Chemical and Biochemical Interrogation of Molecular Specificity in Modular Polyketide Synthases by Jonathan D. Mortison A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2010 Doctoral Committee: Professor David H. Sherman, Chair Professor John Montgomery Associate Professor Anna K. Mapp Assistant Professor Jason E. Gestwicki Acknowledgements I would like to thank my advisor, Dr. David H. Sherman, for the opportunity to join his research group at the University of Michigan. It has been a tremendously rewarding experience to work with him and explore many exciting projects at the interface of chemistry and biology. During my graduate career, his enthusiasm for science and perpetual optimism created an exceptionally positive atmosphere for learning. I am also grateful to have accompanied David on dive expeditions to gather samples for our natural products collection. These trips to Papua New Guinea (2007) and Costa Rica (2009) were wonderful opportunities both culturally and scientifically, allowing me to gain a unique view of these rich and biodiverse marine environments. I express thanks to my committee members, Dr. John Montgomery, Dr. Anna Mapp and Dr. Jason Gestwicki for serving on my thesis committee and providing helpful conversations during the course of my graduate work. I would also like to acknowledge co-workers and collaborators, especially Dr. Jeff Kittendorf and Dr. Sathish Rajamani for their work and guidance during the molecular cloning and biochemistry portion of my doctoral studies. Additional thanks go to graduate student Chris Rath for useful discussions on mass spectrometry and ongoing collaborations with FT-ICR MS studies. Many thanks also go to the other members of the Sherman group who have helped me in various ways throughout my time in the laboratory. I would like to thank the University of Michigan Regents, the Rackham Graduate School, and Eli Lilly & Co. for financial support during my graduate work in the form of ii pre-doctoral fellowships. Thanks also go to the Department of Chemistry and the support staff who have helped in many phases of my graduate career. Special thanks goes to my family and friends who have provided exceptional support to me during my time in graduate school. I am exceptionally grateful to have all of you in my life. Nick and Amy, you have been with me since the start of graduate school and we have shared many great experiences together. You are both truly special friends. Caleb and James, you are both great friends and have provided plenty of laughs and fun times over the years. Finally, I would like to thank my Mom, my brothers Eugene and Ben, my cousins Liz and Chris, and my aunt and uncle Stephanie and Bill. You have all been such important influences in my life, and I cannot thank you enough for all you have given me. iii Table of Contents Acknowledgements ......................................................................................................... ii List of Figures ................................................................................................................ vii List of Schemes .............................................................................................................. ix Abstract .......................................................................................................................... xi Chapter 1 Polyketide Natural Products and Type I Modular Polyketide Synthases..........................1 1.1 Introduction .................................................................................................................1 1.2 The Erythromycin and Pikromycin/Methymycin Polyketide Synthases ....................4 1.3 Previous Work in the DEBS and Pik PKS Systems....................................................6 1.4 Experimental Plan .......................................................................................................9 1.5 References .................................................................................................................11 Chapter 2 Preliminary Efforts Toward the Synthesis of the DEBS Pentaketide Chain-Elongation Intermediate.....................................................................................................................13 2.1 Iterative Organocatalytic Aldol Strategy...................................................................13 2.2 Chelation-Controlled Aldol Strategy.........................................................................15 2.3 References .................................................................................................................20 Chapter 3 Synthesis of the DEBS Pentaketide Chain-Elongation Intermediate..............................22 3.1 Synthesis of the Left Fragment .................................................................................22 3.2 Synthesis of the Right Fragment ...............................................................................23 3.3 Chemoenzymatic Thioesterification Strategies.........................................................25 3.4 C9 Protecting Group Optimization............................................................................26 3.5 Final Aldol Coupling and Deprotection ....................................................................27 iv 3.6 Discussion .................................................................................................................28 3.7 Experimental .............................................................................................................28 3.8 References .................................................................................................................38 Chapter 4 Biochemical Interrogation of Molecular Specificity in the Erythromycin and Pikromycin PKSs ................................................................................................................................40 4.1 Biochemical Analysis of DEBS Modules 5 and 6 ....................................................40 4.2 Cross Reactivity Between the DEBS and Pik Systems.............................................44 4.3 Chemoenzymatic Synthesis of 6-DEB ......................................................................49 4.4 Discussion .................................................................................................................50 4.5 Experimental .............................................................................................................56 4.6 References .................................................................................................................62 Chapter 5 Toward a Convergent and Diversity-Oriented Synthesis of Complex Polyketide Chain- Elongation Intermediates and Their Analogues ..............................................................65 5.1 Toward the Synthesis of Unique Pik Hexaketide Analogues....................................65 5.2 Toward a Convergent Strategy for Synthesis of PKS Intermediates ........................69 5.3 Experimental .............................................................................................................72 5.4 References .................................................................................................................81 Chapter 6 Future Directions.............................................................................................................83 6.1 Kinetic Analysis of Late-Stage Polyketide Assembly in the Erythromycin PKS ....83 6.2 FT-ICR Mass Spectrometry Analysis of Loading, Extension and Processing of the Non-Native DEBS Pentaketide in PikAIII......................................................................86 6.3 Completion of Synthetic Platform for Natural and Non-Natural Polyketide Chain- Elongation Intermediates.................................................................................................87 6.4 Furthering Understanding of the Structural Requirements of Macrocyclization in the Pik TE..............................................................................................................................90 v 6.5 Frontiers and Opportunities in Chemoenzymatic Synthsis .......................................94 6.6 References ...............................................................................................................106 vi List of Figures 1.1 Some representative polyketide-based natural products .............................................1 1.2 PKS condensation mechanism ....................................................................................4 1.3 Illustration of the PKS from erythromycin biosynthesis (DEBS)...............................5 1.4 Illustration of the PKS from methymycin/pikromycin biosynthesis (Pik)..................6 4.1 Illustration of the engineering of DEBS monomodules ............................................40 4.2 Radio-TLC of reaction products for Ery5-TE...........................................................41 4.3 LC-MS chromatogram in selective ion mode (SIM) of Ery5-TE .............................42 4.4 Radio-TLC analysis of Ery5 and Ery6 ......................................................................44 4.5 Radio-TLC of non-cognate pairing of Pik/DEBS pentaketide SNACSs ..................45 4.6 SIM LC-MS chromatogram of Ery6 reactions..........................................................46 4.7 Radio-TLC of Ery6 incubated with Pik hexaketide SNAC ......................................47