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INFORMATION TO USERS While the most advanced technology has heen used to photograph and reproduce this manuscript, the qualify of the reproduction is heavily dependent upon the qualify of the material submitted. For example: • Manuscript pages may have indistinct print. In such cases, the best available copy has been filmed. • Manuscripts may not always be complete. In such cases, a note will indicate that it is not possible to obtain missing pages. • Copyrighted material may have been removed from the manuscript. In such cases, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, and charts) are photographed by sectioning the origined, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or as a 17”x 23” black and white photographic print. Most photographs reproduce acceptably on positive microfilm or microfiche but lack the clarify on xerographic copies made from the microfilm. For an additional charge, 35mm slides of 6”x 9” black and white photographic prints are available for any photographs or illustrations that cannot he reproduced satisfactorily by xerography. 8703560 Houck, David Renwick STUDIES ON THE BIOSYNTHESIS OF THE MODIFIED-PEPTIDE ANTIBIOTIC, NOSIHEPTIDE The Ohio State University Ph.D. 1986 University Microfilms I nternetionsi!300 N. Zeeb R oad, Ann Arbor, Ml 48106 STUDIES ON THE BIOSYNTHESIS OF THE MODIFIED-PEPTIDE ANTIBIOTIC, NOSIHEPTIDE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By David Renwick Houck, B.S., M.S. ***** The Ohio State University 1986 Dissertation Committee: Approved by Heinz G. Floss f ^ Robert M. Mayer David J. Hart Department of Chemistry To my family ii ACKNOWLEDGEMENTS I am very fortunate to have studied with an exceptional scientist such as Dr. Heinz G. Floss. As my academic advisor for five years. Dr. Floss has provided an excellent atmosphere for both my scientific training and my research. Merck & Co. is acknowledged for granting me a paid, educa tion leave of absence. I would like to thank Paul Keller, John Beale, and Jonathan Lee for their help with acquisition of the NMR data. Dr. Edith W. Miles was very generous with the gift of tryptophan synthase. Diego Belinzoni and Rosangela Casati taught me techniques of organic synthesis. Kay Kampsen was a very efficient typist and ensured that all the deadlines were met. My parents are responsible for starting my academic studies, and Dr. Lawrence Wittle, of Alma College, first stimulated my interest in biochemistry. My wife, Allyson, has been incredibly devoted and patient during my preoccupation with science. Allyson's literature searches were invaluable for the completion of this work, as was her moral support. Ill VITA April 10, 1956 ................. Born - Detroit, Michigan May, 1978 ....................... B.S., Alma College, Alma, Michigan September, 1981 ................ M.S., Purdue University West Lafayette, Indiana October, 1981 to Present ...... Research Biochemist Merck and Co. Inc., Rahway, New Jersey December, 1984 to Present ...... Recipient of a Merck Grant for Ph.D. studies at the Department of Chemistry, The Ohio State University PUBLICATIONS 1. Houck, D., Chen, L.-C., Keller, P., Beale, J., Floss, H.G. (1986) "Biosynthesis of Nosiheptide" J . Amer. Chem. Soc., in press. 2. Houck, D.R., Kobayashi, K., Williamson, J.M. and Floss, H.G. (1986) "Stereochemistry of Méthylation in Thienamycin Biosynthesis" J. Amer. Chem. Soc. 108, 5365. 3. Asano, Y., Woodard, R.W., Houck, D.R., Floss, H.G. (1984) Arch. Biochem. Biophvs. 231, 253. 4. Miles, E.W., Houck, D.R. and Floss, H.G. (1982) J. Biol. Chem. 257, 14203. 5. Houck, D.R. and Floss, H.G. (1981) J. Natural Products, 44, 759. FIELDS OF STUDY Biochemistry, enzymology and natural products IV TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ..................................... Ü i VITA .................................................. iv LIST OF TABLES ....................................... V Ü LIST OF FIGURES ...................................... v i ü LIST OF ABBREVIATIONS ................................ X Ü INTRODUCTION ......................................... 1 RESULTS ............................................... 7 Fermentation ..................................... 7 High Performance Liquid Chromatography ......... 10 Nosiheptide Isolation ........................... 14 Chloramphenicol Inhibition of Nosiheptide Biosynthesis .............. 16 Incorporation of ^“C-Labeled Amino Acids into Nosiheptide .................................... 19 Incorporation of ^®C-Enriched Amino Acids into Nosiheptide .................................... 22 Incorporation of [U-^®Ca]Glycerol into Nosi heptide ........................................ 44 DISCUSSION ............................................ 62 EXPERIMENTAL SECTION ................................. 79 Materials ........................................ 79 Organism and Fermentation....................... 80 Isolation of Nosiheptide ........................ 81 Nosiheptide Assay and HPLC ...................... 83 Feeding Experiments ............................. 84 Preparation of 4-Methyl-[3 ' “C]tryptophan .... 87 Preparation of [2-^®C]Indole ............... 88 Preparation of L-[2,l'-i3C2]Tryptophan ........ 90 ^ ^C-NMR Spectroscopy............................ 91 V Table of Contents (continued) Page APPENDIX .............................................. 93 LIST OF REFERENCES .................................... 99 VI LIST OF TABLES TABLE Page 1. Effect of Medium Components on Nosi heptide Biosynthesis .................. 11 2. Incorporation of ^“C-labeled amino acids into nosiheptide ....... 20 3. couplings observed in the reverse INEPT spectrum of nosiheptide biosyn thesized from L-[3-i3C]serine......... 31 4. ^^C-NMR assignments and enrichments of nosiheptide derived from ^^C-labeled precursors ............................. 43 5. The carbon connectivities observed in the 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [ U - ^ ]glycerol ... 55 6. ^^C-NMR assignments of nosiheptide (40-70 mg/0.4 ml, DMSO-dg, 320°K)...... 61 VI 1 LIST OF FIGURES FIGURE Page 1. Nosiheptide and the sequence of its residues ................................. 2 2. Thiostrepton and the sequence of its residues (top) .......................... 3 3. The HPLC profile of a crude extract (tetrahydrofuran) of streptomyces actuosus grown in synthetic medium 13 4. The effect of chloramphenicol on nosi heptide production in synthetic medium. Chloramphenicol was added (10 pg/ml) at 30 h(x) or 54 h (A) or not at all (oscontrol) ............................. 18 5. The HPLC profiles of crude nosiheptide- precipitates from fermentations con taining [^“C]tryptophan; labeled at the CHg group (top) or ring-C7a (bottom traces). The fluorescence detector was in series with radioactive flow detector. 21 6. Natural abundance of the ^^C-NMR spectrum of nosiheptide .......................... 24 7. An expansion of the ^^C-NMR spectrum of nosiheptide biosynthesized from DL-[3-^^C]cysteine ...................... 25 8. The sites of ^^C-enrichemnt (•) in nosi heptide biosynthesized from DL-[3-^^C]cysteine ...................... 26 9. An expansion of the ^ ^C-NMR spectrum of nosiheptide biosynthesized from L-[3-i3C]serine. Stars indicate enriched signals ........................ 28 Vlll List of Figures (continued) Figure Page 10. The ^H-NMR spectra of nosiheptide biosyn thesized from L-( 3 - serine. The reverse INEPT sequence was run with t =1.4 msec (top) or x=1.6 msec (middle trace). The ^^C-NMR satellites are bracketed immediately above the main proton signal (bottom trace) .......................... 30 11. The coupling observed between pyridine C3 and C4 in the spectrum of nosiheptide biosynthesized from L - [3- i3g ]serine ......................... 32 12. The transfer of carbon-13 (A) from L-[3-i3c]serine to nosiheptide ......... 33 13. An expansion of the ^^C-NMR spectrum of nosiheptide biosynthesized from L-[CHa-^^C]methionine. The intense signal at 65.9 ppm corresponds to indole C4'................................ 34 14. The ^H-NMR (A) and reverse INEPT (B) spectra of nosiheptide biosynthesized from L-[CHa-^^cImethionine. With a delay of t =1.6 msec, the pulse sequence for B uncovers the ^^C-satellites of the protons at indole C4'.................... 35 15. The downfield region of the ^^C-NMR spec trum of nosiheptide biosynthesized from DL-[1-^^C]serine. The stars denote enriched signals ........................ 37 16. The transfer of (•) from DL-[1-^®C]- serine to nosiheptide .................. 38 17. The synthesis of L-[2,1'-^^Cj]tryptophan .. 41 18. The ^^C-NMR spectrum of nosiheptide biosynthesized from L-[2,l'-^^Cjl- tryptophan .............................. 42 IX List of Figures (continued) Figure Page 19. A portion of the ^®C-NMR spectrum of nosiheptide biosynthesized from [U-^’Cglglyeroi (top) and a natural abundance spectrum (bottom) ........... 47 19. Continued ................................ 48 20. The 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^^Cglglycerol. Coupled signals appear at the same double-quantum frequency (FI, abscissa) and are listed in Table 5 .............. 53 21. An expansion of the 2D-INADEQUATE spectrum (Fig. 20). Coupled thiazole-carbons are bracketed. Note the weak signals for pyr C4-C5 at 4000 Hz

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