Oxidation of Complex Molecules:
From Nature to the Flask
A thesis presented
by
Quentin Michaudel
to
The Scripps Research Institute Graduate Program
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Chemistry
for
The Scripps Research Institute
La Jolla, California
May 2015
© Copyright 2015 by Quentin Michaudel
All rights reserved.
ii
Thesis acceptance form goes here
iii
Dedicated to my parents for always supporting my dreams,
to Clément and Louise with the hope that they follow their own,
and to the rest of my family and friends for always being around.
In memory of the victims of the Charlie Hebdo shooting on 7 January 2015.
iv
If I have seen further it is by standing on the shoulders of giants.
— Isaac Newton.
v Acknowledgements
Phil: Thank you for giving me a chance, first as a visiting student, then as a graduate student. It was quite a ride, but if I had to start my PhD again, I would not change a thing. I guess, for a PhD more than anything, the journey matters at least as much as the destination. The almost 6 years I spent in the lab taught me how to be an independent researcher, how to write papers and deliver talks and how to collaborate with other scientists, particularly those from other fields or from industry. I have been a privileged witness of your success and I hope that I can carry forward some of your secret recipes to fulfill my dream of becoming a successful professor one day.
Prof. Ryan Shenvi: Thank you for all the advice you gave me along the way, whether it was simply on how to set up a reaction or in helping me choose a post-doctoral position. Thank you also for giving me the opportunity to TA one of your classes.
Prof. Jin-Quan Yu: Thank you for watching over my developments in graduate school and dispensing some always-insightful opinions on my organometallic chemistry.
Prof. Scott Snyder: Thank you for agreeing to fill in for Prof. Nicolaou; I am glad that I can keep a strong Corey influence in my committee. Even if I have known you for a shorter time than the rest of my committee, I always enjoyed talking with you about chemistry or anything else.
Prof. Brad Moore: I had an amazing time working with Robin, Aki and you, as well as our other collaborators at Michigan and the Salk Institute. This collaboration taught me so much, not only in terms of the science, but also about the practical aspects of working with people that have different expertise than mine. Thank you for agreeing to serve on my thesis committee; I am glad to have an external committee member with whom I already share a scientific bond.
Aki, Robin: I had a great time collaborating with you and the folks at Michigan and Salk. My biology/enzymology definitely improved a lot thanks to you.
Scott, Alicia, John, Brad: I got the chance to be part of many interesting collaborations with BMS and to work with all you. I finally met most of you at the symposium there, which was a great event. Working on real-life problems with your respective teams was extremely educational.
vi Taylor: With Yoshi and Phil of course, you are the only person that has been in the lab for all my PhD! Thank you for coping with my terrible handwriting on all the order sheets I filled and for always providing some help when I needed some.
Tom: Thank you for all the life and chemistry advice that you provided me when I started as a young visiting student and all throughout my PhD. In particular, thank you for taking the time in your busy professor schedule to help me choose a post-doc position. I hope I won’t disappoint you at Cornell!
Noah: Along with Tom, you certainly were a model I wanted to emulate when I started grad school. Even if we only briefly overlapped in the lab, this period of time influenced my views on chemistry and grad school a lot.
Will: You were a great hoodmate, as much for chemistry knowledge than for fun moments and I hope we can keep in touch in the future, especially since I am following your footsteps in term of academic path!
Hans: Even though I did not always understand your jokes or obscure references, working next to you in the office, as well as in the lab was a great experience. You’re probably one of the smartest people I have ever met!
Dave Sarlah: Thank you for all the chemistry tips and ideas, as well as all the chemicals you let me borrow from the KC lab. Your work ethic and passion always inspired me.
Brady: Thank you for teaching me how to use your glove box and all your chemistry advice. You’re a truly unique character that I won’t forget anytime soon.
Nick: It was great to share with you all the similar miseries we encountered while working on C–H oxidations of triterpenes; as well as going out in Hillcrest bars with you. I wish you the best for your post-doc.
Rodrigo: I would probably not have been able to work as hard during my PhD if you hadn’t been pushing yourself like crazy every night across the hall. I’m glad you got the job that you deserved.
Emily, Dane, KFoo: You were a great group of students to follow and work along in the lab. I hope we will stay in touch in the future and I know you will do great in your respective companies.
vii
Paul, Dave, Tim, Ian, Cindy, Jonathan: I interacted with all of you in different ways, but I have learned from all of you guys. I am proud to be following you on the Baran lab family tree. Cindy, thanks for having been a great host at BMS!
Ryan G: You were a truly nice baymate and officemate and I am wishing you the best for your career. I’m sure you’ll do great in whatever field you choose.
Nathan: I got the chance to know you better during our Jersey trip and as a roommate in Lake Arrowhead. Good luck for your post-doc with Prof. Corey, I’m sure it will be memorable!
Brandon: You were “un put*** d’enc*** de ta mère”, but I might not have survived my PhD without your coffee machine! I hope you will improve your French and learn words other than the cursing ones.
Matt: You are one of the nicest people I met in the US and I hope you will find the path to your dream job soon, because you really deserve it! Thanks for teaching me basketball tactics and football rules. My best to Sarah, Keene and you!
J-Lo: Thanks for sharing your music and Apple mania, as well as for being a considerate, and sometimes crazy, baymate. You’ve been very successful so far, I have no doubt it will continue.
Ruben: My knowledge of the US and Mexican culture definitely improved through all the interesting (to say the least) talks we had in the office. I will miss those at Cornell!
Ming: Your unconditional love for the Mannschaft and the Bayern Munich amused me as much as your chemistry knowledge amazed me. I know that your PhD experience might be rough on you sometimes, but hang in there, the end of the tunnel is near!
Art: Ruben and you are definitely forming an entertaining pair in the lab. My idea of Oxford’s finest has slightly changed after interacting with you for almost three years, but you’ll come down as Maoecrystal V’s champion!
Yiyang: You’re carrying the oxidase phase torch now and I’m waiting to read all the amazing science that you will come out with. I will think of you everytime Beyoncé is playing on the radio!
viii Marcus: I didn’t have the chance to really work with you, but I’m sure that your organometallics training will prove fruitful in the Baran lab.
Jacob, Sebastian, Roro, Hang, Leah: I didn’t get to know you guys as well as your predecessors, but I’m sure that the Baran and Shenvi labs are in good hands with your generation. At least, I hope so!!
Devon: Visiting Paris with you for two days remains one of the excellent memories of my grad school time. And the story of your one week of employment after your defense is in the top 10 of the funniest stories I have ever heard!
Greg: You’re an extremely nice and funny person and I hope we’ll stay in touch in the future. Good luck in the Romesberg lab!
Dan, Kanny: After all these years, we crossed the finish line around the same time! Congratulations on your new jobs!
Peter, Kelvin: You guys were great classmates and I hope we’ll stay in touch!
Shun Su: You were the most influential person in my chemistry learning phase and I would not have accomplished anything I’ve done during my PhD without your always patient and positive teaching during my internship. Your scientific curiosity and your problem solving skills are truly inspiring. Probably 95% of the way I do or think about chemistry comes from you, so I owe you a huge thank you and I hope we will keep in touch, because I still have a lot to learn!
Ke Chen: I don’t know how many times you jokingly swore at me, but you were really helpful and influential in my early days in the lab.
IY, Chad, Darryl: Thank you for all the chemistry tricks that you taught me! Especially IY, thanks for teaching me how to do a good prep plate, there aren’t many other skills that served me more in grad school! Prof. Chad, I’m looking forward to working near your lab at Cornell!
Rune, Lars, Erik and Mikkel: You guys are the best Danes I know! I hope I can visit you in the near future!
ix Kutti, Danny, Jochen, Isabelle: Despite the fact that Germany beat us during the last world cup, you guys were great coworkers and very knowledgeable! You helped me keep some of my European ways of doing chemistry!
Nakka, Kentaro: Thank you for the tour of Tokyo and all the great Japanese words and cultural facts that you taught me in the lab. Ken, I’m still indebted for the three weeks where you drove me to work almost every day, because my car was broken. I hope I can visit soon, I miss Japan and Japanese food!
Hiroki, Takeshi, Ippei, Yuta, Yuki, Shuhei, Kawa, Atsushi, Shota: You definitely set the bar very high in terms of hard work and it was a great challenge to try to match your work intensity.
Alex S. Your commitment to the Bulls is certainly impressive! Thanks for organizing the baskball pick-up games and some fun parties.
Tony, Emily: You guys are very nice people to be around! Good luck for the end of your PhDs.
Sarah: You are an amazing person to work around because of your always-positive attitude. You are very inspirational for me and I wish you the best at Temple.
J-Green: You’re the perfect impersonation of the BFG! I’m glad you’re having a blast at Takeda.
Bart R: You were here only for a short time, but I already miss the interesting talks we had during our lunches at the Sanford Cafeteria. I will visit you in Bruxelles!
Abraham: Your knowledge of organometallics was unmatched in the lab and I benefited from it tremendously!
Victor, Marta, Jose, Albano: It seems like our lab has always been a popular destination for Spanish students. I had a great time working and having lunch or dinner with all of you and I might see you back in the old world.
Cornelius, Julian, Felix: The same applies to you guys!
César: You were a fun coworker, César Césarez, and I hope we can catch up during a conference sometimes!
x Cecil: You were a very pleasant undergrad and a great soccer player! Thank you for all the tips about Cornell.
Jinghan, Qianghui: Thank you for initiating me to Chinese culture. Good luck being Professors back in China (Jinghan, I’m sure you’ll get a position as soon as you apply), I’m looking forward to reading your papers.
Yeh, Eran, Dr Oberg, Evan, Justin, Aaron, Tian, Myles, Joel: I have known you for a more or less short time, but I wish you all good luck for your post-doc and your next (hopefully real) job!
Fabien, Lucile: Thanks for helping me as soon as I landed in California and when I had to decide where to do my PhD.
Tanja Gulder, Elena, Marion 1, Marion 2, Gloria: I have been very lucky to have all these adoptive moms or aunts to make my life easier so far from home!
Sheena, Dan: Wether it was at a SOF event, for Sheena’s birthday or playing soccer with Dan, I always had a great time with you guys!
Bastien: You didn’t stay in San Diego as long as you thought you would, but we had a lot of good moments when you were here.
Sophie: You got to be my adoptive mom for the longest time since you stayed in San Diego. Your homemade cakes and pastries, birthday parties and coffee breaks and more generally your happy mood was really a sunshine in my grad school experience.
Cyril: I will miss your often sarcastic, but always-insightful opinions on science and life in general when I am freezing my butt on the east coast! Your cooking too!
Marietta: You have been a great friend since we met and even after your move to Boston and then Berkeley. Thank you so much for all the advices pre-, during and post-grad school and for coming to my defense. I am looking forward to attending your vet school graduation! And I’m still planning on visiting you guys up in San Fran!
Damien, Guillaume: You both were amazing post-docs to work with and friends in and outside the lab. While being very different, you both brought hard- work and intellectual contribution to finish the projects we were working on and you taught me a lot on collaborations.
xi Florian: While we don’t agree on everything (far from that), your friendship has always been a strength for me. Spending a day with you in Portland was as fun as unexpected. I hope we will see each other soon.
Pascal, Julien, Marine, Pauline G., François, Paul A., Arthur D., Lucas, Camille, Nor, Olivier P., Alex M., Baptiste, Dimy, Sophie, Dan, Marion J., Aloïs, Samantha, JR, Alvaro, Nathalie, Ferdie, Florian, Michel, Bénédicte, Caroline, Marie-Laure, Marie-Blandine (Andy), Martin, Pu: You guys all spent some time in San Diego at some point during my PhD and that really made me so happy. Even if it was only for a few days, I felt not as far from France during those periods of time. My five-year PhD would have been even harder without the support (via internet or in person) of my friends from France. Pilipon, Geekou, LP and others, you still have a few months to come visit, otherwise, you’ll have to face the cold of upstate New York!!
“Worldwide” (soccer team): Playing soccer every Sunday was one of my rare weekly moment of fun outside of the lab and I was always really looking forward to it. We better win the shirt for my final league!!
Pejman, Chris, Anne, Maryann, Michelle, Josh: Thank you roomies for all the good moments outside the lab!
Yi Ning: The Beckman building is much too quiet since you’re gone, but I’m glad that you finally got a great job at Merck. Your contagious smile and laugh are deeply missed here and I hope I can come down to New Jersey to visit your new home.
Eddie: You’re probably the nicest coworker one can dream of. Hardworker, efficient and always so keen and humble. I wish you the best with Annie and I hope I can be at your wedding this summer.
Justin: We started grad school together, we were almost roomates for two and a half years, then fully roomates for the rest of our PhD, we went out all around Hillcrest, we watched basketball and TV shows and we did so many more things. I would probably not have been able to go through grad school without all those fun moments and I would definitely not feel as “Americanized”;)
Jaclyn: For almost four years, you helped me keep my sanity in check despite the days and nights in the lab I spent in the lab. You’re an
xii amazingly caring person and I’m sure that you will make a great nurse practitioner. Thanks also to the rest of your family for being so nice and welcoming.
Jessy: You’re one the rare people I met that is at least as competitive as me when it comes to board games, which made our game nights even more fun! Thanks also for all our dinner outings, especially the raclette nights, and for the amazing trip to Japan for your wedding!
Justine: I have really known you for only 6 months or so, but I feel like it has been much more time than that! Your help and support in the last leg of my PhD were crucial to keep my stress level manageable. Your personality, at the same time bold, kind and sarcastic, never ceases to amaze me. I am looking forward to our trip across the US very much!!
Yoshi: I owe you at least half of my PhD and I will be forever indebted to you for that. You never counted your hours to answer any questions I had, to give me some suggestions for my chemistry or to correct my English writing. On top of that, you’re an amazing friend outside the lab with whom I share so many passions: soccer, manga, travels… Thank you so much for not only inviting me to your wedding in Japan, but also planning an unforgettable trip. I feel almost sorry to have won our last bet, but I’m sure a great job is waiting for you at the corner. I will miss our (sometimes never- ending) scientific, philosophical, or random-topic bickering. I hope we’ll stay in touch and that you’ll come visit on your way to Montréal.
Mamie, Papy, Véronique, Fred: Thank you so much for coming to my graduation, it meant a lot to me!
Maman, Papa, Clément, Louise: You always supported my choices and dreams despite the fact that they sent me about 10,000 km from you! Luckily, Skype and your several visits made the distance easier. Clément and Louise, I hope that my journey will inspire you to some extent to fulfill your own dreams and I will always be proud of you.
xiii Table of contents
Introduction 1
Chapter 1: Unraveling the intriguing mechanism of EncM, a ‘favorskiiase’ enzyme with a unique flavin-mediated dual oxidation 3
1.1 Introduction 4
1.2 Chemical synthesis of substrate analogs 6
1.3 Crystal structures of FAD-bound EncM and co-crystallization
with substrate analogs 8
1.4 Enzymatic transformation of substrate 1.4 with EncM 11
1.5 Postulated mechanism of the dual oxidation catalyzed by EncM 15
1.6 Conclusion and future outlook 18
1.7 Acknowledgements 20
1.8 Distribution of credit 20
1.9 Experimental procedures and data 21
1.9.1 General 21
1.9.2 Experimental procedures and data of synthetic intermediates 29
1.9.3 X-ray crystallographic data 87
1.10 References/bibiliographic notes 87
1.11 Appendix: copies of NMR spectra 90
Chapter 2: Intermolecular Ritter-Type C−H Amination
3 of Unactivated sp Carbons 117
2.1 C–H amination precedents in the literature 118
2.2 Goal and inspiration of this project 119
xiv 2.3 Reaction optimization 120
2.4 Scope of the reaction 121
2.4.1 Alcohol and ketone substrates 121
2.4.2 Hydrocarbon substrates 124
2.5 Synthesis of enantiopure aminomenthol 2.37 124
2.6 Postulated mechanism 125
2.7 Conclusion and outlook 127
2.8 Acknowledgements 128
2.9 Distribution of credit 128
2.10 Experimental procedures and data 129
2.10.1 General 129
2.10.2 Experimental procedures and data of synthetic intermediates 130
2.10.3 X-ray crystallographic data 149
2.11 References/bibliographic notes 149
2.12 Appendix: copies of NMR spectra 152
Chapter 3: Improving Physical Properties via C–H Oxidation:
Chemical and Enzymatic Approaches 179
3.1 Academia–Industry Symbiosis in Organic Chemistry 180
3.2 Aliphatic C–H oxidation, a well-suited tool to probe the physical properties of natural product drug candidates 183
3.3 Betulin and betulinic acid, two natural products with a potent bioactivity in vitro hampered by a poor solubility in water 184
3.4 Innate reactivity of the lupane skeleton towards electrophilic oxidants 185
xv 3.5 Investigation of the C16 selectivity through DFT calculations 187
3.6 Guided C–H oxidation strategies 189
3.6.1 Oxidation of the A ring via iridium catalysis 189
3.6.2 Oxidation of the C ring via hydroxyl radical species 191
3.7 Enzymatic C–H oxidations 193
3.8 Panel of C–H oxidations used on the lupane framework 193
3.9 Solubility assays with the new oxidized compounds 194
3.10 Conclusion and outlook 195
3.11 Acknowledgments 196
3.12 Distribution of credit 197
3.13 Experimental procedures and data 198
3.13.1 General 198
3.13.2 Experimental procedures and data of synthetic intermediates 199
3.13.3 X-ray crystallographic data 349
3.14 References/bibiliographic notes 349
3.15 Appendix: copies of NMR spectra 354
Appendix: Curriculum vitae 385
xvi List of figures
Note: the figure titles have sometimes been abbreviated for clarity.
Figure 1.1 Overview of the Streptomyces maritimus enterocin biosynthetic pathway and proposed EncM catalysis. 5
Figure 1.2 Synthesis of smaller analogs of compound. 7
Figure 1.3 Crystal structure of EncM. 10
Figure 1.4 Reversed-phase HPLC analysis. 11
Figure 1.5 EncM-catalyzed transformation of substrate 1.4. 11
Figure 1.6 Proof of structure of diastereoisomers. 13
Figure 1.7 18O-labeled enzymatic transformation of substrate 1.4. 15
Figure 1.8 Ultraviolet–visible spectra of the oxidized flavin of EncM. 16
Figure 1.9 Synthesis of flavin-N5-oxide (1.27). 17
Figure 1.10 Proposed catalytic mechanism of EncM. 19
Figure 1.11 Purification of EncM. 29
Figure 1.12 Comparison of catalytic tunnels of EncM and the homologous 6-HDNO . 30
Figure 1.13 Basic surface-patch and tunnel entrance of dimeric EncM. 31
Figure 1.14 In vitro reconstitution of the enterocin biosynthetic pathway. 32
Figure 1.15 Comparison of EncM active-site electron densities. 33
Figure 1.16 Positive ion mode ESI MS and MSn measurements. 34
Figure 1.17 Observed relative amounts of compounds 1.17/1.17’. 35
Figure 1.18 Positive ion mode ESI MS measurements of compounds 5/5’ and
18 18 7/7’ produced by EncM in presence of 50% O2 gas or 60% H2 O. 36
xvii Figure 1.19 Positive ion mode ESI MS/MS measurements of single
18O-labeled compounds. 36
Figure 1.20 Internal cavity of EncM with bound substrate analog 1.4. 37
Figure 1.21 Time-course of EncM-catalyzed formation of 1.15/1.15’. 38
Figure 1.22 UV-Vis spectrum of EncM-bound flavin. 38
Figure 1.23 UV-Vis spectrum of EncM after complete flavin reduction. with dithionite. 39
Figure 1.24 UV-Vis spectra of EncM-Flox[O] and EncM-Flox. 40
Figure 1.25 Potential pathways for the oxygenation of enolate 1.29. 41
Figure 1.26 Time-course of EncM UV-Vis spectral changes during anaerobic single turnover of 1.4. 41
Figure 1.27 Alternative EncM mechanism. 42
Figure 1.28 Synthesis of substrates 1.4 and 1.6. 45
Figure 1.29 Chromatogram of (±)-1.4. 52
Figure 1.30 Chromatogram of (–)-1.4 after chiral HPLC resolution. 52
Figure 1.31 Chromatogram of (+)-1.4 after chiral HPLC resolution. 53
Figure 1.32 Synthesis of substrate 1.11. 61
Figure 1.33 Synthesis of substrate 1.14. 63
Figure 1.34 Synthesis of substrate 1.7. 65
Figure 1.35 Synthesis of product 1.15’. 74
Figure 1.36 Syntheses of intermediate 1.23’ from 1.15’ and (+)-1.18. 78
Figure 1.37 1H NMR spectra comparison of 1.23’ synthesized from (+)-1.18 and 1.15'. 80
xviii Figure 1.38 13C NMR spectra comparison of 1.23’ synthesized from (+)-1.18 and 1.15'. 80
Figure 1.39 Syntheses of intermediate 1.23 from 1.15 and (–)-1.18. 81
Figure 1.40 1H NMR spectra comparison of 1.23 synthesized from (–)-1.18 and 1.15. 82
Figure 1.41 13C NMR spectra comparison of 1.23 synthesized from (–)-1.18 and 1.15. 82
Figure 1.42 Circular dichroism spectrum for 1.15, enzymatically prepared. 83
Figure 1.43 Normalized circular dichroism spectrum for 1.15. 84
Figure 1.44 Normalized circular dichroism spectrum for 1.15’. 84
Figure 1.45 Synthesis of N-oxide 1.27. 85
3 Figure 2.1 (A) Overview of Csp –H amination methods (B) Reported synthesis of dihydrooxazinium salt 2.2. (C) Reaction optimization. 119
Figure 2.2. Scope of the Csp3–H amination for alcohol and ketone substrates. 122
Figure 2.3 Scope of the Csp3–H amination for hydrocarbon substrates. 123
Figure 2.4 Hydrolysis of acetamide products: synthesis of aminomenthol 2.37. 125
Figure 2.5 A postulated mechanism for the reaction. (B) Kinetic isotope effect.
(C) Radical clock experiment. 127
Figure 2.6 Synthesis of 6-epi-dihydrojunenol (2.10). 137
Figure 2.7 Reaction optimization with substrate 2.4. 145
Figure 2.8 Reaction optimization with menthol (2.1). 146
Figure 2.9 Kinetic isotope experiment with compounds 2.21 and d12-2.21. 147
Figure 3.1 Academia–industry symbiosis in organic chemistry. 181
xix Figure 3.2 Diversification of the lupane core via C–H oxidation. 184
Figure 3.3 Analysis of site selectivities on 3.12 for TFDO. 188
Figure 3.4 Divergent synthesis of oxidized compounds from A) betulinic acid and B) betulin. 190
Figure 3.5 Microbial oxidations of betulinic acid (3.2). 193
Figure 3.6 Summary of aliphatic C−H oxidation panel. 194
Figure 3.7 Advanced intermediates 3.22 and 3.23 synthesized for BMS. 196
Figure 3.8 Biosynthesis of compound 3.9. 287
Figure 3.9 LCMS of 2α,7β-dihydroxybetulinic acid (3.9). 291
1 Figure 3.10 H NMR (CD3OD) of 2α,7β-dihydroxybetulinic acid (3.9). 292
1 Figure 3.11 Expanded region of the H NMR (CD3OD) spectrum of 3.9. 292
1 1 Figure 3.12 H– H COSY NMR of 2α,7β-dihydroxybetulinic acid (3.9). 293
13 Figure 3.13 C NMR (CD3OD) of 2α,7β-dihydroxybetulinic acid (3.9). 293
13 Figure 3.14 Expanded section of the C NMR (CD3OD) spectrum of 3.9. 294
13 Figure 3.15 C DEPT NMR (CD3OD) of 3.9. 294
1 Figure 3.16 H NMR (CDCl3) of 2α,7β-dihydroxybetulinic acid (3.9). 295
Figure 3.17 Biosynthesis of compound 3.10. 295
Figure 3.18 Characteristic NOE couplings for compound 3.10. 299
Figure 3.19 LCMS chromatogram of 7β,15α-dihydroxybetulinic acid (3.10). 300
1 Figure 3.20 H NMR (CD3OD) of 7β,15α-dihydroxybetulinic acid (3.10). 301
Figure 3.21 Expanded region of the 1H NMR (CD3OD) spectrum of 3.10. 301
13 Figure 3.22 C NMR (CD3OD) of 7β,15α-dihydroxybetulinic acid (3.10). 302
13 Figure 3.23 Expanded region of the C NMR (CD3OD) spectrum of 3.10. 302
xx 13 Figure 3.24 C DEPT NMR (CD3OD) of 3.10. 303
1 1 Figure 3.25 H– H COSY NMR (CD3OD) of 3.10. 303
Figure 3.26 FaSSIF and FeSSIF solubility of compounds 3.1 to 3.10. 305
Figure 3.27: Activation enthalpies for reactions of TFDO on equatorial C6–H and C16–H of 3.12, and des-acetoxy betulin 3.24. 307
xxi List of tables
Note: the table titles have sometimes been abbreviated for clarity.
Table 1.1 Data collection, phasing, and refinement statistics. 43
Table 1.2 Crystal data and structure refinement for 1.6. 46
Table 1.3 Atomic coordinates and equivalent isotropic displacement parameters for 1.6. 47
Table 1.4 Bond lengths [Å] and angles [°] for 1.6. 48
Table 1.5 Anisotropic displacement parameters for 1.6. 49
Table 1.6 Hydrogen coordinates and isotropic displacement parameters for 1.6. 50
Table 1.7 Crystal data and structure refinement for 1.4. 53
Table 1.8 Atomic coordinates and equivalent isotropic displacement parameters for 1.4. 54
Table 1.9 Bond lengths [Å] and angles [°] for 1.4. 55
Table 1.10 Anisotropic displacement parameters for 1.4. 59
Table 1.11 Hydrogen coordinates and isotropic displacement parameters for 1.4. 60
Table 1.12 Crystal data and structure refinement for 1.7. 66
Table 1.13 Atomic coordinates and equivalent isotropic displacement parameters for 1.7. 68
Table 1.14 Bond lengths [Å] and angles [°] for 1.7. 69
Table 1.15 Anisotropic displacement parameters for 1.7. 72
Table 1.16 Hydrogen coordinates and isotropic displacement parameters for 1.7. 73
Table 1.17 Hydrogen bonds for 1.7 [Å and °]. 74
Table 3.1 Relative solubility enhancement of the oxidized compounds. 195
xxii Table 3.2 Comparison of 1H NMR data for (+)-C23-hydroxybetulinic acid (3.5). 203
Table 3.3 Comparison of 13C NMR data (+)-C23-hydroxybetulinic acid (3.5). 203
Table 3.4 Crystal data and structure refinement for 3.12. 206
Table 3.5 Atomic coordinates and equivalent isotropic displacement parameters for 3.12. 207
Table 3.6 Bond lengths [Å] and angles [°] for 3.12. 208
Table 3.7. Anisotropic displacement parameters for 3.12. 210
Table 3.8 Hydrogen coordinates and isotropic displacement parameters for 3.12. 211
Table 3.9 Crystal data and structure refinement for 3.14. 214
Table 3.10 Atomic coordinates and equivalent isotropic displacement parameters for 3.14. 215
Table 3.11 Bond lengths [Å] and angles [°] for 3.14. 216
Table 3.12 Anisotropic displacement parameters for 3.14. 218
Table 3.13 Hydrogen coordinates and isotropic displacement parameters for 3.14. 219
Table 3.14 Crystal data and structure refinement for 3.4. 225
Table 3.15 Atomic coordinates and equivalent isotropic displacement parameters for 3.4. 225
Table 3.16 Bond lengths [Å] and angles [°] for 3.4. 226
Table 3.17 Anisotropic displacement parameters for 3.4. 230
Table 3.18 Hydrogen coordinates and isotropic displacement parameters for 3.4. 230
Table 3.19 Hydrogen bonds for 3.4 [Å and °]. 232
Table 3.20 Crystal data and structure refinement for 3.16’. 234
Table 3.21 Atomic coordinates and equivalent isotropic displacement
xxiii parameters for 3.16’. 235
Table 3.22 Bond lengths [Å] and angles [°] for 3.16’. 236
Table 3.23 Anisotropic displacement parameters for 3.16’. 241
Table 3.24 Hydrogen coordinates and isotropic displacement parameters for 3.16’. 243
Table 3.25 Crystal data and structure refinement for 3.15’. 250
Table 3.26 Atomic coordinates and equivalent isotropic displacement parameters for 3.15’. 251
Table 3.27 Bond lengths [Å] and angles [°] for 3.15’. 253
Table 3.28 Anisotropic displacement parameters for 3.15’. 257
Table 3.29 Hydrogen coordinates and isotropic displacement parameters for 3.15’. 258
Table 3.30 Hydrogen bonds for 3.15’ [Å and °]. 259
Table 3.31 Crystal data and structure refinement for 3.18’. 261
Table 3.32 Atomic coordinates and equivalent isotropic displacement parameters for 3.18’. 262
Table 3.33. Bond lengths [Å] and angles [°] for 3.18’. 264
Table 3.34 Anisotropic displacement parameters for 3.18’. 272
Table 3.35 Hydrogen coordinates and isotropic displacement parameters for 3.18’. 274
Table 3.36 Hydrogen bonds for 3.18’ [Å and °]. 276
Table 3.37 Crystal data and structure refinement for 3.6. 278
Table 3.38 Atomic coordinates and equivalent isotropic displacement
xxiv parameters for 3.6. 279
Table 3.39 Bond lengths [Å] and angles [°] for 3.6. 279
Table 3.40 Anisotropic displacement parameters for 3.6. 283
Table 3.41 Hydrogen coordinates and isotropic displacement parameters for 3.6. 283
Table 3.42 Torsion angles [°] for 3.6. 285
Table 3.43 Selected failures of non-directed oxidation attempts on 3.12. 287
Table 3.44 Energies with UB3LYP/6-311++G(d,p), with geometries and thermal corrections to enthalpies (TCH) and Gibbs free energies (TCG) at 298K and UB3LYP/6-31G(d). 309
Table 3.45 Energies with UB3LYP/6-311++G(d,p), with geometries at 298K and UB3LYP/6-31G(d). 310
xxv List of abbreviations
Ac acetyl
ACP acyl carrier protein
ADP adenosine diphosphate
ATP adenosine triphosphate nBu n-butyl sBu sec-butyl tBu t-butyl
Bn benzyl c concentration (for optical rotation) cod cyclooctadiene calc’d calculated (for mass spectrometry analysis)
DCE dichloroethane
DCM dichloromethane
DMDO dimethyldioxirane
DMAP 4-dimethylaminopyridine
DMF dimethylformamide
DMSO dimethylsulfoxide
E glutamic acid
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
ESI-TOF electrospray ionization-time of flight
Et ethyl
FAD flavin adenine dinucleotide
xxvi Glu glutamic acid
His histidine
HPLC high-performance liquid chromatography
HRMS high-resolution mass spectrometry hν light
IR infrared
LCMS liquid chromatography–mass spectrometry
LDA lithium diisopropylamide
Leu leucine
Me methyl
MeCN acetonitrile
NMR nuclear magnetic resonance
N asparagine
Pd/C palladium on charcoal or palladium on carbon
Ph phenyl
Phen phenanthroline
PIDA phenyliodonium diacetate
PKS polyketide synthase ppm parts per million iPr isopropyl
PTLC preparative thin-layer chromatography pyr pyridine
Q glutamine
xxvii rt room temperature
Rf retention factor
r.m.s.d. root mean squared deviation
Ser serine
SM starting material
TBAF tetra-n-butylammonium fluoride
TBS tert-butyldimethylsilyl
TEMPO (2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl
TFA trifluoroacetic acid
TFDO methyl(trifluoromethyl)dioxirane
THF tetrahydrofuran
TLC thin-layer chromatography
TMS trimethylsilyl
Trp tryptophan
Tyr tyrosine
δ chemical shift
ν˜ wavenumber
ν˜ max wavenumber at maximum intensity
€ Y tyrosine
€
xxviii Abstract
The concept of oxidation lies at the center of all aspects of science since molecular complexity and function stem from the incorporation of oxygen atoms into organic compounds. Described in this thesis are three projects related to the concept of oxidation in organic chemistry, particularly in the context of complex-molecule synthesis.
Although these projects are seemingly disparate, each of them explores one facet of the wide range of oxidation chemistry from Nature to the flask. While this may be an oversimplification, there are two complementary approaches when developing oxidation chemistry: (i) inventing novel oxidation methods in the flask and using them to mimic
Nature's creation of natural products; and (ii) understanding Nature's oxidation machinery to spark the discovery of new types of reactivity in the flask. Both approaches have been pioneered by several notable chemists, including E. J. Corey and Derek Barton.
Consequently, selected quotes from Barton's book "Reason and Imagination: Reflections on Research in Organic Chemistry" were used to support the underlying principles of this thesis.
Chapter 1 focuses on the investigation of the mechanism of EncM, a very unique enzyme that catalyzes a oxygenation–dehydrogenation dual oxidation of a poly(β- carbonyl) chain en route to the natural product enterocin. The crystal structure of EncM with bound substrate mimics and isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin- oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin.
xxix Chapter 2 discusses the