Combined Biosynthetic and Synthetic Production of Valuable Molecules: A Hybrid Approach to Vitamin E and Novel Ambroxan Derivatives Bertrand T. Adanve Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2015 © 2015 Bertrand T. Adanve All Rights Reserved ABSTRACT Combined Biosynthetic and Synthetic Production of Valuable Molecules: A Hybrid Approach to Vitamin E and Novel Ambroxan Derivatives Bertrand T. Adanve Chapter 1. Introduction Synthetic chemistry has played a pivotal role in the evolution of modern life. More recently, the emerging field of synthetic biology holds the promise to bring about a paradigm shift with designer microbes to renewably synthesize complex molecules in a fraction of the time and cost. Still, given nature’s virtuosity at stitching a staggering palette of carbon frameworks with ease and synthetic chemistry’s superior parsing powers to access a greater number of unnatural end products, a hybrid approach that leverages the respective strengths of the two fields could prove advantageous for the efficient production of valuable natural molecules and their analogs. To demonstrate this approach, from biosynthetic Z,E-farnesol, we produced a library of novel analogs of the commercially important amber fragrance Ambrox®, including the first synthetic patchouli scent. Likewise, we produced the valuable tocotrienols from yeast-produced geranylgeraniol in a single step, the first such process of its kind. The novel acid catalyst system that allowed for this unique regioselective cyclization holds promise as an asymmetric proton transfer tool and could open the door to facile asymmetric synthesis of vitamin E and other molecules. Chapter 2. Z,E-Farnesol Biosynthesis To produce the scarce and costly Z,E-farnesol biosynthetically, we over- expressed the four reported Z,E-FPPS (from acid-fast bacteria M. tuberculosis, T. fusca, C. glutamicum, and C. efficiens) into E. coli and the two with the best reported kinetics (those from M. tuberculosis and T. fusca) into yeast. Though our engineered yeast strains did not produce any detectable Z,E-farnesol, they surprisingly produced high titers of E,E-farnesol. In E. coli, the Z,E-FPPS from M. tuberculosis led to the best production titers of Z,E-farnesol (8.0 mg/L in flasks and 2.9 mg/L at bench scale), allowing us to produce and isolate 50 mg of the material. Chapter 3. Novel Ambroxan Analogs from Biosynthetic Farnesol In a first demonstration of the hybrid approach where the biosynthesized intermediate is not part of the target molecule’s biosynthetic pathway, microbe-produced Z,E-farnesol was homologated and subsequently cyclized with the halonium donors BDSB and IDSI to yield halo-benzohydrofurans, the 3 carbon halogenated versions of the commercially relevant 9-epi-Ambrox®. From this halogenated intermediate, the known compound was derived along with a library of novel analogs with improved organoleptic properties, among which the first synthetic patchouli scent. Chapter 4. Geranylgeraniol Biosynthesis We engineered yeast with a set of fusion enzyme constructs, including a first of its kind triple fusion enzyme construct, which led to geranylgeraniol (GGOH) production with no squalene side product despite no repression of squalene synthase (ERG9). With this triple fusion-engineered strain, we produced 27 mg of isolated GGOH in a very rudimentary, homemade fermenter. Chapter 5. Tocotrienols in Single Step from Biosynthetic Geranylgeraniol In a second demonstration of the hybrid biosynthetic synthetic approach, we coupled the yeast-derived geranylgeraniol with trimethylhydroquinone to produce the potent vitamin E α-tocotrienol in a single step C–C coupling with concomitant regioselective cycloetherification of the most proximal vinyl, which constitutes the shortest synthesis of the tocotrienol. Likewise, we synthesized the three other members of the tocotrienol family. Chapter 6. Novel Chiral Acid for asymmetric proton transfer: Adanve acids applied to Chiral vitamin E synthesis We developed a new class of chiral Bronsted acids (Adanve acids), materials that are highly tunable, easy to synthesize, and easy to handle as well as recover. In a preliminary exploration of the ability of these chiral acids to induce asymmetric proton transfer, we investigated their usage to impart asymmetric formation of chromans, namely the important vitamin E family. Our early findings suggest that a host of factors play a role in the ability of this novel acid catalyst system to induce asymmetric chroman formation. CONTENTS Chapter 1. Introduction 1.1 The Eminence of Synthetic Chemistry and Its Limitations ................................2 1.2 The Emergence of Synthetic Biology and Its Limitations ..................................4 1.3 The Hybrid Biosynthetic and Synthetic Approach..............................................7 1.4 The Hybrid Approach Applied to 9-epi-Ambrox® and tocotrienols ................11 1.5 Conclusion.........................................................................................................13 1.6 References .........................................................................................................15 Chapter 2. Z,E-Farnesol Biosynthesis 2.1 Introduction .......................................................................................................18 2.2 Z,E-Farnesylpyrophosphate synthases (Z,E-FPPS)...........................................22 2.3 Z,E-farnesol in E. coli........................................................................................22 2.4 Z,E-farnesol in yeast..........................................................................................25 2.5 High-ATCC200589/tHMG1: A Yeast Background Strain For Terpenoid Production .........................................................................................................27 2.6 Bench Scale Production Optimizations Without Appropriate Fermenter .........29 2.7 Titer Yield Calculation......................................................................................29 2.8 Conclusion.........................................................................................................30 2.9 References .........................................................................................................31 2.10 Experimental Section ........................................................................................32 Chapter 3. Novel 9-epi-Ambrox® Analogs from Biosynthetic Z,E-farnesol 3.1 Introduction to Ambrox®..................................................................................59 3.2 Farnesol Homologation .....................................................................................61 i 3.3 Cyclization to Halo-hydrobenzofurans .............................................................63 3.4 Derivatization ....................................................................................................64 3.5 Discovery of Novel Structure and Ambergris-type Scent.................................64 3.6 Inspiration for a Synthetic Patchouli Scent .......................................................65 3.7 Conclusion.........................................................................................................66 3.8 References .........................................................................................................68 3.9 Experimental Section ........................................................................................69 Chapter 4. Geranylgeraniol Biosynthesis 4.1 Introduction .....................................................................................................116 4.2 Double Enzyme Fusion Exploration ...............................................................118 4.3 Triple Enzyme Fusion Advent ........................................................................119 4.4 Fusion Constructs in Opposite Backgrounds ..................................................121 4.5 Enzyme Order in the Fusion Constructs..........................................................121 4.6 Bench Scale Production Optimizations Without Appropriate Fermenter .......122 4.7 Titer Yield Calculation....................................................................................122 4.8 Conclusion.......................................................................................................123 4.9 References .......................................................................................................124 4.10 Experimental Section ......................................................................................125 Chapter 5. Tocotrienols in a Single Step from Biosynthetic Geranylgeraniol 5.1 Introduction ....................................................................................................138 5.2 Designing the Catalyst System.......................................................................141 5.3 Choosing the Catalyst: Improving the In(III)-Promoted Synthesis of Tocopherol: from Phytyl Acetate to Phytyl Trifluoroacetate.........................142 ii 5.4 Synthesis of Polyprenyl-chromans, including α-Tocotrienol.........................143 5.5 Accessing the Rest of the Tocotrienol Family: Effect of Deactivating Protection Groups...........................................................................................146 5.6 Next Generation Approach: Type II Adanve Acids.......................................147 5.7 Conclusion......................................................................................................150