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I The synthesis of papyracillic acids: Application of the tandem chain extension-acylation reaction II Diastereoselective synthesis of beta-methyl-gamma-keto esters through the utility of an L- serine auxiliary
Jennifer R. Mazzone University of New Hampshire, Durham
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Recommended Citation Mazzone, Jennifer R., "I The synthesis of papyracillic acids: Application of the tandem chain extension- acylation reaction II Diastereoselective synthesis of beta-methyl-gamma-keto esters through the utility of an L-serine auxiliary" (2011). Doctoral Dissertations. 644. https://scholars.unh.edu/dissertation/644
This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. I. THE SYNTHESES OF PAPYRACILLIC ACIDS: APPLICATION OF THE
TANDEM CHAIN EXTENSION-ACYLATION REACTION
II. DIASTEREOSELECTIVE SYNTHESIS OF p-METHYL-y-KETO ESTERS
THROUGH THE UTILITY OF AN L-SERINE AUXILIARY
BY
Jennifer R. Mazzone
B.A., Assumption College, 2006
DISSERTATION
Submitted to the University of New Hampshire
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
in
Chemistry
December, 2011 UMI Number: 3500790
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DEDICATION
This dissertation is dedicated to my parents
Donna and Frank Mazzone
The admiration I have for you both far exceeds what I am able to express in words. Thank you for teaching me many lessons that could not be learned in a classroom.
in ACKNOWLEDGEMENTS
The entire Department of Chemistry at the University of New Hampshire has contributed to my graduate studies and facilitated the completion of this dissertation. I am especially indebted to my mentor and advisor, Professor Charles K. Zercher for accepting me into his research group and guiding me through this immense journey. Dr. Zercher's wealth of knowledge in the field of organic synthesis is a constant source of personal amazement. His consistent guidance, his dedication to teaching in the classroom and laboratory are gifts, which can never be repaid.
I am thankful to my committee members, Prof. Margaret E. Greenslade, Prof.
Richard P. Johnson, and Prof. Arthur Greenberg for their constructive criticism and insight into my research. I had the pleasure of taking classes with Prof. Gary R. Weisman and Prof. Roy P. Planalp. Their courses are among the best classes I attended. In addition the expertise and advice of faculty members, Prof. Edward H. Wong and Prof. Sterling A.
Tomelini are counted as valuable resources.
Thank you to Cindi Rohwer, Peg Torch, Jane Austin, Kristen Blackwell, and Bob
Constantine for keeping the chemistry department running smoothly! I am especially thankful to Dr. Patricia Wilkinson for her assistance with the NMR spectrometer and other instrumentation. Her friendly encouragement and advice was always appreciated.
Thank you to Prof. Glen P. Miller and John Briggs for running several of our samples on their X-ray crystal diffraction instrument. Thank you to Andy Hanneman, from the
Department of Molecular, Cellular, and Biomedical Sciences, for taking time to run a mass spec, for us on very short notice.
iv I am fortunate for my current group members, Carley Spencer, Deepthi Bhogadhi,
Matt Mower, and Peter Moran. They have offered support, discussed chemistry, and kept me company during many late nights in lab. Additional colleagues and friends, Justin
Massing, Barbra Li, Leon Wong, Nick Bencivenga, Joe Dun, Matt Young, and Greg
Bubnis have contributed to my experience as well. Thank you to Rajesh Thamatam for his advice and suggestions. His dedication to research inspired and strengthened my work ethic.
Early on the path as a graduate researcher, the most memorable experiences were followed by the support of my former group members. At pivotal moments Alex
Jacobine, Ian Taschner, and Tim Henderson offered indispensable advice and moral encouragement. I am especially thankful to Aida Ajaz for her friendship and steady support. I am most grateful she was brave enough to leave her home country and start a new life at UNH. We were destined to be lifelong friends.
I am most grateful to my parents, Donna and Frank Mazzone. Their personal sacrifice and example imparted high values on education and lifelong learning. I am thankful to my sister, Katherine Mazzone. Despite the miles that separate us, Katie was the one person who could make me laugh during the chaos and stress of this vast journey.
Finally, I would like to thank the National Institute of Health, the University of
New Hampshire Graduate School, and the Department of Chemistry for funding this research.
v TABLE OF CONTENTS
DEDICATION iii
ACKNOWLEDGEMENTS iv
LIST OF SCHEMES xiv
LIST OF FIGURES xxi
LIST OF TABLES xxiii
LIST OF ABBREVIATIONS xxv
ABSTRACT xxvii
CHAPTER PAGE
I. INTRODUCTION 1
Proteases 1
Peptide Isosteres 3
Zinc Carbenoid-Mediated Chain Extension Reaction 9
Synthesis of Ketomethylene Isosteres using the Chain Extension Reaction... 17
Formation of a-Functionalized y-Keto Carbonyls 18 i Formation of (3-Functionalized y-Keto Carbonyls 27
vi Natural Product Synthesis using the Chain Extension Reaction 31
II. SYNTHESES OF PAPYRACILLIC ACIDS: APPLICATION OF THE
TANDEM CHAIN EXTENSION-ACYLATION REACTION 39
Papyracillic Acids 39
Synthetic Approach 42
Tandem Chain Extension-Acylation Reaction using Maleic Anhydride 44
Other Anhydrides 54
Carbodiimide-Mediated Heterocycle Formation 59
Synthesis of Spiro-Fused Ketal Cores 64
Equilibration Studies 70
Stereochemical Assignment for the Major Diastereomer 74
Synthesis of Papyracillic Acid C 80
Synthesis of 4-ep/-Papyracillic Acid B and Papyracillic acid B 99
III. DIASTEREOSELECTIVE SYNTHESIS OF B-METHYL y-KETO ESTERS
THROUGH THE UTILITY OF AN L-SERINE AUXILIARY 109
Synthesis of B-Substituted-y-Keto Esters 109
Previous Attempts at Controlling the Stereochemistry 112
Amino Acid-Derived Systems 114
Hydroxyl-Directed Simmon-Smith Cyclopropanation Reactions 118
Synthesis and Results of L-Serine Derived Auxiliary 120
Quantifying the Diastereoselectivity 127
vii Elucidation of the Stereochemistry 129
Attempts at Synthesizing Hydroxyl L-Serine Auxiliary 138
Boc-Protecting Group Problems 141
Lactic Acid Derived System 145
Future Application for the L-Serine Derived Auxiliary 147
EXPERIMENTAL SECTION 149
General Experimental Section 149
Detailed Experimental Section 151
Compound 35c. Methyl 4,4-dimethyl-3-oxopentanoate (methyl pivaloylacetate) 151
Compound 42e. Allyl 3-oxobutanoate 152
Compound 90. 1,1-Diiodoethane 153
Compounds 134. 3-Methoxyfuran-2,5-dione (3-methoxymaleic anhydride) 153
Compounds 144. £)-5-(Methoxycarbonyl)-8,8-dimethyl-4,7-dioxonon-2-enoic acid.. 154
Compounds 155. (2£,4£)-4-Hydroxy-5-(methoxycarbonyl)-8,8-dimethyl-7-oxonona-2,4-
dienoic acid 154
Compound 152. (£)-4J-Dioxo-5-(2-oxooxazolidine-3-carbonyl)oct-2-enoic acid 155
Compound 165. 5-(Methoxycarbonyl)-8,8-dimethyl-4,7-dioxononanoic acid 156
Compound 166. Methyl 2-(2-hydroxy-5-oxotetrahydrofuran-2-yl)-5,5-dimethyl-4-
oxohexanoate 156
Compound 167. Methyl 2-(fert-butyl)-2-hydroxy-7-oxo-l,6 dioxaspiro[4.4]nonane-4-
carboxylate 156
Vlll Compound 171. 4-Ethoxy-4-oxobut-2-ynoic acid 157
Compound 182. Ethyl 2-(l,3-dicyclohexyl-2,5-dioxoimidazolidin-4-
ylidene)acetate 158
Compound 183. Ethyl 2-(l,3-diisopropyl-2,5-dioxoimidazolidin-4-
ylidene)acetate 159
Compound 185. 3-Phenylpropiolic acid 160
Compound 186. 5-Benzylidene-1,3-dicyclohexylimidazolidine-2,4-dione 160
Compound 194. Dimethyl 2-methoxybut-2-enedioate 160
Compound 195. 2-Methoxybut-2-enedioic acid 161
Compound 198a-C. Methyl 2,6-dimethoxy-2-methyl-8-oxo-l-oxaspiro[4.4]non-6-ene-4-
carboxy late 162
Compound 200. Methyl 2-hydroxy-9-methoxy-2,3-dimethyl-7-oxo-l,6-
dioxaspiro[4.4]non-8-ene-4-carboxylate 164
Compound 201. (£)-3-memoxy-5-(methoxycarbonyl)-6-methyl-4,7-dioxooct-2-enoic
acid 164
Compound 203a-d. Methyl 2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-
ene-4-carboxylate 166
Compound 204. (5/?,7/?,85*,9/?)-9-(Hydroxymethyl)-4,7-dimethoxy-7,8-dimethyl-l,6-
dioxaspiro[4.4]non-3-en-2-one 167
Compound 205. (2i?,3S,4£5/?)-2,9-Dimethoxy-2,3-dimethyl-7-oxo-l,6-
dioxaspiro[4.4]non-8-ene-4-carboxylic acid 170
Compound 206. 2,2-Dimethyl-l,3-dioxane-4,6-dione (Meldrum's acid) 171
Compound 208. 5-( 1 -Hydroxyethylidene)-2,2-dimethyl-l,3-dioxane-4,6-dione 171
IX Compound 210. Allyl 2-hydroxy-9-methoxy-2,3-dimethyl-7-oxo-l,6-
dioxaspiro[4.4]non-8-ene-4 carboxylate 172
Compound 211. (2R,3SAS,5R)-A\\yl 2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-
dioxaspiro[4.4]non-8-ene-4-carboxylate 171
Compound 218. (57?,7J/?,85,9i?)-7-hydroxy-9-(hydroxymethyl)-4-methoxy-7,8-dimethyl-
l,6-dioxaspiro[4.4]non-3-en-2-one (4-e/?z'-papyracillic acid C) 175
Compound 219. (2R,3SAS,5R)-Ally\ 9-methoxy-2,3-dimethyl-7-oxo-2-(2,2,2-
trichloroethoxy)-l ,6-dioxaspiro[4.4]non-8-ene-4-carboxylate 175
Compound 220. (2«,35,45',5/?)-9-Methoxy-2,3-dimethyl-7-oxo-2-(2,2,2-
trichloroethoxy)-l,6-dioxaspiro[4.4]non-8-ene-4-carboxylic
acid 176
Compound 221. (5i?,77?,85,9/?)-9-(Hydroxymethyl)-4-methoxy-7,8-dimethyl-7-(2,2,2-
trichloroethoxy)-l ,6-dioxaspiro[4.4]non-3-en-2-one 177
Compound 225, 130. (5tf,7i?,8i?)-4,7-dimethoxy-7,8-dimethyl-9-methylene-l,6-
dioxaspiro[4.4]non-3-en-2-one (4-epi-papyracillic acid B) and
(55,,7i?,85)-4,7-dimethoxy-7,8-dimethyl-9-methylene-l,6-
dioxaspiro[4.4]non-3-en-2-one (papyracillic acid B) 178
Compound 228. ((2^3S,4/?,5i?)-2,9-Dimethoxy-2,3-dimethyl-7-oxo-l,6-
dioxaspiro[4.4]non-8-en-4-yl)methy 1 methanesulfonate 179
Compound 229. (5tf7#,8S,9S)-9-(Iodomethyl)-4,7-dimethoxy-7,8-dimethyl-l,6-
dioxaspiro[4.4]non-3-en-2-one 180
Compound 235. (5#,7i?,8£9S>4J-Dimethoxy-7,8-dimethyl-9-((phenylselanyl)methyl)-
l,6-dioxaspiro[4.4]non-3-en-2-one 181
Compound 225a. Benzyl-(4^-((fe^butoxycarbonyl)amino)-3-oxopentanoate 182 Compound 255b. fe^Butyl-(2S)-(3-(benzyloxy)-3-oxopropanoyl)pyrrolidine-l-
carboxylate 183
Compound 255c. Benzyl-(4S)-(methoxycarbonylamino)-3-oxo-5-
phenylpentanoate 183
Compound 257a. 3-Benzyloxy-3-oxopropanoic acid (monobenzyl malonate) 184
Compound 267b. 3-Ethoxy-3-oxopropanoic acid (monoethyl malonate) 185
Compound 258a,b. Benzyl-(5S)-((fe^butoxycarbonyl)amino)-3-methyl-4-
oxohexanoate 185
Compound 266. N-(fert-Butoxycarbonyl)-L-serine 186
Compound 267. #-(?er/-Butoxycarbonyl)-0-benzyl-L-serine 187
Compound 268. JV-(tert-Butoxycarbonyl)-C-(terf-butyldimethylsilyl)-L-serine 188
Compound 269. iV-(ter/-Butoxycarbonyl)-L-serine methyl ester 190
Compound 270. Af-(fe7"7-Butoxycarbonyl)-0-(methoxyrnethoxy)-L-serine methyl
ester 191
Compound 271. {S)-3-tert-Buty\ 4-methyl 2,2-dimethyloxazolidine-3,4-
dicarboxylate 192
Compound 272. L-Serine methyl ester hydrochloride 193
Compound 274. JV-[(Methoxy)carbonyl]-L-serine methyl ester 193
Compound 275. JV-[(Methoxy)carbonyl]-<9-ethyl-L-serine methyl ester 194
Compound 276. 7V-(/er?-Butoxycarbonyl)-0(methoxymethoxy)-L-serine 195
Compound 277. (S)-3-(fer^Butoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic
acid 196
Compound 278. A4(Methoxy)carbonyl]-(3-ethyl-L-serine 196
XI Compound 281. (S)-Benzyl 5-(benzyloxy)-4-((fer?-butoxycarbonyl)amino)-3-
oxopentanoate 197
Compound 282. (5>Benzyl 4-((fert-butoxycarbonyl)amino)-5-((fert-
butyldimethylsilyl)oxy)-3-oxopentanoate 198
Compound 283. (S)-Benzyl 4-((/ert-butoxycarbonyl)amino)-5-(methoxymethoxy)-3-
oxopentanoate 198
Compound 284. (S)-tert-Butyl 4-(3-(benzyloxy)-3-oxopropanoyl)-2,2-
dimethy loxazolidine-3-carboxylate 199
Compound 285. (S)-Benzyl 5-ethoxy-4-(methoxycarbonylamino)-3-
oxopentanoate 199
Compound 286. (S)-Ethyl 5-(benzyloxy)-4-(benzyloxycarbonylamino)-3-
oxopentanoate 200
Compound 287. (^-Benzyl 4-((benzyloxycarbonyl)amino)-5-((fer7-
butyldimethylsilyl)oxy)-3-oxopentanoate 200
Compound 288. (5S)-Benzyl 6-(benzyloxy)-5-((ter/-butoxycarbonyl)amino)-3-methyl-
4-oxohexanoate 201
Compound 289. (5S)-Benzyl 5-((benzyloxycarbonyl)amino)-6-((terf-
butyldimethylsilyl)oxy)-3-methyl-4-oxohexanoate 202
Compound 304. Ethyl 5-(2-(benzyloxy)-l-(((benzyloxy)carbonyl)amino)ethyl)-5-
hydroxy-4-methyl-2-pentyltetrahydrofuran-3-carboxylate 203
Compound 329. (5)-Methyl 2-(benzyloxy)propanoate 204
Compound 330. (5)-2-(Benzyloxy)propanoic acid 205
Compound 331. (S)-Benzyl 4-(benzyloxy)-3-oxopentanoate 205
xii LIST OF REFERENCES 207
APPENDICES 227
Appendix A. Spectra 227
Appendix B. X-Ray Crystal Structures 366
Appendix C. NMR Data for Spiro-fused Ketals 371
Appendix D. Line Fit Data for L-Serine-Derived Systems 376
xm LIST OF SCHEMES
Scheme 1. General example of an enzymatic hydrolysis on a peptide bond 1
Scheme 2. SrruVmediated C-C bond formation for the synthesis of ketomethylene
isosteres 5
Scheme 3. Synthesis of a structural analog of fibril-forming decapeptide
SNFGAILSS 6
Scheme 4. Synthesis of hydroxyethylene isostere A-762611 highlighting the key
synthetic intermediate lactone 15 7
Scheme 5. Synthetic approach for the formation of peptide isosteres for the inhibition
of human B-secretase 8
Scheme 6. Different approaches for the synthesis of y-keto esters (a) Bieraugel's
method (b) Reissig's method (c) Saigo's method (d) Dowd's method 10
Scheme 7. Fortuitous discovery of the zinc-mediated chain extension reaction 11
Scheme 8. Examples of other B-keto carbonyl precursors subjected to zinc-mediated
chain extension rendering their respective y-keto homologues 13
Scheme 9. Proposed mechanism for the chain extension reaction 15
Scheme 10. Delocalization of zinc anion rendering a more reactive zinc enolate 16
Scheme 11. Synthesis of tripeptide ketomethylene isostere 18
Scheme 12. Electrophiles used during the tandem-chain extension reaction 19
Scheme 13. Comparison between the Reformatsky dimer 68 and intermediate 51 21
xiv Scheme 14. Zimmerman-Traxler closed transition state models for the favored Z-
enolate of the chain extension aldol reaction 22
Scheme 15. Zimmerman-Traxler closed transition state models for the less favored E-
enolate of the chain extension aldol reaction 23
Scheme 16. Open transition state for favored Z-imide enolate 25
Scheme 17. Intramolecular cyclization during chain extension-aldol reaction 25
Scheme 18. Equilibrium between open chain and closed hemiketal isomers 26
Scheme 19. Preparation of 1,1-diiodoethane (90) and a,a-diiodotoluene (93) 28
Scheme 20. Synthesis of B-Ph y-keto ester using substituted carbenoid derived from
a,a-diiodotoluene (93) 30
Scheme 21. Chain extension-oxidation-elimination reaction for the synthesis of (+)-
Patulolide A (99) and (-)-Patulolide B (100) 32
Scheme 22. Formal synthesis of (+)-Brefeldin A 33
Scheme 23. Synthesis of B-proline derivatives using the chain extension-imine capture
reaction 34
Scheme 24. Synthesis of phaseolinic acid derivative (113) through the chain extension-
CAN oxidation reaction 35
Scheme 25. Synthesis of ketomethylene isostere 116 through the use of the tandem
zinc-mediated chain extension-aldol reaction 36
Scheme 26. Synthesis of novel hydroxy-cyclopropane peptide isostere
121 37
Scheme 27. Proposed biosynthetic pathway for the formation of papyracillic acid A
(123) 40
xv Scheme 28. Nucleophilic attack of papyracillic acid A (123) by cyctenine 41
Scheme 29. Synthetic route for the synthesis of papyracillic acid A, B, and C
highlighting the tandem chain extension-acylation reaction as the key
step 43
Scheme 30. First example of the tandem chain extension-acylation reaction using
acetic anhydride (136) as an electrophile 44
Scheme 31. Initial studies of the tandem chain extension-acylation reaction using
maleic anhydride 46
Scheme 32. Keto-enol tautomerization of £-alkene 144 38
Scheme 33. B-Keto imide during the chain extension-acylation
reaction 49
Scheme 34. Possible mechanism for the isomerization of Z- to is-alkene 53
Scheme 35. Proposed strategy for the acylation reaction using a masked anhydride
(160) 54
Scheme 36. Preliminary results using masked anhydrides during chain extension
reaction 56
Scheme 37. Tandem chain extension-acylation reaction using succinic anhydride
(164) 57
Scheme 38. Unanticipated DCC-mediated heterocycle formation 58
Scheme 39. Comparison between the 6-endo-dig and 5-exo-dig cyclization
pathways 60
Scheme 40. Intra versus intermolecular cyclization 62
xvi Scheme 41. Attempted DCC-mediated cyclization using phenylacetylene derived
carboxylic acid 64
Scheme 42. Synthesis of 3-methoxymaleic anhydride (134) 65
Scheme 43. First example of the tandem chain extension-acylation reaction for the
synthesis of spiro-fused cyclic ketal core 66
Scheme 44. Conversion of crude hemiketals to ketals 64
Scheme 45. Comparison of Z- and i?-isomers derived from maleic anhydride (138) and
3-methoxymaleic anhydride (134) 68
Scheme 46. One-step synthesis of spiro-fused ketal core of papyracillic acid 69
Scheme 47. Equilibrium between cw-isomer (200a) and trans-isomer (200b) 71
Scheme 48. Synthesis of spiro-fused methyl ketals 203a, 203b, and 203c 75
Scheme 49. Unsuccessful attempts at converting methyl ester 203a to a carboxylic
acid 82
Scheme 50. Revisited synthetic approach for the synthesis of papyracillic acid C 82
Scheme 51. Facile preparation of allyl acetoacetate (42e) 83
Scheme 52. Synthesis of allyl ester 211 through the chain extension-acylation
reaction 84
Scheme 53. Optimized reaction conditions for Pd(0) mediated deallylation of allyl
ester 211 to carboxylic acid 205 88
Scheme 54. Reduction of carboxylic acid 205 to hydroxymethyl 204 through a mixed
anhydride intermediate 214 89
Scheme 55. Optimization of the reduction of carboxylic acid 205 to alcohol 204 using
a one step method 90
xvii Scheme 56. Hydrolysis of methyl ketal 204 to hemiketal 218 93
Scheme 57. Trichloroethyl-derived ketal as a route for the synthesis of papyracillic
acidC 94
Scheme 58. Acetic acid-mediated zinc insertion-elimination reaction 96
Scheme 59. Analysis of equilibrium for hemiketal 218 97
Scheme 60. Thermally induced equilibrium of 4-epi-papyracillic acid C (218) 99
Scheme 61. Attempted Chugeav elimination of xanthate ester 226 100
Scheme 62. Synthesis of iodomethyl 229 via a Finkelstein-type reaction 101
Scheme 63. Unsuccessful attempts at synthesizing exo-cyclic methylene 225 101
Scheme 64. Attempted synthesis of exo-cyclic methylene through the Grieco
elimination 103
Scheme 65. Synthesis of a 1:1 mixture of 4-epj-papyracillic acid B (225) and
papyracillic acid B (130) 105
Scheme 66. Kashima's stereoselective synthesis of P-substituted-y-keto esters 110
Scheme 67. Proposed mechanism for the chain extension reaction using a methyl-
substituted carbenoid 112
Scheme 68. Unsuccessful enantioselective synthesis of p-methyl-y-keto ester 113
Scheme 69. Unsuccessful diastereoselective synthesis of P-methyl-y-keto
ester 114
Scheme 70. Different synthetic approach towards controlling diastereoselectivity.... 115
Scheme 71. Chain extension reaction at lower temperatures (-42 °C) 118
Scheme 72. Homoallylic alcohol directed cyclopropanation using a methyl-substituted
carbenoid 119
xvm Scheme 73. Homoallylic alcohol as a directing group during the chain extension
reaction 120
Scheme 74. Retro synthetic analysis for the synthesis of P-methyl-y-keto esters 120
Scheme 75. Synthesis of O-Benzyl 267 and O-TBS L-serine 268 121
Scheme 76. Synthesis of 0-MOM 270 and oxazolidine L-serine methyl ester 271... 122
Scheme 77. Synthesis of O-ethyl L-serine methyl ester 275 123
Scheme 78. Diastereomeric ratio obtain from oxazolidine-derived L-serine
auxiliary 126
Scheme 79. Forzato's synthesis of optically active 4-methylparaconic esters and
acids 130
Scheme 80. Synthesis of cis and trans 4-methylparaconic acid derivative 299 131
Scheme 81. Optical rotation for c/s-paraconic acid benzyl ester derivative 132
Scheme 82. Hydrogenolosis of benzyl ester 299 to paraconic acid 300 133
Scheme 83. Synthesis of carboxylate salt 302 in an attempt to obtain X-ray grade
crystals 134
Scheme 84. Tandem chain extension-aldol reaction with L-serine derived system
286 135
Scheme 85. CAN-mediated oxidation of hemiketal 304 136
Scheme 86. Unsuccessful attempt at synthesizing free hydroxyl P-keto ester 314 138
Scheme 87. (a) Proposed cyclization generating benzyl alcohol 315 and lactone 316
(b) Proposed Lewis acid promoted hydrolysis of benzyl ester 317 141
Scheme 88. Zinc carbenoid-mediated degradation of Boc-protecting group 142
Scheme 89. Attempted ethyl carbamate 324 formation from Boc-pyrollidine 144
xix Scheme 90. Proposed route for allylic directed cyclopropanation using a methyl-
substituted carbenoid 145
Scheme 91. Synthesis of lactic acid-derived P-keto ester 331 146
Scheme 92. Chain extension reaction using a lactic acid-derived system 331 147
Scheme 93. StereocontroUed synthesis of papyracillic acid C (131) 148
Scheme 94. P-Methyl hydroxyethylene peptide isostere 148
xx LIST OF FIGURES
Figure 1. (a) Tetrahedral intermediate of aspartic protease (b) Tetrahedral
intermediate of serine protease 2
Figure 2. Peptide isostere replacement of amide bond 4
Figure 3. Z- and E- Geometries for the zinc derived imide enolate 24
Figure 4. Structural comparison between papyracillic acid A (123) and penicillic
acid (124) 39
Figure 5. Recently isolated papyracillic acid B (130) and C (131) 41
Figure 6. Coupling constants for cis- and /r 144 48 Figure 7. Coupling constant for isomerized E-alkene derived from a P-keto imide 52 Figure 8. Electron withdrawing and electron donating substituent effects 64 Figure 9. X-Ray crystal structure for the major isomer of the tandem chain extension-acylation reaction 70 Figure 10. Time study showing the increase of the trans-isomer (200b) and the decrease of the cw-isomer (200a) 72 Figure 11. X-Ray crystal structure for the major diastereomer 76 Figure 12. X-Ray crystal structure for epimer of the major diastereomer 77 xxi Figure 13. Chemical shift comparison between major 203a and minor 203a diastereomers 78 Figure 14. Stereochemical assignment of minor diastereomer 203c 79 Figure 15. Deallylation reaction monitored by lH NMR spectroscopy 87 Figure 16. Stereochemical comparison between 130 reported, 130 synthesized, 225 and 238 107 Figure 17. Illustration of the a- and P-positions of 1,4-dicarbonyl backbones 109 Figure 18. Determination of diastereomeric ratio using MestReNova line fitting.... 128 Figure 19. - Different zinc-enolate geometries 137 Figure 20. Zinc carbenoid-mediated conversion of /-butyl carbamate 322 to ethyl carbamate 322 143 xxn LIST OF TABLES Table 1. Conversion of p-keto esters to y-keto esters using chain extension reaction 12 Table 2. Tandem-chain extension aldol reaction showing high diastereoselectivity for the syn-isomer 20 Table 3. Conversion of P-keto esters and amides to the respective P-methyl y-keto substrates using a methyl-substituted carbenoid 29 Table 4. Failed attempt at converting crude hemiketal 140 to ketal 141 47 Table 5. Optimization of tandem chain extension-acylation reaction using P-keto imide 146 51 Table 6. Cycloreversion temperature (°C) for three masked anhydrides 55 Table 7. Investigation of different reaction conditions for heterocycle formation 61 Table 8. Conversion cis- to trans-isomev 200b through an intermediate enol 202 73 Table 9. Attempted reduction of methyl ester 203a to hydroxymethyl 204 81 Table 10. Stereochemical assignment of allyl ester 211 85 Table 11. Comparison between *H NMR data for synthesized alcohol 204 and reported papyrcillic acid C (131) 91 Table 12. Optimization of reaction conditions for the hydrolysis of ketal 216 to hemiketal 217 92 Table 13. Unsuccessful zinc insertion-elimination conditions 95 xxm Table 14. Comparison between *H and 13C NMR data for hemiketal 218 and papyracillic acid C (131) 98 Table 15. Hypothesized epimerization between 4-epz-papyracillic acid B (225) and papyracillic acid B (130) 106 Table 16. Synthesis of amino acid-derived p-keto esters through the Masamune- Brooks 116 Table 17. Ratio of diastereomers using amino acid-derived systems 117 Table 18. Hydrolysis of L-serine methyl esters to carboxylic acids 276-278 123 Table 19. Synthesis of L-serine derived P-keto esters 281-287 via Masamune- Brooks reaction 124 Table 20. Diastereoselective synthesis of P-methyl-y-keto esters 288-291 using an L- serine auxiliary 125 Table 21. Unsuccessful Montmorillonite KSF aminal deprotection attempts... ..139 Table 22. Attempted bismuth(III) bromide-mediated aminal deprotection 140 Table 23. lH NMR chemical shift and coupling constant data for esters 211, 203a, 203b, and 203c 371 Table 24. 13C NMR chemical shift data for esters 211, 203a, 203b, and 203c 372 Table 25. 'H NMR chemical shifts and coupling constant data for compounds 225, 130 synthesized, 130 reported and 238 373 Table 26. 13C NMR chemical shift data for compounds 225, 130 synthesized, 130 reported, and 238 374 Table 27. Summary of MestReNova line fit data for L-serine-derived systems 376 xxiv LIST OF ABBREVIATIONS Ac Acetyl AIBN Azobis(isobutyronitrile) Boc /-Butyloxycarbonyl Bn Benzyl CAN Ceric Ammonium Nitrate CDI Carbonyldiimidazole Cbz Carbobenzyloxy DBU l,8-Diazabicyclo[5.4.0]undec-7-ene DCC Dicyclohexylcarbodiimide DCM Dichloromethane DIPEA Diisopropylethylamine DMF Dimethylformamide DIBAL Diisobutylaluminum Hydride EDC 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide) HOBT Hydroxybenzotriazole LDA Lithium Diisopropylamide LiHMDS Lithium Hexamethyldisilazide MBM Monobenzylmalonate MEM Monoethylmalonate MMM Monomethylmalonate MOM Methoxymethyl Ms Methanesulfonyl, Mesyl xxv PG Protecting Group Ph Phenyl Pmb p-Methoxybenzyl TBDMS t-Butyldimethylsilyl TFA Trifluoroacetic THF Tetrahydrofuran TMS Trimethylsilyl Ts Tosyl xxvi ABSTRACT I. SYNTHESES OF PAPYRACILLIC ACIDS: APPLICATION OF THE TANDEM CHAIN EXTENSION-ACYLATION REACTION II. DIASTEREOSELECTIVE SYNTHESIS OF p-METHYL-y-KETO ESTERS THROUGH THE UTILITY OF AN L-SERINE AUXILIARY by Jennifer R. Mazzone University of New Hampshire, December 2011 The development of the zinc-mediated tandem chain extension-acylation reaction for the synthesis of spiro-fused ketals was successfully accomplished. The incorporation of a methyl-substituent into the y-keto ester backbone, through a methyl substituted carbenoid, allowed for the synthesis of papyracillic acid B and 4-epz-papyracillic acid C. Comparison between the reported and experimental spectral data for papyracillic acid B and 4-epz-papyracillic acid C permitted structural and stereochemical reassignment. Diastereoselective syntheses of P-methyl-y-keto esters were achieved through the utility of an L-serine auxiliary. Preparation of a series of L-serine derived precursors were prepared and studied. xxvii CHAPTER I INTRODUCTION Proteases Degradation of peptide bonds is mainly attributed to a large class of enzymes called proteases. Eight sub-groups organize the protease family, consisting of hundreds of identified and characterized enzymes possessing specific function.1 The serine and aspartyl proteases are two of the most widely studied protease classes to date.2 The mechanism of action for enzymatic hydrolysis, which has been widely studied, is believed to involve nucleophilic attack on the peptide bond 1 (Scheme l).3 Tetrahedral intermediate 2 is generated, which upon collapse will release peptide fragments 3. 2 O R2 Enzymatic O R Hydrolysis OH + H2N 1 1 H R R1 H R Scheme 1. General example of an enzymatic hydrolysis on a peptide bond 1 The ability to specifically stabilize the transition state of hydrolysis is inherently unique for each protease (Figure 1). Aspartyl proteases (4) have two aspartic residues in the active site, responsible for activating nucleophilic attack on the peptide as well as hydrogen bonding to the resulting tetrahedral intermediate.4 The hydroxyl group of a serine residue, in conjunction with a histidine and aspartic residues, are responsible for the nuleophilic attack through a "charge relay" system of serine proteases (5).5 Two amino groups serve to stabilize the tetrahedral intermediate. While both aspartate and serine proteases perform critical biological services in human systems, such as digestion, homeostasis, and immune response, enzymatic hydrolysis also play a role in the life cycle of other organisms. (b) Serine Protease 5 (a) Aspartate Protease 4 Ser-rl .N-Gly ^2 |_| '-0-'' R2 v^R4 P o R3 Ser-0 H H'" H- H o' b I Asp^O HO ^Asp H 6 Asp^( O Figure 1. (a) Tetrahedral intermediate of aspartic protease (b) Tetrahedral intermediate of serine protease Among the most notable aspartate proteases is HIV-1 protease, which is critical for replication of the HIV-1 virus. The X-ray crystal structure of the HIV-1 protease 2 shows a 99 amino acid aspartyl protease homodimer with one active site that has C2- symmetry. Interception of the HIV-1 protease with a protease inhibitor that is non- hydrolysable has been proposed as a possible therapeutic method for the inhibition of this virus' replication. One strategy for protease inhibition has relied on the synthesis of peptide isosteres. Peptide Isosteres Bioisosteres mimic biologically active molecules, possessing the same number of atoms and valence electrons, through agonist or antagonist action.7 Common bioisosteres have been developed from the manipulation of various peptides.8 The amide bond in 6, also referred to as a peptide bond, can be replaced by a methylene unit to produce a ketomethylene isostere 7 (Figure 2). The ketone functionality 7 can resist hydrolysis by an aspartyl protease through formation of an oxyanion tetrahedral intermediate.9 The hydroxyethylene isostere 8 imitates the tetrahedral geometry of the transition state during hydrolysis by replacing the carbonyl unit with an sp3 hybridized carbon. Both the ketomethylene and hydroxyethylene isostere offer a non-hydrolysable tetrahedral geometry suitable for resting in the active site of an aspartyl protease; however, the conformational flexibility gained through the replacement of the peptide bond is a major drawback. The cyclopropyl peptide isostere 9 replaces the peptide bond with a cyclopropane ring. Unlike the other two isosteres 7 and 8, cyclopropane peptide isosteres 9 are conformationally rigid, but lack the Lewis basic oxygen functionality proven to be an important feature when designing a bioisostere for protease inhibition.33 Since there 3 are several drawbacks to these original bioisosteres, which were designed to combat the HIV-PR and other enzymatic protease cleavage, ongoing efforts towards the synthesis of novel peptide isosteres are still being pursued. Figure 2. Peptide isostere replacement of amide bond Many research groups have reported syntheses of peptide isosteres.10 Efforts have been made to develop synthetic approaches to bioisosteres that are both versatile and high yielding. Skredstrup et al. have accomplished the synthesis of ketomethylene isosteres through a samarium iodide (Smb.) promoted C-C bond formation (Scheme 2).11 iV-Acyl oxazolidinone 10 tethered to a peptide was subjected to an electron deficient alkene 11 in the presence of Sml2, which induced a C-C coupling reaction in moderate to high yields. Most reported examples showed ketomethylene isostere 12 with no substitution at both the a and P positions. Although the chemistry allowed for the generation of 1,4- dicarbonyls with a methyl-substituent at either the a or P positions, poor diastereoselectivity was observed. 4 ^^CONHf-Bu ° II 11 ° NHf BU Peptide-^N O Sml2 (4-6 equiv) Peptide^^^jf " H20 (8-12 equiv) 10 THF, -78 °C 12 40-94% Scheme 2. Sml2-mediated C-C bond formation for the synthesis of ketomethylene isosteres Identification and characterization of fibrillar amyloid fragment decapeptide SNNFGAILSS, used as a distinguishing histopathological marker for type II diabetes, has been known for some time. More recently, efforts by Skredstrup et al. have focused on applying the Sml2-mediated coupling reaction for the synthesis of a fibril-forming decapeptide SNNFGAILSS structural analog (Scheme 3).12 Ketomethylene isostere 13 replaces the glycine residue of polypeptide SNNFGAILSS with a methylene unit. It is believed that structural variations of this decapeptide SNFGAILSS, such as 13, will elucidate the mechanism by which p-sheet fibrils are produced. This application of a ketomethylene isostere emphasizes the desire for a facile method possessing regio and stereochemical selectivity. 5 o o benzyl acrylate O steps A A Sml2, H20 OBn »• Peptide Peptide^^N 0 THF, -55 °C, 18 h O 72% Scheme 3. Synthesis of a structural analog of fibril-forming decapeptide SNFGAILSS Formation of peptide isosteres with high stereoselectivity is often desired. Wagaw et al. at Abbot laboratories have identified a potent and selective HIV protease inhibitor referred to as A-792611 14 (Scheme 4).13 In order to have access to this hydroxyethylene isostere for further studies, they devised a synthetic approach reliant on a diastereoselective reaction that would control the stereochemistry at the C-3 position of lactone 15. A lithium hydroxide-mediated hydrolysis of ethyl ester 16 followed by an acetic acid decarboxylation produced enol 17. Diastereoselective protonation of enol 17 from the re-face furnishes the syn-lactone 15 stereochemistry in a 5:1 ratio of diastereomers. Subsequent chemistry produced hydroxyethylene isostere 14 in 46% overall yield and 97:3 dr. 6 C02Et a. LiOH, H20 *• BocHN b. AcOH, 60 °C 82% O fiu u OH NHC02Me BocHN K' BnN N \—' O Bn A-762611 14 Scheme 4. Synthesis of hydroxyethylene isostere A-762611 highlighting the key synthetic intermediate lactone 15 Recently, a novel application of peptide isosteres has centered on the inhibition of human P-secretase in an effort towards understanding the neurodegenerative disorder Alzheimer's disease. Xu et al. identified a hydroxyethylene isostere and several structural derivatives suitable for studying the active site of the P-secretase enzyme.14 Similar to the Wagaw et al. synthesis, the key synthetic intermediate is functionalized lactone 18 (Scheme 5). Upon hydrolysis of lactone 18, carboxylic acid 19 has been converted to a variety of different amides through standard l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) coupling conditions. While both synthetic methods (Scheme 4 and 5) offer a diastereoselective route to hydroxyethylene isosteres, multiple synthetic steps render each approach less desirable. In addition, novel 7 applications for peptide isosteres have increased the demand for facile and stereoselective synthetic methods. O 0A. . ^/"\ .... OTBSi -« , LiHMDS, CH3I Q V" a. LiOH, THF „ ,,., , , \-l : -* \J I ^ BocHN^A^^^OH BocHN-/ -78 °C BocHN^ b. TBDMSCI 4 \ \ imidazole, DMF \/ O 18 T 19 Scheme 5. Synthetic approach for the formation of peptide isosteres for the inhibition of human P-secretase Among some of the most therapeutically relevant proteases are the HIV PR and the human P-secretase, which function to perpetuate the HIV-1 viron and Alzheimer's disease, respectively. Peptide isosteres, such as ketomethylene and hydroxyethylene isosteres, have displayed therapeutic inhibition of variety of aspartyl proteases. Many researchers have developed synthetic approaches towards a variety of peptide isosteres; however, common drawbacks to some of these synthetic methods are the lengthy reaction schemes, lack of regiochemical selectivity, and stereochemical control. With novel applications for peptide isosteres on the rise, facile synthetic methods with regio and stereochemical control are continually desired. 8 Zinc-Carbenoid Mediated Chain Extension Reaction Several research groups have successfully prepared y-keto carbonyls (Scheme 6). One important strategy involves formation of a donor-acceptor cyclopropane, and a number of variations on this strategy have been developed. Bieraugel's approach inserts a carbene into enamine 20 rendering cyclopropyl intermediate 21, and subsequent fragmentation generates y-keto ester 22.15 Reissig's method subjects silyl enol ether 23 to a carbenoid in order to generate a hydroxysilyl cyclopropane intermediate 24 that can be isolated or quenched in situ to give y-keto ester 25.16 In a similar vein, Saigo's approach also utilizes a silyl enol ether 26 in conjunction with a carbenoid to afford a hydroxysilyl cyclopropane intermediate 27.17 Ring opening of the cyclopropane provides access to P- substituted y-keto ester backbone 28. Lastly, Dowd's protocol uses a radical-mediated process initiated by the cleavage of C-Br bond 29, subsequent intramolecular cyclization 1 R furnishes cyclopropane alkoxy radical 30. Facile fragmentation of 30 affords a resonance-stabilized radical, which can be quenched to provide an a-substituted y-keto ester 31. While each example offers a unique route for the synthesis of y-keto esters, several limitations diminish the applicability for more complex systems. Thus a new method must tolerate a wide array of readily available starting materials with an opportunity for regiospecific incorporation of functionality in both the a and p position of the y-keto carbonyl backbone. 9 N O «N-R30 O •CHo + 2 (a) H OR R1 OR2 R1 OR2 R' O 20 21 22 O TMS. ,TMS OR2 O O O (b) O 2 H+orF-^ RA ^ ^R 1 2 R1 R OR Cu(acac)2 O 23 24 25 O TMS. O O O O O (c) + 2 2 1 2 H 1 OR R^OR R OR R 3 CuS04 R3 R O 26 27 28 3 AIBN •o O O R /^ II II 2 H' OR RlAX'^OR2 n-Bu3SnH_ OR2 3 R O Br 29 30 31 Scheme 6. Different approaches for the synthesis of y-keto esters (a) Bieraugel's method (b) Reissig's method (c) Saigo's method (d) Dowd's method In 1997, Brogan and Zercher reported the discovery of a zinc-carbenoid mediated homologation reaction, later referred to as the zinc-mediated chain extension reaction.19 In an attempt to cyclopropanate the terminal olefins of cyclic ketal 32 using Furukawa- modified Simmons-Smith conditions,20 the desired P-keto ester 33 was not observed (Scheme 7). The y-keto homolog 34 was obtained instead. 10 o o -K- O' 0 O 33 O o Et2Zn or O 0 CHo2'l2 32 O o. o o o 34 < Scheme 7. Fortuitous discovery of the zinc-mediated chain extension reaction Several different P-keto esters 35a-h were subjected to carbenoid, derived from diethylzinc and diiodomethane, rendering their respective y-keto ester homologues 36a-h in good yields (Table 1). The conversion of P-keto esters to y-keto esters proceeded cleanly and efficiently using a 5 equivalent excess of the ethyl(iodomethyl)zinc reagent. Additionally, the chain extension reaction conditions were found to tolerate conjugated and electron deficient alkenes without unwanted cyclopropanation byproducts. Broadening the reaction scope in order to encompass other P-keto carbonyls was also investigated. 11 o o O a. EtoZn, CH lo OR2 — I2 t R1 OR' b. NH4CI O 0 °C, CH2CI2 35a-h 36a-h Entry R1 R2 Yield (%) la Me -OMe 81 2b Me -Ot-Bu 74 3c t-Bu -OMe 71 4d Ph -OEt 58 5e z-Pr -OEt 68 6f Ph(CH2)2- -OEt 73 7g CH2=CH(CH2)2 -OMe 74 8h PhCH=CH- -OMe 68 Table 1. Conversion of P-keto esters to y-keto esters using chain extension reaction The same protocol used to convert p-keto esters to y-keto esters was applied for the synthesis of other y-keto carbonyl systems (Scheme 8). The scope of the chain extension reaction has been expanded to include P-keto amides (37),21 P-keto phosphonates (38), P-keto imides (39), a-carboxyester imides (40), ' and a- carboxydiimides (41).25 The conversion of p-keto amides (37) to y-keto amides (42) was rapid and proceeded in good yields (42-88 %), comparative to that of P-keto esters (35). While tertiary amides were found to yield a cleaner conversion to desired product, secondary amides were converted to their desired y-keto counterparts in lower yields. Additionally, P-keto phosphonates (38) homologated to provide y-keto phosphonate 12 backbones (43) in an efficient manner. Most recently, p-keto imides (39), a-carboxyester imides (40), and a-carboxydiimides (41) were converted to y-keto imines 44, 45, and 46, further broadening the scope of this homologation reaction. O O OR2 R,J^.R3 E,2Zn | n.Jk^.N.^ 2 R CH2I2 O 37 42 O O o 2 OR2 *" ii CH2I2 O 38 43 R2 o o o o V-\ y~/ CH2I2 O O 39 R2 " 44 R2 o o o o y-^ CH2I2 O O 40 R2 45 O O o o X XX A Et2Zn l X^ NP M. ^ CH2I2 °\_^ J S R2 46 Scheme 8. Examples of other p-keto carbonyl precursors subjected to zinc-mediated chain extension rendering their respective y-keto homologues 13 The proposed mechanism for the conversion of P-keto esters to their respective y- keto homologues through the zinc-carbenoid mediated reaction has been studied both experimentally and computationally (Scheme 9).26 Initially, one of the methylene protons between the 1,3-dicarbonyl 47 is deprotonated rendering zinc enolate 48. The enolate 48 undergoes alkylation with the zinc-carbenoid generating a homoenolate 49. An intramolecular cyclization into the keto functionality provides a donor-acceptor cyclopropane intermediate 50. Fragmentation of the strained cyclopropane ring leads to an organometallic intermediate 51, similar to a Reformatsky-type intermediate,27 which is believed to be the resting state of the homologation reaction specifically for P-keto esters, amides, and phosphonates. While intermediate 51 possesses anionic character regiospecifically in the a-position of the 1,4-dicarbonyl, alkylation reactions are not feasible. 8 The lower reactivity of intermediate 51 prompted Aiken to probe the nature of the intermediate 51 through NMR spectroscopy studies.26" These experiments revealed intermediate 51 exists as a C-bound zinc-enolate oligomer, which renders this enolate less nucleophilic than that of a typical enolate. Further discussion of the reactivity of intermediate 51 will follow (see Formation of a-Functionalized-y-Keto Carbonyls). 14 0 0 a. Et2Zn, CH2I2 2 R1'1J^^R2 Ri^Y" 47 b. NH4CI 52 0 1 1 + H+ ZnX r 'V, i 0' 0 J R1' ^AR2 O XZrf" %^ 48 i i 2 :tZnCH2l 51 R . ' O 0 ZnX O' 0 R1'JJvY^R2 R1^>^R2 XZrv 49 50 Scheme 9. Proposed mechanism for the chain extension reaction where R2 = ester, amide or phosphonate In contrast to P-keto esters, amides, and phosphonates, P-keto imide-derived systems proceed in a slightly different mechanistic fashion. The imide functionality of the P-keto imide 53 is more electrophilic in comparison to ester or amide substrates (Scheme 10). Upon exposure to the chain extension conditions, intermediate 51 is produced with anionic character in the a-position; however, the zinc-carbon bond appears to be weaker than the corresponding intermediate formed in the reaction of P-keto esters, possibly due to preferential chelation of the zinc between the two oxygens. In comparison to carbon- bound zinc enolates, oxygen-bound zinc enolate 55 would be expected to be more reactive. This hypothesis is supported by the facile formation of a-methyl-y-keto imide 56 along with other byproducts during the chain extension reaction. ' Such byproducts are believed to arise from alkylation of enolate 55 with excess carbenoid, a process which 15 has never been observed when using P-keto esters or amides even after prolonged reaction times. O O O O XZn" O Et2Zn O R1 R \\ CHo2'l2 Zn 53 EtZnCH2l O O 56 Scheme 10. Delocalization of zinc anion rendering a more reactive zinc enolate In summary, a fortuitous discovery of a chain extension reaction has evolved into a facile method for the conversion of 1,3-dicarbonyls to 1,4-dicarbonyls in one step. The reaction conditions are relatively mild, tolerating a wide array of different functionality. While the homologation of P-keto esters and amides is predictable, P-keto imides pose a slightly greater challenge due to their highly reactive nature. Application of the chain extension reaction has centered on the synthesis of ketomethylene isosteres. 16 Synthesis of Ketomethylene Isosteres using the Chain Extension Reaction Significant effort has been invested in the application of the zinc-mediated chain extension reaction to the synthesis of peptide isosteres. Theberge and Zercher reported a concise synthesis of several ketomethylene isosteres (Scheme ll).31 L-Alanine-derived P-keto ester 57 was subjected to the zinc carbenoid, which produced amino acid derived y-keto ester 58 in good yields. Hydrogenolysis followed by an EDC coupling furnished the tripeptide mimic 59. Subsequent transformations involving manipulation of either the N or C termini allows access to a variety of peptide mimics. The formation of the central ketomethylene backbone was achieved in only one step from easily synthesized starting material. This example illustrates the efficient and versatile nature of the zinc carbenoid- mediated chain extension reaction for the synthesis of peptide isosteres. The regio- and stereo specific incorporation of substituents in the a and p position of ketomethylene isostere 59 has been developed (vide infra), further displaying the efficiency of the chain extension reaction for the synthesis of peptide mimics and other biologically significant natural products. 17 O H ° ° OBn BoC H X X a.Et2Zn,CH2l2 BoC b. NH4CI O 57 58 a. H2, Pd/C b. EDO, HOBT .Ph OCH, HoN O H II « H 9 BoC OCHq Th 59 Scheme 11. Synthesis of tripeptide ketomethylene isostere Formation of a-Functionalized y-Keto Carbonyls Studies of the reaction mechanism explicitly demonstrated that organometallic intermediate 51 has anionic character a to the ester, amide, or imide, which mandates the regiospecific incorporation of electrophihc substitutions. Capitalizing on this opportunity, efforts have been invested in the development of tandem chain extension protocols. Various electrophiles have successfully been used to functionalize the a-position of the y- keto ester backbone (Scheme 12). Electrophiles such as aldehydes 60,32 ketones 61,32 excess carbenoid 62,33 iminium ions 63,34 imines 64,35 and iodine 6523b'30a'36 have been incorporated into a tandem-chain extension reaction. In addition to being used for the synthesis of functionalized peptide isosteres, several tandem processes have been used as 18 a key step in the synthesis of natural products. Furthermore, effort has been invested in the development of stereoselective a-functionalization in order to apply the chain extension reaction towards more challenging synthetic targets. .R ^ ^OH o"J3-V QR4^0H Scheme 12. Electrophiles used during the tandem-chain extension reaction ("Only applicable for esters and amides) Among the first tandem protocols to be developed was the tandem chain extension-aldol reaction (Table 2).32 The zinc enolate, generated after subjecting p-keto ester 35a,c to the Furukawa reagent, has been quenched with several different aldehydes. This regiospecific functionalization at the a-position of a y-keto ester backbone resulted 19 in high diastereoselectivity. In the case of esters and amides, formation of the ,sy«-isomer 66a,c was favored over the anti-isomer 67a,c. While the zinc-organometallic intermediate of the chain extension-aldol reaction is similar to the Reformatsky reaction, the high syn-selectivity was unprecedented. R3»OH Ri ,*OH jf A a-Et2Zn'CH2'2 ^^ n R R1^^\DR2 - o 6 b. R3CHO 35a,c 66a,c 66a,c Entry R1 R2 R3 Yield % synranti la Me OMe Ph 61 15:1 2a Me OMe t-Bu 85 >20:1 3c f-Bu OMe Ph 97 12:1 4c f-Bu OMe Ar 61 9:1 Table 2. Tandem-chain extension aldol reaction showing high diastereoselectivity for the .syrc-isomer The low reactivity of intermediate 51, derived from P-keto ester or amide precursors, is believed to be due to its oligimeric nature, which further supports the similarity between 51 and the Reformasky dimer 68 (Scheme 13).37 Computational studies of the Reformasky reaction support the dissociation of the dimeric species upon addition of an electrophile, affording an oxygen-bound zinc enolate.38 The O-bound zinc enolate can exist in two different geometries, the Z-enolate 69 and the .E-enolate 70. In a 20 similar fashion, intermediate 51 could undergo dissociation to provide two possible O- bound enolate geometries, Z-enolate 71 and £-enolate 72. The ketone-functionality of intermediate 51 may participate in the dissociation of the oligomer by chelating to the Zn(II) thereby favoring formation of a Z-enolate. The Zn(II) metal is proposed to stabilize the enolate while activating the electrophile in a closed chair-like transition state.39 2 R 0 Ri r> Z-enolate E-enolate ' O-V v Zn- O' X OZnX OZnX X^-Zn .O + r^OR2 R1 OR2 68 69 70 X i Zn O XZrY' ^ / \ O .0'' O O 1 OZnX R R 1 2 2 R ^_^^OR 2 OR OR 51 71 71 Scheme 13. Comparison between the Reformatsky dimer 68 and intermediate 51 A Zimmerman-Traxler model has been used to predict stereochemistry for systems that proceed through closed chair-like transition states.40 If Z-enolate 71 reacts with an aldehyde in a chair-like transition state, the facial selectivity of the aldehyde dictates the sterochemical outcome (Scheme 14). When the R -substituent of the aldehyde approaches in the pseudo-equatorial position 73, the ^-stereochemistry is observed. However, if the aldehyde approaches with the R -substituent in the pseudo- axial position 74, the arcfr'-stereochemistry 67 will be obtained. The undesired 1,3-diaxial interactions renders the anti-isomer less favored. The high syn-selectivity of the tandem 21 chain extension-aldol suggests that the formation and reaction of a Z-enolate is occurring. A similar analysis can be made for the is-enolate . R3\,*OH O JV* 0RS X -* A,-V i syn R Zn o 66 O °" '° HAR3 R1^V_^OR2 Z-enolate 71 .R3^ ^OH - TT-'O H-/ O-.. ... ' -X i | z n 67 O 2 R3 ,0R 74 Scheme 14. Zimmerman-Traxler closed transition state models for the favored Z-enolate of the chain extension-aldol reaction Two transition states can be depicted for the reaction between is-enolate 72 and an aldehyde, 75 and 76 (Scheme 15). Transition state 75, where the aldehyde is approaching with the R -substituent in the pseudo-equatorial position, is predicted to be favored over transition state 76. However, transition state 75 would lead to the anft'-isomer, which is experimentally observed in minor quantities. This supports the notion that the Z-enolate is operative in the aldol reaction. Studies by Aiken clearly demonstrate the necessity of the ketone for high ^yn-stereocontrol, which suggests that ketone complexation is key to selective Z-enolate formation.263 22 OR3YOH anti o O OR2 O OZnX H 67 R" 3 H^R 75 OR2 E-enolate 72 OR3YOH syn lAvLoR^ o Scheme 15. Zimmerman-Traxler closed transition state models for the less favored E- enolate of the chain extension aldol reaction Not all 1,3 dicarbonyl precursors are believed to proceed through a closed transition state model. Tandem chain extension-aldol reactions involving P-keto imide precursors have been studied. The diastereoselectivity of this reaction favors the anti- isomer over the syn-isomer. Carbon-bound zinc enolate 54 is in equilibrium with two enolate geometries (Figure 3). The Z-enolate 55 is strongly favored over the is-enolate 77 due to the undesired A1'3 strain observed in the is-geometry.41 If Z-enolate 55 reacts through a closed transition state, svrc-products are expected; therefore the dominance of the anti-isomer is rationalized by a change to an open transition state. 23 O XZn" ;0 R1 A^NY0 o\. , o Zn X cr Favored Z-Enolate 54 55 Figure 3. Z- and E- Geometries for the zinc derived imide enolate An excess of Lewis acid has been proposed to be necessary for the reaction via an open transition state, since a single equivalent of zinc chelating between the two oxygens of the imide is unable to serve in the activation of the aldehyde. 2 Thus a second equivalent of zinc is required to activate the aldehyde. When the re-face of Z-enolate 55 attacks the aldehyde's re-face through an open transition state, 78 is produced (Scheme 16). This facial approach of the aldehyde leads to an undesired steric interaction between the R2-substituent and the oxazolidinone ring, which decreases the likelihood of the syn- aldol product 79. Whereas transition state 80, generated from the enolate attacking an aldehyde from the si-face, minimizes undesired steric interaction thus leading to the anti- aldol product 81. Lai, a former group member, observed the anti-aldol product as the only isomer formed during the homologation-aldol reaction with P-keto imide substrates.233 24 _ R2 *0H 0 R X H „>\—\ I /—\ syn o o II O,o 79 78 Zn Zn X Z-Enolate R^OH 55 °x ,° anti Zn o o 80 81 Scheme 16. Open transition state for favored Z-imide enolate Recent results suggest that if the R^substituent is a leaving group, such as an alkoxide or oxazolidinone, cyclization of the aldol product will occur. Lin,23 Henderson,25 and Sadlowski24 all confirmed cyclization upon exposure of P-keto imide derivatives 81 to chain extension-aldol conditions (Scheme 17). The aldol intermediate 82 is proposed to cyclize into the imide carbonyl extruding oxazolidinone as a leaving group. The result of this intramolecular acylation is a y-lactone derivative 83. Such results further confirm the diverse chemistry available from chain extension reactions of imide derivatives. O O 0 ,0. R OZnX A.JLA,, a. Et2Zn, CH2I2 o o V O N ^-^ R' „ R1 V_J b. 0 V 81 ll ^ o o H^R2 81 83 R1=-OMe,S-N o Scheme 17. Intramolecular cyclization during chain extension-aldol reaction 25 The diastereoselectivity of the tandem chain extension-aldol reaction is frequently difficult to quantify. Both open chain syn (66) and anti-aldol (67) products exist in equilibrium with their respective closed hemiketal forms (Scheme 18). The open chain syn-aldol product (66) typically favors the hemiketal isomers in which trans relationship exists between the substituents at the C-4 and C-5 (84 and 85) position. The anti-aldol product (67) also exists in open and closed (hemiketal) forms. Hemiketals 86 and 87, derived from the anti-aldol products, have a cis relationship between the substituents at the C-4 and C-5 position, which may give, rise to steric congestion and decrease the amount of hemiketal form in the equilibrium mixture. o RV0H H?i<0v-R2 5/VR2 ^"Y ^~ '^OR3 • ?rOR3 u o o 66 84 85 Rl>OH H0«xCL 2 RVO. ,R2 n 1,J R H0 ° V OR3 R O + *O R "' T o o o 67 86 87 Scheme 18. Equilibrium between open chain and closed hemiketal isomers Exploitation of the enolate character in the a-position has allowed for regiospecific incorporation of various substituents within the y-keto carbonyl backbone. Various electrophiles have been studied and extensive discussion of the zinc-mediated homologation-aldol reaction was provided. Stereocontrol for many of the tandem reactions has been achieved and will be discussed later in this document in more detail 26 (see Natural Product Synthesis using the Chain Extension Reaction). In more recent years, functionalization of the P-position within the y-keto carbonyl core has been studied in an effort to broaden the scope and versatility of the chain extension reaction. Formation of B-Functionalized Y-Keto Carbonyls Applying the chain extension reaction towards the synthesis of peptide isosteres has relied on the incorporation of functionality in the a-position of the 1,4-dicarbonyl unit. More recently, expansion of this methodology for the synthesis of various natural and unnatural products has centered on functionalization at the P-position. As predicted by the proposed mechanism of the reaction, the p-methylene of the product arises from insertion of the zinc-carbenoid, derived from diiodomethane, into a 1,3-dicarbonyl. Functionalization at the P-position, therefore, relies on formation of a substituted zinc- carbenoid derived from gew-diiodoalkanes. Many methods have been reported for the synthesis of gew-diiodoalkanes.43 Exposure of 1,1-dichloroethane (88) and ethyl iodide (89) to catalytic aluminum chloride results in a facile preparation of 1,1-diiodoethane (90) (Scheme 19).44 Subjecting benzaldehyde (91) to trimethylsilyliodide (92) at room temperature will result in the formation of a,a-diiodotoluene (93).45 Both 1,1-diiodoethane (90) and a,a-diiodotoluene (93) were successfully employed in the chain extension reaction for the formation of P- substituted y-keto carbonyls. 27 CI cat. AICI3 CI + ^N •- reflux 88 89 90 CHCI3 »- 93 , Scheme 19. Preparation of 1,1-diiodoethane (90) and a,a-diiodotoluene (93) Substituted carbenoids derived from gem-diiodoalkanes have been employed in the synthesis of substituted cyclopropanes;46 however, formation of substituted donor- acceptor cyclopropanes had not been explored. In 2007, Lin et al. reported the first synthesis of p-substituted-y-keto carbonyls using a methyl-substituted carbenoid (Table 3).47 P-Keto esters and amides (35a-d, 42e-i) were exposed to a methyl-substituted carbenoid derived from diethylzinc and 1,1-diiodoethane (90), rendering the donor- acceptor cyclopropane intermediate 94. Subsequent fragmentation of the cyclopropane intermediate 94 followed by an acidic work-up generated P-methyl-y-keto esters or amides 95a-i in one step. The P-methyl-y-keto esters 95a-f were produced cleanly in good yield, whereas the P-methyl-y-keto amides 95g-i were produced in slightly lower yields. All attempts to increase the efficiency of the conversion of P-keto amide 42i to P- methyl-y-keto amide 95i were unsuccessful, which suggested the R -group of a P-keto tertiary amide is limited to less sterically bulky substituents. 28 X Zn o o O a. Et2Zn O O R2 -*• R1 1 2 R b. CH3CHI2 (90) R R O 35a-d 94 95a-i 42e-i Entry R1 R2 Yield (%) la Me -OMe 76 2b Me -0/-Bu 88 3c /-Bu -OMe 82 4d Ph -OEt 84 5e Me -OCH2CH=CH2 74 6f Me -OBn 80 7g Me -NMe2 67 8h Me i-O 74 9i z'-Pr -NMe2 incomplete conversion Table 3. Conversion of p-keto esters and amides to the respective P-methyl y-keto substrates using a methyl-substituted carbenoid Incorporation of other substituents at the P-position was desired. ^-Butyl acetoacetate (35b) was subjected to a phenyl-substituted carbenoid, derived from diethyl zinc and a,a-diiodotoluene (93), which provided P-phenyl-y-keto ester (96) in 74% yield (Scheme 20). Installation of a phenyl substituent at the P-position was successful; however, the reaction of the phenyl-containing carbenoid was, in general, less efficient 29 than the methyl-substituted carbenoid. In order to further expand this methodology and understand its scope and limitation, other substituted-carbenoids must be investigated. XX0M ^i^ JUyy b. PhCHI2 (93) ph O I 42b 74% 96 Scheme 20. Synthesis of P-Ph y-keto ester using substituted carbenoid derived from a,a- diiodotoluene (93) While regiospecific incorporation of functionality at the p-position of a 1,4- dicarbonyl backbone was successfully accomplished, further expansion of this methodology remains of interest. The method described above is the first example of a substituted zinc-carbenoid used for the formation of a substituted donor-acceptor cyclopropane. Tandem chain-extension-aldol reactions using a methyl-substituted carbenoid were investigated by Jacobine and will be discussed further (see Chapter II). Control of the stereochemistry of the P-methyl-substituent was studied as a part of this thesis work and will be described in detail (see Chapter III). Application of the chain extension methodology to the synthesis of peptide isosteres and natural products is greatly augmented by the development of regiospecific a- and P-functionalization. 30 Natural Product Synthesis using the Chain Extension Reaction Tandem chain extension processes have been used as key steps in the synthesis of a variety of natural products. In an attempt to showcase the tandem chain extension- oxidization-elimination reaction,36 Ronsheim efficiently synthesized two macrocylic compounds (Scheme 21). Exposure of macrolactone 97 to zinc-carbenoid in benzene at room temperature induced ring expansion and the intermediate enolate was quenched with iodine (I2) to afford iodinated macrocycle 98. Under kinetically controlled conditions, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) eliminated iodide from the macrocyclic backbone to provide (+)-Patulolide A (99) in 48%. When thermodynamically-controlled elimination conditions were utilized, (-)-Patulolide B (100) was formed in 66%. It is interesting to note that the initial insertion of the carbenoid into the macrolactone 97 is slower in comparison to acyclic 1,3-dicarbonyls. Such chain extension reactions were sluggish in dichloromethane (CH2CI2), but benzene quickly facilitated conversion to the product. 31 a. Et2Zn, CH2I2 benzene »• b. I2 c. Na2S203 97 98 a. Et2Zn, CH2I2 benzene Thermodynamic DBU (1 equiv) b. I2 Kinetic Control Control Et20, -15 °C c. Na2S203 d. DBU (excess) room temp. (-)-Patulolide B (100) (+)-Patulolide A (99) 66% 48% Scheme 21. Chain extension-oxidation-elimination reaction for the synthesis of (+)- Patulolide A (99) and (-)-Patulolide B (100) Another application for the tandem chain extension-oxidation-elimination reaction was the formal synthesis of (+)-Brefeldin A (101) (Scheme 22). Lin synthesized advanced intermediate 102 through the use of a StereocontroUed conjugate addition-intramolecular cyclization reaction and a ring closing metathesis reaction. Subsequent exposure of 102 to chain extension-oxidation-elimination conditions allowed access to bicyclic lactone 103, which possessed two E-alkenes in 46%). Subsequent reduction of the ketone and deprotection of the MOM group would provide (+)-Brefeldin A (101) as reported by Weber et al.50 32 ' H H? O £\^0 HO"' \^r^ H (+) •Brefeldin A (101) o o /^JL^AQ y a. Et2Zn, CH2I2 MOMO- MOMO"- b. I2 c. DBU 102 103(46%) Scheme 22. Formal synthesis of (+)-Brefeldin A In more recent years the tandem chain extension protocols have expanded to encompass novel electrophiles, such as imines. Jacobine developed the tandem chain extension-imine capture reaction in an attempt to synthesis p-proline derivatives (Scheme 23).35 The organometallic enolate, resulting from the treatment of P-keto ester 35 with carbenoid, efficiently captured JV-Boc imine 104. Subsequent exposure of compound 105 to trifluoroacetic acid (TFA) liberated the amine, inducing cyclization to a cyclic imine. Reduction with sodium cyanoborohydride (NaBH3CN) provided P-proline 106. Similarly, 7V-phosphinoylimine 107 was captured resulting in formation of an a- substituted y-keto ester 108. A one-pot protocol was developed for the deprotection of the phosphinoyl group and reduction of the cyclic imine, which afforded the diastereomeric P-prolines 109. X-ray crystal structure analysis of the chain extension-imine capture product 105, derived from the iV-Boc imine 104, allowed for the a«/7-stereochemical assignment. Interestingly, the nitrogen protecting group controlled the symanti stereochemistry at the C-2 and C-3 positions. This versatile methodology allows for 0- 33 proline derivates with specific stereochemistry around the pyrrolidine ring to be achieved through specific selection of protecting group on the imine. H R3 „NHBoc 1 .N. O3 n R n a. Et2Zn, CH2I2 O "y 1 TFA "V h~ OR2 \JT R1 OR2 b. ...Boc J 2. NaBH3CN 7T N ' 0 6 105 106 O O R^H 04 R1'^^-OR2~ 1 H 35 .33 N R3 NHP(0)Ph2 a.SOCI2 RV VR3 abt2Zni. Et2Zn'UH2, CH'22I2, , 19 Y OR2 MeQH - \J 1 R2 b. N^P(0)Ph2 R ^^Y b.NaBH3CN >^° II ° O R3^H 108 109 107 Scheme 23. Synthesis of P-proline derivatives using the chain extension-imine capture reaction Tandem protocols involving a methyl-substituted carbenoid have also been pursued as efficient routes towards highly substituted y-lactones (Scheme 24).51 A serendipitous discovery by Lin revealed a novel oxidative cleavage reaction of hemiketals mediated by eerie ammonium nitrate (CAN). Jacobine further developed a protocol that involved sequential tandem chain extension-aldol reaction and CAN oxidation in order to target substituted y-lactones. Treatment of methyl 4- methoxyacetoacetate (110) with a methyl-substituted carbenoid produced a P-substituted organometallic enolate, which was quenched with hexanal to afford 111 in 51%. Subjecting compound 111 to CAN led to oxidative cleavage of a cyclic reactive intermediate 112, in which the Celv is proposed to chelate between the hemiketal oxygen 34 and the methoxy group. Phaseolinic acid derivative 113 was formed in high yield. Elucidation of the stereochemistry of y-lactone 111 was achieved by comparison with previously synthesized phaseolinic acids.52 It is interesting to note that the stereochemistry of the chain extension-aldol reaction of P-keto esters usually favors formation of the syn-isomer; however, the influence of the p-stereocenter gave rise to the unexpected anti-aldol product 111 as the major isomer. O O a. Et2Zn b CH O ^ CAN MeO^lX - 3CHI2 ^^ — OMDMoe 1 MeO^ A J^ ^OMe 110 c. Hexanal H20, MeCN O O 111 (51%) 113(77%) |V n ~ Ce \^0N / \-OMe O 112 Scheme 24. Synthesis of phaseolinic acid derivative (113) through the chain extension- CAN oxidation reaction The predominant application of the zinc-mediated homologation reaction has been the synthesis of peptide mimics.31'53 Control of the absolute stereochemistry in the a-functionalization of the y-keto backbone during a tandem-chain extension protocols has relied heavily on chiral P-keto imides derived from oxazolidinones (Scheme 25).54 Lin subjected L-alanine-derived P-keto imide 114 to the zinc-carbenoid in the presence of formaldehyde to provide a-hydroxymethyl-y-keto imide (115) in good yield. With the 35 core backbone of the peptide mimic established in one step, subsequent chemistry allowed access to ketomethylene isostere 116, which exists in equilibrium with hemiketal 117. A significant modification to the traditional chain extension-aldol protocol, in which the aldehyde was inserted prior to chain extension of the P-keto imide 114, was necessary for the synthesis of 115. When p-keto imide 114 was stirred with carbenoid in the absence of an aldehyde, rapid homoenolate formation was observed leading to cyclopropane byproducts. HOs Bn, Cbz O O O a. Et2Zn cbz O \ '",—v steps Pmb' ^^"^N O *• Pmb' ^r^^Y V \_J c.CH2l2 s 0 0 68% 114 Bn 115 H Me2NOC^ n \s n Me2NOC. ^ 0 ! O Me2NUU. Q Boc. H o - O = H o - o = 116 117 Scheme 25. Synthesis of ketomethylene isostere 116 through the use of the tandem zinc- mediated chain extension-aldol reaction In an attempt to optimize the formation of the cyclopropane products, Taschner discovered an unusual rearrangement.30b Further exploration led to the development of a novel approach for the formation of an unprecedented peptide isostere (Scheme 26). Amino acid derived P-keto imide 118 was subjected to an excess of diethylzinc and diiodomethane in a 1:2 ratio, respectively. Rapid consumption of starting material produced homo-enolate 119 that underwent an intramolecular cyclization into the imide 36 carbonyl. Cyclopropane isomerization followed by intramolecular acylation afforded bicyclic lactone 120. Amidation of lactone 120 with benzyl amine produced a novel hydroxy-cyclopropyl peptide isostere 121. In comparison with cyclopropyl peptide isostere 122, novel hydroxy-cyclopropyl isostere 121 maintains restricted rotation about the mimicked amide bond while offering a H-bonding functionality that would mimic the tetrahedral intermediate formed in hydrolysis of a peptide. Further exploitation of this methodology is currently under investigation within the Zercher research group and will be reported shortly.55 Pmb O O Q OH Pmb i ,N Cbz Cbz' N^Xbu_z - n XIY O R1 118 121 120 A ^.ZnX Pmb O { J—\ R1 O O cyclopropyl peptide 119 isostere Scheme 26. Synthesis of novel hydroxy-cyclopropane peptide isostere 121 In summary, the zinc-mediated chain extension reaction is a versatile method for the preparation of natural and unnatural products. Many tandem protocols have been successfully used as key steps in the synthesis of these targets. The exceptional regiocontrol allows for incorporation of functionality at the a- and or P-position of a 1,4 dicarbonyl, which is highly desired when targeting peptide isosteres. Ongoing research directed toward the development of tandem chain extension-acylation reactions are 37 underway and will be discussed in detail in chapter II. The stereochemical control at the a-position for many of the tandem reaction sequence has been successfully accomplished through the use of chiral oxazolidinones. Control of the stereochemistry of the P- substituent will be discussed in chapter III. 38 CHAPTER II SYNTHESES OF PAPYRACILLIC ACIDS: APPLICATION OF THE TANDEM CHAIN EXTENSION-ACYLATION REACTION Papyracillic Acids Many fascinating natural product targets originate as metabolites from fungi. Papyracillic acid A (123), initially isolated from the ascomycete fungus Lachnum papyraceum, possesses a spiro-fused ketal core (Figure 4).56 Structural similarities between papyracillic acid A (123) and penicillic acid (124), a classical mycotoxin, suggest an equilibrium between the hemiketal and open-chained form. In fact, papyracillic acid A (123) was reported to exist in a 1:1:2:4 mixture of unspecified isomers. / Ho HO>(X^O _ Wr\ h^n _ X\ = / \> O C02H -\° ° O C02H A (123) 124 Figure 4. Structural comparison between papyracillic acid A (123) and penicillic acid (124) 39 In addition to the structural comparisons, the biosynthetic pathway for the formation of papyracillic acid A (123) is proposed to mimic that of penicillic acid (124) (Scheme 27).57 Oxidation of a quinone, such as 125, could result in the formation of seven-membered lactone 126. Subsequent reduction could generate the open-chain form 127 of papyracillic acid A. [oxid] Scheme 27. Proposed biosynthetic pathway for the formation of papyracillic acid A (123) Nucleophiles, such as amino acid residues, were proposed to attack the C-3 position of the lactone ring 123a or the a,p-unsaturated ketone of the open-chain form 123b, a process which was proposed to give rise to cytotoxic effects. Further investigation, by Shan et al, suggested the cytotoxicity was dependent upon the a,P- unsaturated ketone of the open-chain form 123b (Scheme 28).5 The mixture of hemiketal 123a and open-chain form 123b was subjected to cysteine 128, which produced sulfur-containing hemiketal 129, presumably through conjugate addition into the a,P-unsaturated ketone of the open-chain 123b. 40 o. HO O JO. or o 37 °C o 6 COoH cVsteine 128 C02R pH7 123a 123b R = H, -CH3 Scheme 28. Nucleophilic attack of papyracillic acid A (123) by cysteine More recent, papyracillic acid B (130) and C (131) were isolated from an endophytic fungus Microsphaeropsis sp. (Figure 5). This fungus was extracted from the branches of the tree Larix decidua, found in Hjerting, Denmark. While the biological activity for only papyracillic acid A (123) has been studied, the absence of the a,P- unsaturated ketone within the structure of 130 and 131 suggests that biological significance is unlikely. Regardless of the biological activity, the spiro-fused cyclic backbone lends itself as challenging synthetic target. No synthetic approach has been reported for any of the papyracillic acids, thus the development of a concise route was targeted. B (130) Figure 5. Recently isolated papyracillic acid B (130) and C (131) 41 Synthetic Approach The zinc-mediated chain extension reaction was anticipated to be the key step in the synthetic approach towards the papyracillic acids as illustrated in the retrosynthetic analysis (Scheme 29). The hemiketal of papyracillic acid A (123) would come about from a hydrolysis of the methyl ketal of papyracillic acid B (130). The exocyclic methylene of 130 would arise from an elimination reaction of the hydroxymethyl functionality of papyracillic acid C (131). Installation of the methyl ketal and manipulation of the hydroxymethyl functionality of papyracillic acid C (131) would come from spiro-fused ketal 132, which would be expected to exist in equilibrium with an open-chain form 133. Careful examination of open-chain form 133 reveals an a,P- substituted y-keto ester, which would potentially be accessible from a tandem chain extension reaction. Exposure of 3-methoxymaleic anhydride 134 to organometallic intermediate 135, derived from the P-keto ester, would constitute the necessary acylation reaction. This tandem chain extension-acylation reaction offers a one-step method for the preparation of spiro-fused ketal structures, where two stereocenters are conveniently established. 42 $ B(130) \y O O XZn" = OR1 O \ 135 134 132 Et2Zn CH3CHI;) \y 0 0 j / kA0Ri Scheme 29. Synthetic route for the synthesis of papyracillic acid A, B, and C highlighting the tandem chain extension-acylation reaction as the key step The proposed synthesis of papyracillic acids through the utility of the chain extension reaction relied on two separate variations, a chain extension reaction using a methyl-substituted carbenoid and a tandem chain extension-acylation reaction. The zinc- mediated homologation reaction using a methyl-substituted carbenoid was developed and reported by Lin and McGinness.47 A tandem chain extension-aldol protocol using a methyl-substituted carbenoid was successfully applied to the synthesis of phaseolinic acid derivatives,51 thereby demonstrating the ability to perform tandem reactions with the methyl-substituted carbenoid. Capturing the chain extension intermediate with anhydrides was relatively undeveloped chemistry, thus a greater understanding of the 43 tandem chain extension-acylation reaction was required prior to the synthesis of the papyracillic acids. Tandem-Chain Extension Acylation Reaction using Maleic Anhydride The scope of the chain extension methodology has been enhanced through tandem reaction protocols, in which various electrophiles have been used to trap the organometallic intermediate. Jacobine recently reported that the chain extension reaction could be used to trap anhydrides in a tandem acylation reaction.35 Methyl pivaloylacetate (35c) was subjected to the chain extension conditions then treated with acetic anhydride (136) (Scheme 30). An a-acylated y-keto ester 137 was produced efficiently and did not require further purification. Successful incorporation of an acyl functionality, regiospecifically in the a-position of the y-keto ester core, confirmed the feasibility of the methodology required for the synthesis of papyracillic acids. u a >r ^ b^T^ >r r 35c ^J^ 137 O 136 Scheme 30. First example of the tandem chain extension-acylation reaction using acetic anhydride (136) as an electrophile Of great relevance to the papyracillic acid project was the exploration of a chain extension-acylation reaction that utilized maleic anhydride (138) as an electrophile 44 (Scheme 31). Due to the low solubility of maleic anhydride (138) in dichloromethane, anhydrous toluene was selected as the solvent and was used throughout the development of the chain extension-acylation methodology. Initial efforts focused on the acylation reaction, therefore the substituted carbenoid was not utilized in these early studies. Treatment of methyl pivaloylacetate (35c) with carbenoid, derived from diethylzinc and diiodomethane, followed by anhydride 138 was expected to give rise to a mixture of open-chain form 139 and hemiketal 140. Analysis of the crude reaction mixture by *H NMR and 13C NMR was challenging due to the complexity of the spectra; however, several key resonances believed to correspond to product were identified. In the lH NMR spectrum, the doublet of doublets at 4.29 ppm, which appeared to intergrate for 1 proton, was consistent with the methine a to the ester of the open-chain form 139. The 13C NMR showed a multitude of new resonances tentatively assigned as evidence for the carboxylic acid, a,P-unsaturated ketone, lactone, and hemiketal. Furthermore, the unsubstituted y- keto ester was not observed, which further confirms that the anhydride 138 was trapping the chain extension intermediate through an acylation reaction. The TLC analysis of the crude reaction mixture showed several overlapping streaky spots, which suggested purification by a column chromatography would be a challenge. 45 35c W/ 139 140 O 138 Toluene, 0 °C - r.t. 18 h Scheme 31. Initial studies of the tandem chain extension-acylation reaction using maleic anhydride The equilibrium between open-chain form 139 and hemiketal 140 was believed to give rise to a complex mixture unsuited for column chromatography. Thus, conversion of hemiketal 140 to ketal 141 was hypothesized to simplify the crude mixture and produced a product more appropriate for chromagraphic purification. Both attempts at converting the mixture of 139 and 140 to ketal 141 resulted in an even more complicated crude reaction mixture, in which the desired product could not be identified (Table 4). The conditions for conversion of hemiketals to ketals are well established; therefore, the unsuccessful formation of 141 prompted a closer look at the crude reaction mixture produced after the chain extension reaction. 46 Ck ^ C02H 139 140 O 141 Q \ \ Entry Conditions Results cat. TFA, CH3OH complex mixture cat. TsOH, (CH30)2C(CH3)2 complex mixture Table 4. Failed attempt at converting crude hemiketal 140 to ketal 141 Isomerization of the Z-alkene to a thermodynamically more stable £-alkene was hypothesized. The H-H coupling constant for a cis-alkene 142 (3J~ 11.0 Hz) is typically less than the trans-alkene 143 (J ~ 15.0 Hz) (Figure 6). Confirmation of an isomerization was conducted through detailed inspection of the coupling constants for the alkene protons. Within the crude reaction mixture overlapping alkene resonances, ranging from 8.12 ppm - 6.67 ppm, made the interpretation challenging. However, measurement of the coupling constant for the dominant alkene resonances revealed a 3J = 15.8 Hz, which was consistent with the is-alkene 144. While purification of a single product, as determined by TLC analysis, was successfully accomplished, the lH NMR spectrum for compound 144 revealed a mixture of keto and enol tautomers. 47 H H H CH, OVH' 3J=15.8Hz H,C HoC J CH, H >fAAflArco2H 3 3 I On Hu , s Jcis= 10.9 Hz J/rans=15.1 Hz 142 143 144 Figure 6. Coupling constants for cis- and trans-alkenes compared to compound 144 Inspection of the !H NMR spectrum for the purified acylated product revealed a mixture of two compounds (Scheme 32). Tautomeric isomers, keto 144 and enol 145, were observed to exist in a 1:1 ratio. The H-H alkene coupling constants for the keto-enol tautomers were J= 15.8 and 15.1 Hz, respectively, which confirmed both isomers had trans-alkenes. The unusually high concentration of enol tautomer 145 was rationalized by the increased conjugation extending between the methyl ester and carboxylic acid functionalities. O^/O OH ^= 145 COoH Scheme 32. Keto-enol tautomerization of is-alkene 144 In an effort to probe the Z- to E'-isomerization, P-keto imide precursor 146 was studied (Scheme 33). After trapping anhydride 138, intermediate Z-alkene 147 was expected to withstand deprotonation at the a-position due to the increased steric strain of enolate 148. While the avoidance of A 1,'3 strain of 148 62 was hypothesized to aid in the 48 prevention of enol formation, much effort was required to optimize the tandem chain extension-acylation protocol for the P-keto imide precursor 146. °yo OOO N a Et oV ^ J^X X - 2Zn, CH2I2 \—^ b.0^°\^0 O C02X X02C 146 \=/ 138 147 Scheme 33. P-Keto imide during the chain extension-acylation reaction As described in Chapter I, the tandem chain extension chemistry using P-keto imide 146 is more difficult to control due to the increased reactivity of intermediate enolate 149; therefore, careful manipulation of the reactions conditions were required in order to optimize the conversion to acylated product (Table 5). Initially, P-keto imide 146 was treated with a 2.5 equivalents excess of ethyl(iodomethyl)zinc, which resulted in formation of a-methylated y-keto imide 150 as the major product along with bicyclic lactone 151. The acylated product 152, formed through the capture of maleic anhydride, was produced in low yield. As expected, both the a-methylated y-keto imide 150 and the bicyclic lactone 151 products, which are produced from the reaction of enolate 149 with excess carbenoid resulting in homoenolate 153 formation. p-Keto imide 146 was then subjected to bis(iodomethyl)zinc formed from a 1:2 ratio of diethylzinc and diiodomethane. a-Methylated y-keto imide 150 was again the major product, but the formation of bicyclic lactone 151 was decreased. The increase in the amount of acylated product 152 was presumably due to the absence of an ethyl ligand on the zinc, which is 49 known to react via a 1,2-addition with maleic anhydride 138. Substantially decreasing the amount of carbenoid to 1 equivalent resulted in the formation of an excess of y-keto imide 154 and a decrease in acylated product 152 formation. Finally, exposure of P-keto imide 146 to 1 equivalent of diethylzinc for 10 min, followed by the addition of diiodomethane and maleic anhydride (138), resulted in the highest conversion to acylated product 152. Interestingly, the use of toluene as a solvent produced more a-methylated 150 than bicyclic lactone 151, which was opposite to the results Taschner observed when dichloromethane was used as the solvent. 50 O 9 ij J Et2Zn o°VN^ N O AAA _^£ \\ 138 *—' Toluene °w° o 146 149 ln 152 ^ZnX 0 r\ V 153 O O V 151 o o 154 o i -\ Vr o o 150 a a ab Entry Et2Zn (equiv) CH2I2 (equiv) BKI (equiv) 150 (%) 151 (%)" 154 (%) 152 (%) 1 2.5 2.5 1 — 62 21 17 2 2.2 4.4 1 .... 62 12 26 3 1 1 1 75 10 —- 15 4 lc lc 1 51 5 8 36 Table 5. Optimization of tandem chain extension-acylation reaction using P-keto imide 146 (aRelative abundance using relative integrations from 'H NMR analysis of the crude reaction mixture; bCoupling constant for alkene protons was 3J= 15.8 Hz; cDiethylzinc and P-keto imide were stirred for 10 min prior to adding diiodomethane) The coupling constant for the alkene protons of the acylated product 152 was 3J = 15.8 Hz, which suggests that isomerization of the Z- to .E-alkene took place (Figure 7). As expected, y-keto imide 153 did not exist in a mixture of keto-enol tautomers, which 51 suggests that enol formation is not a requirement for isomerization of the alkene. After purification by preparative TLC, 152 was isolated as one isomer, which was easily characterized. Unfortunately, p-keto imide 146 did not capture the anhydride as efficiently as the P-keto ester precursors. OV'H Figure 7. Coupling constant for isomerized £-alkene derived from a P-keto imide Although no specific studies were conducted, the mechanism for the Z- to E- isomerization was considered (Scheme 34). One feasible route for isomerization may involve iodide, a byproduct of the chain extension conditions, which could serve as a nucleophile. A 1,4-addition to a,P-unsaturated ketone 155 could produce enolate 156. Upon bond rotation, enolate 157 could expel iodide and generate the isomerized intermediate 158. Subsequent protonation with an acid source would generate is-alkene 159. Currently, an iodide-promoted isomerization is speculative. Further mechanistic studies are necessary to definitively conclude the source of isomerization. 52 R O Y° .0 oV ,^X^r'- i RiAAiAco2x W O C02X R 0O^ 0 0 O C0 X 155 2 157 156 .e R R2 o Y° O Y° c iAA1]/^/ °2x O O 159 158 Scheme 34. Possible mechanism for the isomerization of Z- to £-alkene In summary, P-keto esters and imides were subjected to the tandem chain extension-acylation reaction using maleic anhydride as an electrophile. Both systems produced complex reaction mixtures that were difficult to purify and analyze. The p-keto esters were more efficient in trapping the anhydride, resulting in minimal recovery of unsubstituted y-keto esters. Controlling the product distribution desired from the P-keto imides was a challenge. A consequence of the reaction was an undesired Z- to E- isomerization. The exploration of other anhydrides was conducted in an effort to circumvent this isomerization and is described below. 53 Other Anhydrides In an effort to avoid alkene isomerization of the maleic anhydride, several strategies were investigated. One route involved the use of masked anhydrides (Scheme 35). Treatment of organometallic intermediate 51 with anhydride 160 was expected to produce acylated product 161. Exposure to heat was expected to induce a retro-Diels- Alder reaction releasing Z-alkene 155 and an equivalent of cyclopentadiene. The cyclopentadiene would be serving to mask and protect the Z-alkene of anhydride 160 from the undesired isomerization during the chain extension reaction. O ZnX Rl /„0 O Heat C02H O C02H 155 Scheme 35. Proposed strategy for the acylation reaction using a masked anhydride (160) The cycloreversion temperatures for three different masked anhydrides were considered (Table 6). Anhydride 160, derived from maleic anhydride and cyclopentadiene, possesses the highest cycloreversion barrier, which may require flash vacuum pyrolysis (FVP) for cycloreversion.65 The anthracene-derived anhydride adduct 162 can be pyrolized at relatively low temperatures (150 °C) on graphite in the 54 microwave. Additionally, the furan-derived anhydride 163 has the lowest required temperature for a retro-Diels-Alder reaction, at approximately 100 °C.67 Preparation of anhydride, 160,162, and 163 were performed in order to further investigate this strategy. 0 o 6' 160 162 163 Cycloreversion High temp. 150 °Ca 100 °C Table 6. Cycloreversion temperature (°C) for three masked anhydrides ("Cycloreversion temperature was achieved supported on graphite under microwave conditions) 1,3-Dicarbonyl was treated with carbenoid followed by the addition of a masked anhydride (Scheme 36). Each of the three anhydrides, 160, 162, and 163, gave complex crude reaction mixtures. Cyclopentadiene-derived anhydride 160 afforded the best results, with many promising resonances appearing in the H NMR spectrum; however, the high-energy barrier for cycloreversion rendered this anhydride less attractive. The anthracene and furan-derived anhydrides 162 and 163 had lower solubility in the chain extension reaction solvent (toluene). Recovery of both anhydrides 162 and 163, along with their corresponding diacids, from the chain extension-acylation attempts was accomplished, which suggested that efficient acylation of the chain extension intermediate had not occurred. More effort would be required in the development and 55 optimization of the reaction conditions in order to form the desired acylated product suitable for cycloreversion. O O II II a. Et Zn, CH I 2 2 2 Complex Mixture b. Anhydride 160 162 Scheme 36. Preliminary results using masked anhydrides during chain extension reaction An aliphatic cyclic anhydride was also studied, even though its potential utility for the synthesis of papyracillic acids would require substantial manipulation (Scheme 37). Similar to maleic anhydride 138, succinic anhydride 164 is also a 5-membered cyclic anhydride; however, no opportunity for isomerization exists because succinic anhydride 164 lacks an alkene. Exposure of p-keto ester 35c to carbenoid and subsequent addition of succinic anhydride 164 produced acylated product 165. While the equilibration between open and closed forms remained a challenge for characterization, the *H and 13C NMR spectra for the purified mixture clearly indicates three separate isomers, suggestive of 165, 166, and 167. The lH NMR spectrum showed a doublet of doublets at 4.05 ppm (J= 8.3, 5.7 Hz), which is presumed to correspond to the methine of the open-chain form 165. The mixture of products was subjected to conditions for acid-mediated ketal formation in an effort to generate spiro-fused methyl ketal 168. Purification by 56 preparative TLC resulted in the isolation of multiple different products, which suggests conversion to the desired methyl ketal 168 is not selective. Further investigation of this method is necessary to fully understand the product distribution. These efforts were not pursued, since an alternative route was developed, as described below. (See synthesis of spiro-fused ketal cores) O^ JD O^/O O O O O O a. Et2Zn, CH2I2 »• O OH ^= HO b- 0^°N^0 O O- 35c 165 166 O 164 HO .0 O^O O 167 \ cat. TsOH (MeO)2C(Me)2 —O Scheme 37. Tandem chain extension-acylation reaction using succinic anhydride (164) The identification of an anhydride that would allow access to a spiro-fused core of papyracillic acids without competitive isomerization of a Z-alkene was desired. The utilization of an alkynyl anhydride was, therefore, proposed; however, the attempted 57 synthesis of alkynyl anhydride 169 lead to an unanticipated outcome (Scheme 38). Ethyl propiolate (170) was subjected to rc-butyl lithium («-BuLi) at low temperatures and then transferred by cannula into a flask containing solid carbon dioxide, which produced carboxylic acid 171 in high yield. Treatment of carboxylic acid 171 with 0.5 equivalents of dicyclohexylcarbodiimide (DCC) was expected to produce alkynyl anhydride 169; instead an unanticipated heterocycle, initially assigned as 172, was produced cleanly in 25% yield. The clean conversion to product 172 along with the rapid DCC-mediated consumption of carboxylic acid 171 prompted further investigation into this unprecedented cyclization. ^ EtO OEt o 169 a. n-BuLi, -78 °C Eto EtO b. C02 THF, 95% 170 EtO 172 Scheme 38. Unanticipated DCC-mediated heterocycle formation 58 Carbodiimide-Mediated Heterocycle Formation Initially only one heterocycle 172 was considered as the product of the DCC- mediated intramolecular cyclization; however, more detailed consideration of the mechanistic pathways suggested additional options (Scheme 39). Activated carboxylic acid 173 could react via a 6-endo-dig cyclization to produce intermediate 174; subsequent tautomerization would produce isourea 175. Alternatively, fragmentation of intermediate 174 would give rise to cumulene intermediate 176, which could react via another intramolecular cyclization and generate a uracil-type heterocycle 172. Another pathway that was considered was the 5-exo-dig cyclization of 172 producing 5-membered intermediate 177. Upon tautomerization, 5-membered isourea 178 could be produced. Lastly, N- to O- acyl migration, generating intermediate 179, followed by a 5-exo-dig cyclization would generate urea heterocycle 180. The !H NMR of the crude reaction mixture of the unidentified heterocycle revealed the presence of only one major product. Identification of this unknown heterocycle was of interest. 59 R O H©R HN N N EtO Y ^ 6-endo-dig Etc/Sf Y* % tautomerization EtO .O UyO 00 173 174 "I O nucleophilic attack followed by .0 EtO tautomerization EtO C HN.y _ II R C II O 176 ^R ^R 5-exo-dig H-N- tautomerization O 6 Go^C BO'\NO o O OEt 178 177 R O N- to O- acyl 5-exo-dig transfer followed by tautomerization EtO Et0^0 O 180 Scheme 39. Comparison between the 6-endo-dig and 5-exo-dig cyclization pathways Different heterocycle derivatives were prepared in order to probe the reaction conditions and elucidate the structure of the product (Table 7). Carboxylic acid 170a was subjected to carbodiimide 181a in the presence of catalytic DMAP. After column chromatography the heterocycle was isolated in 25% yield. Under more dilute reaction conditions, in the absence of DMAP, carboxylic acid 170b and carbodiimide 181b reacted to produce heterocycle in 30% yield. The heterocycles derived from DCC were viscous oils, unsuited for growing a crystal, thus heterocyclic derivatives possessing 60 subsitutents that would aid in crystallization were pursued. Carboxylic acid 170c was treated with carbodiimide 181c, producing, after purification, a 63% yield of z-Pr-derived heterocycle as a crystalline white solid, which was submitted for X-ray analysis. Unfortunately, the results obtained from X-ray analysis were inconclusive; however, the formation of five-membered ring was tentatively supported. Currently, all attempts at growing an X-ray grade crystal have not been fruitful. O R DMAP s6?0\\A + *N=C=N •- Heterocycle 2 RI R DCM, 1h, r.t. 170 181a,b Entry R1 R2 170 181 (equiv) DMAP Molarity (M) Heterocyle Yield (%)" (equiv) (equiv) la -C02Et Cy 2 1 02 01 182 25% 2b -C02Et Cy 2 1 0 06 182 30% 3c -C02Et (Pr 1 1 0 06 183 63% Table 7. Investigation of different reaction conditions for heterocycle formation ("Isolated yield after column chromatography) A second mechanistic pathway that was considered would arise from an intermolecular reaction. While the intramolecular pathway was speculated to be the favored route, an intermolecular process was also viable. An experiment was designed to probe the mechanism of cyclization (Scheme 40). Carboxylic acid 170 and dicyclohexyl urea 184 were stirred and treated with diisopropylcarbodiimide 181c. Incorporation of the cyclohexyl moieties into the heterocyclic core would only be possible through an 61 intermolecular cyclization to produce 183. Heterocycle 182 would result from an intramolecular cyclization. Analysis of the crude reaction mixture by !H NMR revealed \ exclusive incorporation of z'-Pr substitutents, which suggests that the intramolecular cyclization was operative. a R R - 'A- ' O C)nn O H H Et02C V Et02C ™. . VVR1 vv*2 NX 2 N=C=N .• Rp11 r0> RR2 O> 181c R2 182 183 R1= R2= Scheme 40. Intra versus intermolecular cyclization (Conclusive evidence for the five-membered ring heterocycles has not been achieved, 182 and 183 were tentatively assigned.) Moran synthesized phenylacetylene-derived carboxylic acid 185 in an attempt to study and expand the scope of the DCC-mediated heterocycle formation (Scheme 41).68 Carboxylic acid 185 was subjected to DCC for several hours at room temperature. The *H NMR spectrum for the crude reaction showed a mixture of products. Purification of the crude mixture resulted in isolation of trace amounts of the desired heterocycle 186 and a second product hypothesized to be the imide 187. As previously mentioned, the activated carboxylic acid, derived from DCC, has the opportunity to undergo an N- to O- acyl migration, which may explain the isolation of imide 187. Perhaps the electron rich phenyl 62 substituent disfavors cyclization, which may explain the trace amounts of heterocycle recovered. DCC DCM, r.t. 185 186 187 Scheme 41. DCC-mediated cyclization using phenylacetylene derived carboxylic acid The results obtained using the phenylacetylene-derived carboxylic acid prompted a closer look at the opportunity for substituents to promote or hinder cyclization (Figure 8). If intermediate 188 is the key intermediate during the DCC-mediated cyclization then the functionality in the R position serves a significant role in cyclization. Ester 170 promotes cyclization while the phenyl 185 shows little evidence for cyclization; thus, the exposure of various different electron withdrawing and donating substituents would be valuable. p-Nitrophenyl 189 and/?-cyanophenyl 190 are two examples of easily prepared carboxylic acid precursors which have electron withdrawing capability, hypothesized to promote cyclization. In contrast, alkyl 191 and /?-methoxyphenyl 192 should disfavor cyclization. The investigation of the reaction is ongoing. 63 o HTAO R2 188 170 Alkyl OoN 189 190 192 Figure 8. Electron withdrawing and electron donating substituent effects In summary, the reaction between carbodiimides and alkynyl carboxylic acids was studied. Unfortunately, at the present time, the connectivity of the heterocycle remains unknown. Efforts to isolate a crystal suitable for X-ray crystal structure analysis are ongoing. Optimization of the reaction conditions will be required to increase the yield of the heterocycle. Furthermore, substituent effects must be studied in order to establish the limitations for cyclization. Synthesis of Spiro-Fused Ketal Cores The electrophile needed to produce the desired spiro-fused cyclic ketal core of the papyracillic acids was 3-methoxymaleic anhydride (134). Sahoo et al. had reported a facile method for the preparation of anhydride 134 (Scheme 42), and this method has 64 been reproduced successfully.69 Dimethyl acetylenedicarboxylate (DMAD, 193) was subjected to anhydrous methanol and then treated with triethylamine to produce dimethylmethoxyfumarate (194) in 98%) yield. The mixture of E and Z-isomers 194 was hydrolyzed to the corresponding dicarboxylic acids 195 in 87% yield. Refluxing the dicarboxylic acids 195 in thionyl chloride resulted in the formation of anhydride 134, with the ^-isomer isomerizing to the Z-isomer in situ. After purification by flash chromatography on silica, 3-methoxymaleic anhydride was obtained in 66% as a yellow solid. 4 M KOH Q/ dryCHsOH ^xJL.0^ Et2N I CH3OH 98% 194 u 87% 9 S0CI HO ° OH * , °^y° reflux )~^ ^J 66ceo%/ V 195 \N 134 Scheme 42. Synthesis of 3-methoxymaleic anhydride (134) The initial investigation into the use of anhydride 134 began by subjecting methyl acetoacetate 35a to the Furukawa-modified carbenoid, followed by treatment of the resulting enolate with anhydride 134 (Scheme 43). TLC analysis indicated consumption of the intermediate enolate and production of a product with a much lower Rf. While the equilibrium that exists between the open-chain form 196 and hemiketal 197 made the 1 1 ^ analysis of the H NMR spectrum challenging, the C NMR spectrum suggested the presence of hemiketal and ketal through resonances at 109.2, 108.1, 108.0, 107.5, and 65 102.5 ppm. The crude product mixture was difficult to analyze by TLC due to severe streaking. This result indicated that flash chromatography purification may be difficult, thus the crude reaction mixture was carried on to the next step without further purification. a Et2z cH z I xx0- - "' ". XXX — b. O ^°\—O " I U 35a ^ >* O C02H )=J 196 O \ 134 Scheme 43. First example of the tandem chain extension-acylation reaction for the synthesis of spiro-fused cyclic ketal core Conversion of hemiketal 197 to ketal 198 was performed in an effort to trap the spiro- fused ketal core as a product mixture suitable for chromagraphic purification. The crude product mixture of open-chain form 197 and 198 was subjected to/7-toluenesulfonic acid (p-TsOH)-catalyzed ketal formation in 2,2-dimethoxypropane (Scheme 44). Chromagraphic purification of the crude mixture resulted in the separation of three diastereomers, which were characterized by NMR spectroscopy. Two of the three diastereomers eluted together as a result of similar Rf values. This result was the first confirmation that the tandem chain extension-acylation reaction was suitable for the synthesis of spiro-fused ketal backbones. 66 O ^f O' HO o ^\ cat. TsOH -^^Y^] ^=^ ^\_Xo (CH3)2C(OCH3)2 O CO2H V=o 196 2 steps, 39% three diastereomers Scheme 44. Conversion of crude hemiketals to ketals As was described earlier, reaction of organometallic intermediate 51 with maleic anhydride 138 resulted in formation of ^-isomer 144 through isomerization under the chain extension conditions (Scheme 45). Although the intermediate enolate 51 efficiently trapped the maleic anhydride 138, irreversible isomerization to the ^-isomer 144 prevented formation of the desired spiro-fused ketal backbone. In contrast, the products formed in the reaction of 3-methoxymaleic anhydride (134) were capable of existing in the spiro-fused ketal form, which require a specific isomeric form. The presence of the methoxy-substituent changes the isomerization characterization of the alkene. It is possible that the methoxy group provides the opportunity for facile interconversion between Z- and ii-alkenes (196 and 199); thereby, facilitating closure to the spiro-ketal. The absence of !H NMR coupling constant method to investigate the EIZ product distribution complicated the analysis. Direct evidence was not available to support the existence of both alkene isomeric forms within the open-chain form. Attempts to identify the presence of an .E-alkene 199 by lK NMR spectroscopy were overshadowed by clean conversion to the desired spiro-fused ketal as the major product. The equilibrium of the crude reaction mixture will be described in further detail shortly. 67 O°Y° ^ O^JN^^CO2H O C02H I O O XZn' 139 144 .0u . o. Oc^; N^O 51 r^ o V o o ° Y° o" 4 134 ^ /^^Y^CO2H O C02H o 196 199 Scheme 45. Comparison of Z- and is-isomers derived from maleic anhydride (138) and 3- methoxymaleic anhydride (134) Integration of a methyl-substituent into the spiro-fused ketal backbone was required for the synthesis of papyracillic acid C (131) (Scheme 46). Sequential treatment of methyl acetoacetate (35a) with a methyl-substituted carbenoid and anhydride 134 resulted in a mixture of products. Unlike the tandem chain extension-acylation reactions described previously, the ratio of products was surprisingly biased towards one compound. Several key resonances in the *H and 13C NMR spectra were used to characterize the major product. *H NMR analysis revealed that the proton a to the methyl ester was a doublet at 3.40 ppm with a coupling constant of J = 12A Hz. The larger J value suggested a zra/w-relationship between the methyl substituent and the ester. The 13C NMR analysis suggested that the major product is a hemiketal 200 on the basis of a strong ketal and hemiketal resonances at 108.2 ppm and 107.0 ppm. Although quantifiably imprecise, the less intense ketone resonance at 212.1 ppm in the ' C NMR spectrum also suggests the major product to be cyclic structure 200 or the minor product is the open chain structure 201. 68 O O aiEt2?n. Iffi - b- CH3CHI2 C0 35a YV° ' 6 C02H \=J 201 O \134 Scheme 46. One-step synthesis of spiro-fused ketal core of papyracillic acid The major diastereomer could be selectively recrystallized from the crude product mixture. Fortunately, slow evaporation from dichloromethane afforded a crystal suitable for X-ray crystal analysis (Figure 9).70 The crystal structure analysis confirmed the existence of a hemiketal product. Consistent with the large coupling constant, a trans relationship between the methyl-substituent and the ester of the furan ring was present. Based upon the stereochemical results of tandem chain extension-aldol reaction, this trans relationship was unexpected. 69 Figure 9. X-Ray crystal structure for the major product of the tandem chain extension- acylation reaction Equilibration Studies Previous studies using the methyl-substituted carbenoid in a tandem chain extension-aldol protocol revealed a stereodirecting influence on the new a-stereocenter by the p-methyl substituent.35 Based on these kinetically controlled results, open-chain isomer 201a was predicted to be the major acylated product, which would lead to the formation of hemiketals 200a (Scheme 47). However, the X-ray crystal structure, in consistent fashion to the !H NMR analysis, confirmed that the major product had a trans- stereochemical relationship between substituents on the furan ring. Enol 202 was proposed as a key intermediate for the interconversion of open-chain isomers 201a and 201b, which subsequently cyclizes to hemiketal 200b. 70 H02C H02C OH C02H 202 Scheme 47. Equilibrium between cz's-isomer (200a) and zra/M-isomer (200b) Since the ^rans-relationship was desired for the synthesis of papyracillic acid C (131), understanding the equilibrium between the cis and trans-isomers 200a and 200b was necessary. A study was conducted in order to correlate reaction time and product distribution (Figure 10). At several different times an aliquot of the crude reaction mixture was quenched, worked-up, and analyzed by H NMR spectroscopy. In the aliquot quenched after 1 h, an approximate 1:1 ratio of trans-cis products were observed. These ratios were assigned on the basis of relative integrations of the doublet at 3.42 ppm (corresponding to the trans-isomer 200b, 3J = 12.4 Hz) and the doublet at 3.38 ppm (corresponding to the cz's-isomer 200a, 3J = 6.7 Hz). At 18 h, the trans-cis ratio has changed to 6:1 and at 24 h a 10:1 ratio is observed. The results from this time study support the notion that cw-isomer was equilibrated to the thermodynamic product, the zrara-isomer. 71 0^°N^O O 0 XZn" HO, n O O O O O v or° \ cis 200a trans cis, 1 1 18h trans cis, 6 1 24 h trans cis, 10 1 3 45 3 40 3 35 3 30 3 25 3 20 3 15 3 10 3 05 3 00 2 95 2 90 2 85 2 80 2 75 2 70 2 65 2 60 ppm Figure 10. Time study showing the increase of the trans-isomer (200b) and the decrease of the cz's-isomer (200a) A mixture of the trans-cis products, which had been isolated in a 1:1 ratio, was subjected to several different conditions in an effort to convert the cw-isomer 200a to the trans-isomer 200b, presumably through the enol intermediate 202 (Table 8). Exposure of the mixture to catalytic hydrochloric acid in ethyl acetate at room temperature for 24h increased the trans-cis ratio to 2:1. Similarly, treatment with catalytic p-TsOH in chloroform at room temperature for 24 h resulted in the same ratio of isomers, (2:1). 72 Finally, microwave-irradiation at 100 °C under acid-catalyzed conditions for 1.5 h converted all of the cz's-isomer to the trans-isomer 200b. o Y cr O conditions OH C0 H FPO 2 202 trans-cis (1:1) Entry Conditions trans-cis ratio 1 cat. HC1, EtO Ac, room temp, 24h 2:1 2 cat. TsOH, CHC13, room temp, 24h 2:1 3 cat. HC1, EtOAc, MW 100 °C, 1.5h trans Table 8. Conversion cis- to trans-isomer 200b through an intermediate enol 202 In summary, the equilibrium between open-chain form and hemiketal, arising from the tandem chain extension acylation reaction, has been studied. The equilibrium is biased toward the closed hemiketal products. The cis- and ^rans-relationship between the methyl-substituent and the ester of the furan ring can be manipulated to favor the thermodynamically more stable zrara'-isomer. Although conditions have been developed for the conversion of the cz's-isomer to the trans-isomer, additional research would be necessary to completely understand the variables involved in the initial product distribution generated after the tandem chain extension-acylation reaction. 73 Stereochemical Assignment for the Major Diastereomer The crude reaction mixture, produced as a result of the tandem chain extension-acylation reaction, was subjected to acid-catalyzed ketal formation (Scheme 48). The optimized reaction conditions that were best suited for this transformation were catalytic p-TsOH in trimethyl orthoformate. Full conversion of the isomers 200 and 201 to a mixture of ketal diastereomers was complete after 4 h. The separation of the diastereomers required flash chromatography on silica using a gradient elution mobile phase consisting of hexane and ethyl acetate. The major diastereomer 203a was isolated in 45% (two steps from 32a) and a minor diastereomer 203b was isolated in 6% (two steps from 32a). The stereochemical assignments for the major 203a and minor 203b diastereomers were made through X-ray crystal structure analysis and will be described shortly. Additionally, another minor diastereomer 203c was isolated in a mixture with the major diastereomer. A tentative stereochemical assignment for this third diastereomer 203c will be also described shortly. While the optimized conditions for tandem chain extension-acylation reaction lead to diastereomer with a /ram-relationship between the methyl and ester attached to the furan ring, identification of one cz's-diastereomer 203d was accomplished during the development of this methodology (see chapter IV for full characterization). 74 Scheme 48. Synthesis of spiro-fused methyl ketals 203a, 203b, and 203c ("Not isolated yield, eluted in a 2:1 ratio with major diastereomer 203a to 203c in 22%) The major ketal diastereomer 203a was isolated as a crystalline white solid in 45%) yield for the two steps. Full characterization of the desired product was 1 1 ^ accomplished by H NMR and C NMR analysis. The coupling constant for the doublet at 3.36 ppm, corresponding to the proton a- to the ester, was J= 12.3 Hz^ which suggests a z'rara-relationship, consistent with the trend observed during the characterization of the major hemiketal isomer. The X-ray grade crystal obtained through slow evaporation from chloroform, confirmed the stereochemical configuration of the major methyl ketal diastereomer was unchanged from the hemiketal (Figure ll).71 75 Figure 11. X-Ray crystal structure for the major diastereomer71 An X-ray grade crystal was also obtained for diastereomer 203b (Figure 12). Once again, the methyl-substituent and the ester functionality on the furan ring exist in a zrara-relationship. This minor diastereomer is an epimer of the major diastereomer at the methyl ketal carbon. 76 Figure 12. X-Ray crystal structure for epimer of the major diastereomer !H NMR chemical shifts for the major 203a and minor 203b diastereomers were compared (Figure 13). The major diastereomer 203a has a doublet at 3.38 ppm and a doublet of quartets at 2.64 ppm corresponding to the methines a- and 6- to the ester respectively. Changing the stereochemistry at the methyl ketal carbon, as observed with minor diastereomer 203b, gave rise to a significant chemical shift difference of the methines a- and B- to the ester. The minor diastereomer 203b has a doublet at 3.13 ppm and a doublet of quartets at 3.05 ppm. This chemical shift change was used as a tool to assign the tentative stereochemistry of the other minor diastereomer 203c. 77 / o, \ o ? -o •2 1 o o \ 203b l—'—i—'—1—'—i—'—i—'—i—'—I—'—I—'—I—'—r 3.5 3.4 3.3 3.2, 3.1 3.0 2.9 2.8 2.7 2.6 3.25 3.20 3 15 3.10 3.05 3.00 2.95 2.90 ppm ppm Figure 13. Chemical shift comparison between major 203a and minor 203b diastereomers Minor diastereomer 203c and major diastereomer 203a eluted off a column as a mixture (Figure 14). Since the stereochemistries of the major 203a and one of the minor diastereomers 203b were unambiguously established through X-ray crystal structure analysis, the chemical shift trend described above was used to assign the stereochemistry of minor diastereomer 203c. The similarities in chemical shift and coupling constants for both resonances arising from the methine protons a and B to the methyl ester within the lH NMR spectrum for the mixture of major 203a and minor 203c suggest the minor diastereomer 203c is likely an epimer of the major diastereomer 203a at the quaternary 78 spiro-fused carbon. The fourth possible trans-diastereomer was not isolated or observed by 'H NMR analysis. Figure 14. Stereochemical assignment of minor diastereomer 203c The synthesis of spiro-fused methyl ketals has been accomplished in two steps from simple starting materials. The stereochemistry of the major ketal diastereomer 203a, along with one minor diastereomer 203b, was assigned from the analysis of an X-ray crystal structure. Comparison between the major 203a and minor 203b stereoisomers suggested a chemical shift trend that was used to tentatively assign the stereochemistry of 79 203c. Next, the subsequent reduction of the methyl ester to a hydroxymethyl was aggressively investigated. Synthesis of Papyracillic Acid C Conversion of the methyl ester to an alcohol through a selective reduction in the presence of the electron rich lactone of 203a was proposed. This functional group transformation would furnish the spiro-fused ketal with a hydroxymethyl functionality and complete the synthesis of papyracillic acid C (131). Several attempts were made to selectively reduce methyl ester 203a (Table 9). In an effort to avoid undesired reduction of the lactone ring, borohydride reducing agents were initially investigated. L-Selectride, sodium borohydride (NaBH4), and super-hydride (lithium triethylborohydride) resulted in recovery of starting material. Super-hydride in refluxing THF appeared to react with the starting material as analyzed by TLC, but the product could not be identified by !H NMR analysis. Treatment of 203a with DIBAL-H resulted in rapid decomposition of the starting material. This result was not unexpected due to anticipated Lewis acid complexation with the electron-rich lactone and the resultant fragmentation of cyclic ketal core. More vigorous reduction conditions were pursued by subjecting methyl ester 203a to lithium aluminumhydride (LAH). At refluxing temperatures, LAH rapidly consumed starting material 203a and produced a mixture of products where over reduction of the lactone ring was evident. Full characterization of this product mixture was not pursued. Alternative routes for the formation of the hydroxymethyl side chain 204 were investigated. 80 Reducing Agent o ^o 203a Entry Reducing Agent Temp (° C) Solvent Time Results 1 L-Selectride -78-21 <3C Ether 3h starting material ! D 2 NaBH4, CeCl3 -78-21 C THF 16 h starting material ^ D j Super-hydride -78-21 ' C THF 7h starting material 4 Super-hydride 65 °C THF 6h starting material 5 LiAlH4 0°C Ether 2h starting material 6 DIBAL-H -78 °C DCM 2h decomposition 7 LiAlH4 65 °C THF 2h mixture Table 9. Attempted reduction of methyl ester 203a to hydroxymethyl 204 Other methods were explored for the transformation of methyl ester 203a to the desired hydroxymethyl functionality of papyracillic acid C (Scheme 49). Literature precedence supports the notion that carboxylic acid functionality should be easier to reduce to alcohol than a methyl ester since the carboxylic acid functionality can be easily converted to an activated ester, easily reduced by standard reduction conditions. A potassium hydroxide-promoted hydrolysis of methyl ester 203a was attempted in an effort to form the carboxylic acid, which could subsequently be reduced. Unfortunately, degradation of the starting material rendered this approach unsuitable. Additionally, 81 treatment of methyl ester 203a with potassium trimethylsilanolate (KOSiMes) resulted in recovery of starting material rather than the desired carboxylic acid. / 0.2 M KOH Complex Mixture ovo o)= Y THF, CH3OH ^wV^o *** y=^o 0 KOSiMe2 Starting Material 203a^ CH2CI2 Scheme 49. Unsuccessful attempts at converting methyl ester 203a to a carboxylic acid The synthetic approach for the synthesis of hydroxymethyl 204 was revisited in order to incorporate functionality that would allow facile access to carboxylic acid 205 (Scheme 50). Metal-mediated deallylation is well established in the literature as a selective and mild method for the synthesis of carboxylic acids from the corresponding allyl carboxyester. Incorporation of an allyl carboxyester into the synthesis of papyracillic acid C required the preparation of allyl acetoacetate (42e). .OO » AA,O ' 42e Scheme 50. Revisited synthetic approach for the synthesis of papyracillic acid C 82 Allyl acetoacetate (42e) was prepared through a facile method starting with Meldrum's acid 206 (Scheme 51). Exposure of Meldrum's acid 206 to pyridine followed by slow addition of acetyl chloride (207) produced adduct 208 in 88%. Treatment of 208 with allyl alcohol (209) in refluxing toluene produced crude P-keto ester. Purification by simple distillation at reduced pressure afforded pure allyl acetoacetate (42e) in 62%. Hnu^O / O^yO a. pyridine ^ O^JL^O ^^-OH O O O O ~b. O "" O O *• ^^^OT /\ ^11 /\ Toluene, reflux CI P.O°L 206 207 208 b X 42e 88% Scheme 51. Facile preparation of allyl acetoacetate (42e) Allyl acetoacetate 42e was subjected to the chain extension-acylation conditions, which produced hemiketal 210 as a mixture of isomeric products. As reported by Lin, efficient conversion of the P-keto ester 42e to the intermediate y-keto ester required two portions of carbenoid (2.5 equiv).47 This excess carbenoid may have been responsible for the increased complexity of the crude product mixture; nonetheless, the major trans- hemiketal 210 could be clearly identified by H NMR analysis. The crude mixture of isomeric products was exposed to catalytic p-TsOW in trimethyl orfhoformate (HC(OMe)3), which converted the hemiketals to methyl ketals. After meticulous separation of diastereomers through flash chromatography on silica, allyl ester 211 was obtained as the major diastereomer in 40%> for the two steps. 83 0 0 a. Et2Zn Hn NU JL^X /s^ ^/ b. CHICHICH3CHI,2 ^0 c. 0^°N^0 42e O \ 2 steps, 40% major diastereomer Scheme 52. Synthesis of allyl ester 211 through the chain extension-acylation reaction The assignment of relative stereochemistry for allyl ester 211 was made by comparison of the !H NMR spectra of 211 with that of methyl ester 203a (Table 10). As previously mentioned, methyl ester 203a was easily crystallized and an X-ray crystal structure was obtained, which allowed for the relative stereochemical assignment to be made for the major diastereomer. Several key resonances were compared and only minor chemical shift differences were observed. The AMX3 spin system (H atoms 5, 6, and 10 respectively) for both methyl ester 203a and allyl ester 211 had identical coupling constants, further supporting the stereochemical assignment. The spectroscopic comparison of allyl ester 211 with the other methyl ester diastereomers was poor. For the 1 1 ^ H and C NMR comparison for ally ester 211 with all three methyl ester diastereomers see Appendix C. Several attempts at inducing crystallization of allyl ester 211 were pursued. Slow evaporation from ethyl acetate afforded a crystal suitable for X-ray crystal structure analysis; see Appendix B for the crystal structure of allyl ester 211. The crystal structure is in agreement with the stereochemical assignment made through NMR chemical shift comparison with methyl ester 203a. 84 l H NMR (400 MHz, CDC13) 203a Hatom Spectrum 1 Spectrum 2 2 5.10 (s, 1H) 5.10 (s, 1H) 11 3.93 (s, 3H) 3.94 (s, 3H) 5 3.38 (d,J= 12.3 Hz, 1H) 3.42 (d, .7=12.3 Hz, 1H) 12 3.24 (s, 3H) 3.26 (s, 3H) 6 2.63 (dq,y= 12.3, 6.7 Hz, 1H) 2.67 (dq,J= 12.3, 6.7 Hz, 1H) 8 1.45 (s,3H) 1.47 (s,3H) 10 1.09 (d, .7=6.7 Hz, 3H) 1.12 (d, .7=6.7 Hz, 3H) 11 _A_ Table 10. Stereochemical assignment of allyl ester 211 (For the purpose of consistency, the atom numbering sequence for the spiro-fused ketals was appointed in accordance with Dai et al60 numbering assignment of papyracillic acid A, B and C) 85 Deallylation of allyl ester 211 to produce the corresponding carboxylic acid 205 was rapid and efficient.75 Initial development of this method used lH NMR spectroscopy to monitor the transformation (Figure 15). Allyl ester 211 and morpholine were suspended in THF-d8 and transferred to an NMR tube. Before the addition of the tetrakis(triphenylphosphine) palladium (Pd(PPli3)4), the 'H NMR spectrum shows several key resonances corresponding to the allyl ester 211. The multiplet at 4.46 - 4.36 ppm is the methylene protons of the allyl group. Additionally, the doublet at 3.29 ppm and the doublet of quartets at 2.51 ppm correspond to the methines a and p to the ester respectively. Upon addition of 10 mol% of Pd(PPha)4, both methine resonances shifted upfield (d, 3.04 ppm and dq, 2.38 ppm), consistent with formation of the carboxylic acid salt. The appearance of a new doublet at 2.81 ppm corresponds to the methylene protons of allyl morpholine 212. Furthermore, the absence of the multiplet at 4.46 - 4.36 ppm for the allylic ester methylene protons confirms the deallylation was completed. 86 / 10mol% Pd(PPh3)4 morpholine THF-d8 0 min Mtlk^_ jj\]\,ji{ AJL , w. 58 56 54 52 50 48 46 44 40 38 36 3 2 30 28 Figure 15. Deallylation reaction monitored by fH NMR spectroscopy (Note: The AA'XX' resonances, approximately at 3.54 - 3.31 ppm and 2.69 - 2.57 ppm, corresponding to morpholine were omitted from the plot for simplification) In an effort to scale up the deallylation protocol to accommodate larger scale conversions of allyl ester 211 to the desired carboxylic acid 205, reaction conditions were altered (Scheme 53). Initial efforts to remove the allyl group with Pd(PPli3)4 were complicated by the appearance of a ligand-derived byproduct which proved to be difficult to remove. The original catalyst loading of 10 mol%> was rather high, thus an investigation of lower catalyst amounts was conducted. The optimized reaction conditions used 0.5 mol% catalyst, which required an increase in reaction time from 5 87 min to approximately 4 h. While the reaction time was increased, formation of undesired phosphine byproduct, resulting from catalyst degradation, was decreased, which allowed for an easier purification of carboxylic acid 205. 0.5 mol% Pd(PPh3)4 morpholine THF, 74% Scheme 53. Optimized reaction conditions for Pd(0) mediated deallylation of allyl ester 211 to carboxylic acid 205 (Degradation of the commercially purchased Pd(PPh3)4 required an increase in catalyst loading from 0.5 mol% to 2 mol %) Reduction of carboxylic acid 205 was necessary in order to obtain desired alcohol 204. The first attempt involved treatment of carboxylic acid 205 with ethyl chloroformate 213 in the presence of sodium carbonate (Na2C03) to produce mixed anhydride 214 (Scheme 54). Analysis of the crude anhydride by *H NMR spectroscopy revealed clean conversion to the product; however, purification was necessary. All attempts to purify mixed anhydride 214 resulted in partial hydrolysis back to the corresponding carboxylic acid 205. Subsequent sodium borohydride-mediated reduction of the anhydride 214 afforded crude alcohol 204. After purification by preparative TLC, hydroxymethyl 204 was obtained as a white crystalline solid in 47%> for the two steps. In addition to the desired product, a second product was isolated in which degradation of the lactone ring was observed. Full characterization of the undesired product was not pursued. While the 88 desired alcohol was successfully synthesized, optimization of the reaction conditions would be necessary to increase the yield and decrease undesired decomposition of starting material. / O / / °\ JL ° ° -°* jo^ r=\ ci'XDEt --o, o )=A M DU /e • x -A ov )=i v L 91 «* V Ni I NaBH4 (6 equiv) ^ V \ No^o —?15— ^V_Ao^o " ^V_>0^0 Na2C03 /Vn THF /^ THF 7^u 2 steps, 47% / O HO 214 )=0 204 EtO Scheme 54. Reduction of carboxylic acid 205 to hydroxymethyl 204 through a mixed anhydride intermediate 214 The development of a one-step method was desired in order to optimize the reduction of carboxylic acid 205 to alcohol 204 (Scheme 55). The previous method involved activation of the carboxylic acid 205 as a mixed anhydride; however, subsequent work-up and isolation of the anhydride resulted in partial conversion back to the carboxylic acid 205. Activation of the carboxylic acid to the acyl imidazole intermediate 215 was performed, followed by direct reduction with sodium borohydride at 0 °C. The desired alcohol 204 was formed and purified by preparative TLC in 83%. The one-step activation-reduction method decreased the opportunity for the intermediate activated ester to hydrolyze back to the carboxylic acid 205. In addition, decreasing the equivalents of sodium borohydride to 3.3 equivalent and decreasing the temperature to 0 °C were found to avoid undesired degradation of the spiro-fused ketal core. 89 •Q^O CDI (1.1 equiv) NaBH4 (3.3 equiv) •- O THF 0 °C, 83% room temp. Scheme 55. Optimization of the reduction of carboxylic acid 205 to alcohol 204 using a one step method The successful synthesis of hydroxymethyl alcohol 204 warranted a closer look at the previously reported data for the isolation of papyracillic acid C (131) (Table ll).60 Based upon the stereochemical assignment of the allyl ester described earlier, the synthesized compound 204 was believed to be epimeric to the reported natural product papyracillic acid C (131) at the spiro-fused quaternary center. Although illustrated in the original natural product isolation paper as a methyl ketal, a closer look at the JH NMR data for papyracillic acid C (131) revealed characterization data that was not consistent with a methyl ketal. For example, no methyl resonance was reported in the *H or 13C NMR spectra for the methyl ketal. Furthermore, the high-resolution electron ionization mass spectrometry (HRMS-EI) m/z of 244.0378 (calcd. for CnHi606, 244.0947) was consistent with a hemiketal structure rather than a methyl ketal. With this evidence it was concluded that the reported structure for papyracillic acid C (131) should be corrected as the hemiketal. Therefore, compound 204, which had been produced in the reduction and possessed the same connectivity as the reported natural product, was neither papyracillic acid C nor an epimer. 90 I -o„_ or 'O -o HO Synthesized through Reported structure for 'H NMR (500 MHz, CDC13) chain extension reaction papyracillic acid C Hatom Synthesized 204 Reported 131 2 5.10 (s, 1H) 5.10 (s, 1H) ' 5 2.64 (dt, J =12.4, 5.2, 5.2, 1H) 2.63 (td, J= 12.6, 5.1, 5.1 Hz, 1H) 6 2.26 (dq,J= 12.3, 6.8 Hz, 1H) 2.28 (dq, J = 12.6, 6.8 Hz, 1H) 8 1.48 (s,3H) 1.59 (s, 3H) 9 3.71 (d, J =5.0 Hz, 2H) 3.75 (d, J=5.1Hz,2H) 10 1.07 (d, .7=6.8 Hz, 3H) 1.14(d,J=6.8Hz, 3H) II-OCH3 3.93 (s, 3H) 3.95 (s, 3H) 12 -OCH3 3.29 (s, 3H) Not Reported Table 11. Comparison between *H NMR data for synthesized alcohol 204 and reported papyrcillic acid C (131) (For the purpose of consistency, the atom numbering sequence for the spiro- fused ketals was appointed in accordance with Dai et al60 numbering assignment of papyracillic acid A, B andC) The final step in the synthesis of papyracillic acid C (131) required the hydrolysis of a methyl ketal to a hemiketal (Table 12). Methyl ketal 216 was subjected to several different reaction conditions. Many of the reaction conditions (Entry 1-4) were not successful in the hydrolysis of methyl ketal 216 and resulted in the recovery of starting material. Even subjecting methyl ketal to microwave-irradiation under reported ketal hydrolysis conditions7 (Entry 4) resulted in recovery of starting material. Fortunately, an 91 excess of acetic acid in a water-THF mixture at reflux 77 appeared to produce hemiketal 217. / O -oN o HO. n. ro^o conditions y=o - R10 wo 216 217 Entry Conditions Results NH4Cl, H20-THF, room temp., 18h starting material TsOH, HzO-THF, room temp.', 18h starting material TsOH, Acetone-H20, room temp., 18h starting material TsOH, Acetone-H20, MW 100 °C, lh starting material a Acetic acid-H20-THF, reflux, 5h 83% Table 12. Optimization of reaction conditions for the hydrolysis of ketal 216 to hemiketal 217 ("Percent conversion observed by !H NMR analysis) Hydrolysis of methyl ketal 204 would produce hemiketal 218. While *H NMR analysis of the crude reaction mixture indicate consumption of starting ketal'204, the presence of 4-epz-papyracillic acid C could not be established. The complexity of the NMR spectrum is believed to be due to the equilibrium between isomeric hemiketal 218 and open-chain form. Papyracillic acid A (123), also a hemiketal, is reported to exist as a mixture of isomeric forms; however, the reported NMR data for papyracillic acid C (131) does not include any evidence of isomeric forms. Access to larger quantities of 204 was necessary to address these inconsistencies. A modification of the reaction conditions for the synthesis of papyracillic acid C (131) was proposed. 92 / / O O \,or=^\ HQ V^°Nfv \ <>o^ ^0 AcOH 'V( cAo H20-THF HO reflux HO 204 4-epi-papyracillic acid C 218 Scheme 56. Hydrolysis of methyl ketal 204 to hemiketal 218 The trichloroethyl ketals have been reported as an protecting group for hemiketals, which can be reacted via a zinc insertion-elimination reaction.78 Efforts were made to incorporate a trichloroethyl-protected hemiketal into the synthesis of papyracillic acid C (Scheme 57). Hemiketal 210 was converted to mono-trichloroethyl ketal 219 through treatment with by catalytic /?-TsOH in trichloroethanol. After column chromatography the major diastereomer was separated from the minor diastereomers in 32% for two steps starting from allyl acetoacetate. Deallylation of allyl ester 219 proceeded as expected of the corresponding carboxylic acid 220 in 93%> yield. Reduction of carboxylic acid 220 furnished the spiro-fused ketal core with a hydroxymethyl functionality 221 in 71% yield. In order to complete the synthesis of papyracillic acid C, elimination of the trichloroethyl group was required. 93 CkC. cue. \^CL n 2 mol% Pd(Ph3)4 »• CI C OH morpholine 3 93% 30% a. CDI b. NaBH4 71% / O CkC \-q. of : O o HO 221 Scheme 57. Trichloroethyl-derived ketal as a route for the synthesis of papyracillic acid C Liberation of the trichloroethyl ketal 221 did not proceed as easily as anticipated (Table X). Several attempts were made using the protocol developed by Isidor et a/.,78 which was specifically designed for the deprotection of trichloroethyl ketals. In the application of this method, trichloroethyl ketal 221 was treated with freshly activated zinc dust in refluxing ethyl acetate; however, only starting material was produced, even after lengthy reaction times (36 h). Similarly, exposure of ketal 221 to zinc dust in tetrahydrofuran also returned unreacted starting material after 48 h at elevated temperatures as confirmed by *H NMR analysis. In order to achieve higher reaction temperatures, ketal 221 was subjected to zinc dust in dioxane, which resulted in return of predominantly starting material with minor decomposition impurities. The zinc dust was 94 replaced with freshly activated granular zinc and stirred with ketal 221 in refluxing dioxane. Disappointingly, starting material was again recovered. CI3C. Zn° solvent Entry Zn° Solvent Temp (°C)e Time Results 1 dusta EtOAc0 77 °C 36 h Starting material5 2 dust" THFd 66 °C 48 h Starting material5 3 dusta Dioxaned 101 °C 24 h Starting materials 4 granularb Dioxaned 101 °C 24 h Starting materials Table 13. Unsuccessful zinc insertion-elimination conditions ("Activated by washing with 5 M HCl for 45 min, filtered, washed with water, ethanol, and ether, dried under reduced pressure (5 mmHg) at b 80 °C over P205 and sonicated, Activated by washing with 5 M HCl for 5 min, filtered washed with water ethanol, and ether, dried under reduced pressure (0.4 mmHg), cDistilled at atmospheric pressure and stored over 3A sieves, dDried over 4A sieves, internal thermocouple probe was used to measure temperature of reaction, Confirmed by TLC and 'H NMR analysis, 8Confirmed by TLC analysis) 95 An alternative protocol for the removal of trichloroethyl groups under relatively mild reaction conditions, reported by Marinier et al., was applied. After 1 h of stirring ketal 221 with granular zinc in 70% acetic acid, TLC analysis indicated evidence for product formation. The reaction was allowed to stir for an additional for 24 h at room temperature. Analysis of the !H NMR spectrum for the crude reaction mixture revealed the desired hemiketal 218 and dichloroethyl ketal 222 as a 1:1 mixture of products. While protonation of the zinc anion, which produced dichloroethyl ketal 222, seemed to compete with the elimination reaction, the desired hemiketal 218 was successfully prepared. CI3C HO HO 218 222 1:1 ratio of products Scheme 58. Acetic acid-mediated zinc insertion-elimination reaction Hemiketal 218 was purified by preparative TLC and analyzed by NMR spectroscopy (Scheme 59). The 'H NMR spectrum showed a mixture of two hemiketal isomers 218 and 223, in a 4:1 ratio, respectively. Careful inspection of both the 'H and C NMR spectra revealed no resonances consistent with the open-chain isomer 224. The absence of evidence for the open-chain isomer 224 suggests the equilibrium is strongly biased towards the hemiketal isomers 218 and 223. 96 / / 218 223 224 Scheme 59. Analysis of equilibrium for hemiketal 218 The major hemiketal isomer 218, synthesized via the chain extension-acylation reaction, was compared with the spectroscopic data reported for papyracillic acid C (131) (Table 14). As previously discussed, Dai et al. illustrated papyracillic acid C (131) as the methyl ketal; however, the spectroscopic data and HRMS clearly suggest papyracillic acid C (131) was, in fact, a hemiketal.60 Comparison between the *H and 13C NMR data for 218 and 131 reveal subtle differences in chemical shift. The major hemiketal isomer 218, synthesized through the chain extension-acylation reaction, was concluded to be 4- epz-papyracillic acid C. 97 / o HQ,, n T HO 'II NMR (500 MHz, CDC13) HO H atom Major 218 Reported 131 2 5.11 (s,lH) 5.10 (s, 111) 5 2.61 (dt,J=12.6, 5.3, 5.3 Hz, 1H) 2.63(td,./= 12.6, 5.1,5.111z, 1H) 6 2.28 (dq,./= 12.6, 6.7 Hz, 1H) 2.28(dq,J = 12.6,6.8Hz, 111) 8 1.58 (s.3H) 1.59 (s, 311) 9 3.74 (d, J- 5.1 Hz, 2H) 3.75 (d, .7-5.1 Hz,2H) 10 1.12 (d,J -6.8 Hz, 311) L.14(d,/-- 6.8Hz,3H) Il-OCTL, 3.94 (s,3H) 3.95 (s, 3H) 13 C NMR (126 MHz, CDC13) C atom Major 218 Reported 131 1 169.3 169.3 2 89.9 89.7 3 177.5 177.4 4 109.1 108.9 5 50.4 50.2 6 42.4 42.3 7 107.8 107.5 8 26.2 25.9 9 59.1 59.2 10 11.9 11.7 11-OCH, 60.0 59.8 Table 14. Comparison between *H and l3C NMR data for hemiketal 218 and papyracillic acid C (131) (For the purpose of consistency, the atom numbering sequence for the spiro-fused ketals was appointed in accordance with Dai et al60 numbering assignment of papyracillic acid A, B and C) 98 Conversion of 4-epi-papyracillic acid C (218) to papyracillic acid C (131) through open-chain form 224 was attempted. Hemiketal 218 was dissolved in CDCI3, placed within a closed reaction vessel, and heated. After heating at 80 °C for 2 h the *H NMR spectrum revealed no resonances consistent with papyracillic acid C. Higher temperatures, trace acid, and longer equilibration time was attempted resulting in consumption of starting material and formation of a new product, which was not papyracillic acid C. Conclusive results were not further pursued; however, further attempts at a thermal equilibration of 4-epi-papyracillic acid C should start with the protection of the hydroxyl functionality. _ HO HO O^ OH HO 218 224 231 4-epi-papyracillic acid C papyracillic acid C Scheme 60. Thermally induced equilibrium of 4-e/?z'-papyracillic acid C (218) Synthesis of 4-gp/-Papyracillic acid B and Papyracillic acid B Synthesis of an exo-cyclic methylene 225, as present in papyracillic acid A (123) and B (130), was expected to arise through elimination of a derivative of hydroxymethyl 204. Several different synthetic strategies were proposed to achieve this transformation. Hydroxymethyl 204 was easily converted to the xanthate ester 226 in reasonable yields. Thermally-induced ^yw-elimination80 of the xanthate ester 226 to product the exocyclic 99 alkene 225 was unsuccessful. Microwave-irradiation of xanthate ester 226 in a closed vessel with 1,2-dichlorobenzene at 235 °C81 for 2h did not pyrolize the ester and surprisingly resulted in recovery of starting material combined with only minor impurities. Next, xanthate ester 226 was subjected to conventional heating in dodecane at 216 °C82 for 24h, resulted in the decomposition of the starting material and did not produce the desired product. Although discouraging, the results were not surprising because thermolysis of 1° xanthate esters typically require higher temperatures to achieve elimination rather than the corresponding 2° or 3° ester.83 Nevertheless, the stability of the spiro-fused system to high temperature was established. -°* .0. a. NaH Heat 0^0 —X— b. CS2 syn-elimination c. Mel 225 Scheme 61. Attempted Chugeav elimination of xanthate ester 226 An alternative approach for the synthesis of papyracillic acid B (130) was considered. Conversion of alcohol 204 to a better leaving group created the opportunity for several different elimination reactions (Scheme 62). Hydroxymethyl 204 was treated with methanesulfonyl chloride 227 in pyridine, which efficiently produced mesylate 228 in high yield.84 The activated mesylate 228 was subjected to modified Finkelstein reaction conditions. The displacement of the mesylate leaving group with iodide was sluggish and required long reaction times. The steric hindrance surrounding the site of 100 displacement may explain the slow SN2 reaction. Nonetheless, iodomethyl 229 was isolated in 32% yield after purification. O II —S-CI II o Nal pyridine227 , 4 h dry acetone 98% reflux, 48 h 32% 229 Scheme 62. Synthesis of iodomethyl 229 via a Finkelstein-type reaction Iodomethyl 229 was subjected to catalytic Pd(PPli3)4 in an effort to obtain exo- cyclic methylene 225 (Scheme 63). Although the Schlenk flask was purged and flushed with nitrogen in order to extrude oxygen from the reaction conditions, rapid degradation of the catalyst, as indicated by a dark brown color change within the reaction vessel, was observed. The reaction did not provide the desired product 225, although recovery of starting material was easily achieved via chromatography. Modest degradation of the starting material was observed by *H NMR analysis of the reaction material. Such results were not unexpected since several literature reports suggest Pd(PPh3)4 was not an efficient Pd(0) source for catalyzing p-elimination reactions.86 An alternative method for the preparation of exo-cyclic methylene 225 pursued a potassium /-butoxide-mediated elimination reaction. TLC and !H NMR analysis confirmed the full consumption of starting material and a complex mixture of products; therefore this method was not further investigated. 101 Pd(PPh3)4 THF / / o X O -a .o„ r vo" ^o KOf-Bu DMSO 229 X 225 Scheme 63. Unsuccessful attempts at synthesizing exo-cyclic methylene 225 Transformation of a 1° alcohol to a terminal olefin driven by a o-nitrophenyl selenoxide yvn-elimination, referred to as the Grieco reaction, has been successfully employed in many natural product syntheses.87 Investigation into this method for the synthesis of exo-cyclic methylene 225 was undertaken. o-Nitrophenylselenocyanate 230 was reduced to the corresponding selenium anion by sodium borohydride. The subsequent addition of mesylate 228 was anticipated to produce selenide 231 (Scheme 64). TLC analysis indicated that a new product was formed; however, the purified product was not consistent with the desired selenide 231. The lack of aromatic resonances within the *H NMR spectrum clearly indicated the selenide 231 was not synthesized, but the A2MRX3 spin system was preserved. The hydroxymethyl and cyanomethyl groups were considered as possible products; however, spectroscopic data suggested neither product was formed. A sample of the unknown product was subjected to electron spray ionization mass spectrometry [(ESIMS) m/z of 299.2 (calcd. for Ci2Hi705ClNa, 299.07)], which allowed the identification of the product as chloromethyl 232. While conclusive evidence indentifying the source of chloride remains unknown, the melting point for the recently purchased o-nitrophenylselenocyanate was 118 - 119 °C, which is significantly lower than the literature reported value (144 °C).88 Perhaps the selenium reagent was 102 contaminated with a chloride salt. At the current time the source of chloride remains unknown. An alternative method for the synthesis of the desired product 225 was still needed. NaBH4 O SeCN -CL .0. )=l a NO, 230 H202 -X DMF, 90 °C, 24 h THF 225 231 ESIMSm/zcalcd. for C12H1705CINa: 299.07; 44% found 299.2 Scheme 64. Attempted synthesis of exo-cyclic methylene 225 through the Grieco elimination The preparation of exo-cyclic methylenes, promoted by a selenoxide syn- elimination derived from diphenyl diselenide, has been reported;89 therefore, investigation into this method's applicability for the synthesis of the alkene present in papyracillic acid B (130) was pursued (Scheme 65). Following the protocol developed by Hosay et al., diphenyl diselenide 233 was reduced with sodium borohydride to produce a phenyl selenide anion 234,90 which reacted readily with mesylate 228 under mild room temperature conditions. After chromatographic purification phenyl selenide 235 was 103 isolated in 64%. Treatment of selenide 235 with hydrogen peroxide resulted in rapid oxidation and production of selenoxide 236. The lH NMR analysis of the unpurified selenoxide 236 revealed a mixture of two disastereomers as a result of the new stereogenic center. The selenoxide 236 was subsequently stirred in toluene at room temperature for 18 h, then the reaction mixture was gradually heated to 110 °C over the course of 6 h. Satisfyingly, 4-epz-papyracillic acid B (225) and a second diastereomer were isolated in a 1:1 mixture. Through comparison to the literature data reported by Dai et al60 the second diastereomer was identified to be papyracillic acid B (130). Since the selenide 235 was isolated cleanly as one diastereomer, epimerization of 4-epz-papyracillic acid B (225) to papyracillic acid B (130) was postulated to occur after the exo-cyclic methylene was produced. 104 235 236 Toluene r.t- 110 °C 24 h 4-ep/-papyracillic acid B papyracillic acid B 225 130 1:1 mixture Scheme 65. Synthesis of a 1:1 mixture of 4-epz-papyracillic acid B (225) and papyracillic acid B (130) The presence of the exo-cyclic methylene was hypothesized to facilitate epimerization of 225 to 130 (Table 15). Fragmentation of the quaternary spiro-fused ketal carbon reveals a 3°, allylic, oxocarbenium ion 237. Interconversion of the two spiroketals would be expected to have a low energy barrier. A study was performed in an effort to determine weather the 1:1 ratio of isomers represented a thermodynamic 105 equilibrium. The 1:1 mixture of 225 and 130 was dissolved in CDCI3, placed within a closed reaction vessel, and heated. At a variety of temperatures H NMR spectra were obtained, which revealed a consistent 1:1 ratio of epimers. The equilibrium was apparently established quickly during the elimination reaction. 225 237 130 Entry Temperature (°C) Time Ratio of 225:130 1 110 °C 2h 1:1 2 130°C 2h 1:1 3 160 °C 4h 1:1 4 210°C 2h 1:1 Table 15. Hypothesized epimerization between 4-epz-papyracillic acid B (225) and papyracillic acid B (130) As previously mentioned, the structure for papyracillic acid B (130), reported by Dai et al60 was compared with one of the synthesized ketal isomers 130 (Figure 16). The *H NMR chemical shifts and coupling constants for 130 (reported and synthetic) were consistent, which suggests both compounds are identical and have the same stereochemistry (For full 'H and 13C NMR characterization data for 130 reported and synthesized, 225, and 238 refer to appendix C). Another ketal isomer 238, reported by 106 Shan et al., 56 was assigned a structure that was epimeric and believed to be the epimer of papyracillic acid B (130) at the spiro-fused quaternary center. The structural and stereochemical identification for ketal 238 was established by Shan et al. through an HMBC and NOESY correlation study. Comparison of the data reported by Shan et al. for ketal 238 with the data acquired for ketal 225 synthesized through the chain extension- 1 1 ^ acylation reaction revealed inconsistent in both the H and C NMR spectra. An X-ray crystal structure of an advanced intermediate en route to ketal 225 was used to assign the stereochemistry; therefore ketal 238 can not be an epimer of papyracillic acid B (130) at the spiro-fused center and has been stereochemically misassigned. 130 130 225 238 Synthesized through the Reported by Dai et al. Reported by Shan et al. tandem chain extension-acylation reaction Figure 16. Stereochemical comparison between 130 reported, 130 synthesized, 225 and 238 (For full lH and 13C NMR characterization data refer to appendix C) In summary, the tandem chain extension-acylation reaction was developed and successfully used for the synthesis of spiro-fused ketals, which were produced in two steps from readily available starting materials. Subsequent functional group transformation on the ketal backbone allowed for the first reported synthesis of papyracillic acids to date. The synthesis of 4-epz-papyracillic acid C and its methyl ketal precursor confirm that papyracillic acid C was misrepresented as the methyl ketal when, 107 in fact, papyracillic acid C was a hemiketal. The synthesis of 4-epz-papyracillic acid B and papyracillic acid B was accomplished as a 1:1 mixture of epimers. Subsequent comparison of NMR data between the synthesized 4-epz-papyracillic acid B and a previously reported methyl ketal epimer revealed a stereochemical misassignment made in the original paper that reported isolation of papyracillic acid A. The successful regiospecific incorporation of an anhydride onto a y-keto ester backbone has further broadened the scope of the chain extension methodology. Other natural products, such as papyracillic acid A, may be targeted using this synthetic route. 108 CHAPTER III DIASTEREOSELECTIVE SYNTHESIS OF p-METHYL-y-KETO ESTERS THROUGH THE UTILITY OF AN L-SERINE AUXILIARY Synthesis of B-Substituted-y-Keto Esters Regiochemical control has been a significant challenge to the efficient synthesis of either a or ^-substituted 1,4-dicarbonyls when starting with the unsaturated dicarbonyls (Figure 17). Standard enolate-alkylation reactions on a 1,4-dicarbonyl, such as 239, 240, or 241, would be expected to lead to a mixture of a and P alkylated products along with dialkylated compounds. The pKa values for ketones (pKa~27), esters (pKa«30), amides (pKa~35), and phosphonates (pKa~27.6) are relatively similar, thus selective enolate formation would be challenging. l In order to address these challenges, a variety of clever methods to circumvent this problem have been developed. II « OR2 O R1 pY* * Ri^^V . R1A^YR03 0 o 6 239 240 241 Figure 17. Illustration of the a- and p-positions of 1,4-dicarbonyl backbones 109 Several different research groups have developed methods for the synthesis of 1,4-dicarbonyls that include regiocontrol of substiutents.92 One interesting approach, developed by Kashima et al, utilized an JV-acylpyrazole chiral auxiliary to control the no formation of the P-stereocenter (Scheme 66). Ketone 242 was treated with LDA in the presence of a-bromo ester 243, which produced a 1,4-dicarbonyl 244 with high diastereoselectivity. Subsequent conversion of 244 to optically active P-substituted-y-keto ester 245 was accomplished through the utility of a Grignard reagent, although in low yields (10 - 51%, 3 examples). While the high sterochemical control was attractive, a facile method for formation of P-substituted-y-keto esters from easily prepared starting materials was still desired. • 1 Q 3 R "MgBr 3 I II OR* RY^^OR* o 245 Scheme 66. Kashima's stereoselective synthesis of P-substituted-y-keto esters As previously discussed in chapter I, regiospecific incorporation of functionality at the P-position of a y-keto ester backbone has been developed. The reaction involves the chain extension of a P-keto ester through exposure of a methyl-substituted carbenoid. The facile and efficient transformation of the 1,3-dicarbonyls 35 to methyl-substituted 1,4- dicarbonyls 95 prompted the desire to control the stereochemistry at the P-position 110 (Scheme 67). The proposed mechanism begins for the chain extension reaction with enolate (48) formation, which subsequently undergoes an alkylation with a methyl- substituted carbonoid 246, derived from diethylzinc and diiodoethane, to produce homoenolate 247. Intramolecular nucleophilic addition of homoenolate 247 into the ketone functionality produces a donor-acceptor cyclopropane 94. Recently, Eger et al. reported computational evidence that a large energy barrier must be surmounted to go from the donor-acceptor cyclopropane 94 back to the homoenolate 247, which suggests the stereochemistry of a methyl-substituent is established during a kinetically-controlled intramolecular cyclization. This assertion renders the donor-acceptor cyclopropane 94 as a significant intermediate to consider when designing a strategy for stereochemical control. The reaction of the racemic carbenoid would be expected to produce diastereomers in the alkylation of the enolate. The ratio and subsequent reactivity of these diastereomers may play a pivotal role in the selective formation of cyclopropane intermediate. Ill O o O a. Et2Zn b. CH CHI OR2 R^OR2 3 2 R1 c. NH4CI O 35 95 A Et2Zn H+ ZnX O' O O XZn-" O 2 R Rr -^ -0R OR2 48 Et2Zn + — -*• H3C-C-ZnX Cri3CHI2 H 246 ZnX O' O R1'^SDR2 247 94 Scheme 67. Proposed mechanism for the chain extension reaction using a methyl- substituted carbenoid Previous Attempts at Controlling the Stereochemistry The establishment of the stereochemistry through the formation of a donor- acceptor cyclopropane lends itself to the possibility of using asymmetric cyclopropanation methods for the stereochemical control at the P-position. Optically active cyclopropanes have been successfully synthesized by reacting allylic alcohols with a zinc carbenoid in the presence of chiral ligands. 4 Lin subjected the methyl-substituted zinc carbenoid to a catalytic amount of 7V,iV-bis(methanesulfonyl) derivative 248 in an attempt to induce enantioselective cyclopropanation (Scheme 68).23b Unfortunately the p- 112 methyl-y-keto ester 95a appeared to be produced in a racemic mixture, as evidenced by an optical rotation of zero. O O a. Et2Zn (5 equiv) O b. 248(0.1 equiv) » 0\ c. CH3CHI2 (5 equiv) O 35a 95a Scheme 68. Unsuccessful enantioselective synthesis of P-methyl-y-keto ester Since Evans auxiliaries (chiral P-keto imides) were successfully employed during the tandem chain extension-aldol reaction, their applicability for controlling stereochemistry in the reaction of a methyl-substituted carbenoid was studied. Lin subjected chiral auxiliary derived P-keto imide 39 to the methyl-substituted zinc carbenoid, which produced y-keto imide 249 (Scheme 69).23b Analysis of the *H NMR spectrum revealed a 1:1 mixture of diastereomers. The Evans auxiliary was not suitable for stereocontrol at the P-position and efforts to modify the imide auxiliary were not likely to be successful;- however, many enantioselective cyclopropanation reactions are available. The pursuit of these methods for P-stereocontrol could, and perhaps should, be considered; however, the studies reported below undertook a different approach. 113 O O O § O O J^uK -J^r^ Y^N\ )—/ b. CH3CH2 o )—I Bn**' Brf' 39 249 Scheme 69. Unsuccessful diastereoselective synthesis of p-methyl-y-keto ester Amino Acid-Derived Systems The unsuccessful attempts at controlling the stereochemistry reported above demanded that a new method for inducing stereochemical control be developed. The introduction of chirality neighboring the ketone of P-keto ester 250 was considered (Scheme 70). The stereochemistry of the methyl-substituent in the homoenolate 251 is unknown, yet an asymmetric environment around the ketone may be capable of influencing the stereochemical course of the intramolecular cyclization that produces donor-acceptor cyclopropane 252. Since the methyl group's stereochemistry is fixed in the donor-acceptor cyclopropane 252, fragmentation to p-methyl-y-keto ester 253 should not compromise the stereochemical fidelity at the P-position. In order to study this hypothesis, chiral ketones were synthesized from iV-protected amino acids. 114 a. Et2Zn b. CH3CHI2 250 O O 252 Scheme 70. Different synthetic approach towards controlling diastereoselectivity Amino acids 254a-c can be easily converted to P-keto esters 255a-c without epimerization of the stereocenter through the Masamune-Brooks reaction (Table 16).95 JV-Protected L-amino acid was activated with carbonyldiimidazole in order to generate an acyl imidazole intermediate 256, which was transferred to a pre-formed solution of the monobenzylmalonate 257a and dibutylmagnesium (The same procedure can be followed by replacing monobenzyl malonate 257a with monoethyl malonate 257b to produce the corresponding ethyl ester). After flash column chromatography, pure amino acid-derived P-keto ester 255 was obtained. L-alanine (254a), L-proline (254b), and L-phenylalanine (254c) were all converted to their corresponding P-keto esters (255a-c) in moderate to high yields. 115 O O o o \ L, O O CDI PG OBn PG' OH N PG 1 1 1 R R 257a Entry PG R1 Yieldb la Boc -CH3 81% 2b Boc -CH/j-CH^-CJh^- 65% 3c -C(0)OCH3 -CH2Ph 63% Table 16. Synthesis of amino acid-derived P-keto esters through the Masamune-Brooks reaction ((a)Monobenzyl malonate 257a can be replaced with monoethyl malonate 277b to produce the corresponding ethyl ester, (b)Isolated yield after column chromatography) L-Alanine-derived p-keto ester 255a was subjected to the methyl-substituted zinc carbenoid, which produced P-methyl-y-keto ester 258a and 258b in a 1:1 ratio of diastereomers (Table 17). The ratio of diastereomers was obtained through relative integration of the newly established methyl protons observed in the ]H NMR spectrum of the crude reaction mixture. Although the ratio of diastereomers was discouraging, the size of the methyl group within the amino acid alanine core was not anticipated to be bulky enough to influence the intramolecular cyclization. The L-proline-derived system 255b afforded a slightly greater ratio of diastereomers; however, an undesired side reaction and the appearance of different rotomeric forms made interpretation of the *H NMR spectrum difficult. Finally, L-phenylalanine-derived system 255c was converted to its P-methyl-y- keto counterpart in a better, but still discouraging, 3:1 ratio of diastereomers. 116 H J J a. Et2Zn OBn PG-NYX^A0Bn b. CH3CHI2 ^ R1 c. NH4CI 255a-c 258a-f Entry PG R1 dra la,b Boc -CH3 1:1 2c,d Boc -CH2-CH2-CH2- 2:1 3e,f -C(0)OCH3 -CH2Ph 3:1 Table 17. Ratio of diastereomers using amino acid-derived systems ("Diastereomeric ratio was obtained by the relative integration of the methyl protons observed in the crude *H NMR for both diastereomers) In an effort to increase the diastereomeric ratio, the chain extension reaction using a methyl-substituted carbenoid was run at decreased temperature. Chain extension reactions that utilize the methyl-substituted carbenoid are typically run at 0 °C in an ice- water bath and then allowed to warm to room temperature; however, during this study, P- keto ester 255a was cooled to -42 °C in an acetonitrile-dry ice bath and then subjected to the methyl-substituted carbenoid. While the *H NMR spectrum of the crude reaction material revealed a modest increase in diastereocontrol to a 2:1 ratio of diastereomers for 258a, it became apparent that the amino acid-derived systems were not offering the stereochemical control that was desired. 117 H || II a. Et2Zn H Boc^YA_AOBn b. CH3CHI2 ^ Boc.N OBn R1 .42 °C 255a 258a dr 2:1 Scheme 71. Chain extension reaction at lower temperatures (-42 °C) In summary, three different amino acid-derived starting materials, originally derived from L-alanine, L-proline, and L-phenylalanine, were subjected to the methyl- substituted carbenoid. Each system produced the corresponding p-methyl-y-keto ester; however, the poor diastereocontrol made this approach undesirable. Modification would be necessary to develop a system that would provide superior diastereoselectivity. Hydroxyl-Directed Simmon-Smith Cyclopropanation Reactions Hydroxyl-directed Simmon-Smith cyclopropanations have been developed as a method for controlling the facial-selective approach of the zinc carbenoid to an olefin.96 Chiral allylic and homoallylic alcohols have demonstrated hydroxyl-directing effects for carbenoid cyclopropanation reactions, typically generating cw-stereochemistry with regards to the carbenoid carbon and alcohol substituents.97 Additionally, Kawabata et al. has observed high stereocontrol when using a homoallylic alcohol to direct the methyl- substituted zinc carbenoid (Scheme 72). 3-Cyclopentenol (259) was treated with a methyl-substituted carbenoid to produce exo-6-methyl-cw-3- hydroxybicyclo[3.1.0]hexane (260) as the only isomer. While more complex homoallylic alcohols provided mixtures of isomers, 3-cyclopentenol (259) provided excellent 118 stereochemical control, thereby demonstrating the advantage of incorporating alcohol directing groups when using zinc carbenoids. H 0H >v .OH Et2Zn \/^S H3C - 259 260 Scheme 72. Homoallylic alcohol directed cyclopropanation using a methyl-substituted carbenoid The successful stereochemical control displayed by homoallylic alcohol directing groups during zinc carbenoid-mediated cyclopropanation reactions prompted the investigation into their applicability to the chain extension reaction (Scheme 73). Deprotonation of an a-mefhylene proton of P-keto ester 261 with diethylzinc produces intermediate 262. Within the structure of intermediate 262 a homoallylic hydroxyl group can be observed. Upon addition of the carbenoid, the homoallylic hydroxyl functionality of 262 was hypothesized to offer high diastereoselectivity in the formation of cyclopropane intermediate 263. The fragmentation of donor-acceptor cyclopropane 263 will destroy two of the cyclopropane stereocenters, but the integrity of the methyl- substituent should be preserved during the formation of 264. 119 X Zn O O o' ' o Et2Zn CH3CHI2 R R! II OBn OBn OBn 2 / ^OR R20' 261 262 263 H+ OBn 264 Scheme 73. Homoallylic alcohol as a directing group during the chain extension reaction Synthesis and Results for L-Serine Derived Auxiliary The development of a diastereoselective synthetic route towards P-methyl-y-keto esters 264 required the incorporation of a hydroxyl functionality into the p-keto ester core 261 (Scheme 74). A convenient precursor for the synthesis for P-keto esters 261 was an amino acid, L-serine 265. In a similar fashion to the other amino acid derived systems, described above, the transformation of L-serine to P-keto ester 261 would begin with a series of N- and O-protections followed by a Masamune-Brooks reaction. O O © ° OBn -> H N_X OBn 3 O0 ^OR2 OH 261 265 Scheme 74. Retrosynthetic analysis for the synthesis of p-methyl-y-keto esters 120 A series of different N- and (3-protected L-serine precursors were prepared (Scheme 75). L-Serine (265) in 1M sodium hydroxide was treated with Boc-anhydride to produce JV-Boc-L-serine (266) in 88% yield.99 Exposure of Boc-serine 266 to sodium hydride and then benzyl bromide resulted in O-alkylation and the production of Obenzyl L-serine 267 in moderate yield.100 Alternatively, Boc-serine 266 was converted to O-tert- butyldimethylsilyl-L-serine 268 in 90% yield for the two steps. 101 H ° a. NaH, 0 °C Boc" NX JL OH b. BnBr 5 DMF, 18 h OBn O u P 50% H3N^o0 °^NaOH_ ^^Xc 267 = b. (Boc)2)0 = 1.) TBDMS-CI ^OH Dioxane OH (2 equiv) 5°C-21°C, 4h imidazole H O 265 88% 266 DMF, 16 h N JL ^ Boc" V^OH 2.) K2C03, H20 V CH3OH,4h OTBS 90% 268 Scheme 75. Synthesis of O-Benzyl 267 and O-TBS L-serine 268 In an effort to decrease the water solubility of the protected L-serine precursors, the carboxylic acid functionalities were converted to methyl esters (Scheme 76). iV-Boc- L-serine 266 was treated with anhydrous potassium bicarbonate followed by slow addition of iodomethane. Methyl ester 269 was generated in 70% yield. The free hydroxyl functionality 269 was subsequently converted to the MOM-protected L-serine 270 in 80% yield. Conversely, the hydroxyl functionality 269 could be tethered to the 121 nitrogen as a part of an oxazolidine ring 271, promoted by catalytic p-TsOH and 2,2- dimethoxypropane. H ° a. DIPEA 0°C Boc^Y^O^ b. MOM-CI ^OMOM DCM, 18 h H ° H ° 80% a. KHC03, 0 °C 270 Boc" Boc"N Y^O- _ b. CH3I DMF, 2 h ^OH 70% Boc 0 266 269 cat. />TsOH o— benzene 59% 271 Scheme 76. Synthesis of O-MOM 270 and oxazolidine L-serine methyl ester 271 The labile nature of the Boc-protecting group was not always suited for some of the synthetic steps, thus alternative TV-protecting groups were studied (Scheme 77). Challenges presented by the Boc-protecting group will be discussed shortly. Treatment of L-serine methyl ester hydrochloride salt 272 with sodium methoxide followed by methyl chloroformate 273 resulted in an 83% yield of methyl ester 274. Exposure of hydroxyl functionality of 274 to triethyloxonium tetrafluoroborate (BF40Et3) and diisopropylethylamine (DIPEA) resulted in the formation of O-ethyl-L-serine methyl ester (275).102 The powerful alkylating ability of BF40Et3 is known to compromise the stability of certain carbamates, such as Boc and Cbz.76 All attempts at O-alkylation with BF40Et3 in the presence of Boc-protection resulted in a complex mixture of products. 122 © ° a. NaOCHg H ° H ° 0 BF.OEto CI / CH3OH O H3N-YAQ ~b. O o O DIPEA O OH DCM, 18 h ^OEt ^OH ^O^CI 63% 273 274 275 272 THF, 16 h 83% Scheme 77. Synthesis of O-ethyl L-serine methyl ester 275 Hydrolysis of the methyl esters to the corresponding carboxylic acids were efficiently accomplished (Table 18). Each N- and O-protected L-serine methyl ester 270, 271, and 275 was treated with lithium hydroxide in a THF, H20, and methanol (1:1:1) solution. The corresponding carboxylic acids 276-278 were obtained in moderate to high yields within approximately 4 h. O H ° 1 LiOH R O' -Ms OH .QR2 THF, H20, CH3OH OR2 Esters R1 R2 Acids Yield (%) 270 Boc MOM 276 78% 271 Boc > 275 -C(0)OCH3 Et 278 80% Table 18. Hydrolysis of L-serine methyl esters to carboxylic acids 276-278 123 The protected L-serine precursors were readily converted to the corresponding P- keto esters 281-287 via the Masamune-Brooks reaction (Table 19). The carboxylic acids were activated with carbonyldiimizaole (CDI) followed by treatment with monomalonate magnesium salt to produce the L-serine-derived p-keto esters. Most of the synthesized P- keto esters were benzyl esters; however, p-keto ester 279 was derived from monoethylmalonate. The p-keto esters 281-287 were purified by column chromatography and thoroughly dried in vacuo at 80 °C in the presence of phosphorus pentaoxide (P2O5) before they were carried on to the chain extension reaction. H ° H 0 O N a CDI N R1 ^OH " ' »• R1 ^^-OR3 b. MBM or MEM, ^OR2 ^OR2 Bu 2Mg THF, 0 °C - r.t. Acids R1 R2 R3 P-Keto Esters Yield (%)a 267 Boc Bn Bn 281 50% 268 Boc TBDMS Bn 282 30% 276 Boc MOM Bn 283 52% 277 Boc Bn 284 47% 0—" 278 -C(0)OCH3 Et Bn 285 67% 279 Cbz Bn Et 286 62% 280 Cbz TBDMS Bn 287 25% Table 19. Synthesis of L-serine derived P-keto esters 281-287 via Masamune-Brooks reaction ("Isolated yield after column chromatography on silica) 124 L-Serine-derived P-keto esters were subjected to the chain extension reaction using a methyl-substituted carbenoid in order to study the diastereomeric ratio of the 0- methyl-y-keto esters 288-291 (Table 20). Benzyl (288), TBDMS (289), and MOM (290) protected serine derivatives produced the highest diastereoselectivity with ratios of diasterereomers of >15:1. The method used for the determination of the diastereomeric ratio will be described shortly (see Quantifying the Diastereoselectivity). In contrast, the ethyl-protected serine derivative 291 generated a disappointing 3:1 ratio of diastereomers. More effort was made towards understanding the stereochemical control provided by the L-serine auxiliaries. O O a. Et2Zn H ,N^^ b. CH CHI N OBn R OBn 3 2 D1 c. NH CI ^OR2 4 CH2CI2, 0 °C - r.t. P-Keto Esters R1 R2 y-Keto Esters dr 281 Boc Bn 288 20:lab 282 Boc TBDMS 289 20:lb 283 Boc MOM 290 15:la a 285 -C(0)OCH3 Et 291 3:l Table 20. Diastereoselective synthesis of P-methyl-y-keto esters 288-291 using an L- serine auxiliary ("Diastereomeric ratio was obtained from Bruker Top Spin line fit program, Diastereomeric ratio was obtained from MestReNova line fit program) 125 The hydroxyl functionality of the L-serine core was hypothesized to serve a crucial role in the high diastereocontrol observed during the synthesis of P-methyl-y-keto esters. In an effort to further study the L-serine auxiliary, oxazolidine-derived L-serine auxiliary 284 was pursued (Scheme 78). The hydroxyl group of the L-serine core was tethered into a cyclic five-membered ring, restricting rotation of the hydroxymethyl group during exposure to the chain extension conditions. As predicted, the diastereocontrol was greatly diminished upon exposure to the methyl-substituted carbenoid as evidenced by the 1:1 ratio of diastereomers 292. While the transition state for this reaction remains unknown, this result suggests the hydroxyl functionality is required for high diastereocontrol. Boc o O a. Et2Zn b. CH3CHI2 OBn OBn c. NH.CI 284 Scheme 78. Diastereomeric ratio obtain from oxazolidine-derived L-serine auxiliary ("Diastereomeric ratio was determined by MestReNova line fit program) In summary, a series of different 0-protected L-serine derived P-keto esters were prepared and subjected to the methyl-substituted carbenoid. High diastereoselectivity was observed for most of the L-serine auxiliaries. While oxazolidine-derived L-serine system established the significance of the hydroxyl functionality to the diastereomeric control, the exact role the hydroxyl group plays during the reaction remained unknown. A more 126 detailed understanding of the stereocontrol, as well as elucidation of the stereochemistry at the P-position, was desired. Quantifying the Diastereoselectivity Obtaining a ratio of diastereomers from a H NMR spectrum of a crude reaction mixture can be challenging. Overlapping resonances can skew effort to access the ratio of diastereomers via integration, resulting in an inaccurate ratio. Rather than relying on relative integration of the crude reaction mixture to determine the ratio of diastereomers for the P-methyl-y-keto esters, a MestReNova line fit simulation was applied (Figure 18). The doublets for the methyl substituent of the major and minor P-methyl-y-keto ester 288 diastereomers, at 1.18 and 1.09 ppm, respectively, were used to establish the ratio of diastereomers. A line fit was applied to each peak of the doublet resonances, which established the area under each curve. The ratio for the major to minor diastereomers produced in the reaction of the O-benzyl L-serine derived 288 was >20:1. Additionally, purification of the crude reaction mixture was required to confirm the chemical shift of the methyl group in the minor diastereomer. The line fit data for each of the L-serine- derived systems is presented in Appendix C. 127 Name O-Benzyl O From 1 069 ppm H To 1 200 ppm OBn Residual Error 6 71 Boc # ppm Height Width(Hz) L/G Area CH O BnO 3 1 11873 100 66 2 21 100 3589 744 2 1 1692 101 16 2 03 1 00 3318 426 288 3 10958 6 30 131 0 00 109 114 4 1 0781 6 30 1 46 0 00 121 375 1 14 1 13 ppm Figure 18. Determination of diastereomeric ratio using MestReNova line fitting L-Serine-derived P-keto esters offered high diastereocontrol in the formation of p- methyl-y-keto esters; however, determination of the absolute stereochemistry for the major diastereomer posed an additional challenge. Attempts to obtain an X-ray grade crystal have been unsuccessful. Therefore, several strategies involving chemical manipulation of the products were proposed and executed in an effort to elucidate the absolute stereochemistry. 128 Elucidation of the Stereochemistry Stereochemical determination of the major diastereomer of the P-methyl-y-keto esters derived from the L-serine auxiliaries relied on subsequent chemical transformation in order to compare the products with known compounds in the literature. Forzato et al. reported the synthesis of enantiopure 4-methylparaconic esters and acids produced from a kinetic enzymatic resolution of the corresponding diastereomeric lactonic esters (Scheme 79). The enzymatic hydrolysis of (±)-cz's-methylparaconic ester 293 by horse liver acetone powder (HLAP) regioselectively opened the lactone ring of (+)-cis 293 to produce (+)-(3R,4S) 294. The unreacted ethyl ester (-)-(3S,4R) 295 was also isolated. The enantiomeric excesses of 294 and 295, determined by chiral gas chromatography, were 38% and 99%, respectively. The absolute configuration for all of the enantiopure paraconic acid derivatives were determined by both circular diachroism and theoretical calculations of the specific rotation followed by comparison with experimental data. 129 0^°\ enzyme H0^° _0H 0^°\ / y^O / ^O *"" '*^0 EtO EtO . EtO (±)-cis 293 (+)-(3fl,4S) 294 (-)-(3S,4R) 295 ee 38% ee 99% acidic hydrolysis HO (-)-(3S,4fl) 296 ee 99% Scheme 79. Forzato's synthesis of optically active 4-methylparaconic esters and acids The synthesis of paraconic acid derivatives through the tandem chain extension- aldol reaction followed by a CAN-mediated oxidation was previously developed in the Zercher research group.5 Application of this chemistry towards the synthesis of 4- methylparaconic acids and esters, which were described by Forzato, would allow for comparison with previously assigned lactones. L-Serine derivative systems (281 and 283) were subjected to the methyl-substituted carbenoid using the same conditions necessary for high diastereocontrol (Scheme 80), followed by the bubbling of formaldehyde gas into the reaction mixture. An aldol reaction between the formaldehyde and the organometallic intermediate was anticipated. While the lK NMR spectrum for the crude reaction material revealed a complex mixture of open chain form 297 and hemiketal 298, the 13C NMR spectrum clearly displayed resonances at 105.0 ppm, consistent with a hemiketal. The complexity of the H and C spectra for the crude reaction mixture may 130 be, at least in part, attributed to the labile nature of the Boc protecting group upon exposure to the chain extension conditions for an extended time period. The removal of the Boc protecting group under the chain extension reaction conditions will be discussed in more detail shortly. Nonetheless, the crude mixture was treated with cerric ammonium nitrate (CAN) in water and acetonitrile for several hours, which produced a mixture of cis and trans paraconic acid derivatives. The major c/s-diastereomer 299 and trace amounts of the /rara-diastereomer 299 were separated by preparative TLC analysis. H ° 9 a. Et2Zn H H "R A N OBn Boc - \/V^- OBn bh- CHOH,CHI3CHIo2 ^ _^ ^^ N c. formaldehyde ^OR1 gas XX 298 BnO 281 (R1= Bn) CAN 1 283 (R = MOM) H20 CH2CN BnO cis and trans 299 Scheme 80. Synthesis of cis and trans 4-methylparaconic acid derivative 299 The optical rotation was determined for the cw-lactones produced from the O- benzyl 281 and O-MOM L-serine derivatives 281 (Scheme 81). The cz's-lactone 21 generated from the O-benzyl derived system 281 produced an optical rotation of [a]D +8.3° (c. 0.02, CH2C12) and the cts-lactone generated from O-MOM derived system 283 21 gave an optical rotation of [a]D +2.3° (c. 0.02, CH2CI2). While the enatiomeric excess 131 cannot be established and the concentration for both compounds was extremely low, both cw-lactones produced a positive optical rotation. Conversion of the benzyl ester to the 4- methylparaconic acid allowed for comparison with the literature to establish the stereochemistry. O O Boc" OBn 1 [ « fD = + 8.3° OBn 0; 281 =0 BnO 0 cis-299 H ° 1 [« fD = + 2.3° Boc' OBn sOMOM 283 Scheme 81. Optical rotation for cz's-paraconic acid benzyl ester derivative Hydrogenolysis of benzyl ester 299, derived from 283, produced carboxylic acid 300 (Scheme 82). An optical rotation was obtained for carboxylic acid 300 and was 21 calculated to be [a]D +25.8° (c. 0.004, CH3OH). A positive specific rotation correlates to (+)-a-methyl-paraconic acid. This result suggests that carboxylic acid 300 has an absolute configuration of 3R,4R, thus the methyl substituent of the major diastereomer, produced by L-serine derived system 283, has an absolute configuration of R. Unfortunately the error associated with specific rotation values becomes increasingly greater as the concentration of the sample is decreased. Alternative methods for determining the stereochemistry of the P-substituent were pursued. 132 / \=0 Pd/C / 'VO / FtOAr ' BnO ttUAC HO c/s-299 300 1 [afD = + 25.8° Scheme 82. Hydrogenolosis of benzyl ester 299 to paraconic acid 300 Elucidating the stereochemistry of the methyl-substituent through X-ray analysis of a serine-derived chain extension product would provide definitive evidence in support of the tentative assignment made through optical rotation; therefore, significant time was invested to access X-ray crystals. cw-Lactone 299, derived from L-serine systems 281 and 283, was stirred with (i?)-methylbenzylamine (301) at room temperature under an inert environment for 12 h (Scheme 83). Subsequent slow evaporation of the solvent resulted in a powdery precipitate unsuitable for X-ray crystal analysis. The solid was dissolved in an assortment of different solvents, such as benzene, chloroform, dichloromethane, and ethyl acetate, in an attempt to obtain crystals; however, each slow evaporation chamber resulted in formation of solids not suited for X-ray analysis. A diffusion chamber was assembled in a final attempt at growing crystals for analysis. Once again, all attempts at slow crystallization resulted in the formation of a powdery solid and were unsuccessful. Efforts are ongoing towards obtaining an X-ray grade crystal of salt 302. 133 H2N '-•^O diethyl ether / 'v^o H3N HO O^ c/s-300 302 Scheme 83. Synthesis of carboxylate salt 302 in an attempt to obtain X-ray grade crystals Amador et al. reported a convenient synthesis of paraconic acid derivatives starting with enantiopure diols. An effort was made to synthesize the optically active paraconic acid derivative called phaseolinic acid through the tandem chain extension- aldol reaction. A comparison of the synthetic product to known optical rotation values would allow assignment of absolute stereochemistry. L-serine derived compound 286 was subjected to the methyl-substituted carbenoid and quenched with hexanal to produce a complex crude reaction mixture, which was purified by preparative thin layer chromatography (Scheme 84). Although the yield of the purified product was low (22%), the establishment of three consecutive stereogenic centers was successfully accomplished in one reaction vessel. The complexity of the *H NMR spectrum was consistent with a mixture of open-chain 303 and hemiketal isomers 304, whereas the 13C NMR spectrum clearly indicated the lack of a ketone resonance and four hemiketal resonance at 105.7, 105.1, 105.0, and 104.9 ppm. The absence of a ketone 13C resonance suggests the aldol- product was biased towards existence in the hemiketal isomer forms 304, with little or no open-chain form 303. 134 H ° ° a B2Zn M O""^ Cbz'N H° H N II b.CH3CHI2 H „ I Cbz Cbz' V^-^OEt . ,. = c. Hexanal = | JJ, Bn° ^OBn 22% BnO^ 286 Scheme 84. Tandem chain extension-aldol reaction with L-serine derived system 286 Hemiketal 304 underwent an oxidative cleavage reaction mediated by eerie ammonium nitrite (CAN) in a water and acetonitrile mix (Scheme 85). Jacobine demonstrated that the CAN-oxidation reaction conditions did not epimerize previously established p-methyl stereocenters,35 therefore a loss of stereochemical fidelity was not a concern. The stereocontrol generated by the L-serine auxiliary during the chain extension-aldol reaction was optimistically anticipated to produce an excess of one diastereomer; however, the identification of the four phaseolinic acid derivatives 305, 306, 307, and 308, following the oxidative cleavage in a 4:1:2:2 ratio, respectively, was disappointing. In order to confirm the absolute configuration of the methyl-substituent, tentatively assigned as R on the basis of the optical rotation data associated with Scheme 82, isolation of one lactone diastereomer followed by optical rotation analysis was necessary. The low yield obtained during the chain extension-aldol reaction in conjunction with the lack of selectivity during the aldol reaction created a challenge for the isolation of adequate material for optical rotation analysis. This method was not further pursued as a viable route for confirming the stereochemistry of the methyl- substituent. 135 H HO OEt Cbz- * ir o BnO H20, CH3CN cis,trans-305 304 0^0 ' VJ " ir OEt o trans,trans-308 cis,cis-307 305:306:307:308 dr 4:1:2:2 Scheme 85. CAN-mediated oxidation of hemiketal 304 Attempts were made towards rationalizing the poor diastereocontrol observed during the chain extension-aldol reaction using an L-serine system. As described in detail in chapter I, the diastereocontrol of the chain extension-aldol reaction relies on both the geometry of the enolate and the facial selectivity of the aldehyde. Typically, the Z-enolate 71 is favored due to the zinc chelation between the two oxygen of the 1,4-dicarbonyl (Figure 19). Jacobine observed a decrease in the diastereocontrol when an additional Lewis basic site was available for chelation with the zinc, perhaps leading to a mixture of E- and Z-enolates 309 and 310.35 The L-serine-derived system, described above, has two additional Lewis basic sites open for complexation with the zinc. The nitrogen could complex with the zinc and thereby compromise efficient Z-enolate formation. Additionally, the hydroxyl moiety could participate in competitive complexation and lead to a mixture of enolate geometries 311, 312, and 313. Moreover, the use of a methyl- substituted carbenoid further complicates the analysis of the chain extension-aldol 136 reaction using an L-serine derived system. Further studies are required for a complete understanding of the complexity associated with this reaction. X Zn o-" No 1 R ^V_j^OR2 71 Zn /> ZnX .Zn Rn-o ;o o o" "O OR2 / OZnX 2 ,0 OR R 309 310 X /.o R nX Zn ZnX .Zn 0--1 ZnX PG-N ,0 O W N O -OR" PG // I RV^/wOZnx PG-NX ,NX OR2 PG' 311 312 313 Figure 19. Different proposed zinc-enolate geometries In summary, attempts were made to elucidate the stereochemistry of the methyl- substituent for the major diastereomer produced by the L-serine-derived systems. Synthesis and optical rotation analysis were accomplished for the cz's-lactone generated by CAN cleavage of the chain extension-aldol product. Additional effort was made to validate the absolute stereochemical assignment; regrettably, subsequent results were not fruitful. The diastereocontrol of the chain extension-aldol reaction using an L-serine 137 system must be revisited in order better understand the limitations of the serine-derived auxiliary. Attempts at Synthesizing Hydroxyl L-Serine Auxiliary As previously described, three different 0-protected L-serine derivatives displayed high diastereocontrol in the chain extension reaction with the methyl- substituted carbenoid. In an attempt to further understand the role of the hydroxyl functionality, attempts were made to synthesize a p-keto ester 314 in which the hydroxyl group was not protected (Scheme 86). The opportunity to subject P-keto ester 314 to the methyl-substituted carbenoid was desired. As an initial attempt, yV-Boc-L-serine (266) was exposed to with the Masamune-Brooks reaction conditions. The JH NMR spectrum of the crude product of a Masumune-Brooks reaction would typically show strong evidence for product formation with few byproducts being observed. Unfortunately, the *H NMR spectrum revealed a complex mixture of products with little evidence to support the formation of the desired P-keto ester 314; therefore, this method was not further pursued. H ° u ° ° N X a. CDI N AA OBn Boc' Y^OH x ^ Boc' Y^^^ \ b. MBM, Bu2Mq v OH 2 a T>H 266 314 Scheme 86. Unsuccessful attempt at synthesizing free hydroxyl P-keto ester 314 138 Shaikh et al. reported a facile aminal deprotection protocol mediated by montmorillonite KSF clay.105 Oxazolidine-containing P-keto ester 284 was subjected to similar reaction conditions in an attempt to fragment the aminal ring and produce P-keto ester 314 (Table 21). Several attempts were made, varying the temperature and time of reaction, with no success at removal of the aminal ring. Deprotection of oxazolidine 284 may require harsher reaction conditions, which have not yet been attempted. Boc O O 20%w/w H O O \ N JL JL Montmorillonite KSF N A A ^cV^OBn Boc'NY^^c O— CH3OH U 3 ^OH 284 314 Entry Temperature Time Results 21 °C 6h starting material 21 °C 24 h starting material W75°C 30 min starting material Table 21. Unsuccessful Montmorillonite KSF aminal deprotection attempts Cong et al. developed a bismuth(III) bromide (BiBr3) promoted chemoselective deprotection of cyclic aminals.106 Several reported examples were similar to cyclic aminal 284, thus this method was pursued (Table 22). Aminal 284 was exposed to 20 mol% BiBr3 in acetonitrile and monitored by TLC for 24 h; however, starting material was recovered (Entry 1). The reaction conditions were altered to include 1 equiv of water (Entry 2) and the reaction was monitored by TLC, eluting with 1:1 hexane - ethyl acetate, for approximately 24 h. The starting material (Rf = 0.61) was slightly consumed 139 and three new products were faintly observed by TLC (Rf = 0.16, 0.36, 0.53); however, the reaction did not proceed to completion. In an effort to fully consume the starting material, aminal 284 was treated with 60 mol% of catalyst in a 10 equiv excess of water (Entry 3). Similar to the TLC result observed previously (Entry 2), three new products were observed. While *H NMR analysis of the crude reaction mixture showed promising results, the major compound was still starting material. Purification of the crude reaction mixture by column chromatography afforded starting material 284 (Rf = 0.61) and minor amounts of benzyl alcohol (Rf = 0.16). The desired product 314 was not isolated. Boc o O H ° ° BiBrq OBn BoC OBn ^OH 284 314 Entry BiBr3 Solvent Temperature Time Results 1 20 mol% CH3CN 21 °C 24 h starting material 2 20 mol% CH3CN, H20 21 °C 24 h mixture 3 60 mol% CH3CN, H20 21 °C 24 h mixture 4 20 mol% CH3CN, H20 MW 75 °C 1.5 h starting material Table 22. Attempted bismuth(III) bromide-mediated aminal deprotection Although the desired p-keto ester 314 was not isolated from the bismuth(III) bromide-mediated aminal deprotection, identification of benzyl alcohol 315 suggests that competitive chemistry may be difficult to control (Scheme 87). Production of benzyl alcohol could result from liberation of the aminal protecting group that releases a free 140 hydroxyl functionality. Nucleophilic attack by the hydroxyl functionality into the neighboring benzyl ester would release benzyl alcohol and produce lactone 316. Additionally, the production of benzyl alcohol 315 may arise from a Lewis acid promoted hydrolysis of complex 317. Subsequent decarboxylation would produce ketone 318. Optimization of the aminal deprotection would be required to definitively confirm the production of lactone 316 or ketone 318. u ° ° Boc' OBn Boc "OH 314 QpoH 0 315 Br3Bi.. ~ Boc O 'O® Boc O (b) OBn M o— o— 317 318 Scheme 87. (a) Proposed cyclization generating benzyl alcohol 315 and lactone 316 (b) Proposed Lewis acid promoted hydrolysis of complex 317 Boc-Protecting Group Problems The Boc-group has been commonly used to protect the nitrogen functionality of amino acid-derived systems during the chain extension reaction. However, Verbicky and Jacobine35 have observed undesired degradation of the /-butylcarboxy intermediate under prolonged exposure to the zinc carbenoid. In reactions associated with the studies described above, Boc-protected P-keto esters 255 and 281 were subjected to a methyl- 141 substituted carbenoid (Scheme 88). For example, Boc-protected proline-derived P-keto ester 255 was treated with carbenoid and a complex mixture of products were produced. The *H NMR spectrum of the crude reaction mixture showed evidence for formation of the desired y-keto ester 319, as well as the ethyl carbamate 320; however, rotomeric forms that contributed to the complexity of the spectrum made conclusive identification of the ethyl carbamate 320 challenging. In a similar fashion, Boc-protected p-keto ester 281 reacted upon prolonged exposure to carbenoid to produce 319 and 320, also as a complex mixture of products. The reaction of Boc groups with Zn(II) salts is not surprising, since Nigam et al. reported selective removal of secondary Boc-protected amines in the presence of primary Boc-protected amines when treated with ZnBr2;107 however, the zinc carbenoid promoted substitution of a /-butyl group with an ethyl substituent was unprecedented. Further investigation was warranted. °Y° O O Et2Zn °Y°o [ CH3CH 2 °Y°o \X>Bn R"N JUOBn ' Ri-NvA/ x^OBn+ RI,NJU 2 R2 R I 0 R2 ' T 319 320 1 2 255, R = R = -CH2-CH2-CH2- 1 2 281, R =H, R =-CH2OBn Scheme 88. Zinc carbenoid-mediated degradation of Boc-protecting group Primary /-butyl carbamate 321 was treated with methyl-substituted carbenoid for an extended reaction time (Figure 20). The conversion from /-butyl to ethyl carbamate 322 was slow. At 24 h, the *H NMR of the crude reaction mixture shows about a 1:1 mixture of /-butyl 321 to ethyl 322. While the reaction was rather sluggish, the 142 conversion to ethyl carbamate 322 produced few byproducts, as evidenced by the clean lH NMR spectrum. Optimization of the reaction conditions may allow for higher conversion to ethyl carbamate. O Et Zn O 2 H CH3CHI2 or >f O O 4 321 322 1 Oh ,; i 50 48 46 44 42 40 3 i 36 34 32 30 28 26 24 22 20 18 16 14 12 10 ppm Figure 20. Zinc carbenoid-mediated conversion of /-butyl carbamate 322 to ethyl carbamate 322 Boc-pyrrolidine 323, a secondary protected amine, was subjected to the methyl- substituted carbenoid for 24 h (Scheme 89). Unlike the control study described above, 143 isolation of ethyl carbamate 324 was not observed. Instead, the H NMR spectrum for the crude reaction mixture showed 2-iodobutane (325) and trace amounts of the starting material 323. 2-Iodobutane (325), a byproduct that may come about from carbenoid reacting with itself, has been previously observed. Although not conclusive, the low mass recovery {ca. 20%) suggests the Boc-protecting group may have been deprotected under the zinc carbenoid conditions and pyrrolidine was lost during the aqueous work-up. Further investigation is necessary to confirm the deprotection of the Boc-protecting promoted by zinc carbenoid. r V a. E«2Zn V N CH3CHI2 N < 7 —-^ < 7 \—/ b. NH4CI \—/ 323 324 325 Scheme 89. Attempted ethyl carbamate 324 formation from Boc-pyrrollidine The use of a Boc-protecting group during zinc carbenoid-mediated chain extension reactions that require long reaction times (12 - 24 h) should be avoided. Degradation of the Boc-protecting group, producing ethyl carbamates or inducing complete removal of the carbamate, compromises the efficiency of the reaction and creates complex crude reaction mixtures that are challenging to interpret and purify. Recent synthetic efforts have utilized alternative protecting groups, such as Cbz and Ts, which have shown more stability towards the zinc carbenoid under prolonged reaction times. 144 Lactic Acid Derived System The high diastereocontrol provided by the L-serine-derived systems encourage the exploration into similar systems that provide the opportunity for directed cyclopropanation. For example, treatment of p-keto ester with diethylzinc would produce an enolate intermediate 326, which would posses an allylic hydroxyl functionality (Scheme 90). The allylic system of 326 is hypothesized to function in a similar fashion to that of the homoallylic hydroxyl group of the L-serine-derived systems. Therefore, the treatment of 326 with 1,1-diiodoethane could lead to P-methyl-y-keto ester 327 with high diastereoselectivity. While the results reported above support the notion that the protected homoallylic alcohol functionality is involved in controlling the diastereoselectivity, the transition state for the reaction of an L-serine-derived system with zinc carbenoid remains unknown. Investigation of an allylic hydroxyl-containing system may offer further insight into the stereocontrol of the chain extension reaction using a methyl-substituted carbenoid. X Zn N O Et2Zn Scheme 90. Proposed route for allylic directed cyclopropanation using a methyl- substituted carbenoid 145 Commercially available methyl lactate (328) was converted to the benzyl ether 329 in 70%) yield (Scheme 91). Subsequent hydrolysis of the methyl ester afforded carboxylic acid 330, suitable for facile conversion to the desired P-keto ester. As previously described for the synthesis of amino acid-derived P-keto esters, the Masamune-Brooks reaction converted carboxylic acid 330 to P-keto ester 331 in 63%> yield after column chromatography. O O O a. NaH LiOH, H20 » O b. BnBr O" OH THF, CH3OH OH THF OBn 88% OBn 70% 328 329 330 a. CDI b. MBM, Bu2Mg THF, 63% O O OBn OBn 331 Scheme 91. Synthesis of lactic acid-derived p-keto ester 331 Lactic acid-derived p-keto ester 331 was subjected to the methyl-substituted carbenoid in order to study the ratio of diastereomers created upon the formation of P-methyl-y-keto ester 332 (Scheme 92). Analysis of the *H NMR spectrum for the crude reaction mixture revealed a 2:1 diastereomeric ratio. The poor diastereocontrol induced by the lactic acid- derived system was in stark contrast to the L-serine-derived systems. A better 146 understanding of the sterocontrol induced by the L-serine systems is necessary in order to understand the lack of sterocontrol observed by the lactic acid derived system. O O a. Et2Zn O OBn yUOBn _^^ VW OBn c- NH4CI OBn ' O 331 332 dr2:1 Scheme 92. Chain extension reaction using a lactic acid-derived system 331 Future Application for L-Serine Derived Auxiliary The incorporation of a methyl-substituted carbenoid into a y-keto ester backbone with control of the absolute stereochemsitry broadens the applicability of this methodology for synthesis of natural products. A fitting example would be for the StereocontroUed synthesis of papyracillic acid C (131) (Scheme 93). Subjecting L-serine- derived system 333 to the chain extension-acylation reaction and subsequent CAN- oxidation should produce bis-lactone 334. The serine auxiliary strategically offers absolute stereocontrol of the methyl group and should be easily removed through the oxidative cleavage. Although several chemical manipulations of the to-lactone 334 would be required, papyracillic acid C (131) could be accessible through this approach. An alternative, and perhaps a more desirable approach, for a StereocontroUed synthesis of papyracillic acid C would involve development of an enantioselective incorporation of the methyl-substituted carbenoid during the chain extension reaction. Exploration 147 towards an enantioselective synthesis of P-methyl-y-keto esters would involve a chiral zinc ligand suitable for providing absolute stereocontrol. 2 R O O 1.) a. Et2Zn ' N ji b.CH3CHI2 O 4 c R1 "v^^^iDR - anhydride vOR3 2.) CAN 333 Scheme 93. StereocontroUed synthesis of papyracillic acid C (131) A better suited application for the L-serine derived auxiliaries would involve the transformation of 1,3-dicarbonyls to 1,4-dicarbonyls mediated by zinc carbenoid for the synthesis of peptide isosteres (Scheme 94).30b'31'54 As discussed in chapter I, a significant application of the chain extension methodology has centered on the synthesis of peptide isosteres. The diastereoselective synthesis of P-methyl-y-keto esters 333 offers a method for the synthesis of hydroxyethylene isosteres 335. Manipulation of the p-methyl-y-keto ester backbone 333 should provide access to the desired hydroxyethylene peptide isostere 335 with a methyl-substituent in the P-position. OR4 333 335 Scheme 94. P-Methyl hydroxyethylene peptide isostere 148 CHAPTER IV GENERAL EXPERIMENTAL SECTION Solvents Anhydrous solvents, passed through drying agent with nitrogen pressure, were obtained from an Innovative Technology Inc. Solvent Delivery System prior to use. Tetrahydrofuran (THF) and dimethylformamide (DMF) obtained from the Solvent Delivery System, were stored over 3 A and 4 A sieves, respectively. Reagents All reagents were received from commercial sources and were used as received unless otherwise stated. Commercially available {3-keto esters and aldehydes were dried, distilled, and stored over 3 A sieves in a desiccators. Amines were dried, distilled, and stored over 4 A sieves. 149 Reactions Reaction glassware and magnetic stir bars were stored in an oven at 200 °C prior to use. Sigma-Aldrich Natural Rubber Septa and Teflon coated magnetic stir bars were utilized, unless otherwise noted. Unless otherwise specified, nitrogen gas was introduced into the reaction vessel through a Tygon® tube with a needle or glass inlet adaptor. Henke Sass Wolf Norm-Ject® plastic syringes and oven-dried 25-cm needles were used for volumetric addition of reagents unless otherwise noted. Chromatography Preparative chromatography was accomplished through the use of Analtech Uniplate Silica Gel GF 1000 microns with UV 254 glass-backed plates. Flash column chromatography was preformed with Silica-P Flash Silica Gel with 40-63 um partial size. Mobile phases were freshly prepared as described in the detailed experimental section. Thin layer chromatography (TLC) analysis was conducted on Whatman glass-backed Silica Gel 60 A 250 um thickness with fluorescent indicator. Development of the TLC plate was accomplished by staining the plate with anisaldehyde or phosphomolybdic acid stain, unless specifically noted. TLC solvent systems were identical to the mobile phase used for column chromatography, unless otherwise specified. Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy was achieved using a Varian Mercury spectrometer operating at 500 or 400 MHz for lU and at 126 or 100 MHz for 13C spectroscopy. All carbon spectra were proton-decoupled. All H resonances were 150 reported downfield relative to TMS (5 0 ppm) reference unless otherwise noted. All 13C resonances were referenced to CDCI3 (8 77.16 ppm) or acetone-d6 (8 206.26) unless otherwise, stated. The following abbreviations were used to denote the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, ABq = AB quartet, m = multiplet, br = broad, app = apparent. Infrared (IR) spectroscopy was conducted using a Thermo Nicolet iSlO FTIR with diamond ATR probe. DETAILED EXPERIMENTAL SECTION Methyl 4,4-dimethyl-3-oxopentanoate (methyl pivaloylacetate) (35c)108 An oven-dried 500-mL, three necked, round-bottomed flask, equipped with a magnetic stir bar, reflux condenser with nitrogen gas inlet adaptor, pressure-equalizing addition funnel, and thermocouple probe, was charged with 1,4-dioxane (150 mL). Sodium hydride (80.0 mmol, 3.2 g, 60%) dispersed in mineral oil) was washed with hexanes (5 x 10 mL) then added in one portion. Methyl carbonate (40.0 mmol, 3.60 g, 3.3 mL) was added directly to the stirring solution via a dropwise fashion through an oven-dried needle over the course of 3 min. Pinacolone (40.0 mmol, 4.9 mL, 4.0 g) dissolved in 1,4- dioxane (50 mL) was added dropwise through the addition funnel to the stirring solution over the course of 2 h. The reaction was brought to 90 °C using a mineral oil bath and warmed for 18 h. The crude mixture was quenched with ammonium chloride (150 mL) 151 and extracted with dichloromethane (3 x 150 mL). The organic layers were pooled, dried with anhydrous sodium sulfate (ca. 20 g), gravity filtered and concentrated to a yellow oil. The crude oil was purified by vacuum distillation (b.p. 67 - 70 °C, 13 mmHg) to afford methyl 4,4-dimethyl-3-oxopentanoate 35c as a clear oil (5.0 g, 80%>). !H NMR 13 (400 MHz, CDC13) 8 3.73 (s, 3H), 3.57 (s, 2H), 1.18 (s, 9H); C NMR (100 MHz, CDCI3) 8 208.2, 168.4, 85.4, 52.4, 43.9, 26.3; IR (neat) v 2970 - 2874, 1747, 1706 cm'1. Allyl 3-oxobutanoate (42e) An oven-dried 100-mL round-bottomed flask, equipped with magnetic stir bar, reflux condenser, rubber septum, and nitrogen gas inlet, was charged with 5-(l- hydroxyethylidene)-2,2-dimethyl-l,3-dioxane-4,6-dione (88.0 mmol, 16.0 g) in anhydrous toluene (275 mL). Allyl alcohol (114.0 mmol, 6.7 g, 7.8 mL) was added directly to the stirring solution via syringe in one portion. The reaction was refluxed for 6 h or until TLC analysis indicated consumption of starting material (Rf = 0.44 - 0.0, streak) and production of product (Rf = 0.63), eluting with a 1:1 hexane - ethyl acetate mobile phase. The crude reaction was concentrated via rotary evaporation (10 mmHg, 45 °C) to afford a dark brown oil. The crude residue was distilled under reduced pressure (40 mmHg, 100 - 104 °C) to afford allyl 3-oxobutanoate 42e as a light yellow oil (7.4 g, 62%). lH NMR (500 MHz, CDCI3) 8 6.00 - 5.85 (m, 1H), 5.35 (m, 1H), 5.30 - 5.22 (m, 2H), 4.65 (m, 2H), 3.50 (s, 2H), 2.27 (s, 3H); 13C NMR (126 MHz, CDCI3) 8 200.4, 166.7, 131.5, 118.7, 65.8, 49.9, 30.1; IR (neat) v 3087 - 2946, 1741, 1714, 1649, 1496 cm"1. 152 1,1-Diiodoethane (90)44 A 100-mL oven-dried round-bottom flask, equipped with magnetic stir bar, a condenser and a nitrogen gas inlet adapter, was charged with dichloroethane (250.4 mmol, 21.0 mL), iodoethane (750 mmol, 60.0 mL), and aluminum trichloride (7.5 mmol, 1.0 g). The mixture was refluxed for 3 h. The crude reaction mixture was allowed to cool, and then an additional portion of aluminum trichloride was added (7.5 mmol, 1.0 g). The mixture was refluxed for an additional 3 h. The crude reaction mixture was quenched with water (20 mL), washed with saturated sodium thiosulfate (3 x 100 mL), and dried with sodium sulfate (ca. 10 g). The dark brown crude mixture was purified by vacuum distillation (5 mmHg, ca. 70 - 75 °C), affording 1,1-diiodoethane 90 as a pale yellow liquid (58.6 g, 83 %>). The product was stored in a 20-mL scintillation vial over copper wire and 3 A ! molecular sieves, wrapped with foil, and kept at 5 °C. H NMR (400 MHz, CDC13) 8 5.23 13 (q, J= 6.7 Hz, 1H), 2.93 (d, J= 6.7 Hz, 3H); C NMR (100 MHz, CDC13) 8 210.9, 39.1. IR (neat) v 2970 - 2846, 2412, 2258, 2129, 1435, 1372, 1226 cm"1. 3-Methoxyfuran-2,5-dione (3-methoxymaleic anhydride) (134) An oven-dried 25-mL round-bottom flask, equipped with magnetic stir bar, reflux condenser, and nitrogen gas inlet adapter, was charged with 2-methoxybut-2-enedioic acid 195 (2.8 mmol, 0.41 g). Thionyl chloride (6 mL) was added directly to the flask via an oven-dried syringe whereupon bubbling occurred. The gaseous solution was stirred for 1 h at room temperature then brought to reflux for 16 h. The solvent was removed by distillation under reduced pressure in a well-ventilated fume hood (30 mmHg, 50 °C) to afford a viscous orange oil. The crude product was purified by flash column 153 chromatography, eluting with (4:1) hexane-ethyl acetate (Rf = 0.3) to provide 3- methoxyfuran-2,5-dione 134 as an off white solid (0.24 g, 66 %). (m.p. 110-116 °C); lU 13 NMR (400 MHz, CDC13) 8 5.86 (s, IH), 4.08 (s, 3H); C NMR (100 MHz, CDC13) 8 162.9, 161.8, 160.8, 98.9, 60.6; IR (neat) v 3126, 1856, 1763, 1639, 1454, 1438, 1340 cm"1. £)-5-(Methoxycarbonyl)-8,8-dimethyl-4,7-dioxonon-2-enoic acid (144) and (2E,4E)- 4-Hydroxy-5-(methoxycarbonyl)-8,8-dimethyl-7-oxonona-2,4-dienoic acid (145) An oven-dried 250-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with anhydrous toluene (50 mL) and cooled to 0 °C in an ice-water bath. Neat diethylzinc (7.5 mmol, 0.93 g, 0.77 mL) was added in one portion via an oven-dried needle followed by dropwise addition of diiodomethane (7.7 mmol, 2.0 g, 0.57 mL) over the course of 3 min. The reaction was stirred at 0 °C for 10 min at which time methyl pivaloylacetate 35c (3 mmol, 0.47 g, 0.44 mL) was added dropwise via syringe. The reaction was stirred for 45 min while warming to room temperature. Maleic anhydride (4.5 mmol, 0.44 g), dissolved in anhydrous toluene (10 mL), was added at room temperature and stirred for 16 h. The reaction was quenched with 3 M hydrochloric acid (ca. 40 mL) and extracted with ethyl acetate (5 x 50 mL). The organic layers were pooled, dried with sodium sulfate (ca. 30 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 35 °C) to afford a viscous brown oil. The crude mixture was purified by flash column chromatography, eluting with (1:1) hexane - ethyl acetate, followed by (1:2) hexane - ethyl acetate mobile phase (Rf = 0.44, streak) affording the title compounds 144 and 145 in a mixture of (1:1) keto - enol isomers as a 154 viscous yellow oil (430 mg, 53%). *H NMR (400 MHz, CDC13) 8 12.39 (br s, IH), 7.22 (d, J= 15.8 Hz, IH), 7.17 (d, J = 15.2 Hz, IH), 6.76 (d, J = 15.8 Hz, IH), 6.67 (d, J = 15.2 Hz, IH), 4.27 (dd, J = 8.1, 5.8 Hz, IH), 3.72 (s, 3H), 3.70 (s, 3H), 3.32 - 3.04 (m, 13 6H), 1.20 (s, 9H), 1.14 (s, 9H). C NMR (100 MHz, CDC13) 8 213.2, 212.3, 194.1, 172.7, 171.1, 169.2, 164.0, 138.9, 135.4, 126.6, 101.7, 53.1, 52.4, 51.9, 45.8, 44.6, 44.1, 36.2, 33.9, 28.9, 26.7, 26.7; IR (neat) v 3457, 2962 - 2876, 1778, 1703, 1649 cm"1. (£)-4,7-Dioxo-5-(2-oxooxazolidine-3-carbonyl)oct-2-enoic acid (152) An oven-dried 50-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged P-keto imide 146 (1 mmol, 0.17 g) and anhydrous toluene (20 mL). The solution was cooled to 0 °C in an ice-water bath followed by cautious addition of diethylzinc (1 mmol, 0.12 g, 0.10 mL). The reaction was allowed to stir for 10 min. Diiodomethane (1 mmol, 0.28 g, 0.08 mL) was added and the reaction was stirred for 15 min while warming to room temperature. Maleic anhydride (1.1 mmol, 0.11 g) dissolved in anhydrous toluene (2 mL) was added dropwise at room temperature over the course of 2 min and the reaction was stirred for 12 h. The reaction mixture was quenched with cautious addition of 3 M hydrochloric acid (ca. 15 mL) and extracted with ethyl acetate (5 X 20 mL). The organic layers were combined, dried with sodium sulfate (ca. 5 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford a brown viscous oil. The crude reaction mixture was purified by preparative thin layer chromatography on silica, eluting with a (1:1) hexane - ethyl acetate mobile phase (Rf = 0.17) and afforded the title compound 152 and y-keto imide l 154 as a clear oil (85 mg, 30%). U NMR (400 MHz, CDC13) product 152: 8 7.24 (d,J = 155 15.8 Hz, IH), 6.74 (d, J= 15.8 Hz, IH), 5.22 (dd, J= 10.4, 3.4 Hz, IH), 4.52 - 4.38 (m, 3H), 4.15 - 3.95 (m, 3H), 3.25 (dd, 7 = 17.9, 10.4 Hz, IH), 2.77 (dd, J = 17.9, 3.4, IH), 2.25 (s, 3H); y-keto imide 154: 8 4.43 (t,J= 8.1 Hz, 2H), 4.01 (t, J= 8.1 Hz, 2H), 3.30 - 13 3.15 (m, 2H), 2.85 - 2.78 (m, 2H), 2.23 (s, 3H); C NMR (100 MHz, CDC13) of both produces 8 207.3, 204.6, 194.6, 172.4, 168.4, 167.8, 154.0, 153.8, 139.1, 131.6, 62.7, 62.3, 52.6, 51.1, 42.6, 42.3, 40.4, 37.1, 30.0, 29.5; IR (neat) v 3522, 3146 - 2930, 1769, 1729, 1711, 1680, 1667, 1647, 1524 cm"1. 5-(Methoxycarbonyl)-8,8-dimethyl-4,7-dioxononanoic acid (165), Methyl 2-(2- hydroxy-5-oxotetrahydrofuran-2-yl)-5,5-dimethyl-4-oxohexanoate (166), and Methyl 2-(fer/-butyl)-2-hydroxy-7-oxo-l,6-dioxaspiro[4.4]nonane-4-carboxylate (167) An oven-dried 100-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with anhydrous toluene (50 mL) and cooled to 0 °C in an ice-water bath. Neat diethylzinc (5.5 mmol, 0.67 g, 0.56 mL) was added in one portion via an oven-dried needle followed by dropwise addition of diiodomethane (5.5 mmol, 1.48 g, 0.42 mL) over the course of 3 min. The reaction was stirred at 0 °C for 10 min at which time methyl pivaloylacetate 35c (2.5 mmol, 0.40 g, 0.37 mL) was added dropwise via syringe. The reaction was stirred for 45 min while warming to room temperature. Succinic anhydride (5.0 mmol, 0.50 g), dissolved in anhydrous toluene (2 mL), was added at room temperature and stirred for 16 h. The reaction was quenched with 3 M hydrochloric acid (ca. 40 mL) and extracted with ethyl acetate (5x50 mL). The organic layers were pooled and concentrated via rotary evaporation (10 mmHg, 30 °C) to 156 half the original volume (ca. 150 mL). The organic layer was washed with saturated sodium bicarbonate (2 x 50 mL). The aqueous layer was cooled to 0 °C in an ice-water bath, acidified to pH 2 with slow addition of concentrated hydrochloric acid, and extracted with diethyl ether (5 x 50 mL). The organic layers were pooled, dried with sodium sulfate (ca. 10 g), gravity filtered and concentrated via rotary evaporation (40 mmHg, 21 °C) to afford a mixture of the titled compounds (165, 166, and 167) in a 3:2:1 l ratio as a light yellow oil (460 mg, 68%). R NMR (400 MHz, CDC13) 8 4.05 (dd, J = 8.3, 5.7 Hz, IH), 3.73 (s, 3H), 3.68 (s, IH), 3.66 (s, 2H), 3.36 - 3.19 (m, 2H), 3.19 - 3.02 (m, 3H), 3.02 - 2.86 (m, 3H), 2.87 - 2.53 (m, 9H), 1.18 (s, 3H), 1.16 (s, 9H), 1.14 (s, 6H); 13C NMR (100 MHz, CDCI3) 8 214.0, 213.7, 206.7, 203.0, 178.6, 178.5, 174.9, 169.3, 128.7, 128.4, 128.3, 125.7, 53.2, 52.9, 52.3, 44.2, 44.1, 43.2, 37.8, 37.5, 37.1, 36.1, 35.6, 31.7, 30.5, 28.2, 28.0, 27.9, 26.7, 26.6, 26.5; IR (neat) v 3531, 2968 - 2873, 1788, 1702 cm"1. 4-Ethoxy-4-oxobut-2-ynoic acid (171) An oven-dried 25-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with ethyl propiolate 170 (1 mmol, 0.1 g, 0.1 mL) in dry tetrahydrofuran (17 mL). The solution was cooled to -78 °C in a dry ice- acetone bath then treated with rc-butyl lithium (1.1 mmol, 2.5 M in hexane, 0.44 mL) and stirred for 45 min. The reaction mixture was transferred by cannula into a separate oven- dried, 25-mL, round-bottomed flask equipped with magnetic stir bar and rubber septum, containing dry ice (ca. 0.5 g). The reaction was stirred 2h at room temperature. The reaction mixture was quenched with 1 M hydrochloric acid (ca. 10 mL) and extracted 157 with ethyl acetate (5x12 mL). The organic layers were pooled, dried with magnesium (ca. 10 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford 4-ethoxy-4-oxobut-2-ynoic acid (170) as a viscous brown oil (130 mg, 92%>). The product was used without further purification; however, an acid base work-up may be l required to remove minor impurities. H NMR (400 MHz, CDC13) 8 9.69 (br s, IH), 4.32 13 (q, J= 7.1 Hz, IH), 1.35 (t, J= 7.1 Hz, 2H); C NMR (100 MHz, CDC13) 8 154.7, 152.0, 76.3, 63.5, 41.2, 13.9; IR (neat) v 3517, 2988 - 2942, 2598, 1715 cm"1. Ethyl 2-(l,3-dicyclohexyl-2,5-dioxoimidazolidin-4-ylidene)acetate (Not optimized) (182) An oven-dried 5-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with carboxylic acid 170 (0.70 mmol, 100 mg) in dry dichloromethane (3.5 mL). Dicyclohexylcarbodiimide (0.35 mmol, 70 mg) and 4-(dimethylamino)pyridine (0.06 mmol, 8 mg) were added to solution as solids in one portion. The reaction was stirred for 4 h at room temperature at which time the reaction was vacuum filtered and quenched with 1 M hydrochloric acid (ca. 2 mL). The aqueous layer was extracted with dichloromethane (3x5 mL). The organic layers were pooled, dried with sodium sulfate (ca. 5 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 21 °C) to afford a viscous brown oil. The crude residue was purified by flash column chromatography on silica, eluting with 3:1 hexane - ethyl ] acetate (Rf = 0.29), to afford the titled compound 182 as clear oil (30 mg, 25%>). H NMR (400 MHz, CDCI3) 8 5.66 (s, IH), 4.31 (q, J= 7.2 Hz, 2H), 3.93 (tt, J= 12.4, 3.9 Hz, IH), 3.76 (tt, J= 12.3, 3.7 Hz, IH), 2.20 - 1.95 (m, 4H), 1.95 - 1.73 (m, 6H), 1.73 - 1.58 158 13 (m, 4H), 1.44 - 1.07 (m, 9H); C NMR (100 MHz, CDC13) 8 162.2, 160.3, 153.3, 134.4, 101.9, 61.8, 53.6, 51.9, 29.4, 25.9, 25.1, 14.2; IR (neat) v 2935, 2858, 1770, 1711, 1661 cm" . Ethyl 2-(l,3-diisopropyl-2,5-dioxoimidazolidin-4-ylidene)acetate (183) An oven-dried 25-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with 4-ethoxy-4-oxobut-2-ynoic acid (XX) (0.7 mmol, 0.10 g) in anhydrous dichloromethane (12 mL). Diisopropylcarbodiimide (0.7 mmol, 0.09 g, 0.11 mL) was added to the reaction via syringe in one portion and allowed to stir at room temperature for 4 h. The reaction was quenched with 1 M hydrochloric acid (10 mL) and extracted with dichloromethane (3 x 20 mL). The organic layers were pooled, dried with sodium sulfate (ca. 10 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 21 °C) to afford a brown viscous oil (0.19 g). The crude residue was purified by flash column chromatography on silica, eluting with (5:1) hexane - ethyl acetate mobile phase (Rf = 0.14). The titled compound 183 was obtained as a crystalline white solid (0.12 g, 63%). (m.p. 54 - 56 °C); : H NMR (400 MHz, CDC13) 8 5.63 (s, IH), 4.41 - 4.23 (m, 4H), 1.43 (d, J= 7.0 Hz, 6H), 1.41 (d, J= 7.1 Hz, 6H), 1.35 (t, J= 7.2 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 165.1, 160.2, 153.2, 134.2, 101.9, 61.8, 45.2, 44.3, 19.9, 19.6, 14.2; IR (neat) v 3067, 2979, 2939,2881, 1763, 1707, 1661 cm"1. 159 3-Phenylpropiolic acid (185) Followed the procedure for the formation of 4-ethoxy-4-oxobut-2-ynoic acid (170), replacing the ethyl propiolate with phenyl acetylene. 3-Phenylpropiolic acid (185) was obtained as a light yellow solid (140 mg, 96%) and was carried on without further ! purification, (m.p. 134 - 135 °C); H NMR (400 MHz, CDC13) 8 7.62 - 7.60 (m, 2H), 13 7.49 - 7.45 (m, IH), 7.40 - 7.37 (m, 2H); C NMR (126 MHz, CDC13) 8 158.9, 133.4, 131.3, 128.8, 119.2, 89.2, 80.2; IR (neat) v 3069 - 2503, 2234, 2200, 1665 cm"1. 5-Benzylidene-l,3-dicyclohexylimidazolidine-2,4-dione (186) Using the same reaction conditions reported for the preparation of heterocycle 183, titled compound 186 was isolated from carboxylic acid 185 and DCC as a white solid (16 mg, 3%). (m.p. 88.5 - 91.5 °C); (Rf = 0.87, purified by preparative thin layer chromatography } on silica, eluting with a 2:1 mobile phase); H NMR (400 MHz, CDC13) 8 7.68 (m, 2H), 7.44 - 7.40 (m, 2H), 7.35 (m, IH), 6.46 (s, IH), 4.07 (tt, J = 12.2, 3.8 Hz, IH), 3.82 - 3.75 (m, IH), 2.35 - 2.24 (m, 2H), 1.87 - 1.67 (m, 9 H), 1.49 - 1.28 (m, 9H); 13C NMR (100 MHz, CDC13) 8 162.4, 142.2, 139.6, 132.5, 130.2, 129.1, 129.0, 107.4, 54.8, 53.1, 34.3, 28.7, 26.0, 25.9, 25.3, 24.7; IR (neat) v 3067 - 2853, 1750, 1739, 1708, 1670 cm"1. Dimethyl 2-methoxybut-2-enedioate (194)69 An oven-dried 100-mL round-bottom flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with acetylene dicarboxylate 193 (30.2 mmol, 5.0 mL, 4.3 g) and dry methanol (44 mL). Triethylamine (17.0 mmol, 2.4 mL, 1.74 g) was added to the reaction drop wise via an oven-dried syringe, giving rise to a 160 deep amber colored solution. The reaction was stirred at room temperature for 2 h then concentrated by rotary evaporation (10 mmHg, 30 °C) to yield dimethyl 2-methoxybut-2- enedioate 194 as a red viscous oil (5.3 g, 100 %>). The product did not require further purification and was carried on as a mixture of E- and Z-isomers in a ratio of 2:1. !H NMR (400 MHz, CDC13) ^-isomer: 8 6.19 (s, IH), 3.95 (s, 3H), 3.85 (s, 3H), 3.76 (s, 3H). Z-isomer: 8 5.21 (s, IH), 3.89 (s, 3H), 3.76 (s, 3H), 3.71 (s, 3H); 13C NMR (100 MHz, CDCI3) ^-isomer: 8 164.7, 163.2, 154.9, 107.7, 57.1. Z-isomer: 8 166.3, 164.0, 162.6, 93.0, 61.1; IR (neat) v 3124-2943, 1688, 1631cm"1. 2-Methoxybut-2-enedioic acid (195) A 50-mL round-bottom flask was charged with dimethyl 2-methoxybut-2-enedioate 194 (3.2 mmol, 0.55 g) in methanol (9 mL) and stirred with a magnetic stir bar. A 4 N aqueous potassium hydroxide solution (8 mL) was added to the reaction and the reaction mixture was allowed to stir for 16 h at room temperature. The resulting yellow solution was cooled in an ice-water bath to 0 °C, acidified to pH 2 with 10 M hydrochloric acid solution, and extracted with diethyl ether (5x15 mL). The organic layers were pooled, dried with sodium sulfate (ca. 10 g), gravity filtered, and concentrated by rotary evaporation (10 mmHg, 21 °C) to afford 2-methoxybut-2-enedioic acid 195 as a creamy white solid (0.4lg, 87 %). The product did not require further purification and was carried on as a mixture of E- and Z- isomers in a 5:1 ratio, (m.p. 137 - 139 °C) !H NMR (400 MHz, acetone-d6) ^-isomer: 8 6.17 (s, IH), 3.93 (s, 3H), Z-isomer: 8 5.34 (s, IH), 13 3.81 (s, 3H); C NMR (126 MHz, acetone-d6) 8 165.8, 164.6, 162.8, 154.7, 107.4, 91.8, 59.9, 56.3, 50.4, 48.9; IR (neat) v 3074 - 2569, 1682, 1641 cm"1. 161 Methyl 2,6-dimethoxy-2-methyl-8-oxo-l-oxaspiro[4.4]non-6-ene-4-carboxylate (198a, 198b, and 198c) An oven-dried 50-mL round-bottomed flask equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with anhydrous dichloromethane (20 mL) and cooled to 0 °C in an ice-water bath. Neat diethylzinc (3 mmol, 0.37 g, 0.31 mL) was added via an oven-dried needle in one portion followed by cautious dropwise addition of diiodomethane (3 mmol, 0.81 g, 0.23 mL) over the course of 1 min. The reaction was stirred for 10 min at 0 °C at which time methyl acetoacetate (1 mmol, 0.12 g, 0.11 mL) was added via syringe in one portion. The reaction was stirred for 30 min while warming to room temperature. 3-Methoxymaleic anhydride (1.5 mmol, 0.18 g), dissolved in dry dichloromethane (2 mL), was added dropwise over the course of 3 min at room temperature and the reaction was stirred for an additional 16 h. The reaction was quenched with 3 M hydrochloric acid (ca. 15 mL) and extracted with ethyl acetate (5 x 20 mL). The organic layers were combined, dried with sodium sulfate (ca. 10 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to a viscous orange oil, which was further dried on a high vacuum line (0.4 mmHg) to afford hemiketals 197 as an orange foam (0.32 g). The product was carried on without further purification. Catalytic /?-TsOH (0.06 mmol, 0.01 g), crude hemiketal 197 (0.62 mmol, 0.16 g), and 2,2-dimethoxypropane (6 mL) were added to a 20-mL scintillation via, equipped with magnetic stir bar, rubber septum and calcium sulfate drying tube. The reaction was stirred at room temperature for 12h, and then concentrated via rotary evaporation (10 mmHg, 30 °C) to afford the titled compounds 198 as a viscous dark brown oil. The mixture of 162 diastereomers were separated by flash column chromatography on silica, eluting with a gradient mobile phase of (5:1), (4:1), (3:1), (2:1), (1:1) hexane-ethyl acetate. Diastereomers 198a and 198b eluted together to afford a yellow crystalline solid in 44 ! mg, 26% (Rf = 0.24, 1:1 hexane - ethyl acetate); H NMR (400 MHz, CDC13) diastereomer 198a: 8 5.17 (s, IH), 3.98 (s, 3H), 3.80 (dd, J= 12.5, 7.7 Hz, IH), 3.67 (s, 3H), 3.29 (s, 3H), 2.53 (app t,J= 12.8 Hz, IH), 2.39 (m, IH), 1.59 (s, 3H); diastereomer 198b: 8 5.09 (s, IH), 3.94 (dd, J= 9.8, 6.5 Hz, IH), 3.85 (s, 3H), 3.68 (s, 3H), 3.32 (s, 3H), 2.43-2.34 (m, 2 H), 1.54 (s, 3H); 13C NMR (100 MHz, CDCI3) both diastereomers 198a and 198b 8 176.8, 176.1, 169.8, 169.5, 168.8, 168.1, 109.9, 109.8, 109.5, 108.0, 90.7, 89.8, 59.9, 59.8, 52.4, 52.5 50.9, 49.6, 48.7, 41.3, 38.3, 22.4, 20.7; IR (neat) v 3100, 3000, 2951, 1776, 1747, 1647, 1438, 1373, 1312, 1235 cm"1. Diastereomer 198c was isolated as a light yellow oil in 22.3 mg, 13%). (Rf = 0.13, (1:1) J hexane - ethyl acetate); H NMR (400 MHz, CDC13) 8 5.16 (s, IH), 3.96 (s, 3H), 3.67 (s, 3H), 3.56 (dd, J= 11.0, 9.3 Hz, IH), 3.36 (s, 3H), 2.85 (dd, J= 13.4, 11.0 Hz, IH), 2.18 13 (dd, J = 13.4, 9.3 Hz, IH), 1.50 (s, 3H); C NMR (100 MHz, CDC13) 175.6, 169.0, 167.9, 111.5, 107.2, 91.0, 60.1, 52.7, 50.4, 49.0, 35.1, 24.9; IR(neat) v 3100, 3000, 2952, 1776,1747, 1647,1438 cm"1. 163 Methyl 2-hydroxy-9-methoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene-4- carboxylate (200) and (£)-3-methoxy-5-(methoxycarbonyl)-6-methyl-4,7-dioxooct-2- enoic acid (201) Method A: An oven-dried 100-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with anhydrous dichloromethane (18 mL) then cooled to 0 °C in an ice-water bath. Neat diethylzinc (2.5 mmol, 0.26 mL, 0.31 g) was added in one portion using an oven-dried 25 cm needle. Methyl acetoacetate (1 mmol, 0.11 mL, 0.12 g) was added in one portion and the reaction was stirred at 0 °C for 15 min. 1,1-Diiodoethane (3 mmol, 0.24 mL, 0.85 g) was added drop-wise over the course of 30 sec, after which the reaction was stirred for 90 min while warming to room temperature. TLC analysis indicated consumption of starting material (Rf= 0.49) and formation of the intermediate enolate (Rf= 0.54), eluting with a (1:1) hexane-ethyl acetate mobile phase. To the clear reaction mixture, 3-methoxymaleic anhydride (1.1 mmol, 0.14 g), dissolved in anhydrous dichloromethane (2 mL), was added dropwise over the course of 3 min. The resulting yellow reaction mixture was stirred for 16 h at room temperature (ca. 21-25 °C). TLC analysis indicated consumption of intermediate enolate (Rf= 0.54) and development of product (Rf= 0.14 - 0.0, streak), eluting with a (1:1) hexane-ethyl acetate mobile phase. The reaction was quenched with 3 M hydrochloric acid (ca. 15 mL) then extracted with ethyl acetate (4 x 15 mL). The organic layers were pooled, dried with anhydrous sodium sulfate (ca. 10 g), gravity filtered, and concentrated via rotary evaporator (10 mmHg, 30 °C) to afford the title compounds 200 and 201 as a brown foamy residue (0.26 g). The crude product was carried on without further purification. Characteristic proton resonances for the major 164 l isomer 200: U NMR (400 MHz, CDC13) 8 5.12 (s, IH), 3.89 (s, 3H), 3.59 (s, 3H), 3.38 (d, J= 12.4, IH), 2.60 (dq, J= 12.4, 6.7 Hz, IH), 1.50 (s, 3H), 1.10 (d,J= 6.7, 3H); All carbon resonances for crude reaction mixture: 13C NMR (100 MHz, CDCI3) 8 212.0, 212.0, 177.6, 177.2, 176.7, 176.4, 173.9, 171.5, 170.6, 170.5, 169.9, 169.7, 169.5, 169.3, 168.1, 163.6, 162.1, 110.8, 108.2, 107.9, 107.0, 106.1, 103.3, 103.0, 101.6, 101.4, 99.0, 96.1, 90.4, 89.4, 60.3, 60.1, 57.4, 56.5, 55.6, 54.3, 52.7, 52.5, 51.0, 45.7, 44.0, 42.9, 42.7, 42.6, 41.0, 40.8, 36.6, 29.8, 28.7, 28.7, 25.9, 24.8, 22.8, 20.9, 16.6, 15.8, 14.2, 12.4, 12.2; IR (neat) v 3434, 3124 - 2954, 1742, 1644 cm"1.' Method B: Followed method A for the formation of titled compound 200. The reaction mixture was quenched with 3M hydrochloric acid (ca. 15 mL) and extracted with diethyl ether (3 x 25 mL). The organic extracts were pooled and washed with 5%> sodium bicarbonate solution (3 x 25 mL). The aqueous layers were cooled to 0 °C in an ice-water bath, acidified to pH 2 with 3M hydrochloric acid, and extracted with diethyl ether (5 x 25 mL). The organic extracts were pooled, dried with sodium sulfate (ca. 15 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 25 °C) to a light yellow solid. The crude product was recrystallized from dichloromethane to afford the title compound 200 as a clear crystalline solid (83 mg, 31%>). (Rf = 0.15, (1:1) hexane - ethyl ! acetate mobile phase); (m.p. 142 - 145 °C); H NMR (400 MHz, acetone-d6) 8 5.32 (s, IH), 3.99 (s, 3H), 3.61 (s, 3H), 3.45 (d, J= 12.4 Hz, IH), 2.58 (dq, J= 12.4, 6.7 Hz, IH), 13 1.52 (s, 3H), 1.15 (d, J= 6.7 Hz, 3H); C NMR (100 MHz, acetone-d6) 8 176.9, 168.6, 168.0, 107.7, 106.8, 90.3, 59.7, 54.4, 51.6, 43.1, 25.6, 11.8. 165 Methyl 2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene-4- carboxylate (203) A 50-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and calcium sulfate drying tube, was charged with crude hemiketal 200 (0.75 mmol, 205 mg) dissolved in 2,2-dimethoxypropane (8 mL, 0.1 M)./>-Toluenesulfonic acid (0.08 mmol, 14 mg) was added as a solid in one portion. The reaction was stirred at room temperature for 12 h. The crude reaction mixture was concentrated via rotary evaporation (10 mmHg, 30 °C) to afford the titled compound 203 as a viscous dark brown oil (204 mg). The mixture of diastereomers were separated by flash column chromatography on silica, eluting with a gradient mobile phase of (5:1), (4:1), (3:1) hexane - ethyl acetate. trans-Major ketal 203a was obtained as a white crystalline solid in 92 mg, 45 % (2 steps). l (Rf = 0.21, 3:1 hexane - ethyl acetate); (m.p. 152-153 °C); U NMR (400 MHz, CDC13) 8 5.13 (s, IH), 3.97 (s, 3H), 3.67 (s, 3H), 3.42 (d, J= 12.3 Hz, IH), 3.28 (s, 3H), 3.19 (dq, J = 12.3, 6.7 Hz, IH), 1.50 (s, 3H), 1.14 (d, J = 6.7 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 176.8, 168.9, 168.2, 109.9, 107.1, 90.4, 60.0, 54.2, 52.4, 49.3, 44.3, 19.9, 11.9; IR (neat) v 3000-2850, 1768, 1740, 1646, 1602 cm"1. trans-Minor ketal 203b was obtained as a white crystalline solid in 13 mg, 6% (2 steps). J (Rf =0.18, (3:1) hexane - ethyl acetate); (m.p. 144 - 148 °C); H NMR (400 MHz, CDCI3) 8 5.14 (s, IH), 3.96 (s, 3H), 3.68 (s, 3H), 3.37 (s, 3H), 3.18 - 2.98 (m, 2H), 1.33 13 (s, 3H), 1.14 (d, J= 6.6 Hz, 3H); C NMR (100 MHz, CDC13) 8 175.8, 169.0, 167.5, 166 113.1, 105.5, 90.7, 60.0, 54.9, 52.5, 50.1, 38.3, 21.3, 13.7; IR (neat) v 3126, 2989, 2953, 2837, 1770, 1739, 1645 cm"1. trans-Minor ketal 203c was obtained as an clear oil in 31 mg, 15 %> (2 steps). (Rf = 0.15, ! (3:1) hexane - ethyl acetate); H NMR (400 MHz, CDC13) 8 5.06 (s, IH), 3.84 (s, 3H), 3.66 (s, 3H), 3.56 (d, J= 12.0 Hz, IH), 2.54 (dq, J= 12.0, 6.7 Hz, IH), 1.44 (s, 3H), 1.12 (d, J= 6.7 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 177.1, 169.8, 169.7, 109.7, 108.1, 89.7, 59.7, 56.5, 52.2, 49.5, 47.2, 18.9, 12.1; IR (neat) v 3000-2850, 1772, 1744, 1648 cm" . Isolation of a fourth isomer was observed during the development of this methodology. cis-Minor ketal 203d was obtained as a light yellow solid in 6%> (2 steps). (Rf = 0.09, l (3:1) hexane - ethyl acetate); U NMR (500 MHz, CDC13) 8 5.16 (s, IH), 4.03 (d, J= 7.3 Hz, IH), 3.94 (s, 3H), 3.68 (s, 3H), 3.29 (s, 3H), 2.62 (pentet, J= 7.3 Hz, IH), 1.45 (s, 13 3H), 1.24 (d, J=7.3Hz, 3H); C NMR (126 MHz, CDC13) 8 176.8, 169.4, 168.1, 111.6, 107.2, 90.5, 59.8, 52.0, 50.6, 49.3, 43.9, 17.5, 11.9; IR (neat) 3000 - 2850, 1776, 1743, 1648, 1610 cm"1. (5i?,7if,85,9i?)-9-(Hydroxymethyl)-4,7-dimethoxy-7,8-dimethyl-l,6- dioxaspiro[4.4]non-3-en-2-one (204) Method A: A 20-mL scintillation vial containing carboxylic acid 205 (0.13 mmol, 35.2 mg), equipped with magnetic stir bar, rubber septum, nitrogen gas inlet needle, was charged with anhydrous tetrahydrofuran (2.5 mL). The solution was cooled in an ice- 167 water bath to 0 °C at which time sodium carbonate (0.14 mmol, 20.0 mg) was added as a solid in one portion. The mixture was stirred vigorously while ethyl chloroformate (0.14 mmol, 13.0 uL), dissolved in tetrahydrofuran (0.1 mL), was added dropwise over the course of 1 min. The reaction was stirred while warming to room temperature for 4 h or until TLC analysis indicated consumption of starting material (Rf = 0.08) and evidence for product (Rf = 0.5), eluting with (1:1) hexane-ethyl acetate mobile phase. The crude reaction mixture was vacuum filtered then concentrated via rotary evaporation (10 mmHg, 30 °C) to afford mixed anhydride 214 as a yellow solid (31.3 mg, 70%>) and was carried on without purification. Characteristic proton resonances used to identify mixed 2 anhydride 214: H NMR (400 MHz, CDC13) 8 5.17 (s, IH), 4.35 - 4.26 (m, 2H), 3.98 (s, 3H), 3.55 (d, J= 12.2 Hz, IH), 3.28 (s, 3H), 2.71 (dq, J= 12.2, 6.7 Hz, IH), 1.51 (s, 3H), 1.34 (t,J= 7.3 Hz, 3H), 1.16 (d, J = 6.7 Hz, 3H). A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with mixed anhydride 214 (0.1 mmol, 31.3 mg) in dry THF (1.5 mL). The solution was cooled to 0 °C in an ice-water bath before sodium borohydride (0.36 mmol, 14.0 mg) was added in one portion. The reaction was allowed to warm to room temperature for 2 h. TLC indicated consumption of starting material (Rf = 0.5) and evidence for product formation (Rf = 0.14), eluting with (1:1) hexane-ethyl acetate mobile phase. The reaction was quenched with cautious addition of saturated ammonium chloride (1 mL) and extracted with dichloromethane (3x2 mL). The organic layers were pooled, dried with sodium sulfate (ca. 1 g), gravity filtered, and concentrated in vacuo (10 mmHg, 25 °C) to afford the titled compound 204 as a viscous orange oil (27.0 mg). The crude material was purified by preparative thin layer chromatography on silica by eluting with an ethyl 168 acetate mobile phase (Rf = 0.48). Hydroxymethyl 204 was obtained as a crystalline white solid (15.6 mg, 60%>). For full characterization of titled compound 204 see Method B below. Method B: A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and a nitrogen gas inlet, was charged with carboxylic acid 205 (0.21 mmol, 57 mg) in dry THF (2 mL). Carbonyldiimidazole (0.23 mmol, 37 mg) was added in one portion and the reaction was allowed to stir at room temperature for 1 h. The reaction was cooled to 0 °C in an ice-water bath and sodium borohydride (0.69 mmol, 26 mg) was added in one portion. The reaction was allowed to stir to warm to room temperature for 30 min or until TLC analysis indicated evidence for product (Rf = 0.48), eluting with an ethyl acetate mobile phase. [The acyl imidazole intermediate 215 decomposed rapidly on the silica TLC plate resulting in a streak (Rf = 0.0 - 0.78)]. The reaction was quenched with cautious addition of 1M hydrochloric acid (2 mL) and extracted with ethyl acetate (3x3 mL). The organic layers were pooled, dried with sodium sulfate (ca. 2 g), vacuum filtered, and concentrated in vacuo (10 mmHg, 21 °C) to afforded a viscous yellow oil. The crude oil was purified by preparative thin layer chromatography on silica, eluting with an ethyl acetate mobile phase (Rf = 0.48) to afford hydroxymethyl 204 as a white ! solid (45 mg, 83%). (m.p. 134 - 136 °C); H NMR (500 MHz, CDC13) 8 5.10 (s, IH), 3.93 (s, 3H), 3.71 (br d, J = 5.2 Hz, 2H), 3.29 (s, 3H), 2.64 (dt, J= 12.4, 5.2 Hz, IH), 2.25 (dq, J= 12.4, 6.8 Hz, IH), 1.76 (br s, IH), 1.48 (s, 3H), 1.07 (d, J= 6.8 Hz, 3H); 13C NMR (126 MHz, CDC13) 8 178.2, 169.5, 109.8, 109.6, 89.8, 59.9, 59.1, 50.2, 49.3, 43.7, 20.2, 11.5; IR (neat) v 1365, 2988-2854, 1763, 1644 cm"1. 169 (2/f,35',45',5i?)-2,9-Dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene-4- carboxylic acid (205)109 A 20-mL scintillation vial, equipped with stir bar, rubber septum, and nitrogen gas inlet, was charged with allyl ester 211 (0.81 mmol, 254 mg) in anhydrous tetrahydrofuran (6 mL). Morpholine (8.13 mmol, 0.71 mL) was added via syringe followed by tetrakis(triphenylphospine) Pd(0) (2 mol%, 19 mg), which was added in one portion as a yellow solid. The reaction was stirred for 4 h or until TLC analysis showed starting material (Rf = 0.41) was consumed and product (Rf = 0.19 - 0.0, streak) was observed, eluting with (1:1) hexane - ethyl acetate mobile phase. The solvent was removed via rotary evaporation (10 mmHg, 30 °C) of afford an orange residue that was dissolved in dichloromethane (5 mL). The organic layer was washed with 2 M hydrochloric acid (3 x 5 mL), dried with magnesium sulfate (ca. 2 g), vacuum filtered through a pad of celite, and concentrated (10 mmHg, 25 °C) to afford a orange foam. The crude solid was suspended in dichloromethane (5 mL) and extracted with saturated sodium bicarbonate (3 x 2 mL). The aqueous layer was washed with dichloromethane (5 mL), cooled in an ice- water bath to 0 °C, acidified with dropwise addition of concentrated hydrochloric acid to pH 2, and extracted with ethyl acetate (5x5 mL). The organic layers were pooled, dried with magnesium sulfate (ca. 4 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford the titled compound 205 as a white solid (186 l mg, 84%). (m.p. 178 - 179 °C); H NMR (400 MHz, CDC13) 8 5.16 (s, IH), 3.97 (s, 3H), 3.48 (d,J= 12.3 Hz, IH), 3.26 (s, 3H), 2.64 (dq, J= 12.3, 6.7 Hz, IH), 1.49 (s, 3H), 1.15 13 (d, J= 6.7 Hz, 3H); C NMR (100 MHz, CDC13) 8 176.7, 172.3, 169.5, 110.0, 107.0, 170 90.7, 60.1, 54.0, 49.4, 44.2, 19.9, 11.8; IR (neat) v 3124.1, 2989.4 - 2944.05, 1750.82, 1646.62 cm"1. 2,2-Dimethyl-l,3-dioxane-4,6-dione (Meldrum's acid) (206)110 Into a 250-mL Erlenmeyer flask, equipped with magnetic stir bar, acetic anhydride (430.30 mmol, 40.6 mL, 43.93 g) and malonic acid (337.31 mmol, 35.1 g) were mixed. Concentrated sulfuric acid (18.76 mmol, 1 mL, 1.84 g) was added drop wise to the solution over the course of 3 min via syringe. Acetone (272.90 mmol, 20.0 mL, 15.85 g) was added to the solution and stirred until all the malonic acid was dissolved, approximately 5 min. The flask was sealed with a septum and refrigerated overnight. The precipitate was vacuum filtered through a fritted funnel to collect the white solid, discarding the yellow filtrate. The solid was washed with a minimal amount of ice cold water affording 2,2-dimethyl-l,3-dioxane-4,6-dione 206 as a crystalline white solid J (28.18 g, 58 %). (m.p. 92 - 95 °C, decomposition); H NMR (400 MHz, CDC13) 8 3.84 13 (s, 2H), 1.79 (2, 6H); C NMR (100 MHz, CDC13) 8 163.2, 106.5, 36.4, 27.8; IR (neat) v 2991-2602, 1689, 1434, 1396 cm"1. 5-(l-Hydroxyethylidene)-2,2-dimethyl-l,3-dioxane-4,6-dione (208)m An oven-dried 250-mL round-bottomed flask, equipped with magnetic stir bar, addition funnel, rubber septum, and nitrogen gas inlet, was charged with Meldrum's acid (139.0 mmol, 20.0 g) in anhydrous dichloromethane (96 mL). The solution was cooled to 0 °C in an ice-water bath, at which time pyridine (347.1 mmol, 28.0 mL, 27.4 g) was added via syringe directly to the stirring solution over the course of 10 min. Acetyl 171 chloride (139.0 mmol, 9.8 mL, 10.8 g) in anhydrous dichloromethane (48 mL) was transferred to the addition funnel and then added to the reaction over the course of 2 h at 0 °C. The reaction was allowed to warm to room temp (22 °C) over the course of 12 h at which time the reaction mixture was a deep red slurry. (Note- A second portion of neat acyl chloride (70 mmol, 5.0 mL, 5.4 g) was added to the reaction mixture if the deep red slurry was not observed). The red slurry was diluted with dichloromethane (30 mL), and poured over 3 M hydrochloric acid (100 mL) containing crushed ice. The aqueous layer was extracted with dichloromethane (2 x 30 mL). The organic layers were pooled, washed with 3 M hydrochloric acid (2 x 30 mL), saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate (ca. 20 g), gravity filtered, and concentrated via rotary evaporation (12 mmHg, 30 °C) to afford the title compound 208 as a brown solid (22.0 g, 88%), which did not require further purification, (m.p. 82 - 84 °C); l¥L NMR 13 (400 MHz, CDC13) 8 2.69 (s, 3H), 1.74 (s, 6H); C NMR (126 MHz, CDC13) 8 194.7, 170.3, 160.6, 105.1, 92.0, 27.0, 23.7; IR (neat) v 2986, 2942, 1778, 1731 1660, 1632 1547 1462 cm"1. Allyl 2-hydroxy-9-methoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene-4 carboxylate (210) An oven-dried, one-necked, 250-mL round-bottomed flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with dry dichloromethane (60 mL) and cooled to 0 °C in an ice-water bath. Neat diethylzinc (7.5 mmol, 0.93 g, 0.77 mL) was added in one portion via an oven-dried needle followed by slow addition of allyl 3-oxobutanoate 42c (3 mmol, 0.43 g, 0.41 mL) and stirred for 10 min. 1,1-Diiodoethane 172 (7.5 mmol, 2.12 g, 0.60 mL) was added to the reaction via syringe over a 3 min period and stirred for 0.5 h while warming to room temperature. The reaction was cooled to 0 °C in an ice-water bath before diethylzinc (7.5 mmol, 0.93 g, 0.77 mL) was slowly added and stirred for 10 min. 1,1-Diiodoethane (7.5 mmol, 2.12 g, 0.60 mL) was added via syringe over a 3 min period and the reaction was stirred under a bed of nitrogen for 2 h while allowing to warm to room temperature. TLC indicated consumption of allyl aceto acetate (Rf = 0.61) and evidence for chain extended intermediate (Rf = 0.56), eluting with a 1:1 hexane - ethyl acetate mobile phase. 3-Methoxymaleic anhydride (3.2 mmol, 0.39 g) dissolved in dry dichloromethane (4 mL) was added dropwise via syringe over the course of 5 min and allowed to stir for 18 h at room temperature. The reaction was quenched with 3 M hydrochloric acid (30 mL) and extracted with ethyl acetate (5 x 40 mL). The combined organic layers were dried with sodium sulfate (ca. 20 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford a brown viscous oil (0.86 g, 100%>). The title compound 210 was carried on without further purification. Characteristic proton resonances for the major isomer 210: ]H NMR (500 MHz, CDC13) 8 5.28 (s, IH), 4.00 (s, 3H), 3.48 (d, J= 12.4 Hz, IH), 2.75 - 2.65 (m, IH), 1.61 (s, 3H), 1.21 (d, J = 6.7 Hz, 3H); All carbon resonance for the crude product mixture: 13C NMR (126 MHz, CDCI3) 8 177.7, 177.2, 176.3, 176.1, 169.3, 168.7, 167.5, 167.1, 131.4, 131.2, 119.5, 119.4, 119.2, 110.7, 108.2, 107.9, 107.6, 106.9, 106.0, 90.6, 89.7, 66.5, 66,1, 66.0, 60.1, 56.4, 55.7, 55.0, 54.2, 45.6, 44.1, 42.8, 33.1, 33.0, 27.2, 26.0, 25.6, 24.8, 22.8, 16.7, 14.2, 12.3, 12.2, 10.9, 10.6, 10.5; IR (neat) v 3399, 3124 - 2924, 1742, 1716, 1644, 1455 cm"1. 173 (2R,3S,4S,5R)-Allyl 2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene- 4-carboxylate (211) A 100-mL round-bottomed flask, equipped with a magnetic stir bar, rubber septum, nitrogen gas inlet, and 4A sieves, was charged with crude hemiketal 210 (3 mmol, 0.86 g) in trimethyl orthoformate (30 mL). p-Toluenesulfonic acid (0.05 mmol, 10 mg) was added as a solid in one portion and the reaction was stirred for 4 h or until TLC analysis indicated consumption of hemiketal (Rf = 0.11) and evidence for product (Rf = 0.43), eluting with (1:1) hexane-ethyl acetate mobile phase. The mixture was vacuum filtered through a pad of basic alumina and magnesium sulfate (ca. 2 g) then concentrated via rotary evaporation (10 mmHg, 30 °C) to afford a brown viscous residue (0.94 g). The major diastereomer was isolated by flash column chromatography on silica, eluting with a gradient mobile phase of (10:1), (8:1), (5:1), (4:1) hexane - ethyl acetate. trans-Major diastereomer 211 was obtained as a viscous clear oil 375 mg, 40%> (2 steps); J (Rf =0.15, (4:1) hexane - ethyl acetate); H NMR (500 MHz, CDC13) 8 5.83 (ddt, J = 17.2, 10.5, 5.9 Hz, IH), 5.27 (dq, J= 17.2, 1.5 Hz, IH), 5.23 (dq, J= 10.4, 1.2 Hz, IH), 5.11 (s, IH), 4.56 (m, 2H), 3.96 (s, 3H), 3.44 (d, J= 12.3 Hz, IH), 3.28 (s, 3H), 2.69 (dq, J= 12.2, 6.7 Hz, IH), 1.50 (s, 3H), 1.14 (d, J= 6.7 Hz, 3H); 13C NMR (126 MHz, CDC13) 8 176.7, 168.8, 167.3, 131.6, 119.0, 109.9, 107.1, 90.5, 66.0, 59.8, 54.2, 49.3, 44.2, 19.9, 11.8; IR (neat) v 2989-2838, 1783, 1744, 1647, 1457, 1373 cm"1. 174 (5/?,7/f,85',9if)-7-hydroxy-9-(hydroxymethyl)-4-methoxy-7,8-dimethyl-l,6- dioxaspiro[4.4]non-3-en-2-one (4-ep/-papyracillic acid C) (218) A 20-mL scintillation vial, equipped with magnetic stir bar, was charged with trichloroethyl ketal 221 (0.02 mmol, 7 mg) in 70%) acetic acid (0.5 mL). Freshly activated zinc granular 20 mesh (14 mg) was added in one portion and the heterogeneous reaction was stirred vigorously for 24 h at room temperature. The aqueous reaction mixture was extracted with ethyl acetate (5x1 mL). The organic layers were pooled, dried with sodium sulfate (ca. 1 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford light yellow residue. The crude product was purified by preparative thin layer chromatography on silica, eluting with an ethyl acetate mobile phase (Rf = 0.4). The titled compound 218 was obtained as a clear solid (2 mg, 40%>) as a mixture of isomers. H and C data reported for only the major hemiketal 218 isomer. !H NMR (500 MHz, CDC13) 8 5.11 (s, IH), 3.93 (s, 3H), 3.74 (d, J= 5.1 Hz, 2H), 2.61 (dt, J = 12.6, 5.4 Hz, IH), 2.28 (dq, J= 12.6, 6.7 Hz, IH), 1.58 (s, 3H), 1.12 (d, J= 6.8, 3H); 13 C NMR (126 MHz, CDC13) 8 177.5, 169.3, 109.1, 107.8, 89.9, 60.0, 59.1, 50.4, 42.4, 26.2, 11.9; IR (neat) 3380, 3125,2925,2854, 1750, 1641, 1456 cm"1. (2R,3S,4S,5R)-Allyl 9-methoxy-2,3-dimethyl-7-oxo-2-(2,2,2-trichloroethoxy)-l,6- dioxaspiro [4.4] non-8-ene-4-carboxylate (219) A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and calcium sulfate drying tube, was charged with crude hemiketal 210 (0.53 mmol, 0.15 g), 2,2,2- trichloroethanol (0.80 mmol, 0.12 g, 0.08 mL), and toluene (5 mL).^-TsOH (0.05 mmol, 0.01 g) was added in one portion as a solid and the reaction was stirred at room 175 temperature for 48 h. The reaction was concentrated via rotary evaporation (10 mmHg, 40 °C) affording a black viscous oil. The crude residue was purified by preparative thin layer chromatography on silica, eluting with a (1:1) hexane - ethyl acetate mobile phase (Rf = 0.57). The titled compound 219 was obtained as a white crystalline solid (68 mg, l 30%). (m.p. 104 - 106 °C); H NMR (500 MHz, CDC13) 8 5.83 (ddt, J= 17.2, 10.4, 6.0 Hz, IH), 5.29 (dq, J= 17.2, 1.5 Hz, IH), 5.24 (dq, J= 10.4, 1.2, IH), 5.24 (s, IH), 4.63 - 4.52 (m, 2H), 4.15, 4.06 (ABq, JAB = 10.4 Hz, 2H), 3.95 (s, 3H), 3.56 (d, J = 12.3 Hz, IH), 2.79 (dq, J= 12.3, 6.7 Hz, IH), 1.60 (s, 3H), 1.26 (d, J = 6.7 Hz, 3H); 13C NMR (126 MHz, CDCI3) 8 176.1, 168.5, 166.9, 131.5, 119.3, 109.9, 107.3, 96.7, 90.7, 74.5, 66.2, 59.8, 54.0, 44.6, 20.8, 11.8; IR(neat) v 3125, 2988-2884, 1787, 1746, 1650 cm"1. (2^,35',45,5i?)-9-Methoxy-2,3-dimethyl-7-oxo-2-(2,2,2-trichloroethoxy)-l,6- dioxaspiro[4.4]non-8-ene-4-carboxylic acid (220) A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with allyl ester 219 (0.76 mmol, 324 mg) in dry tetrahydrofuran (6 mL). Morpholine (7.6 mmol, 0.67 mL) was added in one portion via syringe and the reaction was purged with nitrogen. Tetrakis(triphenylphospine) Pd(0) (2 mol%>, 18 mg) was added in one portion as a solid and the reaction was stirred for 3 h at room temperature under nitrogen. A solution of 1M hydrochloric acid (6 mL) was added and extracted with ethyl acetate (4x8 mL). The organic layers were pooled, washed with 1 M hydrochloric acid (2x6 mL), dried with sodium sulfate (ca. 5 g), vacuum filtered, and concentrated in vacuo (10 mmHg, 30 °C) to afford a light brown crystalline solid. The solid was suspended in dichloromethane (ca. 15 mL) and washed with saturated 176 sodium bicarbonate (3x10 mL). The aqueous layer was cooled in an ice-water bath to 0 °C, acidified with dropwise addition of cone, hydrochloric acid to pH 2, and extracted with ethyl acetate (5 x 20 mL). The organic layers were pooled, dried with sodium sulfate (ca. 15 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford the titled compound 220 as a white solid (275 mg, 93%>). (m.p. 196 - 198, r decomposition); H NMR (500 MHz, CDC13) 8 5.18 (s, IH), 4.15, 4.06 (ABq, JAB = 10.4 Hz, 2H), 3.96 (s, 3H), 3.58 (d, J= 12.3 Hz, IH), 2.74 (dq, J= 12.3, 6.7 Hz, IH), 1.60 (s, 3H), 1.27 (d, J = 6.7 Hz, 3H); 13C NMR (126 MHz, CDC13) 8 176.0, 171.7, 169.1, 109.9, 107.2, 96.6, 90.9, 74.5, 59.9, 53.7, 44.6, 20.7, 11.7; IR (neat) v 3130, 3005 - 2877, 1748, 1725,1648, 1454 cm"1. (5JR,7JR,85,9/?)-9-(Hydroxymethyl)-4-methoxy-7,8-dimethyl-7-(2,2,2- trichloroethoxy)-l,6-dioxaspiro[4.4]non-3-en-2-one (221) A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with carboxylic acid 220 (0.60 mmol, 223 mg) in tetrahydrofuran (6 mL). Carbonyldiimidazole (0.63 mmol, 102 mg) was added as a solid in one portion and stirred at room temperature for 2 h. The reaction was cooled to 0 °C in an ice-water bath, sodium borohydride (1.98 mmol, 75 mg) was added in one portion as a solid, and the reaction was stirred for 30 min. TLC analysis indicated full consumption of the carboxylic acid starting material (Rf = 0.0 - 0.2, streak) and production of product (Rf = 0.75) eluting with an ethyl acetate mobile phase. A solution of 1 M hydrochloric acid (6 mL) was cautiously added at 0 °C and extracted with ethyl acetate (3x8 mL). The organic layers were pooled, washed with saturated sodium bicarbonate (2x8 mL), dried 177 with sodium sulfate (ca. 3 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford a the titled compound 221 white foam (158 mg, 71%), which ! did not require further purification. H NMR (500 MHz, CDC13) 8 5.11 (s, IH), 4.16, 4.07 (ABq, JAB = 10.4 Hz, 2H), 3.91 (s, 3H), 3.73 (broad d, J= 6.1 Hz, 2H), 2.79 (dt, J =11.6, 6.1 Hz, IH), 2.29 (dq, J= 11.6, 6.7 Hz, IH), 1.57 (s, 3H), 1.17 (d, J= 6.7 Hz, 3H); 13C NMR (126 MHz, CDC13) 8 127.7, 169.5, 109.8, 109.5, 96.9, 89.7, 74.5, 59.7, 59.0, 49.9, 44.3, 21.0, 11.3; IR (neat) 3456, 3127, 2987, 2939, 2882, 1758, 1644, 1456 cm"1. (5i?,7i?,8/?)-4,7-dimethoxy-7,8-dimethyl-9-methylene-l,6-dioxaspiro[4.4]non-3-en-2- one (4-epi-papyracillic acid B) (225) and (5S,7i?,8S)-4,7-dimethoxy-7,8-dimethyl-9- methylene-l,6-dioxaspiro[4.4]non-3-en-2-one (papyracillic acid B) (130) A 5-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and thermalcouple, was charged with selenide (235) (0.06 mmol, 24 mg) in dichloromethane (0.4 mL). The solution was cooled to 0 °C in an ice-water bath and charged with a solution of 30%o hydrogen peroxide (14.3 ul) and water (12.3 pi). The ice-water bath was removed and the reaction was allowed to stir for 15 min at room temperature then cooled to 0 °C and stirred for an additional 15 min. The reaction mixture was diluted with dichloromethane (1 mL) and washed with saturated sodium bicarbonate (2x1 mL). The organic layer was dried with sodium sulfate (ca. 1 g), vacuum filtered, and concentrated (40 mmHg, 21 °C) to a light orange residue. The residue was suspended in toluene and stirred for 18 h at room temperature then heated slowly to 110 °C over the course of 6 h. The solvent was removed in vacuo (10 mmHg, 35 °C) to afford a viscous orange oil (22 mg). The crude residue was purified by preparative thin layer chromatography on silica, 178 eluting with hexane followed by (1:1) hexane - ethyl acetate (Rf = 0.71), to afford a 1:1 mixture of 4-epz-papyracillic acid B (225) and papyracillic acid B (130) as a viscous clear ! oil (10 mg, 70%). H NMR (500 MHz, CDC13) 4-ep/-papyracillic acid B (225) 8 5.22 (s, IH), 5.13 - 5.11 (m, 2 H), 3.93 (s, 3H), 3.29 (s, 3H), 2.89 - 2.83 (m, IH), 1.56 (s, 3H), 1.16 (d, J= 6.8 Hz, 3H); papyracillic acid B (130) 8 5.22 - 5.18 (m, 2H), 5.07 (s, IH), 3.87 (s, 3H), 3.31 (s, 3H), 2.71 - 2.66 (m, IH), 1.51 (s, 3H), 1.16 (d,J= 6.8 Hz, 3H); All 13 carbon resonances for 225 and 130 C (500 MHz, CDC13) 8 178.2, 176.7, 170.3, 169.1, 148.8, 148.5, 110.0, 109.4, 109.4, 109.0, 107.6, 107.2, 91.3, 88.6, 59.8, 59.7, 49.6, 49.5, 48.8, 46.2, 20.1, 19.0, 10.7, 9.9. IR (neat) v 2923, 2853, 1767, 1642, 1455 cm"1. ((2i?,35',4i?,5/?)-2,9-Dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-en-4- yl)methyl methanesulfonate (228) A 20-mL scintillation vial, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with hydroxymethyl 204 (0.17 mmol, 44 mg) in pyridine (2 mL). Methanesulfonyl chloride (0.15 mmol, 22 mg) was added dropwise and the reaction was stirred at room temperature for 2 h. A second portion of methanesulfonyl chloride (0.15 mmol, 22 mg) was added to the reaction mixture then stirred for an additional 2h. The reaction was cooled to 0 °C in an ice-water bath then quenched with 1 M hydrochloric acid (4 mL) and extracted with ethyl acetate (3x2 mL). The organic layers were combine, dried with sodium sulfate (ca. 2 g), vacuum filtered, and concentrated to a viscous yellow oil (56 mg, 95%). The title compound 228 was carried on without further J purification. H NMR (500 MHz, CDC13) 8 5.10 (s, IH), 4.32 (dd, 7 = 10.2, 4.7 Hz, IH), 4.21 (app t, J= 9.9 Hz, IH), 3.94 (s, 3H), 3.29 (s, 3H), 2.99 (m, IH), 2.95 (s, 3H), 2.10 179 (dq, J= 12.5, 6.8 Hz, IH), 1.47 (s, 3H), 1.09 (d, J= 6.8 Hz, 3H); 13C NMR (126 MHz, CDC13) 8 177.9, 169.4, 109.6, 107.7, 89.6, 67.6, 59.9, 49.3, 47.8, 44.9, 37.2, 20.0, 11.5; IR (neat) v 2979-2851, 1769, 1646, 1455, 1376 cm"1. (5i?,7JR,85,95)-9-(Iodomethyl)-4,7-dimethoxy-7,8-dimethyl-l,6-dioxaspiro[4.4]non-3- en-2-one (229) An oven-dried 50-mL round-bottomed flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with mesylate 228 (0.17 mmol, 57 mg) in anhydrous acetone (6 mL). Sodium iodide (2.55 mmol, 0.39 g) was added as a solid in one portion. The reaction was refluxed for 48h or until TLC analysis indicated consumption of starting material (Rf = 0.57) and development of product (Rf = 0.48) eluting with (1:1) hexane - ethyl acetate mobile phase. Reaction mixture was quenched with water (ca. 12 mL) and extracted with diethyl ether (3x12 mL). The organic layers were combined, dried with magnesium sulfated (ca. 5 g), vacuum filtered, and concentrated in vacuo (10 mmHg, 21 °C) to provide a yellow oil. The crude oil was purified by preparative thin layer chromatography on silica, eluting with a (1:1) hexane - ethyl acetate mobile phase (Rf = 0.48) to afford the titled compound 229 as a light yellow l solid (20 mg, 32%). (m.p. 92 - 94 °C); K NMR (500 MHz, CDC13) 8 5.13 (s, IH), 3.96 (s, 3H), 3.28 (s, 3H), 3.22 - 3.10 (m, 2H), 2.90 (ddd, J= 12.2, 10.3, 4.0 Hz, IH), 2.00 (dq, J= 12.2, 6.8 Hz, IH), 1.45 (s, 3H), 1.08 (d, J= 6.8 Hz, 3H); 13C NMR (126 MHz, CDCI3) 8 177.4, 169.6, 108.7, 108.4, 91.3, 59.7, 51.0, 49.3, 48.6, 20.2, 11.5, 2.8; IR (neat) v 3116, 2971 - 2837, 1768, 1646 cm"1. 180 (5i?,77f,85,95)-4,7-dimethoxy-7,8-dimethyl-9-((phenylseIanyl)methyl)-l,6- dioxaspiro [4.4] non-3-en-2-one (235) A 5-mL round-bottomed flask, equipped with magnetic stir bar, reflux condenser, rubber septum, and nitrogen gas inlet needle, was charged with diphenyl diselenide (0.13 mmol, 41 mg) in ethanol (1 mL). Sodium borohydride (0.15 mmol, 5.7 mg) was added cautiously as a solid and the reaction was refluxed for 30 min at which time the solvent was removed via rotary evaporation (10 mmHg, 25 °C). The resulting light yellow solid was charged with mesylate 228 (0.12 mmol, 40 mg) in tetrahydrofuran and stirred for 5 h at room temperature. TLC indicated a mixture of selenide (Rf = 0.61) and mesylate (Rf = 0.26), eluting with (1:2) hexane - ethyl acetate mobile phase. A second batch of phenyl selenide anion (0.13 mmol, 41 mg) was prepared following the same procedure described above. The reaction mixture was transferred directly to the flask containing the second portion of phenyl selenide anion and allowed to stir at room temperature for 18 h. The solvent was removed via rotary evaporation (10 mmHg, 25 °C) and the residue was partitioned between ethyl acetate (1 mL) and water (1 mL) then the aqueous layer was extracted with ethyl acetate (3x1 mL). The organic layers were pooled, dried with sodium sulfate (ca. 1 g), vacuum filtered, and concentrated to an orange viscous oil. The crude residue was purified by preparative thin layer chromatography on silica, eluting with a (1:2) hexane - ethyl acetate mobile phase (Rf = 0.61). The titled compound 235 J was obtained as an off-white oil (30 mg, 64%). H NMR (500, CDC13) 8 7.46 - 7.36 (m, IH), 7.29 - 7.23 (m, 3H), 5.08 (s, IH), 3.88 (s, 3H), 3.25 (s, 3H), 3.00 (dd, .7= 12.6, 4.2 Hz, IH), 2.91 (dd, J= 12.6, 9.6 Hz, IH), 2.76 (ddd, J= 12.1, 9.6, 4.2 Hz, IH), 2.07 (dq, J 13 = 12.1, 6.8 Hz, IH), 1.45 (s, 3H), 1.04 (d, J= 6.8 Hz, 3H); C NMR (126 MHz, CDC13) 181 8 177.7, 169.7, 132.5, 131.6, 129.3, 127.2, 109.2, 108.9, 90.7, 59.6, 49.3, 48.8, 48.2, 23.9, 20.4, 11.5; IR(neat) 3124, 2985, 2940, 2834, 1769, 1645, 1579, 1477 cm"1. Benzyl-(45)-((^rf-butoxycarbonyl)amino)-3-oxopentanoate (255a) An oven-dried 50-mL round-bottom flask, equipped with magnetic stir bar and nitrogen gas inlet, was charged with jV-(fert-butoxycarbonyl)-L-alanine 254a (3.0 mmol, 0.57 g) and carbonyldiimidizole (3.3 mmol, 0.54 g) in anhydrous tetrahydrofuran (20 mL). The solution was stirred under nitrogen until bubbling stopped, approximately 15 min. A separate oven-dried 100-mL round-bottom flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with freshly prepared monobenzyl malonate 257a (1.7 g, 9 mmol) and dissolved in anhydrous tetrahydrofuran (40 mL). The solution was cooled to 0 C before adding dibutylmagnesium (4.5 mmol, 4.5 mL, IM in heptanes), stirred until clear, and allowed to warm to room temperature. The acyl imidazole solution was transferred via syringe into the magnesium malonate solution. The reaction was stirred for 24 h or until TLC analysis showed amino acid starting material (Rf = 0.15 - 0.0, streak) was consumed and evidence for product was observed (Rf = 0.73). The crude reaction mixture was quenched with saturated ammonium chloride solution (ca. 40 mL) and extracted with diethyl ether (3 x 100 mL). The organic layer was dried with sodium sulfate, gravity filtered, and concentrated to a viscous yellow oil. The crude residue was purified by flash column chromatography on silica, eluting with 15:2 hexane-ethyl acetate, to yield the titled compound 255a a colorless oil (0.78 g, l 81 %). H NMR (400 MHz, CDC13) 8 7.39 - 7.31 (m, 5H), 5.18, 5.16 (ABq, JAB = 12.2 Hz, 2H), 5.07 (br s, IH), 4.38 - 4.34 (m, IH), 3.63, 3.58 (ABq, JAB = 16.0 Hz, 2H), 1.44 182 lj (s, 9H), 1.33 (d, J= 7.2 Hz, 3H); C NMR (100 MHz, CDC13) 8 202.4, 166.9, 155.4, 135.4, 128.8, 128.7, 128.6, 80.4, 67.5, 55.6, 46.0, 28.5, 17.2. IR (neat) v 2978, 1713, 1639, 1499 cm"1. ^^-Butyl-(25)-(3-(benzyloxy)-3-oxopropanoyl)pyrrolidine-l-carboxylate (255b) Using the same procedure as described for the conversion of ./V-Boc-L-alanine 254a to P- keto ester 255a, titled compound 255b was isolated from N-Boc-L-proline 254b as an off-white solid in 65 % (Rf = 0.60, purified by column chromatography on silica, 15:2 ! hexane-ethyl acetate mobile phase); H NMR (400 MHz, CDC13) rotomer 1:87.38- 7.30 (m, 5H), 5.18 (s, 2H), 4.29 - 4 .25 (m, IH), 3.68 - 3.38 (m, 4H), 2.20 - 1.81 (m, 4H), 13 1.30 (s, 9H); rotomer 2: 8 4.38 (br q, IH), 1.45 (s, 9H); C NMR (100 HMz, CDC13) 8 202.9, 202.6, 167.2, 166.9, 154.9, 153.8, 135.5, 135.3, 128.6, 128.5, 128.4, 80.8, 80.2, 67.2, 67.1, 65.6, 65.1, 47.0, 46.8, 46.4, 45.3, 29.8, 28.5, 28.3, 24.5, 23.8. IR (neat) v 2959 -2856,1747,1690 cm"1. Benzyl-(4S)-(methoxycarbonylamino)-3-oxo-5-phenylpentanoate (255c) Using the same procedure as described for the conversion of iV-Boc-L-alanine 254a to P- keto ester 255a, titled compound 255c was isolated from N-(methoxycarbonyl)-L- phenylalanine 254c as a colorless oil in 63%> (Rf = 0.18, purified by column chromatography on silica, eluting with 15:2 hexane - ethyl acetate mobile phase); lH NMR (400 MHz, CDC13) 8 7.38 - 7.32 (m, 5H), 7.27 - 7.11 (m, 5H), 5.27 (br d, IH), 5.17 - 5.11 (m, 2H), 4.62 (br q, IH), 3.61 (s, 3H), 3.57 - 3.46 (m, 2H), 3.12 (dd, J= 14.1, 13 6.1 Hz, IH), 2.95 (dd, J= 14.1, 7.3 Hz, IH); C NMR (100 MHz, CDC13) 8 201.7, 183 166.8, 156.7, 136.0, 135.4, 129.4, 129.0, 128.8, 128.7, 128.6, 128.4, 67.5, 61.0, 52.7, 47.2, 37.1. IR (neat) v 3030-2958, 1705, 1604, 1528 cm"1. 3-Benzyloxy-3-oxopropanoic acid (monobenzyl malonate) (257a) A 100-mL oven-dry round-bottom flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged anhydrous toluene (50 mL). 2,2-Dimethyl- l,3-dioxane-4,6-dione 206 (12.5 g, 86.73 mmol) was added in one portion as a solid followed by the addition of benzyl alcohol (17.5 mL, 168.5 mmol) via an oven dried syringe. The solution was refluxed for 24 h at 106 °C. The reaction mixture was concentrated to half the initial volume and partitioned between diethyl ether (100 mL) and 5 %> aqueous sodium carbonate (175 mL). The aqueous layer was additionally washed with diethyl ether (2 X 100 mL) then acidified to approximately pH 2 with IN hydrochloric acid. The acidified aqueous solution was extracted with ethyl acetate (5 x 75 mL). The organic layers were combined, dried with magnesium sulfate (ca. 10 g), and concentrated by rotary evaporation (10 mmHg, 30 °C) to a viscous cloudy oil. The oil was put on the high vacuum pump (0.4-0.0 mmHg) for approximately 2 h to afford title compound 257a as a crystalline white solid (10.40 g, 62%). (m.p. 46 - 50 °C); !H NMR 13 (400 MHz, CDC13) 8 7.40-7.32 (m, 5H), 5.20 (s, 2H), 3.48 (s, 2H); C NMR (100 MHz, CDCI3) 8 171.9, 166.8, 135.2, 128.9, 128.7, 128.6, 67.9, 41.1; IR (neat) v 3025, 2960, 2947, 1713, 1710, 1497, 1455, 1431, 1402 cm"1. 184 3-Ethoxy-3-oxopropanoic acid (monoethyl malonate) (257b) Followed the procedure for the formation of monobenzyl malonate 257a, replacing the benzyl alcohol with absolute ethanol. Monoethyl malonate 257b was obtained as a clear ! oil (9.5 g, 65%), which did not require further purification. H NMR (400 MHz, CDC13) 8 13 10.88 (br s, IH), 4.24 (q, 2H), 3.45 (s, 2H), 1.30 (t, 3H); C NMR (100 MHz, CDC13) 8 171.7, 167.1,62.1,41.1, 14.0; IR (neat) v 3200, 2987, 1709, 1469 cm"1. Benzyl-(5iS)-((terf-butoxycarbonyl)amino)-3-methyl-4-oxohexanoate (258a and 258b) An oven-dried 100-mL round-bottomed flask, equipped with magnetic stir bar and nitrogen gas inlet, was charged with L-alanine-derived P-keto ester 255a (0.32 g, 1.0 mmol) and suspended in anhydrous dichloromethane (20 mL). The solution was cooled to 0 °C then diethylzinc (5 mmol, 5 mL, 1 M in hexanes) was added via an oven-dried needle dropwise over the course of 3 min. The reaction was stirred for 10 min. 1,1- Diiodoethane (5 mmol, 0.4 mL) was added to the solution and allowed to stir for 2 h. The crude reaction was quenched with cautious addition of saturated ammonium chloride (ca. 20 mL) and extracted with dichloromethane (3 x 25 mL). The organic layers were pooled, dried with sodium sulfate (ca. 10 g), and concentrated via rotary evaporation (lOmmHg, 21 °C) to afford a 1:1 ratio of diastereomers, as determined by the integration of the *H spectra of the crude reaction mixture, as a light yellow oil. The diastereomers were separated by flash column chromatography on silica, eluting with (20:1) hexane - ethyl acetate. 185 Diastereomer 258a was obtained as a colorless oil in 110 mg, 32%) (Rf = 0.15). 'H NMR (400 MHz, CDC13) 8 7.41 - 7.28 (m, 5H), 5.22 (br d,J= 6.6 Hz, IH), 5.09, 5.06 (ABq, JAB = 12.3 Hz, 2H), 4.53 (m, IH), 3.22 (m, IH), 2.90 (dd, J= 17.1, 9.0 Hz, IH), 2.41 (dd, J= 17.1, 4.8 Hz, IH), 1.43 (s, 9H), 1.34 (d, J= 7.2 Hz, 3H), 1.19 (d, J= 7.3 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 211.5, 172.0, 155.2, 135.8, 128.7, 128.4, 128.3, 79.7, 66.5, 53.3, 38.9, 36.5, 28.5, 18.1, 17.2; IR (neat) v 3370, 3067, 3034, 2977, 2935, 2881, 1728, 1705 cm"1. Diastereomer 258b was obtained as a colorless oil in 80 mg, 24%> (Rf = 0.11). 2H NMR (400 MHz, CDCI3) 8 7.42 - 7.28 (m, 5H), 5.29 (br d, J= 8.8 Hz, IH), 5.11, 5.06 (ABq, JAB = 12.3 Hz, 2H), 4.60 - 4.41 (m, IH), 3.35 - 3.24 (m, IH), 2.86 (dd, J= 16.9, 8.8 Hz, IH), 2.41 (dd, J= 17.1, 4.8 Hz, IH), 1.45 (s, 9H), 1.32 (d, J= 7.1 Hz, 3H), 1.12 (d, J = 13 7.0 Hz, 3H). C NMR (100 MHz, CDC13) 8 212.5, 172.0, 155.3, 135.7, 128.7, 128.5, 128.4, 79.8, 66.7, 54.4, 38.2, 38.2, 28.5, 17.4, 16.9; IR (neat) v 3367, 2978, 2935, 1709, 1499 cm"1. N-(tert-Butoxycarbonyl)-L-serine (266)" L-Serine (95.16 mmol, 10.0 g), dissolved in 1 N sodium hydroxide (211 mL), was stirred in a 500-mL round bottom flask, which was equipped magnetic stir bar and an addition funnel. The solution was cooled to 0 °C before di-tert-h\xtyl dicarbonate (114.2 mmol, 24.4 mL) in dioxane (106 mL) was added dropwise through the addition funnel. The biphasic solution was stirred vigorously at ca. 5 °C for 30 min then allowed to warm to room temperature for 4 h. The reaction was monitored by TLC analysis by eluting with 186 (8:1:1) rc-butanol-water-acetic acid, until the starting material was completely consumed from the base line and the product was formed (Rf = 0.63). The crude reaction was concentrated to remove all volatile components, cooled in an ice-water bath, acidified by slow addition of 1 N potassium bisulfate to pH 3, and then extracted with ethyl acetate (5 x 100 mL). The organic layers were pooled, dried with magnesium sulfate, vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C). The residue was dried overnight on a high vacuum line (0.4 mmHg) to yield 7V-(fert-butoxycarbonyl)-L- serine 266 as a sticky clear foam (17.14 g, 88 %>), which was carried on without further ! purification. H NMR (400 MHz, acetone-d6) 8 5.95 (br d, J= 8.2 Hz, IH), 4.26 (m, IH), 3.94 (dd, J= 11.1, 4.4 Hz, IH), 3.85 (dd, J= 11.1, 3.9 Hz, IH), 1.42 (s, 9H); 13C NMR (100 MHz, acetone-dg) 8 172.4, 156.2, 79.3, 63.0, 56.5, 28.4; IR (neat) v 3391, 2979, 2935,1689 cm"1. Af-(terf-ButoxycarbonyI)-0-benzyl-L-serine (267)100 An oven-dried 100-mL round-bottomed flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with dry dimethylformamide (150 mL). The solution was cooled to 0 °C in an ice-water bath before adding sodium hydride (52.6 mmol, 2.2 g, 60 %> suspension), which has been washed with hexanes (5x10 mL), as a white powdery solid in one portion. JV-(tert-Butoxycarbonyl)-L-serine 266 (23.9 mmol, 4.90 g), dissolved in dry dimethylformamide (5 mL), was added slowly via an oven-dried syringe in order to maintain a controlled evolution of hydrogen gas. The resulting foamy mixture was stirred for 20 min, then benzyl bromide (26.3 mmol, 3.2 mL, 4.58 g) was added drop wise via an oven-dried syringe over the course of 3 min. The reaction was 187 stirred for 16 h at which time TLC analysis indicated full consumption of amino acid starting material at the base line and formation of product (Rf = 0.59), eluting with a (1:1) hexane-ethyl acetate solvent system. The solvent was removed by rotary evaporation (10 mmHg, 35 °C) and the yellow residue was dissolved in water (50 mL) and extracted with diethyl ether (2 x 25 mL). The aqueous solution was acidified to pH 3 with 3 M hydrochloric acid and extracted with ethyl acetate (5 x 40 mL). The organic extracts were combined, dried with sodium sulfate (ca. 20 g), gravity filtered, and concentrated by rotary evaporation (10 mmHg, 35 °C) to half the original volume (ca. 100 mL). The organic layer was washed with 5%> LiCl solution (3 X 50 mL), dried with sodium sulfate (ca. 10 g), gravity filtered and concentrated via rotary evaporation (10 mmHg, 35 °C) to afford a light yellow oil. The viscous oil was put on a high vacuum pump (0.4 mmHg) overnight or until JV-(ter/-butoxycarbonyl)-0-benzyl-L-serine (267) was obtained as an off white solid (3.47 g, 50 %>). (m.p. 57 - 59 °C) The product was used without further : purification. H NMR (400 MHz, acetone-d6) 8 7.49 - 7.17 (m, 5H), 6.03 (br d, J= 8.2 Hz, IH), 4.56, 4.54 (ABq, JAB = 12.1 Hz, 2H), 4.41 (m, IH), 3.91 (dd, J= 9.6, 4.5 Hz, 13 IH), 3.77 (dd, J= 9.6, 3.7 Hz, IH), 1.41 (s, 9H); C NMR (100 MHz, acetone-d6) 8 171.5, 155.6, 138.5, 128.4, 127.7, 127.7, 78.8, 72.9, 70.2 54.1, 27.9; IR (neat) v 3264, 2984-2850, 1735, 1647 cm"1. Ar-(ter^-Butoxycarbonyl)-0-(te/"f-butyldimethylsilyl)-L-serine (268)101 An oven-dried 50-mL round-bottom flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with N-(ter/-butoxycarbonyl)-L-serine 266 (9.75 mmol, 2.0 g) in dry dimethylformamide (5 mL). Imidazole (46.8 mmol, 3.19 g) and 188 te^butyldimethylsilyl chloride (23.4 mmol, 3.50g) were sequentially added as solids in one portion at room temperature and the reaction was stirred for 16 h. The reaction mixture was diluted with toluene (100 mL) and ethyl acetate (100 mL), then washed with 10 % aqueous citric acid (100 mL), saturated sodium bicarbonate (100 mL), water (100 mL), and saturated sodium chloride (100 mL). The organic layer was dried with sodium sulfate (ca. 20 g), gravity filtered into a 250-mL round-bottom flask, and concentrated by rotary evaporation (10 mmHg, 30 °C) to produce a viscous residue, which was suspended in methanol (50 mL). Potassium carbonate (24.0 mmol, 3.30 g), dissolved in water (30 mL), and a magnetic stir bar were added to the flask and the reaction mixture was readily stirred for 4 h at room temperature. The reaction was concentrated via rotary evaporation (10 mmHg, 30 °C) until all the volatiles were removed, and the crude residue was diluted with water (50 mL). The aqueous solution was cooled to 0 °C in an ice-water bath, acidified to pH 4 with 10 %> citric acid, extracted with ethyl acetate (5 x 50 mL), dried with sodium sulfate (ca. 20 g), gravity filtered, and concentrated (10 mmHg, 30 °C) to give 7V-(terf-butoxycarbonyl)-0-(fcr^-butyldimethylsilyl)-L-serine 268 as a viscous light yellow oil (2.30 g, 90 %»). The product was used without further purification. lH NMR (500 MHz, acetone-d6) 8 5.75 (br d, J= 8.4 Hz, IH), 4.26 (m, IH), 4.04 (dd, J= 10.2, 3.6 Hz, IH), 3.90 (dd, J= 10.2, 3.6 Hz, IH), 1.40 (s, 9H), 0.86 (s, 9H), 0.05 (s, 3H), 0.05 (s, 13 3H); C NMR (126 MHz, acetone-d6) 8 176.7, 160.7, 84.0, 68.9, 61.0, 33.2, 30.8, 23.4, 0.7, -0.8; IR (neat) v 3434, 2956, 2931, 2886, 2858, 1703, 1504 cm"1. 189 Af-(fert-Butoxycarbonyl)-L-serine methyl ester (269) An oven-dried 100-mL round-bottomed flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with iV-(fer?-butoxycarbonyl)-L-serine 266 (48.7 mmol, 10.0 g) in dry dimethylformamide (44 mL). The solution was cooled to 0 °C in an ice-water bath, then anhydrous potassium bicarbonate (53.6 mmol, 7.4 g) was added in one portion. The solution was vigorously stirred for approximately 10 min before adding methyl iodide (97.4 mmol, 6.1 mL, 13.9 g) in one portion via an oven-dried syringe. The solution was again stirred for 30 min at 0 °C at which point the mixture solidified. The solidified reaction mixture was allowed to warm to room temperature for approximately 1 h while monitoring by TLC, eluting with (1:1) hexane-ethyl acetate, until the starting material was consumed from the base line and product was formed (Rf = 0.38). The crude reaction was vacuum filtered, then partitioned between ethyl acetate (50 mL) and water (50 mL). The organic layer was washed with saturated sodium chloride (2 X 50 mL), dried with magnesium sulfate (ca. 10 g), vacuum filtered, and concentrated on the rotary evaporator to provide iV-(ter/-butoxycarbonyl)-L-serine methyl ester 269 as a light amber oil (7.42g, 70 %>), which was used without further purification. [H NMR (500 MHz, CDC13) 8 5.57 (br d, J= 7.3 Hz, IH), 4.42 - 4.34 (m, IH), 3.96 (dd, J= 11.1, 3.4 Hz, IH), 3.88 (dd, J= 11.1, 3.6 Hz, IH), 3.78 (s, 3H), 1.45 (s, 9H); 13CNMR(126 MHz, CDCI3) 8 171.5, 155.9, 80.4, 63.5, 55.8, 52.7, 28.4; IR (neat) v 3376, 2977, 2975, 1749, 1710, 1661, 1505 cm"1. 190 Ar-(fer^-Butoxycarbonyl)-0-(methoxymethoxy)-L-serine methyl ester (270) An oven-dried 50-mL round-bottomed flask, equipped with a magnetic stir bar, oven- dried addition funnel, and nitrogen gas inlet adaptor, was charged with N-(tert- butoxycarbonyl)-L-serine methyl ester 269 (22.8 mmol, 5.0 g), dry dichloromethane (15 mL) and hexane (22 mL). The solution was cooled to 0 °C in an ice-water bath before diisopropylethylamine (45.6 mmol, 7.8 mL, 5.89 g) was added dropwise directly to the stirring solution via an oven-dried syringe. Methyl chloromethyl ether (45.6 mmol, 3.5 mL, 3.71 g) and dry dichloromethane (7 mL) was added to the addition funnel and slowly dropped into the stirring 0 °C reaction mixture over the course of 20 min. The reaction was monitored by TLC, eluting with (1:1) hexane-ethyl acetate solvent system, until full consumption of amino acid starting material (Rf = 0.38) was observed and conversion to product was observed (Rf = 0.5). The reaction mixture was diluted with water (10 mL) and ethyl acetate (100 mL). The organic layer was washed with 1 M hydrochloric acid (15 mL), 1 M sodium bicarbonate (15 mL), saturated sodium chloride (15 mL), dried with sodium sulfate (ca. 20 g), gravity filtered, and concentrated by rotary evaporation (10 mmHg, 30 °C) to afford A^-(ter^-butoxycarbonyl)-0-(methoxymethoxy)-L-serine methyl ester 270 as a viscous orange oil. The resulting oil was further dried on a high vacuum line (0.4 mmHg) to produce a sticky orange solid (4.8 g, 80 %>). lH NMR (400 MHz, acetone-de) 8 6.11 (br d, J= 8.6 Hz, IH), 4.58, 4.57 (ABq, JAB = 6.6 Hz, 2H), 4.39 (m, IH), 3.92 (dd, J= 10.1, 4.7 Hz, IH), 3.79 - 3.74 (m, IH), 3.72 (s, 3H), 3.29 (s, 3H), 13 1.42 (s, 9H); C NMR (100 MHz, CDC13) 8 171.0, 155.5, 96.5, 78.8, 67.7, 54.7, 53.5, 51.8, 27.9; IR(neat) v 3357, 2978-2890, 1716 cm"1. 191 (S)-3-tert-Butyl 4-methyl 2,2-dimethyIoxazolidine-3,4-dicarboxylate (271) An oven-dried, 100-mL, three-necked round-bottomed flask, equipped with a stir bar, Claisen distillation head with a vacuum adaptor and 50-mL collecting flask, reflux condenser with a nitrogen gas inlet adaptor, and rubber septum, was charged with dry benzene (61 mL). 7V-(fert-Butoxycarbonyl)-L-serine methyl ester 269 (18.3 mmol, 4.0 g), dissolved in 2,2-dimethoxypropane (36.6 mmol, 4.5 mL, 3.81 g), was added to the flask via syringe through the rubber septum. The rubber septum was removed, p- toluenesulfonic acid (0.26 mmol, 0.045 g) was added to the flask in one portion as a solid, and the open neck was fitted with a thermometer and Teflon coated adaptor. The solution was refluxed in an oil bath at 110 °C for 30 min, then the reflux condenser was replaced by a glass stopper. The solution was slowly distilled over the course of 4 h or until full consumption of amino acid starting material (Rf = 0.23) and development of product (Rf = 0.78), eluting with 1:1 hexane-ethyl acetate, was observed by TLC analysis. The resulting amber solution was partitioned between saturated sodium bicarbonate (50 mL) and diethyl ether (50 mL). The organic layer was washed with saturated sodium chloride (50 mL), dried with magnesium sulfate (ca. 5 g), vacuum filtered, and concentrated via rotary evaporation (40 mmHg, 21 °C) to yield (S)-3-tert-hutyl 4-methyl 2,2-dimethyloxazolidine-3,4-dicarboxylate 271 as a viscous amber oil (2.8 g, 59 %>). The product was isolated in an approximate 1.4:1 ratio of rotomers and was used without further purification. *H NMR (500 MHz, CDC13) mixture of rotomers (1.4:1): 8 4.49 (dd, J= 6.8, 2.5 Hz, IH), 4.38 (dd, .7= 7.1, 3.1 Hz, 2H), 4.17 - 4.12 (m, 2H), 4.06 - 4.01 (m, 2H), 3.76 (m, 6H), 1.67 (br s, 4H), 1.64 (br s, 2H), 1.50 (br s, 11H), 1.42 (br s, 12H); 13C 192 NMR (126 MHz, CDC13) 8 171.6, 171.2, 152.0, 151.1, 95.0, 94.3, 80.8, 80.2, 66.2, 65.9, 59.2, 55.2, 28.3, 28.2, 26.0, 25.1 24.9, 24.3; IR (neat) v 2979 - 2884, 1759, 1709 cm"1. L-Serine methyl ester hydrochloride (272) A 250-mL round-bottomd flask, equipped with a magnetic stir bar, was charged with 2,2- dimethoxypropane (189 mL). L-Serine (18.9 mmol, 2.0 g) was added to the stirring solution in one portion as a solid. Concentrated hydrochloric acid (20 mL) was added drop wise via an addition funnel over the course of 30 min. The solution was stirred for 16 h at room temperature. The crude solution was concentrated by rotary evaporation (10 mmHg, 50 °C) to afford a dark brown solid. The solid was dissolved in a minimum amount of methanol, and then poured into an Erlenmeyer flask containing diethyl ether (500 mL). The precipitated was collected by vacuum filtration and recrystallized from a methanol-ether solution to yield L-serine methyl ester hydrochloride 272 as a white solid { (2.77 g, 94 %). (m.p. 161 - 162 °C); U NMR (400 MHz, D20) 8 4.32 - 4.30 (m, IH), 13 4.15 - 4.11 (m, IH), 4.05 - 4.00 (m, IH), 3.88 (s, 3H); C NMR (100 MHz, D20) 8 169.1, 59.4, 54.8, 53.8; IR (neat) v 3401, 2980, 2958, 2849, 1747, 1618, 1513 cm"1. N- [(Methoxy)carbonyl] -L-serine methyl ester (274) An oven-dried 250-mL round-bottomed flask, equipped with a magnetic stir bar, pressure-equalizing addition funnel, rubber septum, and nitrogen gas inlet, was charged with sodium methoxide (38.6 mmol, 2.1 g) and dry methanol (180 mL). L-Serine methyl ester hydrochloride 272 (12.9 mmol, 2.0 g) was added in one portion as a solid. The solution was cooled in an ice-water bath to 0 °C. Methyl chloroformate (15.4 mmol, 1.5 193 g, 1.2 mL), dissolved in dry tetrahydrofuran (80 mL), was added via an addition funnel over the course of 3 min. The solution was allowed to come to room temperature while being vigorously stirred for 16 h under a bed of nitrogen gas. The crude solution was concentrated to half the original volume (ca. 130 mL), cooled to 0 °C in an ice-water bath, acidified to pH 4 with 1 M potassium bisulfate solution, and extracted with ethyl acetate (5 x 25 mL). The organic layers were pooled, dried with magnesium sulfate (ca. 10 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to give N-(terf-butoxycarbonyl)-L-serine methyl ester 274 as a light yellow oil (1.9 g, 83%>), [ which was carried on without further purification. H NMR (400 MHz, CDC13) 8 6.09 (br d, J= 7.6 Hz, IH), 4.48 - 4.36 (m, IH), 3.97 (dd, J= 11.1, 3.6 Hz, IH), 3.88 (dd, J = 11.1, 3.6 Hz, IH), 3.78 (s, 3H), 3.70 (s, 3H); 13C NMR (100 MHz, CDCI3) 8 171.3, 157.1, 62.7, 56.1, 52.5, 52.4; IR (neat) v 3346, 2957, 2850, 1698, 1527 cm"1. 7V-[(Methoxy)carbonyl]-0-ethyl-L-serine methyl ester (275)102b An oven-dried 250-mL round-bottom flask, equipped with a magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with anhydrous dichloromethane (150 mL). (5)-Methyl 3-hydroxy-2-((methoxycarbonyl)amino)propanoate 274 (8.25 mmol, 1.46 g) dissolved in anhydrous dichloromethane (15 mL) was added to the flask via an oven dried syringe. The solution was cooled to 0 °C in an ice-water bath for 10 min. Triethyloxonium tetrafluoroborate solution (9.1 mmol, 9.1 mL, 1 M in hexane) was added slowly via an oven dried syringe. Diisopropylethylamine (9.1 mmol, 1.6 mL, 1.18 g) was added to the reaction by syringe and the solution was stirred at room temperature and monitored by TLC. The crude solution was washed with 1 M hydrochloric acid (3 x 194 50 mL), 1 M potassium bicarbonate (3 x 50 mL), and saturated sodium chloride (50 mL). The organic layer was dried with sodium sulfate (ca. 20 g), gravity filtered, and concentrated by rotary evaporation (10 mmHg, 21 °C) to afford JV-[(methoxy)carbonyl]- O-efnyl-L-serine methyl ester 275 as an amber oil (1.0 g, 63 %). lU NMR (400 MHz, CDC13) 8 5.56 (br d, J= 7.3 Hz, IH), 4.47 (m, IH), 3.85 (dd, J= 9.7, 3.1 Hz, IH), 3.77 (s, 3H), 3.70 (s, 3H), 3.65 (dd, J= 9.7, 3.1 Hz, IH), 3.49 (q, J= 7.1 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 171.1, 156.7, 70.1, 66.9, 54.4, 52.5, 52.4, 14.9; IR (neat) v 2956 - 2876, 1708, 1522 cm"1. A^-(fcrf-Butoxycarbonyl)-0-(methoxymethoxy)-L-serine (276)112 A 100-mL round-bottomed flask, equipped with a magnetic stir bar, was charged with N- (tert-butoxycarbonyl)-0-(methoxymethoxy)-L-serine methyl ester 270 (3.5 mmol, 0.93 g), tetrahydrofuran (12 mL), and methanol (12 mL). Lithium hydroxide (8.8 mmol, 0.37 g), dissolved in water (12 mL), was added to the stirring solution in one portion. The reaction was vigorously stirred for approximately 45 min. The aqueous solution was cooled to 0 °C in an ice-water bath, acidified to pH 3-4 with concentrated hydrochloric acid, and immediately extracted with ethyl acetate (5 X 30 mL). The organic layers were pooled, dried with sodium sulfate (ca. 20 g), gravity filtered, and concentrated by rotary evaporation (10 mmHg, 30 °C) to afford the titled compound 276 a light yellow solid (0.7 g, 78%>), which was carried on without further purification; 2H NMR (400 MHz, acetone- d6) 8 5.99 (br d, J= 6.9 Hz, IH), 4.60 (app s, 2H), 4.38 (m, IH), 3.97 (dd, J= 10.0, 4.4 Hz, IH), 3.78 (dd, J= 10.0, 3.6 Hz, IH), 3.30 (s, 3H), 1.43 (s, 9H); 13C NMR (100 MHz, 195 acetone-d6) 8 174.0, 155.9, 96.6, 80.4, 68.1, 55.5, 53.9, 28.4; IR (neat) v 3343, 2978 - 2891,2827, 1693,1510 cm"1. (5)-3-(terM3utoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic acid (277) Using the same reaction conditions reported for the preparation of 7V-Boc-0-MOM-L- serine 276, titled compound 277 was isolated from methyl ester 271 in a 1:1 mixture of rotomers as a light yellow oil (2.1 g, 99 %>), which was carried on without further [ purification. H NMR (400 MHz, CDC13) 8 10.82 (br s, 2H), 4.53 (dd, J= 6.4, 2.0 Hz, IH), 4.41 (dd, J= 7.0, 2.9 Hz, IH), 4.22 - 4.10 (m, 4H), 1.67 (br s, 4H), 1.63 (br s, 3H), 13 1.51 (br s, 12H), 1.43 (br s, 11H); C NMR (100 MHz, CDC13) 8 176.4, 175.4, 152.8, 151.3, 95.3, 94.7, 81.6, 80.8, 67.9, 66.2, 65.9, 59.2, 28.4, 28.3, 26.1, 25.0, 25.0, 24.4; IR (neat) v 3464, 2981 -2938, 1701 cm"1. N-[(Methoxy)carbonyl]-0-ethyl-L-serine(278) Using the same reaction conditions reported for the preparation of TV-Boc-0-MOM-L- serine 276, titled compound 278 was isolated from methyl ester 275 as a clear oil (0.33 g, l 80 %), which was used without further purification. H NMR (400 MHz, CDC13) 8 11.09 (br s, IH), 5.79 (br d, J= 8.5 Hz, IH), 4.58 - 4.45 (m, IH), 3.91 (dd, J= 9.6, 3.0 Hz, IH), 3.76 - 3.65 (m, 4H), 3.53 (q, J= 7.0 Hz, 2H), 1.17 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCI3) 8 174.5, 157.1, 69.9, 67.0, 54.2, 52.6, 14.7; IR (neat) v 3384, 2981 - 2884, 1707,1724 cm"1. 196 (5r)-Benzyl5-(benzyloxy)-4-((fc^-butoxycarbonyl)amino)-3-oxopentanoate (281) An oven-dried 50-mL round-bottomed flask, equipped with magnetic stir bar and nitrogen gas inlet needle, was charged with carbonyldiimidizole (1.86 mmol, 0.30 g) and anhydrous tetrahydrofuran (10 mL). TV-Boc-O-Bn-L-serine 267 (1.69 mmol, 0.50 g), stored over 3 A sieves in anhydrous THF (2 mL), was transferred to the reaction via syringe and stirred for approximately 20 min. A separate oven-dried 250-mL round- bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with freshly prepared monobenzyl malonate 257a (5.07 mmol, 0.98 g) and dissolved in anhydrous tetrahydrofuran (20 mL). The solution was cooled to 0 °C in an ice-water bath before adding dibutylmagnesium (2.5 mmol, 2.5 mL, IM in heptanes), then allowed to warm to room temperature. The acyl imidazole solution was transferred by cannula into the magnesium malonate mixture. The reaction was stirred for 36 h or until TLC analysis indicated amino acid starting material was consumed and evidence for product (Rf = 0.33) was observed. The crude reaction mixture quenched with 1 M hydrochloric acid (ca. 20 mL) and extracted with diethyl ether (3 x 30 mL). The organic layers were pooled, washed with 5 % sodium bicarbonate (3x15 mL), dried with sodium sulfate (ca. 10 g), gravity filtered, and concentrated by rotary evaporation (40 mmHg, 21 °C) to a viscous yellow oil (0.72 g). The crude residue was purified by flash column chromatography on silica, eluting with 6:1 hexane - ethyl acetate mobile l phase (Rf = 0.33), to afford the title compound 281 as a clear oil (0.36 g, 50%). H NMR (400 MHz, CDC13) 8 7.41 - 7.18 (m, 10H), 5.48 (br d, J= 7.4 Hz, IH), 5.14, 5.12 (ABq, JAB = 12.3 Hz, 2H), 4.46, 4.44 (ABq, JAB = 12.0 Hz, 3H), 3.88 (dd, J= 9.7, 3.6 Hz, IH), 13 3.68 - 3.55 (m, 3H), 1.43 (s, 9H); C NMR (100 MHz, CDC13) 8 200.9, 166.8, 155.6, 197 137.5, 135.6, 128.8, 128.7, 128.6, 125.8, 128.2, 128.0, 80.4, 73.7, 69.5, 67.3, 60.1, 46.9, 28.5; IR (neat) v 3423, 3065 - 2870, 1746, 1709 cm"1. (iS)-Benzyl 4-((fer^butoxycarbonyl)amino)-5-((fer^butyldimethylsilyl)oxy)-3- oxopentanoate (282) Using the same procedure as described for the conversion of iV-Boc-O-Bn-L-serine 267 to P-keto ester 281, title compound 282 was isolated from 268 as a clear oil in 0.59 g, 30% (Rf =0.1, purified by column chromatography on silica, eluting with 9:1 hexane - ethyl acetate mobile phase); Proton resonances are referenced to CHCI3 (8 7.26 ppm) due ! to overlapping resonance with TMS (8 0.00 ppm). H NMR (500 MHz, CDC13) 8 7.39 - 7.29 (m, 5H), 5.42 (br d, J= 7.0 Hz, IH), 5.16 (app s, 2H), 4.43 - 4.35 (m, IH), 4.05 (dd, J= 10.4, 3.3 Hz, IH), 3.84 - 3.76 (m, IH), 3.67, 3.64 (ABq, JAB = 16.3 Hz, 2H), 1.45 (s, 13 9H), 0.84 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H); C NMR (126 MHz, CDC13) 8 206.6, 172.2, 160.9, 141.0, 134.2, 134.0, 134.0, 85.8, 72.8, 68.7, 67.0, 52.7, 34.0, 33.9, 31.4, 23.4, 0.0; IR (neat) v 3358, 3068 - 2934, 1716 cm"1. (5)-Benzyl 4-((ter*-butoxycarbonyl)amino)-5-(methoxymethoxy)-3-oxopentanoate (283) Using the same procedure as described for the conversion of TV-Boc-O-Bn-L-serine 267 to P-keto ester 281, title compound 283 was isolated from 276 as a colorless oil (0.72 g, 52%>). (Rf = 0.23, purified by column chromatography on silica, eluting with 8:1 hexane- ethyl acetate mobile phase); JH NMR (400 MHz, CDCI3) 8 7.44 - 7.23 (m, 5H), 5.55 (br d, J= 7.7 Hz, IH), 5.17, 5.15 (ABq, JAB = 12.2 Hz, 2H), 4.57 - 4.45 (m, 3H), 4.00 (m, 198 1J IH), 3.75 - 3.60 (m, 3H), 3.28 (s, 3H), 1.49 (s, 9H); C NMR (100 MHz, CDC13) 8 200.8, 166.8, 155.6, 135.5, 128.8, 128.6, 128.6, 96.8, 80.5, 67.4, 67.2, 60.0, 55.7, 46.8, 28.5; IR (neat) v 3353, 2976, 2826, 1715, 1613, 1500 cm"1. (S)-tert-Buty\ 4-(3-(benzyloxy)-3-oxopropanoyl)-2,2-dimethyloxazolidine-3- carboxylate (284) Using the same procedure as described for the conversion of JV-Boc-O-Bn-L-serine 267 to p-keto ester 281, titled compound 284 was obtained from 277 as a colorless solid in a complex mixture of rotomers (1.1 g, 47 %>). (Rf = 0.29, purified by column chromatography on silica, eluting with 4:1 hexane - ethyl acetate mobile phase); !H NMR (500 MHz, CDC13) 8 7.55 - 7.12 (m, 10H), 5.23 - 5.09 (m, 4H), 4.59 - 4.28 (m, 2H), 4.20 - 3.88 (m, 4H), 3.76 - 3.49 (m, 4H), 1.68 - 1.39 (m, 30H); I3C NMR (126 MHz, CDCI3) 8 201.1, 201.0, 179.6, 175.8, 172.4, 167.0, 166.7, 152.5, 151.2, 135.3, 128.6, 128.5, 95.4, 94.7, 88.4, 81.3, 67.3, 67.2, 67.0, 66.6, 66.1, 65.5, 65.3, 65.0, 59.5, 48.9, 46.6, 45.5, 41.6, 28.3, 28.2, 26.7, 26.1, 25.7, 25.5, 24.9, 24.5, 23.6; IR (neat) v 3444, 2980, 2937,1700, 1694 cm"1. (S)-Benzyl 5-ethoxy-4-(methoxycarbonylamino)-3-oxopentanoate (285) Using the same procedure as described for the conversion of JV-Boc-OBn-L-serine 267 to P-keto ester 281, title compound 285 was isolated from 278 as a light yellow oil (0.40 g, 67 %>). (Rf = 0.35, purified by column chromatography on silica, eluting with 6:1 l hexane - ethyl acetate mobile phase); U NMR (400 MHz, CDC13) 8 7.40 - 7.29 (m, 5H), 5.75 (br d, J= 7.0 Hz, IH), 5.16 (app s, 2H), 4.58 - 4.48 (m, IH), 3.84 (dd, J= 9.7, 3.8 199 Hz, IH), 3.70-3.62 (m,5H), 3.58 (m, IH), 3.43 (q, J= 7.0 Hz, 2H), 1.11 (t, .7=7.0 Hz, 13 3H); C NMR (100 MHz, CDC13) 8 200.6, 166.8, 156.8, 135.5, 128.8, 126.6, 128.5, 69.7, 67.3, 60.4, 52.6, 46.9, 29.9, 15.0. IR (neat) v 3065 - 2873, 1722, 1498, 1455 cm"1. (5)-Ethyl 5-(benzyloxy)-4-(benzyloxycarbonylamino)-3-oxopentanoate (286) Using the same procedure as described for the conversion of ./V-Boc-O-Bn-L-serine 267 to p-keto ester 281, titled compound 286 was isolated from 279 as a light yellow oil (1.1 g, 50%). (Rf = 0.34, purified by column chromatography on silica, eluting with 6:1 J hexane - ethyl acetate mobile phase); H NMR (400 MHz, CDC13) 8 7.40 - 7.19 (m, 10H), 5.85 (br d, J= 7.6 Hz, IH), 5.09 (app s, 2H), 4.58 (m, IH), 4.48, 4.44 (ABq, JAB = 11.9 Hz, 2H), 4.13 (q, J= 7.1 Hz, IH), 3.90 (dd, .7= 9.7, 3.5 Hz, IH), 3.66 (dd, .7= 9.6, 13 4.3 Hz, IH), 3.55, 3.52 (ABq, JAB = 16.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H); C NMR (100 MHz, CDCI3) 8 200.5, 166.9, 156.2, 137.4, 136.4, 128.8, 128.7, 128.5, 128.4, 128.2, 128.0, 73.7, 69.4, 67.3, 61.7, 60.5, 47.0, 14.3; IR (neat) v 3432, 3065, 3033, 2977, 2932, 2870,1717, 1715 cm"1. (5)-Benzyl 4-((benzyloxycarbonyl)amino)-5-((fer^butyldimethylsilyl)oxy)-3- oxopentanoate (287) Using the same procedure as described for the conversion of V-Boc-O-Bn-L-serine 267 to P-keto ester 281, titled compound 287 was isolated from 280 as a light yellow oil (0.66 g, 25%o). (Rf= 0.1, purified by column chromatography on silica, eluting with 9:1 hexane - ethyl acetate mobile phase). Proton resonances are referenced to CHCI3 (8 7.26 ppm) due to overlapping resonance with TMS (8 0.00 ppm). *H NMR (500 MHz, CDC13) 8 200 7.45-7.24 (m, 10H), 5.77 (br d, J= 7.0 Hz, IH), 5.20 - 5.11 (m, 4H), 4.54-4.51 (m, IH), 4.10 (dd, J= 10.5, 3.4, IH), 3.83 (dd, J= 10.5, 4.4 Hz, IH), 3.69, 3.67 (ABq, JAB = 16.2 13 Hz, 2H), 0.87 (s, 9H), 0.5 (br s, 6H); C NMR (126 MHz, CDC13) 8 205.9, 172.1, 161.5, 141.8, 140.9, 134.2, 134.0, 134.0, 133.9, 133.8, 72.8, 68.7, 67.3, 52.7, 31.4, 23.8, 0.0; IR (neat) v 3067, 3034, 2953, 2929, 2884, 2856, 1715, cm"1. (55)-Benzyl 6-(benzyloxy)-5-((/er/-butoxycarbonyl)amino)-3-methyl-4-oxohexanoate (288) A 10-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with dry dichloromethane (4.5 mL) and cooled to 0 °C in an ice-water bath. Neat diethylzinc (0.59 mmol, 0.06 mL, 0.07 g) was added in one portion via an oven-dried 25 cm needle and plastic syringe. JV-Boc-OBn-L-serine- derived P-keto ester 281 (0.23 mmol, 100 mg), dissolved in dry dichloromethane (0.5 mL), was added drop-wise over the course of 1 min. The reaction was stirred for 15 min at which time 1,1-diiodoethane (0.59 mmol, 0.05 mL, 0.18 g) was added drop-wise by syringe. The reaction was stirred for 1 h at room temperature, then cooled to 0 °C in an ice-water bath before a second portion of diethyl zinc (0.59 mmol, 0.06 mL, 0.07 g) and 1,1-diiodoethane (0.59 mmol, 0.05 mL, 0.18 g were added via syringe. The reaction mixture was allowed to stir for an additional 2 h at room temperature. TLC analysis indicated consumption of starting material (Rf = 0.57) and appearance of product (Rf = 0.69), eluting with a (1:1) hexane-ethyl acetate mobile phase. The reaction was slowly quenched with saturated ammonium chloride solution (ca. 4 mL) and extracted with dichloromethane (3x5 mL). The organic layers were pooled, washed with brine (ca. 10 201 mL), dried with sodium sulfate (ca. 1 g), gravity filtered, and concentrated via rotary evaporation (10 mmHg, 21 °C) to yield a yellow oil in a >20:1 ratio of diastereomers. The major diastereomer was isolated by preparative thin layer chromatography, eluting with (1:1) hexane - ethyl acetate (Rf = 0.69), to afford the titled compound 288 as a ! colorless oil in 56 mg, 55 %. H NMR (400 MHz, CDC13) 8 7.38 - 7.25 (m, 10H), 5.46 (br d,J= 8.3 Hz, IH), 5.08, 5.05 (ABq, JAB = 12.4 Hz, 2H), 4.65 (m, IH), 4.50 (app s, 2H), 3.87 (dd, J= 9.6, 3.7 Hz, IH), 3.71 (dd, J= 9.6, 4.4 Hz, IH), 3.31 (m, IH), 2.82 (dd, J= 16.8, 7.7 Hz, IH), 2.38 (dd, .7= 16.9, 6.3 Hz, IH), 1.47 (s, 9H), 1.18 (d, J= 7.2 Hz, 13 3H); C NMR (100 MHz, CDC13) 8 209.4, 172.0, 155.5, 137.7, 136.0, 128.7, 128.5, 128.3, 128.3, 127.9, 127.8, 80.0, 73.4, 69.6, 66.5, 58.1, 39.3, 36.6, 28.5, 17.1; IR (neat) v 2954, 2923, 2854, 1736, 1720, 1709 cm"1. (55)-Benzyl 5-((benzyloxycarbonyl)amino)-6-((ter^-butyldimethylsilyl)oxy)-3-methyl- 4-oxohexanoate (289) Using the same procedure as described for the conversion of p-keto ester 281 to P- methyl-y-keto ester 288, title compound 289 was isolated from 282 as a pale yellow oil (34 mg, 51%>). (Rf = 0.78, purified by preparative thin layer chromatography on silica, l eluting with 3:1 hexane - ethyl acetate mobile phase); H NMR (500 MHz, CDC13) 8 7.36 - 7.32 (m, 10 H), 5.69 (br d,J= 7.3 Hz, IH), 5.15 - 5.06 (m, 5H), 4.59 (m, IH), 4.10 - 4.04 (m, 4H), 4.59 (m, IH), 4.05 (dd, .7= 10.3, 3.5 Hz, IH), 3.85 (dd, J= 10.5, 4.7 Hz, IH), 3.31 (m, IH), 2.80 (dd, J= 16.7, 6.6 Hz, IH), 2.40 (dd, J= 16.7, 7.1 Hz, IH), 1.19 (d, J= 7.2 Hz, 3H), 0.84 (s, 9H), 0.02 (app d, 6H); 13C (126 MHz, CDC13) 8 209.0, 171.9, 166.6, 156.0, 135.0, 135.4, 128.7, 128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 202 67.4, 67.1, 66.5, 66.0, 63.2, 63.1, 61.8, 60.0, 47.2, 39.5, 36.4, 25.9, 18.3, 16.9, 15.4, 0.1, - 5.5. Ethyl 5-(2-(benzyloxy)-l-(((benzyloxy)carbonyl)amino)ethyl)-5-hydroxy-4-methyl-2- pentyltetrahydrofuran-3-carboxylate (304) A 25-mL oven-dried round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with L-serine P-keto ester 286 (0.25 mmol, 100 mg) and anhydrous dichloromethane (5 mL). The solution was cooled to 0 °C in an ice-water bath at which time neat diethylzinc (0.75 mmol, 80 pi) was added slowly via an oven-dried needle. The reaction was stirred for 10 min at 0 °C then 1,1- diiodoethane (0.75 mmol, 60 pi) was added dropwise over the course of 30 sec and stirred for an additional 1 h at 0 °C. Neat diethyl zinc (0.75 mmol, 80 pi) was added at 0 °C to the mixture and after 10 min, 1,1-diiodoethane (0.75 mmol, 60 pi) was added via syringe and stirred for 2 h while warming to room temperature. Hexanal (0.28 mmol, 34 pi) was in one portion at room temperature via syringe and the reaction was stirred for 16 h. Saturated ammonium chloride solution (ca. 10 mL) was added to the reaction and extracted with dichloromethane (3 x 10 mL). The organic layers were pooled, washed with brine solution (2x10 mL), dried with sodium sulfate (ca. 5 g), vacuum filtered and concentrated to a light yellow oil (133 mg). The crude residue was purified by preparative thin layer chromatography on silica, eluting with hexane then (3:1) hexane - ethyl acetate mobile phase (Rf = 0.55), producing a mixture of hemiketals 304 as a clear ! oil (30 mg, 22%). Characteristic proton resonances: H NMR (500 MHz, CDC13) complex mixture of isomers 8 4.21 - 4.11 (m, 5H), 3.55 - 3.50 (m, 3H), 2.69 - 2.60 (m, 203 13 2H); All carbon resonances for the mixture of isomers: C NMR (126 MHz, CDC13) 8 174.3, 174.1, 173.0, 172.1, 165.7, 156.4, 156.0, 137.7, 137.3, 136.8, 136.6, 128.7, 128.6, 128.5, 128.2, 128.2, 128.12, 127.8, 105.7, 105.1, 105.0, 104.9, 80.9,, 80.0, 79.3, 74.0, 73.5, 73.5, 73.0, 71.8, 70.3, 67.0. 67.0, 61.4, 61.2, 60.9, 57.1, 57.1, 53.7, 53.2, 43.0, 37.1, 32.1, 31.8, 30.3, 25.5, 22.8, 22.7, 14.4, 14.3, 14.2, 14.1, 11.1, 10.0; IR (neat) 3447, 3032 -2859,1710,1708, 1510 cm"1. (5)-Methyl 2-(benzyloxy)propanoate (329) An oven-dried 50-mL round-bottomed flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet needle, was charged with sodium hydride (40.0 mmol, 1.6 g, 60%o dispersed in mineral oil) and dry tetrahydrofuran (15 mL) then cooled to 0 °C in an ice-water bath. Methyl lactate (20.0 mmol, 2.1 g, 1.9 mL), dissolved in dry tetrahydrofuran (5 mL), was added dropwise over the course of 5 min and the reaction was stirred for 30 min at 0 °C. Benzyl bromide (30.0 mmol, 5.1 g, 3.6 mL) was added dropwise over the course of 5 min and the reaction was stirred for an additional 4 h while warming to room temperature. The reaction was quenched with saturated ammonium chloride (ca. 20 mL) and extracted with diethyl ether (3 x 25 mL). The organic layers were pooled, washed with brine (2 x 10 mL), dried with magnesium sulfate (ca. 5 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford the titled compound 329 as a viscous clear oil (2.61 g, 67%>). The product was carried on l without further purification. U NMR (400 MHz, CDC13) 8 7.42 - 7.17 (m, 5H), 4.68 (A of AX, J= 11.7 Hz, IH), 4.43(XofAX,J= 11.7 Hz, IH), 4.04 (q, J= 6.9 Hz, IH), 3.69 204 13 (s, 3H), 1.41 (d,J= 6.9 Hz, 3H); C NMR (100 MHz, CDC13) 8 173.6, 137.5, 128.4, 127.9, 127.8, 73.9, 71.9, 51.8, 18.6; IR (neat) 3040-2876, 1721 cm"1. (iS)-2-(Benzyloxy)propanoic acid (330) A 250-mL round-bottomed flask, equipped with magnetic stir bar, was charged with (S)- methyl 2-(benzyloxy)propanoate (329) (10.3 mmol, 2.0 g), tetrahydrofuran (34 mL), methanol (34 mL), and water (34 mL). Lithium hydroxide (20.6 mmol, 0.87 g) was added as a solid in one portion and the reaction was stirred vigorously for approximately 45 min. The reaction was cooled to 0 °C in an ice-water bath, acidified to pH 2 with concentrated hydrochloric acid, and extracted with ethyl acetate (5 x 60 mL). The organic layers were pooled, dried with magnesium sulfate (ca. 15 g), vacuum filtered, and concentrated via rotary evaporation (10 mmHg, 30 °C) to afford a light yellow oil. The crude residue was purified by flash column chromatography on silica, eluting with a 2:1 hexane - ethyl acetate mobile phase (Rf = 0.29, streaky), to afford the title compound 330 l as a clear oil (1.1 g, 61%). U NMR (500 MHz, CDC13) 8 7.39 - 7.28 (m, 5H), 4.71 (A of AX, J= 11.6 Hz, IH), 4.54 (XofAX,J= 11.5 Hz, IH), 4.12 (q, J= 6.9 Hz, IH), 1.50 (d, J= 6.9 Hz, 3H); 13C NMR (126 MHz, CDCI3) 8 177.9, 137.1, 128.7, 128.3, 128.2, 73.7, 72.3, 18.4; IR (neat) v 3484, 3090, 3065, 3032, 2987, 2939, 2877, 1721 cm"1. (S)-Benzyl 4-(benzyloxy)-3-oxopentanoate (331) An oven-dried 50-mL round-bottom flask, equipped with magnetic stir bar and nitrogen gas inlet, was charged with (5)-2-(benzyloxy)propanoic acid 330 (4.7 mmol, 0.51 g) and carbonyldiimidizole (5.2 mmol, 0.84 g) in anhydrous tetrahydrofuran (30 mL) and stirred 205 at room temperature for 15 min. A separate oven-dried 250-mL round-bottom flask, equipped with magnetic stir bar, rubber septum, and nitrogen gas inlet, was charged with freshly prepared monobenzyl malonate (2.7 g, 14.1 mmol) and dissolved in anhydrous tetrahydrofuran (60 mL). The solution was cooled to 0 C before adding dibutylmagnesium (7.1 mmol, 7.1 mL, IM in heptanes), stirred until clear, and allowed to warm to room temperature. The acyl imidazole solution was transferred via syringe into the magnesium malonate solution. The reaction was stirred for 36 h or until TLC analysis showed lactic acid starting material (Rf = 0.33 - 0.0, streak) was consumed and evidence for product was observed (Rf = 0.50), eluting with a (3:1) hexane - ethyl acetate mobile phase. The crude reaction mixture was quenched with saturated ammonium chloride solution (ca. 50 mL) and extracted with diethyl ether (3 x 150 mL). The organic layer was dried with sodium sulfate (ca. 10 g), gravity filtered, and concentrated to a viscous yellow oil (1.7 g). The crude oil was purified by column chromatography on silica, eluting with a (3:1) mobile phase (Rf = 0.50), affording the titled compound 331 as l a clear oil (0.73 g, 50%). 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G., Application of ring-closing metathesis for the synthesis of peptidomimetics as inhibitors of HCV NS3 protease. Org. Lett. 2007, 9 (16), 3061-3064. 226 APPENDIX A. Spectra 227 x •W6 2-00'2 '^19'E 228 E'9Z- 6'Efr- ^i -=f KS8- oE •"" D. •-I Q. T80Z 229 I-£Z'£ I-9Z-Z I" I-9£'Z ^r60'X ^60'T !F00T \ 230 6"6fr- 8'S9- E O- rsu- S-I£I- o /•99I- fr'OOZ- 0> 231 p£8'£ POO'I 1 Ea . o ON 232 0 6E- oE iH Q. in n. o o 101Z- O ON 233 F'» -OO'l 234 £fr'09- ^9'86- oE •H Q. i-H Q. O 99'09T- 9S'I9T- S8'Z9T- o o 235 „/-6ZH -"^•6£'0T iP "'Z ^2- S9'T ID Q. j if 8Z'I j,/- Zfr'T ^ 9fr'T oi Tf OO'I 236 C9Z-y- 682-^ 6££ — r9£ —~ 9'M?-^ 8'Sir-' 6'TS-\_ t-'ZS-7= V£SV ZIOI- 2£ KS£I- 6'8£T- o 3 0>9I- 2'69I- vin- —i VZll- Tt>6T- o o E2IZ- Zl\Z- 237 £6S Z9'£ — ZS'O rcczz >v ^og'e t-0S'8 I-8l7'I Wl -6Z'I Tf + 238 S'6Z-^ 0'0£^^ TZ£-\. KOfr-v E'Zfr-^ 9'Zfr-^ IIS- 9ZS- £'Z9 ZZ9 > oE t-l OL 9'IEI- I'6£T- 8-£ST^- TT in 8V9T- fr'89I- fr'ZZI- 9*0Z" EYOZ- 239 OO'T r- 'I V© v© 240 V© °.B Z'SZT-v E8ZT-0- fr'8ZT-> Z'8ZIV o ^© E'691 6't-ZI-v. S'8ZI^v 0 £0Z IT) Z'90Z *© ^•nz-> VvlZ-' 241 _L_ f-68'Z Hi-26'1 P66'0 o > 242 o ZT9- T"W- fr'9Z- o£ •"< Q. rzsi- 6>SI- o o 243 182 4^ / // M b-H l-r+1-W h-T-H en O en en -3- ^r o o en o en en d TT ID rr rH I I l I 14 13 12 11 10 6 ppm Note: Structural configuration has been tentatively assigned, see discussion in Chapter n, DCC-mediated heterocycle formation. ui o rn ID 182 4^ J L JL»UU— ~ - T" T T" T T ~r T" T" T" I -r -r~ 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 -10 ppm Note: Structural configuration has been tentatively assigned, see discussion in Chapter LI, DCC-mediated heterocycle formation. ^ f%A^_ X ° M .. AAA, ,A., 182 J' -1.0 -1.5 -2.0 to 4^ ON -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 "I -1— -1 l -i— 5.5 5.0 4.5 4.0 3.5 3.0 2.5 - 2.0 1.5 ppm Note: Structural configuration has been tentatively assigned, see discussion in Chapter LT, DCC-mediated heterocycle formation. 183 to 4^ --a J o Ln m o 1 —T— 14 13 12 11 10 6 ppm Note: Structural configuration has been tentatively assigned, see discussion in Chapter LI, DCC-mediated heterocycle formation. fN fSJ r\j m en to rsi en en ^r W t-i r-i \i V 183 to 4^ oo 11 1 T" T~ T~ T" T T T I T" ~T" T T" 1 1 ' 1 1 1 1 1 1 1 1 1 ' 1 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 ppm Note: Structural configuration has been tentatively assigned, see discussion in Chapter LI, DCC-mediated heterocycle formation. 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CL p8S'S \ o as •y \J 360 8XS- 6'XZ- 6'EZ- ^ o£ 8'ZZX-\ 6'ZZX—^\y = S'ZEX- 9£ZX- \ o as •y \J 361 --H^ZT'E H^TO'T Tf-90'T I-16't' I o © \J -> 362 E'ZZ- Z'EZ- E CL Z8ZX E8ZX > 6'ZZX- © 363 ZZ'E ^ ilr- EO'Z J3E- ZO'T - ooz I*- 6E'Z ^PS9>T en 364 X'Z9- 6XZ- Z'Z9X- o o X'SOZ- \J 365 APPENDIX B. X-Ray Crystal Structures 366 (2J?,3^4L5,5i?)-Methyl2-hydroxy-9-methoxy-2,3-dimethyl-7-oxo-l,6- dioxaspiro[4.4]non-8-ene-4-carboxylate (200) HCl /x r= 367 (2i?,3L?,4^,5/?)-Methyl2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8- ene-4-carboxylate (203a) O —Q\ „c\ r -o o o "077 368 (2L?,3^,45,5^)-Methyl2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8- ene-4-carboxylate (203b) 369 , (2Jf?,3^,45 ,5i?)-allyl2,9-dimethoxy-2,3-dimethyl-7-oxo-l,6-dioxaspiro[4.4]non-8-ene- 4-carboxylate (211) / O =0 o \ 370 APPENDIX C. Spectral Data for Spiro-fused Ketals 371 !H NMR (400 MHz, CDC13) H-atom 211 203ab 203bb 203cc 5.10 (s, IH) 5.10 (s, IH) 5.14 (s, 1H) 5.06 (s, IH) 11 3.94 (s,3H) 3.93 (s, 3H) 3.96 (s,3H) 3.84 (s, 3H) 3.42 (d, IH) 3.38 (d, IH) 3.56 (d, IH) 3.18 (m, IH) J= 12.3 Hz / = 12.3 Hz J= 12.0 Hz 12 3.26 (s,3H) 3.24 (s,3H) 3.37 (s, 3H) 3.66 (s, 3H) 2.67 (dq, IH) 2.63 (dq, IH) 2.54 (dq, 1H) 2.98 (m, IH) J= 12.3, 6.7 Hz /= 12.3, 6.7 Hz J= 12.0, 6.7 Hz 1.47 (s,3H) 1.45 (s,3H) 1.33 (s,3H) 1.44 (s,3H) 1.12 (d,3H) 1.09 (d,3H) 1.14 (d,3H) 1.12 (d, 3H) 10 .7=6.7 Hz, .7=6.7 Hz J=6.6Hz J=6.7Hz Table 23. !H NMR chemical shift and coupling constant data for esters 211, 203a, 203b, and 203c, (a) All resonances reported downfield to TMS (0 ppm), (b) Stereochemical assignment was confirmed through X-ray crystal structure analysis, W Stereochemistry for 203c was tentatively assigned based upon comparison with the 'H NMR chemical shift trends for 203a and 203b, see Chapter II 13 a C NMR (100 MHz, CDCl3) C-atom 211 203ab 203bb 203cc 1 168.8 168.8 169.0 169.8 2 90.5 90.3 90.7 89.7 3 176.7 176.7 175.8 177.1 4 107.1 106.9 105.5 108.1 5 59.8 54.1 54.9 56.5 6 44.2 44.2 38.3 47.1 7 109.9 109.8 113.1 109.7 8 19.9 19.7 21.3 18.9 9 167.3 168.0 167.5 169.7 10 11.8 11.8 13.7 12.1 11 54.2 52.3 52.5 52.2 12 49.3 49.2 50.1 49.5 13 a Table 24. C NMR chemical shift data for esters 211, 203a, 203b, and 203c, <> All resonances referenced to CDC13 (77.16 ppm), W Stereochemical assignment was confirmed through X-ray crystal structure analysis, W Stereochemistry for 203c was tentatively assigned based upon comparison with the !H NMR chemical shift trends for 203a and 203b, see Chapter II X H NMR (500 MHz, CDC13) 225a 130a 130a'b 238c>d H-atom Synthesized Synthesized Reported by Dai et al.60 Reported by Shan et al.59 2 5.22 (s, IH) 5.07 (s, IH) 5.07 (s, IH) 5.01 (s, IH) 2.62 (ddq, IH) 6 2.89-2.83 (m, IH) 2.71-2.66 (m, IH) 2.70 (m, IH) J= 6.8, 3.0 Hz 8 1.56 (s,3H) 1.51 (s, 3H) 1.51 (s,3H) 1.43 (s,3H) 5.12 (d, lH),J=3.0Hz 9 5.13-5.11 (m,2H) 5.22-5.18 (m,2H) 5.20 (m, 2H) 5.10 (d, lH),J=3.0Hz 1.16 (d,3H) 1.16 (d,3H) 1.16 (d,3H) 1.08 (d,3H) 10 J=6.8Hz J=6.8Hz .7=6.8 Hz J= 6.8 Hz 11 3.93 (s, 3H) 3.87 (s, 3H) 3.87 (s,3H) 3.81 (s, 3H) 12 3.29 (s, 3H) 3.31 (s,3H) 3.31 (s,3H) 3.23 (s, 3H) Table 25. !H NMR chemical shifts and coupling constant data for compounds 225, 130 synthesized, 130 reported and 238, (a) All resonances reported downfield of TMS (0.0 ppm), (b) Dai et al.60 isolated from the endophytic fungus Microsphaeropsis sp., internal strain number 7291, (c) Published structure for 238 derived from papyracillic acid A, (d) Shan et al.59 made stereochemical assignment through NOSEY correlation 13 C NMR (126 MHz, CDC13) 1(/ 9 225a-b 130a'b 130 238 C-atom Synthesized Synthesized Reported by Dai et at.60 Reported by Shan et a/.59 1 169.1 170.3 170.2 170.1 2 91.3 88.6 88.4 88.1 3 176.7 178.2 178.2 178.0 4 107.6 107.2 107.3 106.8 5 148.5 148.8 148.8 148.5 6 46.2 48.8 48.8 48.5 7 109.0 109.4 109.3 109.1 8 20.1 19.0 18.6 18.7 9 109.4 110.0 109.8 109.1 10 9.9 10.7 10.6 10.3 11 59.7 59.8 59.8 59.6 12 49.6 49.5 49.5 49.2 Table 26. 13C NMR chemical shift data for compounds 225, 130 synthesized, 130 reported, and 238,(a) Isolated as a 1:1 mixture of epimeric isomers, 225 and 130, (^Resonances were referenced to CDC13 (7. 16 ppm) APPENDIX D. Line Fit Data for L-Serine-Derived Systems 376 MestReNova Line Fitting The H NMR spectra for each crude mixture of L-serine-derived-y-keto esters (288, 289, 290, 291, and 292) were analyzed using MestReNova line fitting program. A multipoint Bernstein Polynomial Fit was applied to the entire spectrum for each system. A multipoint baseline correction was only applied to the expanded portion, which included the resonances of interest. The line fit was applied and manually adjusted for each peak of interest corresponding to the major and the minor diastereomers. Unless otherwise mentioned, the doublet for the methyl group was used to establish the ratio of diastereomers. OBn Entry Compound R1 R2 Calculated ratio Reported dr 1 288 Boc Bn 29.9717:1 >20:1 2 289 Boc TBDMS 26.23483:1 >20:1 3 290 Boc MOM 15.22531:1 15:1 4 291 -C(0)OCH3 Et 2.87561:1 3:1 5 292 Boc 1.19627:1 1:1 o—" Table 27. Summary of MestReNova line fit data for L-serine-derived systems 377 3 w g § Name: 0-Benzyl as — From: 1.069 ppm H re =* To- 1.200 ppm OBn IS'? Boc'N oo re Residual Error 6.71 oo s # ppm Height Width(Hz) L/G Area CH3 O w BnO l 1 1.1873 100.66 2.21 1.00 3589 744 o 2 1.1692 101.16 2.03 1 00 3318 426 I 288 (/I ;3 1.0958 6.30 1.31 0.00 109.114 00 4 1.0781 6 30 1.46 0.00 121.375 P6 ,, cr = o re cr o 3 <^_ 85 3 5" c ST 20 1 19 1.18 1.17 1.16 1.15 1.14 1 13 1 12 1.11 1.10 1 09 1 08 1.07 ppm g'S a- — | Name: O-TBDMS O j From: 1.105 ppm ^ iv OBn i To: 1.212 ppm Boc'" ?| i Residual Error. 4.89 * 2" CH O ^ c # ppm Height Width(Hz) L/G Area s TBDMSO 3 1 o 1 1.1949 41.23 2.04 0.73 1031.040 j v3 » res UJ 2 1,1802 45.78 2.16 0.64 1189.292 J 289 re as B" a" 3 1.1260 3.68 0.89 1.00 42.315 ' 1T. ©a 4 1.1121 3.50 0.94 1.00 42.318 : * ¥ o s re B * o a°3s xV^ S" £ 00 —i—i—i—i—i—i—i—i—j—i—i—i—!—•—i—i—i—i—i—i—i—i—i—i—i—i—i—i—j—i—i—i—\—i—i—i—i—i—i—i i i—r 1.210 1.205 1.200 1,195 1.190 1.185 1.180 1.175 1.170 1.165 1.160 1.155 1.150 1.145 1.140 1.135 1130 1.125 1.120 1.115 1.110 1.1C ppm 'ill 3 ^n cr i ^ W T" <* J- 3 i N © *< Name 0-MOM X — From 1 094 ppm O (Jt To 1 194 ppm o re ^ Residual Error 5 84 OBn as ^ H # ppm Height Width(Hz) L/G Area Boc' -?• © A- 1 1 1813 43 33 1 19 100 833 378 / CH3 O -t- S MOMO re »?• 2 1 1633 43 06 1 22 0 96 841 680 _. o 3 1 1221 3 86 0 71 1 00 44 361 Q * UJ 290 O ° 00 4 1 1054 4 62 0 88 1 00 65 657 1 o a" o 3 3 © as ? re o 3 re O ~r T ~r T" "T" 1 195 1 190 1 185 1 180 1 175 1 170 1 165 1 160 1 155 1 150 1 145 1140 1135 1130 1125 1120 1115 1110 1105 1100 1095 ppm (55)-Benzyl 6-ethoxy-5-((methoxycarbonyi)amino)-3-methyl-4- oxohexanoate (291) CD o V=C xo so o ^f ,-in. ^1- SO r*4 O i— SO ^r (N SO SO OO E^ in r^ 00 r^ ^.in' 0—0 o o OS •S3 J: in so CN r^ rs in o o—* o t^ o 00 o o o o r*l o m o D o rt O -H r-. O -H -» SO rs| c- Os "S SO OO O oo 00 oo tN o so •* '— '— — — — cs B 5 an 01-1 SO OO OO so in oo o. E ^ 3 — O OS '5 *soosiso(Nr^(Nt^o ^ s w oo~-^-or--r--oos I - o mr^^j--H^-.ors|oo CN c--soin^mc^— SSbS cs N (N rj fsi rj oi rsi Z PM H Pi =tfc T-I cs fi ^f in so r^ oo Note: The doublet of doublets corresponding to one of the methylene protons for both the major and minor diastereomers were used to establish the ratio. The doublet corresponding to the methyl overlapped with resonances associated with the ethyl group. 381 a 'S 3 <3 re i S" <••. < Name. Oxazolidine <-< I From: 0.768 ppm © To: 1.090 ppm »X =w ! Residual Error 0 573 N C I # ppm Height Width(Hz) L/G Area Boc O at *«. c , 1 1.0562 16.09 2.22 1 00 576.143 s 4^. re ^—^ ' 2 1.0382 15.87 2.21 1 00 564.289 UJ cr O—' CH 3 O re | 3 0.9812 5 82 4.58 0.63 400.967 3 re a N »asi t 4 0 9642 6.03 4.43 0.61 399.654 cr << UJ 292 © © oo 15 0.8760 11.17 3.26 0 68 552.639 6 0.8586 10 72 3.46 0.64 557.884 3 as *o 7 0 8158 17 06 2.21 1.00 607.185 re 3 K> 8 0.7977 16.63 2.25 1,00 604.324 V© n ^M 4- O X o O" cf-K »9 O ^ ^—• K> S» 108 1.06 1.02 100 0.98 0 94 0.92 0.90 0.88 0.86 0 84 0.82 0.80 0 78 ppm