5.42 178.5 8.71, (C-25)178.5 NH-8 (Leu) 158 methyl groups was overlapping with the H-35 methyl peak so the COSY60 correlations (Figure 4-44) were utilized to confirm assignment. Then, the TOCSY spectrum allowed the spin system construction (Figure 4-45), building from the H-31 methine proton (δH 4.04) to the methyl doublet at H-35 (δH 1.9) on one side and from the H-31 methine proton to a shielded methine proton at H-30 (δH 1.11, δC 43.1) along to the methylene and methyl protons of H-33a/b (δH 1.39, 0.98) and H-34 (δH 0.77). This spin system also showed extension through to the H-29 and H-28 methylene protons, which was then confirmed with COSY60 correlations of the H-29 methylene (δH 1.70) to the distinctive (Figure 4-46) H-30 methine. Figure 4-41. Correlations confirming the β-OH-Leucine amino acid characteristic of these compounds along with the unique substitution pattern in the PKS portion of dentigerumycin F. As well, the correlations that link the amino acid constructed portion of the molecule with the polyketide constructed portion of the molecule are seen. Again, HMBC correlations (Figure 4-41 and 4-47 ) between the C-30 resonating at δC 43.1 and the methyl at H-35 (δH 1.19) along with the deshielded C-31 resonating at δC 70.8 and a methylene proton from H-33 (δH 0.98) confirmed the adjacent nature of the tetrahydropyran 159 substitutions. This unique ring pattern distinguished (4.2) as being a novel linear piperazic acid-containing free amide. As in dentigerumycin G (4.1), the absolute configuration of the molecule was assigned using the crystal structure of incarnatapeptin A (4.3) Vida infra as they are produced from the same biosynthetic pathway along with the similarity in NMR resonances for the amino acids and PKS-NRPS link. Figure 4-42. 1H-13C HMBC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 demonstrating correlations from the methyl singlet in the PKS unit to the adjacent quaternary carbons. C-26, 78.2 C-27, 100.2 C-25, 178.5 H-32, 1.64 160 Figure 4-43. Full 1H-13C HMBC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5. Figure 4-44. Expansion of the 1H-1H COSY60 NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 wherein the correlations connecting the neighboring protons of the oxane ring are seen. 0.77; 1.36 0.77; 0.98 1.19 (H-24); 2.45 (H-22) 1.20 (H-35); 4.04 (H-31) 1.67 (H-13); 6.19 (H-12) 1.44 (H-23); 2.45 (H-22) 0.77 (H-34); 1.11 (H-30); 1.70 (H-29) 1.11 (H-30); 4.04 (H-31) 161 Figure 4-45. 1H-1H TOCSY NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5. Figure 4-46. Expansion of the 1H-13C HSQC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 demonstrating the characteristic H-30 to C-30 1J correlation. H-31, 4.04 H-34, 0.77 H-33, 0.98 H-29, 1.70 H-28, 1.97 H-28, 2.15 H-35, 1.20 H-30, 1.11 H-30: 1.11; 43.1 H-21, 4.12 H-24, 1.19 H-23, 1.44 H-22, 2.45 162 Figure 4-47. Expansion of 1H-13C HMBC NMR spectrum of dentigerumycin F recorded at 600 MHz in pyridine-d5 demonstrating correlations from the methyl doublet at H-35 into the oxane ring and from the methylene at H-33 into the same carbons of the oxane ring. Figure 4-48. 2D J-resolved spectrum for dentigerumycin F. This helped confirm the identity of the substituents on the oxane ring via the triplet splitting pattern at H-34. Recorded at 600 MHz in pyridine-d5.H-35, 1.20 C-30, 43.1 C-31, 70.8 H-33, 0.98 H-34 H-34 H-34 163 8089-13: Dentigerumycin F (4.2) Pyridine-d5 DMSO-d6 Hemiketal Form Hemiketal Form Olefinic Form Residue Position δC/δN δH (J = Hz) δC/δN δH (J = Hz) δC/δN δH (J = Hz) L-Piz Amide 1 C 173.4 172.8 172.8 2 CH 51.5 5.50 m 50.3 4.82 dd (1.5, 5.8) 50.3 4.82 dd(1.49, 5.78) 3 a/b CH2 26.3 1.75, 2.42 m 24.7 1.76, 2.06 24.7 1.76, 2.06 4 a/b CH2 21.7 1.43, 1.40 m 21.3 1.45, 1.50 21.3 1.45, 1.50 5 a/b CH2 47.7 2.67, 2.95 m 46.7 2.58, 3.00 46.7 2.58, 3.00 NH-1 NH -300.3 5.5 -301.0 4.99 m -301.0 4.99 m CONH2 -275.8 8.27, 8.09 -274.3 7.56/7.20 -274.3 7.56/7.20 D-Piz 2 6 C 173.9 172.0 172.0 7 CH 49.4 6.11 dd (2.9; 6.4) 47.3 5.53 m 47.3 5.53 m 8 a/b CH2 25.8 1.88, 2.15 m 24.6 1.76,1.99 m 24.6 1.76,1.99 m 9 a/b CH2 21.4 1.62, 1.46 m 20.7 1.41,1.50 m 20.7 1.41,1.50 m 10 a/b CH2 47.4 3.01, 2.77 m 46.2 2.58, 2.96 m 46.2 2.58, 2.96 m NH-4 NH -300.9 5.67 -300.1 5.11 m -300.1 5.11 m N-OH-L-Ala C11 C 173.5 171.9 12 CH 54.2 6.19 m 53.3 5.53 m 53 5.55 m 13 CH3 16.1 1.67 d (6.8) 14.6 1.33 d (6.9) 14.8 1.35 d (6.8) N5 -197.6 -204.0 N5-OH 11.57 v. broad 9.54 br.s D-Piz 3 14 C 172.9 171.6 171.6 15 CH 49.8 6.24 47.8 5.47 m 47.8 5.47 m 164 16 a/b CH2 25.4 2.48, 1.90 m 24.9 2.1, 1.77 24.9 2.1, 1.77 17 a/b CH2 21.9 1.78, 1.43 m 21.6 1.49, 1.34 21.6 1.49, 1.34 18 a/b CH2 46.9 3.27, 2.98 m 45.3 2.86 m 45.3 2.8 m NH-6 NH -300.0 5.75 -300.0 5.1 m -300.0 5.08 m β-S-OH-L-Leu 19 C 174.2 170.9 170.9 20 CH 52.3 6.40 m 50.1 5.49 m 49.8 5.42 m 21 CH 77.4 4.12 dd (2.7, 8.4) 75.8 3.39 m 75.6 3.41 m 22 CH 29.6 2.45 m 27.8 1.85 m 28.8 1.69 m 23 CH3 16.0 1.44 d (6.87) 15.1 0.82 d (6.87) 14.7 0.82 d (6.87) 24 CH3 21.1 1.19 d (6.87) 20.2 0.83 d (6.87) 20.2 0.82 d (6.87) NH-8 NH -264.9 8.71 d (9.38) -264.9 7.70 d (9.38) -267.0 7.23 d (10.02) 21-OH Not visible PKS 25 C 178.5 176.6 172.3 26 C 78.2 76.9 75.2 27 C 100.2 98.9 154.1 28 a/b CH2 28.0 1.95 m 28.0 2.1, 1.9 m 93.1 4.81 29 a/b CH2 26.0 1.70 m 22.8 1.63, 1.53 m 24.1 1.68, 1.46 m 30 CH 43.1 1.11 m 41.7 0.97 m 38.6 1.26 m 31 CH 70.8 4.04 m 69.0 3.59 m 74.1 3.35 m 32 CH3 21.7 1.64 s 20.7 1.21 s 23.7 1.27 s 33 a/b CH2 24.6 1.36, 0.98 q (6.9) 23.7 1.43, 0.99 m 24.1 1.06, 0.98 m 34 CH3 10.7 0.77, t (6.9) 10.7 0.82 t (6.9) 11.3 0.87 t 35 CH3 19.4 1.19, d (6.5) 18.6 0.99 d (6.5) 19.1 1.14 d (6.5) 26-OH Not visible 5.45 5.45 27-OH Not visible 5.88 s Table 4-2. NMR shifts of dentigerumycin F in both pyridine-d5 and DMSO-d6. Both hemiketal and dehydrated forms are seen in DMSO-d6. 165 4.3.3 Investigations into the Equilibria Observed in Dentigerumycins F And G Dentigerumycin F (4.2) and G (4.1) were initially isolated using HPLC conditions that included 0.05% TFA to remove peak tailing. The samples were also left at room temperature for up to thirty-six hours while removing the transfer solvent and solvent traces. NMR analysis of the natural products isolated under these conditions revealed multiple resonances for some atoms of the molecules (Figure 4-49 a, Figure 4-51 and 4-52). There was initial uncertainty if the signals were from separate atoms or different molecules. Obtaining a 1H spectrum quickly after removing from storage at 4 ℃ demonstrated that specific chemical shifts were changing in intensity in cold temperatures (Figure 4-49 b). Next, a 1H NMR spectrum from the Figure 4-49. Comparison of proton spectra of 4.1 collected at various times and temperatures a) 1H NMR spectrum obtained after isolation and sample preparation. b) 1H NMR spectrum obtained immediately after storage in the fridge. c) 1H NMR spectrum obtained after the sample was let sit at RT. The pink box highlights the H-35 methyl doublet, while the orange boxes highlight the methyl singlet at C-32. Recorded at 600 MHz in DMSO-d6. 1H spectrum immediately after isolation 1H spectrum immediately after storage at 4oC 1H spectrum after storage at RT 0.99 1.14 1.2 1.27 a b c 166 sample sitting at room temperature revealed a return to the mixture of signals (as revealed by key proton peaks) (Figure 4-49 c). Significant changes to some δH and δC in the polyketide portion and the hydroxy leucine of the molecule were shown to be reversible depending on the temperature of the sample and the solvent in which they were stored (Tables 4.1 and 4.2; Figures 4-50 to 4-52). Cold temperatures favored the hemiketal form or natural form of 4.1: resonances were seen for a quaternary carbon at δC 98.8 (C-27); a methyl singlet δC 21.5, δH 1.20 (C-32); a methyl doublet δH 0.99 (C-35); a methylene δC 26.54, δH 1.62 and 1.54 (C-28); and a Figure 4-50. Oxane systems of dentigerumycin G (4.1) in DMSO-d6, showing the resonances seen for each form. H-35 highlighted in pink, and H-32 highlighted in orange to match Figure 4-49. methine at δC 69.1, δH 3.56 (C-31). When samples of 4.1 were left at room temperature (~26 ℃) for the time needed to collect a data set (~20 hrs) or longer (3 or more days), those same resonances changed. In the dehydrated form, quaternary carbon C-27 was seen to resonate at δC 152.9; the C-32 methyl singlet changed to δH 1.27, δC 23.5; the methyl doublet at C-35 was deshielded ( δH 1.27); C-28 now had an olefinic proton (δH 4.84) attached to a very deshielded carbon (δC 94.6), and the C-31 methine was found at δC 74.1, δH 3.35. The molecule was thus 167 seen to equilibrate between a hemiketal at C-27 and an olefin at C-27/C-28 depending on the sample’s temperature. Figure 4-51. 1H-13C HMBC of room temperature dentigerumycin G (4.1) (isolated with TFA as HPLC pH buffer) showing HMBC correlations of both equilibrating forms. Recorded in DMSO-d6 at 600 MHz. Hemi-ketal form Vinyl ether form 168 Figure 4-52. Full 1H-13C HMBC of room temperature dentigerumycin F (4.2) showing the HMBC correlations for both equilibrating forms. Recorded in DMSO-d6 at 600 MHz. The DMSO-d6 (freezing point: 18.5 ℃) solvent prevented obtaining NMR samples at cooler temperatures. Due to this, 2D spectra were challenging to obtain without resonances of both forms present. In order to attempt to obtain spectra at colder temperatures, the DMSO-d6 solvent was removed via lyophilizer, after which CD3OD was added to the NMR tube containing the sample. Proton spectra of this sample showed that all residual TFA from initial isolation was also removed through this process. NMR data of this sample recorded at room temperature in CD3OD showed a majority of the dehydrated form of the molecule, with the C-31 methine correlation δC 74.1, δH 3.35 displaying an intense peak as well as C-28 as deshielded olefinic proton (Figure 4-53; Figure 4-56 to Figure 4-57). With the addition of 50 µL of D2O to CD3OD and data collection at 10 ℃, the equilibrium favours the hemiketal form in an approximately 2:1 fashion. The C-31 methine correlation is observed at δC 69.1, δH 3.56 and the Vinyl ether form Hemi-ketal form 169 correlation at C-28 as δH 4.81, δC 93.1 is no longer present in strong intensity (Figure 4-54; Figure 4-56 to Figure 4-57). Figure 4-53. Expansion of 1H-13C HSQC of dentigerumycin G (4.1) in CD3OD (at 600 MHz) and spectrum recorded at RT demonstrating a strong olefin signal at C-28. C-28 (4.81, 93.1) C-31 (3.35, 74.1) 170 Figure 4-54. Expansion of 1H-13C HSQC of dentigerumycin G (4.1) in CD3OD with 50 uL of D2O added and spectrum recorded at 10 oC where the olefinic correlation at C-28 is not seen. Recorded at 600 MHz. Figure 4-55. Expansion of overlapping 1H-13C HSQC spectra of 4.1 in CD3OD. Red/pink correlations are found in CD3OD let sit at RT. Teal correlations are found in CD3OD after sitting in the fridge for a week. Black correlations are found in CD3OD with 50 uL of D2O added with spectra recorded at 10 oC. Recorded at 600 MHz. C-31 (3.56, 69.1) 4.81, 93.1 C-31 C-31 171 Figure 4-56. Overlapping 1H-13C HMBC NMR spectra of dentigerumycin G (4.1) in CD3OD. Red correlations are found in CD3OD let sit at RT. Black correlations are found in CD3OD with 50 uL of D2O added with spectra recorded at 10 oC, 600 MHz. Distinct differences are observable. Figure 4-57. Scheme of equilibrium seen in both (4.1) and (4.2) when isolated using 0.05% TFA as a pH buffer during HPLC purification and stored in DMSO-d6. Not shown in a caption: CD3OD with all acid removed favors the dehydrated form, addition of small amounts of deuterated water without the addition of further acid favors the hemiketal form once again. 172 Overall, both the primary amide (4.2) and the free acid (4.1) were seen to engage in an equilibrium between hemiketal and vinyl ether forms when isolated with 0.05 TFA% in the HPLC solvent. In DMSO-d6 at RT, there is an approximate 1:1 ratio between the two forms seen in data sets for 4.1 collected after prolonged RT exposure (Figure 4-55). In CD3OD, without the presence of water, the equilibrium more strongly favours the dehydrated form. With the addition of deuterated water to the CD3OD and chilling of the sample, the hemiketal form is dominant. More experiments are required to understand the mechanism driving this transformation. 4.2 and 4.1 were also seen to be undergoing an occasional equilibrium between a hemiketal and ketone form (weakly) as seen in Figure 4-58. The hemiketal/ketone equilibration, similar to that shown in verucopeptin,239 was minor and visible in the NMR spectra of 4.1 only through the appearance of very weak HMBC correlations from the methyl singlet at C-32 ( δH 1.30, δC 21.3) into a ketone carbonyl shift, δC 206.9. While verucopeptin has been previously shown to demonstrate an equilibrium between a ketone and hemiketal form239,240 (a molecular property recently harnessed by researchers to synthesize verucopeptin and some novel derivatives180); the equilibrium between a hemiketal and an vinyl ether form at room-temperature has never been demonstrated for an oxane ring with piperazic-containing compounds or any natural products that I could uncover. As mentioned, isolation of (4.1) and (4.2) with formic acid (FA) as pH modifier proved to produce NMR evidence of only the stable hemiketal form (Appendix B). 173 Figure 4-58. The tautomerization in 4.1 between a hemiketal and ketone form. 4.3.4 Structure Elucidation of Incarnatapeptin A (4.3) After working on the structures of the more abundant 4.1 and 4.2, the proton spectrum of incarnatapeptin A (4.3) was collected at multiple times throughout 2-D data set collection with multiple 1H spectra showing no fluctuations in proton peaks. This observation suggested that incarnatapeptin A (4.3) had a unique polyketide region compared to 4.1 and 4.2, which drove interest in solving the structure despite the minimal material available. In order to corroborate that this HPLC peak was indeed a Piz-containing compound, initial work done to solve the structure with available material was able to show exchangeable protons at δH 4.74 (δN -301.1), and 5.12 (δN -300.5) through a one-bond correlation between proton and nitrogen resonances in the 1H-15N HSQC spectrum (Figure 4-61). 174 Figure 4-59. 1H spectrum for author isolated incarnatapeptin A (4.3) in DMSO-d6 at 600 MHz. 175 Figure 4-60. 13C spectrum collected by author for incarnatapeptin A in DMSO-d6 at 150 MHz. Incarnatapeptin A (4.3) gave a [M - H]- ion at m/z 781.4097 and a [M + Na]+ ion at m/z 805.4075 in the HRESIMS appropriate for a molecular formula of C35H58N8O12 that requires 11 sites of unsaturation. Examination of the NMR data confirmed that 4.3 is a piperazic acid-containing nonribosomal peptide-polyketide (NRP-PK) hybrid molecule with the 1H, gTOCSY/gHSQC/gNHSQC/gHMBC/gNHSQC-TOCSY NMR resonances indicating the presence of multiple piperazic-acid spin systems, an N-hydroxylated alanine, a β-OH-leucine, and a region of clear PKS origin. 176 Figure 4-61. 1H-15N HSQC spectrum for author isolated incarnatapeptin A in DMSO-d6, at 600 MHz showing the Piz N-H correlations. Figure 4-62. 1H-15N HSQC spectrum collected by author for incarnatapeptin A in DMSO-d6 at 600 MHz. Piz N-H correlations Piz N-H correlations 4.71, -301.1 5.12, -300.5 5.07, -299.9 177 Figure 4-63. Chemical shift assignment for the three Piz amino acids of incarnatapeptin A. In the 1H-1H TOCSY spectrum (Figure 4.65), exchangeable protons were shown to have typical Piz spin systems with δH 4.74 (NH-1) demonstrating correlations into H-5a/b, H-4a/b, H-3a/b, and H-2a/b; δH 5.07 (NH-4) showing correlations into H-10a/b, H-9a/b, H-8a/b, and H-7a/b; while δH 5.12 (NH-6) had correlations into the methylene protons of H-18, H-17, H-16, and H-15 (Figure 4.63). A carboxylic acid-like 1H broad resonance at δH 13.26 suggested the presence of a free acid. With spectra recorded from more material isolated by David Williams (Figure 4.62; Appendix C), the third exchangeable proton resonance was confirmed at 5.07 (δN -299.4) in the 1H-15N HSQC, and piperazic acid spin systems were supported with a 1H -15N HSQC-TOCSY spectrum (Figure 4.64). With the initial materials, the HSQC spectrum (Figure 4.66) supported this compound as a Piz-containing natural product with typical deshielded 178 resonances C-5 (δC 45.6), C-10 (δC 46.50) and C-18(δC 46.35) for methylene carbons attached to the Piz N-H’s. Figure 4-64. 1H-15N HSQC-TOCSY spectrum collected by author for incarnatapeptin A in DMSO-d6 at 600 MHz. It illustrates the characteristic Piz spin systems. 2.2, 1.76, 1.4-1.3 2.9, 2.8, 2.6 4.71, -301 5.12, -300.5 & 5.07, -299.9 179 Figure 4-65. Expansion of 1H-1H TOCSY spectrum for author isolated incarnatapeptin A, recorded in DMSO-d6 at 600 MHz, demonstrating the Piz spin systems for all three piperazic acids. Also seen are the correlations between the methyl (H-13) and the α-methine (H-12) of the N-OH-Ala amino acid. Figure 4-66. 1H-13C HSQC spectrum for author isolated incarnatapeptin A in DMSO-d6 at 600 MHz, where the three Piz α-methine resonances with 1J correlations into their respective carbon resonances are shown. Piz 3 and Piz 2 have unusually deshielded α-methines in line with other characterized Piz-containing natural products. In turquoise; the similar proton-carbon correlations of the bicyclic PKS ring. H-12, H-13 Piz 2 α-methine Piz 3 α-methine Piz 1 α-methine (H33, C33) 4.11, 66.5 3.98, 72.6 H31, C31 Piz 2; Piz 3 N-H Piz 1 N-H Piz 1 α-methine Piz 2 α-methine Piz 3 α-methine H-4 H-4 H-3 H-3 H-5 H-5 H-4 H-4 H-3 H-3 H17 H16 H16 H18 H-9 H-8 H-8 H10 H10 H17 H17 H16 H16 H-9 H-8 H-8 180 Figure 4-67. Connecting the core amino acids for incarnatapeptin A. The presence of an N-hydroxylated alanine - identical to that seen in incarnatapeptin C and D -was confirmed via COSY60, HMBC, and 1H-15N LRHSQC correlations. In the COSY60 spectrum, a methyl doublet at δH 1.35 (H-13) correlating to a deshielded α-methine proton resonating at δH 5.59 (H-12) suggested an Ala amino acid (Figure 4-69). An 1H-13C HMBC correlation between the methyl doublet (δH 1.67, H-13) and the deshielded α-carbon (δC 54.2, C-12) confirmed this assignment (Figure 4-74). A hydroxymate-like proton resonance at δH 9.53 (Figure 4-59) suggested the presence of an N-OH-Ala, which was confirmed by 1H-15N LRHSQC correlations between the methyl doublet and the shielded nitrogen (δH 1.67, δN -200.6) (Figure 4-71). tROESY correlations between adjacent α-methine’s and either the nitrogen-attached Piz proton or the nitrogen-attached hydroxyl of Ala were instrumental in building the correct placement of the amino acids (Figure 4-67 and Figure 4-68). At this juncture, the 181 limited material did not provide sufficient data to link N-OH-Ala and Piz 2. However, the similarity of the amino acid data of 4.3 to 4.1 strongly suggested that the 4.3 had the same amino acid sequence as 4.1, for which evidence linking the N-OH-Ala and Piz 2 were present. Subsequently, a tROESY correlation between the α-methine of N-OH-Ala and Piz 2 was confirmed from data collected on 4.3 material that David Williams obtained (Appendix C). Thus, the core amino acid sequence of incarnatapeptin A (4.3) was confirmed as a free acid Piz, Piz, N-OH-Ala, Piz (Figure 4.67). Figure 4-68. tROESY correlations between the protons of the nitrogen-attached hydroxyl/proton and α-methine proton leading to the assigned structure of 4.3. Recorded in DMSO-d6 at 600 MHz. Piz 3 (5.47, H-15) N-OH, 9.55 Piz 2 (5.54, H-7) Piz 1 (4.71, N-H) 182 Figure 4-69. 1H-1H COSY spectrum for author isolated incarnatapeptin A in DMSO-d6 at 600 MHz. Figure 4-70. 1H-13C HMBC spectrum for author isolated incarnatapeptin A in DMSO-d6 at 600 MHz. 1.35, 5.59 1.35, 5.59 1.35, 53.4 H-13, C-12 1.35, 172.0 5.59, 14.5 H-12, C-13 5.59, 172.0 (C-11) H-12 H-13 183 Figure 4-71. 1H-15N LRHMQC spectrum for author isolated incarnatapeptin A in DMSO-d6 at 600 MHz demonstrating the correlation between the methyl group of the N-OH-Ala and the nitrogen resonance of the hydroxamate moiety. Further analysis of the 2D data revealed the standard Piz-containing 19-membered depsipeptides β-OH-Leucine with methyl doublets at C-23 (δC 15.13, δH 0.82) and C-24 (δC 20.2, δH 0.86) showing HMBC correlations from δH 0.86 into C-21 (δC 75.82) (Figure 4.74), and the g1H-1H COSY spectrum revealing three adjacent methines at H-20 (δH 5.50), H-21 (δH 3.36), and H-22 (δH 1.75) as well as a cross peak from H-20 to an amide proton (NH-8; δH 7.29) (Figure 4.72 and 4.73). Analysis of the 2D data demonstrated that the PKS linkage between the β-OH Leu and the oxane ring was present with a methyl singlet at C-32 (δC 21.16, δH1.12) showing HMBC correlations into a carbinol carbon at C-26 (δC 76.40) (Figure 4.74). No other amino acid signatures were present; thus, 4.3 was seen to be a linear peptide like 4.1 and 4.2. 1.35, -200 H13, N-OH 184 Figure 4-72. 1H-1H COSY spectrum for author isolated incarnatapeptin A in DMSO-d6 at 600 MHz. Shown are the proton correlations in the conserved leucine residue. Figure 4-73. 1H-1H TOCSY spectrum for author isolated incarnatapeptin A, recorded in DMSO-d6 at 600 MHz. The correlations supporting the construction of the leucine amino acid are seen. NH-8, H-20 H-20, H-21 NH-8, H-20 H-20, H-21 185 Figure 4-74. Full 1H-13C HMBC spectrum for author isolated incarnatapeptin A, recorded in DMSO-d6 at 600 MHz. and showing key HMBC correlations. Figure 4-75. Building up fragments of incarnatapeptin A to help confirm the structure of the PKS portion. H-32, C-26 H-32, C-27 H-31, C-33 H-13, C-12 H-34 & H-35, C-30 186 Considering an m/z: [M-H] = 781.4097, mass analysis of the solved fragments seen in Figure 4.75 left a small fragment C with eight carbons, thirteen protons, and two oxygen atoms to be accounted for. Initially, however, due to the low amount of compound, the low S/N ratio in the mass spectrum cast uncertainty on the accuracy of this m/z: [M - H] = 781.4097. Nevertheless, it was immediately evident that there was a substantial difference in the oxane ring's substitution pattern, with resonances demonstrating a somewhat symmetrical PKS ring system. Indeed, there was a third methyl doublet in the overall structure (N-OH-Ala methyl doublet, C-35 methyl doublet); this methyl doublet at δH 1.09 was showing COSY correlations into a deshielded methine proton at δH 4.11 (δC 66.53). This correlation was very similar to the H-35/H-31 relationship, as shown in the COSY spectrum in Figure 4.76 and the deshielded carbon at δC 66.53 suggested connection to an electronegative oxygen. Figure 4-76. Expansion of 1H-1H COSY60 spectrum for incarnatapeptin A demonstrating the similar oxane correlations. Recorded in DMSO-d6 at 600 Mhz. H-33 H-31 H-34 H-35 187 In summary, the data set for 4.3 demonstrated two carbinol-like carbons C-31 (δC 72.58) and C-33 (δC 66.53) each with a methine quartet H-31(δH 3.98) and H-33 (δH 4.11) (Figure 4-66 in turquoise). The methine quartets resonating at δH 3.98 (H-31) and δH 4.11 (H-33) each demonstrate 1H-1H COSY correlations into methyl doublets H-35 (δH 1.14) and H-34 (δH 1.09), respectively (Figure 4-76). Additionally, the H-34 and H-35 methyl doublets demonstrated HMBC correlations to the methine carbon at C-30 (δC 34.03) (Figure 4-74, blue arrow), setting up the tentative spin system seen in Figure 4-77. As will be discussed, the bond shown in red had no immediate supporting data. Additionally, two secondary carbons at δC 25.2 (C-28) and δC 18.4 (C-29) were observed with methylene protons (H-28a/b, 1.84/1.75; H-29a/b, 1.88/.54) demonstrating HMBC correlations into C-30 (δC 34.0) and C-31 (δC 72.6). In addition, Figure 4-77. Left-hand panel: COSY and HMBC correlations confirming the hydroxy-leucine amino acid and the PKS portion of the molecule. Center panel: configuration in bicyclic ring. Right-hand panel: 3D representation of the bicyclic ring. the methylene protons of H-28 (δH 1.84, 1.75) showed strong HMBC correlations into the quaternary carbon at δC 98.5 (C-27) (Figure 4.77). With the available NMR data, two possible oxane ring variants were proposed (Figure 4-78). Strong evidence supported an oxygenated 188 ethyl side chain at C-33 and C-34. Consideration of the calculated mass of 782.42 Daltons led to the further conclusion that this hydroxylated ethyl side chain would, in fact be involved in forming a bicyclic system. However, with the available NMR data, it was difficult to determine which of two possible variants, 1) 2,6-dioxabicyclo[2.2.2]octane moiety or 2) a single ring with a hydroxylated ethyl side chain was the correct structure. Figure 4-78. Initial NMR data could not distinguish between a) A possible monocyclic variant for 4.3’s PKS portion given initial data or b) A possible bicyclic variant for 4.3’s PKS portion given the initial data. LR-HSQMBC NMR experiment was conducted in order to see 4J and 5J coupling. An HMBC correlation between H-33 to C-27 or C-26 would have demonstrated connection between the C-33 oxygen and the C-27 quaternary center, thus confirming a bicyclic system. However, this correlation was not present. On the alternate structure, the absence of the correlation did not confirm the single ring. Therefore, in an attempt to see this correlation, a LR-HSQMBC NMR experiment,216 which can detect atoms with four to five bonds separating them was conducted in the hopes of seeing a correlation either between H-33 to C-27 or between H-33 a b 189 to C-26. No signal between the methine proton at H-33 and the quaternary carbon at C-27 or C-26 was seen. Residual trifluoroacetic acid (TFA) in the NMR sample prevented consistent observation of the hydroxyl proton resonances. It was thus difficult to ascertain if an artifact of the isolation process was preventing detection of a hemiketal hydroxyl proton resonance or if it was truly absent from the structure. To overcome this second limitation, David Williams carried out a methyl esterification of the natural product to generate a methyl ester for 4.1 (Appendix D) and purified it by HPLC without TFA. NMR data obtained for the methyl ester of incarnatapeptin A had visible hydroxyl protons (Appendix D) with: 1H-1HCOSY correlations between H-21 (δH 3.36) of the 3-β-hydroxy leucine and an exchangeable doublet without nitrogen attachment at δH 4.73; the appearance of an exchangeable singlet at δH 4.91 (O-10H-10) at as well as additions of methoxy resonances for the free ester (δH 3.67, δC 51.97 with HMBC correlations into δC 170.9) and the transformed N-OH (δH 3.74, δC 63.34 with g15NLRHMQC correlations into N-5 (δN -173.0)). Hydroxyl protons for the typical hemiketal at C-27 were not visible, nor was there a hydroxyl proton with correlations into C-33. However, a definitive assignment was still not obtainable from the NMR data, and there were multiple stereocenters to assign. Fortunately, the methyl ester (Appendix D) appeared to be crystallizable, and the crystals subsequently obtained were suitable for single-crystal X-ray diffraction analysis (Appendix E). The structure determined by X-ray diffraction (ORTEP Diagram in Figure 4-79) was consistent with the NMR data for 4.3, which confirmed that the PKS portion of the natural product contained an unusual 2,6-dioxabicyclo[2.2.2]octane moiety along with confirming the absolute configuration of 4.3. 190 Figure 4-79. X-ray crystal figure produced for the methyl ester of incarnatapeptin A. . To the best of our knowledge, this 2,6-dioxabicyclo[2.2.2]octane moiety has not been reported in a natural product or from synthesis. Interestingly, the X-ray structure confirmed that the chiral carbons of C-31 and C-33 in the bicyclic moiety were both R-configured (Figure 4.77), resulting from the relative trans configuration for the attached methyl branches of C-35 and C-34, respectively. With both C-31 and C-33 having an R-configuration and thus the same Cahn-Ingold-Prelog hierarchy, challenges arose in assigning the absolute configuration of the stereocenters at C-27 and C-30. Application of revised CIP rules, following the Cahn-Ingold- 191 Prelog rules for central chirality, along with analysis of the Xray data through ChemDraw™ helped to assign configuration at C-27 (R) and C-30 (R).241,242 8089-11: Incarnatapeptin A (4.3) DMSO-d6 Residue Position δC/δN δH (J in Hz) L-Piz 1 1 C 172.0 2 CH 50.5 4.90, bdd (5.5, 1.8) 3 a/b CH2 24.7 1.78/2.15 m 4 a/b CH2 20.8 1.29/1.52 m 5 a/b CH2 46.4 2.60/3.0 m NH-1 NH -301.1 4.71, d (12.3) COOH OH 13.26 D-Piz 2 6 C 172.5 7 CH 48.4 5.54 d (5.8) 8 a/b CH2 25.5 1.99/1.78 m 9 a/b CH2 20.8 1.48/1.51 m 10 a/b CH2 46.5 2.6/2.9 m NH-4 NH -300.5 5.12 d (12.6) N3 C N-OH-L-Ala C11 CH 172.0 12 CH3 53.4 5.59 q (7.1) 13 C 14.5 1.35 d (7.1) N5 -200.6 N5-OH 9.55 s D-Piz 3 14 C 171.1 15 CH 48.4 5.47 dd (6.3, 1.4) 16 a/b CH2 24.5 2.12/1.75 m 17 a/b CH2 21.1 1.48/1.51 m 18 a/b CH2 45.6 2.8 m NH-6 NH -299.9 5.07 d(11.16) β-OH-L-Leu 19 C 172.6 20 CH 49.6 5.50 m 21 CH 75.8 3.36 dd (8.6, 2.1) 22 CH 28.1 1.75 m 192 23 CH3 15.1 0.82 d (6.8) 24 CH3 20.2 0.86 d (6.8) NH-8 NH -264.9 7.29 d (9.7) C21-OH Not visible PKS 25 C 172.0 26 C 76.4 27 C 98.5 28 a/b CH2 25.2 1.84/1.75 m 29 a/b CH2 18.4 1.88/1.54 m 30 CH 34.0 1.39 m 31 CH 72.6 3.98 q (6.5) 32 CH3 21.2 1.12 s 33 CH 66.5 4.11 q (6.4) 34 CH3 18.5 1.09 d (6.4) 35 CH3 18.3 1.14 d (6.5) 26-OH Not visible Table 4-3. NMR assignments for incarnatapeptin A (4.3) in DMSO-d6, recorded at 600 MHz. 4.3.5 Incarnatapeptin B The cyclic relative of incarnatapeptin A (4.3), the structure elucidation of incarnatapeptin B (4.4) was completed by David Williams after he isolated the molecule from Streptomyces Incarnatus NRRL 8089 extracts as a very minor metabolite243 during his work to purify more of 4.3 and help establish the bicyclic nature of the PKS-derived moiety. The structure of 4.4 is given here as it is important to the discussion of the putative biosynthetic pathway and bioactivity. The data used to elucidate the molecule can be found in the 2020 Organic Letters paper where it was published.243 193 4.4 Biosynthetic Proposal for the Incarnatapeptins and Dentigerumycins F and G As discussed in Section 3.3.4, analysis of the genome of Streptomyces incarnatus NRRL-8089 (using antiSMASH) allowed us to identify an ~90 kb gene cluster contained within the CP011498.1 contig as the putative incarnatapeptin biosynthetic gene cluster (Scheme 3-1, Figure 4.80). The gene cluster was identified as the sole gene cluster encoding a KtzT homolog within the full genome. The arrangement of genes in this gene cluster are highly related to the gene clusters for the piperazic acid-containing natural products polyoxypeptins, the aurantimycins (Figure 2-5) and verucopeptin.173,180 Specifically, as seen in Figure 3-22, Streptomyces incarnatus NRRL-8089’s CP011498.1 gene cluster contains the same number of PKS modules as both the polyoxypeptins and the aurantimycins, with similar to identical domains (Table 3-9). The CP011498.1 gene cluster also contains a similar number of NRPS modules with similar to identical domains (Table 3-9) to the gene clusters of the 194 Figure 4-80. Biosynthetic proposal for the incarnatapeptins and dentigerumycins F and G. characterized molecules. The similarity in the gene clusters is expected due to the overall similarities in these molecules' structures, where a similar PKS-derived oxane moiety is linked to amino acids through a highly conserved β-OH-Leu. One unique feature of 4.3 and 4.4 is the polyketidic portion of these Piz-containing compounds. The transformation which leads to the unusual bicyclic system of incarnatapeptins A (4.3) and B (4.4) does not have a literature precedent. Additionally, the gene cluster is producing both the bicyclic moiety of 4.3 and 4.4 along with the vicinal methyl/ethyl substituents of dentigerumycins F (4.2) and G (4.1). 4.3 and 4.4 have a modified C-33/C-34 compared to 4.1 and 4.2 (Figure 4.81a), this difference could arise from either pre- or post-assembly modifications. As seen in Figure 4.81b, possible biosynthetic origins of this bicyclic 195 Figure 4-81. Visualizing putative biosynthesis pathways a) Visualizing potential PKS extender units within the structure of the incarnatapeptins and dentigerumycin F and G. b) Possible origins of the oxygenated side chain of the incarnatapeptins. a b 196 system could be either 1) a post-assembly hydroxylation of an ethyl sidechain derived from an ethylmalonyl-CoA extender unit, 2) pre-production of a hydroxyl-containing polyketide extender unit, or 3) pre-production of a carbonyl-containing extender unit that was reduced in post-assembly. In all three routes, the bicyclic moiety of the incarnatapeptins would be formed through, likely, spontaneous dehydration. This dehydration could involve the formation of the second six-membered ring through nucleophilic attack of the oxygen of the side chain hydroxyl onto the electrophilic carbon of an oxonium ion intermediate (Figure 4-80, oxonium ion intermediate not shown due to space limitations). If the two different extender units are constructed in a pre-assembly manner, 4.3 and 4.4’s C-29, C-30, C-33, and C-34 chain may come from a 3-hydroxy butyryl-CoA extender unit; whereas 4.1 and 4.2’s C-29, C-30, C-33, and C-34 could likely come from an ethylmalonyl-CoA extender unit (Figure 4.81a and Figure 4.82a). It is difficult to say with certainty the exact transformations and genes responsible without further labelling or knock-out studies. However, there is a suite of genes between AKJ15838.1 and AKJ15843.1 in the CP011498.1 contig from Streptomyces incarnatus NRRL 8089 (Figure 4.82b) that are associated with the construction of unusual PKS branched-chain extender units.118,182,244 It is common for ethylmalonyl-CoA to be constructed from crotonyl-CoA through a crotonyl-CoA carboxylase/reductase (CCR) which is present at AKJ15838.1.118 This alone would not explain the biosynthesis of both extender units. Given that the machinery is present to construct ethylmalonyl-CoA, the hydroxylation of C-33 may occur with the oxidation of the alkyl chain through a cytochrome P450 in post-assembly (Figure 4.81b). However, the only available cytochrome P450 is quite a distance upstream on the other side of the transpose used to hypothesize an approximate putative cluster boundary (Table 3-10). Given the rest of the genetic 197 Figure 4-82. Proposed biosynthetic pathway for the PKS extender units leading to the incarnatapeptins and dentigerumycin F and G. a) Two different extender units maybe loaded for the incarnatapeptins and dentigerumycins F and G. b) The CCR suite found in the CP011498.1 contig of Streptomyces incarnatus NRRL 8089 c) Possible biosynthetic pathway leading to the two unique extender units. machinery present between AKJ15838.1 and AKJ15843.1, I believe a plausible pathway is seen in Figure 4.82C with an acetoacetyl-CoA constructed through AKJ15839.1, a ketoacyl- ACP synthase III, whereby the carbonyl is transformed into an alcohol through the 3-hydroxybutyryl-CoA dehydrogenase found at AKJ15840.1. Once the 3-hydroxybutyryl-CoA is biosynthesized, dehydration would lead to the crotonyl precursor. Then, through the action of the crotonyl-CoA carboxylase/reductase found at AKJ15838.1, the ethylmalonyl-CoA extender unit would be produced (Figure 4.82C). These proposed transformations mirror a series of characterized biosynthetic transformations in the divergolides245, which adds plausibility to the pre-assembly of two distinct extender units, as seen in Figure 4.82. 198 Also for consideration are the feeding studies for polyoxypeptin, which demonstrated that polyoxypeptin’s unusual extender unit (at the same loading position as the incarnatapeptins’ C33/34) was coming directly from an isoleucine (Ile) pathway.246 If the incarnatapeptins follow this biosynthetic logic, in this case, one would imagine that threonine was the precursor to a hydroxylated extender unit that was loaded onto the AKJ15835.1 PKS module as in Figure 4.81b-2. However, there is some possibility that those feeding experiments simply reflect a crotonyl pathway derivative whose branching chain is via a fatty acid source with Ile a known precursor.118 On the other hand, the published (but possibly truncated) polyoxypeptin gene cluster does not possess a CCR suite and, as such, could have a unique route to this PKS extender unit (as opposed to gene clusters that do show CCR genes). Nevertheless, given the presence of the CCR suite and other genes that reflect transformations seen in the divergolides, in the case of the incarnatapeptins, where I have access to the full putative gene cluster, I think the evidence points away from a threonine-based extender unit. 4.5 Bioactivity 4.5.1 Antimicrobial Assays The incarnatapeptins A, dentigerumycins F and G were tested for antimicrobial activity in a minimum inhibitory concentration assay against E.Coli 8161 and MRSA. Incarnatapeptin A and dentigerumycin G demonstrated no antimicrobial activity. Incarnatapeptin B (4.4) was not tested due to limited material. Dentigerumycin F had conflicting results. Initially, due to availability, samples of dentigerumycins F and G that were initially collected when optimizing HPLC conditions were tested in the antimicrobial assays. When these samples were tested, dentigerumycin F demonstrated potent antimicrobial activity against E.Coli cultivar 8161 (MIC = 1 ug/mL or 1.27 nMol, Table 4-4a) and some activity against MRSA (Table 4-4b). However, 199 when dentigerumycins F collected in optimized HPLC conditions was retested, it showed no antimicrobial activity. This was a puzzling result. However, given the very small distance between the HPLC peaks (less than 1 minute) called 8089.Et.B.C.13 (dentigerumycin F) and 8089.Et.B.C.14 (even in the optimized HPLC conditions), I believe that it is highly likely the initial incarnatapeptin C sample tested contained enough of the compound named 8089.Et.B.C.14 to produce these results, and this is the likely antimicrobial compound. Unfortunately, the tiny amount of the pure fraction of 8089.Et.B.C.14 that was collected was placed into pyridine-d5 for NMR data collection, where, as previously discussed, it underwent an unsolved degradation that left it unsuitable for bioassay testing. See Appendix F for excel sheets with complete assay data. a Dentigerumycin F concentration E. coli 8161 % alive b Dentigerumycin F concentration MRSA % alive Blank Blank 20 mg/ml 0 64 mg/ml 0 4 mg/ml 0 32 mg/ml 2 2 mg/ml 0 16 mg/ml 3 1 mg/ml 0 8 mg/ml 4 500 ng/ml 2 4 mg/ml 4 250 ng/ml 96 2 mg/ml 3 50 ng/ml 99 1 mg/ml 75 10 ng/ml 97 0.5 mg/ml 70 2 ng/ml 95 0.25 mg/ml 80 0.4 ng/ml 124 0.125 mg/ml 76 DMSO + cells 100 DMSO + cells 100 Table 4-4. Antibacterial MIC results for dentigerumycin F that were not reproducible upon subsequent testing with a purer aliquot of the compound. a) E. coli 8161 results b) MRSA results 4.5.2 Bioactivity of Incarnatapeptins in the Prostate Cancer Assay Incarnatapeptins A (4.3, discussed in this thesis work) and B (4.4, isolated by David Williams) were tested for in vitro cytotoxicity to LNCaP and PC3 human prostate cancer cell lines. 4.1 and 4.2 were not tested in this assay due to the interest in the compounds with the 200 bicyclic ring system. Initially, both 4.3 and 4.4 were tested against both PC3 and LNCaP cells at a series of concentrations. PC3 cell growth was not impacted by incarnatapeptin A (4.3) or B (4.4) at 30 µM, whereas the growth of LNCaP cells appeared to be impacted by 4.4 at 15 and 30 µM as indicated by reduced cell density at 24 h (Figure 4-83). Due to the limited amount of 4.4 that was available, we were only able to quantify the LNCaP response at 30 uM (Figure 4-83 & 4-84). Using a marker that stains the DNA of nonviable cells revealed that incarnatapeptin B induced a 17-fold increase in non-viable LNCaP cells, whereas 4.3 had no apparent effect at 72 h (Figure 4-83 & 4-84). These limited data results suggest modest potency but selective sensitivity of LNCaP cells to incarnatapeptin B (4.4) compared to PC3 cells (Figure 4-85). Interestingly, the macrocyclic depsipeptide 4.4 was active at 30 uM, but the linear peptide analog 4.3 was inactive. If additional 4.4 becomes available, further studies are warranted to determine IC50 values, underlying mechanisms, and additional SAR. 201 Figure 4-83. LNCaP and PC3 cells were exposed for 24 hours to increasing concentrations of incarnatapeptins A (4.3) and B (4.4). Micrograph scale bars: 100 µm. a b 202 Figure 4-84. Incarnatapeptin A (4.3) induced cell death of LNCaP prostate cancer cellsand LNCaP. Incarnatapeptin B (4.4) induced cell death of LNCaP prostate cancer cells. Cell viability was measured using NucGreen Dead 488 Ready Probe and the number of positive green stained cells was counted. 203 Figure 4-85. Viability of LNCaP cells treated with incarnatapeptins A (4.3) or B (4.4) was measured using NucGreen Dead 488 Ready Probe and the number of positive green stained cells was counted. Fold induction of positive (dead) cells after 72h of exposure is shown. 204 4.6 Summary and Conclusion Figure 4-86. Combining genome mining and Piz 15N NMR-guided fractionation for the isolation of the incarnatapeptins and dentigerumycins F and G. Figure 4-87. Structures of incarnatapeptin A (4.3), and dentigerumycins F (4.2) and G (4.1). 205 Throughout Chapters 2, 3 and 4, I have described the development of a new natural product discovery method combining genome mining and nitrogen NMR to target a specific functional-group of interest and the structure elucidation of the molecules discovered through this method as represented in Figure 4-86. First, genome mining was used to select strains that produced the desired moiety. Next, a specific nitrogen NMR signature was used to assess the laboratory growing conditions for Piz natural product by specific bacteria. Finally, the nitrogen NMR signature was used as a guide throughout the isolation process. This targeted genome mining/nitrogen NMR approach resulted in the discovery of four novel Piz-containing NRPS/PKS hybrid natural products, three of whose structures were elucidated as part of this thesis work (Figure 4-87). The incarnatapeptins A (4.3) and B (4.4) contain a never before seen bicyclic ring system, and incarnatapeptin B proved to be a selective cytotoxin. Into the future, assay results point to the presence of an unsolved incarnatapeptin produced by Streptomyces incarnatus NRRL 8089 that has potent activity against E. coli. Combining the work that has already been completed with a bioassay-guided approach could uncover this powerful antibiotic. Dentigerumycin F (4.2) and G (4.1) were shown to contain an unusual equilibrium at the hemiketal functional group, knowledge of which could prove useful for studies of these normally stable 1-hydroxyoxane rings. Furthermore, there are very few families within these NRPS-PKS hybrids that show a divergence in the substitution pattern of the oxane ring when all the members of the family are derived from the same bacterial source (Chapter 2). There is only one other, the oleamycins, which look like they diverge in the likely loaded PKS extender unit. Characterizing the biosynthetic pathway for this crossroads could help both in engineering efforts and the ability to predict chemical phenotypes of biosynthetic gene clusters during genome mining efforts. 206 Broadly, the nitrogen NMR-guided approach could be expanded to target other nitrogen-containing natural products. Specifically, this method could be used to prospect for natural products containing nitrogen-nitrogen bond functional groups whose representation in the solved chemical space is inconsistent with possible compounds bioinformatically predicted from sequenced bacterial genomes. As this nitrogen NMR-guided approach is a targeted method, each moiety needs to be considered for a unique NMR signature. Early analysis determines that many unusual nitrogen-nitrogen bond functional groups contain such signatures (Appendix G) and that the solved chemical space could be rationally expanded using this targeted method. Finally, this method can also be used to target other Piz-containing molecules and could be enhanced by a tandem bioassay-guided approach. 4.7 Experimental Section 4.7.1 General Experimental Procedures Optical rotations were measured on a Jasco P-1010 Polarimeter with sodium light (589 nm). The 1H, 13C and 15N NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5mm CPTCI cryoprobe. 1H resonances are referenced to the residual DMSO-d6 ( 2.50), pyridine-d5 ( 7.22), ; CD3OD ( 3.31), and 13C resonances are referenced to the DMSO-d6 solvent peak ( 39.51); pyridine-d5 ( 123.9); CD3OD ( 49.2). The 15N NMR spectra are in reference to CH3NO2 (0 ppm). Low resolution ESIMS were recorded on Bruker HCT Ultra PTM Discovery System and high resolution ESIMS were recorded on a Waters / Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. Labelled 15N2-L-Orn was obtained from Cambridge Isotope Laboratories, Inc and unlabelled L-Orn was obtained from either Sigma-Aldrich or Fisher Scientific. All other media components were obtained from Fisher Scientific. All solvents used for HPLC were Sigma-Aldrich HPLC grade 207 additionally filtered through a micron filter. Reversed-phase HPLC purifications were performed on a Waters 1525 Binary HPLC pump attached to a Waters 2998 Photodiode Array Detector and equipped with Empower Software. Dentigerumycin F (4.2): Isolated as a clear oil; [α]25D -14.13’ (0.47 g/100 mL, MeOH); 1H (both pyridine-d5 and DMSO-d6, 600 MHz) and 13C NMR (both pyridine-d5 and DMSO-d6, 150 MHz); data can be found in Table 4.2, HRESIMS m/z: [M+Na]+ Calcd for C35H61O11N9Na, 806.4388; Found 806.4369. Dentigerumycin G (4.1): Isolated as a clear oil; [α]25D -8.39’ (0.73 g/100 mL MeOH); 1H (both DMSO-d6 and CD3OD, 600 MHz) and 13C NMR (both DMSO-d6 and CD3OD,150 MHz), data can be found in Table 4.1; HRESIMS m/z: [M+Na]+ Calcd for C35H60O12N8Na, 807.4228; Found 807.4237. Incarnatapeptin A (4.3): Isolated as a clear oil; [α]25D +27.4° (c 2.8, MeOH); 1H (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), data can be found in Table 4.3; HRMS (TOFESI) m/z: [M-H]- Calcd for C35H57N8O12, 781.4096; Found 781.4097. HRMS (TOFESI) m/z: [M+Na]+ Calcd for C35H58N8O12Na, 805.4072; Found 805.4075. 4.7.2 Bioassay Experimental Methods 4.7.2.1 MIC Experimental Methods E. coli 8161 and MRSA strains were inoculated into liquid growth media in falcon tubes. A dilution series of incarnatapeptin C and D dissolved in DMSO was placed into separate falcon tubes containing the human pathogen, along with a positive antibiotic control (dilution series of rifamycin) and a 100% control of DMSO. All falcon tubes were incubated at 37oC for 24 hours and then the growth of the pathogen was determined using optical density readings. The optical 208 density reading of the DMSO negative control was utilized as a baseline for which to measure the inhibition of growth of the tubes containing the positive control and the natural products. As such, the percentage of pathogens which were still alive was determined by OD readings over DMSO as 100% control. 4.7.2.2 Prostate Cancer Assay Experimental Methods LNCaP and PC3 prostate cancer cell lines were plated in 24-well plates and incubated with 0, 7.5, 15.0 or 30.0 µM incarnatapeptin A (4.3) (Incarn A) or incarnatapeptin B (4.14) (Incarn B) for 24 h. Cell viability was determined by incubation for 30 min with the NucGreen Dead 488 Ready Probe (Thermo Fisher Scientific, Waltham, MA, USA). Between 500 to 2000 cells per field were counted using the AxioVision AC fluorescence inverted microscopy (Zeiss, Dubling CA, USA). 209 Chapter 5: Bacterial Library Construction for Bioassay-guided Discovery 5.1 Chapter Overview This chapter will discuss the curation of a mini-library towards the discovery of bioactive natural products. The results of testing the mini-library across multiple biological activity assays will be shown. From those results, it will be seen that bioassay-guided fractionation was utilized as a tool to uncover molecules responsible for the bioactivity in multiple bacterial strains. 5.2 Strain Selection of Marine Bacteria The goal of this research effort was to uncover novel bioactive natural products with a focus on bacterial metabolites. One of the challenges of natural product discovery, particularly in bacteria, is that, given no other indication of what an individual bacterial strain is producing, one must look through hundreds and even thousands of strains to find new or interesting molecules.247 High-throughput sequence techniques are routinely employed to simultaneously look for a feature of interest from banks of hundreds or thousands of bacterial extracts and already isolated molecules.247 I focused on producing a smaller library populated with bacterial strains predicted to have a higher than average tendency to produce novel molecules, based on the origin of the bacteria from diverse ecosystems,11,248,249 or prediction that the strain should be “biosynthetically talented”.250 To support the search for novel bacterial natural products of putative biosynthetic interest, a mini library was constructed from bacterial strains curated from the larger Andersen marine bacteria library along with several terrestrial strains isolated by a member of Julian Davies’s lab. The strains were generally selected to meet at least one of several criteria: (1) initial, promising anti-microbial screening results, (2) strains of genera such as Salinispora sp. which had previously been shown to produce diverse secondary metabolites,248 (3) isolation from an invertebrate or other organism,251 or (4) under-representation of that species 210 in natural product isolation attempts.233 For a comprehensive list of the curated strains, see Table 5.1. 5.3 Bioassays for Guiding Fractionation Each of the strains listed in Table 5-1 was grown in small quantities, the crude extracts prepared into a 96 well plate (2 mg/well) and then sent for biological assays. Positive results were received in several bioassays. Bioassay-guided fractionation methodology was then applied to pursue isolation of pure bioactive compounds. The application of these methods led to the isolation of pure compounds in three of the seven assays: phosphatase inhibition, IL-6 anti-inflammatory and anti-microbial assays. When screening crude extracts in biological assays, the identity and abundance of the active compounds are unknown. Initial screening is seeking benchmark activity values for each assay. As the separation process should increase the proportion of the active component in a fraction, assay results should show sustained or increasing activity values after each fractionation step. Bacterial strains can demonstrate variations in metabolite production, yet structure elucidation is dependent on sufficient quantities of a compound. These quantities are obtained by culturing multiple batches of a strain. Therefore, to be of continued interest, extracts from subsequent batches of a strain should be able to sustain levels of activity like that seen in the initial screening. 211 Table 5-1. List of strains selected for mini library. RJA number, species name if known, location or species origin, accession number if 16S RNA gene deposited. NAME Species Isolated From: Source* Accession number NAME Species Isolated from: Source* 1 22 DCA3500 Streptomyces sp. Cactus soil T.S 38 RJA3119 Salinispora sp. Dominican # 2 M.S. 2 DCA3201 Streptomyces phaeochromogenes Cactus soil T.S EU594471 39 RJA3990 Streptomyces cellulosae Georgia Straight #1 M.S. 3 DCA3503 Streptomyces sp. Cactus soil T.S 40 RJA4055 Streptomyces drozdowiczii. Georgia Straight #3 M.S. 4 DCA3504 Streptomyces sp. Whistler forest soil T.S GQ924538 41 RJA4089 unknown Hamigera M.I. 5 DCA3505 Streptomyces sp. Mushroom soil T.S GQ924538 42 RJA4095 unknown Hamigera. Ansell Point, B.C. M.I. 6 7 DCA3506 Streptomyces sp. Peltigera malacea Lichen GQ924538 43 RJA4096 unknown Hamigera. Ansell Point, B.C. M.I. 7 DCA3507 Streptomyces sp. Lobaria pulmonaria Lichen DQ221755 44 RJA4104 Alpha proteobacteria B14 Hamigera. Ansell Point, B.C. M.I. 8 DCA3508 Streptomyces sp. Flavocetraria baltimorensis Lichen AB222068 45 RJA4108 unknown Hamigera. Ansell Point, B.C. M.I. 9 10 11 DCA3509 Streptomyces sp. Flavocetraria baltimorensis Lichen DQ231567 46 RJA4125 unknown Syringella amphispicula, B.C. M.I. 10 RJA471 Kocuria Sp. Indian Arm Indian Arm, March-May 06; Sediment Sample 18, fresh M.S. 47 RJA4127 unknown Syringella amphispicula, B.C. M.I. 11 RJA479 Kocuria Sp. Indian r Indian Arm Indian Arm, March-May 06; Sediment Sample 8, fresh M.S. 48 RJA4128 unknown Syringella amphispicula, B.C. M.I. 12 RJA494w Kocuria Sp. Indian r Indian r , arch- ay 06; edi ent a ple 20, fresh M.S. 49 RJA4134 unknown Syringella amphispicula, B.C. M.I. 13 RJA494y Kocuria Sp. Indian Arm Indian Arm, March-May 06; Sediment Sample 20, fresh M.S. 50 RJA4145 unknown Syringella amphispicula, B.C. M.I. 14 RJA496 Kocuria Sp. Indian Arm Indian Arm, March-May 06; edim nt Sample 9, fresh M.S. 51 RJA4150 Algoriphagus winogradsky 87% Hamigera. Ansell Point B.C. M.I. 15 RJA515 Kocuria Sp. Indian Arm Indian Arm, March-May 06; ediment Sample 5, fresh M.S. 52 RJA4193 unknown M.I. 16 RJA526 Kocuria Sp. Indian Arm Indian Arm, March-May 06; ediment Sample 3, fresh M.S. 53 RJA4198 Arthrobacter sp. KOPRa25422 98% Boltenia villosa, B.C M.I. 17 RJA529 Kocuria Sp. Indian Arm Indian Arm, March-May 06; ediment Sample 19, fresh M.S. 54 RJA4209 unknown Boltenia villosa, B.C M.I. 18 RJA538 Kocuria Sp. Indian Arm Indian Arm, March-May 06; ediment Sample 7, fresh M.S. 55 RJA4220 unknown Styela Clava, B.C. M.I. 19 RJA544 Kocuria Sp. Indian Arm Indian Arm, March-May 06; ediment Sample 3, fresh M.S. 56 RJA4231 unknown Styela Clava, B.C. M.I. 20 RJA2910 Streptomyces Bamfield # 5 M.S. 57 RJA4235 unknown Styela Clava, B.C. M.I. 21 RJA2998 Salinispora sp. Dominicana #1 M.S. 58 RJA4241 unknown Styela Clava, B.C. M.I. 22 RJA3002 Salinispora sp. Papau New Guinea # 3 M.S. JF922126 59 RJA4249 Streptomyces sanglieri Styela lava, . . M.I. 23 RJA3003 Salinispora sp. Papau New Guinea # 3 M.S. 60 RJA4254 unknown Cliona Californiana, B.C. M.I. 24 RJA3004 Salinispora sp. Papau New Guinea # 3 M.S. 61 RJA4265 unknown Cliona Californiana, B.C. M.I. 25 RJA3005 Salinispora sp. Papau New Guinea # 3 M.S. 62 RJA4270 unknown Cliona Californiana, B.C. M.I. 26 RJA3006 Salinispora sp. Papau New Guinea # 3 M.S. JF922127 63 RJA4274 unknown Ophlitaspongia, B.C M.I. 27 RJA3107 Salinispora sp. Papau New Guinea # 3 M.S. JF837441 64 RJA4276 unknown Cliona Californiana, B.C. M.I. 28 RJA3109 Salinispora sp. Papau New Guinea # 3 M.S. JF837442 65 RJA4296 unknown Phorbas Sp. B.C. M.I. 29 RJA3110 Salinispora sp. Papau New Guinea # 3 M.S. 66 RJA4300 unknown Phorbas Sp. B.C. M.I. 30 RJA3111 Salinispora sp. Papau New Guinea # 3 M.S. 67 RJA4303 Streptomyces sp. HB149 89% Phorbas Sp. B.C. M.I. 31 RJA3112 Salinispora sp. Dominicana #2 M.S. 68 RJA4374 Streptomyces sp RJA4020 & 4241 Fusant 32 RJA3113 Salinispora sp. Dominicana #2 M.S. 69 RJA4375 Streptomyces sp RJA4020 & 4241 #3 Fusant 33 RJA3114 Salinispora sp. Papau New Guinea # 3 M.S. 70 RJA4377 Streptomyces sp RJA4020 & 4241 #5 Fusant 34 RJA3115 Salinispora sp. Dominican #1 M.S. 71 RJA4379 Streptomyces sp RJA4020 & 4173 #1 Fusant 35 RJA3116 Salinispora sp. Papau New Guinea #3 M.S. 72 RJA4380 Streptomyces sp RJA4020 & 4173 #3 Fusant 36 RJA3117 Salinispora sp. Dominican #2 M.S. 73 RJA4382 Streptomyces sp RJA4020 & 4173 #5 Fusant 37 RJA3118 Salinispora sp. Papau New Guinea #3 M.S. 74 RJA4384 Streptomyces sp RJA4020 & 4242 #11 Fusant *T.S = Terrestrial Strain; M.S. = Marine Strain; M.I. = Marine Invertebrate *T.S = Terrestrial Strain; M.S. = Marine Strain; M.I. = Marine Invertebrate 212 5.3.1 Phosphatase Assay Regulation of cellular function is critical to life. Reversible phosphorylation is an important mechanism of cellular regulation with dephosphorylation performed by phosphatases. There are several phosphatases of medical interest: PP1-c and calcineurin. Both of these proteins belong to the serine/threonine phosphatase class of enzymes, with the PP1 catalytic subunit dependent on binding with regulatory subunits for activity and specificity of action, while calcineurin has a calmodulin-binding catalytic subunit and a Ca2+-binding regulatory subunit.252 PP1-c inhibitors are often associated with cytotoxic activity. Calcineurin is involved in the immune system’s responses to foreign bodies. Thus, calcineurin inhibitors, such as cyclosporin A, are universally important in modern transplant surgery, yet there are only a handful of known compounds, all with harmful side effects.9 5.3.2 Anti-Inflammatory Assays Interleukin-6 (IL-6) is a keystone cytokine involved in regulating the immune response, capable of causing diseased states if it is either over-produced or under-produced.253 On the one hand, the absence of IL-6 will allow pathogens and pathogen-related diseases to proliferate. On the other hand, elevated levels of IL-6 have been shown to be involved in both chronic inflammation diseased states such as psoriasis and arthritis and the development of certain cancers such as B cell malignancy and myeloma.253,254 Thus, natural products that can act as lead compounds for inducing IL-6 pro-inflammatory or anti-inflammatory responses have many potential applications. 5.3.3 Anti-microbial Assays The antibacterial technology first harnessed in penicillin has vastly decreased the health threat that once marked bacterial infections.12 However, over-utilization of these natural products 213 has saturated the environment, leading to widespread antibiotic resistance to currently used medicines in human pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and E. coli.255 This developing resistance drives the search for novel antibiotics and natural products with antibacterial activity that could be developed into new medicines. Additionally, there are 1.5 million yearly deaths from fungal infections worldwide, both from primary fungal infections and secondary fungal infections in immunosuppressed individuals.256,257 This is in part due to the low number of antifungal drugs that can be clinically prescribed. Despite this, the search for novel antifungal compounds is underrepresented in natural product discovery programs, while new drugs are needed.257 As such, Candida albicans is an excellent model fungal pathogen for use in bioassay-guided fractionation efforts. Several qualitative assays may be used to assess the production of antibacterial compounds, which can also guide the isolation process. The standard disc-diffusion antimicrobial assay is quick and relatively easy.258,259 The bioautography assay260,261 combines the rapid results of thin-layer chromatography’s separations with an assortment of biological tests.262,263 It is particularly useful in targeting antibacterial/antifungal activity in TLC-Agar-Overlay bioautography assays.71,260 5.4 Results from Bioassay-guided fractionation 5.4.1 Phosphatase Assay Results Extracts from five strains demonstrated PP-1c inhibition of interest in the pNPP assay. An interesting result in the pNPP assay will be any crude extract that shows at least 30% inhibition of activity while extracts demonstrating around 50% inhibition marking a strain of particular interest. RJA3005, a Salinispora arenicola strain, showed PP-1c and potent calcineurin inhibition in the pNPP assays and was scaled-up with initial results demonstrating a positive 214 increase in activity upon fractionation. This strain will be discussed further in Chapter 6. Prior work in the Andersen lab demonstrated that Streptomyces sp. RJA4178 is a Streptomyces strain whose extracts demonstrated PP-1c and calcineurin inhibition. Through bioassay-guided fractionation efforts (Scheme 5-1), thienodolin (5.1) 264 was found as a very weak calcineurin inhibitor, along with a family of known thiazole indole derivatives (5.2, 5.3, 5.4), one of which was also a very weak calcineurin inhibitor. Interestingly, none of these compounds had been reported in Streptomyces species previously, with 5.3 being an unpublished natural product discussed in a patent for dye compounds,265 and 5.2 and 5.4 being found in thermophilic bacteria.266–268 Scheme 5-1. Isolation scheme of Streptomyces sp. RJA4178. Darker green above is associated with a more pronounced activity. (5.1) (5.4) (5.2) (5.3) 215 Figure 5-1. Compounds isolated from Streptomyces sp. RJA4178. Darker green above is associated with a more pronounced activity. 5.4.2 Anti-Inflammatory Assay Results Positive anti-inflammatory results are considered of interest in the IL-6 whole-cell assay if the crude extract matches or exceeds the 88% inhibition displayed by the dexamethasone control. The crude extract of RJA4249 showed 97.9% IL-6 inhibition and was the focus of my efforts. RJA4249 was demonstrated to be a Streptomyces sanglieri species through 16s rRNA sequencing completed by the author. It was seen that the Streptomyces sp. RJA4249 was producing the known compound amicoumacin (5.5) which had only previously been reported from Bacillus species.269 It was also producing various diketopiperazines ( 5.6 - 5.8) and several known cycloheximide analogues (5.9 and 5.10).270 Through bioassay-guided fractionation efforts (Scheme 5-2), the fraction 4249.DCM.CDE.D.A.6 (5.11) was identified as the known natural product cycloheximide. Cycloheximide is a potent antifungal and antibiotic natural (5.2) (5.1) 216 Scheme 5-2. Isolation scheme of the Streptomyces sanglieri RJA4249 leading to the known compounds; amicoumacin (5.5), cycloheximide (5.11), and cycloheximide analogs. Green above is associated with activity. product that inhibits protein synthesis of proteins. While it was previously used as a medicine in cases of severe fungal-infections,271 and for the potential treatment of Hodgkin’s disease at low-doses, the observations of alteration in protein metabolism at the required doses along with nausea after prolonged use272 resulted in discontinued use of cycloheximide as a common drug. Recent research has suggested that low doses of cycloheximide work well in conjunction with other medicine for anti-cancer therapy, but it has yet to be incorporated as a treatment.273 (5.5) (5.11) 217 Figure 5-2. Compounds isolated from Streptomyces sanglieri RJA4249. Compounds highlighted in green were found through apparent activity. Green above is associated with activity. Nevertheless, cycloheximide is routinely utilized in scientific labs and agricultural applications.274 The fractions were retested along with a quantity of purchased cycloheximide; this demonstrated that there were equivalent anti-inflammatory responses, thus describing the origin of Streptomyces sp. RJA4249 potent anti-inflammatory results. 218 5.4.3 Anti-microbial Assay Results: Fusant Strains The strains RJA4374-4384 are bacterial protoplast fusants, arising from a genetic technique that allows for homologous recombination between closely related strains.275 These were prepared from various Andersen library Streptomyces strains. In all cases, a Streptomyces caeruleus RJA4020 (JF510466) isolated from Georgia Strait cold waters in Vancouver, British Columbia249 and displaying no activity, was fused with a Streptomyces strain displaying potent anti-MRSA activity by Dr. D. Dalisay (Figure 5.3). These fusants demonstrated different morphology both from the parents and each other and, despite the strong antibacterial activity of some of the parents, the fusants lacked this trait (Figure 5.3). Given the potent antifungal activity of the extracts, a bioautography assay was chosen as the means to rapidly guide262 the isolation of the antifungal compound. Figure 5-3. Fusant strains. a) Identity and chemistry being produced from parent strains. b) The change in observed activity led to interest in the fusant strains. This bioautography assay-guided fractionation (Scheme 5-3) allowed the identification of the active components of fusant RJA4374 as members of the potent known antifungal antimycin family,276 including antimycin A3ab (4374.Hex.C.E.4.1 (5.12)) and antimycin A2ab (4374.Hex.C.E.8.1 (5.13)). The isolation of these antimycins suggests that the fusant’s de novo b elaiophylin nigericin a 219 antifungal activity most likely arises from the novel expression of a silent gene cluster, possibly through insertion or deletion of a regulatory gene during the homologous recombination involved in the fusant process. Scheme 5-3. Isolation scheme of the fusant RJA4374 leading to the known antifungal, antimycin. The darker green above is associated with a more pronounced activity. (5.12) (5.13) 220 Figure 5-4. Antimycins responsible for the antifungal activity of the fusant RJA4374. There was seen to be some difference in activity. The darker green above is associated with a more pronounced activity. 5.5 Summary and Conclusion A mini-library of marine and terrestrial bacterial were grown and assessed for potential bioactive drug leads through a small number of bioassays. Several of the bacterial cultural extracts demonstrated positive hits in the bioassays. Subsequently, the bioassays were used to guide the fractionation of the bacteria’s complex metabolite pool. These bioassay-guided fractionations yielded known compounds. Several thiazole natural products not previously found from Streptomyces species were revealed. Amicoumacins were shown to be produced by Streptomyces species in addition to the original Bacillus producer. It was shown that the de novo antifungal activity of generated fusant strains of cold water bacteria had a novel expression of antimycins. Finally, through the construction of the mini-library, a Salinispora arenicola species from the RJA collection was seen to be producing some previously unknown natural products which will be discussed in Chapter 6. 221 Chapter 6: Salinispora arenicola RJA3005: Chemistry and Feeding Studies 6.1 Chapter Overview Crude extracts from cultures of Salinspora arenicola RJA3005 were initially investigated due to promising bioassay results (Chapter 5), however, no metabolites could be isolated that had these activities following isolation. Nonetheless, NMR examination of purified fractions of the extract revealed natural products 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4). 6.1 has been previously seen as the product of feeding an engineered Amycolatopsis mediterranei S699 strain bacterial mutant (-AHBA synthase) with synthetic aromatic precursors.277,278 6.4 has been previously seen as a shunt product of: an Amycolatopsis mediterranei S699 mutant engineered without multiple sections of the wild-type’s post-PKS modification genes and an A. mediterranei S699 mutant with a rifF deletion.277,279 As a product of these mutants, 6.4 was named SY4b but not completely characterized. As the natural product seen here, I will complete its characterization and refer to the natural product 6.4 by its chemical name 3-acetamide-5-hydroxyphenylpyrone. Following structure elucidation of 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one, derivatization was performed in an unsuccessful attempt to isolate related metabolites that were challenging to isolate. However, this derivatization allowed separation of the 6.1’s tautomers, one of which was subsequently utilized to enable a Mosher’s ester analysis. Consideration of the possible biosynthetic pathway leading to the isolated natural products led to the hypothesis that none of the known literature pathways to a 3,5-dihydroxy aromatic substitution pattern could be reasonably followed to make this molecule. To interrogate the biosynthetic assembly of 6.1’s 3,5-dihydroxy aromatic substitution pattern, I carried out 222 feeding studies with 1,2-13C2-acetate and U-13C6-glucose to track the 13C coupling patterns. The analysis of these coupling patterns strongly suggested that the aromatic moiety of the natural product 6.1 is being produced through PEP and erythrose building blocks. 6.2 Salinispora: A Marine Obligate Genus and it’s Natural Products Salinispora is a marine obligate bacteria genus in the Micromonosporaceae family, Actinomycetales order, and the secondary metabolite prolific, Actinobacteria class and phylum. The first described members of this genus were reported in the literature some 15 years ago.28 Indeed, the Salinispora and Micromonospora genera are very closely related, both exhibiting a leathery orange appearance until black spores develop. The significant phenotypic difference between the genera is Salinispora's higher salt requirement for growth.280 Initially, two Salinispora species were reported, Salinispora arenicola and Salinispora tropica,28 although a third, Salinispora pacifica, was quickly proposed65 and later, confirmed.29,281 Very recently, six new Salinispora species have been characterized, all demonstrating a closest relationship to Salinispora pacifica.282 Salinispora arenicola, Salinispora tropica, and Salinispora pacifica species are distinguishable in part due to a different distribution of secondary metabolites and their corresponding gene clusters,283,284 despite an overlap in ecological ranges.65,281 All three are prolific producers of secondary metabolites, boasting genomes (~6 Mb) rich with natural product gene clusters, and the differential distribution of secondary metabolite genes means that they produce many different natural products.67 Salinipora pacifica uniquely produces cyanosporaside A, salinipyrone A, lomaiviticin A, enterocin, and pacificanone A.285 Salinispora tropica uniquely produces the potent and specific anti-cancer drug salinosporamide A (Marizomib),31 sporolide A, salinilactam, sioxanthin, and antiprotealide.285 Salinispora arenicola, perhaps the most abundant and widely dispersed of the species, produces arenicolide A, saliniquinone, cyclomarin A, 223 cyclomarazine, arenimycin, arenamide A, staurosporines, mevinolin and the related saliniketal A and rifamycin B.67,285 6.2.1 Biosynthesis of Rifamycin and Saliniketal Rifamycin is a major metabolite of S. arenicola strains.280 Additionally, S.arenicola strains produce multiple natural products, such as saliniketal A and salinisporamycin that share a biosynthetic pathway with rifamycin (Figure 6.1).286,287 Key to all of these biosynthetic pathways is the starter unit 3-amino-5-hydroxybenzoic acid (AHBA), a glucose-derived metabolite loaded onto the PKS modules in a specialized loading module.68,132,278,288 AHBA has a dedicated suite of genes adjacent to rifamycin PKS genes (Figure 6.1, Table 6.1, Figure 6.2) and is not made from shikimic acid.68,132,289,290 Instead, AHBA biosynthesis, or the aminoshikimate pathway, closely mirrors the shikimic acid pathway. The shikimate pathway produces 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) through phosphoenolpyruvate (PEP) and erythrose building blocks. The aminoshikimate pathway utilizes AHBA synthase, a PLP-dependent aminotransferase, to ultimately produce a 3-amino-3-deoxy-D-arabinoheptulosonate-7-phosphate (aminoDAHP) through PEP and iminoerythrose building blocks. The AHBA synthase catalyzes the formation of the iminoerythrose and the amino dehydroshikimate equivalent to form AHBA (Figure 6.2) in the final step of the pathway.132 In the shikimic acid pathway, the carbonyl functionality introduced in the first step is maintained until the final step, at which time it is reduced to an alcohol forming shikimate (Figure 6.3).132 224 Gene Function orf1 Hypothetical function rifG Aminodehydroquinate synthase rifH AminoDAHP synthase rifI Aminoquinate dehydrogenase rifK AHBA synthase rifL Oxidoreductase rifM Phosphatase rifN Kanosamine kinase rifJ (far apart from the rest of the genes) AminoDHQ dehydratase Table 6-1. Gene cluster of required genes for production of AHBA. 225 Figure 6-1. AHBA starter unit leading into the polyketide modules. Shared pathway leading to the rifamycins and saliniketals, depending on the carbon bond that is cleaved. Adapted from various sources.68,132,278,288,289 226 Figure 6-2. Key steps during the biosynthesis of AHBA from glucose.132 Please see Table 6.1 for enzyme functions. Figure 6-3. The final step in the shikimate pathway. This is a large difference between the shikimic acid pathway and the aminoshikimate (AHBA) pathway as the latter continues to aromatization. 227 6.3 Isolation and Structure Elucidation of Metabolites from Salinispora arenicola RJA3005 Salinispora arenicola RJA3005, a marine obligate strain isolated by Dr. Doralyn Dalisay from marine sediment collected in Papua New Guinea, was selected for further investigations following bioassay screening of the previously mentioned mini-library (Table 5.1). This strain showed promising activity in a phosphatase inhibition assay run (Table 5-2). Subsequent bioactivity-guided isolation efforts (Scheme 6-1) revealed final pure compounds that did not demonstrate the same bioactivity as the initial complex fractions. However, chemical prospecting of varying fractions revealed several related natural products. S. arenicola RJA3005 was grown as lawns on MM1 solid agar prepared with seawater. After three weeks of growth, the solid agar was harvested and soaked in EtOAc. The EtOAc extracts were filtered, dried under vacuum, and subsequently partitioned between EtOAc and water. The resulting brown gum was subjected to size exclusion chromatography on a Sephadex LH-20™ column (4:1 MeOH:DCM) followed by step-gradient separation on a C18 flash column with the compounds of interest eluting with 4:1 H2O:MeOH (Fraction C) and 3:2 H2O:MeOH (Fraction E). Fraction C was a yellowish, oily solid that was subjected to semi-preparative reverse-phase HPLC (17:83 MeCN:H2O with 0.05% Formic Acid (FA)) to reveal several diketopiperazines, tryptophan derivatives, shikimate pathway metabolites and 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) whose structure elucidation follows in Section 6.3.1. Fraction E was a light brown oily solid that was further separated via semi-preparative reverse-phase HPLC (17:83 MeCN:H2O with 0.05% FA) to yield N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4) whose structure elucidation is found in Section 6.3.2. 228 Scheme 6-1. Isolation scheme for Salinispora arenicola RJA3005 culture extract. 6.3.1 Structure Elucidation of 3,5-dihydroxyphenylpyrone (6.1) 3,5-dihydroxyphenylpyrone (6.1) gave a [M + H]+ ion in the HRESIMS at m/z 293.1046 appropriate for a molecular formula of C15H16O6 that requires eight sites of unsaturation. 1H, 13C, 1H-1H gCOSY60, 1H-13C gHSQC, 1H-13C gHMBC and 1H-1H tROESY NMR data recorded in DMSO-d6 (See Table 6.2) revealed 6.1 as a small metabolite that contains a disubstituted (6.1) (6.4) 229 benzene ring attached to a pyrone functionality. The interesting feature of 6.1 was the relatively shielded aromatic protons at δH 6.16 (H-2 and H-6) and δH 6.09 (H-4) exhibiting a 4JH-H = 2.2 Hz which, in the context of aromatic protons, is usually indicative of meta coupling. Moreover, the proton at δH 6.16 was integrated for two protons (Figure 6.6), indicating some symmetry in the aromatic region. Confirming the symmetry was a quaternary carbon at δc 157.9 (Figure 6.6 and 6.7) that was more abundant than other quaternary carbons in the 13C spectrum. Both proton chemical shifts at δH 6.16 and δH 6.09 demonstrated HMBC correlations into the carbon at δc 157.9 (C-3 and C-5) (Figure 6-11). As well, a hydroxyl proton at δH 9.15 was also demonstrating HMBC correlations into δc 157.9. 3J HMBC correlations were seen from the protons at δH 6.16 (H-2, H-4) to the resonances δc 104.8 (C-2 and C-6) and 101.5 (C-4) in the meta position to the proton thus helping to confirm the substitution pattern of the benzene ring. Figure 6-4. 1H spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. 230 Figure 6-5. 1H spectrum of 6.1 demonstrating integration, recorded in DMSO-d6, at 600 MHz. Figure 6-6. 13C spectrum of 6.1, recorded in DMSO-d6, at 150 MHz. H-2, H-6 231 Figure 6-7. Downfield expansion of 13C spectrum of 6.1, recorded in DMSO-d6, at 150 MHz. Figure 6-8. Expansion of 13C spectrum of 6.1, recorded in DMSO-d6, at 150 MHz. A methine singlet at δH 6.16 (δc 100.0) anchored the elucidation of a pyrone ring (Figure 6-11) within the molecule with HMBC correlations into resonances δc 164.2 (C-9), δc 164.9 (C- 232 11) and δc 96.6 (C-12) (Figures 6-11, 6-14, 6-16). A methyl singlet at δH 1.75 (δc 8.5) demonstrated HMBC correlations into a carbonyl at C-13 (δc 165.2) and the resonance δc 100.0 (C-10, δH 6.16) (Figure 6-18). The methyl singlet at H-12’ helped to link the benzene ring and the pyrone ring through unusual (and very weak) 6J, 7J, and 8J HMBC correlations to C-8 (δc 45.4), C-8’ (δc 14.8), and C-7 (δc 74.7) (Figure 6-17). More importantly, the methyl doublet at C-8’ (δH 1.75, δc 14.8) showed HMBC correlations into the same resonance δc 164.2 (C-9) as the methine singlet at H-10 (Figures 6-17, 6-18). Additionally, the methine doublet of doublets at H-7 (δH 4.58, δc 74.7) demonstrated HMBC correlations into the quaternary carbons of the benzene (δc 145.5 (C-1)) and the pyrone ring (δc 164.2 (C-9)) along with the aromatic resonance δc 104.8 (C-2/6) (Figure 6-18). In the tROESY spectrum, H-7 showed strong through-space coupling to Figure 6-9 Figure 6-10. Numbering and structure of 6.1. 233 Figure 6-11. Selected HMBC correlations for the aromatic and pyrone regions of the 6.1 demonstrating key correlations. the aromatic H-2 or H-4 proton (Figure 6-20) and the methine quartet at H-8 demonstrated strong through-space correlations to both the methine singlet of the pyrone ring at H-9 and one of the aromatic protons at H-2 or H-4. Taken all together, this data aided in the assignment of 6.1. 6.1 is the first recorded isolation of this natural product from a wild-type bacterial culture, and indeed as a biosynthesized molecule, as previous reports of 6.1 were from a mutated rifamycin producer Amycolatopsis mediterranei S699 (-AHBA synthase) that was then fed 3,5-dihydroxybenzoic acid.277 It was noticed that although there was a sharp alcohol peak for OH-7 (δH 5.32), no such sharp peak was seen for OH-11, consistent with a possible keto-enol isomerization. Work was ongoing to isolate several other clearly related compounds. However, isolation was challenging, so derivatization of the fraction containing the compounds was conducted to enable separation. Neither methylation nor acetylation allowed isolation of the related compounds; however, methylation helped separate the isomers of 6.1, which are seen as two methyl ethers - 6.2 and 6.3 (Figure 6-21). 234 Figure 6-12. 1H-13C HSQC spectrum of 6.1, showing 1J 1H-13C coupling, recorded in DMSO-d6, at 600 MHz. Figure 6-13. 1H-1H COSY60 spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. Few correlations were seen, which is in keeping with the many quaternary carbons of the molecule. H-10 H-2, H-6 H-4 H-7 H-8’ H-12’ H-8 235 Figure 6-14. Full 1H-13C HMBC spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. Figure 6-15. Expansion of 1H-13C HMBC resonances for 6.1 demonstrating the aromatic protons correlations into the quaternary carbons of C-3 and C-5. Recorded in DMSO-d6, at 600 MHz. H-2,4,6; C-3,5 OH-3,5; C-3,5 236 Figure 6-16. Expansion of 1H-13C HMBC spectrum of 6.1, showing select scalar couplings of protons into adjacent carbons. Recorded in DMSO-d6, at 600 MHz. Figure 6-17. Expansion of 1H-13C HMBC spectrum of 6.1, showing select scalar couplings between the methyl singlet on the pyrone ring to the quaternary carbons as seen in Figure 6-18 below. Recorded in DMSO-d6, at 600 MHz. H-12’; C-7 H-8’; C-7 H-10; C-9, C-11 H-10; C-12 H-10; C-8 OH-3,5; C-3,5 OH-3,5; C-2,6 OH-3,5; C-4 H-12’; C-10 H-12’; C-13 H-12’; C-12 H-8’; C-8 H-8’; C-9 H-12’; C-8 H-12’; C-8’ 237 Figure 6-18. Key 1H-13C HMBC and 1H-1H tROESY correlations connecting the two rings of 6.1. Figure 6-19. Full 1H-1H tROESY spectrum of 6.1, recorded in DMSO-d6, at 600 MHz. 238 Figure 6-20. Expansion of the 1H-1H tROESY spectrum showing select through-space correlations that can be seen in Figure 6-18 and the diagram insert. Recorded in DMSO-d6, at 600 MHz. H-10; H-8 H-2 or 6; H-8 H-6; H-7 239 Figure 6-21. Structures of the natural product and methylation derivative which allowed separation of the pyrone tautomers. 6.3.1.1 Derivatization of 3,5-dihydroxyphenylpyrone (6.1) In order to complete the methylation of the alcohol on the pyrone, a gentle method for methylation of natural products using trimethylsilyldiazomethane was employed (Scheme 6-2).291 Upon completion of the methylation, the methyl ethers of the respective isomers were separated via reversed-phase HPLC (step-gradient: 0-60 min 17:83 MeCN:H2O; 61mins to 120 mins 23:77 MeCN:H2O). The earlier eluting isomer 6.3 was more abundant, while the later eluting isomer 6.2 was less abundant. Most chemical shifts were identical to the natural product 6.1, with the major differences appearing in the pyrone ring. 240 Scheme 6-2. Methylation of the tertiary alcohol at C-11 with trimethylsilyldiazomethane leading to separation of 6.1’s tautomers. Figure 6-22. 1H spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, 600 MHz. 241 Figure 6-23. 13C spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, at 150 MHz. Figure 6-24. Chemical shifts for the natural product and its methylation derivatives demonstrating key changes. 242 Figure 6-25. 1H-1HCOSY60 spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1). No changes were seen from the original natural product. Recorded in DMSO-d6, at 600 MHz. Figure 6-26. 1H-13C HSQC spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1), demonstrating a key change at the methine of C-10. Recorded in DMSO-d6, at 600 MHz. C-10: 95.0, 6:48 243 Figure 6-27. 1H-13C HMBC spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1) demonstrating changes in chemical shifts through the correlation from the methyl singlet at H-12’ to the carbonyl at C-13. Recorded in DMSO-d6, at 600 MHz. Figure 6-28. 1H-1H tROESY spectrum for methylation of the 2-pyrone isomer (6.2) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, at 600 MHz. Key differences between the 2-pyrone (6.2) and the 4-pyrone (6.3) isomers were seen at C-11, C-13 and C-10 (Figure 6-35). In the 2-pyrone isomer (6.2) spectra, carbon shifts at δc H-12’ to C-13 (165.4) 11-OMe to C-11 (166.2) 244 166.2 (C-11), δc 165.4 (C-13) and δc 95.0 (C-10) (Figure 6-24 to 6-28) displayed a closer match to the dominant 2-pyrone form seen in the natural product [ C-11 (δc 164.9), C-13 (δc 165.2) and C-10 (δc 99.7)] (Figure 6-24). As such those spectra were assigned as the 2-pyrone form (6.2). In the other set of spectra collected for the derivatized isomers, resonances at δc 179.9 (C-11), δc 162.3 (C-13), and δc111.6 (C-10) (Figures 6-29 to 6-34) were observed. The deshielded resonance δc 179.9 (Figure 6-33) was assigned to the C-11 carbon in the 4-pyrone structure in 6.3 because it was a chemical shift more characteristic of a cyclic ketone adjacent to double bonds.292,293 Additionally, in 6.3 the C-10 resonance of δc 111.6 (Figure 6-32) matched an α carbon of an α,β unsaturated ketone293,294 whereas the resonance δc 95.0 of C-10 in 6.2 (Figure 6-26) is more characteristic of the β carbon of a conjugated lactone.293,294 Figure 6-29. Proton spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, at 600 MHz. There are large impurities gained while removing solvent traces on the lyophilizer. Given the small amount of material the impurities are relatively intense. The proton spectrum also shows chemical shift changes at H-10. 245 Figure 6-30. 13C spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, at 150 MHz. Figure 6-31. 1H-1H COSY60 spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). No changes are seen from the natural product. Recorded in DMSO-d6, at 600 MHz. 246 Figure 6-32. 1H-13C HSQC spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). Again C-10 shows chemical shift changes, not just from the natural product but also from the other methylated tautomer. C-10 is demonstrating a significant deshielding. Recorded in DMSO-d6, at 600 MHz. Figure 6-33. 1H-13C HMBC spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). The correlation from H-12’ to C-13 allows the observation that C-13 carbon is significantly deshielded. Recorded in DMSO-d6, at 600 MHz. C-10: 111.6, 5.99 H-12’(1.68) to C-11 (179.9) H-12’(1.68) to C-13 (162.3) 13 OMe to C-13 (162.3) H-10 (5.99) to C-9(164.7) 247 Figure 6-34. 1H-1H tROESY spectrum for methylation of the 4-pyrone form (6.3) of 3005.Et.D.C.4 (6.1). Recorded in DMSO-d6, at 600 MHz. Figure 6-35. Overlay of the 1H-13C HMBC’s of the 6.2 and 6.3. Black is the later eluting 2-pyrone (6.2), red is the earlier eluting 4-pyrone (6.3). The distinguishing δC 179.9 is visible in the red HMBC. Recorded in DMSO-d6, at 600 MHz. 248 DMSO-d6 3,5-dihydroxy (6.1) 2-Pyrone Methyl Ether (6.2) 4-Pyrone Methyl Ether (6.3) Position δC/δN δH (J =Hz) δC/δN δH (J = Hz) δC/δN δH (J = Hz) 1 C 145.4 NV 145.2 2 CH 104.8 6.16 d (2.2) 105.6 6.19 d (2.2) 105.9 6.17 3 C 157.9 158.6 158.6 4 CH 101.5 6.09 t (2.2) 101.6 6.11 d (2.2) 101.6 6.09 5 C 157.9 158.6 158.6 6 CH 104.8 6.16 d (2.2) 105.6 6.19 d (2.2) 105.9 6.17 7 CH 74.7 4.36 dd (4.0, 8.5) 73.6 4.40 dd (4.0, 8.5) 74.8 4.47 dd (4.0, 8.5) 7-OH OH 5.32 d (4.0) 5.34, d (4.0) 5.40 d (4.0) 8 CH 45.4 2.64 q (~7.1) 45.8 2.78 q (~7.1) 46.6 2.81 q (~7.1) 8’ CH3 14.9 0.85 d (7.1) 16.2 0.94 d (7.1) 14.9 1.02 d (7.1) 9 C 164.2 165.9 164.7 10 CH 100.0 5.99 s 95.0 6.48 s 111.6 5.99 s 11 C 165.2 166.2 179.9 11-OH/OMe OH NV 56.3 3.88 s 12 C 96.6 98.3 100.4 12’ CH3 8.5 1.75 s 9.2 1.83 s 6.5 1.69 s 13 C 165.2 165.4 162.3 13- OH/OMe OMe 55.4 3.90 s 3 & 5 -OH OH 9.15 Table 6-2. NMR assignments for 6.1, 6.2 and 6.3. Recorded in DMSO-d6 at 600 Mhz. 6.3.1.2 Assignment of Configuration C-7 and C-8 are stereogenic carbons that required assignment because 6.1, whose shifts and coupling constants were close to identical to those seen in the engineered compounds,277 had not previously been reported as produced from a wild-type bacteria. Given the secondary alcohol 249 present at C-7, a Mosher’s ester protocol was employed. The acid chloride reagents generally react with secondary alcohols to give the Mosher’s esters.295 Given the ability of the natural product to tautomerize and the desire to keep the subsequent NMR analysis as simple as possible, the methyl ether 6.3 was used to perform the Mosher’s ester analysis. Since about 1 mg of 6.3 was available, an NMR tube-based Mosher’s ester procedure was undertaken. This method completes the Mosher’s ester reaction in the NMR tube without purification.296 Two separate reaction tubes were set-up, one each for the R or S α-methoxy-α-trifluormethylphenylacetic acid (MPTA), with deuterated pyridine-d5 employed as reaction and NMR solvent (Figure 6-36).296 As 6.3 was residing in an NMR tube from previous data analysis, careful transfer was utilized to split 6.3 into two tubes. One tube was treated with the S-MTPA chloride, and the other tube was treated with the R-MTPA chloride (Figure 6-36). The tubes were shaken and then monitored by 1H NMR until it appeared the reaction was complete at 4 hrs. Although the MTPA chlorides and acids were in large excess and swamped the proton spectrum, protons at H-2, H-4, H-6, H-7, H-8 and H-8’ had correlations into distinct carbons that were not present in the excess reaction compounds (Figures 6-37 to 6-42). Aiding the assignment was the distinct coupling patterns at H-7 (doublet of doublets) and H-8 (quintet) (Figures 6-38 and 6-41). With this prior information and HSQC and gCOSY60 data, these protons were readily distinguishable, were assigned, and ΔδS-R (Table 6.3) calculated. As seen in Table 6.3, protons at H-2, H-4, and H-6 and H-8 demonstrated positive changes in their chemical shifts when comparing the S-ester to the R-ester. This occurs when they are in the same plane as the phenyl moiety of Mosher’s ester in the R-ester and experience anisotropic shielding (Figure 6.43). Conversely, the methyl proton at H-8’ and the methine proton at H-10 experience negative changes when comparing the S-ester to the R-ester. This occurs when they are in the same plane as the phenyl moiety in the S-ester (Figure 250 6.43). These results directly support that C-7 is in the S configuration, which aligns with previous work done on configuration of the engineered compounds.277 In the Mosher’s ester analysis completed here, C-8’ presented a strong negative difference between the S-ester and the R-ester (Table 6-3), suggesting an R configuration at C-8 (Figure 6.43).295 Although these results can only provide possible support for the configuration at the C-8 stereocenter, in the 2015 paper examining the configuration of these compounds, they use computational evidence to assign the equivalent of C-8 in 6.3 as being in the R configuration. The chemical shifts at C-7 (H-7) and C-8 (H-8), as well as the coupling constants between H-7 and H-8, are nearly identical between the engineered compound and the natural product. The likelihood of the C-8 of 6.1 being in the R configuration is therefore strong and is represented as such here. 251 Figure 6-36. Mosher’s ester reaction with 6.3, a derivative of 6.1. Two separate NMR tubes containing small amounts of 6.3 were each reacted with either the S-Mosher’s chloride or the R-Mosher’s chloride. Due to the priority rules, the chloride and the esters have the opposite configuration. 252 Figure 6-37. 1H-13C HSQC spectrum for S-Mosher’s Ester(made from R-chloride) of 6.3. Recorded in pyridine-d5, at 600 MHz. Figure 6-38. 1H-13C Expansion of HSQC spectrum for S-Mosher’s Ester (made from R-chloride) of 6.3. Recorded in pyridine-d5, at 600 MHz. H-10 H-4 H-2, 6 H-7 H-8 H-8’ H-8 H-7 253 Figure 6-39. 1H-1H Expansion of COSY60 spectrum for S-Mosher’s Ester (made from R-chloride) of 6.3. Connecting squares demonstrating key COSY correlations used to confirm chemical shift assignment. Recorded in pyridine-d5, at 600 MHz. 254 4-Pyrone Methyl Ether (6.3) (DMSO-d6) Measured chemical shift changes to 6.3 under Mosher’s Ester (pyridine-d5) Calculated difference R-Chloride S-Chloride Position δC/δN δH S-MPTA R-MPTA ΔδS-R 1 C 145.2 2 CH 105.9 6.17 7.109 7.103 +0.006 3 C 158.6 4 CH 101.6 6.09 7.068 7.066 +0.002 5 C 158.6 6 CH 105.9 6.17 7.109 7.103 +0.006 7 CH 74.8 4.47 5.1276 5.1287 -0.001 7-OH OH 5.40 Ester Ester 8 CH 46.6 2.81 3.233 3.231 +0.002 8’ CH3 14.9 1.02 1.2893 1.3007 -0.011 9 C 164.1 10 CH 111.6 5.99 6.5714 6.5786 -0.0072 11 C 164.7 No protons below this are shown as they were too complex to determine Table 6-3. Results from Mosher’s ester NMR Analysis. 13C shifts in DMSO-d6 did not change in pyridine-d5 as expected since carbon chemical shifts are not as influenced by solvent effects. 1H shifts are reported as seen in pyridine-d5. Figure 6-40. 1H-13C HSQC spectrum for R-Mosher’s ester (made from S-chloride) of 6.3. Recorded in pyridine-d5, at 600 MHz. H-10 H-4 H-2, 6 H-7 H-8 H-8’ 255 Figure 6-41. Expansion of 1H-13C HSQC spectrum for R-Mosher’s ester (made from S-chloride) of 6.3. Recorded in pyridine-d5, at 600 MHz. Figure 6-42. Expansion of 1H-1H COSY60 spectrum for R-Mosher’s ester (made from S-chloride) of 6.3. Recorded in pyridine-d5, at 600 MHz. H-8 H-7 256 Figure 6-43. Newman projections of the Mosher’s esters demonstrating in yellow the areas of the molecule 6.1 influenced by the anisotropic field of the phenyl moiety of the ester. 6.3.2 Structure elucidation of 3-acetamide-5-hydroxyphenylpyrone (6.4) 3-acetamide-5-hydroxyphenylpyrone (6.4) gave a [M + H]+ ion in the HRESIMS at m/z 334.1297 appropriate for a molecular formula of C17H19O6N that requires 9 sites of unsaturation. Analysis of the 1H, 13C, 1H-1H gCOSY60, 1H-13C gHSQC, 1H-13C gHMBC and 1H-1H tROESY NMR data recorded in DMSO-d6 (See Table 6.3) revealed 6.4 as a natural product that contains a disubstituted benzene ring attached to a pyrone functionality initially thought to be of complete PKS origin. The 1H NMR spectrum of 6.4 was remarkably similar to 6.1 save for distinctive aromatic peaks (δH 6.89, 7.14 and 6.39) where the chemical shifts showed greater deshielding more typical of the standard aromatic region and with three peaks of equal intensity. Furthermore, the gHSQC (Figure 6.47) demonstrated that the three aromatic protons were coupled to varying carbons (δH 6.89, δC 108.5; δH 7.14, δC 106.1; and δH 6.39, δC 109.3). The HMBC spectrum demonstrated that the quaternary carbons at C-3 (δC 168.3) and C-5 (δC 157.0) 257 were also no longer identical. Furthermore, the 1H-15N HSQC spectrum (Figure 6.52) demonstrated an N-H proton at δH 9.80, which was demonstrating weak HMBC correlations into a carbonyl at δC 140.3 (Figures 6.50 and 6.51). A new methyl singlet at δH 2.04 (δC 24.2) was also showing HMBC correlations into the quaternary carbon at C-3. These pieces of data led to the assignment of an acetamide functional group as the second substituent of the benzene ring. The rest of the data showed either extreme similarity or identical chemical shifts to 6.1 and was assigned as such. The molecule 6.4 has previously been shown to be a product of a rifamycin producer Amycolatopsis mediterranei S699 that a) underwent extensive mutation to eliminate a 21kb DNA fragment290 of the rifamycin gene cluster called the post-PKS modification genes,278 or b) was a rifF (an acetyl-CoA acetyltransferase) deletion mutant.279 However, to the best of my knowledge, 6.4 has not been previously isolated as a natural product from a wild-type bacteria. Figure 6-44. Numbering and structure for 3-acetamide-5-hydroxyphenylpyrone (6.4). 258 Figure 6-45. 1H spectrum of 6.4, recorded in DMSO-d6, at 600 MHz. Figure 6-46. 13C spectrum of 6.4, recorded in DMSO-d6, at 150 MHz. 259 Figure 6-47. 1H-1H COSY60 spectrum of 6.4 demonstrating similar to identical correlations as 6.1recorded in DMSO-d6, at 600 MHz. Figure 6-48. 1H-13C HSQC spectrum of 6.4 demonstrating similar carbon-proton correlations from H-7 to H-12’ as 6.1 but unique carbon-proton correlations for aromatic H-2, H-4 and H-6, recorded in DMSO-d6, at 600 MHz. H-10 H-6 H-4 H-7 H-8’ H-12’ H-8 H-3’’ H-2 OH-7, H-7 H-7, H-8 H-8, H-8’ 260 Figure 6-49. Full 1H-13C HMBC spectrum of 6.4, recorded in DMSO-d6, at 600 MHz. Figure 6-50. Key 1H-13C HMBC correlations for the ring systems of 6.4. 261 Figure 6-51. Expansion of the 1H-13C HMBC spectrum of 6.4 demonstrating key correlations leading to assignment of the asymmetric aromatic ring, recorded in DMSO-d6, at 600 MHz. Figure 6-52. Expansion of the 1H-13C HMBC spectrum of 6.4 demonstrating the correlations leading to the assignment of an acetamide functionality, recorded in DMSO-d6, at 600 MHz. H-3’’, C-3 (2.04, 168.3) NH, C-3 (168.3) NH, C-4 NH, C-2 OH, C-5 (157.9) OH, C-4 (106.1) OH, C-6 (109) NH, C-3’ 262 Figure 6-53. 1H-15N HSQC spectrum of 6.4 demonstrating the N-H correlation of the acetamide functionality, recorded in DMSO-d6, at 600 MHz. Figure 6-54. 1H-1H tROESY spectrum of 6.4, recorded in DMSO-d6, at 600 MHz. 9.80, -247.1 263 3-acetamide-5-hydroxyphenylpyrone (6.4) DMSO-d6 Position δC/δN δH (J = Hz) 1 C 145.1 2 CH 108.5 6.89 br.s 3’ HNCOCH3 140.0 3’’ HNCOCH3 24.2 2.01 3-NH HNCOCH3 -247.1 9.80 3 C 168.3 4 CH 106.1 7.14 br.t (1.68) 5 C 157.0 5-OH OH 9.33 6 CH 109.3 6.39 br. s 7 CH 74.8 4.42 dd (4.0, 8.5) 7-OH OH 5.42 d (4.0) 8 CH 46.6 2.66 q (~7.1) 8’ CH3 14.9 0.84 d (7.1) 9 C 164.1 10 CH 100.7 5.99 s 11 C 164.7 11-OH OH NV 12 C 96.8 12’ CH3 9.02 1.77 s 13 C 165.2 Table 6-4. NMR assignments for 6.4. Recorded in DMSO-d6 at 600 MHz. 264 6.4 Biosynthesis of Salinispora arenicola RJA3005’s 6.1 Analysis of the possible biogenesis of 6.1 led to the conclusion that a 3,5-dihydroxybenzoic acid starter unit was likely because the pyrone moiety has largely been shown to form spontaneously from PKS extender units (Figure 6-55).297 Analysis of the literature suggested that direct incorporation of a 3,5-dihydroxybenzoic acid as a starter Figure 6-55. Likely pathway to the biosynthesis of 6.1. However, the biosynthetic pathway leading to the aromatic starting unit is uncertain. unit for a polyketide synthase system had not previously been shown. Vancomycin and kendomycin show PKS III production of a 3,5-dihydroxyphenyl-2-oxoacetic acid precursor which then led to the production of extensively substituted aromatic precursors (Figure 6-56).298,299 As a note, during biosynthetic characterizations of kendomycin, an initial proposal Figure 6-56. Vancomycin and kendomycin A use similar PKS III pathways to produce their aromatic starting units. 265 suggested that a 3,5-dihydroxybenzoic acid was uniquely prepared via a PKSIII module and then modified for incorporation into the natural product300,301, but this proposal was later modified. Figure 6-57. Asukamycin and manumycin incorporation of a 3,5-dihydroxy moiety during the production of their aromatic starting unit. Asukamycin and manumycins also show incorporation of a heavily modified 3,5-dihydroxy moiety in biosynthesis of their starting unit but use a combination of acetate and glycerol building blocks (Figure 6-57).302 Perhaps the most well-known example of a 3,5-dihydroxy aromatic moiety is resorcinol in plants; however, this occurs through cyclization of the terminal units (Figure 6-58).106 As a note, this strategy for production of resorcinol’s 3,5-dihydroxy Figure 6-58. Biosynthetic strategy leading to resorcinol’s 3,5-dihydroxyphenyl moiety. substitution pattern is also found in microbes due to the cyclization of PKS units at the terminal end of fatty acid chains.303,304 The unusual presentation of 6.1 was that both sides of the molecule had previous literature characterization from terminal cyclization of polyketide fragments. Consideration of these factors led to investigations into the biosynthesis of 6.1 using carbon-13 labelled precursors to investigate the three possible hypotheses (Figure 6-59) for the origin of an 266 aromatic starting unit. Lack of incorporation of acetate into the aromatic portion of 6.1 would rule out both the PKSIII or the manumycin pathways. Figure 6-59. Three possible pathways to 6.1's proposed 3,5-dihydroxybenzoic acid starting unit with possible precursor carbons shown in the benzene ring. 6.4.1 Labelled Precursor Feeding Studies 6.4.1.1 1,2-13C2-acetate Feeding Experiments If 6.1 was being produced entirely through a PKS pathway, then acetate would necessarily be incorporated throughout the molecule, including the benzene ring. As such 1,2-13C2-acetate was fed into cultures of Salinispora arenicola RJA3005. With doubly carbon-13 labelled acetate carbon-13 splitting patterns would be expected in the 13C carbon NMR spectra. Analysis of those splitting patterns should reveal how the labelled building blocks were 267 incorporated.133 As acetate is incorporated into the PKS extender units through the citric acid cycle this determines the expected patterns (Figure 6-60). Salinispora arenicola RJA3005 was Figure 6-60. Doubly labelled acetate leads to varying 13C incorporation patterns in PKS propionate extender units. Adjacent carbons from yellow and green highlights would lead to coupling patterns in the NMR spectra. producing 6.1 in relatively small quantities, so 2L of poured agar medium was required to collect enough metabolites for appreciable carbon NMR data. 1,2-13C2-acetate was introduced into the solid agar of RJA3005. After growth, extraction and separation, several hundred micrograms of 6.1 was subjected to NMR analysis. Analysis of the carbon-13 coupling patterns observed in the 13C NMR spectrum obtained from labelled 6.1 saw incorporation of the 1,2-13C2-acetate into the alkyl/pyrone portion of the molecule. Specifically, the strong singlet peak seen at C8’ and C12’ would come from incorporation of C-3 labelled propionyl-CoA units (Figure 6-60) overlapped with the resonance of the unlabelled molecules. The doublet coupling patterns seen at C8’ (δC 14.9) ((J8’8 = 34 Hz) and C12’ (δC 8.4) ((J12’12 = 47.9 Hz) ) (Figure 6-61) demonstrated 268 incorporation of fully labelled propionyl-CoA units derived from succinyl-CoA in the citric acid cycle (Figure 6-60), with splitting from a labelled neighbor at C-8 and C-12. Accordingly, it Figure 6-61. 1,2-13C2-acetate feeding demonstrates doublet splitting patterns for coupling between C-8’/C-8 and C-12’/C-12 overlapping the 13C resonances of C-8’ and C-12’ from the unlabelled molecules of 6.1 as well as the singlets for C-8’ and C-12’. 13C spectrum recorded in DMSO-d6, at 150 MHz. would be expected that C-8 (δC 45.3) and C-12 (δC 99.6) would be split by either two labelled neighbors if complete incorporation was seen in the propionyl-CoA units or by one neighbor if incorporation as seen in C-1 and C-2 of the propionyl-CoA units (Figure 6-60). The resonance at δC 99.6 (C-12) was not appreciably discernable. However, the resonance δC 45.3 (C-8) demonstrated a complex splitting pattern consistent with being split by two neighbors in some molecule and only one in others (Figure 6-62). In Figure 6-62, the coupling constant for J89 was assumed to be the same as J88’ since C-9 was not visible to measure, in reality there would J8’8 J12’12 269 as C1-C7 Figure 6-62. Acetate labelling incorporated into C-8 (δC 45). Showing a complex pattern likely arising from the overlap of greater labelling of adjacent C-9 and C-8 leading to a doublet and occasional labelling at C-9, C-8 and C-8’ leading to a triplet. Both labelling patterns can arise from the production of propionate via acetate. The center peak arises from the unlabelled, uncoupled C-8 and from the center of the triplet. 13C spectrum recorded in DMSO-d6, at 150 MHz. likely be slightly different coupling constants for J89 and J88’ which adds to the complexity of the splitting pattern. There would also be expected signal at δC 45.3 from unlabelled molecules. These results, showing incorporation of propionate units into alkyl portion of the molecule, mirrors the labeling patterns previously seen during rifamycin feeding studies.279,286 Additionally, the same carbon-13 spectrum of 6.1 demonstrating splitting patterns in the alkyl and pyrone portions of the molecule, failed to show incorporation of 1,2-13C2-acetate into the benzene portion of the molecule as carbon resonances for C-1 to C-7 demonstrated only carbon singlets (Fig 6-63). This result indicated the need for further feeding studies. + = 270 Figure 6-63. Carbon-13 spectrum of 6.1 with added 1,2-13C2-acetate demonstrating lack of coupling in C-2,4&6; C-3 & 5; and C-7. Recorded in DMSO-d6, at 150 MHz. 6.4.1.2 U-13C6-Glucose Feeding Experiments Although the 3,5-dihydroxy substitution pattern has not previously been shown to derive from a shikimate acid precursor, the presence of 6.4 in Salinspora arenicola RJA3005’s culture extract pointed to the presence of mutations within the rifamycin pathway. The presence of this shunt product suggested the possibility that the aromatic starter unit in 6.1 could also be a rifamycin shunt product. The aromatic nature of the AHBA starting unit in rifamycin and the lack of incorporation of 1,2-13C2-acetate into the aromatic portion of 6.1 drove the investigation of this possibility. AHBA and shikimic acid follow the same biosynthetic pathway, as seen in Figure 6.2, save for the introduction of the nitrogen and a final aromatization via the AHBA synthase. Both AHBA and shikimic acid are derived from glucose through erythrose 4-phosphate (erythrose) and phosphoenolpyruvate (PEP) precursors. As such, U-13C6-glucose was added to the agar medium during growth of Salinispora arenicola RJA3005. As glucose is incorporated 271 into both acetate and shikimic acid metabolites, the expectation was that labelling would be found throughout the molecule (Figure 6-64). Analysis of the splitting patterns in the NMR 13C spectrum of 6.1 after it was fed universally labelled glucose required consideration of genesis of the 3,5-dihydroxyphenyl moiety from either acetate precursors or PEP/erythrose (Figure 6-64). Incorporation of glucose-derived 13C labelled acetate, either through a PKS III or through a shared acetate/glycerol origin (Figure 6-59), would see a preponderance of sharp doublets (Figure 6-65) for all the aromatic resonances and C-7 of 6.1. Incorporation of Figure 6-64. Glucose is a central metabolite that is incorporated into many molecules. However, when incorporated into acetate molecules through the citric acid cycle (TCA), it is incorporated in various ways, as seen in green here. Glucose is used to construct shikimic acid after being incorporated into two precursors, erythrose and PEP, which are incorporated into aromatic molecules stemming from shikimic acid as shown in red and blue above. The aminoshikimate pathway producing AHBA uses the same building blocks as the shikimic pathway leading to the same incorporation patterns as shikimic acid. glucose-derived 13C labelled PEP and erythrose, would see complex splitting throughout the molecule, mainly due to the different splitting patterns from the incorporation of the three-carbon PEP and the four-carbon erythrose in magnetically equivalent C-2 and C-6 in 6.1’s benzene ring (Figure 6-65). 272 Figure 6-65. When examining the different pathways possible for biosynthetic construction of an aromatic ring, as seen in 6.1, both a PKS and a shikimate pathway were considered. All PKS units would need to incorporate as acetyl units which would when coupled together, lead to many doublets due to labelling shown in green. An aromatic ring built from universally labelled glucose through the shikimate or AHBA synthase pathways would have complex splitting patterns from multiple coupling partners, as seen in red and blue above. Indeed, subsequent extraction, separation, and NMR analysis revealed multiple 13C carbon incorporation sites throughout the entire molecule. As glucose is incorporated into the shikimic acid pathway and the citric acid cycle, careful interpretation of the coupling patterns was required. Incorporation into C-8, C-8’ and C-12’ demonstrated a series of doublets matching the incorporation of 13C with 1,2-13C2-acetate addition in Section 6.4.1.1 above. If the 3,5-dihydroxyphenyl moiety were derived from acetate, then a predominance of doublets would be expected for coupling partners C-2 (δC 105) and C-3 (δC 158); C-4 (δC 101) and C-5 (δC 158); C-6 (δC 105) and C-1 (δC 145) with C-7 (δC 74) demonstrating a singlet peak (Figure 6-65). Instead, coupling patterns for the aromatic resonances at C-2 & C-6, C-3 & C-5, C-1, C-4 as well as at C-7, showed distinct complex splitting patterns in line with the incorporation of labelled erythrose and PEP building blocks. The resonance at δC 158 demonstrated a specifically intricate coupling pattern. Analysis of the coupling tree (Figure 6-66) demonstrated the overlap of a triplet of doublets from C-3 (ortho coupling from C-2 and C-4 and meta coupling from C-5; 273 Figure 6-66. U-13C6-Glucose feeding experiments demonstrated complex coupling patterns around 158ppm in the 13C spectrum. The most likely explanation for this complex coupling pattern is an intact fragment from an erythrose precursor (as seen in red dots) through the shikimate pathway that led to two different coupling patterns as seen in trees for a) C-3 and b) C-5. c) Overlapping coupling patterns of C-3 and C-5 lead to the observed 13C spectrum. Recorded in DMSO-d6, at 150 MHz. J32=~60Hz ; J34= ~60 Hz; J35 = ~4 Hz) and a doublet of doublet of doublets from C-5 (ortho coupling from C-4, meta coupling from C-3 and para coupling from C-2; J54 = ~60 Hz; J53 = ~4 Hz; J52 = ~1 Hz). In Figure 6-67, the coupling pattern for C-4 (δC 101.4) with a triplet of doublets from ortho coupling with C-5 (J45 = ~60 Hz) and C-3 (J43 = ~60 Hz) and meta coupling with C-2 (J42 = ~4 Hz) is shown. However, there is a doublet seen overlapping with the C-4 signal, the origins of which is unclear. Interestingly this doublet appears to be very similar to the a b c + = 274 Figure 6-67. In the 13C spectrum, the resonance at δC 101.5 (C-4) in the U-13C6-glucose feeding experiment demonstrated complex coupling patterns. a) Triplet of doublet splitting pattern as would arise from a labelled erythrose building block (as seen in red dots). b) The origin of the doublet with a J = 60 Hz is unclear. c) Overlapping coupling patterns of A and B lead to the observed 13C spectrum. Recorded in DMSO-d6, at 150 MHz. nearby doublet belonging to C-10 (δC 100). Next, in Figure 6-68, the coupling pattern for the resonance at δC 104.8, assigned to both C-2 and C-6 of 6.1 is shown. Given the origin of C-2 from erythrose and C-6 from PEP, each would have one ortho coupling partner with approximately the same coupling constant. Both should experience fine aromatic splitting, a b c + = C-4 C-10 275 which, when overlapped, leads to a very broad peak that can be seen to be due to multiple fine splitting in Figure 6-68. Analysis of the carbon resonance C-1 (δC 145) was difficult given the low signal of the quaternary carbon, yet a doublet of doublets (J16 = ~60 Hz and J17 = ~ 46 Hz) (Figure 6-69) could be discerned. This doublet of doublets would be expected from two coupling partners derived from a three-carbon PEP precursor. Finally, C-7 (δC 74.7), demonstrated a doublet of doublet arising from its neighbor at C-1 (J71 = ~46 Hz) and fine splitting from the labelled carbon at C-6 (J76 = ~3 Hz). Such coupling at C-7 was only possible if it was part of a three-carbon fragment from PEP. The coupling pattern analysis led to the conclusion that the 3,5-dihydroxyphenyl moiety in 6.1 was likely produced through PEP and erythrose building blocks. At this stage, it is difficult to know whether this is through a modified aminoshikimate or shikimate pathway. However, the presence of 6.4 suggests there is undoubtedly a mutation somewhere in Salinispora arenicola RJA3005’s rifamycin gene cluster. It has also been shown that the aminoshikamate pathway that produces aminoDAHP on its way to AHBA can produce the DAHP precursor of the shikimate pathway.132 276 Figure 6-68. 13C spectrum from the U-13C6-Glucose feeding experiments demonstrated a broad doublet containing multiple fine splitting around 104.8 ppm. Due to the symmetry of the molecule the 104.8 ppm chemical shift arises from both C-2 and C-6. The most likely explanation for this complex coupling pattern is a) An intact fragment from a PEP precursor (as seen in blue dots) leads to a doublet (60 Hz) of doublet (3 Hz) of doublets (1Hz) for C-6 and b) a erythrose precursor (as seen in the red dots) leading to a doublet (60 Hz) of doublets (3 Hz). c) Here the overlapping coupling patterns of C-2 and C-6 leading to the observed broad doublet with fine splitting at δC 104.8 in the 13C spectrum are seen. Recorded in DMSO-d6, at 150 MHz. a b ) c + = 277 Figure 6-69. 13C spectrum from the U-13C6-Glucose feeding experiments demonstrated difficult to interpret weak signals at C-1 at 145 ppm. However, there is a strong suggestion of a doublet of doublet. The most likely explanation for this splitting pattern is an erythrose precursor (as seen in the blue dots) leading to a doublet (60 Hz) of doublets (46 Hz). Recorded in DMSO-d6, at 150 MHz. Figure 6-70. 13C spectrum from U-13C6-Glucose feeding experiments demonstrating a doublet with fine splitting for C-7 at 74.7 ppm. The most likely explanation for this splitting pattern is an erythrose precursor (as seen in the blue dots) leading to a doublet (46 Hz) with long range coupling lead to a second doublet (3 Hz). Recorded in DMSO-d6, at 150 MHz. 278 6.4.2 Bioinformatic Analysis of the Salinspora arenicola RJA3005’s Draft Genome It is difficult to predict the exact biosynthetic pathway from the above evidence. Analysis of the draft genome of Salinspora arenicola RJA3005 indicates the presence of both the rifamycin gene cluster and the AHBA synthase cluster with high similarity to other Salinspora arenicolas (Figure 6-67), although both are part of incomplete gene clusters in the draft genome for RJA3005. The closest AHBA synthase homolog resided on contig 17 of RJA3005’s draft genome sequence. Contig 17 also bears other genes required for AHBA biosynthesis. It bears 100% similarity to other Salinispora arenicola with rifamycin homologous gene clusters. The only place of difference is a tiny gene (171 bp) with homology to hypothetical proteins in BLASTP searches. Figure 6-71. Partial gene cluster comparison from the contig 17 of Salinispora arenicola RJA3005 as produced by antiSMASH. The AHBA synthase homolog is identical to other S. arenicola strains, save for one tiny gene whose closest homolog is a hypothetical protein. The yellow box is highlighting this solitary gene. 279 6.5 Bioactivity There was enough of 6.1 to test the natural products in several bioassays. 6.1, 6.2 and 6.3 were subjected to the phosphatase assay with no activity. They were also subjected to anti-bacterial assays with no activity. They were tested against the human pathogen Candida albicans with no activity. There were some initial weak results from 6.1 as an antifungal against specific plant pathogens in a modified disc diffusion assay however they were considered too weak as a pure compound to consider pursuing. There were very small amounts of 6.4 isolated and as such it was not tested in any bioassay. 6.6 Summary and Conclusion This project demonstrated the isolation of novel natural products through chemical prospecting from an initial survey of a biosynthetically talented Salinispora arenicola RJA3005 in a phosphatase assay. Although 6.4 and 6.1 have previously appeared in the literature, both compounds were only found from a mutated bacterial rifamycin-producer. 6.4 was shown to be a metabolic offshoot of a mutated rifamycin pathway. Additionally, 6.1 was only produced after a mutant was fed synthesized 3,5-dihydroxybenzoic acid, as such this is the first instance of it being shown as a true natural product. Intriguingly, feeding studies suggest that the resorcinol substitution pattern is formed via a modified shikimic acid or AHBA synthase pathway, which would be the first demonstration of such production. Recently, a Salinispora arenicola of Brazilian origin was shown to be producing a related molecule to both 6.1 and 6.4 except without any aromatic substitutions,305 lending support to the feeding study results reported herein. Initial bioinformatic analysis of a draft genome found only a small variant in the AHBA synthase gene cluster present in Salinispora arenicola RJA3005. Given the results from the 280 bioinformatic analysis, further knock-out studies of the AHBA synthase gene cluster of S. arenicola RJA3005 should be completed to confirm the results of the feeding studies. Furthermore, additional stable isotope feeding studies with labelled PEP, erythrose, DAHP and aminoDAHP could be undertaken to confirm the splitting patterns seen with the feeding of universally-labelled glucose. If indeed 6.1 is produced from a slightly modified AHBA synthase then the characterization of this modification along with the wild mutation leading to 6.4 could support engineering efforts of these types of molecules. 6.7 Experimental Section 6.7.1 General Experimental Procedures All solvents used for partitioning and flash chromatography were Sigma-Aldrich HPLC grade, used without further treatment. For HPLC work, Sigma-Aldrich HPLC grade solvents were filtered through Milli-Q filter paper prior to use. Distilled water was used to prepare all growth media for terrestrial bacteria. Seawater collected by M. Leblanc was used to prepare growth media for all marine bacteria. Milli-Q H2O was used for partitioning, reversed-phase Sep-Pak and HPLC. The 1H, 13C and 15N NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual DMSO-d6 peak (chemical shift  2.50) and 13C chemical shifts are referenced to the DMSO-d6 solvent peak ( 39.51). The 15N NMR spectra are in reference to CH3NO2 (0 ppm) with a variability of 1 ppm. Low resolution electrospray-ionization mass spectrometer (LRESIMS) were recorded on Bruker HCT Ultra PTM Discovery System and high resolution electrospray-ionization mass spectrometer (HRESIMS) were recorded on a Waters / Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. Merck Type 5554 281 silica gel plates and Whatman MKC18F plates were used for analytical thin layer chromatography (TLC). Sephadex™ LH-20 purchased from Sigma was used for gel filtration chromatography. para-Anisaldehyde stain with subsequent heat application was used to visualize TLC spots. Sep-Pak SPE Silica and C18 cartridges (2 g, 5 g, and 10 g) were purchased from Waters. All media components were obtained from Fisher Scientific. Reversed-phase HPLC purifications were performed on a Waters 1525 Binary HPLC pump attached to a Waters 2998 Photodiode Array Detector and equipped with Empower Software. 6.7.2 Isolation of metabolites Salinispora arenicola RJA3005 was isolated from marine sediment collected in Papua New Guinea by Dr. Doralyn D. Dalisay. Bacterial cultivation was carried out using nutrient rich media - Marine Medium 1 (MM1- soluble starch: 10.0 g; yeast extract: 4.0 g; peptone: 18.0 g; sea water (marine strains) or distilled fresh water (terrestrial strains): 1.0 L; KBr: 0.001 g and FeSO4.7H2O: 0.0004 g. Solid media had agar added (2.0 g/L). Salinispora arenicola RJA3005 was grown for 14 days on solid agar until a thick mat of orange leathery textured bacterial colonies had formed. After 14 days, the agar and mycelia were cut into small squares and immersed in EtOAc for extraction. The EtOAc soaked agar was filtered through filter paper to separate the agar from the supernatant (3x, ~2 L solvent per 8 L growth medium). The EtOAc portions were then combined and the solvent removed by evaporation in vacuo to reveal a thick red/brown oily-solid, with large portions of the extract only soluble in MeOH until further separation. As such, the crude was dissolved in a 9:1 mixture of H2O:MeOH (v/v). Subsequently, this crude extract was sequentially partitioned between first hexane/ H2O and then CH2Cl2/H2O after which time the aqueous partition had MeOH removed under reduced pressure to leave a 282 deep golden aqueous solution containing reddish brown oily-solids. At this time, EtOAc was added to completely dissolve the oily-solids and the EtOAc-soluble partition was removed and dried under reduced pressure to reveal a reddish golden viscous oil. The EtOAc-soluble partition was the active fraction in the phosphatase inhibition assay and was selected for further separation. This partition was then passed through a Sephadex® LH-20 size exclusion column (4:1 MeOH/ CH2Cl2) and fractioned based on TLC staining of smaller test tubes. Active fractions were then separated using step-gradients on flash chromatography (H2O to MeOH), using C18 Sep-paks (2 g, 5 g, 10 g depending on initial amount of material grown). The fraction eluting in 4:1 H2O:MeOH and 3:2 H2O:MeOH (v/v) contained the NMR peaks of interest and were further separated using semi-preparative reversed-phase HPLC (InertSustain C18, 17:83 MeCN:H2O with 0.05% FA). 3005.Et.CD.C.4: 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) : Isolated as a white powder; [α]25D -8.79° (c 3.5 g/100 mL, MeOH); 1H (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Table 6-2; HRMS (TOFESI) m/z: [M+H]+ Calcd for C15H17O6, 293.1025; Found 293.1046. 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-methoxy-3-methyl-2H-pyran-2-one (6.2): Isolated as an off-white powder; 1H (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6,150 MHz) 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-2-methoxy-3-methyl-4H-pyran-4-one: (6.3): Isolated as an off-white powder; 1H (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6,150 MHz); 283 3005.Et.CD.E.4 : N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4): Isolated as a white powder; 1H (both DMSO-d6 600 MHz) and 13C NMR (both DMSO-d6 150 MHz), see Table 6-3; HRMS (TOFESI) m/z: [M+H]+ Calcd for C17H20O6N, 334.1291; Found 334.1297. 6.7.3 Methylation 1.3 mg (0.0045 mmol) of 6.1 was dried in a vial and left overnight on the lyophilizer to remove any residual waters. A 5:1 (625 µL:125 µL) Toluene(anhydrous):MeOH v/v solution was added to the vial under inert conditions. Purchased trimethylsilyldiazomethane (TMSCHN2, 3.35 µL, 0.6 mmol) was added dropwise to the stirring solution. The solution was left to stir overnight under inert conditions and monitored by TLC. Upon completion, the products were concentrated by removing the solvent and purified by reversed-phase HPLC (InertSustain C18; step gradient 0-60 min 17:83 MeCN:H2O followed by 61 mins to 120 mins 23:77 MeCN:H2O; methyl ester of the 4-pyrone isomer eluting between 55-57 min and the methyl ester of the enol isomer eluting between 75-80 mins) 6.7.4 Mosher’s Ester Compound 6.1 (1.3 mg) was divided into two clean NMR tubes which were dried completely on a lyophilizer under the vacuum of an oil pump. Deuterated pyridine (0.5 mL) was added to each NMR tube and either R-(-)-MTPA-Cl (3R, 10 µL) or S-(+)-MTPA-Cl (3S, 10 µL ) were added into the NMR tube immediately under a N2 gas stream and the top sealed with parafilm. Then the NMR tube was shaken carefully to mix the sample and MTPA chloride evenly. The reaction NMR tube was permitted to stand at room temperature and monitored every 284 1-2 h by 1H NMR. The reaction was found to be complete after 4 h. Given the complex mixture and small amount of sample, COSY and HSQC experiments were recorded in order to assign the chemical shifts for both the (R)-MTPA ester derivative and (S)-MTPA derivative. NMR assignments (as was possible) are found in Table 6.3. 6.7.5 Feeding studies 1,2-13C2-acetate feeding: 1500 mg of 1,2-13C2-acetate was filter sterilized into 2 L of autoclaved MM1 agar medium prepared with sea water. This agar media, containing the labelled precursor, was poured into 6 stainless steel trays (37 mm X 20 mm) and Salinispora arenicola RJA3005, growing in liquid medium for 5-7 days, was spread over the surface of the cooled agar. The trays were covered with sterile aluminum foil and left to grow for 14 days. After 14 days and the formation of thick orange mat of bacterium, the agar and mycelia were cut into small squares and harvested into EtOAc. After a minimum of 24 hrs, the EtOAc extract was filtered through cotton wool and then remove under reduced pressure to reveal a deep yellow gold extract. After separation utilizing C18 Sep-Pak (10 g, step-gradient 4:1 H2O:MeOH to 3:2 H2O:MeOH) the fraction eluting with 3:2 H2O:MeOH was placed onto the HPLC and separated by conditions previously determined for 6.1. U-13C6-glucose feeding: 850 mg of U-13C6-glucose was filter-sterilized into 2 L of autoclaved MM1 agar medium prepared with sea water. This agar media, containing the labelled precursor, was poured into 6 stainless steel trays (37 mm X 20 mm) and Salinispora arenicola RJA3005, growing in liquid medium for 5-7 days, was spread over the surface of the cooled agar. The trays were covered with sterile aluminum foil and left to grow for 14 days. After 14 days and the formation of thick orange mat of bacterium, the agar and mycelia were cut into small squares and 285 harvested into EtOAc. After a minimum of 24 hrs., the EtOAc extract was filtered through cotton wool and then removed under reduced pressure to reveal a deep yellow gold extract. After separation utilizing C18 Sep-Pak (10 g, step-gradient 4:1 H2O:MeOH to 3:2 H2O:MeOH) the fraction eluting with 3:2 H2O:MeOH was placed onto the HPLC and separated by conditions previously determined for 6.1. 286 Chapter 7: Conclusion The work presented in this thesis was conducted in an effort to discover novel natural products with potential therapeutic properties and to investigate the biosynthesis of natural products. Bacteria are a relevant source for exploration, given that they are the origin of many current pharmaceutical medicines. Both marine and terrestrial bacterial sources were explored utilizing a variety of discovery methods, including a 15N NMR-guided approach developed through this thesis work and the robust bioassay-guided fractionation. 15N NMR-guided discovery of Piz-containing natural products: To summarize: In Chapters 2, 3 and 4, I demonstrated the development of a novel method for the targeted discovery of Piz-containing natural products. Chapter 2 describes the bioinformatic methodology utilized to harness the power of characterized genetic sequences to select for bacterial producers of desired natural products. Chapter 3 details the 15N NMR experiments used to track Piz’s diagnostic N-H correlation and N-H connected spin system from crude bacterial culture extract to the isolated natural product. 15N2-L-Orn, a stable isotope labelled precursor of Piz, was fed to the bacterial producer to amplify the N-H NMR signal, allowing its observation from crude extract to a single molecule. This amplified signal allowed observation of minute amounts of a Piz-containing natural product in the crude bacterial extract of a genome-mined bacterium. Thus, linking genome mining with NMR for targeted discovery allowed for the isolation of several novel very minor metabolites. Chapter 4 details the structure elucidation of those metabolites, including the structure elucidation of a novel moiety in chemical space. One of the members of this unique family was a bioactive natural product. Identification of novel drug leads is the first step in drug discovery that can lead to 287 pharmaceuticals development. Thus, Chapters 2, 3 and 4 demonstrate that natural product discovery methods rationally targeting moieties with rich associated bioactivity or structural uniqueness can lead us to our desired goals. In future directions: This 15N NMR-guided methodology is being applied to isolate Piz-containing natural products from diverse bacterial sources. Additionally, although this method was targeted to Piz compounds, nitrogen-nitrogen bond containing natural products and other nitrogen-containing moieties are underrepresented in elucidated natural products compared to their numbers in sequenced genomic databases. Utilizing genome mining to select species producing molecules of interest followed by careful analysis of unique 15N NMR features would allow the elucidation of more significant numbers of these compounds. Coupled with feeding labelled precursors and additional nitrogen NMR experiments, various 15N NMR features, including N-H correlations and longer-range nitrogen correlations, could be exploited for targeted isolation of N-N bond containing natural products. Furthermore, Piz-containing natural products seem to be generally contained to the Actinobacteria bacterial phylum and have been most often found from Streptomyces species. There have been characterized gene clusters for many of the different families of natural products that contain Piz. Through bioinformatics analysis, it is also seen that there are many gene clusters for which the natural product phenotype is unknown. Within these characterized gene clusters and orphan gene clusters, there appears to be diversity of gene cluster organization found in members of different Actinobacteria genera and, to some degree, amongst the more closely related Streptomyces species. Utilizing this Piz-targeted 15N NMR strategy to uncover Piz-containing natural products from various bacterial strains could reveal potential drug leads and new natural product families. 288 Finally, this method demonstrates how developing our understanding of biosynthetic pathways can couple with chemical knowledge for natural product discovery, expanding our natural product library in specific ways. Therefore, this method adds a tool to future discovery processes. As the chemical library grows and genotype linked to chemotype, understanding of the nuances in bacterial secondary metabolite gene cluster organization and genomic organization is enhanced. This information can, in turn, be utilized to uncover diversity present in whole families of natural products. Pursuing diverse members of interesting natural product classes may, in turn, lead to novel bioactivities of related compounds or similar bioactivities with novel mechanisms of action. Bioassay-guided discovery of natural products: To summarize: in Chapter 5, the isolation and structure elucidation of secondary metabolites from crude microbial extracts identified the biologically active natural products in their respective bioassays. In addition, the appearance of novel activity in manufactured bacterial fusants from several wild-type strains was pinpointed to the de novo expression of a known antifungal. This result could, in the future, help build an understanding of the mechanisms driving secondary metabolite operon expression due to genetic recombination events. To summarize: in Chapter 6, the bioassay-guided exploration of a small library of crude extracts also led to the isolation and structure elucidation of natural products from wild-type members of the Salinispora genus. Several natural products were uncovered that had not previously been reported in the literature as naturally-derived molecules, although bioactivities for them are not clear. These natural products had previously been reported in the literature as the products of genetic intervention in the rifamycin pathway in the Amycolatopsis mediterranei S699 strain. These molecules not only serve as potential lead compounds but also expand the 289 known chemical library. Feeding studies were undertaken to understand the natural biosynthetic pathway of these natural products. The results of these feeding studies potentially point to modifications in the AHBA synthase cassette and the rifamycin pathway in the Salinispora arenicola RJA3005 strain. Given the importance of rifamycin as a potent antibiotic, the AHBA synthase gene cluster's potential modification may be of particular interest for the future scope of this work. Knock-out studies would be necessary to unequivocally link 6.1 with the rifamycin gene cluster in Salinispora arenicola RJA3005. Previously, the incorporation of the 3,5-dihydroxybenzoic acid moiety into the rifamycin pathway was achieved by feeding synthetic precursors. In other pathways, the incorporation of this moiety has been the foundation for highly modified starting units produced through PKS III mechanisms. These results point to a novel microbial mechanism for biosynthesizing this moiety. It is also interesting to observe that the rifamycin gene cluster, a highly conserved gene cluster found in diverse bacterial species, could be subject to modifications that prevent rifamycin production as a main secondary metabolite. It could also be utilized in understanding how wild mutations can occur in this sizeable modular synthase. A further avenue for future exploration is comparing both the rifamycin and AHBA synthase gene clusters and secondary metabolite production of the other Salinispora strains retrieved from the same or similar environments detailed in the curated mini-library. As our understanding of bacterial community metabolic pathways has been increasing over the last decade, this could build knowledge on how bacterial communities may be incorporating secondary metabolite gene cluster modifications. In the future, this could inform bacterial strain selection processes in the search for novel chemistry. To conclude: 290 This thesis work demonstrates how genomic knowledge and chemical knowledge can be utilized in tandem for bacterial natural product discovery. Furthermore, it indicates that harnessing discovery methodologies tailored to our research goals can be a valuable strategy. 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Prod. 2012, 75 (5), 915–919. https://doi.org/10.1021/np300026p. 314 Appendices Appendix A NMR of Unsolved Fractions of Streptomyces incarnatus NRRL 8089 Figure A- 1. 1H NMR spectrum of 8089.Et.B.C-14, recorded at 600 MHz in pyridine-d5. Figure A- 2. COSY60 NMR spectrum of 8089.Et.B.C-14, recorded at 600 MHz in pyridine-d5. 315 Figure A- 3. 1H-15N HSQC NMR spectrum of 8089.Et.B.C-14, demonstrating Piz N-H correlations recorded at 600 MHz in pyridine-d5. Figure A- 4. 1H-13C HSQC NMR spectrum of 8089.Et.B.C-14, demonstrating unique proton-carbon correlations at 600 MHz in pyridine-d5. 316 Figure A- 5. 1H-13C HMBC NMR spectrum of 8089.Et.B.C-14, demonstrating unique proton-carbon correlations at 600 MHz in pyridine-d5. Figure A- 6. Expansion of the 1H-13C HMBC NMR spectrum of 8089.Et.B.C-14, demonstrating unique proton-carbon correlations at 600 MHz in pyridine-d5. 317 Figure A- 7. 1H-13C HSQC NMR spectrum of 8089.Et.B.C-14, demonstrating unique proton-carbon correlations at 600 MHz in pyridine-d5 in black. Red correlations are from 8089.Et.B.C-13. Figure A- 8. 1H-13C HMBC NMR spectrum of 8089.Et.B.C-14, demonstrating unique proton-carbon correlations at 600 MHz in pyridine-d5 in black. Red correlations are from 8089.Et.B.C-13. 318 Appendix B NMR Spectra of Dentigerumycin F and G Isolated with Formic Acid taken by Dave Williams, Unpublished Figure A- 9. 1H NMR spectrum of dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. Figure A- 10. 13C NMR spectrum of dentigerumycin F (4.2) recorded at 150 MHz in DMSO-d6. 319 Figure A- 11. 1H-13C HSQC NMR spectrum of dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. Figure A- 12. 1H-1H gCOSY NMR Spectrum of Dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. 320 Figure A- 13. 1H-1H gTOCSY NMR Spectrum of Dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. Figure A- 14. 1H-13C gHMBC NMR Spectrum of Dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. 321 Figure A- 15. tROESY NMR Spectrum of Dentigerumycin F (4.2) recorded at 600 MHz in DMSO-d6. Figure A- 16. 1H NMR Spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. 322 Figure A- 17. 13C NMR spectrum of Dentigerumycin G (4.1) recorded at 150 MHz in DMSO-d6. Figure A- 18. 1H-13C HSQC NMR spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. 323 Figure A- 19. 1H-1H gCOSY NMR spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. Figure A- 20. 1H-1H gTOCSY NMR spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. 324 Figure A- 21. 1H-13C gHMBC NMR spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. Figure A- 22. tROESY NMR spectrum of Dentigerumycin G (4.1) recorded at 600 MHz in DMSO-d6. 325 Figure A- 23. 1H NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Appendix C NMR spectra of Incarnatapeptin A as taken by Dave Williams, Published Figure A- 24. Expansion of 1H NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 326 Figure A- 25. Expansion of 1H NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Figure A- 26. Expansion of 1H NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 327 Figure A- 27. 13C NMR spectrum of Incarnatapeptin A (4.3) recorded at 150 MHz in DMSO-d6. Figure A- 28. Expansion of 13C NMR spectrum of Incarnatapeptin A (4.3) recorded at 150 MHz in DMSO-d6. 328 Figure A- 29. Expansion of 13C NMR spectrum of Incarnatapeptin A (4.3) recorded at 150 MHz in DMSO-d6. Figure A- 30. Expansion of 13C NMR spectrum of Incarnatapeptin A (4.3) recorded at 150 MHz in DMSO-d6. 329 Figure A- 31. 1H-15N HSQC NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Figure A- 32. 1H-1H TOCSY NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 330 Figure A- 33. g1H-1HCOSY60 NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Figure A- 34. 1H-13C HSQC NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 331 Figure A- 35. 1H-13C HSQC-TOCSY NMR spectrum of incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Figure A- 36. g1H-13C HMBC NMR spectrum of incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 332 Figure A- 37. tROESY NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. Figure A- 38. LR15NHMQC NMR spectrum of Incarnatapeptin A (4.3) recorded at 600 MHz in DMSO-d6. 333 Appendix D Experimental and NMR Spectra of the Methyl Ester* of Incarnatapeptin A taken by Dave Williams, Published. *The methyl ester is not in the thesis work but the crystal structure is essential for stereochemical assignment. EXPERIMENTAL: Methyl ester of Incarnatapeptin A (A.1): 2.6 mg of incarnatapeptin A (4.3) was treated with 0.3 ml of 0.6 M trimethylsilyldiazomethane in hexanes, 0.3 mL of anhydrous MeOH and 0.5 mL of C6H6. The reaction mixture was sealed and left stirring for 16 h at rt. After evaporation of the reagents, the sample was purified via C18 reversed-phase HPLC using an InertSustain, 5 µm, 25 x 1 cm column with 11:9 MeCN/H2O as eluent to yield 1.1 mg of product with a positive ion HRMS (TOFESI) m/z: [M+Na]+ Calcd for C37H62N8O12Na, 833.4385; Found 833.4390. 1H (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6,150 MHz). 334 Figure A- 39. 1H NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 600 MHz in DMSO-d6. Figure A- 40. Expanded 1H NMR spectrum of the methyl ester of incarnatapeptin A (A.1) with peak picking recorded at 600 MHz in DMSO-d6. 335 Figure A- 41. Expanded 1H NMR spectrum of the methyl ester of incarnatapeptin A (A.1) with peak picking recorded at 600 MHz in DMSO-d6. Figure A- 42. 13C NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 150 MHz in DMSO-d6. 336 Figure A- 43. Expanded 13C NMR spectrum of the methyl ester of incarnatapeptin A (A.1) with peak picking recorded at 150 MHz in DMSO-d6. Figure A- 44. Expanded 13C NMR spectrum of the methyl ester of incarnatapeptin A (A.1) with peak picking recorded at 150 MHz in DMSO-d6. 337 Figure A- 45. Expanded 13C NMR spectrum of the methyl ester of incarnatapeptin A (A.1) with peak picking recorded at 150 MHz in DMSO-d6. Figure A- 46. 1H-13C HSQC NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 600 MHz in DMSO-d6. 338 Figure A- 47. 1H-13C gHMBC NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 600 MHz in DMSO-d6. Figure A- 48. 1H-15N gHSQC NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 600 MHz in DMSO-d6. 339 Figure A- 49. 1H-15N LRHMQC NMR spectrum of the methyl ester of incarnatapeptin A (A.1) recorded at 600 MHz in DMSO-d6. Figure A- 50. tROESY NMR spectrum of the methyl ester of incarnatapeptin (A.1) recorded at 600 MHz in DMSO-d6. 340 Appendix E Crystal Data and Experimental Data provided by Dr Brian O. Patrick Crystal Data and Experimental Experimental. Single colourless blade-shaped crystals of ra178 were recrystallised from methanol by slow evaporation. A suitable crystal 0.22×0.14×0.05 mm3 was selected and mounted on a mylar loop in oil on a Bruker APEX II area detector diffractometer. The crystal was kept at a steady T = 90(2) K during data collection. The structure was solved with the XT (Sheldrick, 2015) structure solution program using the Intrinsic Phasing solution method and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with version 2018/3 of XL (Sheldrick, 2015) using Least Squares minimisation. Crystal Data. C37H62N8O12, Mr = 810.94, triclinic, P1 (No. 1), a = 9.4700(2) Å, b = 11.1262(3) Å, c = 12.0492(3) Å, = 65.774(2)°, = 67.771(2)°, = 76.655(2)°, V = 1067.41(5) Å3, T = 90(2) K, Z = 1, Z' = 1, (CuK ) = 0.787, 34680 reflections measured, 5446 unique (Rint = 0.0868) which were used in all calculations. The final wR2 was 0.0929 (all data) and R1 was 0.0407 (I > 2(I)). 341 Compound Methyl ester of Incarnatapeptin A Formula C37H62N8O12 Dcalc./ g cm-3 1.262 /mm-1 0.787 Formula Weight 810.94 Colour colourless Shape blade Size/mm3 0.22×0.14×0.05 T/K 90(2) Crystal System triclinic Flack Parameter 0.04(14) Hooft Parameter 0.12(12) Space Group P1 a/Å 9.4700(2) b/Å 11.1262(3) c/Å 12.0492(3) /° 65.774(2) /° 67.771(2) /° 76.655(2) V/Å3 1067.41(5) Z 1 Z' 1 Wavelength/Å 1.54178 Radiation type CuK min/° 4.246 max/° 56.286 Measured Refl. 34680 Independent Refl. 5446 Reflections with I > 2(I) 4663 Rint 0.0868 Parameters 543 Restraints 3 Largest Peak 0.167 Deepest Hole -0.238 GooF 1.053 wR2 (all data) 0.0929 wR2 0.0860 R1 (all data) 0.0547 R1 0.0407 342 Structure Quality Indicators Reflections: Refinement: A colourless blade-shaped crystal with dimensions 0.22×0.14×0.05 mm3 was mounted on a mylar loop in oil. Data were collected using an Bruker APEX II area detector diffractometer equipped with an Oxford Cryosystems low-temperature device operating at T = 90(2) K. Data were measured using and scans of 1.0 ° per frame for between 15 and 60 s using CuK radiation (microfocus sealed X-ray tube, 45 kV, 0.60 mA). The total number of runs and images was based on the strategy calculation from the program APEX3. The maximum resolution that was achieved was = 56.286° (0.93 Å). The diffraction pattern was indexed and the unit cell was refined using SAINT (Bruker, V8.38A, after 2013) on 9880 reflections, 28% of the observed reflections. Data reduction, scaling and absorption corrections were performed using SAINT (Bruker, V8.38A, after 2013). The final completeness is 98.80 % out to 56.286° in . A multi-scan absorption correction was performed using SADABS-2016/2 (Bruker,2016/2) was used for absorption correction. wR2(int) was 0.0935 before and 0.0779 after correction. The ratio of minimum to maximum transmission is 0.8453. The /2 correction factor is not present. The absorption coefficient of this material is 0.787 mm-1 at this wavelength ( = 1.542Å) and the minimum and maximum transmissions are 0.815 and 0.964. The structure was solved and the space group P1 (# 1) determined by the XT (Sheldrick, 2015) structure solution program using Intrinsic Phasing and refined by Least Squares using version 2018/3 of XL (Sheldrick, 2015). All non-hydrogen atoms were refined anisotropically. All C—H 343 hydrogen atom positions were calculated geometrically and refined using the riding model, while all N—H and O—H hydrogen atoms were refined freely. The Flack parameter was refined to 0.04(14). Determination of absolute structure using Bayesian statistics on Bijvoet differences using the Olex2 results in 0.12(12). Note: The Flack parameter is used to determine chirality of the crystal studied, the value should be near 0, a value of 1 means that the stereochemistry is wrong and the model should be inverted. A value of 0.5 means that the crystal consists of a racemic mixture of the two enantiomers. Table 7-1: Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for ra178. Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom x y z Ueq O1 11222(4) 2612(4) 3001(3) 29.8(10) O2 9981(4) 4361(4) 3566(3) 28.9(9) O3 7760(3) 5561(3) 1164(3) 21.5(8) O4 6121(4) 7999(5) 3000(4) 38.5(11) O5 2849(4) 8746(4) 4412(3) 24.1(9) O6 1558(4) 6658(4) 3558(3) 25.7(9) O7 933(4) 5572(3) 7584(3) 25.6(9) O8 -2602(4) 4721(4) 9412(4) 26.3(9) O9 -124(4) 1634(3) 7280(3) 23.3(9) O10 2384(4) 153(4) 6854(4) 30.4(10) O11 4038(4) 2413(3) 7347(3) 23.9(9) O12 3412(4) 2860(3) 5522(3) 23.8(9) N1 10948(4) 6767(4) 1102(5) 21.4(11) N2 10038(5) 5893(4) 1125(4) 20.4(10) N3 6469(4) 8033(4) 1037(4) 19.9(10) N4 5810(5) 7891(5) 216(4) 22.5(11) N5 2953(4) 7908(4) 3759(4) 19.2(10) N6 -1231(5) 5717(4) 5669(4) 21.4(10) N7 -294(4) 6112(4) 6134(4) 18.0(10) N8 802(5) 2977(4) 7800(4) 19.3(11) C1 10560(6) 3853(5) 2768(5) 20.4(13) C2 10616(6) 4508(5) 1377(5) 21.9(13) C3 12225(6) 4411(5) 413(5) 26.0(14) C4 13252(6) 5275(6) 430(6) 28.2(14) C5 12506(5) 6685(6) 199(5) 27.3(14) C6 8620(6) 6328(5) 1037(5) 20.8(13) C7 8152(5) 7801(5) 749(5) 19.7(13) C8 8909(6) 8635(6) -655(5) 22.6(13) 344 Atom x y z Ueq C9 8286(6) 8447(6) -1566(5) 26.5(14) C10 6539(6) 8707(6) -1121(5) 25.9(13) C11 5566(6) 8097(5) 2195(5) 22.3(13) C12 3844(5) 8368(5) 2387(5) 21.0(13) C13 3437(6) 9838(6) 1723(6) 32.0(15) C14 1678(5) 7262(5) 4172(5) 18.0(12) C15 404(5) 7359(5) 5374(5) 19.3(12) C16 -806(6) 8495(5) 4990(5) 22.2(13) C17 -1767(6) 8121(6) 4428(5) 24.8(13) C18 -2403(6) 6809(5) 5335(5) 26.5(13) C19 62(5) 5275(5) 7207(5) 18.9(12) C20 -557(5) 3925(5) 7910(5) 17.9(12) C21 -1370(6) 3700(5) 9335(5) 21.6(13) C22 -1943(6) 2318(5) 10133(5) 25.9(13) C23 -3243(6) 2109(6) 9800(6) 36.4(16) C24 -2412(8) 2059(7) 11571(6) 43.9(17) C25 915(6) 1948(5) 7471(5) 18.5(12) C26 2420(6) 1026(5) 7424(5) 21.9(13) C27 3814(6) 1844(5) 6569(5) 21.3(13) C28 5287(6) 1077(5) 6014(5) 26.6(14) C29 6474(6) 2087(6) 5071(6) 30.8(15) C30 5854(6) 3401(6) 5295(5) 28.4(14) C31 5434(6) 3098(6) 6724(5) 26.2(14) C32 2458(6) 249(5) 8785(5) 28.6(14) C33 4396(6) 3948(6) 4913(5) 27.0(14) C34 4610(7) 4485(7) 3504(6) 40.4(17) C35 5125(6) 4242(6) 7167(6) 35.2(16) C36 11276(7) 1905(6) 4301(6) 35.7(16) C37 3601(6) 8080(6) 5397(5) 29.3(14) Table 7-2: Anisotropic Displacement Parameters (×104) ra178. The anisotropic displacement factor exponent takes the form: -2 2[h2a*2 × U11+... +2hka* × b* × U1 Atom U11 U22 U33 U23 U13 U12 O1 36(2) 26(2) 30(2) -11.3(18) -18.8(19) 8.1(19) O2 29(2) 29(2) 26(2) -11(2) -6.4(18) 0.3(18) O3 12.5(19) 22(2) 32(2) -11.4(17) -6.4(16) -2.4(16) O4 14(2) 77(3) 31(2) -27(2) -8.2(18) -1.1(19) O5 18.2(19) 29(2) 32(2) -18.4(19) -8.9(17) 1.8(16) O6 16.3(19) 32(2) 28(2) -13(2) -2.4(17) -4.0(16) O7 24(2) 25(2) 29(2) -4.1(17) -14.2(17) -6.1(16) O8 19.1(19) 29(2) 35(2) -21.0(18) -7.6(17) 6.0(16) O9 16.2(19) 24(2) 30(2) -10.4(18) -6.4(16) -3.5(16) O10 23(2) 28(2) 43(3) -21(2) -8.3(19) 3.7(17) O11 14.1(19) 29(2) 27(2) -10.3(18) -3.5(16) -5.9(16) 345 Atom U11 U22 U33 U23 U13 U12 O12 15.0(19) 25(2) 24(2) -2.8(17) -3.8(16) -2.1(16) N1 9(2) 26(3) 30(3) -10(2) -7(2) -0.6(19) N2 13(2) 20(3) 27(3) -8(2) -9(2) 2.8(19) N3 11(2) 27(3) 26(3) -13(2) -7(2) 1.5(19) N4 17(2) 27(3) 29(3) -12(2) -12(2) 1(2) N5 13(2) 27(3) 22(3) -13(2) -3.8(19) -4.9(19) N6 18(2) 27(3) 26(3) -11(2) -13(2) 0(2) N7 16(2) 20(2) 19(3) -6(2) -5.5(19) -5(2) N8 12(2) 20(3) 27(3) -12(2) -6(2) 3.4(19) C1 15(3) 18(3) 28(3) -8(3) -8(2) 0(2) C2 16(3) 21(3) 27(3) -10(3) -9(2) 6(2) C3 21(3) 25(3) 29(3) -12(3) -10(3) 10(2) C4 17(3) 34(4) 32(3) -14(3) -8(2) 4(3) C5 10(3) 37(4) 31(3) -9(3) -8(2) 2(2) C6 14(3) 26(3) 20(3) -10(3) -4(2) 4(3) C7 3(3) 30(3) 27(3) -12(3) -5(2) 2(2) C8 13(3) 24(3) 27(3) -7(3) -4(2) -1(2) C9 14(3) 35(4) 26(3) -9(3) -6(2) 2(2) C10 16(3) 32(3) 27(3) -6(3) -10(2) 0(2) C11 13(3) 25(3) 29(3) -10(3) -6(3) -3(2) C12 11(3) 28(3) 20(3) -7(3) -4(2) -1(2) C13 18(3) 32(4) 33(4) -3(3) -7(3) 0(3) C14 15(3) 18(3) 22(3) -9(3) -9(2) 5(2) C15 14(3) 21(3) 20(3) -6(2) -5(2) -2(2) C16 20(3) 21(3) 25(3) -9(3) -6(2) 2(2) C17 20(3) 27(3) 31(3) -11(3) -13(3) 2(2) C18 17(3) 29(3) 33(3) -7(3) -13(3) 1(2) C19 11(3) 21(3) 22(3) -10(3) -4(2) 5(2) C20 14(3) 19(3) 19(3) -9(2) -5(2) 4(2) C21 15(3) 21(3) 28(3) -12(3) -6(2) 5(2) C22 23(3) 25(3) 26(3) -10(3) -1(3) -3(2) C23 24(3) 36(4) 44(4) -14(3) -2(3) -8(3) C24 59(4) 37(4) 25(3) -7(3) -1(3) -14(3) C25 17(3) 17(3) 17(3) -6(2) -3(2) 1(2) C26 16(3) 24(3) 29(3) -13(3) -9(2) 3(2) C27 17(3) 22(3) 23(3) -7(3) -7(2) 0(2) C28 19(3) 25(3) 32(3) -9(3) -10(3) 6(2) C29 18(3) 34(4) 31(3) -11(3) -2(3) 5(3) C30 14(3) 30(3) 33(4) -8(3) 0(2) -5(2) C31 14(3) 31(3) 34(4) -12(3) -5(3) -5(2) C32 18(3) 25(3) 32(4) -1(3) -6(3) -1(2) C33 16(3) 25(3) 30(3) -6(3) -1(2) -3(2) C34 31(3) 47(4) 29(4) -4(3) -7(3) -1(3) C35 23(3) 40(4) 44(4) -16(3) -10(3) -6(3) C36 48(4) 27(4) 33(4) -4(3) -25(3) 2(3) 346 Atom U11 U22 U33 U23 U13 U12 C37 19(3) 47(4) 28(3) -18(3) -10(3) -1(3) Table7-3: Bond Lengths in Å for ra178. Atom Atom Length/Å O1 C1 1.339(6) O1 C36 1.452(6) O2 C1 1.204(6) O3 C6 1.239(6) O4 C11 1.225(6) O5 N5 1.414(5) O5 C37 1.463(6) O6 C14 1.232(6) O7 C19 1.236(6) O8 C21 1.433(6) O9 C25 1.241(6) O10 C26 1.414(7) O11 C27 1.422(6) O11 C31 1.463(6) O12 C27 1.417(6) O12 C33 1.465(6) N1 N2 1.427(6) N1 C5 1.474(6) N2 C2 1.456(7) N2 C6 1.347(6) N3 N4 1.426(6) N3 C7 1.480(6) N3 C11 1.354(6) N4 C10 1.464(7) N5 C12 1.469(7) N5 C14 1.369(7) N6 N7 1.434(6) N6 C18 1.477(7) N7 C15 1.457(7) N7 C19 1.360(6) N8 C20 1.465(6) N8 C25 1.328(7) C1 C2 1.513(7) C2 C3 1.540(7) C3 C4 1.527(8) C4 C5 1.522(8) C6 C7 1.522(8) C7 C8 1.528(7) C8 C9 1.527(7) C9 C10 1.531(7) C11 C12 1.532(7) C12 C13 1.527(8) 347 Atom Atom Length/Å C14 C15 1.520(7) C15 C16 1.542(7) C16 C17 1.527(7) C17 C18 1.513(8) C19 C20 1.518(7) C20 C21 1.530(7) C21 C22 1.537(8) C22 C23 1.520(8) C22 C24 1.533(8) C25 C26 1.548(7) C26 C27 1.545(7) C26 C32 1.517(8) C27 C28 1.528(7) C28 C29 1.545(8) C29 C30 1.536(8) C30 C31 1.517(8) C30 C33 1.534(7) C31 C35 1.503(8) C33 C34 1.497(8) Table 7-4: Bond Angles in ° for ra178. Atom Atom Atom Angle/° C1 O1 C36 115.1(4) N5 O5 C37 110.6(4) C27 O11 C31 112.5(4) C27 O12 C33 111.7(4) N2 N1 C5 110.1(4) N1 N2 C2 118.4(4) C6 N2 N1 119.8(4) C6 N2 C2 121.5(4) N4 N3 C7 118.8(4) C11 N3 N4 120.0(4) C11 N3 C7 119.7(4) N3 N4 C10 110.2(4) O5 N5 C12 115.4(4) C14 N5 O5 115.2(4) C14 N5 C12 119.4(4) N7 N6 C18 109.4(4) N6 N7 C15 119.2(4) C19 N7 N6 120.3(4) C19 N7 C15 120.2(4) C25 N8 C20 123.3(4) O1 C1 C2 110.6(5) O2 C1 O1 124.3(5) 348 Atom Atom Atom Angle/° O2 C1 C2 125.1(5) N2 C2 C1 109.1(4) N2 C2 C3 109.5(4) C1 C2 C3 113.8(4) C4 C3 C2 109.7(4) C5 C4 C3 110.3(4) N1 C5 C4 113.3(5) O3 C6 N2 121.0(5) O3 C6 C7 122.2(4) N2 C6 C7 116.7(4) N3 C7 C6 108.8(4) N3 C7 C8 108.6(4) C6 C7 C8 112.4(4) C9 C8 C7 112.3(5) C8 C9 C10 108.9(4) N4 C10 C9 113.3(4) O4 C11 N3 121.0(4) O4 C11 C12 123.3(5) N3 C11 C12 115.6(5) N5 C12 C11 110.9(4) N5 C12 C13 111.3(4) C13 C12 C11 110.2(4) O6 C14 N5 120.5(5) O6 C14 C15 121.5(5) N5 C14 C15 118.0(5) N7 C15 C14 110.7(4) N7 C15 C16 110.6(4) C14 C15 C16 109.5(4) C17 C16 C15 111.2(4) C18 C17 C16 109.7(5) N6 C18 C17 114.3(4) O7 C19 N7 121.1(5) O7 C19 C20 119.6(4) N7 C19 C20 119.2(4) N8 C20 C19 104.9(4) N8 C20 C21 108.1(4) C19 C20 C21 110.4(4) O8 C21 C20 107.1(4) O8 C21 C22 111.5(4) C20 C21 C22 113.7(4) C23 C22 C21 112.7(5) C23 C22 C24 111.0(5) C24 C22 C21 110.4(5) O9 C25 N8 124.8(5) O9 C25 C26 118.3(5) 349 Atom Atom Atom Angle/° N8 C25 C26 116.7(5) O10 C26 C25 106.3(4) O10 C26 C27 108.7(4) O10 C26 C32 109.9(4) C27 C26 C25 110.1(4) C32 C26 C25 109.0(4) C32 C26 C27 112.6(4) O11 C27 C26 105.7(4) O11 C27 C28 110.8(4) O12 C27 O11 109.7(4) O12 C27 C26 107.5(4) O12 C27 C28 107.5(4) C28 C27 C26 115.6(4) C27 C28 C29 107.6(4) C30 C29 C28 107.5(4) C31 C30 C29 107.2(5) C31 C30 C33 108.4(4) C33 C30 C29 109.3(4) O11 C31 C30 106.5(4) O11 C31 C35 107.0(4) C35 C31 C30 117.9(5) O12 C33 C30 107.0(4) O12 C33 C34 107.7(5) C34 C33 C30 116.4(5) Table 7-5: Torsion Angles in ° for ra178. Atom Atom Atom Atom Angle/° O1 C1 C2 N2 -174.0(4) O1 C1 C2 C3 -51.3(6) O2 C1 C2 N2 7.1(7) O2 C1 C2 C3 129.7(5) O3 C6 C7 N3 16.5(7) O3 C6 C7 C8 -103.8(5) O4 C11 C12 N5 -25.6(7) O4 C11 C12 C13 98.1(6) O5 N5 C12 C11 78.2(5) O5 N5 C12 C13 -44.9(5) O5 N5 C14 O6 169.7(4) O5 N5 C14 C15 -8.1(6) O6 C14 C15 N7 39.0(6) O6 C14 C15 C16 -83.1(6) O7 C19 C20 N8 -60.7(6) 350 Atom Atom Atom Atom Angle/° O7 C19 C20 C21 55.6(6) O8 C21 C22 C23 53.2(6) O8 C21 C22 C24 -71.4(6) O9 C25 C26 O10 13.9(6) O9 C25 C26 C27 131.5(5) O9 C25 C26 C32 -104.5(5) O10 C26 C27 O11 -164.2(4) O10 C26 C27 O12 78.8(5) O10 C26 C27 C28 -41.3(6) O11 C27 C28 C29 -66.4(5) O12 C27 C28 C29 53.4(5) N1 N2 C2 C1 70.5(5) N1 N2 C2 C3 -54.7(6) N1 N2 C6 O3 -173.8(5) N1 N2 C6 C7 7.4(7) N2 N1 C5 C4 -51.7(6) N2 C2 C3 C4 52.7(5) N2 C6 C7 N3 -164.6(4) N2 C6 C7 C8 75.0(6) N3 N4 C10 C9 53.7(6) N3 C7 C8 C9 -50.6(6) N3 C11 C12 N5 157.5(4) N3 C11 C12 C13 -78.8(6) N4 N3 C7 C6 -70.9(6) N4 N3 C7 C8 51.8(6) N4 N3 C11 O4 167.9(5) N4 N3 C11 C12 -15.1(7) N5 C14 C15 N7 -143.2(4) N5 C14 C15 C16 94.6(5) N6 N7 C15 C14 -71.0(5) N6 N7 C15 C16 50.5(6) N6 N7 C19 O7 176.2(4) N6 N7 C19 C20 -0.5(6) N7 N6 C18 C17 53.1(6) N7 C15 C16 C17 -48.8(6) N7 C19 C20 N8 116.1(5) N7 C19 C20 C21 -127.6(5) N8 C20 C21 O8 174.2(4) N8 C20 C21 C22 -62.1(5) N8 C25 C26 O10 -170.5(4) N8 C25 C26 C27 -52.9(6) N8 C25 C26 C32 71.2(6) C1 C2 C3 C4 -69.7(6) C2 N2 C6 O3 -0.2(7) C2 N2 C6 C7 -179.0(4) 351 Atom Atom Atom Atom Angle/° C2 C3 C4 C5 -54.1(6) C3 C4 C5 N1 54.7(6) C5 N1 N2 C2 53.6(6) C5 N1 N2 C6 -132.6(5) C6 N2 C2 C1 -103.2(5) C6 N2 C2 C3 131.6(5) C6 C7 C8 C9 69.9(5) C7 N3 N4 C10 -53.5(6) C7 N3 C11 O4 1.8(8) C7 N3 C11 C12 178.8(4) C7 C8 C9 C10 53.8(6) C8 C9 C10 N4 -55.4(6) C11 N3 N4 C10 140.3(5) C11 N3 C7 C6 95.4(5) C11 N3 C7 C8 -142.0(5) C12 N5 C14 O6 25.7(7) C12 N5 C14 C15 -152.0(4) C14 N5 C12 C11 -137.9(5) C14 N5 C12 C13 99.0(5) C14 C15 C16 C17 73.4(6) C15 N7 C19 O7 2.7(7) C15 N7 C19 C20 -174.1(4) C15 C16 C17 C18 52.0(6) C16 C17 C18 N6 -55.6(6) C18 N6 N7 C15 -51.7(5) C18 N6 N7 C19 134.7(4) C19 N7 C15 C14 102.6(5) C19 N7 C15 C16 -135.9(5) C19 C20 C21 O8 60.0(5) C19 C20 C21 C22 -176.3(4) C20 N8 C25 O9 -3.5(8) C20 N8 C25 C26 -178.9(4) C20 C21 C22 C23 -68.0(5) C20 C21 C22 C24 167.3(4) C25 N8 C20 C19 -133.7(5) C25 N8 C20 C21 108.5(5) C25 C26 C27 O11 79.8(5) C25 C26 C27 O12 -37.3(6) C25 C26 C27 C28 -157.4(4) C26 C27 C28 C29 173.4(5) C27 O11 C31 C30 20.4(5) C27 O11 C31 C35 147.4(4) C27 O12 C33 C30 20.0(6) C27 O12 C33 C34 145.8(4) C27 C28 C29 C30 13.1(6) 352 Atom Atom Atom Atom Angle/° C28 C29 C30 C31 51.6(5) C28 C29 C30 C33 -65.7(6) C29 C30 C31 O11 -73.0(5) C29 C30 C31 C35 166.8(5) C29 C30 C33 O12 48.6(6) C29 C30 C33 C34 -71.8(6) C31 O11 C27 O12 -71.1(5) C31 O11 C27 C26 173.3(4) C31 O11 C27 C28 47.4(5) C31 C30 C33 O12 -68.0(5) C31 C30 C33 C34 171.6(5) C32 C26 C27 O11 -42.2(5) C32 C26 C27 O12 -159.2(4) C32 C26 C27 C28 80.7(6) C33 O12 C27 O11 45.6(5) C33 O12 C27 C26 160.0(4) C33 O12 C27 C28 -74.9(5) C33 C30 C31 O11 44.9(6) C33 C30 C31 C35 -75.3(6) C36 O1 C1 O2 -3.1(7) C36 O1 C1 C2 178.0(4) C37 O5 N5 C12 -115.8(4) C37 O5 N5 C14 98.8(5) Table 7-6: Hydrogen Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for ra178. Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom x y z Ueq H8 -2581.84 5065.79 9909.07 39 H10 1390(110) 400(90) 6780(90) 110(30) H1 10990(60) 6450(50) 1950(50) 22(15) H4 6040(70) 7030(70) 320(60) 37(19) H6 -610(80) 5550(70) 4850(70) 70(20) H8A 1670(80) 3210(60) 7920(60) 51(19) H2 9921.86 4066.68 1239.54 26 H3A 12672.26 3479.62 644.63 31 H3B 12154.22 4707.86 -460.44 31 H4A 13429.95 4906.52 1271.35 34 H4B 14255.37 5275.82 -245.13 34 H5A 13154.52 7210.27 275.09 33 H5B 12459.11 7083.47 -688.44 33 H7 8445.52 8086.78 1312.01 24 H8B 8734.53 9580.86 -753.54 27 H8C 10027.29 8389.38 -891.83 27 353 Atom x y z Ueq H9A 8580.59 7533.54 -1565.38 32 H9B 8723.18 9068.63 -2451.14 32 H10A 6263.58 9652.05 -1221.74 31 H10B 6130.7 8534.88 -1681.39 31 H12 3587.63 7862.11 1977.5 25 H13A 2332.03 10000.54 1863.73 48 H13B 3989.65 10100.77 799.69 48 H13C 3728.68 10356.09 2080.18 48 H15 850.6 7560 5902.5 23 H16A -1485.27 8708.61 5754.89 27 H16B -287.14 9294.52 4347.67 27 H17A -2618.25 8814.07 4295.45 30 H17B -1124.78 8054.79 3584.93 30 H18A -3135.96 6920.17 6136.28 32 H18B -2978.55 6558.47 4938.55 32 H20 -1254.65 3823.87 7510.16 21 H21 -630.61 3811.97 9690.05 26 H22 -1068.1 1655.25 9935 31 H23A -4110.19 2761.74 9963.2 55 H23B -3563.11 1215.45 10332.08 55 H23C -2892.29 2215.24 8891.55 55 H24A -1533.27 2132.7 11774.2 66 H24B -2751.98 1167.7 12067.98 66 H24C -3249.98 2712.19 11790.95 66 H28A 5089.93 599.53 5558.98 32 H28B 5674.67 421.78 6711.91 32 H29A 7471.48 1768.93 5228.89 37 H29B 6622.39 2209.54 4174.09 37 H30 6643.26 4053.57 4792.7 34 H31 6270.46 2478 7030.68 31 H32A 1576.26 -278.7 9276.46 43 H32B 2417.02 863.37 9193.44 43 H32C 3406.74 -339.09 8762.49 43 H33 3857.76 4660.89 5267.88 32 H34A 5118.52 3791.32 3145.07 61 H34B 5241.22 5233.37 3074.13 61 H34C 3608.41 4783.36 3371.43 61 H35A 4377.29 4902.21 6805.89 53 H35B 6079.78 4644.44 6877.85 53 H35C 4714.51 3925.77 8107.05 53 H36A 11687.6 986.34 4406.35 54 H36B 10240.48 1920.75 4912.43 54 H36C 11936.89 2331.63 4463.59 54 H37A 3077.83 7296.14 6038.46 44 H37B 3555.2 8686.98 5815.47 44 354 Atom x y z Ueq H37C 4673.48 7812.08 4997.98 44 Table 7-7: Hydrogen Bond information for ra178. D H A d(D-H)/Å d(H-A)/Å d(D-A)/Å D-H-A/deg O8 H8 O31 0.84 1.96 2.786(5) 168.4 O10 H10 O9 0.95(10) 1.85(9) 2.566(5) 130(8) N4 H4 O3 0.90(7) 2.24(6) 2.897(6) 129(5) N8 H8A O11 0.99(7) 2.17(6) 2.865(5) 126(5) C37 H37C O4 0.98 2.22 2.991(7) 134.3 –––– 1-1+x,+y,1+z Citations O.V. Dolomanov and L.J. Bourhis and R.J. Gildea and J.A.K. Howard and H. Puschmann, Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., (2009), 42, 339-341. Sheldrick, G.M., A short history of ShelX, Acta Cryst., (2008), A64, 339-341. Software for the Integration of CCD Detector System Bruker Analytical X-ray Systems, Bruker axs, Madison, WI (after 2013 355 Appendix F Experimental Data for MIC of Incarnatapeptins 356 357 Appendix G Other N-N Bond Natural Product Functional Groups 358 Examples of Nitrogen-Nitrogen Functional Groups found in Natural Products. Part 1 of 2 Reported 15N NMR chemical shifts are described as well as the availability of genomic information Citations N-N moiety Structure of N-N moiety Genome Mining Natural products containing this moiety+++15N NMR in various solvents referenced to NH3(l) (CH3NO2)Experiment Known ClustersN-nitrosoNot reported N/A Yes FraginAlanosineDopastinStreptozocin++ (in DMSO-d6)δ NHCONCH3NO 162.9(-217.6)15NHMBC Yes Streptozocinδ NHCONCH3NO 267.1(-113.4)δ NHCONCH3NO 3.10N -hydroxytriazene Not reported Yes TriascinsYesBraznitidumine++ (in DMSO-d6)δ NNH not reported15NHSQC Munroniamide δ NNH 55 (-325.5) No Ostrerine Aδ NNH 2.00 BraznitiduminePiperazic Acid Linear HydrazinesPiperazic Acid See Chapter 3 for Piz nitrogen NMR Nitrogen NMRNitrosohydroxylaminesNitrosoureasCyclic Hydrazine +++ 306,307 ++308,309 359 Examples of Nitrogen-Nitrogen Functional Groups found in Natural Products. Part 2 of 2 Reported 15N NMR chemical shifts are described as well as the availability of genomic information Citations N-N moiety Structure of N-N moiety Genome Mining Natural products containing this moiety+++15N NMR in various solvents referenced to NH3(l) (CH3NO2) Experiment Known ClustersN-nitrosoElaiomycin B++ (in DMSO-d6)δ CONNHCO 144.1 (-236.4)δ CONNHCO 140.9 (-239.6)15NHSQC Fosfazinomycinsδ CONNHCO 10.6515NHMBC Yes HydrazidomycinsGeralcin A++ (in DMF-d7) Agaritineδ CONNHCO 133.5 ( -247) Anthglutinδ CONNHCO 137.0 (-243.5) Xanthodermineδ CONNHCO 10.44 PygmeineGeralcin B++ in (CD2Cl2) Spinamycinδ CONNHCO 147.2 (-233.3) Elaiomycinδ CONNHCO 142.3 (-238.2) Geralcinδ CONNHCO 8.01peptide s56-p1++ (in H2O+D2O)R=NNH2 361.4 (-19)15NHMBC Yes KatorazoneR=NNH2 121 (-259.5) YoropyrazoneR=NNH2 not reportedHydrazonesHydrazides Nitrogen NMR ++310–312
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Andersen, Raymond J.
Ryan, Katherine S.
Chemical and genetic investigations of marine and terrestrial bacteria towards bioactive natural product discovery
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