AN ABSTRACT OF THE DISSERTATION OF Chris R. Melville for the degree of Doctor of Philosophy in Chemistry presented on April 4, 1995. Title: Secondary Metabolism in Steptomyces murayamaensis Redacted for Privacy Abstract approved: Professor Stein J. Gould A multidisciplinary approach to unraveling the biosyntheses of secondary metabolites from the kinamycin producer Streptomyces murayamaensis is presented. The antineoplastic and antimicrobial kinamycins, originally believed to contain a cyanamide moiety, were determined to be diazo compounds by X-ray crystallographic analysis of 125. This corrects the misinterpreted X-ray structures of 119 and 113. The structure revision encompasses 19 natural products including 105, 108, 109, 110, and the subsequently isolated kinamycin J, 118. Synthetic studies to understand the transformations between the putative biosynthetic intermediates 11 and 127 were undertaken. A series of benz[a]anthracenes related to 11 were investigated, culminating in the preparations of 24 and 29. During this work, unexpected reductive deoxygenations of the diols 49 and 53 were observed. A second series of compounds prepared to mimic an anticipated biological ring contraction reaction culminated in the preparation of 84 and of 90. An unexpected neighboring group assisted nitrile solvolysis affording either 79 or 80 (depending on the reaction conditions) was also observed. The structure of the benzo[b]fluorene 126 was determined crystallographically, and a series of structurally similar compounds related to the kinamycins were prepared. Of these, 129 was found to be produced by Streptomyces murayamaensis, and was incorporated into 105. Other potential biosynthetic intermediates including 121, 132, and 147a were also prepared. Several compounds in this structural family failed to elicit NMR spectra, which was found to result from the the presence of diamagnetic species such as 149. The synthetic work also revealed unanticipated addition and elimination processes that afforded compounds 162 and 163. Feeding experiments revealed the kinamycin co-metabolite 18 was generated by a complex biosynthetic sequence. A structurally related metabolite, 194, was also discovered. Additional structure work provided the structures of 174, 191, 175, and of 213. Feeding experiments revealed that 213 was generated by an unexpected biosythesis involving a Krebs Cycle intermediate. Compound 214 was found to co-occur with - and 216 was incorporated into - 213. The relevance of this to the biosynthesis of 220 is also discussed. ©Copyright by Chris R. Melville April 4, 1995 All Rights Reserved Secondary Metabolism in Streptomyces murayamaensis by Chris R. Melville A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed April 4, 1995 Commencement June 1995 Doctor of Philosophy dissertation of Chris R. Melville presented on April 4. 1995 APPROVED: Redacted for Privacy Professor of Chemistry ,t c arge majord Redacted for Privacy Chairman of the Department of hemistry Redacted for Privacy Dea Graduate hoot I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Redacted for Privacy Chris R. Melville, Author Acknowledgments I am grateful to my major professor Dr. Steven Gould for his support, and the latitude I enjoyed to pursue a variety of projects. I am particularly grateful to have had the chance to include primary precursor feeding experiments, and X-ray crystallography in my graduate training. I am also grateful to Dr. J. D. White for including me in his group meeting, which was an invaluable part of my synthetic training. Dr. Douglas Keszler is thanked for introducing me to the world of X-ray crystallography, and specifically for serving as a resource for each of the crystal structures. In my initial synthetic work Dr. Makarand Gore was a valuable resource, and Drs. Dwight Weller and Kevin Gable were helpful in addressing many general questions. For useful discussions and help with culturing the bacteria I am grateful to Dr. Martha Cone. For useful discussions, and contributive perspectives, I would like to especially thank Dr. Annapoorna Akella, Brian Nowak-Thompson, Marc Kirchmeier, and Dr. Hyunik Shin. Thanks also to my additional committee members - Drs. Constantine, Gleicher, and Mathews - for their roles in meeting the requirements of this degree. Table of Contents Pale 1. The Kinamycin Producer Steptomyces murayamaensis: Early Structure and Biosynthetic Investigations 1 Isolation, Structure, and Bioactivity of the Kinamycins 2 Primary Precursor Feeding Experiments 4 Isolation of Minor Metabolites 6 Identification of an Acyl Transferase Activity, and Identification of Kinamycins E and F 10 References 12 2. Benz[a]anthracenes 15 Syntheses of X-14881E and Tetrangulol 18 A Benz[a]anthracene Model System 22 Protecting Group and Redox Chemistry of Tetrangulol 25 Protection and Dehydrogenation of Ochromycinone 31 A Re-evaluation of the Synthetic Targets 37 Experimental 42 Table of Contents Continued References 67 3. An Attempted Biomimetic Ring Contraction 72 Preparation of Cyanoaldehyde 54 76 An Unexpected Olefination Product 88 Acid-Aldehyde Intermediates 91 Knoevenagel Condensations Affording the Cinnamate 94 Esterification with 2-(Trimethylsilyl)ethanol 95 Cyanophthalide Annelations 96 Reactions of the Ester Dione 87 101 Experimental 105 References 124 4. Reactivity and Structures of the Kinamycins 127 C3 Regiochemistry 128 Absolute Configuration 130 Cyanamide Substituent 131 Table of Contents Continued C1 Oxidations 134 Methylation 137 Base Catalyzed Acylation 138 Acid Catalyzed Acylation 140 X-ray Crystallography 141 Kinamycin Structure Revision: Ramifications and Perspective 149 Experimental 155 References 164 5. Benz[b]fluorenones and Fluorenes 171 Kinafluorenone Triacetate 172 Kinobscurinone, Synthesis and Characterization 177 Kinobscurinone Feeding and Detection 193 Possible Nitrogen Containing Precursors to Prekinamycin 196 Determination of the Primary Nitrogen Source 205 Other Benz[b]fluorene Chemistry 208 Table of Contents Continued 9-Fluorene Derivatives 214 Experimental 216 References 230 6. Additional Benz[a]anthraquinone Derived Natural Product Structures 235 Murayaanthraquinone 236 Antibiotic PD116744 242 Murayaanthraquinone B 247 Kinamycin J 253 Experimental 258 References 265 7. Murayaquinone Pathway: Structural and Biosynthetic Studies 269 Murayalactone Isolation 270 Rationale for Biosynthetic Feeding Experiments 280 Sodium [1,2-13C2]Acetate Feeding 282 Table of Contents Continued Labeled Butyrate Feedings 285 Sodium [1.13c,1802jAcetate Feeding 286 Interpretation of the Observed Labeling 291 Experimental 294 References 305 8. Structure and Biosynthesis of Sodium tris(4-hydroxy-3-nitrosobenzamide) Ferrate (1-) 311 Isolation and Structure of 213 312 Acetate Feeding Experiments 318 Possible Post Aromatization Precursors 326 Biosynthesis 328 Experimental 335 References 343 Bibliography 346 List of Figures aagg Figure 1.1. D ring substituents of 2-8 3 Figure 4.1. Possible Kinamycin F Acetonides 129 Figure 4.2. Spectral Data for Model Cyanamides 132 Figure 4.3. D ring substituents of 2-8, 19, 20, and 102-110 144 Figure 4.4. ORTEP Representation of 1-0-(2-(S)-Methylbutyryl)k inamycin D 148 Figure 4.5. D ring substituents of 113, 114, and 112, 115-118 152 Figure 5.1. ORTEP Representation of Kinafluorenone Triacetate, 137 174 Figure 6.1. ORTEP Representation of Murayaanthraquinone, 174 238 Figure 7.1. INADEQUATE Spectrum of [1,2-13C2]Acetate Derived Murayaquinone 284 Figure 7.2. 180 Isotope Shifts Observed by 13C NMR 289 Figure 7.3. Inadequate Spectrum of [1-13C, 1802]acetate derived 18 290 Figure 7.4. Observed 13C and 180 Labeling Pattern 291 List of Figures Continued Figure 7.5. Ramifications of Observed Labelings on the Biosynthesis of 18 292 Figure 7.6. Possible Mechanisms to Account for the Observed Labelings of 18 293 Figure 7.7. A Possible Biosynthesis of 18 295 Figure 8.1. INADSYM Spectrum of 213a 320 Figure 8.2. 13C NMR Spectrum of 213a 321 Figure 8.3. INAD2D Spectrum of 213a 322 Figure 8.4. COSYX Spectrum of 213a 323 Figure 8.5. Acetate Labeling of Selected Tricarboxylic Acid Cycle Intermediates 324 Figure 8.6. Partial 13C NMR spectrum of 213c showing resonances for C-5 (8 120.8), C-1 (8 119.6), and C-3 (8 158.7) 327 List of Tables Page Table 3.1. Long Range HETCOSY of 67 77 Table 3.2. Benzylic Bromination Selectivity 85 Table 3.3. Modified HPLC Gradient Elution Protocol 97 Table 3.4. Components of Cyanophthalide Addition Reactions 98 Table 3.5. Deprotonation of Cyanophthalide 100 Table 3.6. NMR Assignments for the Aminopyridine 90 104 Table 4.1. Modeling of Possible Acetonides 130 Table 4.2. Initial Structure Solutions of 1-(2-0-(S)­ Methylbutyryl)kinamycin D 143 Table 4.3. Crystal and Collection Data for 1-0-(2-(S)­ (Methylbutyryl)kinamycin D 146 Table 4.4. 1-0-(2-(S)-Methylbutyryl)kinamycin D Structure Solutionsc 147 Table 4.5. Behavior of Shift/Error (max.) 147 Table 4.6. Number of Poorly Fitting Reflections 147 List of Tables Continued Table 4.7. B Factors 149 Table 4.8. Fourier Peak Heights 149 Table 4.9. Diazonium Bond Lengths and Angle 149 Table 5.1. Crystal and Collection Data for 137 175 Table 5.2. Samples Exhibiting ESR Spectra, and Their Experimental g Values 187 Table 6.1. Crystal and Collection Data for 174, C26H16N207 239 Table 6.2. NMR Spectral Assignments for