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A synthetic approach to tetrodotoxin : via oxidative amidation of a phenol Chau, Jaclyn 2012

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   A Synthetic Approach to Tetrodotoxin: via Oxidative Amidation of a Phenol   by   Jaclyn Chau    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE  in   THE FACULTY OF GRADUATE STUDIES  (Chemistry)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    August 2012     © Jaclyn Chau, 2012     ii   Abstract  This dissertation describes synthetic efforts towards tetrodotoxin. Contact with tetrodotoxin brings upon paralytic shellfish poisoning (PSP) for the unfortunate victim.  The syndrome can be fatal in extreme cases and symptoms include nausea, numbing of the lips and body, throat constriction, slurred speech, and loss of coordination.  Our approach stems from the oxidative amidation of a phenol as a key step.  This technology installs a necessary nitrogen- functionality of the natural product at an early stage; ultimately the route was optimized to allow the synthesis to proceed in a practical manner.             iii  Preface   A portion of the research described in Chapter 1 appeared in Chau, J. and Ciufolini, M. A. The Chemical Synthesis of Tetrodoxin: An Ongoing Quest. Marine Drugs 2011, 9, 2046-2074.  Chau, J. is responsible for: the performance of each of the experiments reported herein; much of the tactical synthetic planning; and the writing of a complete draft of this dissertation. M.A.C. provided: the overall synthetic strategy; many helpful tactical and technical suggestions; and a thorough editing of this document.                    iv  Table of Contents  Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Figures ................................................................................................................................. v List of Schemes .............................................................................................................................. vi List of Abbreviations ...................................................................................................................... x Acknowledgements .................................................................................................................... xviii 1. Introduction ................................................................................................................................. 1 2. Retrosynthetic Considerations .................................................................................................. 19 3. Synthetic Work ......................................................................................................................... 24 References ..................................................................................................................................... 42 Appendix A: Experimental Protocols ........................................................................................... 46  v  List of Figures  Figure 1. The marine toxin tetrodotoxin, 1 ..................................................................................... 1 Figure 2. Saxitoxin (STX), 2........................................................................................................... 2 Figure 3. Spectral properties of compound 176 ............................................................................ 39 Figure 4. Possible hydrogen bonding between osmium salt and the acidic NH ........................... 40   vi  List of Schemes Scheme 1. Equilibria exhibited by 1 ............................................................................................... 3 Scheme 2. Proposed biosynthetic origins of 1 ................................................................................ 4 Scheme 3. Formation of the Singer acetate 4 from 1 ...................................................................... 4 Scheme 4. Opening moves of Kishi's synthesis of 1 ...................................................................... 6 Scheme 5. Completion of Kishi's total synthesis of 1 ..................................................................... 7 Scheme 6. Early stages of Isobe's total synthesis of 1 .................................................................... 8 Scheme 7. Isobe's total synthesis of 1 ............................................................................................. 9 Scheme 8. Isobe's total synthesis of 1 ........................................................................................... 10 Scheme 9. Completion of Isobe's total synthesis of 1 ................................................................... 11 Scheme 10. Second Isobe synthesis of 1 ...................................................................................... 12 Scheme 11. Second Isobe synthesis of 1 ...................................................................................... 13 Scheme 12. Second Isobe synthesis of 1 ...................................................................................... 14 Scheme 13. Sato's total synthesis of 1 .......................................................................................... 15 Scheme 14. DuBois' total synthesis of 1 ....................................................................................... 16 Scheme 15. Completion of DuBois' total synthesis of 1 ............................................................... 17 Scheme 16. Oxidative amidation of phenols ................................................................................ 19 Scheme 17. The oxidative amidation of phenols using oxazolines as trapping agents ................ 20 Scheme 18. The oxidative amidation of phenols using sulfonamides as trapping agents ............ 21 Scheme 19. Possible mechanism for the bimolecular oxidative amidation of phenols ................ 22 Scheme 20. Retrosynthetic analysis .............................................................................................. 23 Scheme 21. Previous preparation of isoxazoline 136 via INOC .................................................. 24 Scheme 22 Undesirable Michael cyclization of synthetic intermediates 137 and 138 ................. 26 Scheme 23 Preparation of cis-nitroketone, 145 ............................................................................ 27 Scheme 24. Large scale preparation of isoxazoline 146 ............................................................... 27 vii  Scheme 25. INOC catalyzed by base and copper(II) .................................................................... 28 Scheme 26. Synthesis of 149 and 150 .......................................................................................... 29 Scheme 27. Bom protection of 149 and 150 ................................................................................. 30 Scheme 28. Product of Bom protection, 153, using commercial materials .................................. 31 Scheme 29. Hypothetical route to diene 155 ................................................................................ 31 Scheme 30. Aromatic product, 158, arising from Wittig reaction of 154 .................................... 32 Scheme 31. Synthesis of diene, 163.............................................................................................. 33 Scheme 32. Osmylation directed by the Kishi effect .................................................................... 34 Scheme 33. Osmylation directed by the Kishi effect .................................................................... 35 Scheme 34. Osmylation directed by the Kishi effect .................................................................... 36 Scheme 35. Synthesis of 171 ........................................................................................................ 37 Scheme 36. Bom deprotection via hydrogenation ........................................................................ 38 Scheme 37. Synthesis of vinyl ether ............................................................................................. 38 Scheme 38. Actual configuration of compounds 171-176............................................................ 40 Scheme 39. Possible hydrogen bonding between osmium salt and the acidic NH ....................... 41 Scheme 40. 1H NMR spectrum of 132.......................................................................................... 48 Scheme 41. 13C NMR spectrum of 132 ........................................................................................ 48 Scheme 42. 1H NMR spectrum of 145.......................................................................................... 51 Scheme 43. 13C NMR spectrum of 145 ........................................................................................ 51 Scheme 44. 1H NMR spectrum of 146.......................................................................................... 53 Scheme 45. 13C NMR spectrum of 146 ........................................................................................ 53 Scheme 46. 1H NMR spectrum of 149.......................................................................................... 55 Scheme 47. 13 CNMR spectrum of 149 ......................................................................................... 55 Scheme 48. 1H HNMR spectrum of 151 ....................................................................................... 57 Scheme 49. 13C NMR spectrum of 151 ........................................................................................ 57 viii  Scheme 50. 1H NMR spectrum of 159.......................................................................................... 59 Scheme 51. 13C NMR spectrum of 159 ........................................................................................ 59 Scheme 52. 1H NMR spectrum of 161.......................................................................................... 62 Scheme 53. 13C NMR spectrum of 161 ........................................................................................ 62 Scheme 54. 1H NMR spectrum of 162.......................................................................................... 64 Scheme 55. 13C NMR spectrum of 162 ........................................................................................ 64 Scheme 56. 1H NMR spectrum of 163.......................................................................................... 66 Scheme 57. 13C NMR spectrum of 163 ........................................................................................ 66 Scheme 58. 1H NMR spectrum of 170.......................................................................................... 69 Scheme 59. 13C NMR spectrum of 170 ........................................................................................ 69 Scheme 60. 1H NMR spectrum of 171.......................................................................................... 72 Scheme 61. 13C NMR spectrum of 171 ........................................................................................ 72 Scheme 62. 1H NMR spectrum of 172.......................................................................................... 74 Scheme 63. 13C NMR spectrum of 172 ........................................................................................ 74 Scheme 64. 1H NMR spectrum of 176.......................................................................................... 77 Scheme 65. 13C NMR spectrum of 176 ........................................................................................ 77 Scheme 66. COSY spectrum of 176 (expansion) ......................................................................... 78 Scheme 67. COSY spectrum of 176 (expansion) ......................................................................... 79 Scheme 68. COSY spectrum of 176 (expansion) ......................................................................... 80 Scheme 69. NOESY spectrum of 176........................................................................................... 81 Scheme 70. NOESY spectrum of 176 (expansion) ....................................................................... 82 Scheme 71. NOESY spectrum of 176 (expansion) ....................................................................... 83 Scheme 72. NOESY spectrum of 176 (expansion) ....................................................................... 84 Scheme 73. 1H NMR spectrum of 169.......................................................................................... 85 Scheme 74. 13C NMR spectrum of 169 ........................................................................................ 85 ix  Scheme 75. COSY spectrum of 169 ............................................................................................. 86 Scheme 76. COSY spectrum of 169 (expansion) ......................................................................... 87 Scheme 77. COSY spectrum of 169 (expansion) ......................................................................... 88 Scheme 78. COSY spectrum of 169 (expansion) ......................................................................... 89 Scheme 79. NOESY spectrum of 169........................................................................................... 90 Scheme 80. NOESY spectrum of 169 (expansion) ....................................................................... 91 Scheme 81. NOESY spectrum of 169 (expansion) ....................................................................... 92 Scheme 82. NOESY spectrum of 169 (expansion) ....................................................................... 93 Scheme 83. NOESY spectrum of 169 (expansion) ....................................................................... 94 Scheme 84. NOESY spectrum of 169 (expansion) ....................................................................... 95 Scheme 85. NOESY spectrum of 169 (expansion) ....................................................................... 96 Scheme 86. Crude 1H NMR of 158 .............................................................................................. 97    x   List of Abbreviations Ac          acetyl add’n addition Am amyl anh anhydrous aq aqueous Ar aryl Bn        benzyl Boc        tert-butoxycarbonyl Bom benzyloxymethyl br broad BRSM based on recovered starting material BSA          bis(trimethylsilyl)acetamide Bu butyl Bz benzoyl °C degrees Celsius CAN           ceric ammonium nitrate cat. catalytic CBS Corey-Bakshi-Shibata Cbz benzyloxycarbonyl CC column chromatography xi  CDI          1,1´-carbonyldiimidazole  cf. confer cm−1 wavenumber(s) CSA         camphorsulfonic acid δ chemical shift (ppm downfield from tetramethylsilane) Cyh cyclohexyl d doublet D dextrorotatory DBU         1,8-diazabicyclo[5.4.0]undec-7-ene DCC         N,N´-dicyclohexylcarbodiimide DCE dichloroethane DCM          dichloromethane DDQ          2,3-dichloro-5,6-dicyano-1,4-benzoquinone de diastereomeric excess DIB (diacetoxyiodo)benzene DIBAL diisobutylaluminum hydride DIPEA            diisopropylethylamine  DMAP      4-(N,N-dimethylamino)pyridine DMF          N,N-dimethylformamide DMP Dess-Martion periodinane DMSO dimethylsulfoxide xii  DMTS dimethylthexylsilyl DNA            deoxyribonucleic acid DOPA 3,4-dihydroxyphenylalanine DPPA       diphenylphosphoryl azide dr diastereomeric ratio E carboxymethyl E entgegen (of an alkene) ee enantiomeric excess ent enantiomeric epi epimeric ESI electrospray ionization Et                ethyl Fmoc fluorenylmethyloxycarbonyl g gram(s) G                guanine (DNA residue) h                hour(s)  [H] reduction HMPA hexamethylphosphoramide HMQC          Heteronuclear Multiple Quantum Coherence HOBt          1-hydroxybenzotriazole  HRMS high resolution mass spectrometry xiii  Hz Hertz (s−1) i iso (as an alkyl group) IBX 2-iodoxybenzoic acid imid            1,3-imidazole INOC intramolecular nitrile oxide cycloaddition i.p. intraperitoneal IR infrared J coupling constant L levorotatory LAH lithium aluminum hydride LDA lithium diisopropylamide HMDS hexamethyldisilizide LN2 liquid nitrogen m meta (as a benzene substituent) m multiplet M molarity Me              methyl mCPBA meta-chloroperoxybenzoic acid MIC minimum inhibitory concentration min minute(s) mol mole(s) xiv  MOM methoxymethyl m.p. melting point Ms            methanesulfonyl MS mass spectrometry or molecular sieves MTr p-methoxyphenyldiphenylmethyl   n normal (as an alkyl group) NBS          N-bromosuccinimide NIS N-iodosuccinimide NMO N-methylmorpholine-N-oxide NMP N-methylpyrrolidin-2-one NMR         nuclear magnetic resonance nOe nuclear Overhauser effect N-PSP N-phenylselenophthalimide NR            no reaction [O]              oxidation p para (as a benzene substituent) P     unspecified protecting group PCC pyridinium chlorochromate Ph              phenyl Phth phthalimido PMB          p-methoxybenzyl xv  PMP p-methoxyphenyl ppm parts per million PPTS pyridinium p-toluenesulfonate Pr propyl psi pounds per square inch (lbs in−2) PSP paralytic shellfish poisoning Pv pivaloyl chloride PyBrOP      bromo-tris(pyrrolidino)phosphonium hexafluorophosphate pyr             pyridine q quartet quant. quantitative R rectus Rf              retention factor rt                  room temperature rxn reaction s singlet S sinister SAM S-adenosyl methionine sat. saturated SEM [2-(trimethylsilyl)ethoxy]methyl SET single electron transfer xvi  SN2 bimolecular nucleophilic substitution spp.  species STX saxitoxin TBAF tetra-n-butylammonium fluoride TBAT tetra-n-butylammonium difluorotriphenylsilicate TBDPS       tert-butyldiphenylsilyl TBS            tert-butyldimethylsilyl t         tertiary (as an alkyl group) TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical TES triethylsilyl Tf               trifluoromethanesulfonyl TFA            trifluoroacetic acid TFAA   trifluoroacetic anhydride THF           tetrahydrofuran Thr            threonine TIPS triisopropylsilyl TLC thin layer chromatography TMS           trimethylsilyl TPAP tetra-n-propylammonium Ts p-toluenesulfonyl TTX tetrodotoxin xvii  UV             ultraviolet var. variety VT variable temperature xs excess Z zusammen (as an alkene)      xviii  Acknowledgements  First and foremost, I wish to thank Prof. Ciufolini for his immense guidance and support.  I am extremely thankful for his wisdom and dedication to teaching.  His vast chemical knowledge and meticulous observations involving chemistry and life in general has made my time in graduate school a valuable experience.   I would like to thank those who funded this research, including the Department of Chemistry, the University of British Columbia, and others who have provided financial contribution to the research of Prof. Marco Ciufolini.  I would also like to thank the members of the Ciufolini group.  Special thanks to Dylan  for helping me proofread this manuscript and to Taka, Josh, Gloria, Veeru, Steven, David and Jimmy for their insightful discussions. Their friendship and advice have made my stay in the group enjoyable and quite entertaining.    1   1. Introduction  Before its identity was known, tetrodotoxin (TTX, 1) made its presence felt by causing an untold number of fatalities. The syndrome caused by contact with 1 and related toxic agents is known as paralytic shellfish poisoning (PSP).1  After eating poisoned shellfish, the victim’s mouth and lips quickly begin to tingle, then, over about thirty minutes, they become numb. The prickling and numbness may spread to the hands and other parts of the body.2  Following these    Figure 1. The marine toxin tetrodotoxin, 1  initial symptoms, difficulty swallowing and throat constriction may appear.  Limbs will become unresponsive, breathing becomes labored and speech becomes incoherent.  Severe nausea may occur.  In fatal cases, complete paralysis will precede death.  It is believed that the unfortunate victim remains nonetheless lucid throughout.  The entire process occurs over several hours, and is complete within half a day.  It is estimated that as little as 2 mg can be fatal to an average person.  The pure toxin can be isolated from the ovaries of the puffer fish (Tetraodon and Fugu spp.),3 though it has also been discovered that 1 is derived from symbiotic microorganisms.4 2  Specifically, bacteria which inhabit dinoflagellates of the genus Alexandrium, Gymnodinium catenatum, and Pyrodinium bahamenese var. compressum are thought to be a source of PSP toxins including 1, though PSP-producing bacteria also reside in the liver of the pufferfish. Furthermore, it is likely that the toxins accumulate in larger organisms through so-called food web transfer.5  With an initial report by Narahashi, Moore and Scott in 1964 exploring the effect of 1 on the conductance of lobster giant axons, 6  it was established that this material derives its extraordinary toxicity from an ability to block the Na+ channel in neurons, though, interestingly, cardiac neurons seem to be largely resistant to the effect.7 Such a mode of action has been well established in the intervening decades.8 The guanidinium moiety of 1 has been implicated, in that it is able to strongly bind ion channels in place of a Na+ ion.9 A structural analogy can be drawn to the related neurotoxin, saxitoxin (STX, 2).   Figure 2. Saxitoxin (STX), 2   TTX has seen wide use in medical research. For example, it was instrumental in the isolation of ion channel proteins.10 Additionally, it has been used medicinally, for example, in the control of pain. The Na+ channel has been implicated in pain transmission, and 1 has proven so 3  useful in the study of neuronal properties that the Na+ channels themselves are classified by their sensitivity to it.11   Scheme 1. Equilibria exhibited by 1   Structurally, 1 is very unusual. Outside of the aforementioned guanidine moiety, its most remarkable feature is the incorporation of an ortholactone group. This rarely seen functional group probably is able to exist due to the highly rigid structure of the toxin.  Given the dispute about which organism(s) are responsible for the production of 1, it should not be a surprise that the biosynthetic origins of 1 also remain unclear. Some of the difficulty in the determination of the biosynthetic production of 1 arises from the greatly diminished amount of the toxin produced by pufferfish raised in captivity.12 Similarly, cultures of the bacteria believed to be responsible for the biosynthesis of 1 produce only very small amounts of the toxin. One theory to explain the lack of toxin generated by bacterial cultures is the necessity of signaling molecules from the host organism. However, it is also possible that the bacteria that produce the bulk of the toxin cannot be cultured. A study aiming to isolate TTX from newts led to the discovery of 1-hydroxy-5,11-dideoxytetrodotoxin, 3. Inferring that this material was a biosynthetic precursor of 1, the authors proposed that 1 derives from the combination of arginine with isopentenylpyrophosphate, rather than a more oxidized precursor (Scheme 2). 13  However, studies attempting to generate radiolabeled 1 through feeding 4  experiments were not successful.14 A detailed review of biosynthetic considerations with respect to 1 is available.15  Scheme 2. Proposed biosynthetic origins of 1   A brief mention of the pioneering structural studies of 1 by Woodward,16 Tsuda,17 and others,18 is in order. One early piece of structural information was the observation that treatment of TTX with aqueous NaOH, followed by Ac2O, yields a quinazoline called the Singer acetate, 4. (Scheme 3). However, the structural formula of the natural product was not known with certainty, and it was estimated to be in the range of C10-12H15-19O8-10N3. Compounding this uncertainty was the rudimentary nature of the analytical methods available at the time. Single-crystal x-ray    Scheme 3. Formation of the Singer acetate 4 from 1  analysis, infrared and primitive 1H NMR spectroscopy, additional degradation work, and isotopic exchange studies eventually yielded enough information to allow determination of the molecular 5  formula and arrangement of the functional groups present in 1. A final puzzling aspect of these investigations was that all available evidence indicated the presence of a carboxy group in the molecule; yet no carbonyl absorption appeared in the IR spectrum of 1.  This led to the correct inference that TTX incorporates an ortholactone moiety (cf. Scheme 1): the first instance of such a functional group being ever detected in any natural or unnatural molecule.  Total syntheses of TTX  The Kishi synthesis of (±)-tetrodotoxin,19 the first synthesis of the natural product, is remarkable for its brevity. Only 28 linear steps from 5 were needed, which compares quite favorably with all later syntheses. The route began with a Diels-Alder reaction between 5 and butadiene to provide diketone 6. This material underwent a Beckmann rearrangement in the presence of MsCl to provide the key tertiary amide at an early stage of the synthesis. Oxidation state adjustments then gave epoxide 7. At this point, acid-catalyzed ether formation, oxidation and selective ketone protection led to 8.  This material then underwent Meerwein-Ponndorf- Verley reduction and acetylation of the emerging alcohol, giving 9. The alkene in 9 served as a lynchpin for the ultimate introduction of three hydroxyl groups through a sequence that commenced with allylic oxidation (SeO2), reduction (NaBH4) and epoxidation (mCPBA). This was followed by release of the ethylene ketal and Baeyer-Villiger oxidation of 12 to give strained lactone 13.   6   Scheme 4. Opening moves of Kishi's synthesis of 1   Substance 13 was primed for a series of transformations which led to 1. To this end, hydrolysis of the lactone and acidification caused transannular carboxylate attack on the epoxide, and dehydration of an intermediate hemiacetal to furnish 14. The latter was subjected to amide cleavage using Meerwein’s salt followed by alkaline aqueous conditions. The guanidine was then installed by a two-step procedure, which set the stage for Lemieux-Johnson oxidation of the vinyl ether to provide 17. Ammonolysis of this compound removed acetate and formyl groups to complete the total synthesis of 1. In summary, Kishi’s landmark synthesis required 28 linear steps starting from 5, producing 1 in 0.66% yield.   7   Scheme 5. Completion of Kishi's total synthesis of 1   Thirty years later, Isobe completed a second total synthesis of 1.20 The route began with glucal 18,21 a derivative of D-glucose. The first major objective was the creation of 21, the precursor for a key Claisen rearrangement. First, 18 was advanced to 19 by dehydration, glucoside installation, and protecting group adjustment. The resultant 19 was then oxidized, treated with iodine, and reduced to provide 20. This material provided an appropriate substrate for coupling with TMS acetylene and vinyl ether formation. The resultant 21 underwent thermal rearrangement to 22, and the emerging ketone 22 was advanced to the kinetic silyl enol ether derivative, in preparation for bis-acetoxylation leading to 23.  A somewhat circuitous sequence then produced MOM enol ether 25. A Rubottom-type oxidation of the latter with mCPBA occurred diastereoselectively, and protection of the intermediate dihydroxyketone yielded 26.  Treatment of this material with H2SO4 and HgO in MeOH induced cleavage of the TBS ether, hemiacetal formation, and alkyne hydration.   8    Scheme 6. Early stages of Isobe's total synthesis of 1   Methyl ketone 27 was then converted into enol silane 28, which underwent intramolecular aldol addition and subsequent dehydration to 29. The next twelve steps of the synthesis involved a series of protecting group and oxidation state adjustments, resulting in primary alcohol 33, which was advanced to the corresponding carbamate 34. The latter was subjected to deprotonation by tert-BuOK, causing transannular 1,4-addition of the NH2 subunit to the unsaturated ester and providing 35 in an efficient manner. At this point, the pendant ester was no longer needed, and it was reduced to an alcohol, which was protected to give 37. The cyclic carbamate was activated as an N-BOC-derivative and cleaved under basic conditions to give 38.  9   Scheme 7. Isobe's total synthesis of 1  The next objective was the elaboration of 38 to aldehyde 41. This material was reached upon epoxidation of the olefin using mCPBA, further protecting group manipulations, and final IBX oxidation. An unusual transannular cyclization occurred when 41 was treated with DBU at high temperature. Thus, the presumed enolate of the aldehyde reacted at the O-terminus with the epoxide to return vinyl ether 42.  Osmylation of the latter occurred with the incorrect facial selectivity, necessitating a redox sequence to produce the desired diastereomer 44 of the ultimate -hydroxylactone.     10   Scheme 8. Isobe's total synthesis of 1   The final stages of the synthesis entailed removal of the acetyl protecting groups, which triggered closure of the ortholactone, followed by a series of protecting group manipulations to yield 46. Finally, after acetonide and N-BOC cleavage using TFA, the emerging 47 was treated with guanylating reagent 48 and HgCl2. The diol moiety was then cleaved using NaIO4. Subsequent BOC, acetyl, and benzoyl protecting group cleavage completed the first asymmetric total synthesis of 1 in 67 steps, producing 1 in 1.22% yield.   11   Scheme 9. Completion of Isobe's total synthesis of 1   Isobe sought to improve his synthesis, publishing a second route the following year. The second effort rectified certain weaknesses, in particular, the relatively high step count. The successful incorportation of an Overman rearrangement to install the nitrogen functionality was beneficial in this regard. The trichloroacetamide thus introduced at an early stage of the synthesis conveniently survived all subsequent operations.  In the early phases of the synthesis, ketone 5322 underwent Diels-Alder cycloaddition with 2-methylbutadiene in the presence of BF3•OEt2. The product was advanced to 55, which upon Boord olefination using Zn-Cu couple afforded 56. Three more steps delivered Overman precursor 57, rearrangement of which at 140 oC in the presence of K2CO3 yielded product 58.  Bromination of the olefin and treatment of 59 with DBU promoted a tandem E2 reaction – SN2’ displacement, whereby the trichloroacetamide cyclized to oxazoline 61. Hydrolysis, selective epoxidation, and acid-catalyzed epoxide opening provided tetraol 63.  12   Scheme 10. Second Isobe synthesis of 1   Unfortunately, both free alcohols of 63 require inversion of configuration to match that of 1. Accordingly, 63 was treated with IBX, followed by a two-step reduction to give 64. Protection and further oxidation (SeO2 followed by NaBH4) provided 65. The next several steps were necessary to convert the olefin of this latter material into the ortholactone carbon and accompanying hydroxyl group of 1. This proved to be a non-trivial proposition.  Eventually a route was charted to α-ketoacid 68, which, upon treatment with H2O2, underwent oxidative cleavage and concomitant epoxide opening by the free carboxylate to give bridged lactone 70.   13   Scheme 11. Second Isobe synthesis of 1   The total synthesis was completed as shown in Scheme 12. First, TES cleavage using TBAF and exhaustive acetylation gave ortholactone 71. Next, oxidative cleavage of the acetonide diol proceeded using periodic acid, and this operation was followed by a few corrective protection steps. Reductive deprotection of the trichloroacetamide and acetyl groups, followed by guanylation, and TFA treatment gave 1. The final compound was obtained as a mixture incorporating a small amount of dehydrated material 75.   14   Scheme 12. Second Isobe synthesis of 1   Sato and collaborators were able to complete another synthesis of this challenging target starting from myo-inositol, 76.23 A series of protection steps produced 77, which served as a substrate for the installation of hydroxymethyl unit, by way of dichloromethyllithium addition to a ketone obtained by Swern oxidation of 77 (cf. 77 to 78).  The action of base on the resultant 78 produced an -hydroxyaldehyde, which was reduced (NaBH4) to give 79.  Benzylation of the alcohols and release of the orthoester protection preceded an interesting, selective TBS protection to provide 81.  This selectivity may be rationalized by invoking preferential reaction from conformer 80, which undergoes silylation at the least hindered equatorial alcohol. Protecting group manipulations and Swern oxidation of 82 then surrendered ketone 83, Peterson reaction of which produced a methylene derivative. Hydroboration-oxidation of the latter, protection of the resulting primary alcohol, and selective excision of the TBS group returned 84. Dess-Martin oxidation set the stage for a second round of dichloromethyllithium addition to provide 86. An interesting transformation developed earlier by the authors ensued upon treatment of 86 with NaN3 in DMSO: first, epoxide formation occurred to generate transient species 87, which was attacked by azide ion at the tertiary center to give aldehyde 88.  15  Cyanohydrin formation took place diastereoselectively to form 89. Nitrile reduction with DIBAL and oxidation of the emerging aldehyde with chromic acid provided lactone 90 directly. A few final steps completed the 33 step synthesis of 1. The same group later disclosed two alternative routes to enantiopure ketone 85.24    Scheme 13. Sato's total synthesis of 1 16    Following Sato’s publication, DuBois described a conceptually novel synthesis of TTX25 that hinged upon a Rh(II) catalyzed nitrene insertion into a C-H bond (technology developed earlier by the same group)26 as a way to install the nitrogen functionality. The planned late-stage execution of this step demonstrates a notable confidence in the reaction.  DuBois’ synthesis began with the conversion of D-isoascorbic acid derivative 92 into aldehyde 95 through well-documented chemical transformations. Aldehyde 95 was then combined with dibenzyloxaloacetate to give 96. The latter compound was converted into diazo derivative 97 in three steps. Treatment of this material with a Rh(II) catalyst promoted carbene C-H insertion to give 98 in a completely diastereoselective manner. Reduction and protecting group adjustment furnished 99.    Scheme 14. DuBois' total synthesis of 1  17   The next subgoal was the synthesis of carbamate 104, which would serve as a precursor for the critical nitrene insertion. Lactone 99 was thus opened using dimethylamine, and the liberated alcohol was oxidized to a ketone. Tebbe methylenation and allylic oxidation produced enone 102. An eight-step sequence, key phases of which included a conjugate addition of a vinylcopper reagent and a stereoselective carbonyl reduction with borane-tert-butylamine complex, afforded 103, and thence key intermediate 104. This carbamate was treated with Rh(II) trifluoroacetamide and (diacetoxyiodo)benzene (“DIB”) to form oxazolidinone 105, displaying the crucial C-N bond of 1. Straightforward manipulations advanced 106 to 107, from which (−)-1 was reached by established methods. The 31 step linear synthesis produced (-)-1   Scheme 15. Completion of DuBois' total synthesis of 1   18   In addition to the foregoing total syntheses, the literature records numerous synthetic studies toward TTX. Space limitations do not allow a thorough illustration of these important investigations, for which the reader is referred to a recent, comprehensive review on the subject.27  It is clear from this article, as well as from the preceding discussion, that a significant body of work has arisen over the past few decades in pursuit of an efficient laboratory synthesis of 1. Though significant strides have been made in this area, the current state of the art – even DuBois’ brilliant synthesis – is unable to outdo Kishi’s route. A more concise avenue to 1 would benefit medical research, in that it might enable the conduct of medicinal chemistry studies that could lead to valuable therapeutic resources. It would be difficult to carry out such investigations using the natural product as the starting point, given its reactivity and its extremely hazardous nature. Synthetic methodology developed in our group appears to offer interesting opportunities in the tetrodotoxin area. The following paragraphs provide an outline of the new technology and illustrate how the latter could be meaningfully harnessed to achieve a concise synthesis of 1.   19  2. Retrosynthetic Considerations  The Oxidative Amidation of Phenols  The oxidative amidation of phenols28 is a process whereby a generic phenol, typically a 4-substituted one such as 108, is converted into a spirodienone, 110 (Scheme 16). Group N in 108 represents a suitable nitrogen nucleophile, while the dashed curve indicates that “N” may be tethered to the aromatic nucleus or it may be independent. In the first case, the reaction will occur in the intramolecular regime; in the second, in the bimolecular mode. The transformation is induced by a hypervalent iodine reagent such as DIB, the action of which upon the substrate produces an electrophilic intermediate, naively rendered in Scheme 16 as cation 109. Capture of 109 by the N functionality produces the ultimate 110, wherein N always emerges in the form of an amide; hence the terminology “oxidative amidation.” This transformation appears to be quite useful in the synthesis of nitrogenous natural products, in that simple phenols may be rapidly converted into products of significant molecular complexity.    Scheme 16. Oxidative amidation of phenols  An early variant of the reaction used an appended oxazoline as the nitrogen nucleophile. This methodology constituted the centerpiece of a synthesis of the natural products FR-901483 and TAN-1251C (Scheme 17). Later work revealed that sulfonamides were also competent 20  trapping agents.29 Such a reaction was used in the total syntheses of cylindricine C30 and putative lepadiformine (Scheme 18).31 The latter mode of reaction also proved useful for the assembly of the core ring system of himandrine.32   Scheme 17. The oxidative amidation of phenols using oxazolines as trapping agents        21   Scheme 18. The oxidative amidation of phenols using sulfonamides as trapping agents   The bimolecular variant of the reaction resorts to acetonitrile as an external nitrogen nucleophile, which presumably captures a cation such as 122 in a Ritter-like fashion (Scheme 19).33  The process was found to occur most efficiently upon slow addition of a CH3CN solution of phenol to a dilute CH3CN solution of DIB and TFA.  34 Best results were obtained when the final concentration of phenol was around 110 mmol/L. It is likely that, at higher concentration, reactive species 122 is captured by the starting phenol 121, leading to undesired polymeric products, while lowering the yield. A slight molar excess of TFA relative to DIB is necessary for rapid and complete dissolution of the hypervalent iodine reagent and efficient conversion. However, larger amounts of TFA lower the yield and complicate isolation procedures. In its current form, the protocol allows practical large-scale preparation of products 124 in an economical and expedient manner.     22   Scheme 19. Possible mechanism for the bimolecular oxidative amidation of phenols   The approach to tetrodotoxin detailed in this thesis is based on a bimolecular oxidative amidation as a key step, according to the retrosynthetic logic developed in the next paragraph.  Retrosynthetic Analysis   Excision of the guanyl segment in 1 and release of the ortholactone reveal a highly oxygenated cyclohexane precursor, rendered in Scheme 20 as protected polyol 125 (P, P’, P” = suitable blocking groups). Recognizing that, in principle, substituents OP could be installed through a stereoselective bis-dihydroxylation of a diene, we recognized compound 126 as a forerunner of 125.  The CHO group in brackets indicates that the formyl substituent in 126 may be expressed, or protected, or latent.  In particular, if one were to conceive a nitrile as a precursor of the aldehyde, and a ketone as that of the exomethylene segment, compound 126 may be further simplified to structure 127. An interesting opportunity materializes at this juncture: substance 127 appears to be available through nucleophilic fragmentation of isoxazoline 128, 23  which is the result of an intramolecular nitrile oxide cycloaddition (“INOC”) of 129. Furthermore, the carbonyl group in the cyclopentane segment of 128 should enable the subsequent introduction of the OH functionality found at the -position of the ester in 127, further simplifying the molecule. A sensible starting point for the generation of 129 would be dienone 130, or an opportune derivative thereof, easily recognized as the product of bimolecular oxidative amidation of phenol 131.   Scheme 20. Retrosynthetic analysis  On paper, this strategy could translate into a total synthesis of 1 in about 25 steps from commercial 131: a significant improvement over all known routes. Although a total synthesis has yet to be achieved, the approach has brought us close to the ultimate goal, as detailed in the following chapter.  24  3. Synthetic Work   Full execution of the above strategy necessitated significant amounts of exploratory work. Former group members, Drs. B. Mendelsohn and C. Benhaim, had determined that ester 132, prepared by them in low yield by an oxidative amidation procedure subsequently shown to be inappropriate, could be elaborated into dienic alcohol 134 in modest yield according to the method of Scheme 21.35  Nitro compound 135 was amenable to conversion into a reactive dipole (not necessarily a nitrile oxide; vide infra) by the method of Torssell (TBS-Cl), resulting in the ultimate formation of isoxazoline 136 in moderate and variable yield. Finally, the nucleophilic cleavage of 136 was problematic, generally proceeding in no more than ca. 40% yield. Clearly, the procedure was entirely unsuitable for a synthetic attack on 1. Consequently, effort was focused on developing a reliable method to readily synthesize 136 in large scale: an objective that demanded the optimization of every single step from 131 to the subgoal isoxazoline.    Scheme 21. Previous preparation of isoxazoline 136 via INOC   25  The first issue to be addressed was the scale-up of the oxidative amidation of 131. The quantities of 132 required for the synthesis ruled against any protocol involving chromatography.  Compound 132 also happens to be quite polar and water soluble, suggesting that a non-aqueous reaction workup may advantageously minimize losses. Fortunately, 132 is a nicely crystalline material which, arguably, could be retrieved from crude reaction mixtures merely by crystallization. A robust procedure for its preparation ultimately emerged as follows. A solution of 131 was added over 3 h (syringe pump) to a solution of DIB in MeCN containing 1.3 equiv. of trifluoroacetic acid (TFA).  TFA serves both as a protonic catalyst and as an agent that transforms MeCN-insoluble DIB into a soluble, more reactive oxidant; probably PhI(OAc)OCOCF3. The oxidation of the phenol to 132 is essentially instantaneous under these conditions. The reaction mixture is then evaporated and the residue is twice filtered through a short plug of silica gel to remove nonpolar byproducts and impurities. Evaporation of the percolate, dissolution of the residue in EtOAc, and cooling to -20 oC result in deposition of crystals of analytically pure 132. This procedure minimizes the use of solvents, it avoids chromatography and aqueous washes, and it performs well on scales of up to ca. 10 g of material. The successful conclusion of this initial investigation induced us to refocus our efforts on the elaboration of 132 to 136. The previously devised route to 136 necessitates a reduction of the dienone to prevent undesirable Michael cyclization of either acid 137 or of 138 itself (Scheme 22). Such a reduction was traditionally carried out with DIBAL, resulting in selective formation (ca. 4:1) of that diastereomer of the alcohol, wherein the OH and NHAc groups are trans (Scheme 22; confirmed by X-ray crystallography of a derivative).36 This step is fraught with   26   Scheme 22 Undesirable Michael cyclization of synthetic intermediates 137 and 138  numerous technical difficulties, due to the very polar nature of alcohol 139, its aqueous solubility, and its ability to coordinate Al(III) species. These properties conspire to severely diminish the overall yield of 139. The search for an improved reductive method revealed that the Corey- Bakshi-Shibata (CBS) procedure suppressed all of the above problems, while selectively producing the cis diastereomer of the alcohol (ca. 8:2; Scheme 23). The observation that DIBAL and CBS reductions produce opposite stereochemical outcomes was initially made by a former group member, Dr. H. Liang.37 A cogent rationale for this phenomenon remains elusive, but the selective formation of the cis stereoisomer was welcome news, in that it subsequently transpired that this diastereomer was more compatible with later steps (vide infra). Nitroketone 145 was obtained from 132 by a series of steps similar to those employed earlier for the synthesis of 135. The cis series was used in all subsequent operations.  27   Scheme 23 Preparation of cis-nitroketone, 145  As indicated earlier, the nitrile oxide cycloaddition sequence required considerable improvement. In order to advance the work of previous group members, optimization of the INOC was necessary.  A remedy was identified in the form of a DeSarlo reaction.38 Pleasingly, treatment of 145 with catalytic amounts of copper (II) acetate and N-ethylpiperidine in freshly distilled chloroform at 30 °C induced slow cyclization to 146 in 60% yield.  The use of purified solvent was essential to the success of this step (see experimental section for details), which was reliably operable on scales of up to six grams. Operation at low substrate concentration improved the yield of the cycloaddition by minimizing polymer formation: best results were obtained when the concentration of the substrate was around 200 mmol/L.   Scheme 24. Large scale preparation of isoxazoline 146  Temperature control was essential to ensure a satisfactory yield. Indeed, heating the reaction to 60 °C (original DeSarlo procedure) resulted in mostly polymeric material, with only a 28  trace amount of 146. Maintaining the reaction at 30 oC maximized the formation of the desired cycloadduct, even though the cycloaddition was slow, requiring around 6.5 days for completion (some starting 145 remained even after this time). On a positive note, isoxazoline 146 was the sole reaction product. Equally important was the choice of base: the use of imidazole (11% yield of 146), DABCO (no product detected), N-methylmorpholine (20% yield of 146) in lieu of N- ethylpiperidine gave inferior results. A plausible mechanism for the process is presented in Scheme 25. DeSarlo postulated that the addition of Cu(II) reduced the induction time for cycloaddition. Moreover, he suggested that Cu(II) catalyzed the cycloaddition pre-equilibrium and that the rate-determining step was the irreversible dehydration step which led to isoxazoline products. It is interesting to note that trans stereoisomer 135 (arising from DIBAL reduction of 132) tolerated higher reaction temperatures when subjected to DeSarlo cyclization, advancing to 136 in 40% yield after only 2.5 days at 60 °C, with marginal formation of polymeric material.   Scheme 25. INOC catalyzed by base and copper(II)  29   With quantities of 146 in hand, we turned to the fragmentation of the tricyclic oxazoline in basic methanol. It was rapidly determined that the reason for the poor results obtained by former group members were squarely attributable to the presence of moisture in the solvent. Indeed, when the reaction was performed with methanol freshly distilled from Mg turnings, the desired 149 was obtained in 83% yield (Scheme 26). The diastereomer 147 reacted equally efficiently. By way of mechanism, we presume that the action of Li2CO3 on MeOH induces the formation of some MeOLi, which then adds nucleophilically to the strained cyclopentanone carbonyl.  This would produce hemiketal 148, which on the basis of precedent35 may be anticipated to fragment rapidly as shown.   Scheme 26. Synthesis of 149 and 150  Fragmentation product 149 was sparingly soluble in common organic solvents, and the majority of it was conveniently purified by way of trituration with Et2O.  Such a treatment caused dissolution of some 149 in the ether phase.  Therefore, the ether extract was concentrated and the residue was subjected to flash column chromatography to retrieve an additional 10-20% of 149.  In this manner, chromatography of the bulk of 149 was avoided.  It is worthy of mention that contrary to 149, the trans-diastereomer (the isomer originating from DIBAL reduction product 133) is readily soluble in common organic solvents, necessitating a full chromatographic purification.  This is one of the respects in which 149 offered advantages over its diastereomer.  30  Even more dramatic was the fact that whereas a subsequent Bom protection of the OH group in 149 proceeded efficiently to afford 151 in 91% yield after 48 h, the analogous protection of 150 was problematic, surrendering the requisite 152 in no more than 35% yield.  Furthermore, for reasons that remain unclear, the blocking of 150 stalled at about 30-35% conversion and none of the remedies thus far examined succeeded in forcing the reaction to completion.  This included the use of excess Bom-Cl, of tetrabutylammonium iodide as a promoter, and operation at higher temperatures.  A final complication was that chromatographic purification of 152 was difficult as its Rf value was very similar to that of the starting material.   Scheme 27. Bom protection of 149 and 150   On a final note, the foregoing protection step required carefully dried 1,2-DCE (freshly distilled from CaSO4) and DIPEA (freshly distilled from CaH2).  A reaction carried out with commercial materials afforded the unanticipated product 153, which we surmise to ensue through in situ hydrolysis of Bom-Cl, addition of 149 to the resultant formaldehyde, and Bom protection of a transient hemiacetal (Scheme 28)  31   Scheme 28. Product of Bom protection, 153, using commercial materials    The successful conclusion of this initial phase of the research had secured a robust avenue to key intermediate 151.  This enabled the launching of an investigation aiming to advance that substance to a more highly oxygenated TTX precursor.  It will be recalled that the synthetic plan called for transforming 154 into 155, which then would undergo two dihydroxylation reactions.  The next subgoal of the work was thus diene 155, which we intended to reach through Wittig reaction of 154.  Thus, 151 was treated with TBAF to release the silyl protecting group and subsequently oxidized to the enone using the Parikh-Doering procedure.  Attempts to carry out a Wittig methylenation of 154 met with failure.  It transpired that contact of 154 with the phosphonium ylide at -78 °C triggered aromatization to 156 in less than five minutes (Scheme 29).   Scheme 29. Hypothetical route to diene 155  32   The mechanism of this pernicious side reaction presumably involves deprotonation of either the ester or the nitrile, followed by expulsion of the acetamide and prototropic isomerization of the resultant 157 (Scheme 30).  A corrective measure would have to suppress enolization, an objective that might be attainable by reduction to non-deprotonatable entity.  However, it was impossible at this stage to determine which functionality, ester or nitrile, was responsible for aromatization.  It was technically easier to reduce the ester: we thus chose to convert 151 into 159 prior to methylenation.  We were not to regret this course of action.   Scheme 30. Aromatic product, 158, arising from Wittig reaction of 154    Reduction of 151 with LiBH4 provided 159 (Scheme 31).  The reaction proceeded uneventfully, but it was sluggish and required occasional addition of borohydride over 24 hours. The emerging primary alcohol was blocked with a second Bom group, at which juncture removal of the TBDPS protection was attempted.  Significant decomposition was observed when 160 was treated with either HF•pyridine or TBAF (the product was obtained in about 20 % yield). However, TBAF buffered with acetic acid gave 161 in good yield (84 % over 2 steps).  The 33  oxidation of 161 to the corresponding ketone was less than straightforward and required further experimentation. Surprisingly, 161 was immune to the action of the Parikh-Doering reagent,  even though the method had performed well with the alcohol arising from deprotection of 151.  Activated MnO2 was moderately successful, giving partial conversion with some loss of material.  On the other hand, Dess-Martin periodinane (DMP) cleanly delivered 162 in 94% yield in only 10 minutes.  It should be stressed that although crude 162 appeared very clean by proton nmr spectroscopy, purification is required before DMP oxidation. Failure to do so causes significant losses to decomposition.  Delightfully, ketone 162 smoothly underwent Wittig olefination giving diene 163 as the sole product. This suggests that the aromatization problem was imputable to the ester functionality.   Scheme 31. Synthesis of diene, 163   The final stage of the present investigation focused on the hydroxylation of 163. Central to our planning was the important observation by Kishi and collaborators that allylic hydroxyl or alkoxy – but not acyloxy – groups exhibit a significant anti-directing effect in osmylation reactions (Kishi effect).39  For instance, cyclohexenol 164 is selectively dihydroxylated to diol 34  165 (Scheme 28, dr = 10:1).  Although several hypotheses have been advanced to rationalize this phenomenon, no universal agreement has yet been reached.40    Scheme 32. Osmylation directed by the Kishi effect    We anticipated that the first osmylation of 163 would take place at the exo-methylene unit, and this for steric reasons.41  The Kishi effect of the neighboring OBom group would direct reaction from the bottom face face of the  bond (as drawn in Scheme 33).  This would set up the correct configuration of the tertiary alcohol.  The stereochemistry of the osmylation of the internal olefin is a more complex issue.  The Kishi effect of the tertiary oxygen substituent (may that be an OH or a protected form thereof) should guide the incoming OsO4 to the top face of the  bond, producing compound 166 with the correct configuration of all oxygenated centers, as required for 1.  On the other hand, the work of Donohoe suggests that acetamides can exert a syn-directing effect in osmylation reactions.42 At the onset of the work detailed below, there was no indication available to us regarding the relative strength of the two opposing effects.  Only experiment could provide a sensible answer.  35   Scheme 33. Osmylation directed by the Kishi effect   Parallel research had afforded us with some compound 168, which is an analog of 163 displaying a primary OMom group in lieu of OBom.  This material constituted a valuable test bed for the osmylation reaction, and indeed, it proved to be a competent substrate for dihydroxylation under Upjohn conditions 43  (cat. OsO4, NMO).  As predicted, only the exomethylene segment reacted, providing a diol, which was protected as an acetonide (2,2- dimethoxypropane, catalytic pTsOH) to give 169.  No evidence could be garnered for the presence of a diastereomeric diol.  Extensive 1D and 2D NMR spectroscopic analysis of 169 confirmed that the Kishi effect had controlled the stereochemical outcome of the reaction.  The nuclear Overhauser correlations (2D-NOESY) shown in Scheme 34 with double-headed arrows were particularly diagnostic, and permitted an accurate assignment of the chemical shifts of all protons in the molecule.   36   Scheme 34. Osmylation directed by the Kishi effect   In a like manner, the Bom substrate 163 underwent Upjohn osmylation and acetonide formation to furnish 170 as the sole product.  Compound 170 was not analyzed as thoroughly as 169; however, the chemical shifts and coupling constants of all ring protons were virtually identical to those of 169, strongly suggesting that the dihydroxylation had occurred in an anti- sense directed by the Kishi effect.  In any event, corroboration of this assignment would be forthcoming at the stage of an even more advanced intermediate. Attempts to dihydroxylate the remaining  bond in 170 using the Upjohn method failed (no reaction), but the traditional stoichiometric osmylation in pyridine as the solvent was successful.  Perhaps due to steric hindrance, the reaction was slow, requiring at least four days for full conversion.  Moreover, the osmate ester underwent cleavage with difficulty.  Multiple treatments with aqueous sodium thiosulfate solution were necessary to induce full release of the osmium.  The emerging diol was then converted to its acetonide, 171, again using 2,2- dimethoxypropane and pTsOH.  While the acetonide protection of the diol arising from 37  dihydroxylation of the exo-methylene proceeded efficiently, acetonide protection of the internal diol proved problematic. Conversion to 171 was poor when the reaction was not performed in thoroughly anhydrous acetone. Best results were obtained when acetone was distilled over calcium sulfate and added immediately to the reaction. Substituting PPTS for pTsOH resulted in no conversion to 171. Furthermore, using a less hygroscopic solvent such as dichloromethane with pTsOH resulted in decomposition of 171.  In any event, the ultimate acetonide emerged as a single stereoisomer. The configuration of 171 was unknown at this stage.  Consequently, the compound is rendered in Scheme 35 without the configuration of the newly installed oxygen atoms.   Scheme 35. Synthesis of 171   The next steps of the synthesis targeted formation of vinyl ether 176 (Scheme 36), which appeared to be an ideal substrate for the introduction of the remaining oxygen functionalities.  The compound was envisaged to ensue through release of the Bom groups in 171, followed by selective oxidation of the primary alcohol, cyclization of the resultant aldehyde to a hemiacetal, and dehydration of the latter.  It should be noted that a related vinyl ether was a valuable intermediate in the Isobe synthesis of 1 (cf. Scheme 8).  In accord with the foregoing, concurrent hydrogenolysis of the Bom groups of 171 in the presence of Pearlman’s catalyst afforded 172 38  (Scheme 36).  Interestingly, conduct of this reaction on scales above 100 mg gave what appeared to be a metastable formaldehyde hemiacetal such as 173 or 174, which could be restored to the expected diol by stirring with silica gel in EtOAc.   Scheme 36. Bom deprotection via hydrogenation   The deprotection product was oxidized selectively at the primary alcohol using DMP to give hemiacetal 175. Dehydration of this compound could be accomplished at -78 oC with Tf2O using 2,6-lutidine base. Weaker dehydrating agents such as MsCl and TFAA did not undergo fruitful reaction with 175.   Scheme 37. Synthesis of vinyl ether     The rigid structure of 176 lent itself nicely to a thorough NMR analysis aiming to determine its precise configuration, thereby ascertaining the stereochemical course of the two 39  prior osmylation steps.  This exercise led to the conclusion that whereas the first reaction had indeed occurred in a Kishi mode, the second one had taken place with Donohoe selectivity.  Indeed, a diagnostic W-type coupling was detected between protons Ha and Hb (Figure 3. 4J = 1.6 Hz), confirming that no isomerization of the nitrile had occurred at any prior stage.  More   Figure 3. Spectral properties of compound 176   significantly, the diagnostic NOESY signals rendered in Figure 3 with double-headed curved arrows are consistent only with the shown configuration.  Evidently, hydrogen bonding of the acidic NH in the acetamide segment to the osmium tetroxide-pyridine complex, as invoked by Donohoe, 44  and as rendered in Figure 4 (formal charges omitted for clarity), constitutes a stronger directing force than Kishi’s stereoelectronic effect.  Therefore, compounds 171-176 are more accurately represented as seen in Scheme 38.   40   Figure 4. Possible hydrogen bonding between osmium salt and the acidic NH   Future work toward tetrodotoxin will have to circumvent the syn selectivity of the second osmylation step.  Possible remedies include the exchange of an N-acetyl group with an alternative blocking unit that lacks H-bond donor ability (e.g., a phthalimide), or temporary N-    Scheme 38. Actual configuration of compounds 171-176   alkylation of the acetamide.  Alternatively, a different syn-dihydroxylation technique, such as the Prevost reaction, 45 might be employed to take advantage of the hydrogen-bonding ability of the amide.   41     Scheme 39. Possible hydrogen bonding between osmium salt and the acidic NH    3. Conclusion  In the preceding pages, we have demonstrated a robust synthesis of key intermediate 170, and have outlined a route by which it might be advanced to the natural product tetrodotoxin. The elaboration of 170 to 176 allowed examination of the configuration resulting from osmium- promoted dihydroxylation of the internal olefin of 170, which proceeded with Donohoe selectivity. 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Soc. 1958, 80, 209.         46  Appendix A: Experimental Protocols  Unless otherwise indicated, 1H (300 MHz) and 13C (75 MHz) NMR spectra were obtained from CDCl3 solutions at room temperature on a Bruker Avance II 300 instrument. Chemical shifts are reported in parts per million (ppm) on the δ scale and coupling constants, J, are in hertz (Hz). Multiplicities are reported as “s” (singlet), “d” (doublet), “t” (triplet), “q” (quartet), “dd” (doublet of doublets), “m” (multiplet), “c” (complex), “br” (broad), “ABq” (AB quartet), “app” (apparent). 2D NMR spectra were recorded at 400 MHz (1H). Infrared (IR) spectra (cm-1) were recorded on a Perkin-Elmer Frontier instrument. High- resolution mass spectra (m/z) were obtained in the electrospray (ESI) mode or atmospheric pressure chemical ionization (APCI) mode on a Micromass LCT mass spectrometer by the UBC Mass Spectrometry laboratory.  Melting points were measured on a Mel-Temp apparatus and are uncorrected.  All reagents and solvents were commercial products and used without further purification except THF (freshly distilled from Na/benzophenone under N2), CH2Cl2 (freshly distilled from CaH2 under N2), CHCl3 (washed with H2O, treated with K2CO3 (s), freshly distilled from Na2SO4 under Ar), MeOH (freshly distilled from magnesium and iodine under Ar), 1,2-dichloroethane (freshly distilled from CaSO4 under Ar), diisopropylethylamine (distilled from CaH2 under Ar),  and acetone (freshly distilled from CaSO4 under Ar). Commercial n-BuLi was titrated against diphenylacetic acid until a yellow color persisted. Flash chromatography was performed on Silicycle 230 – 400 mesh silica gel. All reactions were performed under argon atmosphere in oven dried flasks equipped with TeflonTM stirbars. All flasks were fitted with rubber septa for the introduction of substrates, reagents, and solvents via syringe. 47   A.1 Preparation of 132 A MeCN (20.0 mL) solution of 131 (9.1 g, 55.0 mmol, 1.0 equiv.) was added over 3 h (syringe pump) to a solution of DIB (23.9 g, 74.0 mmol, 1.3 equiv.) and TFA (6.4 mL, 82.5 mmol, 1.5 equiv. vs. DIB) in MeCN (480 mL), at room temperature under argon with good stirring. At the end of the addition, the mixture was concentrated under reduced pressure and the residue was diluted with toluene (10.0 mL). The suspension was concentrated and the procedure was repeated twice to azeotropically remove all residual TFA. The brown residue was filtered through a silica pad (55.0 g) using first 300 mL of Et2O (removal of brown tar and iodobenzene) and then 300 mL of Et2O/ MeCN (2.5:1, elution of the product). Concentration afforded a brown solid, which was re-filtered through fresh silica gel using the same procedure. The solid residue was taken up with 20.0 mL of EtOAc and kept at −20 °C for 5 h. The resulting precipitate was essentially pure product 132. A total of 7.44 g (33.3 mmol, 61%) of 132 was obtained as an off- white solid. A recrystallized sample (EtOAc/Hex = 2:1) of 132 had m.p. 100-102 °C. 1H NMR (acetone-d6): δ 7.54 (br s, 1H); 7.17 (d, J = 10.3, 2H); 6.15 (d, J = 10.3, 2H); 3.62 (s, 3H); 3.02 (s, 2H); 1.88 (s, 3H). 13C NMR (acetone-d6): δ 184.3,169.4, 169.0, 148.8, 128.1, 53.3, 51.1, 41.7, 22.5. IR (film, cm-1): ν 3200; 1738; 1666. HRMS: calc. for C11H13NO4Na [M + Na] + 246.0737; found 246.0742. m.p. 100-102 °C. 48   Scheme 40. 1H NMR spectrum of 132   Scheme 41. 13C NMR spectrum of 132   49   A.2. Preparation of 145 To a dry THF (135 mL) solution of 132 (6.0 g, 27 mmol, 1.0 equiv.) under argon in a 500 mL roundbottom flask immersed in a cold-water bath, was added the (S)-CBS catalys (0.075 g, 0.27 mmol, 0.01 equiv.). BH3 (~10 M in methyl sulfide, 2.7 mL, 27 mmol, 1.0 equiv.) was carefully added dropwise via syringe to the rapidly stirring solution. The mixture was stirred for 45 minutes at room temperature. Following, the septum was removed and MeOH (3.3 mL, 81 mmol, 3.0 equiv.) was carefully added dropwise via syringe at 0 oC. The reaction was warmed to room temperature and the solvent was removed under reduced pressure by rotary evaporation, followed by drying under the high vacuum pump until all residual solvent was removed. The residue was taken up in dry dichloromethane (100 mL) and to this solution was added imidazole (1.83 g, 27 mmol, 1.0 equiv.) followed by TBDPSCl (7.0 mL, 27 mmol, 1.0 equiv.) at room temperature under argon. The mixture was stirred for 12 hours (overnight) and 0.05N HCl (100 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (2x100 mL). The organic layers were combined, dried with Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in THF (100 mL), followed by the addition of H2O (100 mL) and LiOH monohydrate (3.4 g, 81 mmol, 3.0 equiv.). The reaction was stirred for 1.5 hours at room temperature and then most of the THF was removed by rotary evaporation. Diethyl ether (100 mL) was added to the aqueous suspension and the layers were separated (to remove organic byproducts from previous steps since our product should be in the aqueous layer as the carboxylate). The aqueous layer was acidified with a 1:1 (vol/vol) mixture of AcOH/H2O (30 mL) and extracted with EtOAc (3x100 mL, check pH of 50  aqueous layer). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. Coevaporation with toluene was carried out multiple times (3-4 times) to remove all residual acetic acid. The residue was taken up in dry THF(70 mL) and to this was added carbonyl diimidazole (CDI) (4.36 g, 27 mmol, 1.0 equiv.) at room temperature under argon and the mixture was stirred for 1 hour. Nitromethane (8.5 mL, 0.16 mol, 6.0 equiv.) and potassium t-butoxide (12.0 g, 0.11 mol, 4.0 equiv.) were added at room temperature and the resulting mixture was stirred for 2 hours. Following, a 1:1 (vol/vol) mixture of AcOH/H2O (30 mL) was added and the THF was removed under reduced pressure.  H2O (70 mL) and dichloromethane (100 mL) were added to the suspension and the layers were separated. The aqueous layer was extracted with dichloromethane (2x100 mL) and the organic layers were combined, dried over Na2SO4, filtered, and concentrated. The residue was purified by gradient column chromatography (1:1 EtOAc/Hexanes-7:3 EtOAc/Hexanes) to obtain pure 7.9 g (16 mmol, 56% over 4 steps) of 145 (dr 4:1) as a light yellow oil.  1H NMR (acetone-d6): δ 7.75-7.72 (m, 4H); 7.53-7.37 (m, 6H); 6.20-6.14 (m, 2H); 5.88-5.82 (m, 2H); 5.57 (s, 2H); 4.61-4.59 (m, 1H); 3.22 (s, 2H); 1.88 (s, 3H); 1.07 (s, 9H). 13C NMR (acetone-d6): δ 195.30, 170.78, 136.62, 134.52, 130.90, 130.16, 129.71, 128.76, 85.15, 64.37, 52.63, 48.39, 27.39, 23.75, 19.77.  IR (film, cm-1): ν 1736, 1700, 1656, 1558. HRMS: calc. for C27H32N2O5SiNa [M + Na] + 515.1978; found 515.1962.   51   Scheme 42. 1H NMR spectrum of 145  Scheme 43. 13C NMR spectrum of 145 52   A.3 Preparation of 146 To a dry chloroform solution (25 mL) of 145 (3.5 g, 7.1 mmol, 1.0 equiv.) was added solid Cu(OAc)2•H2O (0.072 g, 0.36 mmol, 0.05 equiv.) followed by N-ethylpiperidine (0.10 mL, 0.71 mmol, 0.10 equiv.) at room temperature under argon. The reaction mixture was stirred for 168 hours at 30 °C and then concentrated under vacuum. The crude residue was dissolved in minimal ethyl acetate and filtered through a short plug of silica gel, rinsing with ethyl acetate. The solvent was removed under vacuum and the reside was purified by flash column chromatography on silica gel (EtOAc/Et2O 1:4) to give 2.0 g (4.3 mmol, 60%) of 146 as a pale yellow foam. 1H NMR: δ 7.71-7.60 (m, 4H); 7.53-7.37 (m, 6H); 6.18 (dd, J = 10.0, 5.9, 1H); 5.60 (d, J = 10.0, 1H); 5.75 (br, 1H); 5.05 (app s, 2H); 4.24 (d, J = 5.9, 1H); 4.06 (d, J = 18.4, 1H); 2.97 (d, J = 18.4, 1H); 1.95 (s, 3H); 1.10 (s, 9H).   13C NMR: δ 191.42, 170.84, 161.68, 135.85, 135.60, 135.55, 134.90, 133.40, 132.61, 132.45, 130.47, 130.38, 128.14, 128.02, 85.15, 63.17, 54.23, 52.32, 26.98, 23.98, 19.10.   IR (film, cm-1): v 1747, 1660, 1599, 1538. HRMS: calc. for C27H31N2O4Si [M + H] + 475.2053; found 475.2059.      53  Scheme 44. 1H NMR spectrum of 146  Scheme 45. 13C NMR spectrum of 146   54   A.4 Preparation of 149 To a dry methanol solution (63.0 mL) of 146 (1.50 g, 3.16 mmol, 1.0 equiv.) at room temperature under argon was added solid Li2CO3 (0.117 g, 1.58 mmol, 0.5 equiv.) in one portion. The reaction was stirred for 1.5 h and the solvent was removed under vacuum to afford a light yellow oil. The residue was purified by column chromatography on silica gel (EtOAc/Hex 1:1) to give 1.32 g (2.60 mmol, 83%) of 149 as an off-white solid. 1H NMR: δ 7.72-7.68 (m, 4H); 7.50-7.39 (m, 6H); 5.84 (br d, J = 10.3, 1H); 5.76 (br s, 1H); 5.72 (dd, J = 10.2, 2.6, 1H); 4.45-4.41 (m, 1H); 4.35 (d, J = 3.1, 1H); 4.07-4.03 (m, 1H); 3.67 (s, 3H); 3.45 (d, J = 16.1, 1 H); 2.87 (d, J = 16.1, 1 H); 1.98 (s, 3H); 1.10 (s, 9H). 13C NMR: δ 170.61, 170.28, 135.91, 135.69, 133.13, 132.00, 130.23, 130.22, 128.05, 127.90, 127.83, 117.43, 71.59, 70.22, 55.09, 51.87, 39.17, 38.67, 26.93, 24.07, 19.25. IR (film, cm-1): ν 2247, 1719, 1639. HRMS: calc. for C28H34N2O5SiNa [M + Na] + 529.2135; found 529.2122. m.p. 166-168 °C.   55  Scheme 46. 1H NMR spectrum of 149   Scheme 47. 13 CNMR spectrum of 149   56   A.5 Preparation of 151 To a 1,2-dichloroethane (DCE) solution (3.0 mL) of 149 (0.303 g, 0.60 mmol, 1.0 equiv.) at room temperature under argon was added BOMCl (0.12 mL, 0.90 mmol, 1.5 equiv.) followed by DIPEA (0.19 mL, 1.08 mmol, 1.8 equiv.). The reaction mixture was heated to 75 °C and stirred for 48 h. After cooling to room temperature, the mixture was diluted with DCM (40 mL) and poured into a saturated solution of NH4Cl (50 mL).  The organic layer was separated and the aqueous layer was extracted with DCM (2x30 mL). The organic layers were combined, washed with H2O (150 mL), dried over Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (Et2O/Hex 7:3) to give 0.341 g (0.55 mmol, 91%) of 151 as a colorless oil. 1H NMR: δ 7.73-7.69 (m, 4H); 7.48-7.30 (m, 11H); 6.27 (br s, 1H); 5.77 (d, J = 10.3, 1H); 5.56 (dd, J = 10.3, 3.42, 1H); 4.74-4.67 (m, 4H); 4.53-4.49 (m, 1H); 4.46-4.43 (m, 1H); 4.16-4.13 (m, 1H); 3.68 (s, 3H); 3.28 (d, J = 15.6, 1H); 2.94 (d, J = 15.6, 1H); 1.99 (s, 3H); 1.10 (s, 9H).  13C NMR: δ 170.85, 170.23, 137.28, 135.99, 135.88, 133.62, 133.03, 130.36, 130.07, 130.00, 129.05, 128.48, 128.05, 127.89, 127.85, 127.80, 118.14, 94.47, 77.46, 76.43, 69.95, 68.25, 54.70, 54.52, 51.95, 39.98, 36.72, 30.39, 26.99, 24.05, 19.30. IR (film, cm-1): ν 2247, 1737, 1665. HRMS: calc. for C36H42N2O6SiNa [M + Na] + 649.2710; found 649.2710. 57  Scheme 48. 1H HNMR spectrum of 151  Scheme 49. 13C NMR spectrum of 151   58   A.6 Preparation of 159 To a THF (3.0 mL) solution of 151 (0.227, 0.36 mmol, 1.0 equiv.) at room temperature under argon was added solid LiBH4 (0.079 g, 3.6 mmol, 10.0 equiv.) and the reaction was stirred for 24 h. The reaction mixture was cooled to 0 °C and a saturated solution of NH4Cl (2.0 mL) was carefully added (GAS EVOLUTION!). The reaction was stirred at room temperature for a further 30 min and then diluted with EtOAc (20 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2x20 mL). The organic layers were combined, dried over Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (EtOAc) to give 0.152 g (0.25 mmol, 70%) of 159 as a colorless oil.   1H NMR: δ 7.73-7.68 (m, 5H); 7.44-7.24 (m, 10H); 6.73 (br s, 1H); 5.82 (d, J = 10.4, 1H); 5.52 (dd, J = 10.4, 3.5, 1H); 4.74-4.45 (m, 5H); 4.35-4.33 (br m, 1H); 4.15-4.12 (br m, 1H); 3.90-3.68 (br m, 2H); 2.42-2.33 (m, 1H); 2.20-2.12 (m, 1H); 1.97 (s, 3H); 1.09 (s, 9H). 13C NMR: δ 170.59, 137.35, 136.02, 135.90, 133.63, 133.09, 131.23, 130.06, 129.96, 128.46, 128.17, 127.94, 127.91, 127.82, 127.77, 119.13, 94.32, 77.43, 77.13, 69.93, 67.32, 58.40, 55.89, 39.21, 36.26, 26.99, 24.23, 19.28.  IR (film, cm-1): ν 2248, 1662. HRMS: calc. for C35H42N2O5SiNa [M + Na] + 621.2761; found 621.2759. 59  Scheme 50. 1H NMR spectrum of 159  Scheme 51. 13C NMR spectrum of 159 60   A.7 Preparation of 161 To a 1,2-dichloroethane solution (7.0 mL) of 159 (0.473 g, 0.79 mmol, 1.0 equiv.) under argon at room temperature was added DIPEA (0.34 mL, 1.98 mmol, 2.5 equiv.) followed by BOMCl (0.22 mL, 1.58 mmol, 2.0 equiv.). The reaction was stirred at 50 °C for 24 hours and then cooled to room temperature, diluted with DCM (20 mL), and poured into a saturated solution of NH4Cl (15 mL). The organic layer was separated and the aqueous layer was extracted with DCM (2x20 mL). The organic layers were combined, washed with H2O (50 mL), dried over Na2SO4, and concentrated under vacuum to afford a pale yellow oil. The crude mixture was taken up in THF (5.0 mL) at room temperature under argon and to it was added a 1 M solution of AcOH in THF (2.0 mL, 1.98 mmol, 2.5 equiv.) followed by a 1 M solution of TBAF in THF (2.0 mL, 1.98 mmol, 2.5 equiv.). The reaction mixture was stirred for 15 hours, and a saturated solution of NaHCO3 (10 mL) was then added. The mixture was diluted with EtOAc (20 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (2x20 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford a pale yellow oil. The residue was purified by column chromatography on silica gel (EtOAc) to give 0.329 g (0.66 mmol, 84% over 2 steps) of 161 as a colorless oil. 1H NMR: δ 7.40-7.28 (m, 10H); 6.64 (brs, 1H); 6.00 (dd, J = 10.2, 4.4, 1H); 5.81 (d, J = 10.2, 1H); 4.87 (brs, 2H); 4.78 (brs, 2H); 4.67 (brs, 2H); 4.62 (brs, 2H); 4.53 (d, J = 3.0, 1H); 4.28- 4.18 (m, 1H); 4.14-4.11 (m, 1H); 3.89-3.63 (m, 3H); 2.58-2.49 (m, 1H); 2.28-2.20 (m, 1H); 1.92 (s, 3H). 61  13C NMR: δ 170.37, 137.38, 136.99, 130.23, 128.60, 128.54, 128.31, 128.02, 127.99, 127.75, 118.49, 94.97, 94.76, 70.28, 70.05, 65.38, 63.95, 54.78, 36.76, 35.85, 24.15. IR (film, cm-1): ν 2244, 1658. HRMS: calc. for C27H32 N2O6Na [M + Na] + 503.2158; found 503.2155.          62  Scheme 52. 1H NMR spectrum of 161  Scheme 53. 13C NMR spectrum of 161    63   A.8 Preparation of 162 To a DCM solution (10 mL) of 161 (0.660 g, 1.37 mmol, 1.0 equiv.) at room temperature was added solid Dess-Martin periodinane (0.874 g, 2.06 mmol, 1.5 equiv.) and the mixture was stirred for 30 minutes. An aqueous solution of Na2S2O3/NaHCO3 (7:1, 20 mL) was added and the mixture was diluted with Et2O (20 mL) and stirred until the layers turned clear (around 15 minutes). The layers were separated and the aqueous layer was extracted with Et2O (2x20 mL). The organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford an off-white oil. The residue was purified by column chromatography on silica gel (EtOAc/Hex 7:3) to give 0.618 g (1.29 mmol, 94 %) of 162 as a colorless oil. 1H NMR: δ 7.42-7.30 (m, 10H); 7.16 (br s, 1H); 6.92 (d, J = 10.6, 1H); 6.07 (d, J = 10.6, 1H); 5.03 (d, J = 7.0, 1H); 4.88-4.83 (m, 4H); 4.73 (d, J = 4.3, 1H); 4.67-4.63 (m, 3H); 4.45 (d, J = 4.3, 1H), 3.93-3.78 (m, 2H); 2.53-2.48 (m, 2H); 1.95 (s, 3H). 13C NMR: δ 190.99, 170.67, 154.32, 137.51, 137.35, 128.82, 128.64, 128.40, 128.26, 128.09, 127.91, 126.80, 117.10, 95.32, 93.95, 71.53, 70.69, 70.43, 64.14, 55.53, 39.78, 38.30, 23.85.  IR (film, cm-1): ν 2246, 1678. HRMS: calc. for C27H30N2O6Na [M + Na] + 501.2002; found 501.1989.   64   Scheme 54. 1H NMR spectrum of 162   Scheme 55. 13C NMR spectrum of 162   65   A.9 Preparation of 163 To a THF solution (3.0 mL) of methyltriphenylphosphonium bromide (0.747 g, 2.09 mmol, 5.0 equiv.) under argon at room temperature was carefully added n-BuLi (2.5 M in hexanes) dropwise (0.50 mL, 1.25 mmol, 3.0 equiv.) and the mixture was stirred for 1 hour. A THF solution (3.0 mL) of 162 (0.200 g, 0.42 mmol, 1.0 equiv.) was added dropwise to the reaction mixture at −78 °C. Upon completion of the addition, the reaction was stirred at −78 °C for 5 min and then warmed to 0 °C and further stirred for 2.5 h. A saturated solution (20 mL) of NH4Cl was added and the reaction was warmed to room temperature. The mixture was diluted with Et2O (20 mL) and the layers were separated. The aqueous layer was extracted with Et2O (2x20 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford a pale yellow oil. The residue was purified by column chromatography on silica gel (Et2O) to give 0.173 g (0.36 mmol, 87%) of 163 as a colourless oil. 1H NMR: δ 7.41-7.28 (m, 10H); 6.91 (brs, 1H); 6.23 (d, J = 10.3, 1H); 5.80 (d, J = 10.3, 1H); 5.32 (brs, 1H); 5.27 (brs, 1H); 4.89-4.78 (m, 5H); 4.71-4.59 (m, 5H); 3.86-3.82 (m, 2H); 2.50- 2.47 (m, 2H); 1.95 (s, 3H);. 13C NMR: δ 170.09, 137.43, 137.32, 136.39, 131.26, 128.60, 128.49, 128.22, 127.99, 127.89, 127.84, 127.78, 119.45, 118.57, 94.92, 91.15, 71.64, 69.95, 69.93, 64.31, 55.64, 38.61, 37.19, 24.18. IR (film, cm-1): ν 2243, 1659. HRMS: calc. for C28H32N2O5Na [M + Na] +499.2209; found 499.2189. 66   Scheme 56. 1H NMR spectrum of 163     Scheme 57. 13C NMR spectrum of 163    67    A.10 Preparation of 170 To an acetone solution (1.0 mL) of 163 (0.054 g, 0.11 mmol, 1.0 equiv.) at room temperature was added solid NMO (0.027 g, 0.22 mmol, 2.0 equiv.) and OsO4 (4 wt. % in H2O) (0.03 mL, 4.7 µmol, 0.05 equiv.) via syringe. The same syringe was used to draw up H2O (0.2 mL) which was added to the reaction mixture and the mixture was stirred for 12 hours. Solid sodium bisulfite (0.011 g, 0.11 mmol, 1.0 equiv.) was added and the mixture was stirred for 30 min, filtered over celite, and the filter cake was washed with acetone. The filtrate was concentrated under vacuum to afford an off-white oil. To a dry acetone solution (1.0 mL) of the crude residue was added solid p-toluenesulfonic acid monohydrate (2 µg, 0.01 mmol, 0.1 equiv.) and 2,2- dimethoxypropane (0.08 mL, 0.66 mmol, 6.0 equiv.) at room temperature under argon. The reaction was stirred for 2 h and quenched with a saturated solution of NaHCO3 (15 mL). The mixture was diluted with DCM (20 mL) and the layers were separated. The aqueous layer was extracted with DCM (2x10 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford a colorless oil. The residue was purified by column chromatography on silica gel (EtOAc/Hex 7:3) to give 0.056 g (0.10 mmol, 90% over 2 steps) of 170 as a colorless oil.  1H NMR: δ 7.40-7.30 (m, 10H); 5.99 (d, J = 10.2, 1H); 5.92 (brs, 1H); 5.83 (d, J = 10.2, 1H); 4.98 (d, J = 6.9, 1H); 4.91 (d, J = 6.9, 1H); 4.75-4.60 (m, 6H); 4.57 (d, J = 3.4, 1H); 4.52 (d, J = 9.3, 1H); 4.33 (d, J = 3.4, 1H); 3.97 (d, J = 9.3, 1H); 3.80-3.68 (m, 2H); 2.64-2.49 (m, 1H); 2.31-2.18 (m, 1H); 1.87 (s, 3H); 1.47 (s, 3H); 1.43 (s, 3H). 68  13C NMR: δ 170.22, 137.64, 137.20, 132.70, 129.59, 128.53, 128.49, 127.99, 127.92, 127.88, 127.77, 118.58, 109.41, 95.19, 94.74, 80.22, 74.75, 70.24, 69.69, 69.00, 63.75, 56.77, 38.08, 35.63, 27.37, 26.23, 23.71. IR (film, cm-1): ν 2243, 1659. HRMS: calc. for C31H38N2O7Na [M + Na] + 573.2577; found 573.2573.        69   Scheme 58. 1H NMR spectrum of 170  Scheme 59. 13C NMR spectrum of 170   70   A.11 Preparation of 171 To a pyridine solution (2.0 mL) of 170 (0.320 g, 0.58 mmol, 1.0 equiv.) was added solid OsO4 (0.221 g, 0.87 mmol, 1.5 equiv.) and the reaction was stirred for 96 h at room temperature. A saturated sodium bisulfite solution (5 mL) and acetone (5 mL) were added to the reaction and the mixture was stirred for 4 h. The reaction mixture was diluted with EtOAc (10 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (2x10 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford a light yellow oil. To a dry acetone solution (2.0 mL) of the crude residue was added solid p-toluenesulfonic acid monohydrate (0.011 g, 58 µmol, 0.1 equiv.) and 2,2-dimethoxypropane (0.43 mL, 3.48 mmol, 6.0 equiv.) at room temperature under argon. The reaction was stirred for 2 h and quenched with a saturated solution of NaHCO3 (30 mL). The mixture was diluted with DCM (30 mL) and the layers were separated. The aqueous layer was extracted with DCM (2x20 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford a light, tan oil. The residue was purified by column chromatography on silica gel (EtOAc/Hex 1:1) to give 0.056g (61 % over 2 steps) of 171 as a clear, colourless oil. 1H NMR: δ 7.39-7.29 (m, 10H); 6.02 (brs, 1H); 5.01 (d, J = 5.4, 1H); 4.90-4.84 (m, 2H); 4.76- 4.54 (m, 7H); 4.44 (d, J = 6.5, 1H); 4.38 (d, J = 6.5, 1H); 4.34 (d, J = 5.4, 1H); 4.00 (d, J = 9.2, 1H); 3.78-3.65 (m, 2H); 2.90-2.77 (m, 1H); 2.20-2.04 (m, 1H); 1.92 (s, 3H); 1.62 (s, 3H); 1.47 (s, 3H); 1.44 (s, 3H); 1.42 (s, 3H).  71  13C NMR: δ 170.20, 137.84, 137.31, 128.45, 128.39, 128.03, 127.94, 127.74, 118.01, 110.23, 110.18, 94.70, 94.59, 81.75, 77.56, 77.13, 71.60, 69.95, 69.28, 67.77, 63.37, 56.32, 37.06, 36.82, 27.11, 26.50, 26.36, 24.79, 24.21. IR (film, cm-1): ν 2243, 1679. HRMS: calc. for C34H44N2O9Na [M + Na] + 647.2945; found 647.2946.      72  Scheme 60. 1H NMR spectrum of 171 Scheme 61. 13C NMR spectrum of 171   73   A.12 Preparation of 172 To a solution of 171 (0.180 g, 0.29 mmol, 1.0 equiv.) in EtOH at room temperature was added Pd(OH)2/C (10 %, 0.647 g, 0.46 mmol, 1.6 equiv.) and the reaction vessel was flushed with H2. The reaction was stirred for 12 h and the mixture was filtered over celite. The filtrate was concentrated under vacuum to give an off-white oil. The residue was purified by column chromatography on silica gel (MeOH/EtOAc 1:9) to give 0.089g (80 %) of 172 as a clear, colourless oil. 1H NMR: δ 6.12 (brs, 1H); 4.66 (d, J = 4.9, 1H); 4.54 (d, J = 9.2, 1H); 4.46 (apps, 2H); 4.23 (d, J = 4.9, 1H); 3.98 (d, J = 9.2, 1H); 3.77-3.71 (m, 3H); 2.73-2.64 (m, 1H); 2.16-2.10 (m, 1H); 2.00 (s, 3H); 1.66 (s, 3H); 1.47 (s, 3H); 1.46 (s, 3H); 1.44 (s, 3H). 13C NMR: δ 170.95, 118.05, 110.47, 110.31, 81.41, 77.60, 75.81, 67.92, 58.00, 46.40, 40.01, 38.77, 26.82, 26.62, 26.42, 24.75, 24.22. IR (film, cm-1): ν 2245, 1658. HRMS: calc. for C18H28N2O7Na [M + Na] + 407.1794; found 407.1797.  74   Scheme 62. 1H NMR spectrum of 172   Scheme 63. 13C NMR spectrum of 172   75   A.13 Preparation of 176 To a DCM solution (1.5 mL) of 172 (0.084 g, 0.22 mmol, 1.0 equiv.) was added solid Dess- Martin periodinane (0.139 g, 0.33 mmol, 1.5 equiv.) at room temperature and the reaction was stirred for 10 minutes. A solution of 7:1 (vol/vol) aqueous Na2S2O3:Na2HCO3 (2 mL) was added and the mixture was diluted with Et2O (5 mL).  The cloudy mixture was stirred rapidly until it turned clear (~15 min.).  The layers were separated and the aqueous layer was extracted with Et2O (2x5 mL).  The organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford an off-white oil. The residue was taken up in DCM (1.5 mL) and to it was added 2,6-lutidine (0.37 mL, 3.30 mmol, 15.0 equiv.) and Tf2O (1.0 M in DCM, 1.32 mL, 1.32 mmol, 6.0 equiv.), respectively, at -78 °C under argon.  The mixture was stirred for 2 hours followed by the addition of a solution of saturated NaHCO3 (aq.) (1.0 mL) and the reaction mixture was warmed to room temperature. The mixture was diluted with water (3.0 mL) and DCM (7.0 mL) and the layers were separated. The aqueous layer was extracted with DCM (2x5 mL) and the organic layers were combined, dried over Na2SO4, and concentrated under vacuum to afford pale, brown oil. The residue was purified by column chromatography on silica gel (EtOAc/Hex gradient 1:1-7:3) to give 0.48g (60 % over 2 steps) of 176 as a clear, colorless oil. 1H NMR (acetone-d6): δ 6.37 (d, J = 6.2, 1H); 5.67-5.65 (m, 2H); 4.94 (d, J = 5.3, 1H); 4.30- 4.27 (m, 2H); 3.89 (d, J = 5.3, 1H); 3.82-3.79 (m, 2H); 2.06 (s, 3H); 1.54 (s, 3H); 1.48 (s, 3H); 1.39 (s, 3H); 1.38 (s, 3H). 76  13C NMR (acetone-d6): δ 170.30, 142.56, 117.39, 112.22, 112.07, 101.69, 99.48, 80.36, 76.37, 74.92, 71.40, 68.28, 50.85, 28.30, 27.06, 26.23, 25.93, 25.25. IR (film, cm-1): ν 2246, 1668. HRMS: calc. for C18H24N2O6Na [M + Na] + 387.1532; found 387.1535. 77   Scheme 64. 1H NMR spectrum of 176   Scheme 65. 13C NMR spectrum of 176   78   Scheme 66. COSY spectrum of 176 (expansion) 79   Scheme 67. COSY spectrum of 176 (expansion) 80   Scheme 68. COSY spectrum of 176 (expansion) 81   Scheme 69. NOESY spectrum of 176      82   Scheme 70. NOESY spectrum of 176 (expansion) 83   Scheme 71. NOESY spectrum of 176 (expansion) 84   Scheme 72. NOESY spectrum of 176 (expansion) 85   Scheme 73. 1H NMR spectrum of 169   Scheme 74. 13C NMR spectrum of 169 86   Scheme 75. COSY spectrum of 169 87   Scheme 76. COSY spectrum of 169 (expansion) 88   Scheme 77. COSY spectrum of 169 (expansion) 89   Scheme 78. COSY spectrum of 169 (expansion) 90   Scheme 79. NOESY spectrum of 169 91   Scheme 80. NOESY spectrum of 169 (expansion) 92   Scheme 81. NOESY spectrum of 169 (expansion) 93   Scheme 82. NOESY spectrum of 169 (expansion) 94   Scheme 83. NOESY spectrum of 169 (expansion) 95   Scheme 84. NOESY spectrum of 169 (expansion) 96   Scheme 85. NOESY spectrum of 169 (expansion)        97  Scheme 86. Crude 1H NMR of 158  LRMS: found for C18H17NO5Na [M + Na] + 350.2.  

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