MECHANISTIC INVESTIGATION OF (DHQD)2PHAL CATALYSIS IN CHLORINATION AND DIHYDROXYLATION REACTIONS USING A NAPHTHALENE-BASED ANALOGUE Calvin Grant By 2018 A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry—Doctor of Philosophy ABSTRACT By Calvin Grant MECHANISTIC INVESTIGATION OF (DHQD)2PHAL CATALYSIS IN CHLORINATION AND DIHYDROXYLATION REACTIONS USING A NAPHTHALENE-BASED ANALOGUE This dissertation is composed of three different projects, each discussed in a chapter. Following are short descriptions of the contained chapters. Because of the low financial potential gains in antimicrobial drug development, companies have not invested much capital into the area, relying on naturally occurring compounds found within the last twenty to thirty years to keep the microbes at bay. In the mean time, microbes have been developing resistance to the most consistently used antimicrobials. Strains such as pseudomonas aeruginosa and methicillin-resistant staphylococcus are becoming more prevalent with the ubiquitous use of antimicrobials in animal feeds, cosmetics, and medications. In order to contribute to the search for new antibiotics, we persued antimicrobial lead compounds by tapping an easily accessible resource - repurposed this dissertation addresses the process of organizing, testing, and analyzing a library for antibacterial assays. A few of the positive antibacterial hits have been analyzed to investigate the mode-of-action of their class. laboratory compounds. The first chapter of In another study, discussed in chapter two, addresses a total synthesis of Alexine, a polyhydroxylated pyrrolizidine found to exhibit antiretroviral activity. While most syntheses of this family of pyrrolizidines utilized amino acids or sugars as templates, this synthesis relied on an aza-payne rearrangement and a one-carbon homologative relay ring expansion after incorporation of all necessary carbons required in the structure of alexine. This synthesis attempt ends with the formation of the initial pyrrolidine via our methodology and a proposal for the completion of the total synthesis. that in In particular, hypothesis regarding investigated. the high enantioselectivities seen In the final chapter, for which the dissertation has been titled, new advancements in halofunctionalization that have been uncovered in the Borhan laboratory are the further the enantioselectivities seen when utilizing (DHQD)2PHAL under optimized conditions suggests the halofunctionalization of unfunctionalized alkenes done by Borhan and coworkers are due to the rigidity of DHQD ligand-linker bonds C-O-C=N facilitated by the sp2 character of pendant oxygen as it donates into the electron deficient aromatic linker. In order to probe this, a naphthalene linker analogue (DHQD)2NAPH was synthesized and used as a catalyst in the optimized reactions. Computational studies were used to confirm the results and confirm the validity of the hypothesis. Copyright by CALVIN GRANT 2018 For my wife, my family, and the God who made all things possible. v ACKNOWLEDGEMENTS To Babak, you made worlds of knowledge accessible to me and trusted me to use it well. To Chrysoula, when I didn’t know where to step, you made all the paths visible and even walked me down some of them. To Crystal, you stood by me when I just stood there and even when I just sat, but your expectations of me and your support never waivered. To my family, through blood and through circumstance, your constant support and laughter was a wellspring of life. To my church, you made my successes your successes and made me a part of the whole. To my God, you provided everything, inside and outside of me, to pull success from a place within me where there was none. vi TABLE OF CONTENTS ix xi xiii LIST OF TABLES LIST OF FIGURES LIST OF SCHEMES KEY TO SYMBOLS AND ABBREVIATIONS Chapter I Antibacterial Assay and Evaluation of Laboratory Compounds I.1. Introduction I.2. Results and Discussion I.2.1. High Throughput Assay Screening of Laboratory Compounds I.2.2. Synthesis and Assay of 3-alkyl-3-hydroxytetrahydrofurans I.2.3. Mode-of-Action Studies of 3-alkyl-3-hydroxytetrahydrofurans Using Hemolysis Assays I.2.4. General Hemolytic Assays xvi 1 1 1 4 4 86 95 101 103 106 121 127 127 127 127 II.1.1. Discovery of Alexine II.1.2. Biological Activity of Alexine 127 II.1.3. Previous Synthetic Approaches to Alexine and Other Members of the Family 131 II.1.4. Retrosynthesis of Alexine 139 141 II.2.1. Synthesis of Alexine 141 149 153 174 I.3. Conclusion I.4. Experimental REFERENCES Chapter II Steps Towards the Total Synthesis of (+)-Alexine via One-Carbon Homologative Relay Ring Expansion II.1. Introduction II.2. Results and Discussion II.3. Conclusion II.4. Experimental REFERENCES vii III.2. Results and Discussion III.1.1. Overview III.1.2. Phase-transfer Catalysis in Asymmetric Halofunctionalization III.1.3. Mechanistic Insights into Asymmetric Chlorocyclization III.2.1. Synthesis of (DHQD)2NAPH III.2.2. Intramolecular Chlorofunctionalization III.2.3. Intermolecular Chlorofunctionalization Chapter III 177 Mechanistic Investigation of (DHQD)2PHAL Catalysis in Chlorination and Dihydroxylation Reactions Using a Naphthalene-Based Analogue 177 177 III.1. Introduction 177 183 186 201 201 205 210 211 217 221 227 232 234 234 249 268 III.2.3.1. Chloroetherification III.2.3.2. Dichlorination III.2.4. Computational Derivations III.2.5. Sharpless Dihydroxylation III.4.1. Synthesis III.4.2. Quantum Mechanics Modeling Experiments III.3. Conclusion III.4. Experimental REFERENCES viii LIST OF TABLES 73 91 93 94 Table I-1: Antimicrobial activity of Squalamine and comparable molecules. Table I-2: Substituted tetrahydrofuran activity against Gram-positive bacteria. 86 Table I-3: EC50 Values of 3,3-alkylhydroxytetrahydrofuran analogues of differing chain lengths. Table I-4: EC50 Values of branched and aromatic 3,3- alkylhydroxytetrahydrofuran analogues on Gram-positive bacteria. Table I-5: Evaluation of hydroxyl group importance in antibacterial activity of 3,3-alkylhydroxytetrahydrofuran analogues and EC50 values on Gram-positive bacteria. Table II-1: Action of naturally occurring alexines on mouse gut digestive glucosidase activity compared with those of DMP and castanospermine. Concenctration (M) of alkaloid giving 50% inhibition. >3.3 x 10-4 required for 50% inhibition. 3 4 Table III-1: Erosion of enantiospecificity in acetolysis from olefin-to-olefin transfer. HFIP = hexafluoroisopropanol, Tf = trifluoromethanesulfonyl, Ts = 4- toluenesulfonyl. es = (eeproduct/eestarting material) x 100%. Table III-2: (DHQD)2PHAL mediated halolactonization. a NR = no reaction after 3 h. Table III-3: Selections from the chlorolactonization substrate scope. Table III-4: Effect of benzoyl substitution on 1,1-disubstituted amide chlorocyclization. Table III-5: Olefinic aryl effects on chlorocyclization of 1,1-disubstituted amides. 190 130 181 188 180 189 ix 191 194 212 214 219 Table III-6: Chlorocyclization of 1,2-disubsituted and trisubstituted allyl amides. aReaction was run in 1-nitropropane in the presence of 300 wt% molecular sieves (4Å). Table III-7: Comparison of Chloramine-T•3H2O/(DHQD)2PHAL to DCDPH/(DHQD)2PHAL in the cyclization of allyl amines. Table III-8: Limited substrate scope of intermolecular chloroetherification by Soltanzadeh, et al. Table III-9: A comparison of intermolecular chloroetherification of aliphatic and aromatic allylic amides using (DHQD)2PHAL and (DHQD)2NAPH catalysts. Table III-10: Limited substrate scope of intermolecular dichlorination by Soltanzadeh, et al. Table III-11: A comparison of intermolecular dichlorination of aliphatic and aromatic allylic amides using (DHQD)2PHAL and (DHQD)2NAPH catalysts. Table III-12: DFT B3LYP 6-31G* energies (kcal) of single (DHQD)2Ar bond rotation. Table III-13: Dihydroxylation of trans-5-decene and trans-stilbene using a variety of 9-O-aromatic DHQD catalysts. Table III-14: Oxidation of trans-stilbene via Sharpless Dihydroxylation with (DHQD)2PHAL and (DHQD)2NAPH ligands. Table III-15: Oxidation of trans-decene via Sharpless Dihydroxylation with (DHQD)2PHAL and (DHQD)2NAPH ligands. 221 226 230 248 249 x LIST OF FIGURES 3 6 72 74 Figure I-1: Teixobactin is a gram-positive antibacterial isolated from β- proteobacteria Eleftheria terrae. Figure I-2: Gram-positive and Gram-negative activity of general repurposed laboratory compounds. Figure I-3: Squalamine, an aminosterol isolated from the stomach of the dogshark Squalus acanthias. Figure I-4: Squalamine and select squalamine mimics. Figure I-5: A. Select polymyxins, cyclic members of the cationic peptide antibiotic class. B. Select polymyxin mimics and their minimum inhibitory concentrations (μg/mL) against Gram-positive and Gram-negative bacteria compared to polymyxin B. Minimum hemolytic concentration (MHC) was also recorded in μg/mL. Figure I-6: Synthesis and antibacterial testing of benzothiazine steroids and their ketone precursors against Gram-positive and Gram-negative strains of bacteria. Figure I-7: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by steroidal compounds from an assay of general lab compounds. Figure I-8: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by select compounds from an assay of general lab compounds.80 Figure I-9: Minimum inhibitory concentration of select compounds from an assay of general lab compounds against Gram-positive and Gram-negative bacteria. Figure I-10: Minimum inhibitory concentration of charged compounds in an assay of general lab compounds against Gram-positive and Gram-negative bacteria. 82 75 76 78 81 xi 102 Figure I-11: Allicin, a compound found in garlic that has shown promising antibacterial activity in Gram-positive and Gram-negative bacteria. Figure I-12: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by select compounds from an assay of general lab compounds. Figure I-13: UV/Vis absorption of hemolysis assay supernatant of 3,3- alkylhydroxytetrahydrofurans at 550 nm. Figure I-14: Red blood cell viability after incubation with 3,3- alkylhydroxytetrahydrofurans. Figure I-15: Minimum inhibitory concentration of general laboratory compounds that exhibited no hemolytic activity. Figure II-1: Alexine structural comparison to DMDP. Figure II-2: Retrosynthetic analysis of (+)-Alexine (II-1) via one-carbon homologative relay ring expansion. Figure II-3: One-carbon homoligative relay ring expansion of aziridinols. Figure III-1: Iodolactonization via phase-transfer catalysis.. Figure III-2: Relative energy of rotation of anisole and dimethoxybenzenes. a Relative energy (Erel) is in kcal/mol. Figure III-3: Relative rotational energy of 1,4-dimethoxypyridazine, a simple model system for (DHQD)2PHAL bond rotation. Figure III-4: DFT calculation (B3LYP 6-31G*) of relative energy of (DHQD)2PHAL (a) and (DHQD)2NAPH (b). =C-O-C dihedral angle and relative energy for each structure is written below. Figure III-5: A comparison of DHQD ether catalysts divided into quadrants. 231 Figure III-6: Possible electron deficient naphthyl linkers to test for the recovery of selectivity in chlorofunctionalization. 233 128 139 140 185 222 224 83 85 96 98 227 xii LIST OF SCHEMES 5 89 90 Scheme I-1: Resazurin is reduced by viable cells to resorufin. Scheme I-2: Synthesis of 3,3-alkylhydroxytetrahydrofuran analogues via one- carbon homologative relay ring expansion. a Yields were calculated in relation to the alkyl halide. Scheme I-3: Synthesis of 3,3-alkylhydroxytetrahydrofuran analogues via nucleophilic addition. aUsed nBuLi as nucleophile. bUnless otherwise mentioned, nucleophiles were generated through magnesium insertion to form the corresponding Grignard reagent. Scheme II-1: Synthesis of pyrrolizidine natural products isoretronecanol (II-8) and trachelanthamidine (II-9). Scheme II-2: Synthesis of (-)-Trachelanthamidine (II-9). Scheme II-3: First synthesis of (+)-Alexine (II-1). This initial synthesis was based on the inherent chirality of D-glucose. Scheme II-4: Synthesis of (+)-Casuarine. Scheme II-5: Attempted synthesis of 3-epialexine analogues. Scheme II-6: Asymmetric synthesis of 1,2-diepi-alexine and 1,2,7-triepi- australine via Sharpless aminohydroxylation. Scheme II-7: Synthesis of aziridinal fragment II-57. Scheme II-8: Synthesis of vinyl halide fragment II-58. Scheme II-9: Initial nucleophilic combination of fragments. Scheme II-10: Dehydrohalogenation of cis-vinyl halide II-58b and lithiation of in situ formed alkyne. 131 132 133 135 137 138 142 143 144 145 xiii 145 147 148 152 178 179 Scheme II-11: Nucleophilic addition using Grignard bases. Scheme II-12: Alkyne reduction. a. Phillips NBSH reduction provided 93% of desired product. b. NBSH reduction of propargyl aziridinol proved more reactive and difficult to partially reduce. c. Palladium on barium sulfate proved sufficient for a quantitative partial reduction to give the desired cis-alkene. Scheme II-13: Silyl exchange and one-carbon homologative relay ring - expansion. Scheme II-14: Remaining steps in the synthesis of alexine. Scheme III-1: Spectroscopically observable haliranium ions. Scheme III-2: Mechanisms of olefin-to-olefin transfer of bromenium ions. Scheme III-3: Comparison of halenium source reactivity exhibited through bromolactonization with NBS and chlorolactonization with NCS. Scheme III-4: Iodolactonization using phase-transfer catalysis. Scheme III-5: Fluorocyclization of benzamides using an anionic phase-transfer catalyst. Scheme III-6: Importance of hydantoin structure in chlorolactonization. Scheme III-7: Evaluation of the importance of phthalazine linker in relation to enantioselectivity. Scheme III-8: Enantioselective cost evaluation of monomer modification in dimeric chlorolactonization catalysts. aThe enantioselectivities of lactone III-19e are documented below the structure of the catalyst used. bEach arrow indicates the enantioselective cost of a specified modification. Scheme III-9: Probing the importance of the phthalazine nitrogens. Scheme III-10: Synthesis of (DHQD)2PHAL. 183 184 186 192 195 197 199 201 xiv Scheme III-11: Sharpless synthesis of dihydroquinidine 9-O-(9’- phenanthryl)ether (DHQD-PHN) using Ullmann coupling. 2 Scheme III-12: Synthesis of naphthyl dimer (DHQD)2NAPH. (a) Attempted Ullmann coupling with 1,4-dibromonaphthalene. (b) Synthesis of 1,4- diiodonaphthalene. (c) Ullmann coupling and reduction to form (DHQD)2NAPH dimer. Scheme III-13: Synthesis of hydroquinidine-1,4-naphthalenediyl diether (DHQD)2NAPH. Scheme III-14: Comparison of (QD)2PHAL and (QD)2NAPH in chlorolactonization and allyl amide chlorocyclization. Scheme III-15: Substate scope for the intramolecular chlorocyclization of allene amides. Scheme III-16: Allene chlorocyclization enantioselectivities. Scheme III-17: Bromoesterification of allyl sulfonamides using (DHQD)2PHAL. Scheme III-18: Regioselectivity challenge associated with asymmetric dichlorination of unactivated olefins. Scheme III-19: Nicolaou asymmetric dichlorination of allyl alcohols. Scheme III-20: Recommended ligand for each olefin class and their enantioselectivity range. 1 Scheme III-21: Dihydroxylation of trans-stilbene and trans-5-decene with (DHQD)2NAPH and (DHQD)2PHAL. 202 204 205 206 209 210 211 218 218 228 229 xv KEY TO SYMBOLS AND ABBREVIATIONS Å Ar B3LYP BenzoPHAL BINOL CN CPA CSA DCDMH DCDPH δ DFT DHQD (DHQD)2NAPH (DHQD)2PHAL DMSO DMDP DME DNA Angstrom Aryl Beck, three-parameter, Lee-Yang-Parr Hybrid Functional 1,4-Benzophthalazine 1,1’-Bi(2-naphthol) Cinchonidine Cationic Peptide Antibiotic Cationic Steroid Antibiotic 1,3-Dichloro-5,5-dimethylhydantoin 1,3-Dichloro-5,5-diphenylhydantoin Delta (chemical shift) Density Functional Theory Dihydroquinidine Hydroquinidine 1,4-naphthalenediyl diether Hydroquinidine 1,4-phthalazinediyl diether Dimethylsulfoxide (2R,3R,4R,5R)-2,5-dihydroxymethyl-3,4- Dihydroxypyrrolidine Dimethoxyethane Deoxyribonucleic Acid xvi EC50 ee EtOAc HFIP LB λ λmax LD50 M mCPBA MeCN MeOH μg μL MHC MIC mg MHz mL mM MOM MOMCl Effective Concentration, 50% Enantiomeric Excess Ethyl Acetate Hexafluoroisopropanol Lysogeny broth, Luria Broth, Luria-Bertani Lamda (wavelength) Maximum Wavelength Lethal Dose, 50% (Median Lethal Dose) Molar meta-Chloroperoxybenzoic acid Acetonitrile Methanol Microgram Microliter Minimum Hemolytic Concentration Minimum Inhibitory Concentration Milligram Megahertz Milliliter Millimolar Methoxymethyl Methoxymethyl chloride xvii MP2 NAM NAPY NBSH nBuLi nm NMR PCC PhSCl PMB PPTS PYDZ QD REDOX RNA rt SER STO TBAI TBSCl TFE THF 2nd Order Møller-Plesset ab initio method N-acetylmuramic acid 2,7-Disubstituted-1,8-naphthyridine 2-Nitrobenzene sulfonyl hydrazide n-Butyl lithium Nanometer Nuclear Magnetic Resonance Pyridinium Chlorochromate Benzenesulfenyl Chloride para-Methoxybenzyl Pyridinium para-Toluenesulfonic Acid 3,6-Disubstituted Pyridazine Quinidine Reduction-oxidation reaction Ribonucleic Acid Room temperature Structure-Enantioselectivity Relationship Slater-Type Orbital Basis Set Tetrabutylammonium iodide t-Butylsilyl Chloride Trifluoroethanol Tetrahydrofuran xviii TLC TMEDA TMS UV wt% Thin Layer Chromatography Tetramethylethylenediamine Trimethylsilyl Ultraviolet light Weight Percent xix Chapter I Antibacterial Assay and Evaluation of Laboratory Compounds I.1. Introduction Many of well-known antimicrobials have been discovered in nature. Penicillin, streptomycin, tetracyclin, chloramphenicol, erythromycin, and vancomycin were all isolated from natural sources or were derived from natural product leads. This may lead to the assumption that the antimicrobial drugs were initially a serendipitous isolation from a natural source. In reality, the inception of antimicrobial drugs outside of the realm of naturopathic medicine began in the laboratory with such compounds as salvarsan, an organic arsenic compound used in the treatment of syphilis, and prontosil, an oral precursor to sulfanilamide and a specific activity against gram-positive cocci. 5 In order to combat the high mortality of the day, Paul Ehrilch envisioned the use of small molecules to combat bacterial diseases, or as he referred to it, “chemotherapy”. Relying on the advancement of knowledge of bacteria and developments in organic chemistry, particularly in dye synthesis, Ehrlich worked to synthesize a compound the microorganisms and a toxic element that would eliminate them. With this in mind, he was able to develop compound 606, also referred to as Salvarsan. This compound was effective against syphilis and therefore represented the first real chemotherapeutic success. that would have a specific affinity for 1 Today, we are in the midst of another crisis, ironically, stemming from the ubiquitous use of antimicrobials in addition to the amazing adaptability of microbes. The number of antibiotic-resistant infections is increasing worldwide. That, along with the low return on investments for companies (antibiotics being short-course treatments typically taken for 2 weeks as opposed to chronic illnesses and their noncurative treatments), creates a perfect storm. 6 This problem has increased in urgency so much that the Infectious Disease Society of America are making regular calls for new antimicrobials and enacting policies to encourage the search for new antimicrobials. 7 8 In light of such dire circumstances, there have been a few advancements. For example, Lewis and coworkers have recently developed new methods to grow previously uncultured organisms and have, consequently, isolated a new antibiotic dubbed teixobactin that kills Gram-positive bacteria without detectable resistance. 9 Teixobactin, shown in Figure I-1, prevents cell wall synthesis by binding III domains. As yet, all Staphylococcus aureus and Mycobacterium tuberculosis mutants have not shown any resistance to teixobactin. to highly conserved lipid II and lipid 2 O H N O N H OH H N O O N H HN HN O O OH O H N O HN O NH O O O NH HN O HN NH NH Figure I-1: Teixobactin is a gram- positive antibacterial isolated from β- proteobacteria Eleftheria terrae. While the pursuit of microbe-produced antibacterials is a wise and promising course, to neglect other avenues in the pursuit of new antimicrobials would not be wise. With this in mind, we decided to submit laboratory-synthesized compounds to an antibacterial assay as another means to search for leads. Synthesis provides an ever-increasing amount of compounds to test, whether they are intermediates in a total synthesis or repurposed compounds from methodologies and mechanistic studies. The structural complexity of each individual compound is limited only by its original purpose and the imagination of the scientists involved. Of course, to attempt an endeavor like this, a library must be created to catalog the compounds and their activity. After submission of these compounds to antimicrobial assays, the hits would be evaluated. Analogues would be synthesized and studied in order to fully understand the contribution their 3 structure makes to their activity. Finally, elucidating the mode of action would determine if this compound could be considered a viable lead. I.2. Results and Discussion I.2.1. High Throughput Assay Screening of Laboratory Compounds In order to test the lab compounds for antibacterial activity, it was important to organize them systemically in a library format that would be conducive to tracking their testing results. To this end, approximately 1,400 compounds were labeled and assigned a number. A portion of each compound was diluted to a concentration of 10 mg/mL in DMSO and stored separately at -20 °C. Gram- positive (specifically, bacillus cereus, bacillus subtilis, micrococcus luteus, and staphylococcus aureus) and Gram–negative bacterias (escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and serratia marcescens), were inoculated into 5 mL portions of autoclaved lysogeny broth (LB) and allowed to incubate in a shaker for 6 to 8 hours. 1 mL of the cell culture was diluted by adding 10 mL of LB. To prepare the well-plates, 170 μL of LB were added to each well, followed by 30 μL of the bacterial cell solution. 10 μl of the 10 mg/ml solutions of laboratory compounds was added to each well in triplicate. A streptomycin control of the same concentration was added to a well to provide a positive control with respect to Gram-positive bacteria. Tetracyclin was used as the positive control with respect to Gram-negative bacteria. The 96 well-plates were incubated at 37 °C for another 6 to 8 hours. 4 In order to test the viability of bacterial cells after incubation with the laboratory compounds, resazurin dye was used. Resazurin is a blue dye that is irreversibly reduced to resorufin, a pink compound by mitochondrial enzymes after intake by living cells, making it a useful REDOX indicator of cell viability. 10 11 12 The metabolism of resazurin to resorufin is an indication of the existence of viable cells and, in our case, an indication that the tested laboratory compound proved ineffective as an antibacterial. 1 μL of a 3.3 mg/mL solution of resazurin in water was added to each well and monitored visually and via the Benchmark Biorad microplate reader (at λ =595 nm, 655 nm) every 5 minutes until 30 minutes had passed. Compounds that maintained high concentrations of resazurin after this incubation were considered active, requiring less than 476 μg/mL to inhibit bacterial proliferation. Upon treatment with resazurin dye, 29 compounds were found to be active. Reduction by viable cells Resorufin O Resazurin HO HO O O N Blue N O Pink O Scheme I-1: Resazurin is reduced by viable cells to resorufin. 5 entry compound Gram (+) Gram (–) entry 9 10 11 12 O 13 HO 14 O 15 HO 16 O compound Gram (+) Gram (–) OAc OAc OAc OAC O O O H H H H O OH OAc O OH OH OH 1 2 3 4 5 6 7 8 O OH HO O OH O O OH OH O O O OH O Figure I-2: Gram-positive and Gram-negative activity of general repurposed laboratory compounds. 6 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) H O OAc 17 18 HO HO 19 O O 20 OH HO 21 O AcO 22 O HO 23 TsHN N 24 Br O H O H O O HO O O H O O OAc H H O O O O H H H H O H OAc O H O H OH 25 TsO 26 O 27 28 29 30 OAc OHC 31 OHC 32 O 7 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry O O O O HO OH O O O H O H OH H H H H O O O H O O O NO2 O2N N NH CO2Et H O O 33 34 35 36 37 38 39 40 41 42 43 O 44 45 46 47 48 8 Gram (+) Gram (–) compound O O O O OCOCH2CH2CH2Br O O CO2CH3 CO2CH3 OH CO2CH3 CO2CH3 CO2CH3 CO2CH3 O O PPh3 Br Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O2N N NH NO2 OH O S S OAc O OH O O HO O H OO Br O OH O HO 49 50 51 52 53 54 55 56 HO 57 O O 58 AcO 59 Ph3PCH2PPh3 2Br 60 61 62 63 64 O O O OO CO2Et O OH O HO O O HO 9 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 65 OO O PPh3 Br O O O OO OH NO2 N N H NO2 O H3CO CO2Me OH O 66 67 68 69 70 71 72 HO2C O O O OH OTHP O OH O O O N N Ph N O OAc OAc O O O 73 74 75 76 77 78 79 80 10 Figure I-2 (cont’d) CO2CH3 CO2CH3 82 H3CO2C O 83 H3CO2C H3CO2C CO2CH3 CO2CH3 CO2CH3 H3CO2C OH OH CO2CH3 O 84 85 O HO 86 OH 87 O O OH O Kaltwasser's acid 88 HO2C HO2C CO2H CO2H O OAc OH OH O O O O O O C2H5O2C C2H5O2C CO2C2H5 CO2C2H5 OH O OAc OAc O O OH 90 91 92 93 94 95 96 11 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 97 HO 98 OH OH CO2H HO O O 99 C2H5O2C CO2C2H5 C2H5O2C CO2C2H5 OH O O 100 O 101 O 102 HO2C 103 HO 104 O 105 O 106 O HO 107 O HO 108 O OH OAc O O O O H3CO2C H3CO2C CO2CH3 CO2CH3 CO2CH3 HO O O 109 110 111 112 O2C O2C –4 2BA+2 CO2 CO2 12 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 113 114 115 116 117 118 O 119 120 OH O HO para-bromocamphor CO2CH3 H O OH O HO O O O OH O 13 121 alpha-bromocamphor 122 123 124 OTs CO2H O O 125 OH O 126 HO O O O 127 128 O O HO EtO OCH3 Figure I-2 (cont’d) entry 129 130 131 132 133 134 135 136 compound O OAc O H3CO O O O HO CO2H CO2H O O2N HO O O NO2 NH N O Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O O O O OH ONP O CO2C2H5 O CO2Et O O O CO2Et O O O DNP treated O O O 137 138 139 140 141 142 143 144 14 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OH 145 146 OO OH O O CO2Me 147 OO OH 148 OO CO2Et O 149 CO2Et CO2Et OO 150 CO2Et CO2Et OO 151 OH Et Et OO 152 OH CO2Et O OAc OCOPhpNO2 OCOPhpNO2 O O O O O O O O DNP O H O O O O H DNP DNP OCOCMe3 OCOCMe3 OCOCMe3 153 154 155 156 157 158 159 160 15 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 161 162 O O O OH PMB O O DNP 163 OCH2Ph 164 165 OH OH O O EtO OEt OH OH O 166 OCH2Ph O O 167 168 OAc O 169 170 171 172 173 DNP DNP CO2CMe3 CO2CMe3 DNP DNP OCMe3 OCMe3 DNP CO2CH3 174 OO CO2H O OO OH OH 175 176 16 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 177 OO CO2Me 185 Geranyl phenyl ether O O O O OAc OH O N CO2H CO2H O O 178 179 180 Br O O 181 O O 182 183 184 O O O O CBr2 HO 186 187 188 189 190 191 192 O OMe OEt O OH MeO CD3 O CD3 D3CO OMe OH OMe O H O O O O H O O O H3CO H3CO 17 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 193 O O O H H H 194 Ph O O Ph O 195 196 197 198 199 200 O CHPh O EtO2C EtO2C O O O O O O O O O O O O O O OAc O O H O H Br N CO2CH3 O CO2H OH O 201 202 O O 203 204 205 O O O O O 206 O O H OMe 207 208 18 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 209 210 211 212 213 214 215 O 216 O OH O CD3 MeO D3CO O O O CD3 CD3 O O O O O OTs O OEt O Br Br MeO 217 218 219 220 221 222 223 224 MeO OMe OHD D3CO O O O OH OH O OH OAc O N O CO2H 19 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 209 210 211 212 213 214 215 O 216 O OH O CD3 MeO O CD3 CD3 O O O O O D3CO O O TsO O Br Br MeO O OEt MeO MeO OHD D3CO O O O OH OH O OH OAc O N O CO2H 217 218 219 220 221 222 223 224 20 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 225 226 227 228 229 230 231 232 OH MeO beta-OH MeO OH O CD3 O OH OH D OMe OCD3 OAc O HN H EtO O O O OEt CHO O O O O O O OMe OH O CD3 OH OH D OMe OMe O O O 233 234 235 236 237 238 239 240 21 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 241 O O O 242 H3CO 243 H3CO O O O OAc OHC HO N S O O O O 244 245 246 247 248 Ts NN H H3CO O O O O O HO NHTs TsHN O S Ph MeO O Ph S O OH HO O S Ph OH S O S Ph MeO HO HO Ph S O Ph S O 249 250 251 252 253 254 255 256 22 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 257 258 259 260 261 262 263 264 O S Ph DNP O S Ph CO2H O O H O OH S S OH O S S O S S OH OH H O S S O O OH O O O O HO O OH O O O 265 266 267 268 269 270 271 272 23 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 273 274 275 276 277 278 MeO OH OH O OH OH OAc O O OH OH OH O O 279 280 OH HO MeO MeO O O O O O O O O O O O O O MeO MeO MeO MeO MeO HO MeO MeO O MeO MeO MeO MeO O MeO MeO O MeO O O H H H H H 281 282 283 284 285 286 287 288 24 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O O 289 O O MeO 290 MeO MeO O MeO MeO MeO Br O O O Br O OH O O O 291 292 293 294 295 296 O O O O OH HO OH O O O O O O O PhS O O O O 297 298 299 300 301 302 303 304 25 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O OH O 305 306 307 H3CO 308 Ph Cl O 309 H I Si OH ONa O Ph 310 Ph O 311 O Br 312 O 313 314 Ph OH O Ph Ph O 315 O Ph Ph O O Ph CO2Et CO2Et OEt O CO2H CO2H O O OH 316 317 318 Ph 319 320 26 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) CO2H CO2Et OH DNP O O O O O O 321 322 323 324 325 326 327 328 329 330 OH O O O 331 Formalin dimedone O O CO2Et CO2Et HO O O O O O O 332 333 334 335 336 27 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry Gram (+) Gram (–) compound Ph Ph O O O O 345 O 337 Ph Cu+2 Ph H O 2 EtO O 338 339 340 O H O H O O Ph Ph O O 341 HO 342 O Ph 343 O 344 O O H O O 346 OH H O O beta-epoxide 347 O AcO OAc AcO OAc OMe MeO O O O O AcO OAc OAc 348 349 350 351 352 28 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O OAc OAc OAc Ph Ph HO OH OH OH OH H O beta-epoxide H O beta-epoxide O O Br Br O O HO OH O O O O O 353 354 355 356 357 358 359 360 O H O H O O Ph O O Ph OO alpha-pulegone oxide O O O O O O O 361 362 363 364 365 366 367 368 29 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O H O O OH D D Cl O O O O NNHCONH2 O O D D O O O O 369 370 371 372 373 374 375 376 O O OH O H NNHCONH2 O H Cis O O HO O O O O OH O O OH 377 378 379 380 381 382 383 384 30 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 385 386 387 388 389 390 391 392 OH OMe OTMS CN OH CN OMe OMe OMe OH OH OMe 1st isomer OH OH OMe 2nd isomer OH OMe OMe O OMe OMe OMe O O O O O N OH O O O 393 394 395 396 397 398 399 400 31 Figure I-2 (cont’d) compound Gram (+) Gram (–) entry compound OAc Gram (+) Gram (–) O DNP O DNP H O TBS O O O Br OMe Br OMe CO2H 409 410 411 412 413 414 415 416 32 entry 401 402 O OH OH O OH O 403 Br O 404 405 Br O O H O O O 406 PhS O O H O O 407 408 Figure I-2 (cont’d) entry 417 418 419 compound OMe OH OMe O CO2H O H H OH Gram (+) Gram (–) entry compound Gram (+) Gram (–) 425 HO OH OH OH 426 Ph O O 427 Ph O DNP 420 H H 428 H CO2Et CO2Et CO2Et OH 421 H H 422 423 424 OH O O O NOH NO2 429 430 431 432 O Ph3C H O O O O CPh3 CPh3 33 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 433 434 435 436 437 OH Ph3C O O H OH OH O OMe O OH O OMe O OMe DNP 438 H CO2Me 439 440 O OH CO2Me O 441 442 443 CO2H O CO2H OH H CO2Et O 444 Cl CO2H O O O O IM O OMe 445 446 447 448 34 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 449 450 451 452 453 454 455 456 OTBS OTMS O IM O O O O IM O CH2OH OH CH2OH OH O O OMe 457 O O 458 CH3CONHNH2 459 CH3CONHN=CHOEt N N O N N O 460 461 462 463 464 OMe O O O OAc OAc OAc 35 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) OAc 465 O 466 O 467 O 468 O 469 EtO 470 O 471 472 O OH O O O O O OAc OAc entry compound Gram (+) Gram (–) OAc OAc O O O O O OMe O O 473 474 475 476 AcO 477 TBSO 478 O 479 O 480 O 36 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OTBS H3COH2C 489 O 481 D3C O 482 CD3 O O O O 483 484 485 586 O PhH2C 487 PhH2C O 488 PhH2C PhH2C O OCH3 OTBS OTBS OTBS impure OCH3 NHTs O 490 O 491 492 493 494 O OCH3 O OCH3 OH OTBS OTBS OTBS 495 MeO O 496 TsHNN OCH3 37 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OTBS 497 OCH3 OTBS 498 O 499 H3CO TsHNN O O O 500 501 502 503 O O 504 O O O OTBS OTBS OTBS OTBS OAc 505 O 506 507 508 O TBSO TBSO H O O 509 Br O H OH OH O O O 510 HO 511 BzO 512 BzO 38 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 513 O 514 515 516 517 O O 518 H3CO O 519 520 O O O O OH O OTBS OH O O O 521 522 EtO2C 523 EtO2C 524 EtO2C 525 EtO2C O O O O CO2Me CO2Me OH CO2Me Br Br Br Br HO CO2Me O Br CO2Me O O CO2Me CO2Me Br 526 Br O Br 527 Br O O 528 Br 39 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O O O O O CO2Me CO2Me CO2Me CO2Me CO2H CO2Me CO2Me CO2Me O O 529 530 531 EtO2C 532 EtO2C 533 HO 534 EtO2C 535 EtO2C 536 O O O O CO2Me CO2Me CO2Me OH O OTBS O OTBS O O Br Br 537 538 O O 539 EtO2C 540 541 542 543 544 O O TBSO O O 40 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 545 O Ph O 546 TBSO 547 548 O 549 O 550 O O O 551 552 O O O O OH O CO2Me CO2Me CO2Me CO2Me OH OH OO CO2Et CO2Et O 553 CO2H 554 D D D D D O O 2,4 DNP O O OH DNP CO2H O CO2Et CO2H 555 556 557 558 559 560 41 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OH O O O H O 561 562 563 OH O 564 MeO2C CO2Me O 565 566 567 568 O O O O O O O O NNHTs OH S O O O S O O O O O O S O O OH OH OH OH 569 570 571 572 573 574 575 576 42 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OH OH C8H17 C8H17 C8H17 C8H17 577 578 579 580 581 HOOC OH D D C8H17 582 TsO OH 583 584 D OH C8H17 Br O CO2Br OH OH S S B OH O O E/Z mixture OH O Ar O O Ar O O Ar O O Ar O 585 586 587 588 589 590 591 592 43 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 593 594 595 596 597 598 AcO 599 600 O Ar O O Ar O ODNBH ODNBH HO OH O O O OH Br OH OH Br OH OH Cl OH OH Cl OH OH O Br O O Br O O O Br OH O O O O Ph 601 602 603 604 605 606 607 608 44 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) NO2 O O O Ar O O Ar O OH OH Ph OH NO2 NO2 609 610 611 612 613 614 6`5 Ph O 616 OH O O O HO O OH Ph O OH AcO Ph OH O O OO Br 617 618 619 620 621 622 623 624 45 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 625 626 627 628 629 630 631 632 O HO O OH OH Ar O O TMS O O C6H11 O Ar O OH O O O Ph O OH OH O Ph O OH OH HO OH 633 634 635 636 637 638 639 640 46 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O 641 CO2Na 642 643 644 645 646 O Ph OH O O H Ph O Ph Ph O Ph Ph 647 Ph O H Ph O 648 O O 649 650 651 652 MeO 653 654 HCl Me2N O O O Ph H O H Ph NNH2 CN H OMe Br O OMe CO2H 655 PhCH2OCH2CH2OH 656 N TsHN 47 Figure I-2 (cont’d) compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) HO OH O entry 657 658 659 TsO OTs O O Me2N HCl 660 O CO2H 661 662 O O O O S O C7H7 665 666 667 668 669 670 O S O C7H7 Ph Ph O Ph Ph OH OH O O Ph Ph OH Ph O O O TMS OtBu AcO OAc 663 671 H2B CN O O 664 672 Ph H O H Ph 48 entry 673 674 OO OH O CO2Et O 675 H CO2Et CO2Et EtO2C 676 677 678 O CO2Et O O S S S S 679 O Ph H Ph 780 O S C7H7 O Figure I-2 (cont’d) compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) CO2CH3 O O O O O O 681 EtO2CO 682 AcO 683 AcO 684 O 685 686 687 49 Br Br OH OH OMe OMe OH OH Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O O O O O 688 689 691 692 693 694 695 O O OH OH OMe OMe O OMe OMe O O OMe OMe OMe OMe O O O O OMe OMe OMe OMe OMe OMe OMe OMe OTs OH H H 696 697 698 699 700 TsO 701 EtO2CO 702 O O 703 O 50 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OAc OAc O O OCH3 OAc OAc O O H3CO 704 705 706 707 O O O O O H H3CO HO H H 708 O 709 O 710 711 712 713 O 714 715 O 716 O O H EtO2CO H 717 AcO 718 HO 719 H H O OH H CO2CH3 CO2CH3 CO2CH3 CO2CH3 CO2CH3 H OH H H H O H OH OAc O O EtO2CO H 51 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 720 O 721 H3CO 722 EtO2CO 723 EtO2CO 724 725 726 EtO2CO 727 H O O H O H OH H H H O H HO Me O H OH O H3CO Br H O H H H3CO O O O O CO2CH3 CO2CH3 CO2CH3 O O O CO2CH3 CO2CH3 O O CO2CH3 H H H O H O OMs H H O H O O O H O H H H 728 729 O O H3CO 730 731 AcO H 732 AcO H3CO 733 734 EtO2CO H H3CO 735 52 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O H OMs H3CO O OH HO H H H O OH O O BnO O H O O O O H3CO H HO H3CO O OH O H H H3CO OMs OH H O O O O O O 736 737 D3C O 738 H3CO O H OH 739 740 O H OH O H3CO HO H 741 HO 742 O H3CO H OH OMOM 743 O H 744 745 746 747 748 749 750 751 53 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 752 O HO H 735 O H O OH H H H H H O OH H OH O H H 754 O 755 HO 756 757 O H3CO 758 O 759 H3CO OH OH O O OH O OH O O CO2CH3 OTBS O 760 761 HO 762 O H O H H O H EtO2CO H 763 764 H3CO O O H3CO H 765 766 H H O O O (Me2N)2OPO 767 AcO H 54 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O H H O O 768 TBSO 769 770 O 771 HO 772 H3CO H O H H O H O Br H Br H3CO Br 773 Br O 774 O H H O H OMs 775 H O HO CO2CH3 O O O CO2CH3 O OH O H3CO MsO O 776 777 H H EtO2CO H O CO2CH3 CO2CH3 OH CO2CH3 778 O 779 EtO2CO 780 781 782 AcO 783 55 H O H H H3CO OH O OH OCH3 H OH O HO H Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 784 H3CO O OAc O H O O O O CO2CH3 O CO2CH3 785 Ar O 786 787 O 788 789 790 BzO 791 HO Cl O O OAc O O H H O H H H O O O OH O H O H 792 HO 793 H3CO TMSO OH O CO2CH3 794 O 795 O 796 797 798 799 AcO AcO HO AcO 56 O O O O O Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O 800 TBDPSO O H 801 HO 802 BrH2CO2CO OH O O O O OHC OH OH O OMe OMe O O OMe OMe OMe OMe O 803 804 805 806 807 O OAc O O OAc OAc O OH O OAc O OTBDPS O OTBS O O O O O OMe 808 O OH OH O O O O 809 810 811 812 813 814 815 57 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) OAc OAc O OAc OAc OAc OH OH O O O O O OMe OMe O O 816 817 818 819 820 821 TsHNN 822 O O 823 O O 824 O 825 826 827 828 829 830 HO 831 H H Br H O O O O H O OMe O O OTBS OTBS OAc OTBS OAc H H H O H OH O 58 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O OTBS O O 832 O 833 MeO H O H H O 834 835 H O H O H H MeO HHO 836 MeO O H OAc O 837 O O O OEt 838 Ph 839 Ph Ph OH Ph O O 840 O Ph3C Ph 841 Ph OH Ph NOH OH Ph Ph O O Ph2HC Ph Ph O Ph Ph O O NHBoc HN O O N H O NH O OCH3 842 843 844 845 846 847 59 Figure I-2 (cont’d) entry 848 849 850 851 compound Ph O O O OCOCH3 O Br HO OH 852 EtO2C COOH O O CO2H CO2R CO2H O O 853 854 855 Gram (+) Gram (–) entry compound Gram (+) Gram (–) O N2 MeO OMe O Br O F2C F2C F2 C CF3 F CF2 C F2 O H3CO2C CO2CH3 N N H OO OH CH2OH O 856 857 508 859 860 861 862 863 60 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 864 865 O O O H H CO2H CO2H 866 OO OAc O OAc O O O O OH O O 867 868 869 870 871 HO OH O HN SO2 N C7H7 OMe O OTBS 872 873 874 875 O O O O OTBS OTBS O 876 877 878 879 61 Figure I-2 (cont’d) compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) 888 889 890 891 892 893 894 EtO O O O CO2Et O O O H O O Ph Ph Ca2+ 2 OTs O O O Ph O 895 O 62 entry 880 881 O O O 882 EtO2C CO2Et 883 884 885 886 887 O O O O O O O O Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) Ph O 896 O 897 898 899 900 901 902 903 OO O O O CO2H Br OH O OAc HO OH O O O O N Ph O HO OH O O O O O O OC2H5 O H O H O OH CO2Me O S S 904 905 906 907 908 909 910 911 63 Figure I-2 (cont’d) compound Gram (+) Gram (–) entry entry 912 913 914 915 916 917 918 O PhS S Ph O OH Ph S O OH OTMS OTMS Ph O N S O H Br OH Ph O O 919 N H CO2Et 920 921 O Gram (+) Gram (–) compound O NH O O O H N O H N CO2Et OBn N Bn CO2Et CONHBn 922 923 924 925 926 927 64 O N EtO2C Ph O OEt CO2Et Ph BnHN CO2Et BnHN CO2Et O OEt BnO Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) Ph OH Ph O O CO2Et O NNHSO2Ar 936 937 938 928 929 930 931 HO 932 933 O O NHCbz O O O O NHCbz HO O O NHCbz O O NHCbz HN HN 934 HN 935 Ph Ph O O O NHBoc O O O NHCbz H O HN HN Ph Ph Cl O Cl O O NHCbz O OH Ph Ph O OH NHCbz O O HN N O O NHCbz HN 939 O O CbzHN 940 942 943 65 OH OHC HO 941 HN Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O HO 944 945 946 HO 947 948 949 950 HO 951 O NH O N H O O NHCbz O O O NHCbz O O OH NH2.HCl Ph Ph O NTs O N H O O NHCbz O O O Ph OH OH C6H11 OBNR OH OH H O H N N CONH2 O HO 952 953 954 955 956 957 958 959 66 Figure I-2 (cont’d) entry 960 compound O O Ph Gram (+) Gram (–) entry compound Gram (+) Gram (–) 968 O O Ph 961 962 963 964 965 966 967 O O O O O Ph OH OH OH O OAc O H H O O NO2 NO2 O 969 O OHOH Br O O Ph ODNB ODNB O O Ph 970 971 972 973 974 HO 975 67 Figure I-2 (cont’d) Gram (+) Gram (–) entry compound Gram (+) Gram (–) compound OH O entry 976 HO2C O OH CO2H OO O HO2C OH O O O O O OO O O 977 978 979 980 O 981 D3C D D O 982 983 CD3 O O D D CONH2 984 985 986 Ph3C O O O O 987 H CH2OMs CH2OMs CH2OMs O OH O S Ph Cl O TMS O H 988 989 990 991 68 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry compound Gram (+) Gram (–) O O O O O MeO H O O H H MeO HHO O MeO HHO MeO MsO O H O H H MeO H3C Ph H O O O Ph H O 992 993 994 995 996 997 998 O OH 999 O H H O 1000 OMe OMe O 1001 Ph COCH3 COCH3 O CO2Et CONHPh NNH2 CO2Et Ph OH N Ph 1002 1003 1004 1005 1006 O 1007 69 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) entry Gram (+) Gram (–) compound NH2 O CO2Et Ph 1016 1008 Cl 1009 1010 O2N 1011 1012 Cl O MeO MeO 1013 CO2Et H N O CO2Et H N O NH2.HCl O CO2H Ph NH CO2H OH O Ph N H NHCbz 1014 MeO 1015 MeO 1017 H2N 1018 1019 CO2Et H N O CO2Et tBuO O CO2Et 1020 HN CO2Et H N O 1021 1022 NH S N H H N O CO2Et N O O N H Ph EtO2C 1023 Cl NH S 70 Figure I-2 (cont’d) entry compound Gram (+) Gram (–) 1024 O N Ph N H EtO2C 1025 Cl N O EtO2C Ph 1026 Cl N O EtO2C C8H17 1027 TsO OH 71 Amongst the biologically active molecules, we noticed a group of steroidal compounds that were active against Gram-positive bacteria. While it is not common, steroidal compounds have been isolated that have shown antibiotic activity. 13 Squalamine, an aminosterol antibiotic shown in Figure I-3, was isolated from the stomach of the dogfish shark Squalus acanthias. This steroid exhibits significant antibacterial activity in both Gram-positive bacteria, such as the Staphylococci strains (1-2 μg/mL for both aureus and faecalis), and Gram- negative bacteria, such as Eschericia coli, Pseudomonas aeruginosa, and Serratia marcescens (1-2 μg/mL, 4-8 μg/mL, and >125 μg/mL, respectively) as shown in Table I-1. This is notable when compared to ampicillin, a known and therapeutically utilized broad-spectrum antibacterial that interestingly exhibits comparable activity to the natural steroid with S. aureus at <1 μg/mL, S. faecalis at <0.25 μg/mL, E. coli at 2.4 μg/mL, P. aeruginosa at 62-125 μg/mL, and S. marcescens at 4-62 μg/mL. OSO3H H2N H H N H N H H OH Figure I-3: Squalamine, an aminosterol isolated from the stomach of the dogshark Squalus acanthias. In order to highlight the importance of the specific combination that makes up squalamine, taurolithocholic acid 3-sulfate, a bile acid salt, and spermidine, a 72 polyamine found in living tissue that has various metabolic functions, were both tested and demonstrated no activity at concentrations less than 500 μg/mL. Individually, neither compound is an effective antibacterial. It should be noted, however, that bile acids have been found to exhibit some protection against bacteria. 14 15 Squalamine was found to be more active against bacterial strains than megainin- II, a cationic peptide antibiotic. For example, where squalamine exhibited a 1-2 μg/mL minimum inhibitory concentration with respect to E. coli, magainin-II required 31-62 μg/mL. This trend spanned all evaluated strains of bacteria and the tested fungus and protozoa. Cationic peptide antibiotics (CPAs) are a class of peptide that adopt α-helical conformations or β-sheet conformations and subsequently, exhibit facially amphiphilic secondary structure that has been O NH NaO3S NH2 O HH S N H N O Ampicillin O H2N OH H N Spermidine NH2 NaO3SO H Taurolithocholic acid 3-sulfate Sample Ampicillin Squalamine Taurolithocholic acid 3-sulfate Spermidine Magainin-II amide E. coli 2-4 1-2 >500 >500 31-62 Antimicrobial activity (MIC), µg/mL Pseudomonas aeruginosa Staphylococcus aureus 62-125 4-8 >500 >500 31-62 <1 1-2 >500 >500 >250 MIC = Minimum Inhibitory Concentration Staphylococcus faecalis <0.25 1-2 >500 250-500 125-250 Serratia marcescens 4-62 >125 >500 >500 >250 Table I-1: Antimicrobial activity of Squalamine and comparable molecules. 73 found to have potent antibacterial properties via two models that exist to represent the modes of action: (1) In the “carpet model”, the amphiphilic peptides adhere to the negatively charged bacterial membrane and remove portions of the membrane once they’ve reached a high enough concentration, compromising the membrane. (2) In the “barrel-and-stave model”, the cationic peptides reach into the anionic cell membrane like the staves of a barrel, creating stable pores. 16 H2N Squalamine N H I-1 N H O N H H H I-2 HO3SO OH H H OH HO Figure I-4: Squalamine and select squalamine mimics. H H H OH N H N H H N H N O N H I-3 OSO3H NH2 NH2 Antibacterial steroids have also been the subject of synthesis.17 18 Savage and coworkers synthesized cationic steroid antibiotics (CSAs) based on the rigid and predictable structure of steroids and the overall amphiphilic nature of the previously mentioned CPAs. CSAs have been classed into two categories: (1) Squalamine and the squalamine mimics synthesized and tested by Regen and 74 coworkers 19 20 featured in Figure I-4 and (2) polymyxin mimics. Polymyxins (shown in Figure I-5A) are cyclic CPAs known for their ability to rupture the outer and inner membranes of Gram-negative bacteria. 21 Similarly, polymyxin mimics were found to be selective for bacterial cells, exhibiting a bacteriocidal propensity not mirrored in its higher hemolytic activity. For example, compound I-5c had a minimum inhibitory concentration of 3.0 μg/mL with Streptococcus pyogenes with a minimum hemolytic concentration of greater than 200 μg/mL (Figure I-5B). H2N O O NH HN HN H2N R1 H2N R2 H N NH O O HN O O O HN O HN HN NH HN R1 = CH3, R2 = iPr Colistin I-4a R1 = H, R2 = Ph Polymyxin B1 I-4b R1 = CH3, R2 = Ph Polymyxin B2 I-4c OH O O NH2 O NH2 A B H2N O H2N O H H O R NH2 NH2 NH2 NH2 O O O Polymyxin B R = -OH, I-5a R = -NHC8H17, I-5b R = -NH(CH2)3NH(CH2)3NH2, I-5c R = -N((CH2)3NH2)2, I-5d nra = not reported E. coli 1.8 36 3.0 6.6 7.3 Salmonella typhimurium Staphylococcus aureus Streptococcus pyrogenes R Minimum hemolytic concentrations nra 43 nra 25 12 26 2.0 0.40 4.6 2.0 19 4.2 2.3 3.0 1.6 nra 100 29 >200 >200 Figure I-5: A. Select polymyxins, cyclic members of the cationic peptide antibiotic class. B. Select polymyxin mimics and their minimum inhibitory concentrations (μg/mL) against Gram-positive and Gram- negative bacteria compared to polymyxin B. Minimum hemolytic concentration (MHC) was also recorded in μg/mL. 75 These compounds have shown broad-spectrum antibacterial activity comparable to polymyxin B in the case of both Gram-positive and Gram-negative bacteria. 22 Shamsuzzaman and coworkers synthesized another class of steroids, featured in Figure I-6, that target internal bacterial pathways instead of directly compromising cell integrity. 23 They found steroidal compounds advantageous because of their low toxicity, the ease with which they penetrate the cell wall, and their high bioavailability. With into in mind, steroidal ketones were modified this benzothiazines. These compounds are known to exhibit antibacterial effects via inhibition of bacterial topoisomerase II (also known as DNA-gyrase), an enzyme X H H H O X OAc I-6a Cl I-6b H I-6c I2,2-aminothiophenol EtOH, reflux, 19-21h H H H N X S X OAc I-7a Cl I-7b H I-7c I-6a 64 64 128 64 I-6b 128 128 128 256 I-6c 256 256 128 128 I-7a 32 32 64 32 I-7b 32 64 64 128 I-7c 21 64 128 32 Chloramphenicol 32 32 32 32 S. aureus S. pyogenes P. aeruginosa E. coli MIC (µg/mL) Figure I-6: Synthesis and antibacterial testing of benzothiazine steroids and their ketone precursors against Gram-positive and Gram- negative strains of bacteria. 76 five fold higher in effective concentration than (Figure the I-7a-c were three desired benzothiazine products necessary for DNA replication.24 Submission of three steroidal ketones to a one- pot α-iodination and subsequent cyclization using 2-aminothiophenol led to formation of I-6). incubated with S. pyogenes, S. aureus, P. Benzothiazines aeruginosa, and E. coli along with their ketone predecessors and a chloramphenicol control. When looking at Figure I-6, it is apparent that the steroidal ketones are not as effective as the benzothiazines (all ketones were two to their benzothiazine counterparts). Compound I-7a proved especially effective with comparative activity to that of chloramphenicol (32 μg/mL with all bacteria except P. aeruginosa, 64 μg/mL). Interestingly, all active steroids tested in our laboratory manifested activity only against Gram-positive strains of bacteria. With regard to the assayed steroids (Figure I-7), diepoxide I-8a was compared with other ketone steroids that lacked the epoxidation of the polar α,β-unsaturation and, in the absence of the oxirane, lacked any notable activity against any of the evaluated bacteria. In a search for assayed steroids with α,β-epoxidation, we found no steroid similar enough for a direct comparison. The tested α,β-oxo-keto-steroids exhibited no activity against either of the tested classes of bacteria. 77 Gram (+) Gram (-) Gram (+) Gram (-) OH OH OH O O O O O O H O O H O H O I-8a I-8b I-8c I-9a I-9b I-10a O O O O O O O O O O O O O O O H3CO H3CO I-10b O O I-10c O O I-10d OH OH I-11a OMe OMe I-11b OMe OMe I-11c MIC ≤ 476 µg/mL MIC > 476 µg/mL Figure I-7: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by steroidal compounds from an assay of general lab compounds. 1,4-benzoquinone based steroids I-9 and I-10 were found to exhibit antibacterial activity for as long as there was no substitution on carbons 2 and 3 of the 78 quinone moiety. This may be due to the reduced electron deficient nature of the olefin as exemplified by the methoxy-substituted steroids I-10c and I-10d. Naturally isolated25 and synthetic26 benzoquinones have been previously used as bacteriocides and have been found to block essential enzymes via combination with sulfhydryl groups of the enzymes themselves or essential bacterial metabolites27. Hydroquinone derived steroid I-11a and like steroids showed Gram-positive inhibition though methyl protection of the diol moiety resulted in complete in loss of observable activity. Such activity has been seen hydroquinone derivatives such as natural products culpin28 and avarol29. We suspect that the mode of action is similar to that of the benzoquinone derivatives, requiring only prior oxidation to become active30. Cyclobutanols, pictured in Figure I-8, have also been found to provide Gram- positive bioactivity. Activity was consistently observed whether or not the aryl substitution persisted. The pendant hydroxyl group seems to be essential for antibacterial activity. Upon oxidation of this group (as seen in compound I-12c) or protection of the hydroxyl group (namely, as an ester as demonstrated by compound I-12f), the activity is lost. While cyclobutanes have been moieties present in many natural products and patented for their use as antivirals31, we have found none used as antibacterials. 79 Gram (+) Gram (-) Gram (+) Gram (-) OH I-12a OH I-12b O I-12c HO I-12d OH C6H11 I-12e O Ar O I-12f MIC ≤ 476 µg/mL MIC > 476 µg/mL Figure I-8: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by select compounds from an assay of general lab compounds. Aryl compounds I-13a-e, when tested, provided Gram-positive activity for diketones and alcohols (Figure I-9). Ester I-13e provided no detectable activity for Gram-negative cells. Even more interestingly, in a comparison of I-13b and I-13c, the loss of a carbonyl seems to have contributed to the new activity in Gram-negative bacteria. Among the tested carboxylic acids, I-14a also exhibited activity against both Gram-positive and Gram-negative bacteria. While it could be suggested that this is due to the amphiphilic nature of the compound, it appears that I-14b, exhibiting singular Gram-positive activity, and I-14c, lacking 80 completely in activity, would be more drastically amphiphilic due to their more extensive hydrophobic regions. Gram (+) Gram (-) Gram (+) Gram (-) O O I-13a O Ph I-13b I-13c Ph O O O Ph I-13d Ph Ph O O O O I-13e Ph OH Ph OH Gram (+) Gram (-) CO2H O OH I-14a Ph3C O I-14b OMe OH O CO2H I-14c MIC £ 476 µg/mL MIC > 476 µg/mL Figure I-9: Minimum inhibitory concentration of select compounds from an assay of general lab compounds against Gram-positive and Gram-negative bacteria. 81 Also among the tested compounds, we found two hits I-15a and I-15b, anionic and cationic, respectively, that inhibited growth in both Gram-positive and Gram- negative bacteria in a subclass of charged molecules (FigureI-10). This type of activity has been seen before 17 32 and theoretically results in the lysis of the cell membrane and, eventually, cell death. This is not to say, however, that charged compounds will always produce activity in bacterial cell lines. Compound I-15c produced no activity in either Gram-positive or Gram-negative bacteria though it is a highly charged molecule. This may be due to the limited hydrophobic portions in the molecule. It would seem likely that this compound is highly soluble in water and would have no considerable effect on the cell membrane. Gram (+) Gram (-) O O Ca2+ 2 Ph Ph I-15a Ph3P Br PPh3 Br O2C O2C I-15b CO2 CO2 I-15c -4 2Ba2+ MIC ≤ 476 µg/mL MIC > 476 µg/mL Figure I-10: Minimum inhibitory concentration of charged compounds in an assay of general lab compounds against Gram-positive and Gram-negative bacteria. 82 Sulfur based compounds had varying activity (Figure I-12). It appeared that sulfur compounds specifically containing S-S and S-N bonds were found to be bacteriocidal against Gram-positive bacteria. Compounds I-16a and I-16b provided activity where I-16c and I-16d did not. Notably, allicin (pictured in Figure I-11), a disulfide compound found in Allium sativum (garlic), has displayed Gram-positive (S. aureus with a 50% lethal dose concentration or LD50 of 12 μg/mL) and Gram-negative (E. coli with an LD50 of 15 μg/mL) bacteriocidal activity 33, 34. Allicin was found to inhibit certain important thiol containing enzymes, such as acetate kinase and phosphotransacetyl-CoA synthetase. For example, allicin has shown partially inhibit DNA and protein synthesis in Salmonella typhimurium while completely shutting down RNA synthesis at bacteriostatic concentrations of 0.2 to 0.5 mM. O S S Figure I-11: Allicin, a compound found in garlic that has shown promising antibacterial activity in Gram-positive and Gram-negative bacteria. Still, disulfides as antibacterials are rare outside of peptide antibiotics35. Hocquellet and coworkers have found that hepcidin 25, a 25 amino acid antibacterial peptide known for its function as an iron regulator in humans 36, does not kill bacteria by destroying their membrane. Instead, the disulfide bonds 83 are necessary in order for peptide to bind to DNA. It is not clear whether these disulfide bonds are solely to maintain the structure of the peptide or if it also participates directly in binding to DNA. Some less obvious hits produced interesting results. Bicyclic compound I-17 exhibited activity in Gram-positive strains. Interestingly, alkynyl compounds produced activity in Gram-positive strains (I-18a and I-19a) and, in some cases, both Gram-positive and –negative strains (I-19b). In order to try to better understand the activity of compound I-18a, we compared it to I-18b. I-18a is biologically active because of the terminal alkyne moiety. Internal alkyne esters I-19a and I-19b differ only in the substitution of C3. The difference is not very great but the results are substantially different. The activity seen in Gram-negative cells in the case of the protected propargyl alcohol I-19b is lost when the CH2OBn is absent. While there are other in the literature with antimicrobial activity,37,38 their complexity complicates the direct association of their activity to the alkynes within. 84 Gram (+) Gram (-) O O O I-17 OH O I-18a OH O I-18b Ph O N S O I-16a O O S S Ph I-16b OH I-16c O O Ph O S PhS O I-16d Gram (+) Gram (-) Gram (+) Gram (-) O OEt I-19a Ph O OEt BnO I-19b MIC ≤ 476 µg/mL MIC > 476 µg/mL Figure I-12: Minimum inhibitory concentration of Gram-positive and Gram- negative bacteria by select compounds from an assay of general lab compounds. 85 I.2.2. Synthesis and Assay of 3-alkyl-3-hydroxytetrahydrofurans In 2004, Borhan and coworkers published work on the sulfoxonium ylide induced one-carbon homologative relay ring expansion of 2,3-epoxyalcohols to form initial stereochemical substituted enrichment.39 The methodology rearrangement of enantiomerically pure epoxyalcohols, followed by sulfoxonium ylide attack on the least substituted side of the generated epoxide. The displacement of DMSO tetrahydrofurans while maintaining relied on Payne the Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus 1 2 3 4 5 6 7 8 9 Streptomycin I-20a HO O 167 250 333 ND HO O O O O O O O O 33 NA NA NA NA NA NA NA 74 NA NA NA NA NA NA NA I-20b BnO I-21a HO BnO I-21b HO BnO I-21c BnO I-22a HO BnO I-22b BnO I-22c BnO I-22d HO HO HO HO <23 <23 ND 167 1000 NA NA NA NA <23 ND ND NA NA NA NA NA NA NA NA *Numbers represent EC50 values (µg/ml). Compounds were added until activity was observed. ND = Active but EC50 not determined NA = No Activity Table I-2: Substituted tetrahydrofuran activity against Gram-positive bacteria. 86 Table I-2 (cont’d) Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus Streptomycin O O Cl 10 11 12 13 14 15 16 17 18 TrO I-23a AcO HO I-23b HO TBSO I-24 TBSO HO HO I-25 TBSO I-26 TBSO I-27 I-28 I-29a I-29b O O Cl OH OH OH 167 NA NA NA NA NA 333 NA NA NA NA NA <23 833 NA NA NA NA OAc O O O 952 1333 666 NA NA NA NA NA NA NA NA NA O O O O HO O HO O HO <23 NA NA NA NA NA NA NA NA NA *Numbers represent EC50 values (µg/ml). Compounds were added until activity was observed. ND = Active but EC50 not determined NA = No Activity intramolecularly (5-endo-tet) by the alkoxide yielded enantiomerically sustained 2,3- disubstituted tetrahydrofurans. This methodology provided a new subclass of molecules to be repurposed and a great opportunity display the utility of this project. Initial tests on substituted tetrahydrofuran compounds by Vaseliou and the Borhan group provided two hits across Gram-positive cell lines (Bacillus cereus, 87 Bacillus subtilis, Micrococcus lureus, and Staphylococcus aureus) featured in Table I-2. Resazurin antimicrobial assays revealed that compound I-20a showed activity comparable to that of streptomycin, prompting the synthesis and evaluation of I-20b to probe the importance of the terminal alkene. The larger number of stereochemical groups and halogen incorporated into the structures of the other tetrahydrofurans tested did not seem to contribute to antibacterial activity, though spirocyclic compound I-27 did manifest activity at very high concentrations (EC50 values of 952 μg/mL for Bacillus cereus, 1333 μg/mL for Bacillus subtilis, and 666 μg/mL for Micrococcus lureus). The promising activity observed in 3,3-substituted tetrahydrofurans I-20a and I- 20b persuaded us to pursue structure-activity relationship studies in order to determine the optimum antibacterial lead compound. We began by varying alkyl chain length in order to determine its importance and the ideal length. One mode of synthesis required the formation of the 2-methyl-2-propen-1-ol dianion, using n-butyl lithium and TMEDA at -78 °C (Scheme I-2). The added alkyl halides reacted selectively with the carbanion forming extended chain 1,1-alkenols. mCPBA epoxidation provided epoxy alcohols I-33 which were subsequently submitted to the sulfoxonium ylide induced relay ring expansion to form the 3,3- alkylhydroxytetrahydrofurans I-20 in moderate yields. 88 1. nBuLi (2.2 equiv) TMEDA (2.2 equiv) hexanes, -78 °C 2. X (0.9 eq) n I-31 Me3SOI (3.0 equiv) nBuLi (3.0 equiv) THF, reflux, 1-3 h OH I-30 O HO n I-20 OH n I-32 mCPBA (2.0 equiv) CH2Cl2 O OH n I-33 I-32 Yielda (%) I-33 Yield (%) I-20 Yield (%) n 5 6 9 11 13 15 40 76 54 89 50 76 23 29 33 18 55 29 45 46 68 57 41 30 Scheme I-2: Synthesis of 3,3- alkylhydroxytetrahydrofuran analogues via one-carbon homologative relay ring expansion. a Yields were calculated in relation to the alkyl halide. of from 3-oxatetrahydrofuran An alternate method of synthesis, pictured in Scheme I-3, required the initial synthesis 3- hydroxytetrahydrofuran via PCC oxidation. The resulting ketone was submitted to multiple metalloalkane 3,3- alkylhydroxytetrahydrofurans. These two synthetic methods resulted in the formation of 3,3-disubstituted THF molecules with alkyl chains ranging from 4 to 17 carbons long. commercially available attacks in order to form other 89 O HO I-34 PCC (1.7 equiv) 3Å MS, CH2Cl2 reflux, 83% O O I-35 M (1.7 equiv) n Et2O, -78 °C to rt nb 2a 3 4 8 10 12 14 O HO n I-20 Yield (%) 25 27 60 15 30 50 58 Scheme I-3: Synthesis of 3,3- alkylhydroxytetrahydrofuran analogues via nucleophilic addition. aUsed nBuLi as nucleophile. bUnless otherwise mentioned, nucleophiles were generated through magnesium insertion to form the corresponding Grignard reagent. Evaluation of the EC50 values of 3,3-alkylhydroxytetrahydrofurans was done in duplicate using a 45 well plate and a Biorad Benchmark microplate reader and based on the metabolism of resazurin dye by viable Gram-positive bacteria after incubation with the tested compound. We found that the activity did vary in accordance with the lengths of the alkyl chains of the tested substrates, with an optimal range of seven to thirteen carbons. The increase in activity seen in the shorter length seemed to increase gradually culminating at a chain length of twelve carbons (I-20j) (EC50 values of 4 μg/mL for Bacillus cereus, <10 μg/mL for Bacillus subtilis, and 12 μug/mL for Micrococcus lureus). Tests of the twelve- I-20j against Staphylococcus areus activity proved carbon substrate inconclusive. We began to see a rapid decrease in activity as the chain lengths exceeded twelve carbons. This rapid drop in activity can be explained by the increasing insolubility of the substrates in the growth media. Though we began to 90 see problems with the determination of antibacterial activity of I-20k, the activity Bacillus strains suggested we had reached the pinnacle of activity for these analogues and were beginning the decline of activity due to both the interaction with the cells and the decreasing solubility of the more hydrophobic lengthy chain substrates in the cell media. 1 2 3 4 5 6 7 4C 5C 6C 7C 8C 9C 10C Streptomycin O O O O O O O HO I-20c HO I-20d HO I-20e HO I-20f HO I-20g HO I-20b HO I-20h 2 3 4 5 Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus 111 >476 133 >476 133 133 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 222 190 111 22 200 222 67 58 167 56 29 32 ND 222 95 42 *Numbers represent EC50 values (µg/ml). ND = Not Determined Table I-3: EC Values of 3,3-alkylhydroxytetrahydrofuran analogues 50 of differing chain lengths. 91 Table I-3 (cont’d) Streptomycin 8 9 10 11 12 13 14 11C 12C 13C 14C 15C 16C 17C O O O O O O O 6 7 8 9 10 11 12 HO I-20i HO I-20j HO I-20k HO I-20l HO I-20m HO I-20n HO I-20o Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus 111 13 4 7 ND 133 14 <10 <19 ND 133 22 12 >476 133 12 ND ND >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 *Numbers represent EC50 values (µg/ml). ND = Not Determined the effects of branching and aromaticity in 3,3- To briefly scan alkyhydroxytetrahydrofuran analogues, compounds I-20p-I-20r were made via the one carbon homologative relay ring expansion of the corresponding starting epoxide using the sulfoxonium ylide as previously done. As illustrated in Table I- 3, we found no measurable effects on tested Gram-positive bacterial strains. 92 Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus Streptomycin 111 7 HO O 4 133 <10 133 12 133 ND I-20j HO I-20p O >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 >476 O O HO I-20q HO I-20r 1 2 3 4 12C iBu iPn hBn *Numbers represent EC50 values (µg/ml). ND = Not Determined Table I-4: EC50 Values of branched and aromatic 3,3- alkylhydroxytetrahydrofuran analogues on Gram-positive bacteria. To test the necessity of the hydroxyl moiety (Table I-5), two active analogues, I- 20i and I-20k, were protected with trimethylsilyl chloride and methyl iodide, respectively. The activity initially seen in hydroxytetrahydrofuran I-20k was completely muted when converted to the methyl ether. The shorter more universally active I-20i seemed to maintain activity even after protection with the silyl group. We suspected that the silyl ether was deprotected upon addition to the cellular medium. Trimethylsilyl ethers have a well-known susceptibility to solvolysis, especially in acidic media. 40 93 The importance of the hydroxyl group and the chain length to the antibacterial activity of the 3,3-alkylhydroxytetrahydrofurans suggested a mode of action similar to that exhibited by surfactants and other amphiphiles, namely, the rupture or dissolution of the cell membrane. 41 11C 13C O O 6 HO I-9i 8 HO I-9k TMSCl (1.1 equiv) imidazole (1.2 equiv) CH2Cl2, 68% NaH (1.5 equiv) MeI (1.5 equiv) THF, 84% O O 6 8 TMSO I-9s MeO I-9t Bacillus cereus Bacillus subtilis Microc. lureus Staph. aureus Streptomycin 1 2 3 4 11C 13C 11C 13C 6 8 6 8 HO I-20i HO I-20k TMSO I-20s MeO I-20t 111 13 7 4 O O O 133 14 <19 <10 133 22 >476 12 133 12 ND 83 O >476 >476 >476 >476 *Numbers represent EC50 values (µg/ml). ND = Not Determined Table I-5: Evaluation of hydroxyl group importance in antibacterial activity of 3,3-alkylhydroxytetrahydrofuran analogues and EC50 values on Gram-positive bacteria. Kubo and coworkers have shown similarly that long chain alcohols are effective antibacterials against Streptococcus mutans. 42 43 Interestingly, amongst the various alcohols tested, the simple thirteen-carbon alcohol, 1-tridecanol, showed inhibitory concentration (6.25 μg/mL). Linalool and the lowest minimum 94 is manifested solely further suggests in Gram-positive cells Geranylacetol gave MICs of >800 and 25 μg/mL, respectively. The fact that this the activity disassembly of the cell membrane generally seen with detergents. I.2.3. Mode-of-Action Studies of 3-alkyl-3-hydroxytetrahydrofurans Using Hemolysis Assays To probe the merits of this hypothesis, we employed a hemolysis assay. 20 μL of each 10 mg/mL alkylhydroxytetrahydrofuran solution was added to 400μL of a buffered solution of red blood cells (5.0 x 107 cells/mL) creating a 420 μL solution that was mixed and allowed to sit for 30 minutes. Triton X-100, a commercially available surfactant, was used as a positive control (20 μL of 10mg/mL solution) with isotonic buffer (20 μL), pure DMSO (20 μL), and streptomycin (20μL of 10mg/mL) serving as negative controls. 100 μL of each solution was placed in a 96 well plate were used to observe cell viability under a microscope and the remaining 100 μL was centrifuged. The supernatant of the centrifuged samples was submitted to UV analysis on a 96 well plate at 550 nm using the Benchmark Biorad microplate reader. Hemolysis of red blood cells results in the release of hemoglobin (λmax = 540 nm) into the solution, giving higher absorptions than samples wherein hemolysis did not occur. 95 The results of the analysis of the supernatant after hemolysis are shown in Figure I-13. The red blood cell control showed little absorption at 550 nm, indicating minimal cell lysis. Addition of 20 μL of pure DMSO caused a small amount of cell lysis as indicated by the moderate absorbance. Studies have shown that DMSO does have the ability to lyse erythrocytes in the absence of saline.44 Conversely, while the presence of saline does not completely prevent the osmolysis of erythrocytes, it greatly reduces it. 20 μL of a 10 mg/mL solution of Triton X-100 was added to the cell solution and provided extensive cell lysis. The streptomycin control produced no notable cell lysis as expected. Figure I-13: UV/Vis absorption of hemolysis assay supernatant of 3,3- alkylhydroxytetrahydrofurans at 550 nm. 96 While our shorter analogues, specifically the 4 to 7 carbon chain analogues (I- 20c-f), exhibited moderate hemolysis, it seemed comparable to that of DMSO. This is likely due to the solvent itself instead of the short chain 3,3- hydroxytetrahydrofuran compounds. As a matter of fact, the first signs of significant activity are not observed until the 8-carbon chain length of I-20g is utilized. Major lysis of the red blood cells occurred with carbon chain lengths between eight and fifteen carbons, a range closely comparable with the antibacterial activity seen in the previous assays (7 carbons to 13 carbons). Figure I-14 illustrates the surviving cell populations resulting from incubation with differing carbon chains. The protected 3,3-alkylhydroxytetrahydrofurans have shown hemolytic activity very similar to the antibacterial activity observed earlier. The methyl-protected alcohol (I-20t) showed little activity while the TMS-protected alcohol (I-20s) lysed the red blood cells efficiently. This is in line with the theory that the trimethylsilyl group was solvolyzed, facilitating the lysis of the red blood cells by the free 11-carbon 3,3-alkylhydroxytetrahydrofuran (I-20i). Based on these results, it is apparent that the mode of action of the 3,3- alkylhydroxytetrahydrofurans is the rupture of the cell, a mechanism commonly seen in detergents. This theory is supported by the fact that addition of high 97 doses of the substrates to Gram-negative cells showed no significant activity while exhibiting substantial activity with Gram-positive cell lines. Viable Red Blood Cells (Control) 4 Carbon Chain RBCs 12 Carbon Chain RBCs 15 Carbon Chain RBCs Figure I-14: Red blood cell viability after incubation with 3,3- alkylhydroxytetrahydrofurans. Gram-positive and Gram-negative bacteria differ in many ways. One of the major differences that directly affect the passage of antimicrobials into the cell is the peptidoglycan cell “wall”. While both bacterial classes have this rigid structure that stabilizes the cytoplasmic membrane, their thicknesses are very different. Gram-positive bacteria possess 40-80 peptidoglycan layers 40-80 nm thick that 98 account for 90% of the dry weight of the cell. Gram-negative bacteria contain one layer of peptidoglycan 7-8 nm thick that account for roughly 10% of the dry weight of the cell. 45 This difference justifies the persistence of the Gram stain by Gram-positive bacteria and inability of the Gram stain to persist in Gram-negative bacteria. It also explains the purpose of the peptidoglycan cell wall: scaffolding for the bacteria to maintain the cell’s structure. 46, 47 The cell wall is not a protective barrier against antimicrobial compounds and in some instances, may even attract antibacterial peptides to the bacteria as a first step in the 48, 49 lipid II. precursor This means bacteriocidal mechanism. For example, the 34 amino acid peptide antibacterial nisin primarily inhibits peptidoglycan synthesis by initially binding to the peptidoglycan the alkylhydroxytetrahydrofurans tested would not be hindered greatly by the cell wall of Gram-positive bacteria. The incorporation of teichoic acid polymers into the cell surface is another difference seen between Gram-positive and Gram-negative bacteria. Teichoic acid is a soluble glycerolphosphate or ribitolphosphate polymer found in most Gram-positive bacteria linked either to the cytoplasmic membrane via a pendant glycolipid anchor (lipoteichoic acid) or covalently linked to N-acetylmuramic acid (NAM) of the peptidoglycan cell wall (wall teichoic acid).50 Because of their phosphate heads, these polymers create a relatively negative charge on the cell envelope. It has been found that the absence of these polymers in the bacterial 99 foothold casing will negatively affect the growth and viability of the cells.51 Consequently, for cationic and hydrogen bonding these polymers provide a antimicrobials to adhere to bacterial cells, promoting the initial entry into the cell membrane.47 A final major contributing difference between Gram-positive and Gram-negative is the outer membrane of Gram-negative bacteria. This is the most significant difference between the two Gram classifications in terms of antimicrobial contact. The outer membrane serves as a layer of protection. The inner leaflet of the outer membrane is made up of Zwitterionic phospholipids while the outer leaflet consists of Zwitterionic phospholipids and lipopolysaccharides.52 Because of their neutral heads, it is more difficult to distinguish between eukaryotic cells and Gram-negative cells using electrostatic interactions. In terms of antibiotics, Savage and coworkers suggest that this layer of protection can be disrupted by the introduction of amphiphilic compounds (specifically cationic antibiotics) that can sensitize the cell to hydrophobic bacteria that, alone, could not traverse the outer membrane.18 Similarly, Helander and coworkers found that lactic acid served to permeabilize Gram-negative bacteria by similarly compromising the outer membrane.53 100 With regard to Gram-positive bacteria, Kubo and coworkers shed light on the specific effects of alcohols as antimicrobials. They note that Gram-negative bacteria show no loss in viability when treated with alcohols of different lengths (MIC > 800 μg/mL) and Gram-positive bacteria are inhibited with chain lengths between 8 and 13 carbons. They suggest that the rapid decrease in activity with longer chains is due to the dispersion of the alcohol into the phospholipid bilayer, resulting in the breaking of the hydrogen bond. investigated Since a compound with a mode-of-action of nonselective cell lysis would not serve well as an effective antibacterial administered to eukaryotic organisms, 3,3- alkylhydroxytetrahydrofurans were not further as a viable antibacterial. I.2.4. General Hemolytic Assays In order to seek out other compounds that exhibit hemolytic activity, the antibacterial hits were submitted to new hemolytic assays. The compounds that were found active against gram-positive and both gram classes of bacteria were submitted to a similar assay to that of 3,3-alkylhydroxytetrahydrofurans. 101 While many of the compounds were found to have slight hemolytic activity, there were a select few that had none. Steroids I-10a and I-11a were found to exhibit little to no cell lysis. This seems reasonable when we consider the mode of action of natural antibiotic benzoquinones and hydroquinones suggested previously. O O OH I-10a OH I-11a O O O N S Ph O I-16a Ph O O S S Ph I-16b Ph O OEt I-19a Gram (+) Gram (-) MEC = 476 µg/mL MEC > 476 µg/mL Figure I-15: Minimum inhibitory concentration of general laboratory compounds that exhibited no hemolytic activity. Sulfide compounds I-16a and I-16b also did not rupture the red blood cell. This fits with the observations by Mirelman and coworkers that disulfides such as allicin inhibit cellular function by reacting with thiol moieties in important enzymes. Finally, internal alkyne I-19a also did not lyse the cells. We also noted that every compound that was found to be effective against both Gram-positive and Gram-negative bacteria (alcohol I-13c, carboxylic acid I-14a, anionic salt I-15a, cationic salt I-15b, and alkyne I-19b) provided hemolysis 102 comparable to that of the Triton X-100 control. To some extent, all of these compounds discouraged cell viability via a compromised membrane. I.3. Conclusion In attempts to combat the increasing resistance of bacteria to antimicrobials, we began to search for hits in a rarely accessed library: synthesized laboratory compounds that have outlived their usefulness in other academic pursuits. Out of found these compounds, we approximately 1400 compounds tested for antibacterial activity, approximately 29 compounds, not accounting for redundancies, were found to be biologically active at the appropriate concentrations against gram-positive bacteria. 5 of those compounds were found to be active against gram-negative bacteria. Among that 3,3-alkylhydroxytetrahydrofurans showed substantial activity. With that, we began to further explore the source of this activity and found that the chain length and the hydroxyl group were responsible for the activity seen. In terms of chain length, the compounds within the range of 7-13 carbons were the ones in which activity was observed. Activity was gradually introduced as the chain length grew from 4 carbons to 7 carbons, suggesting the appropriate chain length was important with respect to the inhibition of cell function. However, activity seemed to quickly subside from 14 to 15 carbons, suggesting the loss of activity was directly connected to the solubility 103 of the substrate in the cellular media. Protection of the hydroxyl moiety completely shut down activity. The combination of these two ideas led us to believe that these 3,3- alkylhydroxytetrahydrofurans were acting as surfactants and destroying the cell membranes of the bacteria. In order to test this hypothesis, we submitted the compounds to a hemolytic assay and found that there was a substantial correlation between the activity exhibited in gram-positive bacteria and the hemolysis of red blood cells, substantial hemolysis occurring between 8 and 15 carbons. In order to rule out unselective membrane rupture as a mode of action, we submitted all other antibacterial hits found in this probe to the hemolysis assay. While many provided moderate hemolysis, only five gave rise to little to no hemolysis. Four of these were expected to be due to mechanisms suggested by other compounds with similar moieties. The fifth, however, an internal alkyne ester, seemed to have precedence only with largely complex compounds wherein other functionalities could be implicated in the antibacterial activity seen. More study of the simple alkynes utilized in these experiments is necessary to fully understand the contribution of the alkyne to the activity seen in those more complex structures. 104 Since new compounds are continually synthesized and utilized in the laboratory, this method of searching for new antibacterial hits is continually evolving to incorporate new and different compounds. The perpetual revision of such a library could prove essential in the fight for new antimicrobials and further increases the probability of quickly finding new weapons in the struggle against antibiotic resistant strains. 105 I.4. Experimental General Antimicrobial Assay. Gram-positive cell lines Bacillus cereus ATCC-1778, Bacillus subtilis ATCC-6633, Micrococcus luteus ATCC-7468D, Staphylococcus aureus ATCC-6538 and Gram-negative cell lines Escherichia coli ATCC-8739, Klebsiella pneumonia ATCC-4352, Pseudomonas aeruginosa ATCC-9027, Serratia marcescens ATCC-14756, Enterobacter aerogenes ATCC- 13048 were inoculated into 5 mL autoclaved LB solution and allowed to stir un an incubator shaker for 6 to 8 hours. 1 mL of the bacterial solution was added to 10 mL of autoclaved LB. 30 μL of the cell solution is added to each well of a 96 well plate after the addition of 170 μL of autoclaved LB. 10 μL of a 10mg/mL solution of the tested compound was added to the assigned well (done in duplicate or triplicate). The 96 well plate was incubated at 37 °C overnight. 1 μL of 3.3 mg/mL Resazurin in water was added to each well after incubation. The plate was analyzed using UV/Visible spectroscopy via Benchmark Biorad microplate reader (595 and 655 nm), scanning incrementally every 5 minutes until the unaffected wells were light pink. R OH Preparation of 1,1-disubstituted alkenyl alcohols (I-32). A flame-dried flask (3-neck) was charged with a stir bar and equipped with an argon balloon. TMEDA (10.32 ml, 8g, 69 mmol) was added in 15ml hexanes (spectro. grade) followed by 106 the addition of nBuLi (30 ml, 2.5M in hexanes, 69 mmol) while maintaining a temperature below -10 oC. After stirring for 30 minutes, 2-methyl-2-propen-1-ol (2.8 ml, 2.38g, 32 mmol) was added at -78 oC. The solution was allowed to stir overnight providing a yellowish-orange opaque texture. At this point the alkyl halide (28 mmol) was added after cooling the suspension again to -78 oC. The mixture was again allowed to stir overnight. The reaction was quenched with saturated ammonium chloride solution. The mixture was extracted (EtOAc 30 ml x 3), dried over sodium sulfate, and concentrated in vacuo. The compound was purified by column chromatography (10% EtOAc in hexanes) to give yields varying from 30-75%. OH 1H NMR (500 MHz, CDCl3) δ 5.02 (s, 1 H), 4.83 (s, 1 H), 4.05 (s, 1 H), 1.91 (d, J = 7.0 Hz , 2 H), 1.79-1.64 (m, 1 H), 0.851 (d, J = 11 Hz, 6 H). OH 1H NMR (500 MHz, CDCl3) δ 4.98 (s, 1 H), 4.84 (s, 1 H), 4.05 (s, 2 H), 2.35 (dd, J = 2.0 Hz, 1.5 Hz, 2 H), 1.53 (m, 1 H), 1.31 (m, 2 H), 0.873 (d, J = 11 Hz, 6 H). OH 1H NMR (500 MHz, CDCl3) δ 7.50-7.24 (m, 5 H), 5.22 (s, 1 H), 5.06 (s, 1 H), 4.22 (s, 2 H), 2.91 (t, J = 10, 2 H), 2.52 (t, J = 10 Hz, 2 H). 4 OH 107 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.84 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (d, J = 7.5 Hz, 2 H), 1.09-1.34 (m, 12 H), 0.865 (t, J = 7.5 Hz, 3 H). 5 OH 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.84 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (t, J = 7.5 Hz, 2 H), 1.34-1.09 (m, 12 H), 0.865 (t, J = 7.5 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 149.3, 108.9, 65.9, 33.0, 31.9, 29.4, 29.3, 27.8, 22.7, 14.1. 8 OH 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.84 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (t, J = 7.5 Hz, 2 H), 1.37-1.08 (m, 18 H), 0.865 (t, J = 7.5 Hz, 3 H). 10 OH 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.84 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (d, J = 7.5 Hz, 2 H), 1.09-1.34 (m, 12 H), 0.865 (t, J = 7.5 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 149.3, 108.9, 65.9, 33.0, 31.9, 29.4, 29.3, 27.8, 22.7, 14.1. 12 OH 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.85 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (t, J = 7.5 Hz, 2 H), 1.16-1.32 (m, 26 H), 0.867 (t, J = 108 7.5 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 149.3, 108.9, 70.0, 34.1, 33.0, 32.8, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.8, 28.2, 27.8, 22.7, 14.1. 14 OH 1H NMR (500 MHz, CDCl3) δ 4.99 (dt, J = 0.5, 1.0 Hz, 1 H), 4.85 (dt, J = 1.0, 1.5 Hz, 1 H), 4.05 (s, 2 H), 2.03 (t, J = 7.5 Hz, 2 H), 1.16-1.32 (m, 26 H), 0.867 (t, J = 7.5 Hz, 3 H). O OH R Preparation of 2-alkyl-2-hydroxymethyl epoxide. The alkene substrate was stirred in dry methylene chloride (50 ml) at 0 oC. mCPBA (2.0 equiv.) was dissolved in the necessary methylene chloride and added via cannula to the stirring solution. The ice bath was left in place and the mixture was allowed to war to room temperature and then left for three hours. The reaction was also monitored via TLC. The reaction was quenched with saturated sodium carbonate. Extraction was done with methylene chloride (30 ml x 3). The organic layer was concentrated in vacuo and dried over sodium sulfate. Purification was achieved via column chromatography (15% EtOAc in hexane). The pure compounds generally resulted in yellowish-brown oils. In the case of the 17-carbon analogue, it resulted in a yellow gel like solid. Yields were approx. 30%. O OH 109 1H NMR (500 MHz, CDCl3) δ 3.53 (d, J = 20 Hz, 1 H), 3.51 (d, J = 20 Hz, 1 H), 2.54 (d, J = 8.0 Hz, 1 H), 2.51 (d, J = 8.0 Hz, 1 H), 1.53-1.52 (m, 1 H), 1.22 (d, J = 9.5 Hz, 2 H), 0.913 (d, J = 11 Hz, 6 H). O OH 1H NMR (500 MHz, CDCl3) δ 3.91 (d, J = 20.5 Hz, 1 H), 3.76 (d, J = 20.5 Hz, 1 H), 3.02 (d, J = 7.5 Hz, 1 H), 2.80 (d, J = 7.5 Hz, 1 H), 2.48 (bs, 1 H), 1.90 (dt, J = 14, 23.5 Hz, 1 H), 1.72-1.56 (m, 1 H), 1.37 (dt, J = 12, 15.5 Hz, 1 H), 1.01 (d, J = 11 Hz, 6 H). O OH 1H NMR (500 MHz, CDCl3) δ 7.49-7.22 (m, 5 H), 3.95 (d, J = 20 Hz, 1 H), 3.85 (d, J = 20 Hz, 1 H), 3.34 (d, J = 8.0 Hz, 1 H), 2.81 (d, J = 8.0 Hz, 1 H), 2.36-2.20 (m, 2 H), 2.10-1.94 (m, 2 H). O OH 4 1H NMR (500 MHz, CDCl3) δ 3.76 (d, J = 20 Hz, 1 H), 3.63 (d, J = 20 Hz, 1 H), 2.87 (d, J = 5.0 Hz, 1 H), 2.65 (d, J = 5.0 Hz, 1 H), 1.82-1.66 (m, 2 H), 1.40-1.14 (m, 2 H), 0.862 (t, J = 13 Hz, 3 H). O OH 5 110 1H NMR (500 MHz, CDCl3) δ 3.75 (d, J = 12 Hz, 1 H), 3.62 (d, J = 12 Hz, 1 H), 2.86 (d, J = 4.5 Hz, 1 H), 2.64 (d, J = 4.5 Hz, 1 H), 1.74 (dt, J = 7.0, 15 Hz, 1 H), 1.62 (bs, 1 H), 1.48 (dt, J = 8.0, 16 Hz, 1 H), 1.35-1.16 (m, 12 H), 0.857 (t, J = 7.0 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 62.8, 59.7, 49.8, 32.0, 31.8, 29.7, 29.4, 29.2, 24.6, 22.6, 14.1. O OH 8 1H NMR (500 MHz, CDCl3) δ 3.76 (d, J = 20 Hz, 1 H), 3.63 (d, J = 20 Hz, 1 H), 2.87 (d, J = 8.0 Hz, 1 H), 2.65 (d, J = 8.0 Hz, 1 H), 1.82-1.66 (m, 2 H), 1.40-1.14 (m, 2 H), 0.862 (t, J = 13 Hz, 3 H). O OH 10 1H NMR (500 MHz, CDCl3) δ 3.76 (d, J = 12 Hz, 1 H), 3.62 (d, J = 12 Hz, 1 H), 2.87 (d, J = 5.0 Hz, 1 H), 2.65 (d, J = 5.0 Hz, 1 H), 1.74 (dt, J = 7.0, 15 Hz, 1 H), 1.48 (dt, J = 8.0, 16 Hz, 1 H), 1.35-1.14 (m, 24 H), 0.860 (t, J = 6.5 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 62.97, 50.06, 32.25, 32.16, 29.97, 29.91, 29.88, 29.85, 29.76, 29.72, 29.58, 24.86, 22.92, 14.35. O OH 12 1H NMR (500 MHz, CDCl3) δ 3.76 (d, J = 12 Hz, 1 H), 3.62 (d, J = 12 Hz, 1 H), 2.87 (d, J = 5.0 Hz, 1 H), 2.65 (d, J = 5.0 Hz, 1 H), 1.74 (dt, J = 7.0, 15 Hz, 1 H), 1.48 (dt, J = 8.0, 16 Hz, 1 H), 1.35-1.14 (m, 26 H), 0.860 (t, J = 6.5 Hz, 3 H). 111 O OH 14 1H NMR (500 MHz, CDCl3) δ 3.91 (d, J = 20.5 Hz, 1 H), 3.79 (d, J = 20.5 Hz, 1 H), 3.02 (d, J = 8.0 Hz, 1 H), 2.80 (d, J = 8.0 Hz, 1 H), 1.92 (dt, J = 7.0, 15 Hz, 1 H), 1.63 (dt, J = 8.0, 16 Hz, 1 H), 1.52-1.26 (m, 30 H), 1.01 (t, J = 10 Hz, 3 H). R O OH was generated dimethylsulfoxonium methylide Preparation of 3-alkyl-3-hydroxytetrahydrofuran. Trimethylsulfoxonium iodide was dried under high vacuum at 40 oC overnight. In preparation of 3-hydroxyTHF derivatives, from trimethylsulfoxonium iodide and nBuLi in THF at -78 oC. Addition of the epoxy alcohol precurser followed by heating to reflux for 3 hours resulted in a darker tinted liquid that was quenched with ammonium chloride after cooling. The mixture was extracted several times with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, and concentrated in vacuo. The crude mixture was purified via column chromatography (10% EtOAc in hexanes) resulting in a yellow oil in most cases and a brown gel in the case of the 17 carbon chain analogue. Yields varied (30-74%). HO O 1H NMR (500 MHz, CDCl3) δ 3.98 (dt, J = 10 Hz, 15 Hz, 1 H), 3.86 (td, J = 6 Hz, 13 Hz, 1 H), 3.72 (dd, J = 1 Hz, 15 Hz, 1 H), 3.51 (d, J = 17.5 Hz, 1 H), 1.97-1.82 (m, 3 H), 1.56 (dd, J = 9.5 Hz, 11.5, 2 H), 0.955 (dd, J = 12 Hz, 15.5 Hz, 6 H). 112 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dt, J = 6.5 Hz, 9 Hz, 1 H), 3.90-3.84 (m, 1 H), 3.68 (d, J = 10 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.93-1.86 (m, 1 H), 1.62 (dd, J = 9.5 Hz, 10.5, 2 H), 1.56-1.46 (m, 1 H), 1.38-1.20 (m, 3 H), 0.891 (d, J = 11 Hz, 6 H). HO O 1H NMR (500 MHz, CDCl3) δ 7.34-7.25 (m, 2 H), 7.26-7.15 (m, 3 H), 4.03 (dt, J = 8 Hz, 10 Hz, 1 H), 3.89 (td, J = 4.5 Hz, 9.0 Hz, 1 H), 3.72 (d, J = 10 Hz, 1 H), 3.57 (d, J = 9 Hz, 1 H) 2.86-2.70 (m, 2 H), 2.02-1.93 (m, 4 H). 13C NMR (125 MHz, CDCl3) δ 142.1, 128.8, 128.5, 126.3, 110.0, 81.3, 79.2, 67.6, 40.2, 31.32. 2 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dt, J = 7.5 Hz, 8.5 Hz, 1 H), 3.87 (td, J = 5.5 Hz, 8 Hz, 1 H), 3.68 (d, J = 9 Hz, 1 H), 3.53 (d, J = 9 Hz, 1 H), 1.90 (dd, J = 5 Hz, 7 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.34-1.18 (m, 10 H), 0.864 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 84.5, 79.1, 67.7, 40.0, 38.2, 32.0, 30.3, 29.4, 24.9, 22.9, 14.3. 3 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (td, J = 7.5 Hz, 8.5 Hz, 16 Hz, 1 H), 3.87 (dt, J = 5 Hz, 7.5 Hz, 1 H), 3.68 (d, J = 9 Hz, 1 H), 3.53 (d, J = 9 Hz, 1 H), 1.90 (dd, J = 6 Hz, 7.5 Hz, 2 H), 1.63 (t, J = 8.5 Hz, 2 H), 1.32-1.18 (m, 18), 0.863 (d, J = 7 Hz, 113 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 31.9, 30.1, 29.5, 29.2, 24.6, 22.6, 14.1. 6 HO O 1H NMR (500 MHz, CDCl3) δ 4.00 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.90-3.84 (m, 1 H), 3.67 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.90 (d, J = 5 Hz, 7.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.34-1.18 (m, 24 H), 0.859 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.5, 79.1, 67.7, 40.0, 38.2, 32.1, 30.3, 29.9, 29.85, 29.81, 29.79, 29.6, 24.9, 22.9, 14.3. 8 HO O 1H NMR (500 MHz, CDCl3) δ 4.00 (dd, J = 7 Hz, 14 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.67 (d, J = 9 Hz, 1 H), 3.53 (d, J = 9 Hz, 1 H), 1.90 (dd, J = 6 Hz, 7.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.32-1.18 (m, 22), 0.860 (d, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 60.4, 39.8, 37.9, 31.9, 30.1, 29.7, 29.6, 29.5, 29.3, 24.6, 22.7, 21.0, 14.2, 14.1. 10 HO O 1H NMR (500 MHz, CDCl3) δ 4.04 (dd, J = 7 Hz, 14 Hz, 1 H), 3.91 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.72 (d, J = 9 Hz, 1 H), 3.57 (d, J = 9 Hz, 1 H), 1.93 (dd, J = 6 Hz, 7.5 Hz, 2 H), 1.66 (t, J = 8.5 Hz, 2 H), 1.38-1.22 (m, 24), 0.897 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.4, 79.1, 67.7, 40.0, 38.2, 32.2, 30.3, 29.92, 29.89, 29.88, 29.82, 29.80, 29.51, 24.9, 22.9, 14.3. 114 12 HO O 1H NMR (500 MHz, CDCl3) δ 4.14 (dd, J = 7 Hz, 14 Hz, 1 H), 4.05 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.92 (dt, J = 5 Hz, 8 Hz, 1 H), 3.73 (d, J = 9 Hz, 1H) 3.58 (d, J = 9.5 Hz, 1 H), 1.36-1.26 (m, 18 H), 0.902 (t, J = 7 Hz, 3 H). Lithium Reagent n-Butyllithium was added to dry diethyl ether and stirred while purging with N2 gas. After lowering the temperature to -78 °C, 3-oxa-THF was added. The reaction was monitored via TLC and diluted with H2O upon completion. NH4Cl was added to the solution before extraction several times with CH2Cl2. The organic layer was dried over sodium sulfate, and concentrated in vacuo. The crude mixture was purified via column chromatography (20% EtOAc in hexanes) to reveal a clear liquid. The yield was low (25%). HO O 1H NMR (300 MHz, CDCl3) δ 4.01 (dd, J = 7 Hz, 14 Hz, 1 H), 3.87 (td, J = 5 Hz, 8 Hz, 12.5 Hz, 1 H), 3.68 (d, J = 9 Hz, 1 H), 3.54 (d, J = 9 Hz, 1 H), 1.90(d, J = 5 Hz, 8 Hz, 2 H), 1.64 (t, J = 8.5 Hz, 2 H), 1.38-1.30 (m, 4 H), 0.908 (t, J = 7 Hz, 3 H). 115 Grignard Reagents Magnesium metal and a very small amount of iodine was charged into a small dry flask with a stir bar and sealed. After purging, dry diethyl ether was added via syringe and the solution was stirred for 30 minutes. The alkyl halide was added and allowed to stir for 30 more minutes. The disappearance of the brownish tint produced by the iodine was a coloremetric gauge to mark initiation of magnesium insertion. The temperature was lowered to -78 °C and 3-oxaTHF was added. The reaction was monitored via TLC and diluted with H2O upon completion. After the solution stilled, NH4Cl was added to the solution before extraction several times with CH2Cl2. The organic layer was dried over sodium sulfate, and concentrated in vacuo. The crude mixture was purified via column chromatography (20% EtOAc in hexanes) to reveal a clear liquid. The yield was low (25%). HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dd, J = 7 Hz, 14 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.62 (t, J = 9 Hz, 2 H), 1.34-1.16 (m, 6), 0.881 (t, J = 6.5 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 32.3, 24.3, 22.6, 14.0. HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.63 (t, J = 9 Hz, 2 H), 1.34-1.20 (m, 8), 0.869 (t, J = 6 Hz, 3 116 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 31.7, 29.7, 24.6, 22.6, 14.0. 5 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.54 (d, J = 9.5 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.63 (t, J = 8.5 Hz, 2 H), 1.33-1.13 (m, 16), 0.862 (t, J = 6 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 31.9, 30.1, 29.59, 29.57, 29.54, 29.3, 24.6, 22.7, 14.1. 7 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.32-1.14 (m, 20), 0.861 (t, J = 6 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 78.9, 67.5, 39.8, 37.9, 31.9, 30.1, 29.64, 29.58, 29.3, 24.6, 22.7, 14.1. 9 HO O 1H NMR (500 MHz, CDCl3) δ 4.00 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.34-1.18 (m, 24), 0.860 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 31.9, 30.1, 29.68, 29.67, 29.64, 29.57, 29.55, 29.3, 24.6, 22.7, 14.1. 117 11 HO O 1H NMR (500 MHz, CDCl3) δ 4.01 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.68 (d, J = 9.5 Hz, 1 H), 3.53 (d, J = 9.5 Hz, 1 H), 1.90 (dd, J = 6 Hz, 8.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.33-1.14 (m, 28), 0.860 (t, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 39.8, 37.9, 31.9, 30.1, 29.68, 29.65, 29.57, 29.54, 29.3, 24.6, 22.7, 14.1. Protected Tetrahydrofuran Analogues TMS-protected undecylhydroxytetrahydrofuran O 6 TMSO Undecylhydroxytetrahydrofuran (1.0 equiv, 1.51 mmol, 365 mg) was dissolved in dried methylene chloride along with TMS chloride (1.1 equiv, 1.66 mmol, 0.21 mL) and imidazole (1.1 equiv, 1.66 mmol, 113 mg). The reaction was stirred at room temperature for 10 hours. The reaction mixture was diluted with dietyl ether and washed with brine (3 x 30 mL). The organic layer was dried over sodium sulfate and concentrated. The crude was purified using column chromatography (10% ethyl acetate in hexanes), yielding the clear product in 69%. 1H NMR (500 MHz, CDCl3) δ 3.94 (dd, J = 8.5 Hz, 16.5 Hz, 1 H), 3.84 (td, J = 4 Hz, 8.5 Hz, 1 H), 3.71 (d, J = 9 Hz, 1 H), 3.52 (d, J = 9 Hz, 1 H), 1.98-1.92 (m, 1 H), 1.86-1.78 (m, 1 H), 1.58 (t, J = 8.5 Hz, 2 H), 1.35-1.18 (m, 18), 0.862 (t, J = 7 Hz, 3 H), 0.109 (s, 9 H). 13C NMR (125 MHz, CDCl3) δ 83.9, 78.8, 67.8, 40.2, 39.6, 32.1, 30.3, 29.88, 29.87, 29.84, 29.6, 24.9, 22.9, 14.3, 2.29. 118 Methyl protected tridecylhydroxytetrahydrofuran O 8 MeO 60% sodium hydride (1.5 equiv, 0.61 mmol, 24 mg) was added to a flame-dried flask with a stirbar. Dry THF was added to the flask before sealing and purging with nitrogen gas. 110 mg of tridecylhydroxytetrahydrofuran (1.0 equiv, 0.41 mmol) was added to the suspension and the solution was left stirring for 30 minutes. Finally, methyl iodide (1.5 equiv, 0.61 mmol, 0.04 mL) was added to the stirring solution and allowed to stir to completion observed via TLC. Water was added to the solution and it was left stirring for 8 hours. The solution was extracted with ethyl acetate. The organic layers were combined and dried over sodium sulfate. The organic solution was concentrated. The pure clear compound was isolated via column chromatography (20% ethyl acetate in hexanes) in 53% yield. 1H NMR (500 MHz, CDCl3) δ 4.00 (dd, J = 7 Hz, 14 Hz, 1 H), 3.87 (dd, J = 9 Hz, 16.5 Hz, 1 H), 3.89 (s, 3 H), 3.67 (d, J = 9 Hz, 1 H), 3.53 (d, J = 9 Hz, 1 H), 1.90 (dd, J = 6 Hz, 7.5 Hz, 2 H), 1.62 (t, J = 8.5 Hz, 2 H), 1.32- 1.18 (m, 22), 0.860 (d, J = 7 Hz, 3 H). 13C NMR (125 MHz, CDCl3) δ 81.2, 78.9, 67.4, 60.4, 55.7, 39.8, 37.9, 31.9, 30.1, 29.7, 29.6, 29.5, 29.3, 24.6, 22.7, 21.0, 14.2, 14.1. General Hemolysis Assay. A buffered solution of rabbit blood cells was diluted with an isotonic phosphate buffer solution (40 mL 0.066 M NaH2PO4, 60 mL 0.0667 M Na2HPO4, pH = 7.0, .46g/100mL NaCl) to 5 x107 cells/mL. An eppindorf tube was filled with 400 μL of the cell solution and 20 μL of 10 μg/mL of the analyzed compound or control. Triton X-100 served as the positive control. 20 μL of pure DMSO was used as a negative control along with a 400 μL sample of the 119 cell solution without any added compounds. The solutions were stirred via vortex and allowed to sit for 30 minutes. 100 μL of the cell solution was added to a 96 well plate for microscopic evaluation. Microscopic photos were taken to keep record of the cell samples. The remaining portion of each cell solution was centrifuged at 11,000 rpm for 5 minutes. The supernatant was added to a 96 well plate to be analyzed by a Benchmark Biorad microplate reader at 550 nm. 120 REFERENCES 121 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Lesch, J. E. 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"Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane" Applied and environmental microbiology 2000, 66, 2001. 126 Chapter II Steps Towards the Total Synthesis of (+)-Alexine via One-Carbon Homologative Relay Ring Expansion II.1. Introduction II.1.1. Discovery of Alexine Alexine (II-1) is a polyhydroxylated pyrrolizidine isolated from Alexa leiopetala in 1988 by Robert Nash and coworkers3. (1R, 2R, 3R, 7S, 8S)-3-hydroxymethyl-1, resemble 2, 7-trihydroxypyrrolizidine, given the common name alexine, was the first example of a pyrrolizidine with a carbon substituent on C3. It was also found to closely (2R, 5R)-dihydroxymethyl-(3R, 4R)-dihydroxypyrrolidine (DMDP) (II-2), a known glucosidase inhibitor found in leguminosae derris and leguminosae lonchocarpus. II.1.2. Biological Activity of Alexine Due to this resemblance to DMDP, alexine was tested for glucosidase inhibition. In initial screenings in 1988, alexine barely performed, inhibiting the hydrolysis of 6 mM p-nitrophenyl-α-D-glucopyranoside and 10 mM p-nitrophenyl-β-D- glucopyranoside by less than 50% at >3.3 x 10-4 M and 10 mM p-nitrophenyl-β- D-galactopyranoside by 50% at 1.5 x 10-4 M. While DMDP exhibited weak the β-anomer inhibition of a-D-glucopyranosidase, disaccharidase and the galactopyranosidase was much more effective (3.0 x 10-4 inhibition of the 127 M, 1.0 x 10-5 M, and 2.0 x 10-6 M, respectively). Additional biological testing by Nash in 1990 showed that alexines as a general class provided a weak activity, if any, with regard to mammalian glycosidases compared to castanosperamine (II- 7).4 HO HO N HO HO H OH HO (+) - Alexine II-1 NH OH HO DMDP II-2 Figure II-1: Alexine structural comparison to DMDP. In an overall sense, the evaluated alexines are not very effective against mammalian glucosidases. It is important to note, however, that there are varying activities that are solely dependent on the variation of particular stereocenters. When comparing alexine (II-1) and 3, 7a-diepialexine (II-3), the change in the two stereocenters decreases a moderate 50% inhibition (5.9 x 10-5M for alexine) in trehalase to no observable inhibition at all (>3.3 x 10-4 M for 3, 7a-diepialexine). Interestingly, 1, 7a- and 7, 7a-diepialexines show an opposite effect when compared to alexine with regards to p-nitrophenyl-a-D-glucopyranoside (9.5 x 10- 5 M, 1.5 x 10-5 M, and >3.3 x 10-4 M, respectively). It is important to note that the alexines have exhibited a more significant inhibitory activity on fungal glucan1,4-a-glucosidase (amyloglucosidase). Alexine (II-1) has 128 found to effectively inhibit specific also been thioglucosidases. While castanospermine seems to have a promiscuous mode of action with regard to enzyme inhibition, the pyrrolizidines seem to be much more selective in their operation. It is, in this regard, apparent that though alexine seems to have inferior activity the activities can potentially be manipulated by synthesizing alternative diastereomers and analogues. towards mammalian glycosides, 129 Entry Inhibitor p-Nitrophenyl-α-D- glucopyranosidase p-Nitrophenyl-β-D- glucopyranosidase p-Nitrophenyl-β-D- galactopyranosidase Trehalase 1 2 3 4 5 6 7 HO HO NH 3.0 x 10-4 1.0 x 10-5 2.0 x 10-6 -- HO OH DMDP (II-2) HO HO N >3.3 x 10-4 >3.3 x 10-4 1.5 x 10-4 5.9 x 10-5 HO H OH Alexine (II-1) HO HO N >3.3 x 10-4 >3.3 x 10-4 H HO OH 3, 7a-Diepialexine (II-3) HO HO N 9.5 x 10-5 >3.3 x 10-4 H HO OH 1, 7a-Diepialexine (II-4) HO HO N 1.6 x 10-5 2.3 x 10-4 HO H OH 7, 7a-Diexpialexine (II-5) HO HO N H OH OH HO 7a-Epialexine (II-6) N OH HO H OH Castanosperamine (II-7) >3.3 x 10-4 3.3 x 10-4 2.8 x 10-6 1.7 x 10-5 -- -- -- -- -- >3.3 x 10-4 >3.3 x 10-4 1.0 x 10-4 >3.3 x 10-4 9.8 x 10-6 Table II-1: Action of naturally occurring alexines on mouse gut digestive glucosidase activity compared with those of DMP and castanospermine. Concenctration (M) of alkaloid giving 50% inhibition. >3.3 x 10-4 required for 50% inhibition. 3 4 130 to pyrrolizidines II.1.3. Previous Synthetic Approaches to Alexine and Other Members of the Family Most approaches involved naturally chiral compounds, specifically amino acids, sugars, and their derivatives. Earlier pyrrolizidine synthesis included the synthesis of necines and necic acids.54 Synthesis of trachelanthamidine (II-9) utilized an racemic unsaturated pyrrolidine ester (II-10) that, after amidation, oxidative esterification, and cyclization, afforded an unsaturated pyrrolizidine (II-12) in 66% yield. This compound produced II-8 after a two-step reduction sequence. In order to form trachelanthamidine, the ester a-carbon of lactam II-13 was flipped under basic conditions, producing the desired compound after the final hydride reduction. isoretronecanol (II-8) and N H CO2Et II-10 H II-8 H N N OH OH 1. (MeSCH2CO)2O pyridine 2. NaIO4 LiAlH4 1. POCl3, NaBH4 2. NaOEt, EtOH 3. LiAlH4 CO2Et N O II-11 S O (CF3CO)2O 31% N O CO2Et OCOCF3 S H CO2Et II-13 N O Raney Ni 66% N O CO2Et S II-12 II-9 Scheme II-1: Synthesis of pyrrolizidine natural products isoretronecanol (II-8) and trachelanthamidine (II-9). 131 (II-16), In order to synthesize trachelanthamidine chirally, 55 Ishibashi turned to L-prolinol (II-14) to provide the initial chirality on which the synthesis would depend (Scheme II-2). N-protection and Swern oxidation gave pyrrolidinal (II-15), followed by Wittig olefination to produce olefin (II-16) unselectively. After N- deprotection of the pyrrolidine nitrogen was amidated using MeSCH2C(O)Cl and triethylamine followed by oxidation to sulfoxide (II-17) with sodium periodate with an overall yield of 49% from L-prolinol. Trifluoroacetic anhydride was used to form pyrrolizidine II-18 in 87% yield through a carbocation sequence similar to the mechanisms of the racemic synthesis that would form a terminal olefin handle for further functionalization. It is important to note the desired stereochemistry of the pendant olefin. Formation of the olefin was regiospecific. that cyclization occurred stereospecifically and gave OH N H II-14 1. ClCO2Et, NaOH 2. (COCl)2, DMSO Et3N, -60 °C O N CO2Et II-15 Ph3PEt+Br- NaCH2S(O)CH3 N CO2Et II-16 1. NaOH 2. MeSCH2C(O)Cl, Et3N 3. NaIO4 S N O H N (CF3CO)2O N S O O II-17 49% overall yield H N OH 1. NaIO4 2. aluminum amalgum 1. OsO4, NaIO4 2. LiAlH4 O II-19 O II-18 87% yield d.r. 4:1 Scheme II-2: Synthesis of (-)-Trachelanthamidine (II-9). (-)-II-9 H H N O N S S 132 1H NMR showed the existence of two diastereomers in a ratio of 4:1. Sulfur was removed by oxidation to the sulfoxide and removal with aluminum amalgam to give compound (II-19). Oxidative cleavage of the olefin followed by global reduction produced (-)-trachelanthamidine (-)-II-9. Similarly, many syntheses of pyrrolizidine natural products begin with simple chiral compounds, usually amino acids, sugars and their derivatives. For example, the first synthesis of (+)-alexine was accomplished using D-glucose as the template, 56 featured in Scheme II-3. HO HO H OBn N3O H OMe H H II-19 TBSCl, DMF 0 °C, 95% HO OBn N3O H TBSO H H II-20 OMe H 1. Tf2O, pyr., -30 °C 2. H2, Pd, EtOAc 3. BnBr, NaOH DMF, 77% TBSO Bn N BnO OMe O II-21 1. TBAF, THF 2. (COCl)2, (CH3)2SO Et3N TBSO OH NBn O II-24 OMe 1. BH3 . SMe2 THF 2. -OH, H2O2 67% TBSO BnO NBn O TBSO BnO NBn O + OMe II-23, 38% OMe II-23', 37% 1. CH2CHMgBr THF 2. TBSCl, DMF 89% O BnO Bn N O II-22 OMe TsCl, pyr. CH2Cl2, 77% Bn H2, 10% Pd/C AcOH:H2O, 72% N O II-25 OMe TBSO HO N O II-26 1. AcOH:H2O 2. NaBH4, EtOH 54% OMe HO H OH OH OH N (+)-II-1 BnO TBSO BnO Scheme II-3: First synthesis of (+)-Alexine (II-1). This initial synthesis was based on the inherent chirality of D-glucose. 133 Methyl-2-azido-3-O-benzyl-2-deoxy-a-D-mannofuranoside (II-19), a compound synthesized from D-glucose by Fleet and coworkers, 57 was submitted to a selective silyl protection of the primary alcohol. Silyl ether II-20 was triflated and the azide reduced, resulting in immediate cyclization to form bicyclic compound II-21 after benzyl protection of the secondary amine. Deprotection and Swern oxidation of II-21 gave aldehyde II-22, which was submitted to nucleophilic addition of vinylmagnesium bromide. Epimeric alcohols II-23 and II-23’ were formed in almost equal amounts (38% and 37%) and subsequently, silyl protected with TBSCl. While compound II-23’ was used to synthesize 7-epialexine, researchers continued with II-23 to make (+)-alexine by hydroborating and oxidizing the terminal alkene to form primary alcohol II-24 in 67% yield. Tosylation of the primary alcohol resulted in spontaneous cyclization to form ammonium II-25. Global benzyl deprotection followed by acid mediated silyl deprotection and hydrolysis of the furanoside provided II-1 in 54% after sodium borohydride reduction of the lactol and ion exchange chromatographic purification. Much of the synthesis of members of the alexine family, including every synthesis of (+)-alexine (II-1), was achieved by the same means as this initial synthesis of alexine and the pyrrolizidines before it. 134 The first achiral to chiral synthesis of a member of the alexine family, featured in Scheme II-4, was the Denmark synthesis of (+)-casuarine (II-27), 58 a pentahydroxypyrrolizidine effective in the inhibition of glucosidase I. Utilizing a tandem [4+2]/[3+2] bicyclization of nitroalkene (II-28), chiral auxillary-protected cis-vinyl ether (II-29), and dipolarophile (II-30), researchers were able to synthesize the key nitroso acetal intermediate that would provide II-27 after reduction, oxidation of C-Si bond, and global deprotection. NO2 1. OG* OBz II-29 BzO II-28 SnCl4, toluene -78 °C 2. Et3N/MeOH Hg(OTFA)2, TFA, AcOH AcO2H, 84% RO OH OH N HO HO H OH II-27 O H N O OBz OG* OBz Ph Ph HO G*OH 99% Recov. + OTDS N OR PhMe2Si R = H II-34 R = Bz II-35 H OR BzCl, pyr, 12 h 87% O OTDS SiMe2Ph II-30 76% (55% after HPLC) OTDS N O O 6 12 3 4 5 OBz O PhMe2Si H II.31 OG* OBz L-Selectride THF, -78 °C 87%, 10:1 1. Raney Ni, MeOH 260 psi H2 2. K2CO3, 64% OTDS H O RO PhMe2Si R = H II-32 R = Ms II-33 N O OG* H OBz OBz Me2O pyr, 1 h 97% Scheme II-4: Synthesis of (+)-Casuarine. As mentioned previously, the chirality is induced using a chiral auxillary during the [4+2] cyclization. This sets stereocenters at C4, C5, and C6, which in turn, along with the rigidity of the newly constructed 6-member heterocycle, establish the stereochemistry at carbons C1, C2, and C3 via the subsequent [3+2] 135 cyclization to form bicyclic ketone II-31. Denmark utilizes a sim ilar method in the synthesis of (+)-1-epiaustraline, 59 incorporating tethered components for the [3+2] cyclization but inducing the intermolecular [4+2] cyclization prior. Pearson and coworkers attempted a racemic synthesis utilizing the cyclization of an azodiene (II-40), 60 forming the desired pyrrolizidine with incorporation of the fully arranged and complete carbon skeleton. Though this synthesis is racemic, it is of note because of introduction of chirality to an achiral substrate followed by the incorporation of all other stereocenters in a process reliant on the originally introduced stereocenter. They propose the formation of triazoline (II-I), which undergoes fragmentation to zwitterion (II-II) and release of nitrogen gas to then recyclize via a slightly favored exo cyclization. The synthesis began with the nucleophilic addition of enyne (II-36) to aldehyde (II-37). This synthesis had the potential to be an asymmetric synthesis of alexine or the C7 diastereomer of alexine with the formation of alcohol II-38. However, the use of a general and nonselective nucleophilic addition produced II-38 a racemic mixture. Red Al alkyne reduction followed by a PhSCl quench resulted in vinyl thiol ether (II-40). Secondary alcohol protection followed by primary deprotection and Mitsunobu led to azide (II-40), the precursor to the key step of this synthesis. Cyclization produced lower yields (50%) and selectivities (70:30 diastereoselectivity) of pyrrolizidine II-41 than would have been preferred. Global 136 deprotection was also low yielding at 50%. All final attempts to use the vinyl sulfide to introduce the remaining hydroxyl groups failed leaving the synthesis incomplete and causing researchers to pursue other methods of synthesis. TBDPSO II-36 TBDPSO TBDPSO 1. n-BuLi 2. OHC OTBS II-37 73% OH 1. Red Al“ 2. PhSCl, 50% 3. TBDPSCl, DMF, imid., 69% TBDPSO TBSO II-38 SPh H OTBDPS N II-II OTBDPS SPh H OTBDPS N N N II-I 90 °C, 15 h sealed tube, 50% TBDPSO SPh OTBDPS TBSO II-39 1. AcOH aq. THF, 61% 2. PPh3, DEAD (PhO)2P(O)N3, 71% SPh OTBDPS N3 II-40 PhS H OTBDPS N PhS H OH N n-Bu4F 50% OTBDPS OH II-42 II-41 Scheme II-5: Attempted synthesis of 3-epialexine analogues. Han and coworkers61 chose a more fragmented synthesis utilizing Sharpless asymmetric aminohydroxylation initial the chirality of the to impose stereoisomers. In this case, the carbon skeleton is pieced together and the oxygens placed before culmination in a double cyclization of the amine to form the alexines of choice, 1,2-diepi-alexine (II-43) and 1,2,7-triepi-australine (II- 44). 137 Aminohydroxylation of achiral ester (II-45) produced amino alcohol (II-46) in 70% yield and excellent regio- (>20:1) and enantioselectivity (>99% ee after recrystallization). After protection and reduction to form aldehyde (II-47), vinylmagnesium bromide was used to form alcohol (II-48) in a diastereomeric ratio of 4:1. Cross metathesis installs the remaining carbons with a primary O O II-45 OEt OMe HO HO ∑ HCl N OH HO H II-43 HO HO ∑ HCl N HO H OH II-44 K2OsO4 . 2Η2Ο (DHQD)2PHAL LiOH, AcNHBr, t-BuOH-H2O 2:1, 4 °C, 8 h, 70% 1. CAN, MeCN-H2O 4:1, 4 °C 2. H2, Pd/C, MeOH, 72% Ac NH O PMPO OEt OH II-46 PMPO BnO N H OH HO II-52 1. NaH, BnCl DMF, 0 °C, 10 h, 78% 2. (Boc)2O DMAP, THF, reflux, 4 h then NH2NH2, MeOH, 4 h, 85% 3. DIBAL, CH2Cl2 -78 °C, 3 h, 90% 1. 3 N HCl, MeOH 2. K2CO3, MeOH 83% 1. CAN, MeCN-H2O 4:1, 4 °C 2. H2, Pd/C, MeOH, 80% PMPO BnO N H OH 1. 3 N HCl, MeOH 2. K2CO3, MeOH 83% PMPO HO II-53 Ac NH O H PMPO OH CH2CHMgBr THF, -50 °C, 1 h, then rt, 1 h, 85% Boc NH PMPO OH OBn II-48 Grubb's 2nd Gen. 4-butenol p-tolylsulfonate CH2Cl2, 65% Boc TsO NH VO(acac)2, TBHP, toluene, 74% PMPO OH OBn II-49 II-47 TsO O NH Boc OH PMPO OBn II-50 TsO O NH Boc OH OBn II-51 Scheme II-6: Asymmetric synthesis of 1,2-diepi-alexine and 1,2,7-triepi- australine via Sharpless aminohydroxylation. tosylate leaving group. Vanadium-mediated e poxidation provided epoxides II-50 and II-51 as an inseparable mixture of diastereomers in a ratio of 2:3, respectively. Acidic deprotection followed by basification led to double cyclization producing pyrrolizidines II-52 and II-53 in 83% yield. Final deprotections removed the PMP and benzyl groups, giving 72% of diepi-alexine hydrochloric acid salt (II-43) and 80% of triepi-australine hydrochloric acid salt (II-44). 138 II.1.4. Retrosynthesis of Alexine While all previous alexine syntheses and most general syntheses of polyhydroxylpyrrolidines were based on the chirality of an amino acid, a sugar, or a derivative of either, we devised a synthesis that would begin with an achiral starting material, with each additional step amenable for the synthesis of any diastereomer of alexine. In our synthesis of alexine, we desired the early incorporation of the necessary carbons with the later stage chiral addition of the hydroxyl groups. A working synthesis of this type would allow for synthesis of alexine analogues with little augmentation of the reaction sequence. We visualized the synthesis culminating in a chiral anti-dihydroxylation and benzyl deprotection to produce the final hydroxylated pyrrolizidine II-1 (Figure II-2). Tosyl deprotection and palladium-mediated amino-cyclization of pyrrolidine (II- 55) will form unsaturated pyrrolizidine (II-54). The construction of the pyrrolidine ring will be achieved via one carbon homoligative relay ring expansion of HO HO N Dihydroxylation HO H OH (+) - Alexine (II-1) H HO II-54 BnO N Pd mediated cyclization OBn Ts N II-55 PO H HO X OBn II-58 + HO NTs OTBS II-57 oxidation addition BnO OH II-56 one carbon homoligative relay ring expansion NTs OTBS Figure II-2: Retrosynthetic analysis of (+)-Alexine (II-1) via one-carbon homologative relay ring expansion. 139 to (II-59) form a 2,3-disubstituted pyrrolidine aziridine (II-56), a methodology developed by Borhan and coworkers (Figure (II- 3). 62 This methodology relies on the basic and nucleophilic nature of a ylide created under basic conditions from sulfoxonium iodide (II-61) to react with an (II-60) with aziridinol stereochemistry determined by the substitution of the aziridinol. Aziridinol deprotonation by the ylide causes formation of aza-Payne rearrangement product (II-B). Subsequent ylide attack creates dianion (II-D). 5-Exo-tet cyclization gives rise to the ring-expanded pyrrolidine (II-E). It should be noted that the aziridine submitted to this methodology in this synthesis is the most complex compound R1 R2 Ts N OH R3 II-59 O I S II-61 NaH, DMSO 80 oC, 24 h HO R3 R2 N Ts R1 II-60 68-82% yield Maintained ee TsN R OH base aza-Payne rearrangement R O S II-C R NTs O NTs 5-exo-tet HO R N Ts II-A II-E Figure II-3: One-carbon homoligative relay ring expansion of aziridinols. II-B S O O II-D submitted to this reaction, producing all of the required carbons for the alexine skeleton after cyclization. This aziridinol (II-56) can be synthesized using oxidation of a simpler disubstituted aziridinol followed by nucleophilic addition of protected allyl alcohol (II- 58) to the aldehyde (II-57). 140 the alkene diol II-63 in 81% yield. the diol produced gave II.2. Results and Discussion II.2.1. Synthesis of Alexine Our synthesis of alexine began with the synthesis of the simple aziridinol II-57 via the reduction of commercially available 2-butyne-1,4-diol with LiAlH4 (Scheme II-7). This reaction produced trans-1,4-butanediol in 54% yield. Decreasing the equivalents of LAH from 2.0 equivalents to 1.2 equivalents and adding Celite® to the work up to prevent the coagulation of the aluminum gel and the sequestration of Initial monoprotection of trans-alkenediol II-63 with benzyl bromide proceeded in 70% of II-64. Though this procedure normally provides higher yields, 63 the reaction was not optimized. We decided it would be more feasible to use a less permanent protecting group and reserve the benzyl protection for a functionality that would endure to the end of the total synthesis, namely the allyl alcohol II-69. In searching for a new protecting group, initial attempts at mono protection using tert- silyl chlorides and dehydropyran provided butyldimethylsilyl chloride provided the highest at 34%. Later optimizations using sodium hydride brought the yield up to 62% approximately doubling the yield. Allyl alcohol II-65 was submitted to a racemic aziridination using chloramine-T and N-bromosuccinimide to provide 55% of aziridinol II-66. Dess-Martin periodinane oxidation gave high yields of aziridinal II-57 (92% to quantitative yield). low yields, of which 141 HO II-62 OH LiAlH4, THF rt, 12 h, 81% HO OH 1.1 equiv BnBr 1.0 Ag2O, DCM 70% BnO OH II-64 II-63 TBSCl (1.0 equiv) NaH (1.0 equiv), THF, rt, 1 h, 62% TBSO II-65 OH Chloram. T (1.1 equiv) NBS (0.2 equiv), MeCN rt, 24 h, 55% TBSO NTs II-66 OH DMP (1.4 equiv) NaHCO3 (1.5 equiv) CH2Cl2 (0.09M) 92% to quant. TBSO NTs O II-57 Scheme II-7: Synthesis of aziridinal fragment II-57. As shown in Scheme II-8, synthesis of vinyl halide II-58 began with a procedure done by Beruben et. al.64 that incorporated the hydroiodination of commercially available ethyl propiolate II-67 (82%) and subsequent DIBAL-H reduction of (Z)- b-iodo ethyl acrylate (II-68), giving cis-allyl alcohol II-69 in 92% yield. In order to avoid elimination and formation of the protected propargyl alcohol, we attempted the acidic benzyl protection of II-69. All attempts at benzyl protection under acidic conditions failed, giving rise to a change in the method for the synthesis of ether II-58. Commercially available propargyl alcohol (II-70) was benzyl protected under basic conditions giving benzyl propargyl ether (II-71). Alkyne iodination gave II- 72 in 71% yield. Utilizing NBSH reduction, II-58a was formed in a decent yield (69%) but produced a substantial amount of overreduced alkyl halide in 142 combination with the desired vinyl iodide (3:4, respectively). In a similar sequence, benzyl propargyl ether was brominated in aqueous potassium O O II-67 NaI, AcOH 82% I O O II-68 DIBAL-H, CH2Cl2 -78 °C to 0 °C 92% OH I II-69 NH CCl3 BnO BF3.OEt2 CH2Cl2 No Conv. OH BnBr, KOH DMSO, 92% II-70 OBn II-71 AgNO3 (0.1 equiv) NIS (1.3 equiv) acetone, 3 h 71% I OBn NBSH (2.0 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH 69% II-72 I I OBn II-58a OBn II-58a NH2 O O S N H NO2 2-Nitrobenzenesulfonylydrazide NBSH 2 : 1.5 reduced/overreduced Br2 (2.0 equiv) KOH (5.2 equiv) H2O, 86% Br OBn NBSH (2.0 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH 37% II-73 OBn Br II-58b 10 : 1 reduced/overreduced Scheme II-8: Synthesis of vinyl halide fragment II-58. hydroxide. NBSH alkyne reduction to produce II-58b proceeded much less efficiently, giving 37% yield with a much lower instance of overreduction (10:1, alkene to alkane). It should also be noted that while iodination of the alkyne utilizes expensive reagents, bromination is significantly more economical and produces a greater overall selectivity in the end. In an exploration of nucleophilic addition of organometallic reagents to aziridinals, 65 Kulshrestha et. al. found that the aziridine substitution and the nitrogen protecting group affect the stereochemistry and selectivity of the resulting 143 alcohol. While this study is thorough, addressing aliphatic, aromatic, and acetylenic organometallics, it does not extend to cis-vinyl organometallics. Therefore, the nucleophilic addition of II-58 to aziridinal II-57 featured in Scheme II-9 would expand on the previous study. OBn X II-58 (1.2 equiv) 1. nBuLi (1.05 equiv) or tBuLi (2.0 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC OBn Br II-58b (1.1 equiv) 1. Mg (5.0 equiv) THF, -78 oC 2. O TsN THF, 0 oC OTBS II-57 BnO HO Ts N OTBS II-56 (0%) No magnesium insersion observed BnO HO Ts N II-56 (0%) + OTBS BnO HO Ts N OTBS II-74 (20-30%) BnO 1. nBuLi (1.05 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC BnO HO Ts N OTBS II-74 (28%) II-71 (1.1 equiv) Scheme II-9: Initial nucleophilic combination of fragments. Initial attempts to nucleophilically introduce vinyl halide II-58 to aziridinal II-57 via Grignard addition failed using both the vinyl bromide II-58b and the vinyl iodide II-58a. Since the vinyl halide was not consumed, it appeared that the vinyl Grignard was not forming. Lithium-halogen exchange resulted in the complete consumption of the vinyl halide though the expected allyl aziridinol II-56 was not observed. Instead, the propargyl aziridinol II-74 formed in 20-30% yield. It appears that alkyl lithium was causing the dehydrohalogenation of the vinyl halide resulting in the formation of II-71 in situ, followed by formation of the 144 alkynyl lithium nucleophile (Scheme II-10). This is a surprising outcome because Beruben and coworkers were able to form the cis-vinyl lithium adduct under similar conditions with the only difference being the substrate protecting group. In order to verify the synthesis of II-74, benzyl protected propargyl alcohol II-71 was submitted directly to the nucleophilic addition to aziridinal II-56, producing II-74 in 28% yield. OBn RLi RLi OBn OBn Br Scheme II-10: Dehydrohalogenation of cis-vinyl halide II-58b and lithiation of in situ formed alkyne. Li Ethylmagnesium bromide as a base was much more reactive (Scheme II-11), giving II-74 and bromoamine II-75 in a ratio of 1:3 favoring the ring opened product. We theorize that the ethylmagnesium bromide disproportionated into diethyl magnesium and magnesium bromide via the Schlenck equilibrium, both a Lewis acid and a source of nucleophilic bromide. This has long been used as a 1. EtMgBr (1.1 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC BnO II-71 (1.1 equiv) BnO HO Ts N II-74 (25%) OTBS + BnO OH NHTs OTBS Br II-75 (69%) BnO II-71 (5.0 equiv) 1. iPrMgBr (5.0 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC HO Ts N OTBS II-74 (78%) BnO Scheme II-11: Nucleophilic addition using Grignard bases. 145 method for preparing dialkylmagnesium compounds. 66 In order to discourage the formation of the dialkylmagnesium, isopropylmagnesium bromide was submitted as a base. The modified reaction provided 78% of II-74 with no formation of the ring-opened II-75. It should be noted that only one diastereomeric product was produced by this addition. This is intriguing because Kulshrestha showed that trans substituted tosyl-protected aziridinals should produce a ratio of 70:30 of syn to anti when acetylene magnesium bromide is the nucleophile of choice. Due to the formation of this single diastereomer, we decided to postpone syn/anti assignment because the new stereocenter is and will be on a freely rotating portion of the synthetic intermediate until after the ring expansion. Initial reduction of complex alkyne II-74 featured in Scheme II-12 was attempted using NBSH reduction. Wulff and coworkers showed the utility of NBSH for the purposes of partial alkyne reduction in the synthesis of Fostriecin. 67 Attempting this partial reduction of II-74 with NBSH was much less effective, providing mixtures of products at different reductive levels-from unreduced to completely saturated. Even after employing observed optimizations, overreduction was a major product. We found that palladium on barium sulfate provided a quantitative yield of II-56. 146 With aziridinol II-56 in hand, it was important to protect the secondary alcohol and free the primary alcohol in order to prepare the compound for one-carbon homologative relay ring expansion. To this end and in attempts to protect and selectively free the primary alcohol, we initially attempted MOM protection with MOMCl in diisopropyl ethylamine only retreaving starting material. More reactive conditions, MOMCl with sodium in dimethoxyethane (DME), led to small amounts of product and deterioration of the starting material. Even methyl etherification using dimethyl sulfate and TBAI iodide and diisopropyl ethylamine a. b. c. TBSO MeO OTBS O II-76 NBSH (2.0 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH rt, 14 h, 93% I TBSO MeO OTBS O II-77 I HO Ts N II-74 HO Ts N II-74 BnO BnO OTBS NBSH (2.2 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH rt, 14 h, 0.08 M OTBS NBSH (2.2 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH rt, 14 h, 0.03 M HO Ts N OTBS OBn II-56 1 HO Ts N OTBS OBn II-56 1 + : + : HO Ts N OTBS OBn II-78 1.5 HO Ts N OTBS OBn II-78 2 HO Ts N II-74 OTBS H2, Pd-BaSO4 (1.0 mol%) quinoline (30.0 mol%) MeOH, quantitative BnO HO Ts N OTBS II-56 OBn Scheme II-12: Alkyne reduction. a. The NBSH reduction performed by Phillips et al. provided 93% of desired product. b. NBSH reduction of propargyl aziridinol proved more reactive and difficult to partially reduce. c. Palladium on barium sulfate proved sufficient for a quantitative partial reduction to give the desired cis-alkene. 147 (0.5%) in a biphasic solution of 30% aqueous sodium hydroxide and light petroleum ether gave no noticeable yield of the desired product. Acidic PMB protections catalyzed by PPTS also showed no conversion. Silyl protection using TBDPSCl and TBSCl were also ineffective. The difficulty in protecting allyl aziridinol II-56 led us to consider protection of propargyl aziridinol II-74 instead. Reactive MOMCl protection with sodium iodide also failed using this substrate. TBSCl provided the only successful protection of the secondary alcohol with 71% yield of diprotected aziridine II-78 (Scheme II- 13). An economic selective deprotection of the primary alcohol proceeded to give 45% yield of aziridinol II-79 before the formation of significant amounts of doubly deprotected aziridinediol. As an analogue of II-56, II-79 was submitted to the one-carbon homologative relay ring expansion and produced pyrrolidine II-80 in 65% yield with what appeared to be two diastereomers (d.r. = 3.5:1). While this is on the lower side of the yields seen in Schomaker’s chemistry, the result is BnO HO Ts N II-74 OTBS TBSCl (1.1 equiv) imidazole (2.2 equiv) DMF, 71% BnO TBSO Ts N II-78 OTBS AcOH, H2O, THF (13:7:3) 45% BnO OTBS Ts N H HO II-80 (CH3)3SOI (5.0 equiv) NaH (5.0 equiv), DMSO, 85 °C, 24 h, 65% BnO TBSO Ts N OH II-79 Scheme II-13: Silyl exchange and one-carbon homologative relay ring expansion. 148 reasonably good in lieu of the complexity of the substrate with no real optomization. Additionally, the diastereomeric ratio is interesting. It is known that the pyrrolidines formed using the one-carbon homologative relay ring expansion generally produce a single diastereomer with little change in the purity of enantiomerically enriched substrates. The fact that pyrrolidine II-80 was produced in two diastereomers suggests that a previous step in which diastereomers could be produced, namely, the nucleophilic addition of the metalloalkyne of propargyl ether II-71 to aziridinal II-57, did produce diastereomers but did not show obvious signs of the two molecules via general spectroscopic means. These ratios are comparible to those seen in tosyl-protected trans-aziridinals with ethynylmagnesium bromide (70:30, syn : anti). 65 secondary TBS group with In tetrabutylammonium fluroride and to remove the tosyl group from the nitrogen with magnesium powder in methanol produced inconclusive results. Further advancements into the total synthesis of alexine (II-1) are still being investigated. II.3. Conclusion Pyrrolizidine syntheses have generally utilized small chiral compounds as initial building blocks in their synthesis and impose subsequent modifications on the original chiral center. More specifically, alexine has had very few syntheses that did not begin with a chiral building block of some sort. Our synthesis was initial attempts to deprotect the 149 designed to impose the desired chirality upon an achiral substrate and cleverly and predictably add carbon skeleton and hydroxyl moieties that would, with little change to the total synthesis, be able to create new and different diastereomeric analogues of alexine. To this end and in attempts to explore cis-vinylmagnesium bromide addition to aziridinals, we attempted the nucleophilic addition of vinyl halide II-58 to aziridinal II-57. Instead of the desired product, propargyl ether incorporation was the only product observed. Utilizing the lithium acetylenide provided the newly desired propargyl aziridinol in low yields. The desire to increase nucleophile reactivity and prevent Grignard disproportionation left us with isopropylmagnesium bromide as the ideal base to provide the necessary acetylene magnesium bromide in adequate yields. Due to the ineffectivity of the partially reduced II-56, alkyne II-74 was submitted to a silyl exchange, protecting the new secondary alcohol and liberating the primary alcohol in preparation for the one-carbon homologative relay ring expansion. This reaction resulted in the complex disubstituted pyrrolidine in comparable yield to that of the more simple substrates initially tested in the development of this method. It should be noted that this is the most complex substrate submitted to this reaction. In order to complete this synthesis, it will be necessary to remove the tosyl protecting group. While it is a necessary protecting group for the efficiency of the relay ring expansion, it is a somewhat tempermental process and must be tested 150 for the most optimal removal after every step beginning with the ring expansion itself. While there are many ways to continue this synthesis, a protection of diol II-81 as a carbonate provides many benefits: (1.) it enables us to find the relative stereochemistry of the unknown chiral center created by the nucleophilic addition by creating a rigidified ring system, (2.) it locks the alkyne in a position that would better educate us in the feasibility of tosyl removal at this stage, (3.) it provides insertion for for the palladium the upcoming the necessary handle aminocyclization, and (4.) it provides us with a predictive model of the aminocyclization based on the stereochemistry of the unknown center. Reduction of the alkyne II-82 to the cis-alkene II-83 provides the ideally prepared susbstrate for a palladium-mediated aminocyclization. With a stereochemically salt-directed aminocyclization, II-84 requires only an anti- dihydroxylation to give a benzyl-protected alexine requiring only a mild deprotection of a primary alcohol. This racemic synthesis can be made asymmetric utilizing simple known methodology.62 Schomaker used Sharpless epoxidation, azide ring opening, and aziridine ring closing followed by tosyl protection to form the necessary chiral aziridinol. Córdova and coworkers 68 rely on the asymmetric aziridination of a,b- unsaturated aldehydes to give the necessary aziridinal. 151 In closing, while this synthesis is not complete, it provides a few colorful steps in the synthesis of alexine from completely achiral starting materials that is amenable to subtle changes to create other analogues. More studies will be undertaken to complete this and, ultimately, the asymmetric syntheses of alexine. OBn Ts N EtO2CO BnO OH H HO II-81 O O H R N O H II-82 H2, Pd-BaSO4 OBn H R N O O O H II-83 Pd0 R = Ts/H OH BnO N HO H OH II-1 anti- dihydroxylation BnO N OH H II-84 Scheme II-14: Remaining steps in the synthesis of alexine. 152 II.4. Experimental HO II-62 OH LiAlH4, THF rt, 12 h, 81% HO II-63 OH Preparation of (E)-2-butene-1,4-diol. THF was cooled to 4 °C in a flame dried flask with a stir bar. Lithium aluminum hydride (1.2 equivalents, 139.39 mmol) was added slowly while monitoring changes in temperature and rate of bubbling. A dry THF solution of 2-butyne-1,4-diol (1.0 equivalent, 116.16 mmol) was added drop wise via cannula slowly while monitoring changes in temperature. The solution was allowed to slowly warm to room temperature and left for 14 hours. After verification of completion of the reaction via TLC in EtOAc (Rf = 0.3), the solution was cooled again to 4 °C. Celite (2.5g for every 10g of diol) was added to the stirring solution. Saturated ammonium sulfate solution was added slowly allowing for evolution of hydrogen gas and maintenance of cooled temperature. A saturated solution of sodium sulfate was slowly added followed by a 15% sodium hydroxide solution. The solution was filtered through a pad of Celite. The pad was washed several times with EtOAc. The solution was concentrated via reduced pressure giving a clear oil. The oil was distilled via krughelror (177 °C, 40 Torr) to give a more homogeneous clear oil. Alternately, the crude residue can be purified via flash column chromatography in ethyl acetate with yields of 81%. 1H NMR (500 MHz, CDCl3) δ 5.9 (dt, J = 1 Hz, 1.5 Hz, 2 H), 4.2 (s, 4 H), 1.6 (brs, 2 H); 13C NMR (62.8 MHz, CDCl3) δ 130.5, 62.8. 153 OH HO II-63 1.1 equiv BnBr 1.0 Ag2O, DCM 70% BnO OH II-64 2-butene-1,4-diol (E)-4-(benzyloxy)but-2-en-1-ol. Preparation of (1.0 equivalent, 124.7 mmol) was dissolved in methylene chloride along with silver oxide (1.0 equivalents, 124.7 mmol) and allowed to stir for minutes. Benzyl bromide (1.1 equivalents, 137.2 mmol) was added to the solution and allowed to stir for 24 hours. The reaction was monitored via TLC and upon completion, filtered through a silica gel plug. Concentration en vacuo was followed by additional filtration through another silica gel plug, yielding a clear oil. The crude was purified via column chromatography (30% EtOAc in hexanes) to give a clear oil in 70% yield. 1H NMR (500 MHz, CDCl3) δ 7.31 (m, 5 H), 5.91 (dtt, J = 16.0 Hz, 5.5 Hz, 1.5 Hz, 1 H), 5.84 (dtt, J = 15.5 Hz, 5.5 Hz, 1.5 Hz, 1 H), 4.51 (s, 2 H), 4.16 (tq, J = 5.0 Hz, 1.0 Hz, 2 H), 4.03 (dq, J = 5.5 Hz, 1.0 Hz, 2 H); 13C NMR (62.8 MHz, CDCl3) δ 132.37, 128.82, 128.64, 128.21, 127.99, 127.89, 72.59, 70.31, 63.32. OH HO II-63 TBSCl (1.0 equiv) NaH (1.0 equiv), THF, rt, 1 h, 62% TBSO II-65 OH (E)-4-((tert-butyldimethylsilyl)oxy)but-2-en-1-ol. Sodium Preparation of hydride (1.0 equivalent, 93.8 mmol) was charged into a flame dried flask with a stir bar and 100 mL of dry tetrahydrofuran. After sealing, the container was purged with nitrogen gas. 2-butene-1,4-diol (1.0 equivalent, 93.8 mmol) was dissolved in 60mL of dry tetrahydrofuran and added dropwise to the suspension 154 and allowed to stir for 45 minutes. Tert-butyldimethylsilylchloride (1.0 equivalent, 93.8 mmol) dissolved in 15 mL of dry tetrahydrofuran was added to the solution in one portion, providing a milky white solution that was allowed to stir for another 45 minutes. After monitoring the consumption of the alcohol using TLC (20% EtOAc in hexanes), 450 mL of ether was added to the solution. 300 mL of 10% K2CO3 solution is used to wash the ether solution followed by 40 mL of a brine solution. The aqueous layers were washed with diethyl ether. Organic layers were combined, dried over sodium sulfate, filtered, and concentrated en vacuo. The crude residue was purified using column chromatography initially using 10% EtOAc in hexanes and after elusion of excess TBS chloride, 20% EtOAc in hexanes was used to elute the desired product in 62% yield of a clear liquid. 1H NMR (500 MHz, CDCl3) δ 5.86 (dt, J = 5.5 Hz, 15.5 Hz, 2 H), 5.78 (dt, J = 3.5 Hz, 15.5 Hz, 2 H), 4.16 (dd, J = 1.5 Hz, 4.5 Hz, 2 H), 4.14 (d, J = 5.5 Hz, 2 H), 1.35 (brs, 1 H), 0.89 (s, 9 H), 0.05 (s, 6 H); 13C NMR (62.8 MHz, CDCl3) δ 131.26,129.15, 63.43, 63.33, 26.18, 18.65, -5.02. TBSO II-65 OH Chloram. T (1.1 equiv) NBS (0.2 equiv), MeCN rt, 24 h, 55% TBSO NTs II-66 OH (3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2- Preparation of yl)methanol. The mono-protected diol, (E)-4-((tert-butyldimethylsilyl)oxy)but-2- en-1-ol (1.0 equivalent, 1.96 mmol) was dissolved in dry acetonitrile along with a stir bar. Chloramine-T trihydrate (1.1 equivalents, 2.16 mmol) dried 60 °C under vacuum was added along with recrystallized NBS (0.2 equivalents, 0.39 mmol) were added to the solution and the flask was sealed. After purging with nitrogen gas, the solution was allowed to stir at room temperature for 19 hours. Upon 155 completion, an equal volume of water was added and the solution was extracted with ethyl acetate. The combined organics were dried over sodium sulfate and concentrated en vacuo. The viscous crude was purified via column chromatography (20% EtOAc in Hexanes) to give a slightly yellow viscous oil in 55% yield. 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.0, 2 H), 7.29 (d, J = 8.5, 2 H), 4.13 (ddd, J = 13.0 Hz, 9.5 Hz, 3.0 Hz, 1 H), 3.96 (ddd, J = 13.0 Hz, 8.0 Hz, 4.5 Hz, 1 H), 3.76 (dd, J = 11.5 Hz, 4.0 Hz, 1 H), 3.58 (dd, J = 11.5 Hz, 6.0 Hz, 1 H), 3.15 (dd, J = 10.5 Hz, 4.5 Hz, 1 H), 3.03 (ddd, J = 8.0 Hz, 4.5 Hz, 3.0 Hz, 1 H), 2.86 (dd, J = 9.0 Hz, 4.5 Hz, 1 H), 2.41 (s, 3 H), 0.78 (s, 9 H), - 0.089 (s, 3 H), - 0.114 (s, 3 H). TBSO NTs OH II-66 DMP (1.4 equiv) NaHCO3 (1.5 equiv) CH2Cl2 (0.09M) 92% to quant. TBSO NTs II-57 O (3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridine-2- Preparation of carbaldehyde. DMP (1.4 equivalents, 0.377 mmol) and sodium bicarbonate (1.5 equivalents, 0.412 mmol) were charged into a flame drid flask with a stir bar. The flask was cooled in an acetone/dry ice bath and a prepared solution (0.09M) of aziridinol II-66 (1.0 equivalents, 0.269 mmol) in dry methylene chloride was added. The flask was moved from a -78 °C bath to a 0 °C bath and allowed to stir without monitoring for 3 hours. After TLC verification of completion, a saturated aqueous solution of sodium thiosulfate was added to the solution. It caused a change in the color of the solution to white opaque and then clear. Saturated sodium bicarbonate solution was then added followed by water. The solution was extracted with methylene chloride and the organics were dried over sodium sulfate to be concentrated en vacuo giving a viscous yellow oil in quantitative 156 yield. 1H NMR (500 MHz, CDCl3) δ 9.54 (d, J = 12.0 Hz, 1 H), 7.83 (d, J = 14.0 Hz, 2 H), 7.32 (d, J = 13.5 Hz, 2 H), 3.83 (dd, J = 19.0 Hz, 6.0 Hz, 1 H), 3.70 (dd, J = 19.0 Hz, 8.0, 1 H), 3.60 (dt, J = 8 Hz, 6.5 Hz, 1 H), 3.18 (dd, J = 11.0 Hz, 6.5 Hz, 1 H), 2.43 (s, 3 H), 0.79 (s, 9 H), - 0.077 (s, 3 H), - 0..91 (s, 3 H). O O II-67 NaI, AcOH 82% I O O II-68 Preparation of (Z)-ethyl 3-iodoacrylate. Sodium iodide (1.0 equivalent, 51 mmol) was dissolved in acetic acid in a flame-dried flask with a stir bar. The solution was purged with nitrogen gas and heated to 80 °C. Ethyl propionate (1.0 equivalent, 51 mmol) was added dropwise while noting the color change in the solution (slightly darker yellow). Water was added to the solution after cooling to room temperature. The solution was extracted with diethyl ether (3 x 50 ml). The organic phase was then washed with 4M potassium hydroxide until the aqueous layer had become neutral. The organic layer was dried over sodium sulfate and concentrated en vacuo. Purification has proven unnecessary since by NMR the compound appears pure. 82% yield. 1H NMR (500 MHz, CDCl3) δ 7.42 (d, J = 9.0 Hz, 1 H), 6.87 (d, J = 9.0 Hz, 1 H), 4.23 (q, J = 7.0 Hz, 2 H), 1.26 (t, J = 7.0 Hz, 3 H); 13C NMR (62.8 MHz, CDCl3) δ 164.8, 130.2, 94.8, 61.0, 14.4. I O O II-68 DIBAL-H, CH2Cl2 -78 °C to 0 °C 92% I OH II-69 157 (Z)-ethyl-3-iodoacrylate (Z)-3-iodoprop-2-en-1-ol. Preparation of (1.0 equivalents, 4.4 mmol) was dissolved in dry methylene chloride in a flame-dried flask with a stir bar. The flask was sealed, purged with nitrogen gas, and cooled to -78 °C. Diisobutylaluminum hydride (2.2 equivalents, 9.8 mmol) was slowly added to the solution and after 10 minutes of stirring, it was warmed to 0 °C and left for 30 more minutes. After verification of completion using TLC (25% EtOAc in hexanes), a saturated solution of Rochelle’s salt was added very slowly over an hour. After bubbling ceased, Glycerine was added and methylene chloride was used to dilute the solution. The solution was allowed to stir for 12 hours. The solution was filtered over Celite and washed with water and then with brine. The organic layer was dried over sodium sulfate and concentrated en vacuo. Reaction yielded 77% of mildly impure (peaks for toluene, the DIBAL-H solvent were apparent) light yellow oil. 1H NMR (500 MHz, CDCl3) δ 6.48 (dt, J = 8.0 Hz, 5.5 Hz, 1 H), 6.35 (dt, J = 8.0 Hz, 1.5 Hz, 1 H), 4.23 (t, J = 4.0 Hz, 2 H), 1.54 (s, 1 H); 13C NMR (62.8 MHz, CDCl3) δ 140.2, 82.9, 66.0. OH BnBr, KOH DMSO, 92% II-70 OBn II-71 Preparation of ((prop-2-yn-1-yloxy)methyl)benzene. Potassium hydroxide (3.0 equivalents, 160.54 mmol) and DMSO were charged into a flask with a stir bar and cooled to 0 °C. Stirring had to be maintained during the process of cooling. Propargyl alcohol (1.0 equivalent, 53.51 mmol) was added at 0 °C and the solution was allowed to stir for 10 minutes, periodically warming to prevent total freezing of DMSO. Benzyl bromide (1.0 equivalent, 53.51 mmol) was also added at 0 °C and the solution was allowed to warm to room temperature. The reaction 158 was allowed to stir for 15 hours. Upon completion of the reaction, water was added to the solution followed by diethyl ether. The aqueous layer was washed with diethyl ether (3 x 50 ml) and the organic layers were combined and dried over sodium sulfate. After concentration, the compound was proven to be quite pure. 92% yield. 1H NMR (500 MHz, CDCl3) δ 7.35 (m, 4 H), 7.29 (m, 1 H), 4.60 (s, 2 H), 4.16 (d, J = 2.5 Hz, 2 H), 2.45 (t, J = 2.5 Hz, 1 H). OBn II-71 Br2 (2.0 equiv) KOH (5.2 equiv) H2O, 86% Br II-73 OBn Preparation of (((3-bromoprop-2-yn-1-yl)oxy)methyl)benzene. Potassium hydroxide pellets (5.2 equivalents, 7.11 mmol) were added to a round bottom flask with a stir bar and dissolved in water. The solution was then cooled to 0 °C and bromine liquid (0.75 equivalents, 1.03 mmol) was added in small portions. The solution was allowed to stir for 15 minutes. The alkyne (1.0 equivalents, 1.37 mmol) was then added drop wise before allowing to stir for 30 minutes at 0 °C. After cooling to room temperature, the aqueous solution was extracted with diethyl ether (3 x 10 ml). The combined organic phase was dried over magnesium sulfate and concentrated en vacuo. Purification was not required. 86% yield. 1H NMR (500 MHz, CDCl3) δ 7.32 (m, 5 H), 4.58 (s, 2 H), 4.18 (s, 2 H). 159 OBn II-71 AgNO3 (0.1 equiv) NIS (1.3 equiv) acetone, 3 h 71% I OBn II-72 Preparation of (((3-iodoprop-2-yn-1-yl)oxy)methyl)benzene. Silver nitrate (0.1 equivalent, 0.14 mmol) and NIS (1.3 equivalents, 1.78 mmol) were added to a solution of the alkyne (1.0 equivalent, 1.37 mmol) in magnesium sulfate-dried acetone. The solution was allowed to stir for 3 hours. Hexanes were added to the solution and filtered over Celite. In the dark, water was added to the solution and the solution was extracted with hexanes. The organics were combined, dried over sodium sulfate, and concentrated en vacuo. Purification of this light yellow oil was not required. 71% yield. 1H NMR (500 MHz, CDCl3) d 7.33 (m, 4 H), 7.29 (m, 1 H), 4.58 (s, 2 H), 4.30 (s, 2 H). OBn NBSH (2.0 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH 37% Br II-73 OBn Br II-58b 10 : 1 reduced/overreduced Preparation of (Z)-(((3-bromoallyl)oxy)methyl)benzene. The alkynyl bromide (1.0 equivalent, 0.755 mmol) was dissolved in a 1:1 mixture of tetrahydrofuran and isopropyl alcohol. Nitrobenzene sulfonic hydrazide (NBSH) (2.0 equivalents, 1.511 mmol) was added to the solution and the flask was sealed and purged with nitrogen gas. Triethylamine (1.5 equivalents, 1.133 mmol) was added to the solution and the flask was purged with nitrogen gas. The solution was allowed to stir for 14 hours. The reaction was quenched with saturated sodium bicarbonate and diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate (3 x 5 ml) and the organic layers were combined and dried over sodium 160 sulfate. The solution was concentrated en vacuo. The crude was purified via column chromatography to yield a slight yellow mixture of the alkene and its overreduced equivalent in a ratio of 10:1 in favor of the alkene with a total yield of 37%. Alkene: 1H NMR (500 MHz, CDCl3) d 7.31 (m, 5 H), 6.48 (dt, J = 6.5 Hz, 4.5 Hz, 1 H), 6.39 (dt, J = 6.5 Hz, 1.5, 1 H), 4.53 (s, 2 H), 4.12 (dd, J = 4.5 Hz, 1.5 Hz, 2 H). Overreduced: 1H NMR (500 MHz, CDCl3) d 7.31 (m, 5 H), 6.48 (dt, J = 6.5 Hz, 4.5 Hz, 1 H), 6.39 (dt, J = 6.5 Hz, 1.5, 1 H), 4.51 (s, 2 H), 3.53 (t, J = 5.0 Hz, 2 H), 3.29 (t, J = 6.0 Hz, 2 H), 2.08 (quint, J = 5.0 Hz, 2 H). I II-72 OBn NBSH (2.0 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH 69% OBn II-58a : 1.5 I 2 reduced/overreduced Preparation of (Z)-(((3-iodoallyl)oxy)methyl)benzene. The alkynyl iodide (1.0 equivalent, 0.967 mmol) was dissolved in a 1:1 mixture of tetrahydrofuran and isopropyl alcohol. Nitrobenzene sulfonic hydrazide (NBSH) (2.0 equivalents, 1.933 mmol) was added to the mixture and the flask was purged with nitrogen gas. Triethyl amine (1.5 equivalents, 1.450 mmol) was added and the solution was allowed to stir for 14 hours. The reaction was quenched with saturated sodium bicarbonate and diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate (3 x 5 ml). The organic layers were combined, dried over sodium sulfate, and concentrated en vacuo. The crude was purified via column chromatography to yield a light yellow mixture of the alkene and the overreduced equivalent in a ratio of 2:1.4 with a total yield of 69%. 1H NMR (500 MHz, CDCl3) d 7.34 (m, 4 H), 7.28 (m, 1 H), 6.48 (dt, J = 6.5 Hz, 4.5 Hz, 1 H), 161 6.39 (dt, J = 6.5 Hz, 1.5 Hz, 1 H), 4.53 (s, 2 H), 4.12 (dd, J = 4.5 Hz, 1.5 Hz, 2 H); 13C NMR (62.8 MHz, CDCl3) d 138.5, 138.0, 128.7, 128.1, 127.9, 83.4, 72.9, 69.8. 1. nBuLi (1.05 equiv) or tBuLi (2.0 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC OBn Br II-58b (1.1 equiv) BnO HO Ts N OTBS II-74 (20-30%) Preparation of 4-(benzyloxy)-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1- tosylaziridin-2-yl)but-2-yn-1-ol. The (Z)-(((3-bromoallyl)oxy)methyl)benzene (1.0 equivalent, 0.434 mmol) was dissolved in dry tetrahydrofuran with a stir bar. After sealing, the flask was purged with nitrogen gas. After cooling to -78 °C, n- butyllithium (1.3 equivalents, 0.564 mmol) was added to the solution and allowed to stir for 1 hour. A solution of the aziridinal (0.9 equivalents, 0.391 mmol) was added to the reaction and monitored via TLC (30% EtOAc in hexanes). Upon completion, 10 mL of ammonium chloride saturated solution was added dropwise to the solution and allowed to stir for 5 minutes. The solution was extracted with ethyl acetate, the organics were combined, washed with brine, and dried over sodium sulfate before concentrating en vacuo. The residue was purified using column chromatorgraphy (20% EtOAc in hexanes) to provide 20-30% of a light yellow liquid. . 1H NMR (500 MHz, CDCl3) d 7.83 (d, 8.5 Hz, 2 H), 7.31 (m, 7 H), 4.74 (dd, J = 5.0 Hz, 7.0 Hz, 1 H), 4.68 (s, 2 H), 4.56 (d, J = 1.5 Hz, 2 H), 4.17 (d, J = 1.5 Hz, 2 H), 3.85 (dd, 4.5 Hz, 11.5 Hz, 1 H), 3.73 (dd, 5.5 Hz, 11.0 Hz, 1 H), 3.14 (m, 2 H), 3.07 (d, 4.5 Hz, 1 H), 2.40 (s, 3H), 0.80 (s, 9 H), - 0.052 (s, 3 H), - 162 0.072 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.8, 128.8, 128.7, 128.3, 128.2, 127.9, 127.8, 127.2, 72.0, 65.6, 61.5, 61.4, 57.5, 51.0, 47.2, 26.0, 21.8, 18.4. The (Z)-(((3-bromoallyl)oxy)methyl)benzene (1.0 equivalent, 0.44 mmol) was dissolved in dry tetrahydrofuran with a stir bar. After sealing, the flask was purged with nitrogen gas. After cooling to -78 °C, tert-butyllithium (2.0 equivalents, 0.88 mmol) was added to the solution and allowed to stir for 30 minutes. A solution of the aziridinal (1.0 equivalents, 0.44 mmol) was added to the reaction dropwise and monitored via TLC (30% EtOAc in hexanes). After stirring overnight (increasing to room temperature slowly), 4 mL of ammonium chloride saturated solution was added dropwise to the solution and allowed to stir for 10 minutes. The solution was extracted with ethyl acetate (4 x 4 mL), the organics were combined, and dried over sodium sulfate before concentrating en vacuo. The residue was purified using column chromatorgraphy (20% EtOAc in hexanes) to provide 20% of a light yellow liquid. . 1H NMR (500 MHz, CDCl3) d 7.83 (d, 8.5 Hz, 2 H), 7.31 (m, 7 H), 4.74 (dd, J = 5.0 Hz, 7.0 Hz, 1 H), 4.68 (s, 2 H), 4.56 (d, J = 1.5 Hz, 2 H), 4.17 (d, J = 1.5 Hz, 2 H), 3.85 (dd, 4.5 Hz, 11.5 Hz, 1 H), 3.73 (dd, 5.5 Hz, 11.0 Hz, 1 H), 3.14 (m, 2 H), 3.07 (d, 4.5 Hz, 1 H), 2.40 (s, 3H), 0.80 (s, 9 H), - 0.052 (s, 3 H), - 0.072 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.8, 128.8, 128.7, 128.3, 128.2, 127.9, 127.8, 127.2, 72.0, 65.6, 61.5, 61.4, 57.5, 51.0, 47.2, 26.0, 21.8, 18.4. 163 BnO II-71 (1.1 equiv) 1. nBuLi (1.05 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC BnO HO Ts N OTBS II-74 (28%) The ((prop-2-yn-1-yloxy)methyl)benzene (1.0 equivalent, 1.37 mmol) was charged into a flame dried flask with a stir bar. After sealing, the flask was purged with nitrogen gas and dry THF was added via syringe. The solution was cooled to -78 °C and n-butyllithium (1.05 equivalents, 1.44 mmol) was added to the solution and allowed to stir for 30 minutes. A solution of the aziridinal (1.0 equivalents, 1.37 mmol) was added to the reaction dropwise and the color changed noted. After stirring overnight (increasing to room temperature slowly), 4 mL of ammonium chloride saturated solution was added dropwise to the solution and allowed to stir for 10 minutes. The solution was extracted with ethyl acetate (4 x 4 mL), the organics were combined, and dried over sodium sulfate before concentrating en vacuo. The residue was purified using column chromatorgraphy (20% EtOAc in hexanes) to provide 28% of a light yellow liquid. 1H NMR (500 MHz, CDCl3) d 7.83 (d, 8.5 Hz, 2 H), 7.31 (m, 7 H), 4.74 (dd, J = 5.0 Hz, 7.0 Hz, 1 H), 4.68 (s, 2 H), 4.56 (d, J = 1.5 Hz, 2 H), 4.17 (d, J = 1.5 Hz, 2 H), 3.85 (dd, 4.5 Hz, 11.5 Hz, 1 H), 3.73 (dd, 5.5 Hz, 11.0 Hz, 1 H), 3.14 (m, 2 H), 3.07 (d, 4.5 Hz, 1 H), 2.40 (s, 3H), 0.80 (s, 9 H), - 0.052 (s, 3 H), - 0.072 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.8, 128.8, 128.7, 128.3, 128.2, 127.9, 127.8, 127.2, 72.0, 65.6, 61.5, 61.4, 57.5, 51.0, 47.2, 26.0, 21.8, 18.4. 164 BnO II-71 (1.1 equiv) 1. EtMgBr (1.1 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC BnO HO Ts N II-74 (25%) OTBS + BnO OH NHTs OTBS Br II-75 (69%) Preparation of 4-(benzyloxy)-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1- tosylaziridin-2-yl)but-2-yn-1-ol. The ((prop-2-yn-1-yloxy)methyl)benzene (1.1 equivalent, 0.613 mmol) was charged into a flame dried flask with a stir bar. After sealing, the flask was purged with nitrogen gas and 15 mL dry THF was added via syringe. Ethylmagnesium bromide (1.1 equivalents, 0.613 mmol) was added to the solution at room temperature and allowed to stir for 1 hour before reducing the temperature to -78 °C. A 16 mL solution of aziridinal (1.0 equivalents, 0.557 mmol) was added to the reaction dropwise and the solution was allowed to stir at the lowered temperature for 10 minutes before raising the temperature to 0 °C. The reaction was left stirring for 2 hours before quenching with 4 mL of saturated ammonium chloride solution. The solution was extracted with ethyl acetate (3 x 10 mL). The organics were combined and washed with water and then with brine before drying over sodium sulfate and concentrating en vacuo. The residue was purified using column chromatorgraphy (20% EtOAc in hexanes) to provide 25% of the light yellow desired product. 1H NMR (500 MHz, CDCl3) d 7.83 (d, 8.5 Hz, 2 H), 7.31 (m, 7 H), 4.74 (dd, J = 5.0 Hz, 7.0 Hz, 1 H), 4.68 (s, 2 H), 4.56 (d, J = 1.5 Hz, 2 H), 4.17 (d, J = 1.5 Hz, 2 H), 3.85 (dd, 4.5 Hz, 11.5 Hz, 1 H), 3.73 (dd, 5.5 Hz, 11.0 Hz, 1 H), 3.14 (m, 2 H), 3.07 (d, 4.5 Hz, 1 H), 2.40 (s, 3H), 0.80 (s, 9 H), - 0.052 (s, 3 H), - 0.072 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.8, 128.8, 128.7, 128.3, 128.2, 127.9, 127.8, 127.2, 72.0, 65.6, 61.5, 61.4, 57.5, 51.0, 47.2, 26.0, 21.8, 18.4. 165 of N-(7-(benzyloxy)-3-bromo-1-((tert- N-((2S,3S)-7-(benzyloxy)-3-bromo-1-((tert- Preparation butyldimethylsilyl)oxy)-4-hydroxyhept-5-yn-2-yl)-4- methylbenzenesulfonamide. butyldimethylsilyl)oxy)-4-hydroxyhept-5-yn-2-yl)-4-methylbenzenesulfonamide was a large byproduct of the attempt to form 4-(benzyloxy)-1-((2R,3R)-3-(((tert- butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2-yl)but-2-yn-1-ol using ethylmagnesium bromide as the base and was isolated in 69% yield. 1H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.5 Hz, 4 H), 7.36-7.25 (m, 7 H), 6.05 (d, J = 8.0 Hz, 1 H), 4.69 (d, J = 5.0 Hz, 1 H), 4.54 (s, 2 H), 4.30 (q, J = 4.0 Hz, 1 H), 4.09- 4.02 (m, 3), 3.81 (dd, J = 5.0 Hz, 12 Hz, 2 H), 3.24 (br s, 1 H), 2.37 (s, 3 H), 0.905 (s, 9 H), 0.073 (s, 3 H), 0.062 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) δ 143.6, 137.2, 137.1, 129.5, 128.5, 128.1, 128.0, 127.6, 83.6, 83.4, 71.9, 65.1, 63.9, 62.0, 57.2, 51.5, 26.0, 25.7, 21.6, 18.2, -5.5, -5.6. BnO II-71 (5.0 equiv) 1. iPrMgBr (5.0 equiv) THF, -78 oC 2. O OTBS II-57 TsN THF, 0 oC BnO HO Ts N II-74 (78%) OTBS Preparation of 4-(benzyloxy)-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1- tosylaziridin-2-yl)but-2-yn-1-ol. The ((prop-2-yn-1-yloxy)methyl)benzene (5.0 166 equivalent, 1.356 mmol) was charged into a flame dried flask with a stir bar. After sealing, the flask was purged with nitrogen gas and 2 mL dry methylene chloride was added via syringe. Isopropylmagnesium bromide (5.0 equivalents, 1.356 mmol) was added to the solution at room temperature and allowed to stir for 30 minutes before reducing the temperature to -78 °C. A solution of aziridinal (1.0 equivalents, 0.271 mmol) was added to the reaction dropwise and the solution was allowed to stir for 2 hours before TLC monitoring. Upon completion, 4 mL of saturated ammonium chloride solution was added and the solution was allowed to slowly return to room temperature. The solution was extracted with methylene chloride (3 x 40 mL) and washed with brine before combining the organic layers, drying over sodium sulfate, and concentrating en vacuo. The residue was purified using a short chromatorgraphic column (5% EtOAc in hexanes to remove the excess propargyl ether before increasing to 20% EtOAc in hexanes) to provide 78% of the light yellow desired product. 1H NMR (500 MHz, CDCl3) d 7.83 (d, 8.5 Hz, 2 H), 7.31 (m, 7 H), 4.74 (dd, J = 5.0 Hz, 7.0 Hz, 1 H), 4.68 (s, 2 H), 4.56 (d, J = 1.5 Hz, 2 H), 4.17 (d, J = 1.5 Hz, 2 H), 3.85 (dd, 4.5 Hz, 11.5 Hz, 1 H), 3.73 (dd, 5.5 Hz, 11.0 Hz, 1 H), 3.14 (m, 2 H), 3.07 (d, 4.5 Hz, 1 H), 2.40 (s, 3H), 0.80 (s, 9 H), - 0.052 (s, 3 H), - 0.072 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.8, 128.8, 128.7, 128.3, 128.2, 127.9, 127.8, 127.2, 72.0, 65.6, 61.5, 61.4, 57.5, 51.0, 47.2, 26.0, 21.8, 18.4. 167 HO Ts N II-74 OTBS NBSH (2.2 equiv) NEt3 (1.5 equiv) 1:1 THF/iPrOH rt, 14 h, 0.08 M BnO HO Ts N OTBS OBn II-56 1 HO Ts N OTBS OBn II-78 1.5 + : Preparation of (Z)-4-(benzyloxy)-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)- 1-tosylaziridin-2-yl)but-2-en-1-ol. A 1:1 solution of tetrahydrofuran and isopropyl alcohol (0.969 mL) was charged into a flask with a stir bar and 4- (benzyloxy)-1-((2R,3R)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2- yl)but-2-yn-1-ol (1.0 equivalents, 0.078 mmol). NBSH (2.2 equivalents, 0.171 mmol) was added to the solution and it was capped. Triethylamine (1.5 equivalents, 0.116 mmol) was added to the solution and it was allowed to stir for 14 hours. The reaction was quenched with 2 mL of saturated sodium bicarbonate solution and diluted with 4 mL of ethyl acetate. The aqueous portion was extracted with ethyl acetate (3 x 3 mL) before the combined organic layers were dried over sodium sulfate and concentrated en vacuo. The mixture of products (desired and overreduced, 1:1.5), though identifiable by NMR, were inseparable via chromatography. Dilution of this reaction to 2.65 mL decreased access to the cis alkene even further (1:2, desired to overreduced). Alkene: 1H NMR (500 MHz, CDCl3) d 7.84 (d, J = 8.5 Hz, 2 H), 7.29 (m, 7 H), 5.79 (dt, J = 6.5 Hz, 6.0 Hz, 1 H), 5.59 (dd, J = 7.0 Hz, 9.5 Hz, 1 H), 4.75 (dd, J = 8.5 Hz, 8.5 Hz, 1 H), 4.51 (d, J = 5.5 Hz, 2 H), 4.17 (d, J = 6.0 Hz, 2 H), 3.69 (dd, J = 4.5 Hz, 11.5 Hz, 1 H), 3.58 (dd, J =6.0 Hz, 11.5 Hz, 1 H), 3.06 (dt, J = 4.5 Hz, 6.0 Hz, 1 H), 2.88 (dd, J = 4.0 Hz, 8.0 Hz, 1 H), 2.42 (s, 3 H), 0.78 (s, 9 H), - 0.099 (s, 3H), - 0.121 168 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 144.6, 130.7, 130.6, 129.8, 128.7, 128.2, 128.0, 127.8, 72.9, 67.1, 66.4, 61.7, 51.8, 47.5, 26.0, 21.9. Overreduced: 1H NMR (300 MHz, CDCl3) d 7.84 (d, J = 13.5 Hz, 2 H), 7.29 (m, 7 H), 4.54 (dt, J = 3.3 Hz, 8.4 Hz, 1 H), 4.49 (s, 2 H), 4.15 (dt, J = 1.8Hz, 6.0 Hz, 2 H), 3.66 (m, 1 H), 3.49 (m, 1 H), 3.16 (m, 1H), 3.07 (m, 1 H), 2.41 (s, 3 H), 0.79 (s, 9 H), - 0.053 (s, 3H), - 0.75 (s, 3 H) HO Ts N II-74 OTBS H2, Pd-BaSO4 (1.0 mol%) quinoline (30.0 mol%) MeOH, quantitative BnO HO Ts N OTBS II-56 OBn of (Z)-4-(benzyloxy)-1-(3-(((tert- 4- Preparation butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2-yl)but-2-en-1-ol. (benzyloxy)-1-((2R,3R)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2- yl)but-2-yn-1-ol (1.0 equivalents, 0.078 mmol) was charged into a flame-dried flask and dissolved in 10 mL of methanol. Distilled quinoline (30 mol%, 0.14 mmol) was added followed by Pd/BaSO4 (1.0 mol%, 0.005 mmol). After purging with hydrogen gas, the solution was allowed to stir for approximately 19 hours to complete conversion. Filtration over Celite and concentration gave a clear residue. NMR showed quantitative conversion to the desired alkene. 1H NMR (500 MHz, CDCl3) d 7.84 (d, J = 8.5 Hz, 2 H), 7.29 (m, 7 H), 5.79 (dt, J = 6.5 Hz, 6.0 Hz, 1 H), 5.59 (dd, J = 7.0 Hz, 9.5 Hz, 1 H), 4.75 (dd, J = 8.5 Hz, 8.5 Hz, 1 H), 4.51 (d, J = 5.5 Hz, 2 H), 4.17 (d, J = 6.0 Hz, 2 H), 3.69 (dd, J = 4.5 Hz, 11.5 Hz, 1 H), 3.58 (dd, J =6.0 Hz, 11.5 Hz, 1 H), 3.06 (dt, J = 4.5 Hz, 6.0 Hz, 1 H), 2.88 169 (dd, J = 4.0 Hz, 8.0 Hz, 1 H), 2.42 (s, 3 H), 0.78 (s, 9 H), - 0.099 (s, 3H), - 0.121 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 144.6, 130.7, 130.6, 129.8, 128.7, 128.2, 128.0, 127.8, 72.9, 67.1, 66.4, 61.7, 51.8, 47.5, 26.0, 21.9. BnO HO Ts N II-74 OTBS TBSCl (1.1 equiv) imidazole (2.2 equiv) DMF, 71% BnO TBSO Ts N II-78 OTBS Preparation of 2-(4-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)but-2-yn-1-yl)- 3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridine. 4-(benzyloxy)-1- ((2R,3R)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1-tosylaziridin-2-yl)but-2-yn-1-ol (1.0 equivalent, 0.097 mmol), TBSCl (1.1 equivalent, 0.107 mmol), and imidazole (2.2 equivalents, 0.214 mmol) were charged into a flask and dissolved in DMF. The reaction was allowed to stir overnight before TLC verification of completion. Water was added at three times the volumetric amount of DMF and the solution was allowed to stir for 20 minutes. Extraction was done with diethyl ether (4 x 10 mL) before combining and drying the organic layers over sodium sulfate. The solution was concentrated under nitrogen gas. The residue was purified using column chromatorgraphy (3% EtOAc in hexanes) to yield 71% of the desired product. 1H NMR (500 MHz, CDCl3) d 7.83 (d, J = 8.5 Hz, 2 H), 7.30 (m, 5 H), 7.24 (d, J = 8.5 Hz, 2 H), 4.51 (d, J = 2.0 Hz, 2 H), 4.35 (dt, J = 1.5 Hz, 6.5 Hz, 1 H), 4.18 (dd, J = 5.0 Hz, 11.5 Hz, 1 H), 4.09 (d, J = 1.5 Hz, 2 H), 3.97 (dd, J = 7.0 Hz, 11.5 Hz, 1 H), 3.15 (dd, J = 4.5 Hz, 6.5 Hz, 1 H), 2.94 (dt, J = 4.5 Hz, 7.0 Hz, 1 H), 2.38 (s, 3 H), 0.85 (s, 9 H), 0.80 (s, 9 H), 0.038 (s, 6 H), 0.017 (s, 3 H), - 0.061 (s, 3 H); 13C NMR (62.8 MHz, CDCl3) d 129.69, 128.69, 128.29, 128.14, 170 128.09, 71. 75, 63.16, 60.55, 57.43, 50.44, 47.25, 26.05, 25.93, 21.80, -4.52, - 4.79. BnO TBSO Ts N II-78 OTBS AcOH, H2O, THF (13:7:3) 45% BnO TBSO Ts N II-79 OH Preparation of 3-(4-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)but-2- yn-1-yl)-1-tosylaziridin-2-yl)methanol. 2-(4-(benzyloxy)-1-((tert- butyldimethylsilyl)oxy)but-2-yn-1-yl)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1- tosylaziridine (0.048 mmol) was charged into a flame-dried flask and dissolved in 1 mL of a 13:7:3 solution of acetic acid, water, and tetrahydrofuran, respectively. The reaction was allowed to stir at room temperature with continuous monitoring to prevent global deprotection using TLC (20% EtOAc in hexanes). After 13 hours, the reaction was quenched using saturated sodium bicarbonate solution. The solution was extracted with ethyl acetate (3 x 10 mL). The organic were combined and dried over sodium sulfate before concentration. The compound was purified via column chromatography (15% EtOAc in hexanes) yielding 45% of selectively silyl deprotected product. It should be noted that there was little diol found at this time and substantial reactant after quenching. 1H NMR (500 MHz, CDCl3) d 7.84 (d, J = 8.5 Hz, 2 H), 7.29 (m, 7 H), 4.51 (d, J = 1.5 Hz, 2 H), 4.18 (m, 2 H), 4.10 (d, J = 2.0 Hz, 2 H), 4.05 (m, 1 H), 3.31 (dd, J = 4.5 Hz, 6.5 Hz, 1 H), 3.08 (ddd, J = 3.0 Hz, 4.5 Hz, 9.0 Hz, 1 H), 3.02 (dd, J = 4.0 Hz, 10.0 Hz, 1 H), 2.39 (s, 3 H), 0.76 (s, 9 H), -0.035 (s, 3 H), -0.13 (s, 3 H); 13C NMR (62.8 171 MHz, CDCl3) d 162.50, 129.83, 128.73, 128.27, 128.23, 127.98, 84.03, 72.62, 71.84, 63.35, 60.81, 57.36, 49.41, 25.85, 21.83, 18.33, -1.39, -4.56. BnO TBSO Ts N II-79 OH (CH3)3SOI (5.0 equiv) NaH (5.0 equiv), DMSO, 85 °C, 24 h, 65% BnO OTBS Ts N H HO II-80 Preparation of 2-(4-(benzyloxy)-1-((tert-butyldimethylsilyl)oxy)but-2- yn-1-yl)-1-tosylpyrrolidin-3-ol. 2.34 mmol of 60% sodium hydride was charged into a 50 mL flame-dried round bottom flask with a stir bar, sealed, and purged with nitrogen gas. 2.25 mL of dry DMSO was added, followed by trimethylsulfoxonium iodide (5.0 equivalents, 2.34 mmol) and allowed to stir for 30 minutes until formation of a milky white solution. ((2R,3R)-3-(4-(benzyloxy)-1- ((tert-butyldimethylsilyl)oxy)but-2-yn-1-yl)-1-tosylaziridin-2-yl)methanol (0.048 mmol) was dissolved in 2 mL of dry DMSO and added to the milky white solution. The reaction was allowed to stir for 30 minutes before increasing the temperature to 80 °C and allowing to stir for 24 hours. The reaction was allowed to cool to room temperature before addition of an equivolumetric amount of saturated ammonium chloride solution. The solution was extracted with ethyl acetate (3 x 30 mL). The organics were combined, washed with brine, and dried over sodium sulfate before concentrating en vacuo. The compound was purified via column chromatography (15% EtOAc in hexanes) yielding 65% of the desired product. Two unseparated diastereomers were observed in NMR at a ratio of 3.5:1. 1H NMR (500 MHz, CDCl3) d 7.73 (d, J = 8.5 Hz, 2 H), 7.31 (m, 5 H), 7.21 (m, 2 H), 172 4.93 (m, 1 H), 4.87 (m, 1 H), 4.51 (s, 2 H), 4.47 (m, 1 H), 4.11 (d, J = 3.5 Hz, 1H), 3.99 (d, J = 2.0 Hz, 2 H), 3.69 (dd, J = 11.5 Hz, 2.5 Hz, 1 H), 3.48 (dd, J = 11.5 Hz, 7.5 Hz, 1 H), 3.39 (d, J = 2.0 Hz, 1 H), 2.93 (s, 3 H), 0.88 (s, 9 H), 0.143 (s, 3 H), 0.113 (s, 3 H). 1H NMR (500 MHz, CDCl3) d 7.73 (d, J = 8.5 Hz, 2 H), 7.31 (m, 5 H), 7.21 (m, 2 H), 4.96 (m, 1 H), 4.89 (m, 1 H), 4.51 (s, 2 H), 4.47 (m, 1 H), 4.11 (d, J = 3.5 Hz, 1H), 3.99 (d, J = 2.0 Hz, 2 H), 3.69 (dd, J = 11.5 Hz, 2.5 Hz, 1 H), 3.48 (dd, J = 11.5 Hz, 7.5 Hz, 1 H), 3.39 (d, J = 2.0 Hz, 1 H), 2.94 (s, 3 H), 0.88 (s, 9 H), 0.132 (s, 3 H), 0.098 (s, 3 H). 173 REFERENCES 174 REFERENCES Nash, R. J.; Fellows, L. E.; Dring, J. V.; Fleet, G. W. J.; Derome, A. E.; Hamor, T. A.; Scofield, A. M.; Watkin, D. J. "Isolation from Alexa- Leiopetala and X-Ray Crystal-Structure of Alexine, (1r,2r,3r,7s,8s)-3- Hydroxymethyl-1,2,7-Trihydroxypyrrolizidine [(2r,3r,4r,5s,6s)-2- Hydroxymethyl-1-Azabicyclo[3.3.0]Octan-3,4,6-Triol], Unique a Pyrrolizidine Alkaloid" Tetrahedron Letters 1988, 29, 2487. Nash, R. J.; Fellows, L. E.; Dring, J. V.; Fleet, G. W. J.; Girdhar, A.; Ramsden, N. G.; Peach, J. M.; Hegarty, M. P.; Scofield, A. M. "2 Alexines [3-Hydroxymethyl-1,2,7-Trihydroxypyrrolizidines] from Castanospermum- Australe" Phytochemistry 1990, 29, 111. Robins, D. J. "Pyrrolizidine alkaloids" Natural Product Reports 1995, 12, 413. Ishibashi, H.; Ozeki, H.; Ikeda, M. "Synthesis of optically active (-)- trachelanthamidine from L-prolinol" Journal of the Chemical Society, Chemical Communications 1986, 654. Fleet, G. W. J.; Haraldsson, M.; Nash, R. J.; Fellows, L. E. "Synthesis from of D-glucose [(1R,2R,3R,7S,8S)-3-hydroxymethyl-1,2,7- trihydroxypyrrolizidine], 3-epialexine and 7-epialexine" Tetrahedron Letters 1988, 29, 5441. Fleet, G. W. J.; Smith, P. W. "Methyl 2-azido-3-O-benzyl-2-deoxy-α-D- mamnofuranoside as a divergent intermediate for the synthesis of polyhydroxylated piperidines and pyrrolidines: synthesis of 2,5-dideoxy- 2,5-imino-D-mannitol [2R,5R-dihydroxymethyl-3R,4R- dihydroxypyrrolidine]" Tetrahedron 1987, 43, 971. Denmark, S. E.; Hurd, A. R. "Synthesis of (+)-casuarine" The Journal of organic chemistry 2000, 65, 2875. Denmark, S. E.; Cottell, J. J. "Synthesis of (+)-1-epiaustraline" The Journal of organic chemistry 2001, 66, 4276. Pearson, W. H.; Hines, J. V. "Total syntheses of (+)-australine and (-)-7- epialexine" The Journal of organic chemistry 2000, 65, 5785. alexine 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Chikkanna, D.; Singh, O. V.; Kong, S. B.; Han, H. "A general asymmetric route for the synthesis of the alexine and australine family of pyrrolizidine 175 11. Schomaker, alkaloids. The first asymmetric synthesis of 1,2-diepi-alexine and 1,2,7- triepi-australine" Tetrahedron Letters 2005, 46, 8865. J. M.; Bhattacharjee, S.; Yan, pure J.; Borhan, B. 2,3-disubstituted "Diastereomerically pyrrolidines from 2,3-aziridin-1-ols using a sulfoxonium ylide: A one- carbon homologative relay ring expansion" Journal of the American Chemical Society 2007, 129, 1996. and enantiomerically 12. Dressel, M.; Restorp, P.; Somfai, P. "Total Synthesis of (+)-Alexine by Utilizing a Highly Stereoselective [3+2] Annulation Reaction of an N-Tosyl- α-Amino Aldehyde and a 1,3-Bis(silyl)propene" Chemistry – A European Journal 2008, 14, 3072. 13. Beruben, D.; Marek, I.; Normant, J. F.; Platzer, N. "Stereodefined Substituted Cyclopropyl Zinc Reagents from Gem-Bismetallics" The Journal of organic chemistry 1995, 60, 2488. 14. Kulshrestha, A.; Schomaker, J. M.; Holmes, D.; Staples, R. J.; Jackson, J. E.; Borhan, B. "Selectivity in the Addition Reactions of Organometallic Reagents to Aziridine-2-carboxaldehydes: The Effects of Protecting Groups and Substitution Patterns" Chemistry – A European Journal 2011, 17, 12326. 15. Cope, A. C. "The Preparation of Dialkylmagnesium Compounds from Grignard Reagents" Journal of the American Chemical Society 1935, 57, 2238. 16. Phillips, G. W.; University, M. S. Synthetic Studies Toward the Total Synthesis of Fostriecin and Some Analogs; Michigan State University, 2006. 17. Deiana, L.; Dziedzic, P.; Zhao, G.-L.; Vesely, J.; Ibrahem, I.; Rios, R.; Sun, J.; Córdova, A. "Catalytic Asymmetric Aziridination of α,β-Unsaturated Aldehydes" Chemistry – A European Journal 2011, 17, 7904. 176 Chapter III Mechanistic Investigation of (DHQD)2PHAL Catalysis in Chlorination and Dihydroxylation Reactions Using a Naphthalene-Based Analogue III.1. Introduction III.1.1. Overview Halogenation is an old reaction first seen in textbooks as early as 1938. 69 Almost 30 years after, 70-72 Olah et. al. proved the existence of the haliranium ion, an intermediate proposed by Kimball and Roberts to explain the specific stereochemistry seen in bromination and chlorination of disubstituted olefins. 73 A haliranium ion can be described as a three membered ring containing a positively charged halogen. This haliranium intermediate (pictured in Scheme III-1) was observed via 1H NMR by Olah et. al., who showed that all bromonium and iodonium and some chloronium exist as cyclized haliranium ions at -60 to -80 °C. Among the observable ions, the 2-chloro-tert-butyl carbocation III-1g stood out as a chloronium equivalent to haliraniums III-1c. It is here that we can initially see the implicit difference between the chloronium and that of the bromonium and iodonium ions. Interestingly, almost 30 years had passed before the publication of an asymmetric halofunctionalization was achieved. In order illustrate some of the reasons that may have contributed to this, the major mechanistic challenges the development of an asymmetric that befell 177 halofunctionalization and the success that address these issues will be discussed. X F SbF5, SO2 (l) VT-NMR X X X X X = I, Br, Cl X = I, Br, Cl III-1a III-1b X = I, Br III-1c X X = I, Br III-1d X X = I, Br III-1e X X = I, Br III-1f Cl III-1g Scheme III-1: Spectroscopically observable haliranium ions. One of the main problems with achieving a selective halofunctionalization was brought to light by Brown and coworkers. 74,75 76 It was observed that the sterically encumbered olefin adamantylidene adamantine participated in rapid olefin-to-olefin transfer with its isolable bromiranium and iodiranium ions. In a general sense, a bromiranium (like the one featured in Scheme III-2) on a catalytic scale is surrounded by a high concentration of alkene that, upon attacking, will create the same product in a nonselective way. This implies that any face selective formation of bromiranium and iodiranium that could lead to enantioenriched halofunctionalization would be compromised because of the racemization due to olefin-to-olefin transfer. 178 H H H H Br H H H H H H H H Br H H H H π complex H H H H Br H H H H transition state H H H H Br H H H H π complex H H H H Br H H H H free free Scheme III-2: Mechanisms of olefin-to-olefin transfer of bromenium ions. Denmark and coworkers were able to shed further light on the olefin-to-olefin transfer mechanism when they showed that they could form the bromiranium from the β-bromotosylate III-2a and capture it enantiospecifically (Table III-1).77 However, in the presence of olefin III-3, they saw substantial erosion of enantiospecificity. The degree of erosion depends on the concentrations of the olefin and the nucleophile as well as the identity of the nucleophile counterion. A more loosely coordinated counter ion, like tetrabutylammonium, was found to provide for a greater retention of stereospecificity because of the increase in trapping rate. Interestingly, when a chlorine containing substrate III-2b was submitted to the same reaction with olefin III-3, they saw retention of enantioenrichment in the trapped product III-4b. 179 nPr nPr + nPr nPr OR X MOAc (2 equiv) HFIP or HFIP/CH2Cl2 rt nPr OAc X nPr III-4a: X = Br III-4b: X = Cl III-2a: X = Br, R = Ts III-2b: X = Cl, R = Tf entry Substrate III-3 0.0 1.0 1.0 1.0 III-3 (equivalence) III-2a III-2a III-2a III-2b M Na Na nBu4N nBu4N es (%) 100 1 28 2 81 3 100 4 Table III-1: Erosion of enantiospecificity in acetolysis from olefin-to-olefin transfer. HFIP = hexafluoroisopropanol, Tf = trifluoromethanesulfonyl, Ts = 4-toluenesulfonyl. It is important to point out a trend in these previously discussed works that would lend to a means by which the problems address can be overcome. From the work of Olah et. al. to Denmark’s acetolysis, it becomes apparent that the halogen subjected to the halofunctionalization has a strong effect on the mechanism itself. While iodine and bromine, the larger and less electronegative halogens, tend to adopt a haliranium intermediate, the smaller and more electronegative halogens have a greater tendency to exist in open carbocation forms. This is especially visible when considering the case of fluorine in the cation synthesis reaction previously addressed (Scheme III-1). When a difluorinated substrated was submitted to antimony pentafluoride in liquid SO2, NMR gave indications of a β-fluoro carbocation interemediate equilibrating between classical fluorinated ions at temperatures as low as -90 °C. Merritt similarly proposes the key fluorinated carbocation as formation of the 180 intermediate in the fluorination of propenyl benzene due to the stereo- and regioselectivity observed.78 Interestingly, chlorine can exist as the chloriranium or “bridged chloronium” or the open carbocation depending on the substrate (Scheme III-1). This has the potential to introduce new mechanisms based on substrate tuning. With this in mind, it is not difficult to resolve that limiting halofunctionalizations to chlorenium sources could prevent the erosion of stereospecificity by eliminating olefin-to-olefin transfer.79 Borhan and coworkers exemplified this in their seminal work on asymmetric chlorolactonization. Using (DHQD)2PHAL as an organic catalyst in chloroform at room temperature, they showed that they could produce lactone III-6a in 35% enantimeric excess. Utilizing NCS in lieu of NBS dramatically increased the enantioselectivity to 65% ee (Table III-2, entry 3). Ph CO2H III-5 (DHQD)2PHAL (0.1 equiv) X+ source (1.1 equiv) Solvent (0.05 M), rt O O Ph X III-6a: X = Br III-6b: X = Cl entry X+ source temp (∞C) NBS NBS NCS NCS DCDMH DCDMH solvent CH2Cl2 CHCl3 CHCl3 CHCl3 CHCl3 CHCl3 % ee 1 22 35 2 65 3 NRa 4 5 71 83 6 Table III-2: (DHQD)2PHAL mediated halolactonization. a NR = no reaction after 3 h. Prdt III-6a III-6a III-6b III-6b III-6b III-6b RT RT RT -40 RT -40 O Cl N N Cl DCDMH III-7 O 181 to give lactone the same context of Interestingly, Borhan and coworkers were able to bring light to another problem with asymmetric halofunctionalization within the lactonization.80 They were able to show that the reactivity of the halenium source is of significant importance. Highly reactive halenium reagents can cause an uncatalyzed background reaction in competition with the catalyzed reaction, eroding the stereoselectivity imbued by the catalyst. In Scheme III-3, alkene acid III-5 was completely consumed in a bromolactonization with NBS that proceeded III-6a after only 5 minutes. However, without catalyst chlorolactonization showed no conversion after 24 hours. In an ideal asymmetric catalysis, there is no background reaction to compete with the selective halofunctionalization. This would seem to suggest that simply switching to chlorenium sources in general would be a solution to both problems, but it is important to note that some chlorenium sources may not be reactive enough. When the Borhan lab attempted to lower the temperature of the selective NCS chlorolactonization in order to further bolster the enantioselectivity, there was no reaction after 3 hours (Table III-2, entry 4). Switching to 1,3-dichloro-5,5- dimethylhydantoin (DCDMH, III-7) at room temperature resulted in an increase in selectivity (71% ee) and lowering the temperature to -40 °C gave still higher enantioselectivities (83% ee). 182 Ph CO2H III-5 NBS (1.1 equiv) DCM, rt, 5 min NCS (1.1 equiv) DCM, rt, 24 h O O Ph III-6a O O Br Ph Cl III-6b Complete conversion by TLC No consumption of SM observed Scheme III-3: Comparison of halenium source reactivity exhibited through bromolactonization with NBS and chlorolactonization with NCS. III.1.2. Phase-transfer Catalysis in Asymmetric Halofunctionalization Phase-transfer catalysis has the potential to address these two issues with halofunctionalization in a different way. It can keep the halenium source and the olefin separate outside of the context of the catalyst, preventing competition from background reactions. Depending on the structure of the starting material relative to that of the product, it also has the potential to reduce the concentration of unreacted olefin in the presence of the haliranium intermediate, preventing olefin- to-olefin to attempt halofunctionalization with phase-transfer catalysis.81 Under optimal conditions addressed in Scheme III-4, trans-4-pentenoic acid III-8 was submitted to a biphasic mixture of aqueous sodium bicarbonate and methylene chloride with stoichiometric molecular iodine in the presence of 30 mol% of a cinchonidine derived ammonium salt III-11, producing exo cyclized iodolactone III-9a in 42% ee from III-8a and endo cyclized iodolactone III-10b in 31% ee from III-8b. transfer. Gao and coworkers were the first 183 PTC III-11, NaHCO3 (aq) I2, CH2Cl2 O OH R 0 °C III-8a: R = o-tolyl III-8b: R = anthracen-9-yl I * R * O + O III-9a: R = o-tolyl III-9b: R = anthracen-9-yl I * * O O R III-10a: R = o-tolyl III-10b: R = anthracen-9-yl entry 1 2 S.M. III-8a III-8b % yield (III-9+III-10) 89 38 X III-9:III-10 22:78 0:100 % ee III-9 % ee III-10 42.0 -- 10.0 31.0 H N R''O N R' III-11 R' = anthracen-9-yl R'' = H X = Cl Scheme III-4: Iodolactonization using phase-transfer catalysis. This system effectively prevents background reaction by separating the olefin and the halenium source without the help of the phase-transfer catalyst (Figure III-1). However, it does not segregate the iodiranium intermediate from the unreacted olefin. Therefore, the propensity for olefin-to-olefin transfer remains high, especially in the presence of the highly reactive iodiranium ion. While this first attempt at asymmetric catalytic halofunctionalization produced only moderate yields, it serves as a platform to further investigate the use of phase-transfer systems to asymmetrically functionalize olefins with larger halogens. 184 R O O O O R I I R O O NaHCO3 (aq) CH2Cl2 I R * * O O H N R''O X R' N I * R * O O R O O I I I I I I Figure III-1: Iodolactonization via phase-transfer catalysis. to fluorocyclize allyl benzamides Toste and coworkers were more successful in their phase-transfer catalysis (Scheme III-5).82 Toste utilized a chiral BINOL phosphoric acid derived anion III- 12 to transport Selectfluor (III-13), a dicationic fluorination reagent, into a III-15. nonpolar solvent Dihydropyran substrates III-14, with products exemplified by III-16a and III- 16b, proved to be highly enantio- and dieasteroselective with enantioselectivities generally ranging from 87% to 97% ee and diastereoselectivities generally over III-15 were similarly effective with 20 enantioselectivities of 92% to 96% ee and diastereoselectivities as high as the previous substrates (>20:1). to 1. Electron deficient olefins III-14 and 185 (R)-III-12 (5-10 mol%) Selectfluor III-13 (1.25 eq.) Proton sponge (1.1 eq.) or Na2CO3 (1.25 eq.) C6H5F, -20 °C, 24 h or C6H5F/hexanes (1:1), 23 °C, 24h R1 R2 O NH R III-14: R1,R2 = dihydropyran III-15: R1,R2 = dihydronaphthalene 86% yield (>20:1 dr) 87% yield (>20:1 dr) O O F O N III-16a 92% ee F O N III-16b 97% ee Cl R1 R2 F O N R III-16: R1,R2 = dihydropyran III-17: R1,R2 = dihydronaphthalene C8H17 Br C8H17 III-12 iPr Ar O P O Ar O OH Ar = iPr iPr F O N III-17a 93% ee F O N III-17b 96% ee 95% yield (>20:1 dr) 80% yield (>20:1 dr) N N BF4 F Cl BF4 Selectfluor III-13 Scheme III-5: Fluorocyclization of benzamides using an anionic phase-transfer catalyst. This halofunctionalization incorporated the controlled separation of the olefin and the halenium source as seen prior with phase-transfer catalysis. However, in this case, Toste and coworkers were also able to prevent olefin-to-olefin transfer not by separating the cationic halogen intermediate from the unreacted olefin, but by utilizing a small halogen with decreased susceptibility to the olefin-to-olefin transfer mechanism. III.1.3. Mechanistic Insights into Asymmetric Chlorocyclization One of the advantages of the Borhan methodology is the ease with which it can be modified. Borhan and coworkers have not only used this as an opportunity to 186 broaden the chlorofunctionaliztion substrate scope, but also as an opportunity to increased understanding of chlorofunctionalization chemistry and mechanism. In the previously addressed chlorolactonization methodology,79 Borhan and coworkers have shown clear implications with regards to substrate scope. Electron rich aryl substituted 4-pentenoic acids III-18b-c (Table III-3) exhibited a loss in enantioselectivity, especially in the case of p-methoxy substituent. This can be attributed to the stabilization of the open carbocationic form. The effect is significantly milder in the case of p-methyl substituted acid III-18b. Para- halogenated aryl substituents slightely lowered yields (III-19d with 80%, III-19e with 81%), but showed no real effect on selectivity. The highly electron deficient trifluoromethyl substrate III-19f showed a significant drop in yield (61%) with little effect on enantioselectivity. Though no insight was given, it seems apparent that the electron-withdrawing propensity of the halogens is deactivating the olefin to some extent. Cyclohexyl substrate III-19h gave a moderate yield (55%) and compromised selectivity (43% ee). This substrate is the only one that saw a significant drop in both yield and enantioselectivity. Apparently, an aryl handle is necessary for the success of this reaction. 187 R CO2H III-18a-h (DHQD)2PHAL (0.1 equiv) DCDPH (1.1 equiv) Benzoic Acid (1.0 equiv) CHCl3:Hex (1:1), -40 ∞C 30-180 min O O R Cl III-19a-h entry % Yield R Ph Cy Product III-19a III-19b III-19c III-19d III-19e III-19f III-19g III-19h % ee 1 89 <5 2 80 3 4 88 5 89 90 6 7 83 43 8 Table III-3: Selections from the chlorolactonization substrate scope. p-OMe-C6H4 p-Me-C6H4 p-Cl-C6H4 p-F-C6H4 p-CF3-C6H4 p-Ph-C6H4 86 99 86 80 81 61 75 55 O Ph Ph Cl N N Cl DCDPH III-20 O Broadening the substrate scope to include unsaturated amides provided new handles for tuning along with new oxazolines and dihydrooxazines products. Utilizing DCDPH with 2 mol% of (DHQD)2PHAL in trifluoroethanol (TFE) at -30 °C, Borhan and coworkers provided substituted oxazolines III-22a-g from 1,1- disubstituted allyl amides III-21a-g.83 The pendant benzoyl moiety represented a new malleable group that could easily removed under acidic conditions. Changes made to this functionality, while able to directly affect the reaction progression and selectivity, would have little permanent effect on the product unless desired. 188 Ph H N Ar O III-23a-g entry 1 2 3 4 5 6 7 Product III-24a III-24b III-24c III-24d III-24e III-24f III-24g (DHQD)2PHAL (2 mol%) DCDPH (1.1 equiv) TFE, -30 οC, 2 h Cl Ar O N Ph III-24a-g % ee 90 93 93 86 98 98 88 % Yield 96 97 90 83 95 98 93 p-NO2-C6H4 p-OMe-C6H4 3,5(NO2)2-C6H3 3,5(NO2)2-4-MeC6H2 Ar Ph p-Br-C6H4 p-tBu-C6H4 Table III-4: Effect of benzoyl substitution on 1,1-disubstituted amide chlorocyclization. When comparing these compounds, it is easy to see that there are both steric and electronic implications. The effectiveness of para-substitution is based a steric component. This becomes evident when comparing unsubstituted benzoyl III-21a with 4-nitro (III-21b) and 4-methoxybenzoyl (III-21c) substrates. The presence of a para-substituent increased the enantioselectivity irrespective of the electronic composition (90%, 93%, and 93%, respectively). This is further demonstrated in the comparison of III-21d and III-21e, differing only in the presence of a methyl substituent in the para position. The meta positioning of the nitro groups in the 3,5-dinitrobenzoyl group produced a notable decreased in selectivity (86%) but the presence of the methyl substituent in III-21e restored full activity, giving an enantioselectivity of 98% ee. 189 Br H N Ar O III-25a-g Br (DHQD)2PHAL (2 mol%) DCDPH (1.1 equiv) TFE, -30 οC, 2 h Ar entry 1 2 3 4 5 6 Product III-26a III-26b III-26c III-26d III-26e III-26f Ar Ph 3-NO2-C6H4 3-OMe-C6H4 4-F-C6H4 4-Br-C6H4 4-Cl-C6H4 % Yield 93 75 72 65 89 94 Cl O N III-26a-g % ee 98 68 93 87 84 87 Table III-5: Olefinic aryl effects on chlorocyclization of 1,1-disubstituted amides. Changes in olefinic aromatic substituents appeared to produce interesting effects on yields and enantioselectivities (Table III-5). For example, the lowest selectivity seen in the tested compounds comes from the meta-nitrophenyl substrate (75% yield, 68% ee). Halogens, while tolerated, were also slightly deficient in enantioselectivity (III-26d at 87% ee, III-26e at 84% ee, and III-26f at 87% ee). Meta-methoxy substituted substrate III-26c gave moderate yields but high enantioselectivity. In this case, it is important to note that meta placement of the methoxy group prevents electron donation through resonance normally seen in ortho and para substituted systems. In this case, we rely only on the inductively withdrawing nature of the methoxy oxygen. There for it can be surmised that electron withdrawing substituents adversely affect the enantioselectivity. This trend seems to be exactly opposite to that of the chlorolactonization. Whether this trend speaks to the need for stabilization of the open β-chloro carbocation or the deactivation of the olefin has not been addressed. 190 O Ar (DHQD)2PHAL (2 mol%) DCDPH (1.1 equiv) TFE, -30 οC, 2 h R2 R1 N H III-27a-n O R2 R1 Ar N entry R2 H H H H H H H H H C6H5 Me H H H Prdt III-28a III-28b III-28c III-28d III-28e III-28f III-28g III-28h III-28i III-28j III-28k III-28l III-28m III-28n Ar 4-Br-C6H4 4-OMe-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-OMe-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 R1 C6H5 C6H5 4-F-C6H4 4-Br-C6H4 4-CF3-C6H4 4-OMe-C6H4 4-Me-C6H4 2-Me-C6H4 2-Me-C6H4 C6H5 C6H5 Cy n-C5H11 CH2Cy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table III-6: Chlorocyclization of 1,2- disubsituted and trisubstituted allyl amides. aReaction was run in 1-nitropropane in the presence of 300 wt% molecular sieves (4Å). III-28a-n Cl Yield (%) ee (%) 91 93 99 85 94 84 93 99 64 92 52a 77a 90a 80a 99 >99 95 93 95 20 60 87 91 86 91 >99 >99 88 Finally, Borhan and coworkers explored the potential of 1,2-disubstituted and trisubstituted allylic amides (Table III-6). These substrates were more robust, maintaining selectivity under a greater variety of changes. The optimal selectivity seen using the p-bromobenzoyl amide protecting group (91% yield, 99% ee) made it the ideal starting point for analysis of these new substrates. Electron deficient rings provided relatively high yields and enantioselectivities (Entries 3- 5). However, electron rich aryl substitution on the olefin led to extensive erosion of enantioselectivities (III-28f at 20% ee and III-28g at 60%). Ortho-methyl substitution (III-28h) better maintained good enantioselectivities (87% ee) and gave an excellent yield (99%). Introduction of a comparable substrate with a p- methoxybenzoyl protection provided a slightly higher enantioselectivity (91% ee 191 of III-28i). Trisubstituted systems performed well with only slightly lower enantioselectivities (III-28j at 86% ee and III-28k at 91% ee). These are likely attributed to the marginal stabilization of the open carbocation intermediate. CO2H (DHQD)2PHAL (0.1 equiv) chlorohydantoin (1.1 equiv) Benzoic Acid (1.0 equiv) CHCl3:Hex (1:1) (0.05M) -40 οC, overnight O O Cl F III-19e F III-18e O R2 R1 Cl N N Cl O O A2 N N A1 O Cl+ Source III-7 III-20 III-29 III-30 III-31 III-32 % Yield % ee 84 89 81 85 78 72 87 81 76 III-7: R1, R2 = Me, Me 82 III-20: R1, R2 = Ph, Ph 50 III-29: R1, R2 = H, H 7 III-30: R1, R2 = Me, Ph Scheme III-6: Importance of hydantoin structure in chlorolactonization. III-31: A1, A2 = Me, Cl III-32: A1, A2 = Cl, Me Interestingly, the compounds with aliphatic olefin substitutions (III-28k-n) did not perform well in TFE. However, 1-nitropropane in the presence of molecular sieves (4 Å) performed with comparably high enantioselectivities (III-28k-n at 88% to >99% ee) when compared to the best performing aryl compounds (for example, III-28a at 91% yield and 99% ee). In order to fully gain an understanding of the chlorine source, Borhan and coworkers initially probed analogues of the 1,3-dichlorohydantoin that has proven so effective (Scheme III-6). Analysis of the methylated analogues (III-31 and III- 32) of the hydantoin chlorine source showed that with the transfer of the chlorine 192 from N-3 of III-31 was much more facile (50% yield) than that of N-1 on analogue III-32 (7% yield), insinuating that the chlorine on N-3 is exclusively delivered to the substrate. Moreover, the low yield observed in the case of III-31 in comparison to that of III-7 suggests that the chlorine on N-1 is instrumental in inductively activating the chlorine on N-3, preparing it for transfer. Additionally, a comparison of hydantoins III-7 with III-20, III-29, and III-30 exhibits a direct correlation between the steric load on C-5 and the enantioselectivity seen in chlorolactonization product. This suggests that the dichlorohydantoin is intimately involved in the enantioselective transfer of chlorine and not just a chlorenium donor. This was confirmed through 1H NMR analysis of C-5 protons when unsubstituted hydantoin III-21 was incubated with (DHQD)2PHAL and benzoic acid in CDCl3 at -40 °C. The singlet representing the C-5 hydrogens was split into a clear AB quartet that coalesced into a singlet as the temperature was warmed to room temperature, indicating the association of the hydantoin with the catalyst. Recently, Borhan and coworkers were able to exclude hydantoins as the sole electrophilic chlorine reagent that could provide the high efficiency in the chlorocyclizations up to this point.84 It was found that the use of Chloramine T- trihydrate in the presence of an HFIP additive at 24 °C could provide comparable yields to the originally optimized hydantoin system (Table III-7). Conveniently, this reaction does not require the cryogenic temperatures required by the original process. 193 1.2 equiv TsNaCl . 3H2O 2 mol% (DHQD)2PHAL 9:1 TFE:HRIP (0.10 M) 24 oC, 15 - 60 min Ar O N Cl R2 R1 R3 or O R3 R2 Ar N Cl R1 Ar NH O R1 R2 R3 III-23a/25a,c,e/27b,e,g,h,j,k,o III-24a/III-26a,c,e/III-28o III-28b,e,g,h,j,k entry 1 2 3 4 5 6 7 8 9 10 11 R1 C6H5 C6H5 C6H5 4-Br-C6H4 4-OMe-C6H4 H H H H H H R2, R3 H, H H, H H, H H, H H, H C6H5, H 4-CF3-C6H4, H 2-Me-C6H4 C6H5, C6H5 C6H5, Me 4-Me-C6H4, H Ar C6H5 4-Br-C6H4 4-Me-C6H4 4-Br-C6H4 4-Br-C6H4 4-OMe-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 4-Br-C6H4 Prdt III-24a III-26a III-28o III-26e III-26c III-28b III-28e III-28h III-28j III-28k III-28g yield/ ee (TsNNaCl) yield/ ee (DCDPH) 85%, 92% ee 87%, 97% ee 92%, 92% ee 87%, 90% ee 86%, 92% ee 93%. >99% ee 90%, 97% ee 95%, 91% ee 84%, 92% ee 64%, 92% ee 86%, 40% ee 96%, 90% ee 93%, 98% ee 90%, 83% ee 89%, 84% ee 72%, 93% ee 93%, >99% ee 94%, 95% ee 99%, 87% ee 92%, 86% ee 52%, 91% ee 73%, 37% ee Table III-7: Comparison of Chloramine-T!3H2O/(DHQD)2PHAL to DCDPH/(DHQD)2PHAL in the cyclization of allyl amines. relationship In order to gain a deeper understanding of the dimeric (DHQD)2PHAL the catalyst and its role in halofunctionalization, Marshall et al. performed structure enantioselectivity (SER) studies of various chlorocyclization reactions, some of which were previously discussed.85 Analogues of (DHQD)2PHAL were synthesized and divided into 5 classifications: (1) the linker, (2) the sterics of the quinoline substituent, (3) the sterics of the quinuclidine substituents, the stereochemistry of the C-9 carbinol center. the quinuclidine nitrogens, and (4) (5) 194 catalyst (10 mol%) DCDPH (1.1 equiv) Benzoic Acid (1.0 equiv) CHCl3:Hex (1:1) -40 οC O O Cl F III-19e N O O N dihydroquinidine (DHQD) III-36 CO2H F III-18e Catalysts: DHQD N N DHQD DHQD N N DHQD DHQD DHQD N N III-33 (DHQD)2PYDZ III-34 (DHQD)2PHAL III-35 (DHQD)2BenzoPHAL ee 80% 84% 85% Scheme III-7: Evaluation of the importance of phthalazine linker in relation to enantioselectivity. linker using the length of the necessary Initial studies addressed the chlorolactonization of 4-p-fluorophenyl-4-pentenoic acid III-18e as the model reaction (Scheme III-7). (DHQD)2PHAL (III-34) gave 84%. Shortening the linker to 2,6-pyridazine (PYDZ) III-33 gave a slightly less selective reaction (80% ee) while lengthening the linker to a 1,4-benzophthalazine (BenzoPHAL) III-35 provided an even more slight (and possibly negligible) increase (85%). The change in enantioselectivity has been attributed to a computational study by Sharpless and coworkers addressing the preferred torsion angle about the C-O- C=N bonds. In the end, they conclude that introducing the larger aromatic linker rigidifies the optimal conformation, leading to stronger enantioinduction. In order to probe the sterics of the quinoline substituents, researchers synthesized pyridazine dimers using other two other well-known commercially 195 featured in Scheme III-8 compared available cinchona alkaloids, namely quinidine (III-37) and cinchonidine (III-38). the approximate This evaluation enantioselective “cost” of each modification of the monomeric units. When comparing (DHQD)2PYDZ (III-33) to (QD)2PYDZ (III-39), researchers found a drop of 5% ee, the “cost” of switching the saturated quinuclidine ethyl group for the vinyl moiety of the quinidine. A similar comparison of (QD)2PYDZ (III-39) with (CN)2PYDZ (III-40) was made, differing solely in the pendant methoxy group of the quinidine. The enantioselectivity fell 11%. In order to verify the additive effect the phthalazine and benzophthalazine compounds were also compared. (DHQD)2benzoPHAL (III-35) provided a 5% higher enantiomeric access than did (QD)2benzoPHAL (III-42). It should be noted that these values are close approximations. For example, while the cost of going from a saturated ethyl group to a vinyl group costs 5% ee, (DHQD)2PHAL (III-34) to (DQ)2PHAL (III-41) decreased the enantioselectivity by 3% ee. 196 O O Cl F III-19e N O N N O N III-37 quinidine (QD) III-38 cinchonidine (CN) CO2H F catalyst (10 mol%) DCDPH (1.1 equiv) Benzoic Acid (1.0 equiv) CHCl3:Hex (1:1) -40 οC III-18e N O O O N III-36 dihydroquinidine (DHQD) Catalysts: DHQD DHQD QD -4% QD CN 11% N N N N N N CN III-33 (DHQD)2PYDZ 80% ee +4% III-39 (DQ)2PYDZ 76% ee +5% III-40 (CN)2PYDZ 65% ee DHQD DHQD N N III-34 (DHQD)2PHAL 84% ee +1% DHQD DHQD N N -3% -5% QD QD N N III-41 (QD)2PHAL 81% ee -1% QD QD N N 80% ee 85% ee III-42 (QD)2BenzoPHAL III-35 (DHQD)2BenzoPHAL Scheme III-8: Enantioselective cost evaluation of monomer modification in dimeric chlorolactonization catalysts. aThe enantioselectivities of lactone III-19e are documented below the structure of the catalyst used. bEach arrow indicates the enantioselective cost of a specified modification. Borhan and coworkers then synthesized a number of catalyst analogues for the purposes of probing the importance of the phthalazine nitrogens (Scheme III-9). It 197 was observed that the fluorinated ether III-43 led to a significant drop in enantioselectivity (-18% ee). Being aware of the fact that fluorine is capable of hydrogen bonding, Borhan and coworkers sought reassurance with Dissertation1,4-disubstitued benzene III-44. This use of this catalyst resulted in the complete erosion of the enantioselectivity. This observation showed that the nitrogens are important in conferring selectivity and not the added electronic alteration provided by the fluorine atoms. Dimer III-44 gave 1% ee. In order to round out the series (QD)2NAPH III-45, one of the most telling of the series, was examined. This catalyst produced near racemic product with 7% ee. While the quinidine (QD) monomer provides an effective catalyst when dimerized with phthalazine, it does not represent the catalyst with the most optimal activity. For this reason, (DHQD)2NAPH (III-46) and (DHQD)2NAPY (III-47) were also tested. The naphthyridine linker (NAPY), remarkably provided a moderate enantioselectivity of the opposite enantiomer (-59% ee). Researchers suggest that this 143% difference in enantioselectivity is most likely the result a very different conformation due to the wide separation of the alkaloids. 198 catalyst (10 mol%) DCDPH (1.1 equiv) Benzoic Acid (1.0 equiv) CHCl3:Hex (1:1) -40 οC CO2H F III-18e Catalysts: F F QD QD QD QD F F III-43 (QD)2F4Ph -18% ee III-44 (QD)2Ph 1% ee O O Cl F III-19e QD QD III-45 (QD)2NAPH 7% ee DHQD DHQD DHQD N N DHQD III-46 (DHQD)2NAPH 6% ee III-47 (DHQD)2NAPY -59% ee Scheme III-9: Probing the importance of the phthalazine nitrogens. (DHQD)2NAPH, the most similar analogue to (DHQD)2PHAL in this sequence provided a 6% ee. The summation of these results affirms the importance of the phthalazine nitrogens as essential to the induction of stereoselectivituy in (DHQD)2PHAL. In light of these findings, especially those seen in (DHQD)2NAPH, Borhan and coworkers suggest two hypotheses: 1. The conformation of the naphthalene analogue of DHQD2PHAL is not as rigid. This could be attributed to the absence of nitrogen lone pairs to repel the oxygen lone pairs, allowing for greater rotation about the C9-O-C=C. Another suggested contributor is the decreased sp2- 199 character of the C9 oxygen associated with the more electron rich naphthalene linker. This is a contrast to the donation of the oxygen into the electron deficient phthalazine linker, giving the oxygen with a greater sp2-character. Calculations done by Sharpless and coworkers seem to support this hypothesis.86 Using direct Hartree-Fock calculation ([6-31G*] basis set), they simulated the simultaneous turn of the methoxy units to the desired C-O-C=N torsion angle of 2,6-dimethoxypyridazine. The geometry was then optimized in Cs-symmetry at each point, fixing only the aforementioned torsion angle. The relative energies of the point MP2 (6-31G*) calculations were recorded. A comparison of the relative energies at 0° and 180° (an increase of approximately 52 kJ/mol) supports the suggestion that the oxygen lone pairs repel the nitrogen lone pairs at 180°. The initial increase of 15 kJ/mol seen when rotating from 0° to 30° underscores the broken conjugation of the oxygen lone pairs with the pyridazine ring. 2. The pthalazine nitrogens may play a role in masking the carbocation generated by the chloronium addition step. Attack of the nucleophile displaces the nitrogen, producing the syn product, a process that could not be ruled out (though unlikely) by experiments done by Yousefi et al. 87 200 III.2. Results and Discussion III.2.1. Synthesis of (DHQD)2NAPH The idea that the phthalazine nitrogens play a key roll in the rigidity of the catalyst, and inadvertently the selectivity of the entire chlorofunctionalization methodology, is an intriguing prospect that we believed can and should be tested. To this end, we began with synthesizing the analogue that most closely represents the original structure of the optimal phthalazine dimer catalyst, (DHQD)2PHAL III-34. This analogue, (DHQD)2NAPH (III-46), because of the electronics of the naphthalene linker, can not be synthesized using the same methods used to synthesize the original catalyst. Synthesis of (DHQD)2PHAL by Sharpless and coworkers began with chlorination of phthalhydrazide III-48 to form electron deficient phthalazine III-49 in 78% yield. Basic coupling of dihydroquinidine III-36 in toluene with azeotropic removal of water gave 88% of the desired (DHQD)2PHAL dimer (Scheme III-10).2 O NH NH O III-48 PCl5 (2.1 equiv) DMF (cat.), 78% Cl N N Cl III-49 III-36 K2CO3, KOH toluene, 135 οC to reflux, 12 h, 88% N O NN N O O O N III-34 N O N OH III-36 N Scheme III-10: Synthesis of (DHQD)2PHAL. 201 Synthesis of (DHQD)2NAPH needed to follow a different method due to the electron rich nature of the naphthalene linker. To this end, we used the copper mediated Ullmann coupling, a method known to be effective in formation of biaryl systems from aryl halides88 and the coupling of aryl halides with heteroatoms.89,90 Sharpless used this coupling to synthesize the phenanthryl ether catalysts featured in the catalyst screening of the asymmetric dihydroxylation (Scheme III- 11). I NaH (1.1 equiv), CuI (1.0 equiv) Pyridine (1.0 equiv) DMSO, 113 ∞C, 70 h, 73% N O N O DHQD-PHN III-51 III-50 Scheme III-11: Sharpless synthesis of dihydroquinidine 9-O-(9’- phenanthryl)ether (DHQD-PHN) using Ullmann coupling.2 Initial attempts to synthesize the (DHQD)2NAPH dimer by Marshall et al. failed when the commercially available 1,4-dibromonaphthalene produced only the monoaddition products III-53 and III-54 (Scheme III-12a). It is known that aryl bromides require higher reaction temperatures, higher concentrations of substrates, and longer reaction times in order to undergo Ullmann coupling than their iodide counterparts. 91 202 III-57. After Consequently, they attempted the synthesis of 1,4-diiodonaphthalene using commercially available 1,4-dibromonaphthalene85 via metal-halogen exchange, producing mixtures of the desired diiodonaphthalene and the single halogen exchange product 1-bromo-4-iodonaphthalene, along with monohalogenated naphthalenes the monohalogenated naphthalene byproducts via column chromatography and crystallization of the remaining 1,4- disubstituted naphthalenes, researchers submitted a 4:1 mixture of dihalide III- 56 to the Ullmann reaction producing a mixture of products and isolating naphthalene dimer III-45 in 9% yield (Scheme III-12c). Hydrogenation of the quinidine dimer provided dihydroquinidine dimer III-45 in 32% yield. removing 203 N OH O N III-37 (3.0 eq) Br Br III-55 a) b) c) Br Br III-52 (1.0 equiv) NaH (3.4 equiv) DMSO, pyidine. (6.0 equiv), CuI (3.0 equiv) reflux, 7 days, 9% 1. n-BuLi (2.5 equiv) Et2O, 40 min 2. I2 (3.33 equiv), 2 h 3. 50% Na2S2O3 45% I QD Br + QD III-53 X + I III-54 X III-56 X = I to Br, 4:1 III-57 X = I, Br III-56 (1.0 equiv) (X = I/Br 4:1) QD QD QD QD Br N OH O N III-37 (3.0 eq) X NaH (3.4 equiv) DMSO, pyidine. (6.0 equiv), CuI (3.0 equiv) reflux, 7 days, 9% III-45 III-54 III-53 QD QD H2, Pd/C EtOAc / iPrOH rt, 24 h, 32% DHQD DHQD III-45 (QD)2NAPH III-45 (DHQD)2NAPH Scheme III-12: Synthesis of naphthyl dimer (DHQD)2NAPH. (a) Attempted Ullmann coupling with 1,4-dibromonaphthalene. (b) Synthesis of 1,4-diiodonaphthalene. (c) Ullmann coupling and reduction to form (DHQD)2NAPH dimer. In order to streamline this synthesis, we made a slight change to the halogen exchange submitting 1,4-dibromonaphthalene to increased equivalents of n- butyllithium and iodine (Scheme III-13). The isolated residue was submitted to a recrystallization, providing pure 1,4-diiodonaphthalene with no single halogen exchange product, though the yields were not as high (28%). 92 We followed with Ullmann coupling of dihydroquinone III-36 to remove the necessity for the low 204 yielding hydrogenation. The coupling, while possible, has proven difficult under the current conditions, giving yields of 8% pure product. We found that heating using microwaves for 72 hours provided almost three times the pure product (22%) than heating to 140 °C for 7 days. We also noticed this reaction gave a cleaner reaction, which provided us with a more efficient purification. I 1. n-BuLi (4.0 equiv) Et2O 2. I2 (4.4 equiv) 3. 50% Na2S2O3 28% I III-58 Br Br III-55 I N OH O N III-36 (3.0 eq) O N OH N III-36 (3.0 eq) III-58 I (1.0 equiv) NaH (3.4 equiv) DMSO, pyr. (6.0 equiv), CuI (3.0 equiv) reflux, 1 week, 8.2% I III-58 I (1.0 eq) NaH (3.4 equiv) DMSO, pyr. (6.0 equiv), CuI (3.0 equiv), mw, 72 h, 22% O O N O N O N III-45 N N O N O N N III-45 O O Scheme III-13: Synthesis of hydroquinidine-1,4-naphthalenediyl diether (DHQD)2NAPH. III.2.2. Intramolecular Chlorofunctionalization Chlorofunctionalization using the Borhan lab methodologies has proven to be valuable with regard to their broad scope, high enantioselectivities and generalized procedure. Because of their success, they can serve as effective tools to probe the importance of the phthalazine nitrogens. 205 Initial SER studies performed by Marshall et al.85 spanned approximately 25 catalysts and five reactions. For our purposes, we will look at 2 catalysts and 3 reactions. Unfortunately, due to the low yield of (DHQD)2NAPH in the previously discussed synthesis (Scheme III-12c), there was not sufficient catalyst to test the 5 previously tested reactions, and therefore, not enough data to do a direct comparison of the optimal (DHQD)2PHAL with its naphthalene analogue. However, the quinidine dimers were in sufficient supply to test against the desired reactions (Scheme III-14). p-F-Ph O III-18e OH catalyst (10 mol%) DCDPH (1.1 equiv) CHCl3 / Hexanes -40 οC, 3 h O p-F-Ph QD2PHAL 81% O Cl : III-19e QD2NAPH2 7% p-Cl-Ph H N Ph-p-Br O III-25f catalyst (10 mol%) DCDPH (1.1 equiv) TFE, -30 οC, 3 h O Cl p-Cl-Ph Ph-p-Br N III-26f : QD2NAPH2 -14% QD2PHAL 86% O Ph catalyst (10 mol%) DCDPH (1.1 equiv) TFE, -30 οC, 3 h Ph N H III-27p Ph N O Ph Cl III-28p QD2PHAL >99% : QD2NAPH2 15% Scheme III-14: Comparison of (QD)2PHAL and (QD)2NAPH in chlorolactonization and allyl amide chlorocyclization. 206 III-18e The chlorolactonization of 4-(para-fluorophenyl)-4-pentenoic acid discussed earlier gave good enantioselectivities (81% ee) when (QD)2PHAL was used as the catalyst. Switching to (QD)2NAPH resulted in a significant drop in enantioselectivities (7% ee). Cyclization of 1,1-disubstituted allylic amide III-25f gave 86% ee in the presence of (QD)2PHAL. The same cyclization oxazoline III- 26f gave in 14% ee of the opposite enantiomer when catalyzed by (QD)2NAPH. Trans-allyl amide III-27p cyclized with excellent enantioselectivity (>99% ee) when catalyzed by the phthalazine dimer (QD)2PHAL. Again, the naphthalene analogue (QD)2NAPH gave highly eroded stereoselectivities (15% ee). The SER studies done by Marshall et al. clearly show a consistent loss of enantioselectivity when using the naphthalene dimer analogue. Allene halocyclization is a handy tool for the modification of organic molecules, providing a stereogenic center along with a vinyl halide with the potential to undertake cross-coupling reactions among other transformations. Ma and coworkers developed an iodoetherification and an iodolactonization that exhibited high region- and stereoselectivities without the use of a catalyst, instead relying on the intrinsic axial chirality of the allene.93 94 Hennecke and coworkers established an asymmetric bromolactonization of allenoic acids using (DHQD)2PHAL as to moderate enantioselectivities (12-66% ee). their catalyst but were met with low 207 in further advancement With this information in hand, we decided to continue our SER studies by the addressing an allenyl chlorocyclization, a chlorocyclization reaction scope developed in the Borhan lab (Scheme III-15).95 When investigating the substrate scope, compounds III-60 and III-61 showed lower enantioselectivities. This is due to the electron deficient nature of the amide benzoyl group. It is theorized that the nucleophilicity of the amide oxygen is reduced to the point of competition with intermolecular processes because of the electron withdrawing nature of the benzoyl ring. This was confirmed by the appearance of TFE-incorporated intermolecular byproduct. Conversely, 1,1- disubstitued allenes III-63-66 performed very well with generally yields and enantioselectivities (90-97% yield, 92-98% ee). These compounds seemed to exhibit a greater eletrophilicity at the site of C-O bond formation. Unfortunately, this can lead to a greater background reaction as exhibited in the case of phenyl substituted III-67 where enantioselectivities fell to 86%. The temperature was therefore lowered to regain selectivity. 208 O NH Ar R (DHQD)2PHAL (10 mol%) DCDMH (1.1 equiv) TFE:HFIP (1:1), 0.025M rt, 1-5 h N O Ar Cl R NO2 N O III-60 NO2 75% yield, 94% ee Cl N O Cl NO2 III-61 62% yield, 94% ee N O Et Cl III-63 90% yield, 94% ee Br N O Cl Me III-64 72% yield, 92% ee N O III-59 Cl 92% yield, 90% ee N O III-62 95% yield, 94% ee Cl N O Cl Me III-65 Br Br N O Cl Me III-66 NO2 N O Cl Ph III-67 89% yield, 86% ee 97% yield, 98% ee 91% yield, 98% ee Scheme III-15: Substate scope for the intramolecular chlorocyclization of allene amides. Under optimized conditions (Scheme III-16), (DHQD)2PHAL-assisted allene amide III-59’ was cyclized to form the corresponding vinyl chloride in 91% yield and 90% ee. Utilizing DHQD2NAPH produced a yield of 85% but a low enantioselectivity of 2% ee remaining consistent with the previously seen deterioration of enantioselectivities due to the absence of the phthalazine nitrogens. 209 H N O III-59' catalyst (10 mol%) DCDMH (1.1 equiv) TFE:HFIP (1:1), 0.025M rt Cl O N III-59 NN DHQD DHQD DHQD DHQD III-34 (DHQD)2PHAL 91% yield, 90% ee III-45 (DHQD)2NAPH 85% yield, 2% ee Scheme III-16: Allene chlorocyclization enantioselectivities. III.2.3. Intermolecular Chlorofunctionalization An overview of previous advancements in asymmetric halofunctionalization illustrates another method used to avoid the selectivity problems resulting from olefin-to-olefin transfer and β-halocarbocation formation. The increased local concentration provided by tethered nucleophiles has been instrumental in the rate enhancement required for routinely higher enantioselectivities seen using a veritety of halenium precursors and nucleophiles. Intermolecular halofunctionalization opens up new direct routes for asymmetric aminohalogenation, haloesterification, dihalogenation, and halohydrin formation. Zhang et. al. exemplified this shift to intermolecular halofucntionalization in 2013 with the bromoesterification of allyl sulfonamides (Scheme III-17).96 Using 20 mol% of (DHQD)2PHAL the same equivalence of camphorsufonic acid (CSA), researchers were able to functionalize alkenes III- 68 providing benzoyl esters III-69 in yields of 52-82% and ee’s of 21-93%. the presence of in 210 R NHTf (DHQD)2PHAL (20 mol%) NBS (1.1 equiv), PhCO2H (1.1 equiv) (+)-CSA (20 mol%), CHCl3, rt R OBz NHTf Br III-69 III-68 yield: 52-82% ee: 21-93% Scheme III-17: Bromoesterification of allyl sulfonamides using (DHQD)2PHAL. increased to include reactions, intermolecular III.2.3.1. Chloroetherification In a recent publication Borhan and coworkers, 97 the chlorination reaction profiles were specifically chloroetherification (Table III-8). This system relies on a mixed solvent system (3:7, MeOH/MeCN) and a lowered temperature (-30 °C) to imbue upon intermolecular reactions a similar selectivity seen in intramolecular chlorination reactions using the same organic catalysts. Under the optimized conditions, allyl amides provided high enantioselectivities of halo-ethers, halo-esters, and halohydrins in both aryl and alkyl substrates. In the originally optimized chloroetherification reactions, it was found that a solution of DCDMH in the optimal methanol/acetonitrile solution with (DHQD)2PHAL provided 56% yield and 84% ee of anti-2-chloro-3-methoxy amide III-71a from the trans-aryl alkene III- 70a and 93% yield and 99% ee of the syn diastereomer (Table III-8, Entries 1 and 2). These compounds also exhibited high regioselectivity (both greater than 99:1) and seemed only to fall short in diastereoselectivity, showing ratios of 3.4:1 211 and 3.3:1, respectively. Both of these results can presumably be attributed to the carbocationic character of the benzylic carbon. Aliphatic compounds III-70c and III-70d (Table III-8, entries 3 and 4) showed greater selectivity with both trans and cis propyl alkenes, giving high diastereo- (>99:1) and regioselectivities (>20:1). While the trans substrate provided adequate enantioselectivity (74%), the cis substrate was yet higher with ee’s of 99%. These regioselectivities are somewhat remarkable as there is not much distinguishing the difference between the two olefinic carbons. R1 H N Ph-p-NO2 R2 O III-70a-d Starting material R1 R2 III-70a III-70b III-70c III-70d Ph H C3H7 H H Ph H C3H7 Entry 1 2 3 4 10 mol% (DHQD)2PHAL 2.0 equiv DCDMH MeOH:MeCN (3:7) 0.01 M, -30 R1 R2 Cl H N OMe O III-71a-d Ph-p-NO2 Product Yield (%) d.r. r.r. e.r. anti syn anti syn 56 93 86 87 3.4:1 3.3:1 >99:1 >99:1 >99:1 92:8 >99:1 99.5:0.5 >20:1 87:13 >20:1 99.5:0.5 Table III-8: Limited substrate scope of intermolecular chloroetherification by Soltanzadeh, et al. Due to the interesting and promising results seen with these substrates, these compounds were also used to explore the comparison of DHQD2PHAL and DHQD2NAPH as catalysts in intermolecular chloroetherification. The reactions 212 were compared at room temperature in order to eliminate possible inconsistency of varying low temperatures that may complicate the comparison of the two catalysts. We believed this would lead to a decrease in the enantioselectivities seen previously in DHQD2PHAL. However, we believed the difference in enantioselectivity between the two catalysts would still be large enough to assess their effectiveness. We had already reasoned that some of the selectivities seen in the literature in would be lower because of the change in temperature. Therefore, the lower diastereoselectivities and enantioselectivities seen the (DHQD)2PHAL- catalyzed reactions were seen as consequences of the higher temperature. With this in mind, there were still stark differences in the effectiveness of the two catalysts. We were able to confirm the deterioration of the enantioselectivities and surprisingly regioselectivities with (DHQD)2NAPH. For example, the cis- propyl allyl amide III-70d was converted to syn-β-chloromethoxy ether III-71d with little regioisomer (94:6) and 85% ee when mediated by (DHQD)2PHAL. III- 70d mediated by (DHQD)2NAPH produced the desired methoxy ether III-71d with no selectivity (0% ee) along with higher quantities of the regioisomer III-72d and a substantial amount of the intramolecular oxazoline byproduct III-74d (regioselectivity was 49:12:39, respectively). 213 R O N H III-70 a-d 10 mol% catalyst 2.0 equiv DCDMH MeOH:MeCN (3:7) 0.02 M, rt NO2 OMe O Cl + Ar R R N H Cl III-71 O Ar N H OMe III-72 Desired Product (CE) Regioisomer (RI) R Cl O Ar N R + Cl III-73 6-member ring (6-memb) O Ar N III-74 5-member ring (5-memb) Entry Substrate 1 Ph O Ar N H III-70a DHQD2PHAL 84% Yield R.I. 0 6 6-memb DHQD2NAPH 100% Yield R.I. 0 22 6-memb 5-memb 0 C.E. 78 C.E. 94 Syn 36 86% ee Anti 58 -32% ee Syn 30 16% ee Anti 48 -8% ee 2 3 4 Ph O Ar N H III-70b 73% Yield R.I. 0 0 6-memb C.E. 100 Syn 62 Anti 38 94% ee -90% ee O N H Ar III-70c O Ar N H III-70d C.E. 87 60% ee C.E. 94 85% ee 75% Yield R.I. 7 2 6-memb 80% Yield R.I. 6 0 6-memb 5-memb 0 5-memb 3 5-memb 0 23% Yield R.I. 0 0 6-memb C.E. 100 Syn 59 Anti 41 10% ee -20% ee 85% Yield R.I. 11 30 6-memb 98% Yield R.I. 12 0 6-memb C.E. 50 15% ee C.E. 49 0% ee 5-memb 0 5-memb 0 5-memb 9 5-memb 39 Table III-9: A comparison of intermolecular chloroetherification of aliphatic and aromatic allylic amides using (DHQD)2PHAL and (DHQD)2NAPH catalysts. Similarly to the previously discussed literature findings, aliphatic substrates submitted to chloroetherification catalyzed by (DHQD)2NAPH were found to have high diastereoselectivity. In each aliphatic compound, whether cis or trans, there was only one detectable diastereomer. Aromatic substrates, on the other hand, 214 the nitrogen that erode inductively reduces were found to produce poor diastereoselectivities of approximately 2:1. These results can be explained in one of two ways: (1) Classical halogenation mechanisms suggest that the formation of a chloriranium intermediate that, in aliphatic, systems should not differentiate between the two carbon sites as a point of nucleophilic attack unless the electrophilicity of the closer carbon. This explains the 5-exo-trig oxazoline byproducts seen in the cyclization of aliphatic systems. This could also explain erosion in regioselectivity seen in the use of (DHQD)2NAPH. Aromatic systems the diastereoselectivity whilst produce β-chlorocarbocations enhancing regioselectivity. (2) These results are also supported by Ashtekar’s nucleophile assisted alkene activation theory,98 a concept that contradicts the driving force for the formation of bridged halonium species often represented in Organic Chemistry textbooks. This paper suggests that reactivity of an unactivated alkene comes from the polarizing of the pi bond through the close proximity of the nucleophile. While this paper addresses appended nucleophiles to establish this point, it is not difficult to justify the use of this theory in explaining the diastereoselectivities seen in these intermolecular reactions. The product III- 71 is dependent on the approach of an external nucleophile to polarize the olefinic carbon distal to the nitrogen. Unactivated aliphatic olefins (represented by propyl allyl amines III-70c and III-70d) produce a small amount of regioisomer III-72c and III-72d, likely due to the closeness in eletrophilicity of the olefinic carbons. The increase in regioisomer seen in the case of (DHQD)2NAPH 215 suggests a change in conformation that allows the external nucleophile an easier approach of the olefinic carbon proximal to the nitrogen. Interestingly, in the case of cis versus trans aliphatic systems, cyclized byproducts ratios vary substantially. Trans-III-70c provides 30% of 6-membered cyclized byproduct III- 73c while cis-III-70d yielded 39% of 5-membered cyclized byproduct III-74d. This suggests that the nucleophilic amide nears the olefin at different positions depending on the alkene isomer. In the case of mildly activated alkenes, namely aryl alkenes, there was a breakdown in diastereoselectivity that could justifiably be attributed to one of two scenarios: (1) the formation of a benzylic carbocation allowing the formation of significant amounts of both diastereomers with any selectivity mitigated by the catalyst or (2) the temporary formation of a six membered ring through cyclization of the pendant amide nucleophile later reopened by an external nucleophile. The first condition is much more easily justified than the second due to the formation of the six-membered oxazoline in the trans-aryl alkene and lack thereof in the cis- aryl alkene. On the other hand, these compounds were highly regioselective, yielding no recognizable regioisomer. This also points to the formation of a benzylic carbocation and no chance of any opposing polarization of the pi bond by a nucleophile. 216 When evaluating the enantioselectivities in the comparison of (DHQD)2PHAL and (DHQD)2NAPH, the results seem comparable to those in the intramolecular cases. There is a drastic loss in selectivity in every instance with the use of the naphthyl catalyst. This is exemplified best with the cis-aryl and aliphatic substrates. III.2.3.2. Dichlorination In order to further explore the difference between the two catalysts, we began to evaluate the dichlorination of the previously tested substrates. Dihalogenation forms of provides a new challenge not previously seen with other halofunctionalization (Scheme III-18). The formation of the chloriranium ion, whether in a facially selective manner or not, is underminded by the low regioselectivity of unactivated alkenes. In order to address this issue, Nicolaou and coworkers relied in major part on a substrate scope limitation of cinnamyl alcohols illustrated in Scheme III-19.99 The increased electrophilicity of the phenyl-adjacent olefinic carbon provides an electron deficient trap for the nucleophilic chloride. 217 Cl R2 R1 III-75 Cl R2 H H R1 Cl Regioselectivity challenge R1 R1 Cl Cl III-76 Cl R2 R2 Cl ent-III-76 Scheme III-18: Regioselectivity challenge associated with asymmetric dichlorination of unactivated olefins. OR2 (DHQ)2PHAL III-79 (20 mol%) p-Ph(C6H4)ICl2 (1.6 equiv) CH2Cl2 (0.05 M), -78 °C R1 III-77 Cl OR2 O R1 Cl III-78 N O NN N O O N (DHQ)2PHAL III-79 N Cl F3C Cl OH Cl III-78a 63% Yield 81% ee Cl OH Cl III-78e 84% Yield 74% ee H3C Cl OH Cl III-78b 65% Yield 44% ee Cl CH3 Cl III-78f 63% Yield 68% ee OH Cl OH Cl F III-78c 75% Yield 48% ee Cl OTES Cl III-78g 32% Yield <5% ee BnO OH Cl III-78d 73% Yield 72% ee Cl Cl III-78h 48% Yield 43% ee OH Scheme III-19: Nicolaou asymmetric dichlorination of allyl alcohols. The procedure used was developed by Borhan, and coworkers,100 using dichlorodimethylhydantoin, (DHQD)2PHAL catalyst, and one hundred equivalents of lithium chloride to decrease byproduct formation in trifluoroethanol at cold temperatures (Table III-10). Soltanzadeh et. al. has been able to transform allyl amides III-70a-d into chiral dihalides III-80a-d with moderate to high 218 enantioselectivities (84:16 to 99.5:0.5). As seen in the chloroetherification, the tested aryl alkenes give moderate to high enantioselectivities (84:16 for trans and 97:3 for cis) with a similar drop in diastereoselectivities, the greater of which was seen in cis-aryl substrates at 15.6:1. Alkyl substrates showed impressively high enantio- and diastereoselectivities with ratios of 90.5:9.5 and 98.2 for trans and cis, respectively, with regard to enantiomer selectivity and greater than 99:1 with regard to both diastereomers. H N Ph-p-NO2 O III-70a-d 10 mol% (DHQD)2PHAL 2.0 equiv DCDMH 100 equiv LiCl TFE, 0.02 M, -30 oC R1 Cl Cl H N Ph-p-NO2 R2 O III-80a-d Starting material R1 R2 Ph H C3H7 H Ph H H C3H7 III-70a III-70b III-70c III-70d Yield (%) d.r. e.r. 63 62 76 91 53:1 84:16 15.6:1 >99:1 >99:1 97:3 90.5:9.5 98:2 R1 R2 Entry 1 2 3 4 Table III-10: Limited substrate scope of intermolecular dichlorination by Soltanzadeh, et al. When this reaction was explored with both catalysts at room temperature (Table III-11), the greatest selectivities were seen with (DHQD)2PHAL, as expected. With regard to the cis- and trans-aliphatic compounds, the major product was the desired dichlorination product III-80c-d with small amounts of trifluoroethanol (TFE) incorporation (1% and 8% for trans and cis, respectively) and no apparent 219 formation of the 5-membered ring III-74. The enantioselectivity was moderate in the trans aliphatic compound (64% ee) and good in the cis at 86% ee. The trans aliphatic allyl amide III-70c in the case of (DHQD)2NAPH was less chemoselective, providing 14% TFE incorporation. The cis aliphatic amide provided much still less chemoselectivity, with greater amounts of TFE incorporation (30%) and, additionally, cyclization to form the 5-membered oxazoline ring (12%). With regards to the enantioselectivity, the expected erosion was observed with (DHQD)2NAPH as the catalyst. The aliphatic amide yielded 12% ee and 16% ee with regards to trans and cis compounds, respectively. 220 R O N H III-70c,d 10 mol% Catalyst 2.0 equiv DCDMH LiCl (100 equiv) TFE, 25 oC NO2 O + Ar CF3 O R Cl R N H Cl III-80 Desired Product (DC) TFE Incorporation O Ar N H Cl III-81 (TFE inc.) Ar R + Cl O N III-74 5-member ring (5-memb) O N H III-70c O N H III-70d DHQD2PHAL 90% Yield TFE inc. 1 5-memb 0 99% Yield TFE Inc. 8 5-memb 0 NO2 D.C. >99 64% ee NO2 D.C. 92 86% ee DHQD2NAPH 55% Yield TFE inc. 14 5-memb 0 >99% Yield TFE inc. 30 5-memb 12 D.C. 86 12% ee D.C. 58 16% ee Table III-11: A comparison of intermolecular dichlorination of aliphatic and aromatic allylic amides using (DHQD)2PHAL and (DHQD)2NAPH catalysts. III.2.4. Computational Derivations In lieu of the aforementioned results, we believed that the hypothesis put forth by the our group suggesting that the removal of the phthalazine nitrogens results in a less rigid catalyst is valid. In order to further prove this hypothesis, we employed computational analysis of the select rotomers about the CAr-O bond in the C9-O-C=C sequence of (DHQD)2NAPH, comparing it to the rotomers of the C9-O-C=N sequence of (DHQD)2PHAL. 221 their single ring analogues With little data available on the torsional strain of 1,4-dialkoxy naphthalene and (p- phthalazine systems, we sought out dimethoxybenzene and 1,4-dimethoxypyridazine) to shed light on their torsional energies. Kollman and Houk,101 in an exploration of the nonplanarity of o- dimethoxybenzenes, showed using STO-3G calculations that the relative energy difference between a p-substituted benzene wherein both methoxy groups are planar (0° and 0°) and one wherein a single methoxy group is orthogonal (0° and 90°) is near zero (Erel = 0.16 kcal/mol)(Figure III-2). It should be noted that this value is lower than the energy of a 90° rotation of anisole (0.94kcal/mol). This low relative energy allows the free and easy rotation of the methoxy group. O deg O deg1 O deg2 O deg2 deg1 O deg1 O O deg2 Erel 0 2.25 III-82 deg 0° 45° 90° deg1 0° 90° III-84 deg2 0° 0° deg1 0° 90° Erel 0 0.16 Erel 0 1.05 0.94 III-83 deg2 0° 0° III-85 deg2 0° 0° 0° 0° 0° Figure III-2: Relative energy of rotation of anisole and dimethoxybenzenes. a Relative energy (Erel) is in kcal/mol. deg1 0° 30° 60° 90° 180° Erel 0 1.90 0.98 0.34 10.14 Sharpless and coworkers, in a discussion of two models for asymmetric induction in osmium-catalyzed dihydroxylation, presented the relative torsion angle energies of 1,4-dimethoxypyridazine.86 Both methoxy units were synchronously rotated along the CAr-O in the N=C-O-CH3 sequence to the desired torsion angle. Using Hartree-Fock calculations (6-31G* basis set) to calculate the relative 222 energies at each optimized point, Sharpless was able to show that the relative energy required to rotate a methoxy group a mere 30° would be approximately 15 kJ/mol (3.6 kcal/mol) (Figure III-3). At 90°, the energies reached as high as 60 kJ/mol (14 kcal/mol). It should be noted that this system is unlike the singular bond rotations addressed Kollman and Houk and should not be directly compared. The synchronous movement of both methoxy groups may have additional destabilizing effects not addressed herein. However, both studies shed light on the possible mechanistic underpinnings that govern the observed catalyst functionality. Pople and coworkers explored the torsional barriers of p-substituted phenols using an STO-3G basis set.102 Understanding that phenols have increased double bond character in their C-O bonds, they sought to highlight the effects specific substituents in the para position would have on the π-donating ability of the attached oxygen. They were able to show that electron-donating moieties, such as another hydroxyl group, decreased the π donation from the phenol oxygen and consequently, decreasing the double bond character and the barrier to rotation. 223 CH3 O τ = 180° N N O O O N N O N N III-45 (DHQD)2PHAL H3C O N N τ = 180° O N N H3C τ = 0° O CH3 τ = 0° III-86 1,4-dimethoxypyridazine Figure III-3: Relative rotational energy of 1,4-dimethoxypyridazine, a simple model system for (DHQD)2PHAL bond rotation. In our attempts to clarify the role of the phthalazine nitrogens, we calculated the relative energies of the rotomers of (DHQD)2PHAL and (DHQD)2NAPH using density functional theory (DFT) B3LYP 6-31G* in vacuum with post solvent = trichloromethane. After initial geometry optimization, all bonds of the molecule were locked save the dihedral angle of N=C-O-C9 in the case of phthalazine and C=C-O-C9 in the case of naphthalene. This bond was rotated by 36° intervals for 360° and the relative energies recorded at each interval. 224 Initial observation of the optimized catalysts revealed that the optimal dihedral angle was not 0° as some may expect, but 13.03° (Figure III-4). With an approximately 109° between the C9-O bond and the oxygen lone pair, the lone pair becomes orthogonal to the plane of the aromatic linker (96° from line of lone pair orbital to the linker C=C or C=N bond). This appears to be the optimal angle by which donation into the ring can occur. Comparison of the two catalysts at this optimal dihedral angle showed that (DHQD)2PHAL was the most stable (-2.65 kcals). (DHQD)2NAPH was more than two time less stable in the initial conformation than its phthalazine counterpart (-1.23 kcals). This is reasonable when considering Pople’s phenol study. The electron deficient phthalazine ring is further stabilized by the lone pair donation of the pendant oxygen atoms. On the other hand, naphthalene is more electron rich and will not be equally stabilized by the π-donation of their analogous counterparts. Though we analyzed the full 360° rotation of the catalysts, we were only interested in the initial rotational conformations, noting that other factors, such as sterics could play a more significant role in the observed energies of conformers of greater than 60° (Table III-12). We found that (DHQD)2PHAL requires 1.4 kcals more energy (13.03° at -2.64 kcal to 49.03° at 0 kcal) to rotate 36° than the more electron rich (DHQD)2NAPH catalyst (13.03° at -1.23 kcal to 49.03° at 0 kcal). While the difference becomes even more drastic when considering the rotation from 13.03° to 85.03° (72° bond rotation requires 6.65 kcals of energy in 225 (DHQD)2PHAL and 4.87 kcals in (DHQD)2NAPH), we believed that the larger differences were due to the addition of alternate destabilizing effects, such as sterics. N O A A N O O O N N A = N, III-34 (DHQD)2PHAL A = CH, III-46 (DHQD)2NAPH A=C-O-C dihedral angle [°] 13.03 49.03 85.03 13.03 49.03 85.03 Eresa -2.64 0 4.01 -1.23 (DHQD)2PHAL (DHQD)2NAPH 0 3.64 Table III-12: DFT B3LYP 6- 31G* energies (kcal) of single (DHQD)2Ar bond rotation. These results confirm that the phthalazine nitrogens provide an electron deficient environment that causes greater π-donation from the pendant ether oxygen lone pairs. This, in turn, rigidifies the catalyst giving the high enantioselectivities exhibited in the chlorofunctionalization reactions. We have not yet addressed the possible lone pair repulsion also suggested in the hypothesis. 226 a b 13.03° -2.64 kcal 85.03° 4.01 kcal 13.03° -1.23 kcal 85.03° 3.64 kcal Figure III-4: DFT calculation (B3LYP 6-31G*) of relative energy of (DHQD)2PHAL (a) and (DHQD)2NAPH (b). =C-O-C dihedral angle and relative energy for each structure is written below. III.2.5. Sharpless Dihydroxylation Asymmetric dihydroxylation is a well studied reaction1 developed and expounded upon by Sharpless and many others. This reaction uses (DHQD)2PHAL and (DHQ)2PHAL ligands, among other ligands with pendant cinchona alkaloids, with 227 catalytic potassium osmate to form a chiral vicinal diol. In evaluating a multitude of alkenes, studies showed that, with the exception of cis-disubstituted olefins, cinchona alkaloid dimerized phthalazines were the preferred ligand linker among the tested systems with trans-disubstituted and trisubstituted alkenes providing the best range of enantioselectivity (90-99.8% ee, pictured in Scheme III-20). Testing of trans-disubstituted susbtrate scope revealed that trans-5-decene and trans-stilbene showed high selectivity using both phthalazine ligands (93-97% and >99.5% ee, respectively).1 Olefin class Preferred ligand ee range PYR PHAL 30-97% PHAL IND PHAL PHAL 70-97% 20-80% 90-99.8% 90-99% PYR PHAL 20-97% NN OR* *RO Ph OR* *RO PHAL-class Ph PYR-class OR* O N IND-class R* = DHQD or DHQ Scheme III-20: Recommended ligand for each olefin class and their enantioselectivity range.1 Since these compounds are easily accessible, they were used to probe the viability of (DHQD)2NAPH in comparison to the phthalazine catalyst. It would be reasonable to believe that, similarly to the halogenation reactions, (DHQD)2NAPH would produce selectivity much lower than that of (DHQD)2PHAL. 228 little (Scheme the dihydroxylation very It was found, however, that the naphthalene linker changed the enantioselectivity III-21). Using Chiral HPLC, of hydrobenzoin produced using (DHQD)2NAPH was highly enantioselective at 93% ee. (DHQD)2PHAL provided still higher selectivity at greater than 99% ee. Even more interestingly, (DHQD)2NAPH produced a slightly more selective diol in the case of trans-5-decene (>99% ee) than (DHQD)2PHAL (98% ee). R R (DHQD)2Ar (8.0 mol%) K2OsO2(OH)4 (1.0 mol%) K2Fe(CN)6 (3.0 equiv), 1:1 t-BuOH/H2O (0.1M), 0 oC MeSO2NH2 (1.0 equiv), K2CO3 (3.0 equiv) OH R OH R OH OH C4H9 (DHQD)2NAPH (DHQD)2PHAL (DHQD)2NAPH OH C4H9 OH (DHQD)2PHAL 93% ee >99% ee 98% ee Scheme III-21: Dihydroxylation of trans-stilbene and trans-5-decene with (DHQD)2NAPH and (DHQD)2PHAL. >99% ee in those seen While dimeric dihydroquinidine napthyl ether has not been published, these results are somewhat consistent with the structure- enantioselectivity studies done by the Sharpless group in 1991.103 The p- (1’-(4- chlorobenzoyl ester, methylnapthyl)), and (9’-phenanthryl) ether, among with quite a few others, were probed using stilbene and trans-5-decene to represent aromatic and aliphatic to substrates the evaluation of catalyst effectiveness with (1’-naphthyl) ether, the phenyl ether, regards the in 229 enantioselectivity (Table III-13). Seemingly, all four catalysts showed an excellent enantioselectivity with stilbene with a slight drop when using the phenyl ether. More interestingly, 9-O-(1’-naphthyl), 9-O-(1’-(4-methylnaphthyl), and 9-O-(9’- phenanthryl) ethers (Table III-13, entries 3,4, and 5) compared favorably against the phenyl ether and p-chlorobenzoyl ester (entries 1 and 2) in the oxidation of trans-decene. The phenyl ether selectivity was significantly lower at 88% ee than that of the naphthyl (94%), 4-methylnaphthyl (95%) and phenanthryl (95%) catalysts, with the p-chlorobenzoyl still lower (79% ee). entry 1 O Cl 2 3 4 5 Me decene (% ee) stilbene (% ee) III-87 III-88 III-89 III-90 III-91 79 88 94 95 95 99 94 99 98 99 O N RO N (DHQD) Table III-13: Dihydroxylation of trans-5-decene and trans-stilbene using a variety of 9-O-aromatic DHQD catalysts. It was reasoned, based on x-ray crystallographic data, that when directly overlapped, the catalysts showed the greatest steric difference in the upper left 230 quadrant (Figure III-5). The steric filling of this quadrant appeared to increase enantioselectivities in the case the aliphatic olefin. In accordance with this observation, the p-chlorobenzoyl catalyst showed much lower selectivity when compared to the naphthyl and phenanthryl catalysts. The phenyl catalyst also showed lower enantioselectivities though not as low as those of the benzoyl catalyst. This point the enantioselectivity imposed by the less sterically bulky (1’-naphthyl) ether when compared to the (1’-(4-methylnaphthyl) and phenanthryl catalysts, the latter of the slight difference illustrated by futher in is which show a slight increase in selectivity. This suggests that filling right quadrants is also important though the absence of sterics there is not as detrimental to enantioselectivity. When this logic is applied to the dihydroxylation of olefins using (DHQD)2NAPH in place of impose similar enantioselectivities on both aromatic and aliphatic olefins since they have similar groups in the most significant quadrants. (DHQD)2PHAL, they should it suggests that 2 3 O 1 Cl 4 2 3 1 4 2 3 Me 1 4 2 3 1 4 2 3 1 4 III-87 III-88 Figure III-5: A comparison of DHQD ether catalysts divided into quadrants. III-89 III-90 III-91 231 III.3. Conclusion In the asymmetric halogenation of alkenes, the specific composition, the difference between phthalazine and naphthalene, is important. There is a marked difference in the enantioselectivity of both intra- and intermolecular halogenation. This difference is directly attributable to the electron deficient nature of the phthalazine ring, leading to an increase in the sp2 character of the ether oxygen and restricting the O-CAr bond. This creates high concentrations of the rigid catalyst conformation responsible for producing the the rotation around high enantioselectivities. When the phthalazine nitrogens are removed, the electron withdrawing nature of the ring is replaced by the electron donating nature of the naphthalene, dispelling the oxygen sp2 character seen previously and lowering the conformational energy. The result is a much less rigid catalyst and greatly diminished ee’s. In the asymmetric dihydroxylation of olefins, the composition, as it corresponds to the electronics of the linker, appears not to be as important as the general structure of the catalyst. This has been shown in singly and doubly substituted linkers that are both electron deficient and electron rich. This is likely due to the face that the cinchonidine-based catalysts are operating as ligands. While it was never assumed that the mechanisms for asymmetric halogenation and dihydroxylation were the same, the research shows that the mechanisms 232 differ even in the importance of basic configuration of (DHQD)2PHAL. It is important to note that (DHQD)2PHAL plays the role of a catalyst in halogenation but the role of a ligand in dihydroxylation. Further study is necessary to explore the importance of rigidity in a catalyst as opposed to a ligand in the case of (DHQD)2PHAL and hopefully in a more generally applicable sense. To this end, (QD)2NAPH can be synthesized to provide another handle for the manipulation of catalyst orientation. In order to further prove the claims that the electron deficiency of the aryl linker lends itself to a more rigid system and higher enantioselectivities, it seems logical to synthesize electron deficient naphthalene catalysts. Catalysts like III-92, III- 93 and III-94 featured in Figure III-6 should regain rigidity and with it, the high selectivities seen in chlorofunctionalization reactions performed in our lab using (DHQD)2PHAL catalysts. NO2 O2N NC F F CN F F *RO OR* *RO OR* *RO OR* NC CN III-92 III-93 III-94 Figure III-6: Possible electron deficient naphthyl linkers to test for the recovery of selectivity in chlorofunctionalization. 233 III.4. Experimental III.4.1. Synthesis Br Br I 1. n-BuLi (4.0 equiv) Et2O 2. I2 (4.4 equiv) 3. 50% Na2S2O3 28% I Preparation of 1,4-diiodonaphthalene. 1,4-dibromonaphthalene (1.0 equivalents, 6.99 mmol) was charged into a flame dried flask with a stir bar, sealed and purged with nitrogen gas. n-Butyllithium (4.0 equivalents, 27.98 mmol) was added slowly to the stirring solution. A yellow precipitate began to form. The suspension was allowed to stir for 10 minutes before the addition of iodine (4.4 equivalents, 30.77 mmol). The solution bubbled and then turned a dark brown. The solution was left to stir overnight in darkness. More diethyl ether was added to the solution and it was transferred to a separatory funnel and washed with 25% w/w aqueous sodium thiosulfate (3 x 20 mL). This was followed by washing with 50 mL of water. The organic phase was dried over sodium sulfate, filtered and concentrated en vacuo. The yellow residue was dissolved in methylene chloride and run through a plug of silica gel. The solvent was completely removed and the residue redissolved in ethanol while warming. The solution was refrigerated overnight. The yellow needle crystals were filtered through filter paper and the solid (28% yield) was transferred to a vial. 1H NMR (500 MHz, CDCl3) δ 8.06 (dd, J = 6.4, 3.2 Hz, 1H), 7.79 (s, 1H), 7.62 (dd, J = 6.4, 234 3.3 Hz, 1H); 13C NMR (126 MHz, CDCl3) δ 138.16, 134.75, 133.05, 128.66, 100.75. I O N OH N I (1.0 equiv) NaH (3.4 equiv) DMSO, pyr. (6.0 equiv), CuI (3.0 equiv) reflux, 1 week O N O N O N N 8.2% isolable yield O Preparation of Hydroquinidine 1,4-naphthalenediyl diether. A stir bar was charged into a flask of dried hydroquinidine. The flask was purged with argon for 30 minutes. Dry DMSO (8.2 mL) was added to the flask and left stirring until the solid was completely dissolved. Sodium hydride was added in one portion. The solution changed from a light brown to an orange hue. The reaction was allowed to stir for 30 minutes. Dry pyridine and copper (I) iodide were both added, changing the solution color to a dark brown, and the solution was stirred for another 45 minutes. Finally, 1,4-diiodonaphthalene was added and the reaction was heated to 120 °C and allowed to stir for 7 days. After cooling to room temperature, 30% ammonium hydroxide was carefully added and the reaction was left stirring for 10 minutes before addition 50 mL of ethyl acetate. The organic layer was washed with 30% ammonium hydroxide until the blue color had dissipated in the aqueous layer. The organic layer was dried over 235 sodium sulfate and concentrated. Two columns, the first with 5% methanol in chloroform and the second in 10% methanol in ethyl acetate to 40% methanol in ethyl acetate, were necessary to purify the compound, a tan solid in 8.2% yield. 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 4.6 Hz, 2H), 8.59 – 8.49 (m, 2H), 8.02 (d, J = 9.1 Hz, 2H), 7.65 (dt, J = 6.4, 3.6 Hz, 2H), 7.47 – 7.34 (m, 6H), 6.08 (s, 2H), 6.03 (s, 1H), 5.30 (s, 0H), 4.01 – 3.93 (m, 1H), 3.93 (s, 5H), 3.25 (td, J = 9.2, 3.9 Hz, 2H), 3.07 (t, J = 10.9 Hz, 2H), 2.96 (d, J = 13.4 Hz, 1H), 2.90 (dt, J = 18.2, 7.2 Hz, 3H), 2.74 (dt, J = 13.2, 8.9 Hz, 2H), 2.30 (t, J = 11.4 Hz, 2H), 1.59 (dtd, J = 35.8, 16.2, 15.0, 7.9 Hz, 7H), 1.44 (td, J = 14.2, 13.3, 7.6 Hz, 1H), 1.27 (p, J = 5.1, 4.3 Hz, 1H), 0.93 (t, J = 7.3 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 157.95, 147.59, 146.41, 144.55, 144.11, 131.97, 126.70, 126.50, 126.03, 121.93, 121.87, 118.27, 105.60, 100.78, 60.53, 55.71, 51.10, 50.19, 37.46, 29.71, 27.19, 26.67, 25.28, 22.04, 11.93. I O N OH N I (1.0 equiv) NaH (3.4 equiv) DMSO, pyr. (6.0 equiv), CuI (3.0 equiv), mw, 72hrs N O N O O N N 22% isolable yield Cleaner Reaction O Preparation of Hydroquinidine 1,4-naphthalenediyl diether. A microwave vial for 2 to 5 mL of solution was flamed dried with a stir bar. Upon addition of the hydroquinidine, the vial was closed with a rubber septum, purged with argon gas, and 2 mL of dry DMSO was added. Sodium hydride was added 236 in one portion and the solution was allowed to stir for 30 minutes. Dry pyridine and copper (I) iodide were added to the stirring solution. The solution stirred for 45 minutes before addition of diiodonaphthalene solution (3 mL DMSO). Argon was bubbled into the solution. The reaction was sealed with the vial cover and put into the microwave. The reaction was set to microwave for a combination of 38 hours at 120 °C. After cooling to room temperature, 4 mL of 30% ammonium hydroxide was carefully added and the reaction was left stirring for 10 minutes before transfer to a separatory funnel and addition 10 mL of ethyl acetate. The organic layer was washed with 30% ammonium hydroxide (10 mL incriments) until the blue color had dissipated in the aqueous layer. The organic layer was dried over sodium sulfate and concentrated. Two columns, the first with 5% methanol in chloroform and the second in 10% methanol in ethyl acetate to 40% methanol in ethyl acetate, were necessary to purify the compound, a tan solid in 22% yield. It should be noted that the reaction was cleaner and more easily separable. 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 4.6 Hz, 2H), 8.59 – 8.49 (m, 2H), 8.02 (d, J = 9.1 Hz, 2H), 7.65 (dt, J = 6.4, 3.6 Hz, 2H), 7.47 – 7.34 (m, 6H), 6.08 (s, 2H), 6.03 (s, 1H), 5.30 (s, 0H), 4.01 – 3.93 (m, 1H), 3.93 (s, 5H), 3.25 (td, J = 9.2, 3.9 Hz, 2H), 3.07 (t, J = 10.9 Hz, 2H), 2.96 (d, J = 13.4 Hz, 1H), 2.90 (dt, J = 18.2, 7.2 Hz, 3H), 2.74 (dt, J = 13.2, 8.9 Hz, 2H), 2.30 (t, J = 11.4 Hz, 2H), 1.59 (dtd, J = 35.8, 16.2, 15.0, 7.9 Hz, 7H), 1.44 (td, J = 14.2, 13.3, 7.6 Hz, 1H), 1.27 (p, J = 5.1, 4.3 Hz, 1H), 0.93 (t, J = 7.3 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 157.95, 147.59, 146.41, 144.55, 144.11, 131.97, 126.70, 126.50, 126.03, 237 121.93, 121.87, 118.27, 105.60, 100.78, 60.53, 55.71, 51.10, 50.19, 37.46, 29.71, 27.19, 26.67, 25.28, 22.04, 11.93. H N O (DHQD)2PHAL (10 mol%) DCDMH (1.1 equiv) TFE:HFIP (1:1), 0.025M rt Cl O N and 0.18 mL of the 0.006 mmol), Preparation of (S)-5-(1-chlorovinyl)-2-phenyl-4,5-dihydrooxazole. A small stir bar was charged into a small test tube followed by N-(buta-2,3-dien-1- yl)benzamide (1.0 equivalent, 0.06 mmol), the desired catalyst (10 mol %, 0.1 equivalents, solvent (trifluoroethanol/hexafluoroisopropanol, 1:1). Finally DCDMH was added and the reactions were allowed to stir until completion, with TLC monitoring occurring every 10 minutes. After completion, 2 mL of saturated sodium thiosulfate were added to the reactions. The reactions were allowed to stir for 2 minutes before addition of methylene chloride. The aqueous layer was extracted with portions of methylene chloride (2 mL x 3). The organics were combined, dried over sodium sulfate, and concentrated en vacuo. The crude compounds were purified using column chromatography (10% EtOAc in hexanes to 20% EtOAc in hexanes). The yields were generated via NMR using MTBE. The enantioselectivity was evaluated using chiral HPLC. Yield for DHQD2PHAL catalyst: 91%. Yield for DHQD2PHAL catalyst: 85%. 1H NMR (500 MHz, CDCl3) d 7.95 (d, J = 7.0 Hz, 2 H), 7.48 (dd, J = 7.0 Hz, 7.5 Hz, 1 H), 1.6 (t, J = 7.5 Hz, 2 H), 5.55 (s, 1 H), 5.37 238 (s, 1 H), 5.19 (dd, J = 6.5 Hz, 9.0 Hz, 1 H), 4.24 (dd, 10.5 Hz, 15.0 Hz), 4.05 (dd, 7.5 Hz, 15.0 Hz, 1 H). R R O N H trans-III-68 or O N H cis-III-68 NO2 NO2 10 mol% catalyst 2.0 equiv DCDMH MeOH:MeCN (3:7) 0.02 M, rt OMe O Cl + Ar R R N H Cl III-69 O Ar N H OMe III-70 Desired Product (CE) Regioisomer (RI) R Cl O Ar N R + Cl III-71 6-member ring (6-memb) O Ar N III-72 5-member ring (5-memb) the tested catalysts for catalytic asymmetric chloroetherification of General procedure unsaturated amides. Small stir bars were added to medium test tubes. The allylic amide was added with (DHQD2PHAL and DHQD2NAPH) into the tubes along with 2 mL of a 7 : 3 mixture of acetonitrile and methanol. Finally, DCDMH was added and the solution was allowed to stir for 3 hours. The reaction was quenched with saturated sodium thiosulfate solution (2 mL). Water (2 mL) and methylene chloride (2 mL) were added. The organic were combined and dried over sodium sulfate before concentrating en vacuo. The crude material was purified using column chromatography (10% EtOAc in hexanes) after documenting crude MTBE-based NMR yields. 239 a-2c-OMe-NO2: nitrobenzamide N-((2R,3S)-2-chloro-3-methoxyhexyl)-4- Cl H N OMe O NO2 Rf : 0.38 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 7.24 (br s, 1H), 4.16-4.10 (m, 2H), 3.60-3.56 (m, 1H), 3.49-3.47 (m, 4H), 1.68- 1.62 (m, 2H), 1.54-1.35 (m, 2H), 0.95 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 165.40, 149.70, 139.86, 128.14, 123.90, 83,90, 61.78, 59.37, 42.92, 33.69, 18.46, 14.09. HRMS analysis (ESI): Calculated for [M-H]¯: C14H18ClN2O4: 313.0955; Found: 313.0953; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 7% IPA-Hexanes, 0.5 mL/min, 254 nm, RT1 (major) = 32.6 min, RT2 (major) = 34.7 min. αD s-2c-OMe-NO2: nitrobenzamide N-((2R,3R)-2-chloro-3-methoxyhexyl)-4- 20 = -30 (c 0.25, CHCl3, er = 87:13) NO2 Cl H N O OMe Rf : 0.25 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 6.79 (br s, 1H), 4.25-4.23 (m, 1H), 4.13-4.08 (m, 1H), 3.61-3.55 (m, 1H), 3.45 - 3.41(m, 4H), 1.68-1.62 (m, 2H), 1.54-1.35 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H); 13C 240 NMR (125 MHz, CDCl3) δ 165.44, 149.74, 139.73, 128.14, 123.90, 82,70, 61.04, 58.27, 43.78, 32.08, 18.90, 14.04. HRMS analysis (ESI): Calculated for [M+H]+: C14H20ClN2O4: 315.1112; Found: 315.1116; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 10% IPA-Hexanes, 1.0 mL/min, 254 nm, RT1 (major) = 12.1 min, RT2 (minor) = 14.0 min. αD a-2b-OMe-NO2: N-((2R,3S)-2-chloro-3-methoxy-3-phenylpropyl)-4- nitrobenzamide 20 = +19.0 (c 0.1, CHCl3, er = 99.5:0.5) Cl H N OMe O NO2 Rf : 0.20 ( 30%EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 9.0 Hz, 2H), 7.88 (d, J = 9.0 Hz, 2H), 7.40-7.33 (m, 5H), 6.82 (br s, 1H), 4.45 (d, J = 4.5 Hz, 1H), 4.25-4.22 (m, 1H), 4.11-4.06 (m, 1H), 3.66-3.61 (m, 1H), 3.34 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 165.26, 149.64, 139.66, 136.87, 137.22, 128.76, 128.12, 127.18, 123.87, 86.33, 62.60, 57.98, 42.54. HRMS analysis (ESI): Calculated for [M+H]+: C17H18ClN2O4: 349.0955; Found: 349.0950; Resolution of enantiomers: DAICEL Chiralcel® OJ-H column, 20% IPA-Hexanes, 1.0 mL/min, 265 nm, RT1 (minor) = 27.0 min, RT2 (major) = 30.1 min. αD 20 = +46.7 (c 0.5, CHCl3, er = 92:8) 241 s-2b-OMe-NO2: nitrobenzamide N-((2R,3R)-2-chloro-3-methoxy-3-phenylpropyl)-4- Cl H N OMe O NO2 Rf : 0.22 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J = 9.0 Hz, 2H), 7.86 (d, J = 9.0 Hz, 2H), 7.41-7.24 (m, 5H), 6.57 (br s, 1H), 4.41 (d, J = 4.5 Hz, 1H), 4.29-4.28 (m, 1H), 4.00-3.95 (m, 1H), 3.56-3.52 (m, 2H), 3.27(s, 3H); 13C NMR (125 MHz, CDCl3) δ 165.32, 149.75, 139.66, 136.83, 123.84, 128.68, 128.13, 127.49, 123.87, 85.03, 63.63, 57.44, 43.80. HRMS analysis (ESI): Calculated for [M+H]+: C17H18ClN2O4: 349.0955; Found: 349.0955; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 10% IPA-Hexanes, 1.0 mL/min, 254 nm, RT1 (major) = 22.8 min, RT2 (minor) = 29.9 min. αD a-4c-NO2: chlorobutyl-2-(4-nitrophenyl)-4,5-dihydrooxazole 20 = -8.0 (c 0.1, CHCl3, er = 99.5:0.5) O N Cl NO2 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.5 Hz, 2H), 8.10 (d, J = 8.5 Hz, 2H), 4.82-4.77 (m, 1H), 4.21 (dd, J = 16.0, 10.0 Hz, 1H), 4.09-4.03 (m, 2H), 1.85-1.83 (m, 1H), 1.69-1.66 (m, 2H), 1.47-1.43 (m, 1H), 0.98 (t, J =7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 161.86, 149.54, 133.11, 129.19, 123.59, 82.03, 63.02, 57.96, 35.85, 19.28, 13.49. 242 t-3c-NO2: 5-chloro-2- oxazine (4-nitrophenyl)-6-propyl-5,6-dihydro-4H-1,3- O N Cl NO2 1H NMR (500 MHz, CDCl3) δ 8.21 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 8.5 Hz, 2H), 4.26 (dt, J = 8.5, 3.0 Hz, 1H), 3.02-3.94 (m, 2H), 3.70 (dd, J = 16.5, 7.0 Hz, 1H), 1.99-194 (m, 1H), 1.71-1.64 (m, 2H), 1.55-1.51 (m, 1H), 1.03 (t, J =7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 153.32, 149.22, 138.64, 128.19, 123.30, 78.85, 52.44, 50.48, 34.48, 18.02, 13.84. HRMS analysis (ESI): Calculated for [M+H]+: C13H16N2O3Cl: 283.0849; Found: 283.0863. R O N H 10 mol% Catalyst 2.0 equiv DCDMH MeOH:MeCN (3:7) 0.02 M, rt NO2 Cl R Cl O N H + Ar Desired Product (DC) CF3 O R Cl O N H Ar TFE Incorporation (TFE inc.) R O Ar N Cl 6-member ring (6-memb) R + Cl Ar O N 5-member ring (5-memb) General procedure for catalytic asymmetric dichlorination of unsaturated amides. The substrate (0.04 mmol, 1.0 equiv) and LiCl (4 mmol, 100 equiv) were suspended in trifluoroethanol (TFE, 1.0 mL) in a small septum-covered test tube equipped with a stir bar. (DHQD)2PHAL (3 mg, 10 mol%) was then introduced. After stirring for 2 min DCDMH (16 mg, 0.08 mmol, 2.0 equiv) was added. The 243 stirring was continued at -30 °C till the reaction was complete (TLC). The reaction was quenched by the addition of saturated aq. Na2SO3 (1 mL) and diluted with DCM (3 mL). The organics were separated and the aqueous layer was extracted with DCM (3 × 3 mL). The organic were combined and dried over sodium sulfate before concentrating en vacuo. The crude material was purified using column chromatography (10% EtOAc in hexanes) after documenting crude MTBE-based NMR yields. 5a: N-((2S,3S)-2,3-dichlorohexyl)-4-nitrobenzamide Cl H N Cl O NO2 Rf : 0.60 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 6.52 (br s, 1H), 4.41-4.38 (ddd, J = 9.5, 4.5, 2.5 Hz, 1H), 4.18-4.15 (ddd, J = 9.0, 4.5, 2.0 Hz, 1H), 4.13-4.07 (ddd, J = 14.0, 7.5, 4.5 Hz, 1H), 3.66-3.61 (ddd, J = 13.5, 8.5, 5.0 Hz, 1H), 1.93-1.80 (m, 2H), 1.62-1.54 (m, 1H), 1.46-1.38 (m, 1H), 0.96 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.77, 149.81, 139.27, 128.22, 123.96, 63.49, 62.76, 44.92, 37.45, 19.67, 13.40. HRMS analysis (ESI): Calculated for [M+H]+: C13H17Cl2N2O3: 319.0616; Found: 319.0609; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 15% IPA-Hexanes, 1.0 mL/min, 254 nm, RT1 (major) = 8.3 min, RT2 (minor) = 10.8 min. αD 20 = +47.1 (c 0.65, CHCl3, er = >99:1) 244 5h: N-((2S,3S)-2,3-dichloro-3-phenylpropyl)-4-nitrobenzamide Cl Cl H N O NO2 Rf : 0.50 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 9.0 Hz, 2H), 7.88 (d, J = 9.0 Hz, 2H), 7.47-7.35 (m, 5H), 6.50 (br s, 1H), 5.18 (d, J = 5.0 Hz, 1H), 4.59-4.55 (m, 1H), 4.14-4.01 (ddd, J = 14.0, 7.5, 4.0 Hz, 1H), 3.52-3.46 (ddd, J = 14.0, 8.5, 4.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 165.53, 149.81, 139.21, 136.71, 129.23, 128.71, 128.17, 127.80, 123.94, 64.97, 64.25, 44.38. HRMS analysis (ESI): Calculated for [M+H]+: C16H15Cl2N2O3: 353.0460; Found: 353.0452; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 15% IPA-Hexanes, 1.0 mL/min, 254 nm, RT1 (major) = 13.5 min, RT2 (minor) = 27.6 min. αD 20 = -11.3 (c 0.6, CHCl3, er = >99:1) 5m:N-((2S,3R)-2,3-dichloro-3-phenylpropyl)-4-nitrobenzamide Cl Cl H N O NO2 Rf : 0.44 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 7.43-7.35 (m, 5H), 6.56 (br s, 1H), 5.02 (d, J = 8 Hz, 1H), 4.59-4.55 (dt, J = 11, 3.5 Hz, 1H), 4.49-4.44 (ddd, J = 14.5, 7.0, 3.5 Hz, 1H), 3.67-3.62 (ddd, J = 13.5, 8.5, 5.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 165.47, 149.77, 139.36, 137.47, 245 129.22, 128.79, 128.21, 127.75, 123.91, 63.98, 63.55, 43.89. HRMS analysis (ESI): Calculated for [M+H]+: C16H15Cl2N2O3: 353.0460; Found: 353.0462; Resolution of enantiomers: DAICEL Chiralcel® AD-H column, 20% IPA-Hexanes, 1.0 mL/min, 254 nm, RT1 (minor) = 10.5 min, RT2 (major) = 11.6 min. αD 20 = +5.6 (c 1.0, CHCl3, er = 90:10) 6a: N-(2-chloro-3-(2,2,2-trifluoroethoxy)hexyl)-4-nitrobenzamide Cl H N OCH2CF3 O NO2 Rf : 0.60 (30% EtOAc in hexanes, UV) 1H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 6.68 (br s, 1H), 4.28-4.25 (m, 1H), 4.15-4.10 (ddd, J = 14.5, 7.0, 4.5 Hz, 1H), 3.99-3.91 (m, 2H), 3.73-3.70 (dt, J = 10.5, 3.5 Hz, 1H), 3.59-3.54 (ddd, J = 13.5, 9.0, 5.0 Hz, 1H), 1.78-1.73 (m, 1H), 1.67-1.60 (m, 1H), 1.45-1.35 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.63, 149.76, 139.46, 128.16, 127.03 (q, JCF = 277.8 Hz), 123.93, 82.57, 67.33 (q, JCF = 34.1 Hz), 60.76, 43.44, 32.18, 18.53, 13.96. HRMS analysis (ESI): Calculated for [M+H]+: C15H19ClN2O4F3: 383.0985; Found: 383.0970 246 K2OsO2(OH)4 (0.004 equiv) OH OH K2CO3 (3.0 equiv) tBuOH/H2O 1:1 (0.1M), 0 οC Ligand (0.008 equiv) MeSO2NH2 (1.0 equiv) K3Fe(CN)6 (3.0 equiv) Preparation of (R,R)-Hydrobenzoin. The required catalysts [(DHQD)2PHAL and (DHQD)2NAPH] (0.008 equiv, 1.1 x 10-2 mmol, 8.5 mg), potassium osmate (0.004 equiv, 5.0 x 10-3 mmol, 2 mg), potassium ferrocyanate (3.0 equiv, 4.07 mmol, 1.340 g), potassium carbonate (3.0 equiv, 4.07 mmol, 563 mg), methanesulfonamide (1.0 equiv, 1.36 mmol), and 13.6 mL of a 1:1 solution of tert-butyl alcohol and distilled water (0.1 M) were combined in a flask with small stir bar and left to stir for 20 minutes. The solution was cooled to 0 °C before addition of stilbene (1.0 equiv, 1.36 mmol, 245 mg). Similar reaction conditions were repeated using AD-mix β (1.4g/mmol, 1.904g). In order to quench the reaction, a saturated sodium thiosulfate solution was added to the solution. Water was added to the stirring solution, followed by methylene chloride. The organic layers were combined and dried over anhydrous sodium sulfate. The opaque residue was purified using column chromatography (20% EtOAc/Hexanes). 1H NMR (500 MHz, CDCl3) δ 7.11-7.22 (m, 10H), 4.68 (s, 2H), 3.05 (br s, 2H); 13C NMR (125 MHz, CDCl3) δ 139.8, 128.1, 127.9, 126.9, 79.1. Enantiomeric excess was determined by HPLC: HP Series 1100, Chiralcel OJ-H Column (hexane/2- propanol = 90:10, flow rate 1ml/min, RT1 (minor) = 15.25 min, RT2 (major) = 17.55 min), αD 28 = +93° (lit.), c = 2.5 in EtOH. 247 Catalytic System Product Yield αD 28 HPLC AD-mix β (DHQD)2PHAL (DHQD)2NAPH 113 mg 43 mg 50 mg +69 +73.2 +79.2 99% ee 65-98% ee 77-99% ee Table III-14: Oxidation of trans-stilbene via Sharpless Dihydroxylation with (DHQD)2PHAL and (DHQD)2NAPH ligands. K2OsO2(OH)4 (0.004 equiv) Ligand (0.008 equiv) MeSO2NH2 (1.0 equiv) K3Fe(CN)6 (3.0 equiv) K2CO3 (3.0 equiv) tBuOH/H2O 1:1 (0.1M), 0 οC OH OH required of The (5R,6R)-5,6-decanediol. Preparation catalysts [(DHQD)2PHAL and (DHQD)2NAPH] (0.008 equiv, 1.1 x 10-2 mmol, 8.5 mg), potassium osmate (0.004 equiv, 5.0 x 10-3 mmol, 2 mg), potassium ferrocyanate (3.0 equiv, 4.07 mmol, 1.340 g), potassium carbonate (3.0 equiv, 4.07 mmol, 563 mg), methanesulfonamide (1.0 equiv, 1.36 mmol), and 13.6 mL of a 1:1 solution of tert-butyl alcohol and distilled water (0.1 M) were combined in a flask with small stir bar and left to stir for 20 minutes. The solution was cooled to 0 °C before addition of stilbene (1.0 equiv, 1.36 mmol, 245 mg). Similar reaction conditions were repeated using DABCO as a catalyst (0.008 equiv, 1.1 x 10-3 mmol, 100μL of a 1.23mg/mL solution, 0.123 mg). In order to quench the reaction, a saturated sodium thiosulfate solution was added to the solution. Water was added to the stirring solution, followed by methylene chloride. The organic layers were combined and dried over anhydrous sodium sulfate. The opaque 248 residue was purified using column chromatography (20% EtOAc/Hexanes). 1H NMR (500 MHz, CDCl3) δ 3.41 (2H,s), 2.03–1.98 (2H, br), 1.47–1.33 (12H, m), 0.94–0.90 (6H, t, J = 6.8 Hz). Enantiomeric resolution was determined via Chiral GC: HP 6890 Series GC System, GAMA DEX 225 Column 30M x 0.25, 0.25 film, Column # 19951-01B (90 °C to 250 °C, 3 °C/min, holding for 60 min, RT1 (minor) = 22.48 min, RT2 (major) = 22.77 min). Catalyst Enantioselectivities (% ee) DABCO (DHQD)2PHAL (DHQD)2NAPH 6% 99% 99% Table III-15: Oxidation of trans-decene via Sharpless Dihydroxylation with (DHQD)2PHAL and (DHQD)2NAPH ligands. III.4.2. Quantum Mechanics Modeling Experiments Full optimization of the all conformations of DHQD2PHAL and DHQD2NAPH truncated structures, including their rotamers locked at 30° increments about the C-O-C=C bond, was performed using density functional calculations at the in the Spartan-10 software running on level B3LYP/6-31G*/SM8(CHCl3) Macintosh and Linux platforms. 1,4-Dimethoxylnaphthalene 249 I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Standard Nuclear Orientation (Angstroms) Atom Y -2.786393 -1.831628 -0.648901 -0.648902 0.603283 -1.831629 0.603282 1.843292 -2.786393 1.835200 3.033812 1.835202 3.976742 3.033810 3.976741 1.843290 -0.549607 -1.746856 -2.351748 -1.442867 Z -0.000075 -0.000066 -0.000044 -0.000029 -0.000019 -0.000055 -0.000013 -0.000005 -0.000054 0.000003 0.000018 -0.000014 0.000028 0.000021 0.000034 0.000004 -0.000043 0.000152 0.894780 0.000083 H C C C C C C C H H C H H C H C O C H H X 1.222840 0.710165 1.413306 -1.413304 0.714167 -0.710161 -0.714167 1.401507 -1.222838 -2.485556 0.706132 2.485555 1.246611 -0.706136 -1.246615 -1.401508 2.778645 3.533071 3.330804 4.581709 250 21 22 23 24 25 26 H O C H H H 3.330786 -2.778643 -3.533076 -3.330768 -4.581712 -3.330828 -2.352062 -0.549610 -1.746856 -2.352007 -1.442862 -2.351806 -0.894260 -0.000025 0.000077 -0.894367 0.000005 0.894671 Nuclear Repulsion Energy = 852.4788794914 hartrees There are 50 alpha and 50 beta electrons. Incremental DFT Cycle 1 2 3 4 5 6 7 8 Energy -618.1244544228 -614.7728026387 -614.7800062000 -614.9323128850 -614.9366783091 -614.9365856275 -614.9366783091 -614.9366860498 9 10 -614.9366860710 -614.9366861992 251 DIIS Error 5.28 x 10-2 4.79 x 10-3 4.84 x 10-3 8.27 x 10-4 3.41 x 10-5 1.16 x 10-4 3.41 x 10-5 5.03 x 10-6 2.01 x 10-6 2.80 x 10-7 11 12 -614.9366862824 -614.9366863036 2.61 x 10-7 4.17 x 10-8 Ground-State Mulliken Net Atomic Charges Charge (a.u.) Atom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 H C C C C C C C H H C H H C H C O 0.128044 -0.217919 0.329123 0.329123 0.058723 -0.217919 0.058723 -0.179911 0.128044 0.146630 -0.132817 0.146630 0.126009 -0.132816 0.126009 -0.179911 -0.513688 252 18 19 20 21 22 23 24 25 C H H H O C H H 26 Sum of Atomic Charges H -0.211011 0.149834 0.167151 0.149832 -0.513689 -0.211011 0.149832 0.167151 0.149833 0.000000 Cartesian Multipole Moments Charge (ESU x1010) Dipole Moment (Debye) 0.0000 0.0000 2.2642 Tot Quadrupole Moments (Debye-Ang) XY YZ -68.5954 0.0003 XX XZ X Y -2.2642 Z 0.0003 0.0000 -0.0003 YY ZZ -69.6703 -85.0054 Traceless Quadrupole Moments (Debye-Ang) QXX 17.4849 QYY 14.2603 QZZ -31.7452 253 QXY 0.0000 QXZ 0.0010 QYZ -0.0009 Octapole Moments (Debye-Ang2) XXX YYY YYZ ZZZ XXY XXZ XZZ -0.0004 -5.0212 -0.0001 -0.0005 -33.0225 0.0031 0.0014 Traceless Octapole Moments (Debye-Ang2) XXX XXY XYZ YZZ YYY XXZ XZZ -0.0024 -360.7399 -0.0072 32.2650 328.4750 0.0392 0.0014 Hexadecapole Moments (Debye-Ang3) XXXX XYYY XXYZ XXZZ XZZZ -1840.1259 0.0000 -0.0040 -374.2399 -0.0043 XXXY YYYY XYYZ XYZZ YZZZ 0.0002 -1523.2672 -0.0013 0.0000 0.0001 Traceless Hexadecapole Moments (Debye-Ang3) XXXX XXYY XYYY XZZZ 1156.3993 2832.4692 -0.0111 -0.1943 XXXY XXYZ XYYZ YYYY 0.0166 -0.4328 -0.0506 325.6009 XYY XYZ YZZ ZZZ XYY YYZ XXYY XXXZ YYYZ YYZZ ZZZZ XXXZ XXZZ XYZZ YYYZ 0.0000 -0.0005 -6.8222 -0.0303 0.0010 -0.0089 -535.9420 -0.0001 0.0050 -312.1990 -98.2987 0.2449 -3988.8685 -0.0055 0.4800 254 YYZZ -3158.0701 YZZZ -0.0472 ZZZZ 7146.9386 1 Gradient of SCF Energy 2 1 2 3 -0.0000263 0.0000954 0.0000398 0.0000001 0.0001118 -0.0000627 -0.0000002 -0.0000018 -0.0000004 7 8 9 3 4 5 6 -0.0000391 -0.0001235 -0.0000945 -0.0000616 -0.0000056 0.0001117 -0.0000011 0.0000003 0.0000009 10 12 11 1 2 3 1 2 3 1 2 3 1 2 0.0001231 -0.0000025 0.0000259 -0.0000060 0.0000321 -0.0000000 0.0000010 -0.0000003 0.0000002 0.0000070 0.0000253 -0.0000048 -0.0000404 -0.0000001 0.0000005 -0.0000068 -0.0000050 -0.0000002 13 14 15 16 17 18 -0.0000060 -0.0000259 0.0000061 -0.0000022 -0.0000408 -0.0000022 -0.0000001 0.0000001 -0.0000001 -0.0000469 0.0000029 -0.0000526 0.0000323 -0.0001662 0.0001179 -0.0000004 0.0000001 0.0000009 19 20 21 22 23 24 0.0000005 0.0000033 -0.0000007 0.0000173 -0.0000136 0.0000164 -0.0000091 -0.0000003 0.0000096 0.0000530 0.0000474 0.0000009 -0.0001669 0.0001183 0.0000163 0.0000000 0.0000002 0.0000097 25 26 -0.0000032 0.0000001 -0.0000136 0.0000173 Max Gradient Component = 1.669 x 10-4 RMS Gradient = 4.912 x 10-5 255 -0.0000002 -0.0000093 Gradient Time: CPU 3.72 s Wall 3.75 s 3 Geometry Optimization Parameters NAtoms 26 NIC 177 NZ NCons NDum 0 1 NFix 0 NCnnct MaxDiss 0 0 0 Optimization Cycle: 1 Atom 1 2 3 4 5 6 7 8 9 10 11 12 H C C C C C C C H H C H Coordinates (Angstroms) Y -2.786393 1.831628 -0.648901 -0.648902 0.603283 -1.831629 0.603282 1.843292 -2.786393 1.835200 3.033812 1.835202 Z -0.000075 -0.000066 -0.000044 -0.000029 -0.000019 -0.000055 -0.000013 -0000005 -0.000054 0.0000003 0.000018 -0.000014 X 1.222840 0.710165 1.413306 -1.413304 0.714167 -0.710161 -0.714167 1.401507 -1.222838 -2.485556 0.706132 2.485555 256 1.246611 -0.706136 -1.246615 -1.401508 2.778645 3.533071 3.330804 4.581709 3.330786 -2.778643 -3.533076 -3.330768 -4.581712 -3.330828 3.976742 3.033810 3.976741 1.843290 -0.549607 -1.746856 -2.3517480 -1.442867 -2.352062 -1.746856 -1.746856 -2.352007 -1.442862 -2.351806 0.000028 0.000021 0.000034 0.000004 -0.000043 0.000152 0.894780 0.000083 -0.894260 -0.000025 0.000077 -0.894367 0.000005 0.894671 Number of degrees of freedom: 72 13 14 15 16 17 18 19 20 H C H C O C H H H O C H H H 21 22 23 24 25 26 Point Group: c1 Energy is -614.936686304 Constraints and their Current Values 18 17 3 2 Dihedral: Value Constraint -0.010 -0.010 257 71 Hessian modes were used to form the next step Hessian Eigenvalues 0.019388 0.025490 0.033569 0.078374 0.134426 0.020757 0.025971 0.034389 0.078564 0.136072 0.160419 0.188097 0.294424 0.308969 0.339062 0.361514 0.458402 0.175073 0.202916 0.296822 0.309720 0.343240 0.375724 0.764108 0.021276 0.026944 0.035798 0.126446 0.142111 0.175490 0.212829 0.297032 0.310320 0.344142 0.384340 0.771759 0.021450 0.027795 0.038869 0.126616 0.142840 0.176540 0.255909 0.298030 0.314779 0.345008 0.385366 0.002295 0.023501 0.028825 0.078054 0.129744 0.150755 0.184067 0.273854 0.298164 0.316166 0.347270 0.387219 0.012401 0.024386 0.031015 0.078079 0.131989 0.153538 0.184165 0.282001 0.305604 0.337017 0.359256 0.432373 1,4-Dimethoxyphthalazine Standard Nuclear Orientation (Angstroms) Atom Y -4.016389 -3.071643 Z -0.000010 -0.000006 I 1 2 H C X 1.241895 0.705224 258 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 H C C C C C C H H N N C O O C H H H C H H H -1.868488 -1.880755 -1.880755 -0.658348 -3.071643 -0.658348 0.638688 -4.016389 -1.868489 1.770827 1.770826 0.638688 0.639628 0.639628 1.918992 2.494401 1.701547 2.494520 1.918991 2.494446 1.701547 2.494474 -0.000031 -0.000017 0.000017 -0.000012 0.000012 0.000004 -0.000019 0.000022 0.000031 -0.000018 0.000001 0.000009 -0.000016 0.000017 -0.000012 -0.888269 0.000156 0.888081 -0.000015 0.888195 -0.000109 -0.888155 2.493205 1.409006 -1.409006 0.706365 -0.705223 -0.706365 1.333532 -1.241895 -2.493205 0.685858 -0.685858 -1.333532 2.685182 -2.685182 3.328246 3.051510 4.397684 3.051238 -3.328247 -3.051455 -4.397684 -3.051293 259 Nuclear Repulsion Energy = 864.6502624925 hartrees There are 50 alpha and 50 beta electrons. Incremental DFT Cycle 1 2 Energy -650.2216189062 -614.7728026387 3 4 5 6 7 8 9 10 11 12 13 -614.7800062000 -614.9323128850 -614.9366783091 -614.9365856275 -614.9366783091 -614.9366860498 -614.9366860710 -614.9366861992 -614.9366862824 -614.9366863036 -647.0053338358 Ground-State Mulliken Net Atomic Charges Charge (a.u.) Atom 260 DIIS Error 5.36 x 10-2 4.78 x 10-3 5.86 x 10-3 1.51 x 10-3 6.14 x 10-4 1.90 x 10-4 6.61 x 10-5 2.26 x 10-5 4.24 x 10-6 1.30 x 10-6 3.42 x 10-7 1.25 x 10-7 2.65 x 10-8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 H C H C C C C C C H H N N C O O C H H H C H 0.140986 -0.127603 0.160903 -0.180929 -0.180929 0.083466 -0.127603 0.083466 0..522553 0.140986 0.160903 -0.390236 -0.390236 0.522553 -0.501180 -0.501180 -0.204327 0.169435 0.157485 0.169447 -0.204326 0.169438 261 23 24 Sum of Atomic Charges H H 0.157485 0.169444 0.000000 Cartesian Multipole Moments Charge (ESU x1010) Dipole Moment (Debye) 0.0000 0.0000 2.0292 Tot Quadrupole Moments (Debye-Ang) XY YZ -67.4048 0.0001 XX XZ X Y -2.0292 Z 0.0000 YY ZZ QZZ QYZ XYY XYZ YZZ -74.5258 -81.3629 -20.7952 0.0000 0.0000 0.0003 11.6279 0.0000 0.0000 Traceless Quadrupole Moments (Debye-Ang) QXX QXY 21.0792 0.0000 QYY QXZ Octapole Moments (Debye-Ang2) XXX YYY YYZ ZZZ XXY XXZ XZZ 0.0000 -27.3481 0.0006 0.0009 Traceless Octapole Moments (Debye-Ang2) -0.2840 0.0003 30.1599 0.0001 0.0000 262 XXX XXY XYZ YZZ YYY XXZ XZZ -0.0001 409.0800 0.0041 131.0990 -540.1790 -0.0028 0.0000 ZZZ XYY YYZ -0.0007 0.0001 0.0035 Hexadecapole Moments (Debye-Ang3) XXXX XYYY XXYZ XXZZ XZZZ -1695.5001 0.0000 0.0000 -336.9152 0.0023 XXXY YYYY XYYZ XYZZ YZZZ 0.0000 -1563.8100 0.0004 0.0000 0.0018 Traceless Hexadecapole Moments (Debye-Ang3) XXXX XXYY XYYY XZZZ YYZZ -1309.4874 4672.2351 0.0005 -0.2261 -2490.1776 XXXY XXYZ XYYZ YYYY YZZZ -0.0006 -0.0313 -0.1125 -2182.0575 -0.1018 XXYY XXXZ YYYZ YYZZ ZZZZ XXXZ XXZZ XYZZ YYYZ ZZZZ -493.1787 0.0077 0.0022 -305.2716 -90.5634 0.3386 -3362.7476 0.0000 0.1331 5852.9252 1 Gradient of SCF Energy 2 1 2 3 263 3 4 5 6 0.0000055 -0.0000148 0.0000119 -0.0000108 -0.0000136 0.0000017 0.0000000 -0.0000000 -0.0000000 0.0000043 -0.0000043 0.0000043 0.0000171 0.0000171 0.0000049 0.0000001 0.0000000 -0.0000002 1 2 3 1 2 3 9 8 7 0.0000148 -0.0000043 -0.0000652 -0.0000136 0.0000049 0.0000589 -0.0000000 -0.0000001 -0.0000003 12 11 10 -0.0000055 -0.0000119 0.0000739 -0.0000108 0.0000017 -0.0000538 0.0000000 0.0000000 0.0000001 13 14 15 16 17 18 -0.0000739 0.0000652 0.0000276 -0.0000538 0.0000589 -0.0000158 0.0000003 0.0000001 0.0000007 -0.0000018 -0.0000276 -0.0000055 -0.0000158 -0.0000062 0.0000100 -0.0000001 -0.0000007 -0.0000105 20 22 23 24 0.0000022 -0.0000115 0.0000032 0.0000099 -0.0000018 0.0000095 -0.0000103 0.0000106 0.0000001 21 19 0.0000115 -0.0000036 0.0000055 -0.0000018 0.0000094 -0.0000062 -0.0000001 0.0000104 -0.0000000 1 2 3 Max Gradient Component = 7.386 x 10-5 RMS Gradient = 2.283 x 10-5 Gradient Time: CPU 4.46 s Wall 5.23 s Geometry Optimization Parameters NAtoms 24 NIC 160 NZ Optimization Cycle: 1 264 NCons NDum 0 1 NFix 0 NCnnct MaxDiss 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Atom H C H C C C C C C H H N N C O O C H H Coordinates (Angstroms) Y -2.786393 1.831628 -0.648901 -0.648902 0.603283 -1.831629 0.603282 1.843292 -2.786393 1.835200 3.033812 1.835202 3.976742 3.033810 3.976741 1.843290 -0.549607 -1.746856 -2.3517480 Z -0.000075 -0.000066 -0.000044 -0.000029 -0.000019 -0.000055 -0.000013 -0000005 -0.000054 0.0000003 0.000018 -0.000014 0.000028 0.000021 0.000034 0.000004 -0.000043 0.000152 0.894780 X 1.222840 0.710165 1.413306 -1.413304 0.714167 -0.710161 -0.714167 1.401507 -1.222838 -2.485556 0.706132 2.485555 1.246611 -0.706136 -1.246615 -1.401508 2.778645 3.533071 3.330804 265 4.581709 3.330786 -2.778643 -3.533076 -3.330768 -1.442867 -2.352062 -1.746856 -1.746856 -2.352007 0.000083 -0.894260 -0.000025 0.000077 -0.894367 Number of degrees of freedom: 66 H C H H H 20 21 22 23 24 Point Group: c1 Energy is -647.005333836 Constraints and their Current Values Value Constraint 0.000 0.000 15 17 9 12 Dihedral: 62 Hessian modes were used to form the next step 0.001482 0.022240 0.030892 0.076513 0.132546 0.181996 0.004489 0.023652 0.033135 0.121466 0.145799 0.183760 Hessian Eigenvalues 0.008163 0.025401 0.034870 0.121596 0.151822 0.191787 0.012866 0.026202 0.037114 0.128577 0.171100 0.199235 0.021094 0.026946 0.075065 0.130773 0.177462 0.236772 0.022009 0.028666 0.075244 0.131641 0.180070 0.248401 266 0.305558 0.314849 0.338116 0.428032 0.305853 0.318101 0.342488 0.439884 0.305882 0.320539 0.346454 0.450207 0.306755 0.321098 0.351732 0.458218 0.280880 0.310155 0.336418 0.374638 0.604588 0.297922 0.310519 0.337515 0.380298 0.615563 267 REFERENCES 268 1. 2. 3. 4. 5. 6. 7. 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