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Olefin Metathesis in Peptidomimetics, Dynamic Combinatorial Chemistry, and Molecular Imprinting


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OLEFIN METATHESIS IN PEPTIDOM IMETICS, DYNAMIC COMBINATORIAL CHEMISTRY, AND MOLECULAR IMPRINTING By TAMMY KARRIE CHENG LOW A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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The views expressed in this dissertation are those of the author and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government.

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This dissertation is dedicated to my pare nts for their unconditi onal love and support.

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iv ACKNOWLEDGEMENTS My sincere gratitude goes out to the many individuals who have supported me throughout the years. I firs t like to thank the Air For ce Academy for giving me the opportunity to pursue my Ph.D. I look forwar d to returning and working in a wonderful teaching environment. Special thanks go to Lt Col Ron Furstenau, a mentor and role model, who taught me a lot about being a good instructor and inspired me to teach. I would like to extend my sincere apprecia tion to my research advisor, Dr. Eric Enholm, for his support, patien ce, understanding, and invalu able help. He provided me all the necessary guidance to complete my dissertation, and allowed me the research freedom to develop my own ideas. Most importantly, he is a caring person who was always concerned for my family, especia lly during the monstrous hurricane season of 2004 in Florida. He has been a great adviso r and I will never forget his encouragement and kindness. I would like to thank my committee members for their constructive feedback and advice. Special thanks go to Dr. William Dolbier. He is one the most sincere and helpful professors I have ever met, who shows true concern and interest toward his students. I thank him for allowing me to drop by his o ffice anytime to discuss mechanisms and research, and for building my confidence in chemistry. I would also like to thank Dr. Ronald Castellano. His excellent teaching st yle and well organized lectures gave me a great start to the Ph.D. program. I am very appreciative for his advice on my research and also for the generous use of the HPLC I sincerely thank Dr. Ion Ghiviriga for

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v helping with the elucidation of the struct ure of my organic compounds, and for sharing his vast NMR expertise, more than I thought I could ever learn about NMR. I also appreciate Dr. Kenneth Sloan for bein g on my committee and providing valuable feedback during my oral qualifier and the prep aration of this dissert ation. I truly have been fortunate to have these individuals on my committee. Graduate school would not have been enjoyable without my fellow Enholm group members Jed Hastings, Sophie Klein, Kalyan Mondal, and Ryan Martin. It has been a blessing to work in a cooperative environmen t, where laboratory discussions are open and free, and everyone is so helpful and genuine ly friendly. I especial ly like to thank Jed, for his patience in helping with my lab e xperiments early on, for exchanging knowledge, and for providing feedback as I prepared for my oral qualifier, fi nal defense, and the writing of my dissertation. It has been a pleasure working with Sophie. Her relaxed, cheerful nature makes any working environment enjoyable. I also thank Kalyan for all his help and assistance. Not only has it been a joy working w ith these individuals, I also appreciate their frie ndship outside of lab. I extend my thanks to all of my supportiv e friends while in graduate school. I would like to thank my thoughtful and car ing friend Heshan Illangkoon, for our many chemistry discussions, the proofreading of my work, and his willingness to help anyone in need. It has also been a pleasure to sh are my AFIT Ph.D. expe rience with Lt Col John Peak. I thank him for his enc ouraging words, assistance, a nd his support. I would also like to acknowledge Dr. Rico Del Sesto and Andy Lampkins for taking the time to read my dissertation and providing me their honest and constructive comments. I am also very grateful to Dr. Joe Cradlebaugh, who provided tremendous advice for the

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vi preparation of my oral and final defense and the reviewing and formatting of this dissertation. Most importantl y, I thank him for his wonderful friendship, especially my last year in gra duate school. Finally, my most heartfelt acknowledgement must go to my parents, sister, and brother for their continuous s upport, encouragement, and kindne ss. I especia lly thank my parents for their inspiration, infinite love, a nd faith. They have made me a better person by being my role models and instilling me with strong values. They taught me determination and responsibil ity at an early age, and pr ovided me the foundation to achieve my life endeavors. I would not have b een in the position to write this dissertation without my parents. Words alone cannot e xpress my gratitude, especially for their tremendous love and belief in me during the Ph.D. period. I am fortunate to have many family and friends who have supported and encouraged me throughout these years in school and my Air Force career. With heartfelt and sincere appr eciation, I thank everyone.

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vii TABLE OF CONTENTS page ACKNOWLEDGEMENTS...............................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x LIST OF SCHEMES........................................................................................................xiii CHAPTER 1 HISTORICAL BACKGROUND.................................................................................1 1.1 Olefin Metathesis....................................................................................................1 1.1.1 Development of Olefin Metathesis and Catalysts........................................1 1.1.2 Mechanism of Olefin Metathesis..................................................................5 1.1.3 Important Types of Metathesis Reactions and Applications........................6 1.2 Peptidomimetics...................................................................................................11 1.3 Dynamic Combinatorial Chemistry......................................................................14 1.4 Molecular Imprinted Polymers.............................................................................18 1.5 Conclusions...........................................................................................................23 2 USE OF CROSS-METATHESIS TO COUPLE L-PHENYLALANINE TO A MACROCYCLIC LACTAM.....................................................................................24 2.1 Introduction...........................................................................................................24 2.2 Results and Discussion.........................................................................................28 2.2.1 Synthesis of Compound 2-6 .......................................................................28 2.2.2 Examining the Reversibility of the CM reaction in Model 2-6 ..................32 2.3 Conclusions...........................................................................................................33 3 OLEFIN METATHEIS OF AMINO AC ID DERIVATIVES WITH CYCLIC SCAFFOLDS..............................................................................................................34 3.1 Introduction...........................................................................................................34 3.2 Results and Discussion.........................................................................................41 3.2.1 Synthesis of Cyclic Scaffolds.....................................................................42 3.2.2 Synthesis of Amino Acid Derivatives........................................................45 3.2.3 CM Reactivity of Dimer with One Amino Acid........................................47

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viii 3.2.4 Generation of Small Libraries and Template Effects.................................53 3.3 Conclusions...........................................................................................................58 4 DYNAMIC COMBINATORIAL LIBRARIES EMPLOYING PEPTIDOMIMETIC DIENES...................................................................................59 4.1 Introduction...........................................................................................................59 4.2 Results and Discussion.........................................................................................63 4.3 Conclusions...........................................................................................................77 5 MODEL STUDY MOLECULAR IMPRINTING OF NERVE GASES.................78 5.1 Introduction...........................................................................................................78 5.2 Results and Discussion.........................................................................................85 5.2.1 Synthesis of N -(5-fluoresceinyl)maleimide 5-7 .........................................85 5.2.2 Synthesis of Compounds 5-13 and 5-15 .....................................................88 5.2.3 Model Study...............................................................................................89 5.3 Conclusions...........................................................................................................93 6 EXPERIMENTALS...................................................................................................94 6.1 General Method and Instrumentation...................................................................94 6.2 Experimental Procedures and Data.......................................................................95 6.2.1 General Procedures for RCM.....................................................................98 6.2.2 General Procedures for Amino Acid Coupling........................................100 6.2.3 General Procedures for Ethylenolysis of 2-6 ............................................103 6.2.4 General Procedures for EDCI Coupling...................................................106 6.2.5 General Procedures for Hydrolysis..........................................................106 6.2.6 General Procedures for CM of Dimer 3-3 with an Amino Acid..............119 6.2.7 General Procedures for Removing Boc Protecting Group with TFA.......134 APPENDIX A HPLC DATA............................................................................................................149 B SELECTED NMR SPECTRAL DATA...................................................................157 LIST OF REFERENCES.................................................................................................178 BIOGRAPHICAL SKETCH...........................................................................................186

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ix LIST OF TABLES Table page 1-1. Differences between traditional and dynamic combinatorial libraries.......................15 2-1. RCM to obtain 2-16 and 2-17 .....................................................................................30 3-1. Yields, melting points and optical rotations of amino acid derivatives......................46 3-2. Yields, melting points, and optical rotati ons of amino acid derivatives without use of HOBt during synthesis.........................................................................................47 3-3. Yields of cyclic and amino acid dimers......................................................................49 3-4. Expected products from CM reaction of dimer scaffold and two or three amino acid derivatives.........................................................................................................56 3-5. CM conditions with and without lithium template.....................................................57 4-1. Series of CM reactions...............................................................................................65 4-2. Proton chemical shifts and J values............................................................................75

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x LIST OF FIGURES Figure page 1-1. Schrocks alkoxy imido molybdenum-based catalyst 1-1 ............................................4 1-2. Ruthenium catalysts......................................................................................................5 1-3. Hydrogen-bonding leads to bent conformation..........................................................13 1-4. Design of putative azasugar peptidomimetic..............................................................14 1-5. A dynamic combinatorial library and its free energy landscape................................15 1-6. Casting and molding process in DCC.........................................................................16 1-7. Dynamic combinatorial chemistry (top) versus virtual combinatorial libraries (bottom)....................................................................................................................17 1-8. Mass spectrometric analysis of the vancomycin dimer mixture.................................18 1-9. A comparison between the Lock and Key model and the MIP model.......................20 1-10. Schematic of molecular imprinting process.............................................................21 2-1. Oxazole-based macrocyclic lactam............................................................................24 2-2. Phenylalanine on a macrocyclic lactam......................................................................26 2-3. Macrocyclic lactam with anchors for CM with amino acids......................................27 3-1. Examples of scaffolds.................................................................................................34 3-2. Dynamic combinatorial libra ries of peptidomimetics................................................35 3-3. Schematic of a DCL and a model of amino acids linked to a cyclic scaffold by olefin CM.................................................................................................................36 3-4. Glycine-based dimer, trimer and tetramer scaffolds..................................................37 3-5. Natural cyclic tetrapeptide..........................................................................................37 3-6. Small dynamic library of cyclic dipeptidomimetics...................................................38

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xi 3-7. A dynamic library of cyclic dipeptidomimetics including cis/trans isomers.............39 3-8. Dynamic library of cy clic tripeptidomimetics............................................................39 3-9. Dynamic library of cyclic tripeptidomimetics including cis/trans isomers...............40 3-10. Dynamic library of cyclic tetrapeptidomimetics......................................................40 3-11. Dynamic library of cyclic tetrapeptidomimetics including cis/trans isomers..........41 3-12. Amino acids used in the library................................................................................47 3-13. Satellites of the alkene protons from cis homodimer 3-30a .....................................52 3-14. Satellites of the alkene protons from trans homodimer 3-30e .................................53 4-1. Formation of [2]catenane............................................................................................62 4-2. Comparison of experiments 1 and 10.........................................................................67 4-3. Comparison of experiments 2 and 11.........................................................................68 4-4. Comparison of experiments 4 and 14.........................................................................69 4-5. Comparison of experiments 7 and 17.........................................................................70 4-6. HPLC spectrum of isolated compound from experiment 3........................................72 4-7. Structures of dimers 4-7a 4-7b and catenane 4-14 ...................................................73 4-8. 13C NMR chemical shifts and the corres ponding protons of isolated compound......74 4-9. Model of catenane 4-14 ..............................................................................................76 4-10. Model of dimer 4-7a .................................................................................................76 4-11. HPLC spectrum of experi ment 3 reaction mixture...................................................77 5-1. Structures of common nerve agents............................................................................79 5-2. Mechanism of acetylcholine in th e transmission of nerve impulses..........................79 A-1. HPLC spectrum of experi ment 1 reaction mixture..................................................150 A-2. HPLC spectrum of experi ment 2 reaction mixture..................................................150 A-3. HPLC spectrum of experi ment 3 reaction mixture..................................................151 A-4. HPLC spectrum of experi ment 4 reaction mixture..................................................151

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xii A-5. HPLC spectrum of experi ment 5 reaction mixture..................................................152 A-6. HPLC spectrum of experi ment 7 reaction mixture..................................................152 A-7. HPLC spectrum of experi ment 10 reaction mixture................................................153 A-8. HPLC spectrum of experi ment 11 reaction mixture................................................153 A-9. HPLC spectrum of experi ment 12 reaction mixture................................................154 A-10. HPLC spectrum of experi ment 14 reaction mixture..............................................154 A-11. HPLC spectrum of experi ment 15 reaction mixture..............................................155 A-12. HPLC spectrum of experi ment 16 reaction mixture..............................................155 A-13. HPLC spectrum of experi ment 17 reaction mixture..............................................156

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xiii LIST OF SCHEMES Scheme page 1-1. Olefin metathesis......................................................................................................... .2 1-2. Proposed intermediates for olefin metathesis...............................................................3 1-3. Metallacyclobutane intermed iate proposed by Chauvin..............................................3 1-4. Dissociative substitution of ruthenium catalysts..........................................................5 1-5. Mechanism of ol efin metathesis...................................................................................6 1-6. Types of olefin metathesis............................................................................................8 1-7. Utilizing RCM to synthesize coumarins.......................................................................8 1-8. Employing ROMP to create new materials..................................................................9 1-9. CM of asymmetric internal olefins.............................................................................10 1-10. Primary and secondary CM metathesis reactions.....................................................11 1-11. Synthesis of C-glycosyl asparagines via CM...........................................................11 1-12. Cyclization of a linear peptide using coupling agents..............................................13 1-13. Use of RCM toward the synthesis of -turn mimetics.............................................14 1-14. Dimerization of monomeric vancomycin derivatives with terminal olefins by metathesis.................................................................................................................18 1-15. An example of the pre-organized approach via covalent interactions......................21 2-1. Synthesis of an analogue of Trienomycin A utilizing RCM......................................25 2-2. Synthesis of a macrocyclic inhibitor..........................................................................25 2-3. Retrosynthethic analysis of 2-6 ..................................................................................28 2-4. Synthesis of 2-10 and 2-12 .........................................................................................28 2-5. Synthesis of diene 2-15 ...............................................................................................29

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xiv 2-6. RCM of 2-15 ...............................................................................................................29 2-7. Synthesis of lactam 2-8 ...............................................................................................30 2-8. CM reaction to obtain 2-6 and 2-19 ............................................................................31 2-9. CM reaction with ethylene gas...................................................................................33 3-1. Combinatorial synthesis of pi perazine-2,5-dione derivatives....................................37 3-2. Synthesis of dimer scaffold........................................................................................42 3-3. Synthesis of trimer scaffold........................................................................................44 3-4. Synthesis of tetramer scaffold....................................................................................45 3-5. Synthesis of amino acid derivatives...........................................................................46 3-6. Olefin CM of dimer scaffold with an amino acid derivative......................................48 3-7. Synthesis of cis homodimer 3-30a .............................................................................51 3-8. Synthesis of trans homodimer 3-30e ..........................................................................52 3-9. CM of dimer scaffold w ith two amino acid derivatives.............................................55 4-1. Comparison of building blocks 4-1 and 4-4 and their library constituents................60 4-2. Retrosynthesis of dipeptide 4-4 ..................................................................................61 4-3. Library of dimers, tetramers, he xamers, oligomers, linear compounds and catenanes..................................................................................................................62 4-4. Synthesis of 4-allylbenzoic acid ( 4-5 )........................................................................63 4-5. Synthesis of dipeptide 4-4 ..........................................................................................64 5-1. Degradation products of VX, following hydrolysis...................................................80 5-2. Formation of hydrogen-bonded complexes................................................................81 5-3. Formation of the MIP.................................................................................................82 5-4. Mechanism of the ROMP polymerization..................................................................82 5-5. Coupling of norbornene moiety to fluorescein maleimide.........................................84 5-6. Synthesis of cross-linking monomer 5-11 ..................................................................85 5-7. Synthesis of fluorescein amino hydrochloride 5-26 ...................................................86

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xv 5-8. Synthesis of N -(5-fluoresceinyl)maleimide ( 5-7 ).......................................................87 5-9. Synthesis of compounds 5-13 and 5-15 ......................................................................89 5-10. Synthesis of succinimide 5-32 ..................................................................................89 5-11. Synthesis of succinimide 5-34 ..................................................................................90 5-12. Oxidation and elimination reactions.........................................................................91 5-13: Hydrogenation of fluorescein maleimide 5-7 ...........................................................92 5-14: Attempted oxidation of fluorescein 5-37 ..................................................................93

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xvi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy OLEFIN METATHESIS IN PEPTIDOM IMETICS, DYNAMIC COMBINATORIAL CHEMISTRY, AND MOLECULAR IMPRINTING By Tammy Karrie Cheng Low August 2006 Chair: Eric J. Enholm Major Department: Chemistry Catalysis based olefin metathesis is a very valuable and useful tool in synthetic organic chemistry. Our research goals consis ted of employing olefin metathesis in the synthesis of peptidomimetics, and studying the feasibility of this method in dynamic combinatorial chemistry and molecular imprinting of bioactive molecules. One of the approaches to developing pep tidomimetics is attaching biologically significant molecules, such as amino aci d chains, to a scaffold. Grubbs second generation ruthenium catalyst was used to couple phenylalanine to a 17-membered lactam using cross-metathesis in 48% yield with an E : Z ratio of 1.2:1. The crossmetathesis product of two phenylalanine ami no acids was isolated in 45% yield and found to be predominantly trans The 17-membered lactam was constructed in 6 steps, including the fundamental ringclosing metathesis reaction. The reversibility of the cross-metathesis of this macrocyclic system was demonstrated, which is essential to the development of a dynamic combinatorial library.

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xvii Olefin metathesis in dynamic combinatorial chemistry is of interest as a method in generating peptidomimetic libraries. Th e olefin cross-metathesis reactivity and selectivity of amino acid derivatives with a cy clic scaffold to generate diketopiperazine peptide derivatives were investigated. Produc t yields were dependent on the amino acid R groups, and whether the amino acid possessed an allyl or homoallyl moiety at the carboxylate side. Stereoselec tivity of the dipeptide derivatives was found to be predominantly trans Larger sized cyclic scaffolds were also synthesized to create a more diversified library. In addition, several pept ide derivatives posse ssing diene functionality were examined and subjected to Grubbs s econd generation ruthenium catalyst. Various conditions, including substrate and catalyst concentrations, as well as diverse metal templates, were explored. Also described is a model study to inves tigate the feasibility of using ring-opening metathesis polymerization in molecular im printing technology capable of detecting bioactive molecules, namely nerve agents. The project involved the synthesis of a fluoresceinyl dye to be used as the detector a nd the investigation of thiols as templates in the molecular imprinted system.

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1 CHAPTER 1 HISTRORICAL BACKGROUND 1.1 Olefin Metathesis Olefin metathesis is a powerful synthetic tool that has found its way into a vast array of applications, ranging from the deve lopment of small molecu le drug candidates to the industrial scale synthesis of petrochemicals.1-7 This catalytic organic reaction is unlike other carbon-carbon bond forming strategies due to the versatility of synthetic transformations it promotes, such as the synt hesis of various sized cycloalkenes from dienes and specialized polymers by the ringopening of cyclic molecules. Olefin metathesis has opened efficient synthetic routes to complex natural products, drug molecules, and new materials as demonstrated by the explosion of metathesis related applications found in the liter ature during the past decade. In 2005, the value of this organic reaction was prestigiously recogni zed by the award of the Nobel Prize in Chemistry to the major contributors of olefin metathesis Yves Chauvin, Robert H. Grubbs, and Richard R. Schrock. 1.1.1 Development of Olefin Metathesis and Catalysts Olefin metathesis was discovered accident ally by researchers in petrochemical companies in the 1950s when they were search ing for heterogeneous catalysts to convert olefins to high-octane gasoline.7,8 Instead of the expected products, the chemists observed newly formed olefins. It was not until the 1960s when researchers at Goodyear Tire & Rubber determined that these new pr oducts were the result of an exchange of

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2 substituents on different olefins, which th ey referred to as olefin metathesis9 as shown in Scheme 1-1. R1 R2R4 R3 R1 R3 R 4 Catalyst R2 Scheme 1-1. Olefin metathesis For years, chemists attempted to expl ain the mechanism behind the novel reaction for the skeletal transforma tion of olefins. Calderon et al .,10 Lewandos and Pettit,11 and Grubbs and Brunck12 initially suggested cyclobutan e, tetramethylene complex, and rearranging metallacyclopentane intermediates as part of the mechanism, respectively, but all proposals later proved to be incorrect (Scheme 1-2).8 It was in 1971 when Chauvin and Hrisson proposed a metalcarbene mechanism which involves the formation of a metallacyclobutan e intermediate (Scheme 1-3).8,13 The debate over the mechanism continued for year s until Katz, Schrock, and Te bbe independently conducted experiments, which supported Chauvins proposal.1,8 During the debate over the olefin metathes is mechanism, several groups continued to develop transition metal carbene complexe s, including Fischer ca rbenes (low oxidation state metals and electron poor carbon centers) and Schrock ca rbenes (high oxidation state metals and electron poor metal centers).1,8 The Fischer carbenes showed little activity for olefin metathesis, and Schrocks early tantalum and niobium complexes proved unsuccessful as well.1,8 These initial studies however paved the road to improved alkylidene complexes which eventually dem onstrated improved r eactivity for olefin metathesis.

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3 R2 R1 R3 R4 M cyclobutane intermediate M R1 R2 R3 R4 tetramethylene complex M R1 R2 R3 R4 M R3 R1 R4 R2 rearranging metallacyclopentane Scheme 1-2. Proposed intermed iates for olefin metathesis LnM R R1 LnM R R1 Scheme 1-3. Metallacyclobutane inte rmediate proposed by Chauvin Despite early advances in catalyst deve lopment, olefin metathesis was not a practical synthetic methodology due to the cataly sts low reactivity, instability, and lack of tolerance towards functiona l groups. It was not until the 1990s when Schrock lab introduced the well-defined alkoxy imido molybdenum-based catalyst 1-1 which made olefin metathesis a usef ul tool (Figure 1-1).14,15 In contrast to many of the early catalytic systems of the 1970s and 1980s, which are often referred to as classi cal or ill-defined catalysts because the propagating species can not be observed, isolat ed, or structurally characterized, the molybdenum alkylidene comple x is highly reactive and leads to desired products in high yields, even with sterically hindered alkenes.1,16 However, the downfall is the catalysts relatively limited tolerance toward polar functional groups such as alcohols and carboxylic acids, and its sensitivity to air and moisture.5

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4 N Mo iPr iPr (F3C)2MeCO (F3C)2MeCO Ph Me Me 1-1 Shrock'scatalyst Figure 1-1. Schrocks alkoxy imi do molybdenum-based catalyst 1-1 In an effort to improve tolerance for functional groups and moisture, Grubbs group examined ruthenium catalysts, which have an oxidation state lower than Schrocks metallaolefins but higher th an the Fischer carbenes.1,17 Despite the development of the ruthenium catalyst [(PPh3)2Cl2Ru=CHCH=C(Ph)2] ( 1-2 ) in 1992, which was stable in protic and aqueous solvents, the catalyst exhibited limited reactivity compared to Schrocks carbene complexes (Figure 1-2).1,18,19 Modifications of the ruthenium catalyst were conducted throughout the years. Eventually in 1996, Grubbs first generation catalyst 1-3 was introduced, which not only displa yed functional group tolerance, but also up to 20-10,000 times greater ac tivity than ruth enium catalyst 1-2 (Figure 1-2).20 In 1999, based on Herrmann and coworkers studies on N-heterocyclic carbenes,21 Grubbs group substituted one of the tricyclohexyl phosphine (PCy3) ligands from 1-3 with a mesityl N-heterocyclic ligand to afford the more stable ruthenium complex 1-4 which is now referred to as Grubbs s econd generation catalyst and is far superior to other catalysts because of its tolerance to air, mois ture, and a wide variet y of functional groups (Figure 1-2).1,6,22,23 As a result of these enhanced pr operties, our research efforts focused on olefin metathesis utilizing Grubbs second generation catalyst 1-4

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5 Ru Ph PCy3 Cl Cl N N Mes Mes Ru Ph PCy3 PCy3 Cl Cl 1-31-4 1-2 Grubbs'first generationcatalyst Grubbs'second generationcatalyst (Cy=cyclohexyl,Mes=2,4,6-trimethylphenyl) Ru PCy3 PCy3 Cl Cl Ph Ph Figure 1-2. Ruthenium catalysts 1.1.2 Mechanism of Olefin Metathesis Commercial availability of ruthenium catalysts 1-3 and 1-4 has made them a practical, standard organic tool ; thus the synthesis of the metal alkylidene complexes will not be discussed. To better apply olefin metathesis towards the synthesis of target compounds and polymers, it is helpful to examine the mechanism that was first introduced by Chauvin. When utilizing Grubbs catalysts 1-3 and 1-4 the first step of the mechanism involves the di ssociation of the PCy3 ligand, followed by the binding of the alkene to the carbene (Scheme 1-4).24,25 The next step is a [2 +2] cycloaddition with the metal catalyst to form the metallacyclobutan e intermediate, which can then undergo a cycloreversion to produce a new metal alkylidene (Scheme 1-5).4,25 The mechanism proceeds as a catalytic cycle where the metal alkylidene undergoes another [2+2] cycloaddition with a second alkene, followe d by the cycloreversion leaving the newly formed olefin with R1 and R2 groups and the metal alkylidene for further catalytic use. Ru L PCy3 Cl Cl R +olefin -olefin +PCy3Ru L Cl Cl R Ru L Cl2R R -PCy3 Scheme 1-4. Dissociative substi tution of ruthenium catalysts

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6 LnM R1 R2 R2 R1 LnM R1 LnM R1 LnM R1 R2 2 + 2cycloreversion cycloreversion 2 + 2 H2CCH2 Scheme 1-5. Mechanism of olefin metathesis Since ethylene gas is released as a byproduct,6 it is possible to shift the equilibrium toward the desired products by deliberately flushing the headspace with inert gas to remove the evolved ethylene.26 The cycle continues until the reaction is quenched, for example, with ethyl vinyl et her (EVE), which reacts with the ruthenium catalyst and forms the Fischer carbene L(PCy3)(Cl)2Ru=CHOEt.24 The formation of the Fischer carbene is virtually irreversib le and the electron rich carben e complex is significantly less reactive than the ruthenium alkylidenes.24,27 1.1.3 Important Types of Metathes is Reactions and Applications As highlighted many times, olefin meta thesis is a versatile technique which includes ring-closing metathesis (RCM), ring-opening metathesis (ROM), crossmetathesis (CM), ring-opening metathesis polymerization (ROMP) and acyclic diene metathesis (ADMET) (Scheme 1-6).6 Discussed in greater detail are the three main metathesis reactions of interest in our studies, RCM, CM and ROMP.

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7 RCM is the cyclization of a diene to ge nerate various sized cycloalkenes, ranging from small 5-membered rings to macrocycles.16 The stereochemistry of the cycloalkene products is dependent on the substrates; for example, small and medium sized rings formed from RCM are in a less strained cis conformation while in contrast, the stereochemistry of non-rigid RCM derived m acrocyclic compounds is difficult to predict and can encompass a mixture of cis and trans stereoisomers.28 RCM reactions are conducted under highly dilute conditions to prevent ADMET polymerization. In addition, heat is often em ployed to improve ring closures due to the entropy of activation required to bring the two ends of the chain together.29 However, higher temperatures can cause the catalyst to decompose; thus a grea ter catalyst loading is required.5 Despite this requirement, RCM has provided a shorter, more efficient synthetic route to natural products, drug mol ecules, and new materials, compared to conventional methods. An example is shown in Scheme 1-7 in which Van et al. utilized RCM to synthesize coumarins in excellent yields, while other methods reported in literature had disadvantages a nd required harsher conditions.30 The reverse reaction of a RCM is ROM, where the cycloalkene breaks open to form two terminal dienes, which can be followed by a CM reaction with other acyclic alkenes to form new products.5 Similar to RCM, ROM requires dilute conditions due to the resulting dienes undergoing polymerization, refe rred to as ROMP. The polymerization is quite practical in the synthesis of specialized polymers and is more widely used than ROM. Highly strained cycloalkenes, such as norbornene, cyclopentene and cyclooctene favor ROMP.16 By reducing ring strain, the reaction is enthalpically driven forward and is not reversible.

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8 RCM -C2H4-nC2H4ADMET ROMP ROM n +C2H4 R1 R2 R1 R1 R1 R2 R2 R2 CM -C2H4 Heterodimer Homodimers Scheme 1-6. Types of olefin metathesis O R1 R2 R3 R4 O CH2Cl2,reflux,4h 1-4 (5mol%) O R1 R2 R3 R4 O 1-5a R1=OMe;R2=R3=R4=H 1-5b R1=R2=R4=H;R3=OMe 1-5c R1=R4=H;R2+R3=O-CH2-O 1-5d R1=R4=OMe;R3=R2=H 1-5e R1=R4=R3=R2=H 1-6a 81% 1-6b 90% 1-6c 83% 1-6d 72% 1-6e 70% Scheme 1-7. Utilizing RCM to synthesize coumarins Grubbs catalyst 1-4 has high functional group tolerance and has been demonstrated in ROMP to generate func tionalized, telechelic and trisubstituted polymers.31 ROMP has been responsible for the s ynthesis of a variety of new materials, from the development of nonlinear optics to biologi cally relevant polymers.32 A recent

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9 application of this polymerization is s hown in Scheme 1-8, where a polymer was synthesized to create biomater ials that can undergo a [2+2] cycloaddition when irradiated with UV light.33 O O Ph O O Ph O O Ph O O Ph n ROMP 1-3 ,CH2Cl279% 1-7 1-8 Scheme 1-8. Employing ROMP to create new materials RCM and ROMP started as the most popular types of metathesis reactions, but due to recent studies and a better understanding of the selectivity and ster eoselectivity of CM, it has become a more useful and versatil e synthetic technique. The concerns over selectivity arose from the mixture of heterodimers, homodimers, and cis / trans stereoisomers that can be generated fro m CM reactions. In addition, employing asymmetric internal olefins in CM can also lead to a greater number of product mixtures (Scheme 1-9). Factors such as sterics a nd electronic effects may also affect CM reactivity and selectivity, and must be consid ered when planning reactions. For example, olefins possessing electron withdrawing or bulky substituents often lead to little or no homodimerization because of the poor reactivity with the catalyst. However, steric effects can favor trans selectivity.34

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10 R1 R3 R2 R3 R1 R3 R1 R4 CM R2 R4 R2 R4 R3 R3 R1 R1 R2 R2 R4 R4 Heterodimers Homodimers Scheme 1-9. CM of asymmetric internal olefins Fortunately, new models a nd methodology were develope d to improve selective CM. For instance, Grubbs group categorized ol efins as Type I, II, III, and IV based on their reactivity to form homodimers by CM with catalyst 1-3 and 1-4 Primary allylic alcohols, protected amines, and esters are examples of Type I alkenes (sterically unhindered, electron-rich) because they read ily form homodimers by CM and also undergo secondary metathesis reactions.26,34,35 The more sterically hindered Type II alkenes (i.e., secondary alcohol s and vinyl ketones) are less reactive, and Type III alkenes are nonreactive (i.e., tertiary allylic carbons). Type IV alkenes (i.e., protected trisubstituted allyl alcohols) are spectators a nd do not participate in the CM reaction. The examples given above are based on the utilization of catalyst 1-4 One strategy toward selective CM involves a two step procedure in which homodimers of Type I alkenes are generated, followed by a secondary metathesis reaction with Type II/III alkenes to preferentially form the heterodimer product, which can favor the trans isomer in the presence of selected func tional groups (Scheme 1-10).34 CM is more widely used now, and an example of a recent a pplication of CM is shown in Scheme 1-11, where Nolen and coworkers were able to synthesize C-gl ycosyl asparagines in good yields with predominantly trans selectivity.36

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11 Cat R1 R2 Cat R2 R1 R1 R1 R1 R1 Scheme 1-10. Primary and secondary CM metathesis reactions O AcO AcO AcO AcO O AcO AcO AcO AcO CO2Me NHCbz 1-4 (20mol%) CH2Cl2reflux,12h CO2Me NHCbz 82% 1-9 1-10 1-11 predominantly E Scheme 1-11. Synthesis of Cglycosyl asparagines via CM 1.2 Peptidomimetics Olefin metathesis is now a common synthe tic tool for the organic chemist. As shown by the examples in the previous section, it is versatile and has been used to make new materials and analogues of natural product s. Other areas of science that have a particular interest in metathesis are peptid e and medicinal chemistry. Peptides, made of amino acid building blocks, are vital biological molecules with vast functionality. For example, they can serve as antibiotics, an algesics, and building blocks of important proteins, such as enzymes. Therefore, peptid es have gained significa nt interest as drug candidates. Unfortunately, natural peptides typically do not make good pharmaceuticals because of their lack of bioavailability a nd susceptibility to premature hydrolysis invivo.37,38 A solution to this downfall is pepti domimetics, which are synthetic molecules that can mimic the peptides topography and functionality, but possess improved pharmaceutical properties.39,40 One strategy to enhance these properties is by synthesizing cyclic molecules that mimic peptides, which are referred as cyclic peptidomimetics. Frequent discovery of

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12 natural cyclic peptides possessing antibiotic, antiviral, antitumor, and therapeutic properties has made them an active area of research.41,42 In addition, cyclic peptides are more stable than linear peptides becaus e of their constrained conformation, which improves receptor selectivity.37 Therefore, there is great interest in the synthesis of mimetics of cyclic peptides for use in me dicinal chemistry and drug development. Peptidomimetics can be designed to be less susc eptible to proteolysis, and biostable with improved ability for absorption in the body.43,44 Several methods exist to synthesize cyclic amino acids and peptidomimetics. One strategy involves cyclization of a linear peptide by conventi onal coupling agents to form a new amide bond (Scheme 1-12).37,45 Some common reagents used to perform this task are dicyclohexylcarbodiimide (DCC), diis opropylcarbodiimide (DIC), and expensive reagents such as HATU or PyBOP.45,46 Racemization of the chiral center is of great concern, and often times racemization suppressants such as 1-hydroxy-7-azabenzotriazole (HOAt) and 1-hydroxybenzot riazole (HOBt) must be employed.45 Cyclization can sometimes be complicated due to difficulties in bringing the two terminal ends together;42 thus peptidomimetics are ofte n designed where hydrogen-bonds can assist in the ring closure by inducing the linear peptid e to turn (Figure 1-3).37,39 It is also possible to synthesize cyclic peptidomimetics by attaching biologically significant molecules, such as amino acids, to a scaffold.43,44,47,48 The scaffold can be designed where the functional groups are pr operly oriented to their corresponding binding sites. For example, Chery and Murphy synthesized po tential HIV protease inhibitors by grafting pharmacophoric gr oups on a azasugar scaffold (Figure 1-4),48 but a rather lengthy synthetic scheme starti ng from D-fructose and involving selective

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13 deprotection was required. Other types of scaffolds have been generated including conformationally constrained bicyclic amino acid motifs39 and pyridine derivatives.44 O OH O couplingreagents H2N O H N O -H2O linearpeptidecyclicpeptide Scheme 1-12. Cyclization of a linear peptide using coupling agents HN N H O O NH R R O N R R H O Figure 1-3. Hydrogen-bonding lead s to bent conformation Above is a brief overview of peptidomimetic s and some strategies used to generate cyclic mimetics. Methodologies used to s ynthesize scaffolds and cyclic peptides often involve intricate synthetic procedures. Recentl y, RCM has been utilized as an alternative method towards generation of cyclic peptidomimetics.37,49,50 An example is shown in Scheme 1-13, where Gmeiner gr oup employed RCM to synthesize -turn mimetics in excellent yields.51

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14 H N OH O OH O O O Orientedintoenzymesubsites Bindstoasparticacids Chargedaminecanhydrogen bondwiththecarbonylgroup ofHIV-proteaseamide backbone 1-12 Figure 1-4. Design of putativ e azasugar peptidomimetic N OMe O NHBoc 1-4( 7mol% ) 88% CH2Cl2,40oC N O OMe O NHBoc O 1-13 1-14 Scheme 1-13. Use of RCM toward the synthesis of -turn mimetics 1.3 Dynamic Combinatorial Chemistry In addition to applications in peptidomimetics, olefin metathesis in dynamic combinatorial chemistry (DCC) is very prom ising. DCC has gained interest in recent years as a powerful methodology for explori ng molecular recognition systems, thereby leading to the discovery of new biologically active molecules, drugs, receptors, and catalysts.52,53 A dynamic combinatorial library (DCL ) is made from molecular building blocks connected via reversible linkage s which interconvert in a thermodynamic equilibrium.52,53 A template, such as a biological molecule, is a dded to the system which can shift the equilibrium to form one majo r constituent in the library (Figure 1-5).54 Unlike traditional combinatorial techniques, where molecules are created en masse and

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15 tested for desired properties, DCC combines the synthesis and selectivity of library constituents in one pot. Key differences between DCC and traditional combinatorial chemistry are shown in Table 1-1. DCC is not only valuable in discovering new properties, but also as a learning tool towards a better unders tanding of molecular recognition, self-assembly, and supramolecular chemistry. Reprinted from Publication: Curr. Opin. Chem. Bio. ; Vol 6; Otto, S.; Furlan, R. L. E.; Sanders, J. K. M.; Recent Developments in Dynamic Combinatorial Chem istry; 321-327; Copyrigh t 2002, with permission from Elsevier. Figure 1-5. A dynamic combinatorial lib rary and its free energy landscape Table 1-1. Differences between traditiona l and dynamic combinatorial libraries COMBINATORIAL LIBRARY DYNAM IC COMBINATORIAL LIBRARY Real set Virtual set Collection of molecules Collection of components Covalent Covalent or non-covalent Non-reversible Reversible Systematic Recognition-directed Preformed by synthesis Self-assembled In absence of target In presence of target There are two types of dynamic combinat orial recognition described by biological aspects of receptors and substrates (Figure 1-6).53,55 In the casting process, the target receptor (TR) influences the organization of the bu ilding blocks into the substrate with specific binding affinity to the TR. In the molding process, the target molecule is a substrate (TS) which results in the molding of the receptor.

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16 The examples in Figure 1-6 demonstrate the addition of the target molecule to the reaction mixture after the library of building bl ocks have assembled. The presence of the target can then shift the equilibrium where onl y the constituents with the highest affinity for the target will be amplified. This is considered a true dynamic combinatorial system.56 The other method, which is sometimes referred as the gene ration of a virtual combinatorial library, involves the addition of the target molecule without formation of a library of interchangeable species, as depict ed by the lock and key metaphor shown in Figure 1-7.56 TR TR Casting Ts Ts Molding Figure 1-6. Casting and mo lding process in DCC There are different types of reversible r eactions that can pote ntially be used in DCC, such as covalent bond formation, non-co valent interactions and intramolecular processes.56 Some reversible reactio ns that have already been studied for use in DCC include disulfide exchange,57 metal-ligand coordination,58 exchange of oximes,59 hydrazones,60 and olefin metathesis.61,62

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17 Reprinted from Publication: Nature Rev. Drug Discov. ; Vol 1, Ramstrom, O.; Lehn, J. M.; Drug Discovery by Dynamic Combinatorial Libraries; 26-36; Copyright 2002, with permission from Nature Review Drug Discovery, MacMillan Magazines Ltd. Figure 1-7. Dynamic combinatorial chemistry (t op) versus virtual combinatorial libraries (bottom) Even with the expanding amount of dynami c combinatorial rela ted research found in literature, very few studies have examined olefin metathesis in DCC. One of the most interesting developments came from Ni colaou group in 2000, where they proved the applicability and significance of DCC via olef in metathesis by synthesizing dimers of vancomycin derivatives (Scheme 1-14).63 The building blocks possessed various olefin chain lengths and different amino acid R groups while the amino acid target chosen had a high affinity to vancomycin.63 As they expected, the dime rs with the shorter tethers were amplified upon addition of the target (Figure 1-8).63 Despite Nicolaous important studies on DCL of vancomycin derivatives, there is still more to learn about the

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18 reactivity, influence of functi onal groups, and stereochemis try of olefin metathesis reactions for use in DCC. HN O HO O HO OH OH O O n N H O H N O H N O NH2 O O N H OH O Cl Cl R O H N O NH HO H H OH OH H O O HO OlefinMetathesis 1-3 Dimers Scheme 1-14. Dimerization of monomeric vanc omycin derivatives with terminal olefins by metathesis A B Reprinted from Publication: Angew. Chem. Int. Ed. ; Vol 39; Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, C.; Labischinski, H.; Endermann, R.; Target-Accelerated Combinatorial Synthesis and Discovery of Highly Potent Antibiotic s Effective Against Vancomycin-Resistant Bacteria; 3823-3828; Copyright 2000, with permission from Wiley. Figure 1-8. Mass spectrometric an alysis of the vancomycin dimer mixture. (A) Absence of a target and (B) in th e presence of a target 1.4 Molecular Imprinted Polymers There has been a great interest in the Enholm group to employ ROMP in molecular imprinted technology, which has only been re ported once in the literature by Steinke

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19 lab.64 Molecular imprinted polymers (MIPs) are useful as mimics of biological receptors, enzymes, and antibodies,65-70 and can be designed as drug delivery or drug separation systems.67 In addition, research has already shown that MIPs can have enhanced catalytic activities compared to co rresponding natural catalytic antibodies.70 In addition to a plethora of studies found in the literature related to biological applications, this technology has also been used for chemical sensors71,72 and environmental analysis.71 The general principle behind molecula rly imprinted technology involves the synthesis of a polymer that possesses a 3-di mensional molecular memory or imprint for a target. For example, in the lock and key metaphor by Emil Fischer, an enzyme (lock) has active sites specific for a particular s ubstrate (key) (Figure 1-9). In MIPs, the polymer acts as an enzyme by having a cavit y with functional groups that can interact covalently or non-covalently w ith the template (Figure 1-9). Like enzymes, the polymer has a specific binding affinity for the template. The general process of molecularly impri nted technology is shown in Figure 1-10, in which a template and functional monomers, which include a polymerizable moiety, interact to form a stable template-functional monomer assembly.69 A cross-linker is added and polymerization proceeds resulting in a rigid template-cross-linked polymer complex. The template is then extracted to give the MIP. There are two main ways a template can interact with the functional monomers the self-assembly approach via non-covalent (i.e. electr ostatic, hydrogen bonding, and hydrophobic) or metal coordination interactio ns, and the pre-organized approach via reversible covalent interactions,69 where both methods have their advantages and disadvantages. With the pre-organized appro ach, the template is fixed in its proper

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20 orientation during polymerization resulting in a pronounced imprint.65 However, only selected reversible covalent reactions, such as the formation and hydrolysis of boronate esters, are suitable for MIP.68,72 An example of the pre-organized approach is shown in Scheme 1-15, in which Wang et al built fluorescent sensors using boronate esters.73 Enzyme Enzyme A:"LockandKey" B:MIP Polymer ActiveSites Active Sites S S S S S=Substrate Figure 1-9. A comparison between the Lo ck and Key model and the MIP model The non-covalent approach is the most wi dely used due to the ease of template removal by breaking non-covalent interactions.67 However, more functional monomers are required to fix the template in place prior to polymerization. If the template is not properly set, then the resulting molecular impr int could have a reduction in the specificity for the template. A solution to both methods is a semi-covalent approach, in which the template is covalently bound during imp rinting and non-covalently bound during rebinding.72 For example, Vulfson group designed a molecular imprint of a tripeptide using this semi-approach (Scheme 1-16).74

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21 Polymer FunctionalMonomers Template Cross-Linking Monomer Polymerization TemplateExtraction MolecularImprintedPolymer Figure 1-10. Schematic of molecular imprinting process Fluorophore B OH OH Fluorophore B O O R1 R HO HO R1 R Scheme 1-15. An example of the pre-organi zed approach via covalent interactions Radical polymerization is the standard me thod for the synthesis of MIPs, where the functional and cross-linking monomers chosen for MIP often possess vinyl or acrylic groups, such as methyl methacrylate ( MMA) and ethylene glycol dimethacrylate

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22 (EDMA). A large variety of these monomers are readily available and have functional groups exhibiting hydrogen bonding, hydrophobicity, and other interactions with the template.66 The radical polymerization can pr oceed via thermal initiators (azobis isobutyronitrile (AIBN) is most commonly used), or photoinitiators.72 O O O N H HN O O O O CO2H CO2H N H O H N N divinylbenze, AIBN O O O N H HN O O O O CO2H CO2H N H O H N N N NaOH/MeOH/D O OH OH O N N Scheme 1-16. An example of a se mi-covalent approach to MIPs In 2003, Steinke group used ROMP rather than radical polymerization to synthesize enantioselective MIPs to ther modynamically control the polymerization reaction.64 The goal was to prevent the formation of MIPs polyclonal cavities, which can decrease selectivity for the template. This was the first time ROMP was demonstrated in

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23 the generation of MIPs. During that same time period, Allias performed a side by side comparison of radical polymerization and ROMP employing Grubbs 1-4 catalyst, and discovered that ROMP required shorter r eaction times and easier work-up conditions.75 Preliminary studies also showed that there was an increase in the affinity for the template using the ROMP strategy, while the MIP cav ity generated by radical polymerization showed poor selectivity for the template. Another benefit of employing ROMP is that heat or photo labile templates which are not acceptable in radical polymerization can now be used in the system. Based on these initia l studies, it appears promising to examine olefin metathesis in molecularly imprinted technology. 1.5 Conclusions Olefin metathesis is a powerful organic synthetic tool, attested by the large volume of metathesis related resear ch found in literature. Grubbs second generation catalyst 1-4 and its tolerance for functiona l groups have made this methodology even more useful. However, there are still areas of olefin metathes is that require more studies in the field of peptidomimetics and MIPs. The work presen ted here will examine the use of olefin metathesis in the 1) attachment of an am ino acid on a macrocyclic scaffold by CM; 2) synthesis of dipeptide mimetics by CM a nd the examination of the reactivity, stereochemistry, and feasibility of CM in D CC; 3) generation of a DCL of macrocyclic compounds employing peptidomimetic dienes an d 4) development of chemical sensors using ROMP in molecularly imprinted technology to dete ct nerve agents.

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24 CHAPTER 2 USE OF CROSS-METATHESIS TO COUPLE L-PHENYLALANINE TO A MACROCYCLIC LACTAM 2.1 Introduction Peptidomimetic research is of paramount importance to the field of medicinal chemistry. One approach toward the synthesi s of peptidomimetics is to use molecular templates or scaffolds to which biologically significant functional groups, such as amino acid chains, are covalently anchored.26,30,33,62 These molecules have an excellent potential for chiral discrimination and di splay stabilization of functional groups. Macrocyclic lactams are excellent choices as scaffolds because many naturally occurring macrocycles possess important biological and medicinal properties.76,77 In addition, the ability to attach multiple f unctional groups on a large ring can increase diversity, essential to drug discovery.78 One example of a macrocyclic scaffold used for drug discovery is shown in Figure 2-1, in wh ich side chains are attached to a rigid macrocyclic lactam in a well-define d orientation toward receptor sites.79 NH HN HN NH O O O O AllO2C NHCbz N O N O MeO2C NHBoc 2-1 Figure 2-1. Oxazole-based macrocyclic lactam

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25 Ring-closing metathesis (RCM) has recently become a useful organic reaction to synthesize macrocylic scaffolds as demonstrated in Scheme 2-1.77 In this example, Peng and Blagg utilized Grubbs first generation catalyst 1-3 to generate an analogue of Trienomycin A, an antibiotic whic h also possesses antitumor activity.77 R NH 2-3 R NH O 2-2 1-3 (12mol%) O trans : cis ,3.5:1 45% CH2Cl2r.t.24h Scheme 2-1. Synthesis of an anal ogue of Trienomycin A utilizing RCM Another example of metathesis derived m acrocyclic lactams is shown in Scheme 22, in which Hu and associates discovered th at 15-17 membered rings provided the best inhibitor properties against an enzy me involved in bacterial biosynthesis.76 O H N O N H H N O OBn n O H N O N H H N O OBn n 1-3 (7mol%) CH2Cl2reflux20h 2-42-5 Scheme 2-2. Synthesis of a macrocyclic inhibitor The examples above demonstrate the im portance of macrocyclic lactams as scaffolds in drug development. As part of our continuing in terest in cyclic peptidomimetics, we envisioned RCM as a m eans to generate macrocyclic scaffolds and cross-metathesis (CM) as a way to anchor am ino acids to the scaffolds. Model system 2-

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26 6 is ideally suited for a CM reaction1,34,80,81 since it has a dumbbell sh ape with two halves connected together by an E -alkene tether (Figure 2-2). We believed that Grubbs welldefined second generation catalyst 1-4 would be an ideal choice to facilitate this reaction due to its high tolerance of functional groups.82-84 O O O O H N O N t -BOC 2-6 Figure 2-2. Phenylalanine on a macrocyclic lactam In addition to examining RCM and CM as a synthetic approach toward peptidomimetics, we also wanted to test the reversibility of the CM reaction in this model system for potential use in dynamic combinatorial chemistry (DCC) of cyclic peptidomimetics.85,86 We envisioned a macrocyclic scaffold possessing several anchoring points where functional groups coul d be attached by a CM reaction, as shown in Figure 2-3. The CM reaction must be revers ible such that functional groups can attach and detach onto the scaffold in a dynamic equilibrium. Olefin metathesis is known to be reversible, though only Miller lab has syst ematically examined the reactivity and selectivity of CM and the effects of remo te functionality for potential use in DCC.62 However, the work conducted by Miller group62 and other researchers61,63 focused on the homodimerization of their substrates. In c ontrast, our focus is on anchoring amino acid derivatives on molecular scaffolds. Dynami c combinatorial libraries (DCL) of cyclic peptidomimetics will be discussed further in Chapter 3. The main focal point of this

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27 chapter will be on the synt hesis of model compound 2-6 by RCM and CM, followed by examination of the reversib ility of the CM reaction. N N N N O O O O R1 R2 R3 R4 R5 R=variousaminoacids SitesforCMreactions Figure 2-3. Macrocyclic lactam with anchors for CM with amino acids In examining the viability of our approach to the synthesis of 2-6 we decided that anchoring the amino acid to the nitrogen atom of a large-ring lact am would function well (Scheme 2-3). Compound 2-6 could be prepared from protected L-phenylalanine 2-7 bearing an allyl ester and allyl lactam 2-8 Each of these halves of the molecule contains a terminal alkene to be used in the CM reaction. Precursor 2-8 was to be prepared by RCM and the alkene would later be re moved by hydrogenolysis. Eventually we envisioned 2-8 as emanating from the SN2 coupling of bromo-amide 2-9 and alcohol 210

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28 O O O N O H N O Br OH + 2-6 O H N O t -BOC + 2-7 2-8 2-9 2-10 Scheme 2-3. Retrosynthethic analysis of 2-6 2.2 Results and Discussion 2.2.1 Synthesis of Compound 2-6 We began our synthesis with a Williamson etherification using commercially available 1,8-octanediol ( 2-11 ) and allyl bromide (0.9 eq), as shown in Scheme 2-4. The desired allyl ether 2-10 was obtained in 70% yield, while the minor product, double Williamson ether 2-12 was isolated in 25% yield. The use of tetrabutylammonium iodide (TBAI) was essentia l in obtaining high yields.87 YO O HO OH NaH,THF,TBAI 2-10: Y=H,70% 2-11 allylbromide 2-12: Y=allyl,25% Scheme 2-4. Synthesis of 2-10 and 2-12 Next, N-Allyl-2-bromoacetamide ( 2-9 ) was readily synthesized in 73% yield from allyl amine ( 2-13 ) and dibromide 2-14 followed by recrystalliza tion in hexane/ether,88 as shown in Scheme 2-5. Deprotonation of 2-10 with NaH, addition of TBAI, and nucleophilic substitution of allyl-bromoacetamide 2-9 gave a 54% yield of the terminal diene 2-15

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29 H N O Br NaH,THF,TBAI, 2-10 N O H O O 2-15 NH2 Br O Br CH2Cl2,0oC,3h 73% 2-132-14 54% 2-9 Scheme 2-5. Synthesis of diene 2-15 Diene 2-15 was reacted with Grubbs catalyst 1-4 under various conditions (Scheme 2-6 and Table 2-1) to give two 17-membered RCM lactams, 2-16 and 2-17 as geometric isomers.3 All reactions were refluxed in CH2Cl2 and monitored by thin layer chromatography (TLC). In every entry, 2-15 was never fully consumed. We were pleased with the excellent trans selectivity of our macrol actam; however, the next synthetic procedure required the hydrogenation of the alke ne making stereoselectivity a non-issue. None the less, the E-isomer 2-16 was isolated as the major product, which was separated from Z-isomer 2-17 by chromatography over silica gel. For entries 6-8, the cis-isomer was not isolated due to th e small scale of the reaction. O O O HN 61% 2-15 1-4 2-16 ( E ): 2-17 ( Z ) 20:1 CH2Cl2reflux Scheme 2-6. RCM of 2-15

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30 After RCM, cycloalkenes 2-16 and 2-17 were hydrogenated using Pd on activated C (10% Pd) as the catalyst to give the corresponding saturated lactam 2-18 in high yields (Scheme 2-7). According to NMR spectra and TLC, the compound was pure enough for use in the next step. Addition of NaH, TBAI, and nucleophilic s ubstitution of allyl bromide, gave 2-8 in 62% yield. Two different conformations of 2-8, due to the rotation about the lactam C-N bond, were present in roughl y equal amounts at ambient temperature and readily detected by 1H NMR and 13C NMR by the doubling of peaks. Table 2-1. RCM to obtain 2-16 and 2-17 Entry Conc (M) 1-4 mol% Time (h) Yield % E : Za 2-16:2-17 1 0.001 10 25 59 93:7 2 0.0014 10 21 45 98:2 3 0.0016 10 24 46 92:8 4 0.0016 10 16 61 96:4 5 0.0018 8 19 60 93:7 6 0.003 14 3.5 48 NA 7 0.007 8 2 35 NA 8 0.013 5 20 18 NA a The reported E / Z ratio is based on isolated compounds NaH,THF,TBAI allylbromide,62% 2-16 O O O N 2-8 H2,PdonC,98% O O O HN 2-18 Scheme 2-7. Synthesis of lactam 2-8 The next step involved the CM of Nallyl lactam 2-8 with the allyl ester of t -Bocphenylalanine 2-7 (Scheme 2-8). To synthesize the amino acid derivative, commercially available t -Boc-L-phenylalanine was treated with 1,3-diisopropylcar bodiimide (DIC), hydroxybenzotriazole (HOBt) a nd allyl alcohol to give 2-7 in 95% yield. We were

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31 pleased that the CM of phenylalanine derivative 2-7 (2 equiv.) and N -allyl lactam 2-8 was observed with 5 mol% of catalyst 1-4 The reaction was heated at 55 C in CHCl3 for 21 h while continuously flushing out ethylene w ith argon to drive the reaction forward. Additional CHCl3 was added as needed to keep the concentration to ca. 1 M. After quenching the reaction with ethyl vinyl ether (EVE), purification by column chromatography gave the desired product 2-6 in 48% yield with an E : Z ratio of 1.2:1. The stereoisomers were not separable by column chromatography and the reported cis / trans ratio was determined by 1H NMR of the isolated compound. Similar to the 13C NMR of N -allyl lactam 2-8 we also observed the doubling of peaks for 2-6 due to the rotation about the lactam C-N bond. Colu mn chromatography of the crude reaction mixture also afforded amino acid dimer 2-19 in 45% yield with a predominantly trans configuration. 2-6 2-19 1-4 2-8 CHCl3 O O O O H N O N t -Boc O H N O t -Boc O N H O t -BOC (48%yield) (45%yield) O H N O t -Boc 2-7 + E : Z ,1.2:1 Scheme 2-8. CM reaction to obtain 2-6 and 2-19 The moderate yields and lack of CM ster eoselectivity in the preparation of the heterodimer 2-6 were expected since both amino acid 2-7 and lactam 2-8 were considered Type I olefins with fast homodimerization reactivity,34 as discussed in Chapter 1.

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32 Interestingly, the dimer of Nallyl lactam 2-8 was not observed. A 2:1 ratio of amino acid 2-6 and lactam 2-8 in the CM reaction should result in a statistical hetero dimer selectivity of 66%, which is slightly highe r than the 52% selectivity we obtained. In regards to the stereoselectivity, the trans homodimers were predominantly formed, which is most likely due to secondary CM reactions to produce the more thermodynamically favored isomer.34 However for heterodimer 2-6 we believed that the bulky lactam may prevent cis / trans isomerization by secondary metathesis reactions; thus resulting in the lack of stereoselectivity.34 2.2.2 Examining the Reversibility of the CM reaction in Model 2-6 We examined the reversibility of CM in this model system for potential use in DCC using compound 2-6 It is known that CM is reve rsible, and was tested in our system.52,85,86 We were able to confirm that 2-6 can be converted back to allyl lactam 2-8 and amino acid derivative 2-7 (Scheme 2-9). Ethylene gas, used without purification, was admitted via a balloon to a stirred solution of compound 2-6 catalyst 1-4 (2 mol%), and CH2Cl2. The solution was first maintained at room temperature overnight. TLC showed the formation of Nallyl lactam 2-8 and allyl ester 2-7 In an attempt to drive the reaction further, the so lution was refluxed in CH2Cl2 for 2.5 h under an atmosphere of ethylene gas, then quenched with EVE upon cooling. Purification by chromatography afforded N -allyl lactam 2-8 in 36% yield and allyl ester 2-7 in 37% yield. Both the amino acid dimer 2-19 and unreacted starting material 2-6 were recovered as well. These studies demonstrated the potenti al of using allyl lactams and allyl amino acids as building blocks for DCL of cyclic peptidomimetics.

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33 2-6 O H N O t -Boc 2-7 + 1-4 CH2Cl2ethylene O O O N 2-8 Scheme 2-9. CM reaction with ethylene gas 2.3 Conclusions In summary, Grubbs second generation ruth enium catalyst was used to couple the amino acid phenylalanine to a 17-membered lactam using CM in 48% yield with an E : Z ratio of 1.2:1. The CM product of two phe nylalanine amino acids was isolated in 45% yield and found to be predominantly trans The 17-membered lactam was constructed in 6 steps, including the fundamental RCM reaction. The reversibility of the CM of this macrocyclic system was demonstrated, which is essential to the deve lopment of a DCL. Based on the results of our model study, the ne xt goal was to generate a small library of cyclic peptidomimetics to determine the sh ift in dynamic equilibrium upon addition of a template.

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34 CHAPTER 3 OLEFIN METATHEIS OF AMINO ACID DERIVATIVES WITH CYCLIC SCAFFOLDS 3.1 Introduction As discussed in Chapter 2, one of th e approaches to the development of peptidomimetics is to attach biologically significant functio nal groups to a scaffold. An example of a scaffold used for drug devel opment is shown in Figure 3-1, where amino acids are linked to a carbohydrate backbone.47 Another important type of scaffold is one that is peptide based. For example, glycine based scaffolds are of interest because it has been shown that they can poten tially act as host molecules.89 O O BnO BnO NH O NH2 Glucose-basednonpeptidescaffold Glycine-basedscaffold BnO N N N O R2N O O O O NR2 O NR2 R=CH2CH2OBn Figure 3-1. Examples of scaffolds By having terminal olefin moieties attached to peptide based scaffolds, there is potential to create a dynamic combinatorial library (DCL) of peptidomimetics via olefin cross-metathesis (CM). Numerous studies have shown the advantages of dynamic combinatorial chemistry (DCC) over traditional combinatorial chemistry. Employing olefin metathesis in DCC can lead to the e fficient synthesis and is olation of biologically significant peptidomimetics. There have b een no reports in literature where cyclic

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35 scaffolds and amino acid building blocks were used to create a dynamic library of peptidomimetics. As part of our ongoing study directed to ward the attachment of amino acid derivatives to cyclic scaffolds by olefin CM,90 our research group is interested in studying the viability of reversible olefin CM fo r DCC of cyclic peptidomimetics. The constituents of our library are made by CM of amino acid precursors with a cyclic scaffold (Figure 3-2). Both the amino aci d precursor and the cyclic molecule must possess a terminal alkene. We are examini ng the reactivity of va rious types of amino acid precursors and cyclic scaffolds. In addi tion, several templates are also being tested on the reaction to select for the be st constituent in the library. BuildingBlocks DynamicLibrary Figure 3-2. Dynamic combinatoria l libraries of peptidomimetics Amino acids can be combinatorially arra nged around a glycine based lactam by a reversible CM reaction, as show n in Figure 3-3, to prepare DCL 3-1 on a cyclic scaffold. To examine the viability of the approach, model compound 3-2 was utilized in anchoring amino acid derivatives to the nitrogen atoms of a lactam.

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36 CMlinkage reversible aa6 a a 7 aa8 aa5 aa4 aa3 aa2 aa1 dynamiccombinatorial library model 3-1 3-2 aa1 N N O O aa2 n n Figure 3-3. Schematic of a DCL and a model of amino acids linked to a cyclic scaffold by olefin CM Miller group recently demonstrated the impor tance of remote functionality of olefin CM using Grubbs first generation catalyst 1-3 for potential use in DCC.62 Here we report product yields and dist ributions of CM reactions of allyl and homoallyl ester amino acid derivatives with a rigid cyclic scaffold us ing Grubbs second generation catalyst 1-4 Several cyclic scaffolds are in cons ideration for these studies dimer 3-3 trimer 34 and tetramer 3-5 (Figure 3-4). Dimer 3-3 is of interest because cyclic dipeptides possess significant medicinal a nd biological characteristics.41,91-93 In comparison to linear peptides, diketopipera zines are conformationally co nstrained and more stable toward hydrolysis, which is critical in drug design.94-96 In addition, th e diketopiperazine peptide derivatives could also be used as bui lding blocks for the synthesis of larger or more complex cyclic peptides.93 There has also been grea t interest in combinatorial synthesis of cyclic dipeptide derivatives.93,97 For example, Loughlin et al demonstrated the solution-phase combinatorial synthesis and evaluation of piperazine-2,5-dione derivatives for cytotoxic activities (Scheme 3-1).97

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37 N N N N O O O O N N N O O O 3-4 3-5 N N O O 3-3 Figure 3-4. Glycine-based dimer, trimer and tetramer scaffolds N N O O Ac Ac O R1 H N N O O Ac Ac R1 O R2 H N H H N O O R1 R2 K t BuO DMF t BuOH 24h K t BuO DMF t BuOH 24h 3-6 3-7 3-8 1) 2)AceticAcid Scheme 3-1. Combinatorial synthesis of piperazine-2,5-dione derivatives We are also interested in synthesizing triallylcyclo -triglycine 3-4 to serve as the trimer scaffold. Similar to diketopipera zines, cyclic tripeptides possess a rigid conformation and display important pharmaceutical properties.50 In addition to cyclic dipeptides and tripeptides, the ability to cr eate a DCL of molecules that mimic cyclic tetrapeptides would be important since th ey can also exhibit phytotoxic, cytotoxic,98 and medicinal properties.42 An example of a natural cyclic tetrapeptide is shown in Figure 35. Comparable to the structures of the dimer and trimer scaffolds, tetrallylcyclo tetraglycine 3-5 can serve as a tetramer scaffold. N HN NH N O O O O Cyclo(Proline-Valine-Proline-Tyrosine) Tyrosinase inhibitor OH 3-9 Figure 3-5. Natural cyclic tetrapeptide

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38 To generate a library of peptidomime tics, various amino acids possessing a terminal olefin moiety can couple with these cyclic scaffolds by olefin CM. For example, the metathesis of two amino acid derivatives with N -allyl glycine scaffold 3-3 can lead to three amino acid dimers and three cyclic CM products (Figure 3-6). The number of amino acid dimers generated by metathesis is given by N *( N +1)/2 excluding E/Z isomers, where N is the number of unequivalent amino acid derivatives in the pool. When cis / trans isomers are considered, then there are 16 total possible produc ts (Figure 3-7). Products from oligomerization of the dimer scaf fold or olefin isomerization are excluded from these projected numbers.99,100 There is great interest in synthesizing cyclic diketopiperazine peptide deriva tives. However, these simple systems for use in DCC are not ideal since at most, only two different amino acids coul d be attached to the dimer scaffold. This methodology would be more su itable towards a tradit ional combinatorial library. The main interest in using the dimer scaffold in these experiments is to better understand CM reactivity, yield, and stereochemis try of the expected products in a simple dynamic combinatorial system. BuildingBlocks DynamicLibrary =allylesteraminoacid=dimerscaffold 3-3 CyclicCMProducts AminoAcidDimers Figure 3-6. Small dynamic library of cyclic dipeptidomimetics

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39 BuildingBlocks DynamicLibrary E E Z E Z Z E E Z E Z Z E E Z E Z Z Z E E E E Z Z Z Figure 3-7. A dynamic library of cy clic dipeptidomimetics including cis/trans isomers Employing the trimer scaffold 3-4 can lead to a larger libr ary. If three amino acid derivatives react with the trimer scaffold in CM coupling, then the library will be composed of ten unique cyclic CM molecule s and six amino acid dimers (Figure 3-8). This excludes any olefin isomerization products or oligomerization of the cyclic scaffold. If stereoisomers are considered, then there ar e 48 isomers of cyclic CM molecules and 12 isomers of amino acid dimers (Figure 3-9). CM of tetramer scaffold 3-5 with four different amino acid derivatives would result in a larger library, where there can potentially be 55 cyclic peptidomimetics (652 including E / Z isomers) and ten amino acid dimers (20 including E / Z isomers) (Figures 3-10 and 3-11). = =allylesteraminoacid =triallylcyclo -triglcine 3-4 BuildingBlocks DynamicLibrary = Figure 3-8. Dynamic library of cyclic tripeptidomimetics

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40 Z Z Z Z Z E Z E E E E E Z Z Z Z Z E Z E E E E E Z Z Z Z Z E Z E E E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E Z Z E E E E Z E E E Z Z Z Z Z E Z E E Z E Z E E E E E Z E Z E E Z Z CyclicMolecules Homodimers ZE ZE ZE ZE ZE ZE E E E E = = Figure 3-9. Dynamic library of cy clic tripeptidomimetics including cis/trans isomers BuildingBlocks DynamicLibrary =allylesteraminoacid =tetramerscaffold = = Figure 3-10. Dynamic library of cyclic tetrapeptidomimetics

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41 CyclicMolecules AminoAcidDimers = = 6 E/Z isomer each/24total 10 E/Z isomers each/120total 10 E/Z isomers each/60total 8 E/Z isomerseach/ 48total 16 E/Z isomers each/192total 12 E/Z isomers each/144total 2 E/Z isomers each/20dimers total 16 E/Z isomers each/64total 652 E/Z isomersof cyclicmoleculestotal 672isomers Figure 3-11. Dynamic library of cycl ic tetrapeptidomimetics including cis/trans isomers 3.2 Results and Discussion In order to study the viability of olefin metathesis for DCC, we first needed to synthesize the various monomers the cyclic scaffolds and amino acid derivatives. It was then necessary to re act the monomers with Grubbs second generation catalyst 1-4 to study the olefin CM selectivity and reactivity. The genera tion of a small library and template effects were also examined.

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42 3.2.1 Synthesis of Cyclic Scaffolds To obtain the Nallyl dimer scaffold 3-3 NaH was added to a solution of commercially available glycine anhydride ( 3-10 ), tetrabutylammonium iodide (TBAI), and DMF (Scheme 3-2). Excess allyl bromide was added and the reaction was stirred at 70 C to give the desired product in 4 h. However, workup of the reaction proved to be cumbersome. Due to the polarity, some of dimer 3-3 remained in DMF during the aqueous workup even after numerous extractions with organic solvents. The best method we found to obtain good yields was to quench the reaction with H2O, then remove the DMF and water in vacuo with heat. The residue was redissolved in EtOAc, leaving behind the sodium salts which were filtered. Concentration in vacuo and purification by chromatography gave the desire produc t as a white solid in good yields. N N O O N N O O H H Br NaH, DMF, TBAI, 77% 3-10 3-3 Scheme 3-2. Synthesis of dimer scaffold Triallylcyclo -triglycine 3-4 was prepared following literature procedures,49,89 with modifications as described in the experime ntal section (Scheme 3-3). Procedures consisted mainly of hydrolysis and typical amino acid coupling. Reichwein and Liskamp used benzotriazol-1-yloxyt ripyrrolidinophosphonium hexafl uorophosphate (PyBOP) as the coupling reagent.49 We chose N(3-Dimethylaminopropyl)N -ethylcarbodiimide hydrochloride (EDCI) as our coupling reagent simply because it was less expensive and we were able to obtain high yields. In addition to EDCI, 4dimethylaminopyridine (DMAP), diisopropylethylamine (DIPEA, Hnigs base), and hydroxybenzotriazole

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43 (HOBt), were used with the coupling reag ent. All of the above hydrolysis and condensation reactions gave high yields compar able with literature, and typically did not require purification by column chromatography. Trichlorophenol 3-19 was coupled to acid 3-16 to afford compound 3-20 .101 After removing the t -Boc protecting group from ester 3-20 with trifluoroacetic acid (TFA), we attempted to cyclize the compound. The r eaction was run in DMF under very dilute conditions (4 mM) to preferentially form the cyc lic trimer rather than the cyclic hexamer. Closure to form a nine member ring is known to be difficult.29 Hioki et al. reported a 11% yield for the cyclic trimer, with the cyclic hexamer as the major product.89 Upon purification by column chromatography, we isol ated a white solid appearing to be either the trimer or the hexamer, according to 1H NMR and 13C NMR. High resolution mass spectroscopy (HRMS) indicated the presence of trimer 3-4 and an unknown compound. A pure sample of trimer could not be isolated even after numerous recrystallizations in various solvent systems, or by column chromatography. Tetramer scaffold 3-5 was synthesized through simple hydrolysis and condensation starting from acid 3-18 (Scheme 3-4). The t -Boc protecting group of trichloro ester 3-23, was removed by TFA. Using similar procedur es for the cyclization of the trimer, the ester was heated for 24 h in dioxane (4 mM ) and pyridine (1.1 eq). Purification by column chromatography afforded tetramer 3-5 in 26% yield. Identical results were obtained using DMF, but this is a less desirable solvent because of the difficulty of removing DMF during workup.

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44 NH2 Br O O O O H N N O HO N O Boc N O HO THF 60% 3-11 3-123-13 3-14 3-13 ,EDCI,DMAP, HOBt,DIPEA 3-13 ,EDCI,DMAP, HOBt,DIPEA Boc N O N N O O HO N O O O N N O O O Cl Cl Cl EDCI,DMAP,HOBt,DIPEA N N N O O O 3-16 3-18 3-20 3-4 1)TFA,CH2Cl22)Py,DMF,100oC 1)Boc2O,THF 2)NaOH/MeOH Boc N O O N O 3-15 1)NaOH/MeOH 2)HCl Boc N O N N O O O 3-17 1)NaOH/MeOH 2)HCl >90% >90% >90% >90% >90% Boc 58% +UnknownCompoundBoc=O O OH Cl Cl Cl 3-19 Scheme 3-3. Synthesis of trimer scaffold

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45 Boc N O N N O O HO 3-19 ,EDCI,DMAP, HOBt,DIPEA 3-18 1)TFA,CH2Cl22)Py,Dioxane,100oC 51% 3-13 ,EDCI,DMAP, HOBt,DIPEA 87% Boc N O N N O O N O O 1)NaOH/MeOH 2)HCl >90% Boc N O N N O O N O HO Boc N O N N O O N O O Cl Cl Cl N N N N O O O O 3-21 3-22 3-23 3-5 26% Scheme 3-4. Synthesis of tetramer scaffold 3.2.2 Synthesis of Amino Acid Derivatives Different amino acid derivatives were s ynthesized to determine the effects of protecting groups, side chains and alkene moieties in olefin CM. Miller group had examined the effect of remote functiona lity but with allyl and homoallylamides.62 Herein, we investigated the allyl and homoa llyl olefin moiety of the carboxylate end of the amino acid. A series of amino acid derivativ es were synthesized by coupling t -Boc or Fmoc protected amino acids with allyl alcohol or 3-buten-1-ol using DIC, DMAP, and HOBt (Scheme 3-5). The starting material was typically consumed within 20 minutes as indicated on TLC. The urea bypr oduct was filtered and purifi cation of this filtrate by column chromatography gave the amino acid products in high yiel ds (Table 3-1 and Figure 3-12). MeOH was used as the solvent to determine the optical rotation of the samples.

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46 O O R1 HN R O OH HN R DIC,DMAP,DCM HOBt,R1OH PG PG 3-24 ,PG=Boc 3-25 ,PG=Fmoc 3-26 ,PG=Boc 3-27 ,PG=Fmoc Boc= O O O O Fmoc= Scheme 3-5. Synthesis of amino acid derivatives Table 3-1. Yields, melting points and optical rotations of amino acid derivatives Entry N -PG-AA R1 ProductaYield (%) mp (C) [ ]D( ) (C, c ) MeOH 1 Boc-Phe 3-24a Allyl 3-26a >95 71-72 -8.05 (25, c = 1.10) 2 Boc-Ala 3-24b Allyl 3-26b >95 oil -35.0 (25, c = 1.04) 3 Boc-Pro 3-24c Allyl 3-26c 95 oil -70.9 (25, c = 1.00) 4 Boc-Met 3-24d Allyl 3-26d >95 oil -32.4 (25, c = 1.04) 5 Boc-Phe 3-24e Homoallyl 3-26e 90 7980.5 -9.01 (25, c = 1.00) 6 Boc-Ala 3-24f Homoallyl 3-26f 98 oil -45.7 (25, c = 1.11) 7 Boc-Pro 3-24g Homoallyl 3-26g 89 oil -72.3 (25, c = 1.24) 8 Boc-Met 3-24h Homoallyl 3-26h 88 oil -23.8 (25, c = 1.10) 9 Boc-Leu 3-24i Homoallyl 3-26i 86 oil -39.2 (25, c = 1.42) 10 Fmoc-Phe 3-25a Homoallyl 3-27a >95 52-54 -20.0 (25, c = 1.06) 11 Fmoc-Pro 3-25b Homoallyl 3-27b 92 oil -49.4 (25, c = 1.25) 12 Fmoc-Gly 3-25c Homoallyl 3-27c 85 78.580 N/A a Allyl Boc-Phe was first discussed in chapter 2 and referred as compound 2-7 For clarity and simplicity in the numbering of our libraries, the compound will be labeled as 3-26a

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47 H2NCH C CH3 OH O H2NCH C CH2 OH O HN C OH O H2NCH C CH2 OH O CH CH3 CH3 H2NCH C CH2 OH O CH2 S CH3 H2NCH C H OH O Phenylalanine (Phe) Alanine (Ala) Proline (Pro) Methionine (Met) Leucine (Leu) Glycine (Gly) Figure 3-12. Amino acids used in the library. As shown in Table 3-2, HOBt was required to prevent racemizat ion of the chiral center. The 1H NMR and optical rotation data of allyl ester phenylalanine 3-26a102 and alanine 3-26b103 were similar to those reported in literature. Full characterization of the other amino acid derivatives is found in the experimental section of Chapter 6. Table 3-2. Yields, melting points, and optical rotations of amino acid derivatives without use of HOBt during synthesis Entry N -PG-AA R1 ProductYield (%) mp (C) [ ]D( ) (C, c ) MeOH 1 Boc-Phe Allyl 3-26a 87 70-71 -2.31 (25, c = 1.00) 2 Boc-Ala Allyl 3-26b 88 oil -13.6 (25, c = 1.08) 3 Boc-Phe Homoallyl 3-26e 56 78-80 -2.20 (25, c = 1.00) 4 Boc-Ala Homoallyl 3-26f 80 oil -33.4 (25, c = 1.00) 3.2.3 CM Reactivity of Di mer with One Amino Acid The CM reactivity of dimer scaffold 3-3 with amino acid derivatives possessing different protecting groups and olefin moieties was examined. Dimer scaffold 3-3 was

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48 allowed to react with an amino acid deriva tive (5 eq.) using 10 mol% Grubbs second generation catalyst 1-4 (Scheme 3-6). The reaction was stirred at reflux in CHCl3 for 10 h while flushing the headspace with argon to re move evolved ethylene. The reaction was quenched with EVE and purification by co lumn chromatography gave the desired products in moderate yield (Table 3-3). We attempted to isolate compound 3-32 the CM product of one amino acid derivative with dimer scaffold 3-3 by column chromatography. Separation and purification of 3-32 from other byproducts was unsu ccessful, but the mass spectrometer data did indicate th e presence of compound 3-32 The low yields of 3-32 were expected due to excess amount of amino acid deriva tives which would favor the homodimer or heterodimer products. In addition, these CM reactions were run on a small scale. N N O O N N O O O O CHCl3,1-4 3-3 O N H O H N O O HN R O O NH R PG PG R R PG PG O O HN R PG n n nn n nn 3-28 ,PG=Boc 3-29, PG=Fmoc 3-30 ,PG=Boc 3-31 ,PG=Fmoc CyclicCMProductsAminoAcidDimers 3-26 ,PG=Boc 3-27 ,PG=Fmoc + N N O O O O H N R PG n NotIsolated 3-32 Scheme 3-6. Olefin CM of dimer sc affold with an amino acid derivative

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49 Table 3-3. Yields of cyclic and amino acid dimers Entry Starting Material N -PG-AA R1, n Cyclic CM Product s Yielda % Amino Acid Dimer Yield % 1 3-26a Boc-Phe Allyl, n=1 3-28a 37 3-30a 38 2 3-26b Boc-Ala Allyl, n=1 3-28b 40 3-30b 42 3 3-26c Boc-Pro Allyl, n=1 3-28c 42 3-30c 31 4 3-26d Boc-Met Allyl, n=1 3-28d 0 3-30d 0 5 3-26e Boc-Phe Homoallyl, n=2 3-28e 44 3-30e 50 6 3-26f Boc-Ala Homoallyl, n=2 3-28f 39 3-30f 66 7 3-26g Boc-Pro Homoallyl, n=2 3-28g 45 3-30g 55 8 3-26h Boc-Met Homoallyl, n=2 3-28h 0 3-30h 14 9 3-26i Boc-Leu Homoallyl, n=2 3-28i 30 3-30i 59 10 3-27a Fmoc-Phe Homoallyl, n=2 3-29a 42 3-31a 48 11 3-27b Fmoc-Pro Homoallyl, n=2 3-29b 46 3-31b 52 12 3-27c Fmoc-Gly Homoallyl, n=2 3-29c 30 3-31c 44 a Isolated yields except 3-31b which is based on NMR Similarly, the cyclic CM products 3-28 and 3-29 were difficult to purify by column chromatography due to the polarity of the compounds. Based on TLC, these compounds have Rf values similar to dimer 3-3 and compound 3-32. Fortunately, we were able to separate the cyclic CM products from the ot her compounds for characterization. For all of the CM reactions, the amino acid starting ma terial was not fully consumed. The amino acid dimers had Rf values close to the amino acid star ting material, but we were able to obtain pure samples by column chromatography.

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50 The yields for the cyclic CM products 3-28 and 3-29 were comparable, whether an allyl or homoallyl olefin moiety was attached to the amino acid. There was also little difference between the yields of Fmoc protected 3-29a and Boc protected 3-28a and 328e However, the yields improved for the amino acid dimers 3-30 e-h and 3-31 a-b, which possess the homoallyl olefin chain, in comparison to dimerized products 3-31 a-d with the allyl chain. A possible reason for th e higher yield is that the ruthenium catalyst is less sterically hindered by the amino aci d moiety and has bette r access to the longer chain terminal olefin.26 Again we saw little difference between the Fmoc 3-31 a-b and Boc protected 3-30e and 3-30g In all cases, CM of methionine derivatives resulted in zero or low yields, which was a surpri se since Grubbs and Mioskowski groups demonstrated the CM of sulfur c ontaining compounds in good yields.23,104 TLC analysis of the reaction mixtur e showed that mostly starting material 3-26d and dimer 3-3 were present even after 2 days of reflux. The stereochemistry of the cyclic CM products was found to be predominantly trans by 1H NMR of the isolated compounds. We examined the 1H NMR of the crude reaction mixture to determine the cis / trans ratio of amino acid dimers 3-30 and 3-31 The chemical shift of the cis isomers was expected to be further downfield than the trans isomers, but the large number of library cons tituents in the crude reaction mixture made the NMR data rather complex. We therefor e isolated the amino acid dimers by column chromatography. Based on the NMR spectra, most of the isolated compounds appeared to be a pure isomer, rather than a mixture of cis and trans isomers. We expected them to be mostly trans based on Grubbs work.34 However, experiments conducted by Miller lab

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51 showed a 3:1 ratio of cis / trans but using Grubbs firs t generation catalyst 1-3 instead of catalyst 1-4.62 To ensure we properly assigned the cis/ trans ratio of amino acid dimers, we examined the satellites of the alkene protons using a 500 MH z spectrometer with deuterated acetone as the solvent. These weak satellites were formed from protons attached directly to the 13C (1% natural abundance), rather than protons a ttached to the more abundant 12C isotope.105 The satellites were located 80 Hz to the right and left side of the of the stronger proton signal. We expected the alkene protons of the trans isomers to have a larger coupling constant ( J = 15-17 Hz) than the cis isomers ( J = 9-11 Hz).106 We independently synthesized two authentic homodimers, where the stereochemistry was known, to confirm the predicted J coupling values of the weak satellites. We first synthesized the cis allyl homodimer 3-30a by coupling ( Z )-2-butene1,4-diol ( 3-33 ) with N -( tert -butoxycarbonyl)-phenylalanine ( 3-24a 3 eq) using DMAP, HOBt, DIPEA, and EDCI in 95% yield (Scheme 3-7). Homodimer 3-30a was also synthesized using DIC as the coupling agen t. However, removal of the urea byproduct was cumbersome, and the yield was 88%. NMR analysis of the weak satellites of the cis alkene protons indicated a patter n of a doublet of a triplet with J = 11 and 6 Hz (Figure 313). O HO NHBoc Ph EDCI, HOBt, DMAP, DIPEA, DCM 95% O NHBoc Ph OH HO O O O BocHN Ph 3-30a 3-24a 3-33 Scheme 3-7. Synthesis of cis homodimer 3-30a

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52 Figure 3-13. Satellites of the alkene protons from cis homodimer 3-30a We also independently synthesized the trans homoallyl dimer by employing the same method as above, but st arting from the less expensive trans -3-hexenedioic acid 334 rather than diol 3-35 (Scheme 3-8). Acid 3-34 was converted to the diester, which was then reduced to the diol by LiAlH.62 Amino acid coupling with Boc-protected Lphenylalanine 3-24a afforded the trans homodimer product 3-30e NMR analysis of trans 3-30e and the weak satellites of the alkene protons indicated a doublet of a triplet pattern with J = 16 and 7 Hz (Figure 3-14). HO O O OH HO OH 3-34 3-35 O O O NHBoc O BocHN Ph Ph 3-30e EDCI, HOBt, DMAP 1) MeOH, H2SO4 85% 2) LiAlH 65% DIPEA, DCM, 3-24a 54% Scheme 3-8. Synthesis of trans homodimer 3-30e NMR analysis of our homodimer samples from the CM reactions indicated the presence of trans isomers, as determined by the J coupling of the weak satellites since we observed a doublet of a triplet pattern with J = 16 and 7 Hz.

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53 Figure 3-14. Satellites of the alkene protons from trans homodimer 3-30e 3.2.4 Generation of Small Libraries and Template Effects After examining the CM reactivity of the amino acid derivatives with the dimer scaffold, we were interested in generating a small library and examining the template effects. As a preliminary study, dimer 3-3 was allowed to react with two or three different amino acid derivatives (2-3 eq) in a cross-coupling reaction at room temperature or at reflux using 1018 mol% of catalyst 1-4 See Scheme 3-9 for a representative reaction. For this particular example, in which two different amino acids were used, we would expect to see three different amino acid dimers and cyclic CM products, and possibly the mono-coupled compounds (Table 3-4). The resulting equilibrium mixture was frozen upon addition of EVE, which significantly reduced th e activity of the catalyst. The catalyst was removed by washing with water soluble tris(hydroxymethyl)phosphine [THP, P(CH2OH)3]107 or through column chromatography. Isolation of each CM product by column chromatography was not feasible due to the number of possible CM products

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54 as well as any unreactive starting materials. We then analyzed the samples by high performance liquid chromatography (HPLC). Several groups have studied lithium salts and their effect on the conformation of peptides.108,109 Sanders group used hydrazone chem istry to prepare a DCL of cyclic peptidomimetics.60 They observed the formation of one major 42-membered macrocyclic compound upon addition of lithium salts. This was a surprising result since Li+ is a small cation that would not be expect ed to coordinate with a large cyclic compound. In our system, the diketopiperazine CM product had a linear configuration where the amino acid derivatives were separated by the cyclic sca ffold. Because of this configuration, we would not expect a small ion to influence the dynamic equilibrium very much. Nevertheless, we were interested in knowing the effects, if a ny at all, of lithium salts on our small dynamic library. Several reaction c onditions were examined to determine the effects of the lithium ion template (Table 3-5). In experiments 1 and 2, the reaction mi xture was refluxed for 11 h without the addition of a template (Table 3-5). These re actions were compared to experiment 3, in which the dimer, amino acid derivatives, a nd catalyst were refluxed for 3 h followed by the addition of the LiClO4. The reaction mixture was then refluxed for another 8 h. Based on HPLC data, there were no significant changes when the template was added to the reaction. We then examined the influence of a pr oline derivative, which possesses a rigid structure compared to other amino acids. Fo r experiments 3-7, the reaction was refluxed in its respective solvent as listed in Table 3-5. There were some changes to the peak patterns and area % of the HPLC traces of samples with and without LiClO4. However,

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55 there was no major increase in concentrati on of one or two peaks after the addition of LiClO4 to conclude that the template influenced the equilibrium in a significant capacity. O O H N PG O O N H PG R2 N N O O O O H N PG O O N H R2 PG N N O O N N O O O O N PG R1H O O N PG R1H O O O N H O N PG PG H R1R1N N O O O O O N H O N PG PG H O O N PG H O O N PG R2H R1R2 R2 R2 R1 1-4, CH2Cl2orCHCl3 3-3 O O HN R1 PG n 3-26a ,PG=Boc O O HN R2 PG n 3-26b ,PG=Boc and (3eq) (3eq) N N O O O O H N PG N N O O O O N PG H R1R2 3-30a 3-30b 3-31a 3-32a 3-32b 3-30a 3-30b 3-34a Scheme 3-9. CM of dimer scaffold with two amino acid derivatives

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56 Table 3-4. Expected products from CM reactio n of dimer scaffold and two or three amino acid derivatives Possible Productsa Entry Starting Material Scaffold Coupled w/ 2 Amino Acids Scaffold Coupled w/ 1 Amino Acid Amino Acid Dimers 1 Dimer 3-3 Boc-Allyl Phe 3-26a Boc-Allyl Ala 3-26b 3-28a (Phe-D-Phe) 3-28b (Ala-D-Ala) 3-33a (Phe-D-Ala) 3-32a (D-Phe) 3-32b (D-Ala) 3-30a (Phe-Phe) 3-30b (Ala-Ala) 3-34a (Phe-Ala) 2 Dimer 3-3 Boc-Homoallyl Phe 3-26c Boc-Homoallyl Pro 3-26g 3-28e (Phe-D-Phe) 3-28g (Pro-D-Pro) 3-33b (Phe-D-Pro) 3-32c (D-Phe) 3-32d (D-Pro) 3-30e (Phe-Phe) 3-30g (Pro-Pro) 3-34b (Phe-Pro) 3 Dimer 3-3 FmocHomoallyl Phe 3-27a FmocHomoallyl Pro 3-27b FmocHomoallyl Gly 3-27c 3-29a (Phe-D-Phe) 3-29b (Pro-D-Pro) 3-33c (Phe-D-Pro) 3-33d (Phe-D-Gly) 3-33e (Gly-D-Pro) 3-32e (D-Phe) 3-32f (D-Pro) 3-32c (D-Gly) 3-31a (Phe-Phe) 3-31b (Pro-Pro) 3-31c (Gly-Gly) 3-34c (Phe-Pro) 3-34d (Phe-Gly) 3-34e (Pro-Gly) a The dimer scaffold is abbreviated as D. In experiment 8, the system was analy zed using Fmoc amino acid derivatives, which were also easier to obser ve by HPLC because of the ar omatic rings (Table 3-5). Grubbs catalyst 1-4 (13 mol%) was added to a solution of the dimer 3-3 Fmoc-L-Phe 327a Fmoc-L-Pro 3-27b Fmoc Gly 3-27c and CHCl3 at room temperature and stirred for 24 h to ensure equilibration. LiCl (3 eq) was added and the reaction stirred for 1 h, followed by another addition of catalyst 1-4 (7 mol%). After stirring at room temperature for 21 h, additional LiCl (4 eq), catalyst 1-4 (7 mol%), and CHCl3 (0.5 mL) were added. The reaction was maintained for another 23 h. In order to observe th e influence of heat, catalyst 1-4 (4%) and CHCl3 (1 mL) were added and reaction heated at 40 C for 23 h.

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57 Aliquots of the reaction mixture were take n at numerous time points to monitor the changes by HPLC. Similar to previous expe riments, minor changes were observed but there was no dramatic shift in the equilibrium. Table 3-5. CM conditions with and without lithium template Experiment Starting Materi al (equivalent) Solvent (Molarity) Catalyst 1-4 (mol%) Template (equivalent) 1 Dimer 3-3 (1 eq) Boc-Allyl Phe 3-26a (2 eq) Boc-Allyl Ala 3-26b (2 eq) CHCl3 (0.5 M) 10% None 2 Dimer 3-3 (1 eq) Boc-Allyl Phe 3-26a (3 eq) Boc-Allyl Ala 3-26b (3 eq) CHCl3 (0.5 M) 10% None 3 Dimer 3-3 (1 eq) Boc-Allyl Phe 3-26a (3 eq) Boc-Allyl Ala 3-26b (3 eq) CHCl3 (0.5 M) 10% LiClO4 (1 eq) 4 Dimer 3-3 (1 eq) Boc-Allyl Phe 3-26a (2 eq) Boc-Allyl Pro 3-26g (2 eq) CH2Cl2 (0.7 M) 10% LiClO4 (1.4 eq) 5 Dimer 3-3 (1 eq) Boc-Homoallyl Phe 3-26e (2 eq) Boc-Homoallyl Pro 3-26g (2 eq) CHCl3 (0.4 M) 16% None 6 Dimer 3-3 (1 eq) Boc-Homoallyl Phe 3-26e (3 eq) Boc-Homoallyl Pro 3-26g (3 eq) CHCl3 (0.2 M) 13% LiClO4 (1.4 eq) 7 Dimer 3-3 (1 eq) Boc-Homoallyl Phe 3-26e (3 eq) Boc-Homoallyl Pro 3-26g (3 eq) CHCl3 (0.3 M) 16% LiClO4 (1.6 eq) 8 Dimer 3-3 (1 eq) Fmoc-Homoallyl Phe 3-27a (3 eq) Fmoc-Homoallyl Pro 3-27b (3 eq) Fmoc-Homoallyl Gly 3-27c (3 eq) CHCl3 (0.2 M) 18% LiCl (7 eq)

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58 3.3 Conclusions Our main objective in this project was to examine the olefin CM reactivity and selectivity of amino acid derivatives with cy clic scaffolds, and its potential for use in dynamic combinatorial chemistry. Having the olefin moiety further from the amino acid functional groups increased the yields of th e amino acid dimers, but there were little differences in yields for the diketopiperazine products. Altering protecting groups on the amino acids from Boc to Fmoc resulted in l ittle changes to the yields as well. The stereochemistry of the isol ated cyclic and amino aci d dimers was found to be predominantly trans

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59 CHAPTER 4 DYNAMIC COMBINATORIAL LIBRARIES EMPLOYING PEPTIDOMIMETIC DIENES 4.1 Introduction In our continuing studies of cyclic pep tidomimetics, we were interested in generating a dynamic combinatorial library (DCL) 52,54,56,110 of macrocyclic compounds by olefin metathesis. Several groups have reported the use of reversible chemical reactions, such as disulfide exchange,57 metal-ligand coordination,58 exchange of oximes59 and hydrazones,60 formal metathesis111 and olefin metathesis in dynamic combinatorial chemistry (DCC).61-63 However, few have examined olefin metathesis as a means to generate a library of cyclic pep tidomimetics. The chemistry involves amino acid building blocks possessing tw o terminal olefin moieties. Cyclic molecules can be formed from these building blocks by ri ng-closing metathesis (RCM), or by crossmetathesis (CM) followed by RCM. A large number of library constituents, such as cyclic and linear compounds, as well as oligom ers can be generated from this method. Upon addition of a template with specific bi nding properties to the library constituents, the equilibrium can shift to amplify one or two major products in good yields. Sanders group used building block 4-1 consisting of L-prol ine, L-phenylalanine and an aromatic linker which can engage in non-covalent interac tions, hydrogen bonding, Lewis acid-base, pi-pi and cation-pi interactions.54,60,112 Hydrazone formation was used to create these libraries of linear compounds, oligomers, and cyclic molecules. They were able to show amplification of one of his library constituents, trimer 4-2 by adding a

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60 lithium metal ion as a template (Scheme 4-1).54 They employed other metals such as KI, RbI and CsI as templates but did not obser ve the same shift toward the trimer.60 However, under the same hydrazone base d library, Sanders group observed the amplification of the trimer using alkylammonium salts.110,113 We were curious to see if similar resu lts could be obtained using a comparable building block but with olefin metathesis rath er than hydrazone exchange. A trimer made up of dipeptide 4-4 would consist of a 51 membered ring (Scheme 4-1). A 48 membered macrocyclic could also be made by replac ing the homoallyl moiety on building block 4-4 with an allyl ester; however, previous studies in our lab have show n that allyl groups are more sluggish toward metathesis than longer chain olefins. N O N O H N NH2 O R1 R1=CH2Ph R2=CH(OMe)2N O NH O HN N O R1 N O NH O O R1 N O HN O NH N O R1 N HN N O NH O O R1 N O NH O O R1 N O HN O O O R1 O versus 42-membered N O N H O O O R1 BuildingBlock 4-1 4-2 4-34-4 WorkdonebySijbrenOtto, RicardoLEFurlan,and JeremyKMSanders O 51-membered LibraryofDimer, Trimer,Hexamer, etc. Li+R2 Scheme 4-1. Comparison of building blocks 4-1 and 4-4 and their library constituents

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61 The proposed building block 4-4 would consist of phenyl alanine, proline, an aromatic linker, and terminal alkene moieties for olefin CM and/or RCM. The retrosynthesis of dipeptide 4-4 is shown in Scheme 4-2. The molecule can easily be synthesized by the coupling of allyl benzoic acid 4-5 and peptide 4-6 which is made from proline 3-24c and phenylalanine 3-26e N O NH O O O NH O N H O O HO O N H O O N O OH Boc Boc 4-4 4-5 4-6 3-24c 3-26e Scheme 4-2. Retrosynthesis of dipeptide 4-4 The CM and/or RCM reactions of dipeptide 4-4 can give a lib rary of linear compounds, cyclic dimers 4-7 trimers 4-8 hexamers and oligomers (Scheme 4-3). The regioselectivity and stereochemistry of the metathesis reactions can also increase the number and types of library constituents as well. For example, the coupling of two molecules can occur in a head to tail or a tail to tail fashion, to form dimers 4-7a and 4-7b respectively. In addition, CM and RCM reactions can give cis or trans alkenes. Catenanes, interlocked molecular rings, can also be generated by intramolecular RCM mediated syntheses under thermodynamic control.114 Two molecules can become interwined and the two-fold RCM can result in the formation of ri ngs covalently linked (Figure 4-1). Several groups have also demonstrated the template or hydrogen bond directed synthesis of catenanes in good yields using olefin metathesis.115-118

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62 4-4 Head-Tail Head-Head Head-Tail Head-Head Catenanes Dimers 4-7 Trimers 4-8 Tetramers,Hexamers,Oligomers,LinearCompounds 4-7a 4-7b 4-8a4-8b N O N H O O O ( E/E E / Z Z / Z) ( E / E / E,E / Z / E Z / E / Z Z / Z / Z ) Scheme 4-3. Library of dimers, tetramers, hexamers, oligomers, linear compounds and catenanes 2 Figure 4-1. Formation of [2]catenane

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63 4.2 Results and Discussion In order to study olefin CM and RCM in DC L of cyclic peptidomimetics, we first needed to synthesize dipeptide 4-4. The molecule can be synthesized by the coupling of 4-allyl benzoic acid (4-5) with peptide 4-6. Allylbenzoic acid 4-5 was synthesized following literature procedures,119,120 starting from commercia lly available 4-iodobenzoic acid (4-9) (Scheme 4-4). The synthesis involves esterification of acid 4-9, followed by the conversion of the aryl iodide to the Gri gnard reagent. Treatment of the magnesium compound with allyl bromide and Cu CNLiCl afforded benzoate 4-11, which was then hydrolyzed to give 4-allyl benzoic acid (4-5) in excellent yield. 121 O HO I O O I O HO O O 4-5 4-9 4-10 4-11 SOCl22) CuCN 2LiCl, allyl bromide NaOH EtOH 98% 94% 89% 1) iPrMgCl Scheme 4-4. Synthesis of 4-allylbenzoic acid (4-5) Dipeptide 4-4 was synthesized by a series of N-(3-Dimethylaminopropyl)-N ethylcarbodiimide hydrochloride (EDCI) amino acid coupling reactions (Scheme 4-5). Boc-homoallyl ester phenylalanine 3-26e was first deprotected with TFA, and then coupled with Boc-L-proline 3-24c to afford compound 4-13. The Boc protecting group was removed with TFA once again, followe d by another EDCI coupling reaction with allylbenzoic acid 4-5 to produce dipeptide 4-4 in good yield.

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64 O O H N O O N O H N O O O O N O NH O O O TFA,DCM 4-13 3-26e 4-4 TFA,DCM EDCI,HOBt, DIPEA, 3-24c 87% O O NH2 HN O H N O O EDCI,HOBt, DIPEA, 4-5 73% 4-6 4-12 Scheme 4-5. Synthesis of dipeptide 4-4 Upon the synthesis of dipeptide 4-4, a series of CM experiments were conducted in various conditions (Table 4-1) to generate a library of peptidomimetics. We were interested in seeing the major compone nts of the reacti on when dipeptide 4-4 was allowed to react with catalyst 1-4, with and without a template. A representative procedure for the CM reactions listed in Tabl e 4-1 (experiment 12) is as follows: under an atmosphere of argon, a solution of LiI (1 eq) and THF (2 mL) wa s added to a solution of dipeptide 4-4 and CH2Cl2 (20 mL). After stirring for 30 minutes, a solution of Grubbs catalyst 1-4 and CH2Cl2 was added drop-wise and the reaction refluxed for 13 h. Upon cooling, the reaction was quenched with EVE. The work-up included an aqueous extraction with tris(hydroxymethyl)phosphine (THP) to remove the ruthenium catalyst.107

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65 Table 4-1. Series of CM reactions Experiment Solvent (mM) Catalyst 1-4 mol% Template (equivalent) Conditions 1 CH2Cl2 (5) 5 None Monitored reaction for 4 days at r.t. 2 CH2Cl2 (36) 5 None Monitored reaction for 4 days at r.t. 3 CH2Cl2 (5) 5 None 15 h at reflux 4 CH2Cl2 (5) 5 None 15 h at reflux 5 CH2Cl2 (5) 5 None 19 h at reflux 6 CHCl3 (5) 5 None 19 h at reflux 7 CHCl3 (5) 5 None 15 h at reflux 8 THF (5) 5 None Monitored reaction for 3 days at r.t. 9 THF (36) 5 None Monitored reaction for 3days at r.t. 10 CH2Cl2 (5) 5 LiClO4 (1.5) Monitored reaction for 4 days at r.t. 11 CH2Cl2 (36) 5 LiClO4 (1.5) Monitored reaction for 4 days at r.t. 12 CH2Cl2 (5) 5 LiI (1 eq) 13 h at reflux 13 CH2Cl2 (5) 5 LiI (1 eq) 15 h at reflux 14 CH2Cl2 (5) 5 LiI (1 eq) 15 h at reflux 15 CH2Cl2 (5) 5 LiI (1 eq) 15 h at reflux 16 CH2Cl2 (5) 5 LiI (1 eq) 15 h at reflux 17 CHCl3 (5) 5 LiI (1 eq) 13 h at reflux 18 CHCl3 (5) 5 LiI (1 eq) 15 h at reflux The samples were analyzed on a Shimadzu high performance liquid chromatography (HPLC) using a reverse phase column. The HPLC conditions consisted of an isocratic (65% acetonitr ile, 35% water) flow rate of 0.5 mL/min and UV detection

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66 at 254 nm. HPLC spectra of selected experi ments can be found in appendix A. To ensure accurate comparison of the HPLC tr aces, aliquots of the reaction mixture from each experiment were co-injected into the HP LC. The peaks were also labeled for ease of comparison and do not necessarily re present known compounds, unless otherwise indicated. For experiments 1-2, the reaction mixtur e was stirred at room temperature in CH2Cl2 for 4 days (Table 4-1). There were only minor changes in the HPLC trace whether the concentration was 5 mM or 36 mM After four days the major component was the starting material. We also examined THF as a solvent because it was later used to dissolve the templates and we wanted to make sure THF was not a contributor to the equilibrium shift. Similarly to experime nts 1-2, starting material was the major component when THF was employed both at concentrations of 5 mM and 36 mM (experiments 8 and 9, respectively). We then studied the effects of LiClO4 and LiI as templates in various conditions. As shown in Figure 4-2, there was a decrease in peak F and an increase in peak J when LiClO4 was employed and the reaction was maintain ed at room temperature (Table 4-1, experiments 1 and 10). However, the star ting material (peak D) was predominantly present in the reaction mixture. No major sh ift in the equilibrium was detected between experiments 2 and 11, when the reaction wa s maintained at a higher concentration (Figure 4-3). Minimal changes to the equilibrium when LiClO4 was employed may be due to reaction conditions at room temperature, rather than lack of template effects. The minor product formation was most likely due to the entropy of activation required to

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67 bring two terminal alkene moieties together for CM or ring closure.29 Therefore, we next conducted experiments where heat was employed. A B Figure 4-2. Comparison of expe riments 1 and 10. A) HPLC spectrum of experiment 1 reaction mixture (CH2Cl2, 5 mM, room temperature, 4 days, no template). B) HPLC spectrum of experiment 10 reaction mixture (CH2Cl2, 5 mM, room temperature 4 days, LiClO4)

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68 A B Figure 4-3. Comparison of expe riments 2 and 11. A) HPLC spectrum of experiment 2 reaction mixture (CH2Cl2, 36 mM, room temperature, 4 days, no template). B) HPLC spectrum of experi ment 11 reaction mixture (CH2Cl2, 36 mM, room temperature 4 days, LiClO4) In experiments 3-5, a solution of dipeptide 4-4, Grubbs catalyst 1-4, and CH2Cl2 was refluxed for 15 h and for 19 h (Table 4-1). There were negligible differences in the HPLC traces between the two reaction times However, we observed new products compared to reactions maintained at room te mperature. There was more of a dramatic change in the HPLC trace when LiI was used as a template and the reaction was refluxed in CH2Cl2 (5 mM) (Figure 4-4). We observed a large decrease of the starting material

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69 (peak D), along with an increase in some of th e larger molecules (peaks G, H, and I). LiI was chosen as the template rather than LiClO4 for these set of e xperiments to better replicate Sanders work. The CM reactions, with and without lithium, were repeated several times as shown in Table 4-1, and for all cases, similar results were obtained. HPLC samples from the olefin metathesis reactions were also co-injected with the starting material to ensure we ha d properly labeled the peaks. A B Figure 4-4. Comparison of expe riments 4 and 14. A) HPLC spectrum of experiment 4 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, no template). B) HPLC spectrum of experiment 14 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, LiI)

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70 We next examined conditions in which a solution of dipeptide 4-4, Grubbs catalyst 1-4, and CHCl3 was refluxed for 13 h and 15 h without the addition of a template (Table 4-1, experiments 6-7). In contrast to experiments conduc ted in refluxing CH2Cl2, there was little amount of starting material present after 13 h or 15 h (Figure 4-5). A B Figure 4-5. Comparison of expe riments 7 and 17. A) HPLC spectrum of experiment 7 reaction mixture (CHCl3, 5 mM, reflux 15 hours, no template). B) HPLC spectrum of experiment 17 reaction mixture (CHCl3, 5 mM, reflux 13 hours, LiI)

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71 We then explored the effects of LiI as a template. As shown by the HPLC traces in Figure 4-5, there was little change to the starting material dipeptide 4-4. However, we did observe a decrease in peak E and an increase in peaks B and F. A series of reactions were also mon itored by HPLC where the dipeptide was allowed to react with catalyst 1-4 for a period of several hours to 24 hours at room temperature to allow equilibration. Afterw ards, different lithium templates (LiI or LiClO4) were added and the reaction maintained for another 24 h. We continued to monitor the reaction after refluxing for another 24 h. For some experiments, additional 5 mol% of the catalyst was introduced every 24 h to ensure the catalyst was not degraded. However, we did not observe any significant ch anges in the HPLC data when the lithium template was added before the catalyst to a llow pre-coordination of the amino acids with the metal. Even though we did not see a significant sh ift in the equilibrium before and after the addition of the template as was seen in Sanders work, we felt it was of interest to identify the major peaks. Attempts were made to isolate the major products by column chromatography. We were able to re cover the starting material, dipeptide 4-4. However, obtaining pure CM products was more difficu lt than anticipated. We did isolate a compound that appeared to be a dimer ma de up of two molecules of dipeptide 4-4 based on high resolution mass spectrometry (HRMS) and 2D NMR [(acetone d6), COSY (Correlated Spectroscopy), HMBC (Heteronuc lear Multiple Bond Correlation), HMQC (Heteronuclear Multiple Quantum Correla tion), NOESY (Nuclear Overhauser and Exchange Spectroscopy)]. The 1H and 13C spectra were obtained on an Inova spectrometer operating at 500 MHz for proton and 100 MHz for carbon. Two

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72 dimensional NMR experiments were conducted on the same spectrometer, and samples were dissolved in deuterated acetone. The HPLC trace of the isolated compound is shown in Figure 4-6. HRMS data of the isolated compound reve aled three possible structures as shown in Figure 4-6. The dimer of dipeptide 4-4 can be formed by the CM of two molecules in a head to head or head to tail fashion, followed by a RCM to give 4-7a and 4-7b, respectively. In addition, the HRMS data also corresponded with the structure of [2]catenane 4-14, in which two rings of dipeptide 4-4 are interlocked within each other (Figure 4-7). Figure 4-6. HPLC spectrum of isol ated compound from experiment 3

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73 N O N H O O O N O H N O O O N O NH O O O N O HN O O O 4-7a 4-7b N O NH O O O N O HN O O O [2]Catenane HeadtoHead HeadtoTail Theoreticalm/z:C52H56N4O8[M]+=865.4171 Found:C52H56N4O8[M]+=865.4174 4-14 Figure 4-7. Structures of dimers 4-7a, 4-7b, and catenane 4-14 The NMR chemical shifts and coupling va lues of the isolated compound are shown in Figure 4-8 and Table 4-2. NMR data prov ed that two dipeptide building blocks were attached in a head to tail fashion because th e alkene protons had two different chemical shifts (5.04 and 5.84 ppm), which ruled out structure 4-7b. If the building blocks were linked in a head to head fashion, then the chemical shifts would be identical.

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74 N O NH O O O (128.2) g q (32.4) (63.5) j/k (169.8) (171.3) e (169.1) (55.2) i o/p (37.3) c (129.8) b (128.4) d (126.7) (138.1) (62.1) h r/t (32.2) s (22.9) (47.4) l (142.6) (37.9) m/n (132.7) f a b Numbersinparenthesisreferto13Cchemicalshifts Figure 4-8. 13C NMR chemical shifts and the corresponding protons of isolated compound To determine if the isolated product was structure 4-7a or catenane 4-14, we examined the NOESY spectrum. Molecular mo dels of the most st able conformation of catenane 4-14 were generated using the HyperChe m program (HyperCube Inc., Version 7). The most likely conformation of catenane 4-14 is shown in Figure 4-9, where the aromatic ring of one molecule is perpendicula r to the alkene of the second ring. Because of the shielding effects of the aromatic ring on the olefinic protons, we would expect the chemical shifts to be approximately 2-3 ppm further upfield.106 However, we did not observe the shielding effects on the olef inic protons. Therefore, catenane 4-14 was ruled out based on the models, leaving the most likely structure to be dimer 4-7a (Figure 4-10). The large J value (15 Hz) also indicated the presence of a trans alkene.

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75 Table 4-2. Proton chemical shifts and J values Proton Chemical Shift (ppm) Spin Coupling (J value in Hz)a a 7.39 m b 7.26 m c 7.22 m d 7.18 m e 6.62 d (7.2) f 5.84 d,t,t (15.3, 6.0, 1.1) g 5.04 dddt (14.3, 7.9, 6.2, 1.6) h 4.55 dd (8.3, 3.5) i 4.28 q (7.3) j 4.27 m k 3.82 m l 3.62 m m 3.38 dd (15.1, 6.6) n 3.22 dd (14.5, 6.2) o 3.02 dd (14.1, 7.6) p 2.66 dd (13.7, 7.7) q 2.25 m r 2.18 m s 1.84 m t 1.76 m a doublet (d), multiplet (m), triplet (t), q (quartet) In addition to dimer 4-7a, we also attempted to isolate some other major CM products by column chromatography. We obtained a compound that corresponded to peak X of the HPLC trace shown in Figure 4-11. HRMS of the compound showed the presence of a dimer made up of two dipeptide 4-4 molecules. However, 1H NMR of the isolated compound appeared oligomeric and 2D NMR confirmed the presence of terminal alkenes. Therefore, we c ould only conclude that there was a mixture of CM products corresponding to peak X.

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76 Figure 4-9. Model of catenane 4-14 Figure 4-10. Model of dimer 4-7a

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77 Figure 4-11. HPLC spectrum of experiment 3 reaction mixture (CH2Cl2, 5mM, reflux 15 hours, no template) 4.3 Conclusions A series of CM reactions were conducted to generate a library of peptidomimetics. The building block utilized wa s a dipepetide molecule cons isting of a phenylalanine, proline, aromatic linker, and two terminal olefin moieties. Lithium templates were introduced into the system to determine the influence on the equilibrium. There was no dramatic shift in the equilibrium where one major product was formed as demonstrated by Sanders group. However, the largest change occurred in our system when the reaction mixture was refluxed in CH2Cl2 (5 mM) for 15 h and LiI was used as the template. We have isolated and characterized dimer 4-7a, which was formed by the CM and RCM of dipeptide 4-4 in a head to tail arrangement with the trans alkene favored. The research group is continuing the proj ect to isolate and purify other peptide molecules.

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78 CHAPTER 5 MODEL STUDY MOLECULAR IMPRINTING OF NERVE GASES 5.1 Introduction Molecular imprinted polymers (MIPs) are useful mimics of biological receptors, enzymes and antibodies.71,72 In addition, their applica tion in chemical sensors is imperative due to recent national concerns over chemical warfare. This current threat involves the use of chemical ag ents that can incapacitate or kill those who are exposed by affecting the functions of the body, such as the nervous system, lungs, blood, or skin. 122 Chemical weapons were first used during World War I by the Germans. During an attack against the Allied troops, the release of chlorine gas resulted in an estimated 5,000 dead and 10,000 disabled.123 Chemical weapons are not just a concern for the military, but also civilians. In 1981, th e Iraqis attacked a Kurdish town with mustard gas, a blister agent that can cause nausea, vomiting, pain, and death.123 Due to recent terrorist attacks on U.S. soil, the ability to detect these deadly agents quickly and accurately has been a priority for national defense. Our group is interested in using molecu lar imprinted technology as a sensor for chemical agents such as the nerve gases, a family of organophos phates that interfere with the nervous system and can cau se injury or death (Figure 5-1).122,123 In a healthy body, a neurotransmitter called acetylcholine is released from the presynaptic neuron and crosses over the synaptic cleft (Figure 52). Upon attachment to a receptor on the postsynaptic neuron, a nerve signal is transmitted.124 Acetylcholine is then released from the receptor, and the enzyme acetylcholines terase hydrolyzes the neurotransmitter to

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79 prevent build up of the chemical in the syna pse and over firing of the nerve. Nerve agents are toxic because they have the abil ity to phosphorylate acetylcholinesterase; thus inactivating the enzyme and causing an accumulation of acetylcholine, which continuously binds to the receptors.122 This essentially means the nerve is constantly in the on position and can lead to a variety of effects, such as vomiting, coma, paralysis of the muscles and respiratory arrest.123 Of the four chemical agents shown below in Figure 5-1, VX (5-1) is the most toxic.122 N S P O O P O F O P O F O P O F N O VX Sarin Soman Tabun 5-1 5-4 5-3 5-2 Figure 5-1. Structures of common nerve agents A Ch Acetylcholine SynapticCleft PresynapticNeuron PostsynapticNeuron AcetylcholineReceptor A Ch A Ch Acetate Choline Acetylcholinesterase Figure 5-2. Mechanism of acetylcholine in the transmission of nerve impulses

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80 Due to VX toxicity, we have chosen 2-(diisopropylamino)ethanethiol ( 5-5 ) as the template. This thiol is a degradati on product of VX and less hazardous to handle.125 Thiol 5-5 can be synthesized from thiourea a nd 2-(diisopropylamino)ethyl chloride following literature procedures.126 N SH 5-5 N S P O O P O O HO 5-6 5-1 Scheme 5-1. Degradation produc ts of VX, following hydrolysis Development of our proposed MIP involves the Michael addition of thiol template 5-5 to the fluorescein maleimide 5-7 which serves as the detector (Scheme 5-2). Fluorescein maleimide is a widely used fluorescent green dye selective toward thiols.127 Compound 5-8 is then coupled to 5norbornene-2-carboxylic acid ( 5-9 mixture of endo and exo), which is required for polymerization. Carboxylic acid 5-9 serves as the functional monomer and is allowed to form hydrogen-bonded complexes with 5-10 Ring Opening Metathesis Polymerization (ROMP) with the complex and cross-linking monomer ( 5-11 ) using Grubbs second generation catalyst 1-4 results in the rigid network (Schemes 5-2 and 5-3). The mechanism of the ROMP with the monomers and dye is shown in Scheme 5-4. Removal of the temp late and the regeneration of the maleimide leave the desired imprint. In other words, a molecular memory is introduced in the MIP, in which the recognition sites possess the corr ect shape and orienta tion of the functional groups to make it selective for thiol template 5-5

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81 O OH O O HO N O O N SH Template 5-5 5-7 O OH O O O N O O O S N O OH O O HO N O O S N FunctionalMonomers O OH O O O N O O O N S O O O O H H O O H O O H 5-9 O O O O Cross-LinkingMonomer Grubbs'2ndGenerationCatalyst 1-4 5-11 Hydrogen-bondedComplexes ROMP 5-8 5-10 O O H 5-9 Cross-linkedpolymer(SeeScheme5-3) Scheme 5-2. Formation of hydrogen-bonded complexes

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82 O OH O O O N O O O N S O O O O H H O O H O O H O OH O O O N O O O O O O O H H O O H O O HRemove Rebind Cross-Linked Polymer Scheme 5-3. Formation of the MIP O O O O X O MLn R 2+2 X O MLn X O MLn R X O R MLn Repeatwith 5-9 or 5-11 4 O O O O 45-11 Cross-LinkedPolym e 5-9 :X=OH 5-10 :X=Fluoresceinylthiolether 2+2 R Scheme 5-4. Mechanism of the ROMP polymerization

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83 Fluorescein maleimide 5-7 was chosen as the dye because it has been extensively used as a thiol reactive dye.127-129 It possesses many useful photophysical properties and is very reactive and sele ctive toward thiol groups.127,130 It absorbs light of mean wavelength 427 nm and emits vi sible green light at 515 nm.127 One research group demonstrated the increase in fluorescence emission when glutathionine, a tripeptide containing a thiol group, was allowed to react with fluorescein maleimide.131 We plan on examining the reaction of our thiol substrates with the dye and determining the effects on the fluorescence emission. Fluorescein maleim ide was also selected as the dye because the norbornene moiety, which is required for ROMP, can be coupled to either one or two of the alcohol groups in one step without m odification to the dye (Scheme 5-5). With only one norbornene molecule attached, th e fluorescein molecule can exist in two tautomeric forms. Cross-linking monomers 5-11 have been made previous ly in our group, starting from commercially available 5-norbornene-2-carboxaldehyde ( 5-16 ).75 The aldehyde can be reduced to alcohol 5-17 which can then react with adipoyl chloride ( 5-18 ) to form diester 5-11 (Scheme 5-6). Ratios of the template/monomer/cross-linking agent will be tested to determine the best polymerization conditions. Binding properties of the MIP will be evaluated using gas chromatography and mass spectrometry methods to ensure whether it is specific to only thiol 5-5.

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84 O O O HO N O O O OH O O O N O O O OH O OH O O HO N O O O O O O O N O O O O O OH MixtureofEndoandExo MixtureofEndoandExo 1eq O O O OH O N O O O Lactone 5-12 5-13 5-14 5-7 5-15 5-9 5-9 O OH Scheme 5-5. Coupling of norbornene moiety to fluorescein maleimide

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85 O O O O Cross-LinkingMonomer 5-11 O H OH NaBH4MeOH Pyridine,CHCl3O Cl O Cl 5-16 45-17 5-18 87% 98% Scheme 5-6. Synthesis of cross-linking monomer 5-11 5.2 Results and Discussion 5.2.1 Synthesis of N-(5-fluoresceinyl)maleimide 5-7 N-(5-fluoresceinyl)maleimide ( 5-7 ) is commercially available, but also very expensive. Therefore, we d ecided to synthesize the maleim ide based on a combination of literature procedures (Schemes 5-7 and 5-8).130,132-135 The synthesis involves 5 major steps: 1) Condensation of commer cially available resorcinol ( 5-19 ) and 4-nitrophthalic acid ( 5-20 ) to form 5,6-nitrofluorescein 5-21 ; 2) Acetylation and crys tallization to isolate isomers 5-23 and 5-24 ; 3) Saponification of isomer 5-23 to give desired 5nitrofluorescein ( 5-25) ; 4) Reduction of 5-25 with sodium sulfide nonahydrate (Na2S9H2O) and sodium hydrosulfide (NaH S) to produce amino hydrochloride 5-26 ; and 5) Reaction of aminofluorescein 5-27 with maleic anhydride 5-28 in acetic acid to give maleic acid 5-29 and cyclization to yi eld fluorescein maleimide 5-7

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86 O O O OH HO N O O OH HO O OH O HO N O O 1)190-200oC 94% 5-20 5-19 5-21 (CH3CO)2O O O O O O N O O O O Recryst. O O O O O N O O O O O O O O O N O O O O 5-22 5-24 :12% 5-23 :24% 1)NaOH/MeOH 2)HCl 97% O O O OH HO N O O 5-25 O O O OH HO N H H H 1)Na2Sx9H2O,NaHS 46% 5-26 pyridine 2)0.6 N HCl 2)CH3CO2H,6%HCl Scheme 5-7. Synthesis of fluorescein amino hydrochloride 5-26

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87 5-26 O O O OH HO N H H O O O OH HO N H O O OH O O O OH HO N O O NaOH, CH3CO2H 5-7 5-27 5-29 O O O CH3CO2H 85% 1)ZnCl2,HMDS,DMF 2)HCl 91% 67% 5-28 O OH O O HO N O O 5-14 Scheme 5-8. Synthesis of N-(5-fluoresceinyl)maleimide ( 5-7 ) Crude nitrofluorescein 5-21 was obtained in good yield by simply heating resorcinol ( 5-19 ) and 4-nitrophthalic acid ( 5-20 ) at very high temperatures. However, to isolate pure diacetate isomers 5-23 and 5-24 several recrystallizations were required which resulted in low yields. Diacetate 5-23 was converted to 5-nitrofluorescein 5-25 through saponification with NaOH/Me OH. Based on work by McKinney et al.133 and Steinbach,135 Na2S9H2O and NaHS were used to reduce 5-25 to aminofluorescein 5-27, rather than hydrogen and Raney nickel. Ou r attempts to isolat e the aminofluorescein 527 over several recrystallizations resu lted in lower than desired yields.

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88 At this point, it was determined to be more time and cost efficient to purchase aminofluorescein 5-27 from Aldrich, and follow procedures from Toru group130 to obtain maleimide 5-7 If large quantities of aminofluorescein 5-27 are required than it would be more cost beneficial to start from compounds 5-19 and 5-20 Maleimide 5-7 was easily synthesized in good yields by reacting aminofluorescein 5-27 in glacial acetic acid and maleic anhydride 5-28 followed by cyclization by use of the Lewis acid Zn2Cl and hexamethyldisilazane (HMDS). 5.2.2 Synthesis of Compounds 5-13 and 5-15 To ensure we could couple the norbornene moiety to the fluoresceinyl dye, we investigated the synthesis of compound 5-13 Carboxylic acid 5-9 was treated with fluorescein maleimide 5-7 in the presence of dicycl ohexylcarbodiimide (DCC) and dimethylamino pyridine (DMAP) Even after filtration and column chromatography, it was difficult to remove the byproduct, dicycl ohexylurea. Similar results were obtained using diisopropylcarbodiimide (D IC). Therefore, we deci ded to use oxalyl chloride (Scheme 5-9). Carboxylic acid 5-9 reacted with oxalyl chlori de and DMF (catalytic) to form the acid chloride which was then dissolved in CH2Cl2. The acid chloride solution was added drop-wise to a solution of maleimide 5-14 CH2Cl2, and NEt3 at 0 C. Dilute conditions (0.03 M) were used to pr event over acylation. Mono-acylated 5-13 was isolated in 22% yield and the di-acylated 5-15 in 6% yield.

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89 O O O OH O N O O O O O O O O N O O O O 5-13: 22% 5-15: 6% 1)(COCl)2,DMF(cat) 2) 5-14 ,NEt3 O OH Scheme 5-9. Synthesis of compounds 5-13 and 5-15 5.2.3 Model Study We selected phenyl maleimide 5-30 as a model to test the reactivity of the Michael addition of benzenethiol ( 5-31 ) and the elimination of the thio l derivative to regenerate maleimide 5-7 Following literature procedures,136 phenylthiol succinimide 5-32 was synthesized in high yields (Scheme 5-10). N O O S N O O benzene,90% 5-30 5-32 5-31 SH Scheme 5-10. Synthesis of succinimide 5-32

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90 We then tested the reactivity of phenyl maleimide 5-30 with dimethylaminoethanethiol salt 5-33 which is similar to our target molecule 5-5 The thiol salt 5-33 was deprotonated with Na2CO3, and the addition to the double bond of the maleimide gave thioether 5-34 in 87% yield (Scheme 5-11). N O O S N 2)NEt3,benzene, 5-30 87% 5-34 HS N Cl H 5-33 1)Na2CO3 Scheme 5-11. Synthesis of succinimide 5-34 The ability to regenerate phenyl maleimide 5-30 after the nucleophilic addition of the thiol was an essential part in the design of the MIP. Since Mich ael-type additions are reversible by heating, we attempted to reform the phenyl maleimide by this method.29 Succinimides 5-32 and 5-34 were refluxed in CHCl3 (b.p. = 62 C) or toluene (b.p. = 111 C) for 36 h and monitored by TLC to obtain phenyl maleimide 5-30 Even with toluenes high boiling point, refluxing only prov ided the starting material succinimides. Bases can also be employed with heating to favor the reversible Michael addition.29 We then refluxed succinimides 5-32 and 5-34 in THF, CHCl3, and toluene with base (triethylamine and t-butoxide) to obtain phenyl maleimide 5-30 but no reaction was observed. Even though Michael additions are known to be reversible, thiol anions are very good nucelophiles and poor leaving groups, therefore favoring the succinimides.29 The thiol needed to be converted to a mo re reactive leaving group. For example, sulfonate esters, such as mes ilates, tosylates, a nd triflates, are excel lent leaving groups

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91 because the resulting sulfona te ion is a weak base.29 We envisioned the oxidation of 5-32 to obtain sulfoxide 5-35 Treatment with heat woul d eliminate the sulfoxide and regenerate the maleimide. Util izing typical oxi dation procedures,137 a solution of mchloroperbenzoic acid (MCPBA), succinimide 5-32 and CHCl3 were stirred at room temperature for 1 h to obtain sulfoxide 5-35 (Scheme 5-12). The compound was refluxed in toluene for 1 h to gi ve the elimination product 5-30 in high yield. For the proposed MIP system, MCPBA could pot entially oxidize the fluoresce in molecule or form epoxides from the alkenes of the polymer b ackbone. The solution was to hydrogenate the polymer double bonds prior to oxidation. The MIP system would be made by a series of experiments in this order: 1) add the thio l to the fluoresceinyl ma leimide; 2) couple the dye with the norbornene moiety to allo w ROMP; 3) hydrogenate the olefins on the polymer backbone to prevent epoxidation; a nd 4) oxidize and elim inate the thiol to regenerate the maleimide. N O O S N O O MCPBA,CHCl387% 5-32 5-30 toluene, N O O S O 5-35 Scheme 5-12. Oxidation and elimination reactions A quick test was performed to ensure hydrogenation condition s would not reduce the aromatic rings on the fluoresceinyl dye One of the most common methods for hydrogenation of olefins involves the use of H2 and palladium on activated carbon (Pd/C) as the catalyst. Palladium is usually preferred as the cata lyst for the reduction of double

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92 bonds in molecules possessing several functional groups, such as esters.138 Based on previous studies in our lab, hydrogenation of olefins can usually proceed in less than one hour at atmospheric pressure.90 We wanted to employ th ese reaction conditions to fluorescein maleimide 5-7 as a model, where we expect to observe the hydrogenation of the maleimide but not the re duction of the double bonds of th e aromatic rings (Scheme 513). For the proposed MIP, hydrogenation of th e maleimide would not be an issue since thiol 5-5 would have been added to the double bond of the maleimide at this point. Pd/C was introduced to a solu tion of fluorescein maleimide 5-7 and ethanol. After evacuating the headspace, hydrogen gas was adm itted via a balloon that was attached to the reaction flask by a three way stopcock, and stirred for a total of 45 min. Observation of the reaction by thin layer chromatogra phy (TLC) indicated the formation of a new product with small amounts of starting material present. Hydrogenation for another 30 min revealed no major changes by TLC. After removal of the solvent and the catalyst, NMR analysis of the crude product indicated selective hydrogenation of the maleimide alkene but the aromatic rings of the molecule were not affected. H2,EtOH 5-75-36 Pd/C O OH O O HO N O O O OH O O HO N O O Scheme 5-13: Hydrogenation of fluorescein maleimide 5-7 We then examined the aff ects of MCPBA on fluorescein 5-37 which is commercially available and less expensive than fluorescein maleimide 5-7 (Scheme 5-

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93 14). Unlike succinimide 5-32 which is soluble in CHCl3, fluorescein 5-36 is a very polar compound. We conducted the oxidation experime nts in EtOH or in an aqueous solution of NaHCO3 (0.3 N) based on literature procedures.139 The reactions were monitored by TLC, and after 1.5 h, we observed mostly st arting material presen t and no decomposition of the fluorescein 5-37 O O O HO OH MCPBA NoChangesObservedby TLC 5-37 Scheme 5-14: Attempted oxidation of fluorescein 5-37 5.3 Conclusions To examine the viability of developing a nerve gas detector using ROMP in molecular imprinting technology, several model studies were conducted. We first investigated the Michael addi tion of thiol compounds to the maleimide. Then several reaction conditions were tested to eliminate th e thiol and regenerate the maleimide. The oxidation/elimination reaction us ing MCPBA and heat appeared to be most reasonable, although hydrogenation of the polym er will be required to elim inate side reactions. We were able to demonstrate the synthesis of fluorescein maleimide and the coupling with 5norbornene-2-carboxylic acid, which is re quired for the ROMP. Several more experiments are required, such as analyzing the fluorescent pr operties of fluorescein type molecules, before we can synthesize the MIP. However, based on preliminary studies, it seems promising that a MIP could be ge nerated to detect thiol compounds using fluorogenic maleimide.

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94 CHAPTER 6 EXPERIMENTALS 6.1 General Method and Instrumentation All moisture and air-sensitive reactions were performed under an atmosphere of argon with flame-dried glassware. Solvents were distilled under N2 from appropriate drying agents according to established procedures. Analytical Thin Layer Chromatography (TLC) was performed on Kiesel gel 20 F-254 pre-coated 0.25mm silica gel plates. UV light, phosphom olybdic acid in ethanol, anisaldehyde in ethanol, permanganate, and vanillin were used as i ndicators. Column flash chromatography was carried out using silica gel 60 (40-60 m). Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Gemini VXR, or Mercury 300 megahertz (MHz) spectrometer. Carbon nucl ear magnetic resonance (13C NMR) spectra were recorded at 75 MHz on the same spectrometers. Two di mensional (2D) NMR experiments were conducted on an Inova 500 MHz spectrometer. Chemical shifts were reported in ppm downfield relative to tetramethylsilane (TMS ) as an internal standard. The coupling constant (J) were reported in hertz (Hz). The following abbreviations were used: s, singlet; d, doublet; t, triplet; m, multiplet. Infrared spectra were recorded using a Bruker Vector 22 IR and reported in wavenumbers (cm-1). High-resolution mass spectroscopy (HRMS) was performed by the Mass Spectrosc opy Laboratory at the University of Florida. Electron ionizati on (EI) was carried out on a Fi nnigan MAT 95Q hybrid-sector mass spectrometer at 70 eV using a direct in sertion probe. Chemi cal Ionization (CI) was carried out at 150 eV using a direct insertion probe in the presence of methane.

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95 Electrospray ionization (ESI) was performed on a Finnigan LC Q Quadrupole Ion Trap. Elemental analyses were performed by the Elem ental Analysis Service at University of Florida, Department of Chemistry. Hi gh Performance Liquid Ch romatography (HPLC) analyses were performed on a Shimadzu LC-6AD. Yields reported re fer to the isolated materials unless otherwise indicated. Melting points were determined with a Barnstead or Thomas Hoover melting point ap paratus and are uncorrected. 6.2 Experimental Procedures and Data N-Allyl-2-bromo-acetamide ( 2-9 ) N H O Br Following procedures similar to litera ture, a flame dried 250 mL round bottom flask was charged with bromoacetyl bromide 2-14 (8.0 mL, 61 mmol) and freshly distilled CH2Cl2 (80 mL) and cooled in an ice bath to 0 C. To the stirred solution was added a solution of allyl amine 2-13 (12 mL, 160 mmol) and CH2Cl2 (40 mL) drop-wise and the mixture was stirred at 0 C for 4 h. Distilled H2O was added and the organic layer was extracted and washed with 1 N HCl, followed by H2O. After drying (anhydrous Na2SO4), concentration gave crude 2-9 as a yellow-orange oil. Recrystallization from hexane and ethyl ether (9:1) at 0 C overnight yielded 2-9 (8.0 g, 73%) as analytically pure white crystals, m.p. = 27 C. 2-9: Rf = 0.39 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 6.62 (s, 1H), 5.85 (ddt, J = 17.2, 10.2, 5.3 Hz, 1H), 5.23 (dq, J = 17.1, 1.5 Hz, 1H), 5.19 (dq, J = 10.0, 1.5 Hz, 1H), 3.96-3.90 (m, 4H); 13C NMR (CDCl3) 165.86, 133.33, 116.82, 42.48, 29.12; IR (neat) 3285, 3082, 1654, 1552, 1430, 1309, 1211, 1132, 990, 925 cm-1; HRMS (CI pos) for C5H9BrNO [M+H]+: calcd 177.9868, found 177.9862.

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96 8-Allyloxy-octan-1-ol (2-10) HO O A flame dried flask flushed with argon was charged with sodium hydride (60% mass) (1.1 g, 28 mmol). The gray powder was washed three times with pentane to remove the protective oil. To the NaH was added 1,8-octanediol (2-11) (3.55 g, 24.3 mmol) dissolved in THF (25 mL). Tetrabutylammonium iodide (TBAI) (0.0251 g, 0.0680 mmol) was added and the mixture stirred for 40 min. A solution of allyl bromide (1.9 mL, 22 mmol) and THF (25 mL) was a dded drop-wise to th e reaction flask and heated at reflux for 12 h. The reaction mi xture was cooled to room temperature, neutralized with saturated ammonium chloride and extracted 3x with EtOAc. The combined organic layers were washed with brine, dried with sodium sulfate (Na2SO4), and concentrated under reduced pressu re. The crude product was purified by chromatography on silica gel with CH2Cl2/MeOH (100:0-98:2) to give colorless oil 2-10 (2.9 g, 70%) and double Williamson etherification product 2-12 (0.62 g, 25%). 2-10: Rf = 0.23 (CH2Cl2/MeOH, 96:4); 1H NMR (CDCl3) 5.91 (ddt, J = 17.1, 10.2, 5.6 Hz, 1H), 5.26 (dq, J = 17.2, 1.6 Hz, 1H), 5.16 (dq, J = 10.7, 1.4 Hz, 1H), 3.95 (dt, J = 5.6, 1.3 Hz, 2H), 3.61 (t, J = 6.5 Hz 2H), 3.41 (t, J = 6.7 Hz, 2H), 1.66 (s, 1H), 1.60-1.49 (m, 4H), 1.31 (s, 8H); 13C NMR (CDCl3) 135.20, 116.93, 71.97, 70.63, 63.10, 32.91, 29.88, 29.60, 29.52, 26.26, 25.84; IR (neat) 3385 (br), 2931, 2856, 1647, 1463, 1348, 1101 cm-1; HRMS (CI pos) for C11H23O2 [M+H]+: calcd 187.1698, found 187.1697. 2-12: Rf = 0.74 (CH2Cl2/MeOH, 96:4); 1H NMR (CDCl3); 5.90 (ddt, J = 17.2, 10.3, 5.65 Hz, 1H), 5.89 (ddt, J = 17.2, 10.3, 5.65, 1H), 5.25 (dq, J = 17.1, 1.7 Hz 1H), 5.24 (dq, J = 17.1, 1.8, 1H), 5.15 (dq, J = 10.4, 1.5 Hz, 1H), 5.14 (dq, J = 10.4, 1.5 Hz, 1H), 3.94 (dt, J = 5.6, 1.5, 2H), 3.93 (dt, J = 5.4, 1.5 Hz, 2H), 3.40 (t, J = 6.7 Hz, 2H), 3.39 (t, J

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97 = 6.7 Hz, 2H), 1.61-1.50 (m, 4H), 1.30 (s, 8H); 13C NMR (CDCl3) 135.20, 116.81, 71.93, 70.59, 29.88, 29.56, 26.26; IR (neat) 3080, 2932, 2856, 1647, 1457, 1420, 1400, 1347, 1264, 1106 cm-1; HRMS (CI pos) for C14H27O2 [M+H]+: calcd 227.2011, found 227.2015. N-allyl-2-(8-allyloxy-oc tyloxy)-acetamide (2-15) H N O O O A flame dried 25 mL round bottom flask under argon was charged with alcohol 210 (2.97 g, 16.0 mmol) and THF (9.5 mL). NaH (60% mass, 0.64 g, 16 mmol) was added portion wise slowly into the stirred so lution and the mixture continued to stir for 20 min. TBAI (0.0259 mg, 0.0701 mmol) and allyl bromo acetamide 2-9 (2.20 g, 12.3 mmol) were added and the mixture was heated at reflux for 4 h. The mixture was diluted with CH2Cl2 and washed with brine. The orga nic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduce d pressure. The crude oil was purified by chromatography on silica gel with he xane/EtOAc (100:0-80:20) to give 2-15 (1.9 g, 54%) as a clear oil. 2-15: Rf = 0.53 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 6.63 (s, 1H), 5.87 (ddt, J = 17.2, 10.7, 5.4 Hz, 1H), 5.82 (ddt, J = 17.0, 10.6, 5.3 Hz, 1H), 5.23 (dq, J = 17.2, 1.6 Hz, 2H), 5.12 (dq, J = 10.5, 1.6 Hz, 2H), 3.94-3.86 (m, 6H), 3.46 (t, J = 6.6 Hz, 2H), 3.38 (t, J = 6.7 Hz, 2H), 1.61-1.49 (m, 4H), 1.28 (s, 8H); 13C NMR (CDCl3) 169.99, 135.31, 134.24, 116.93, 116.61, 72.12, 72.04, 70.67, 70.42, 41.34, 29.98, 29.71, 29.63, 29.56, 26.36, 26.22; IR (neat) 3426, 3335, 3081, 2932, 2856, 1738, 1676, 1526, 1447, 1432, 1342, 1277, 1111, 997, 921 cm-1; HRMS (CI pos) for C16H30O3N [M+H]+: calcd 284.2226, found 284.2225.

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98 6.2.1 General Procedures for RCM Trans-saturated lactam 2-16 and Cis-saturated lactam 2-17 O HN O O O N H O O To a stirred solution of diene 2-15 (377 mg, 1.33 mmol) and CH2Cl2 (750 mL) was added a few crystals of butylated hydroxyt oluene (BHT). A solution of Grubbs 2nd generation catalyst 1-4 (108 mg, 0.127 mmol) in CH2Cl2 (100 mL) was added and heated at reflux for 16 h. The reaction was cooled to room temperature, quenched with ethyl vinyl ether (EVE, ca. 3 mL), and maintained for 1 h. The solution was concentrated under reduced pressure and the residu e chromatographed on silica gel with hexane/EtOAc (9:1-7:3) to give the E isomer 2-16 (200 mg, 58%) and Z isomer 2-17 (8.2 mg, 2.4%) as a brown oil. 2-16: Rf = 0.35 (EtOAc/hexane, 4:1); 1H NMR (CDCl3) 6.59 (s, 1H), 5.80 (dd, J = 14.8, 4.4 Hz, 1H), 5.72 (dd, J = 14.8, 4.3 Hz, 1H), 3.99-3.89 (m, 6H), 3.53 (t, J = 5.7 Hz, 2H), 3.46 (t, J = 5.7 Hz, 2H), 1.65-1.29 (m, 12H); 13C NMR (CDCl3) 169.95, 130.11, 127.92, 71.92, 70.25, 69.91, 69.33, 39.95, 29.18, 28.91, 28.39, 28.10, 26.08, 25.27; IR (neat) 3419, 2929, 2856, 1683, 1525, 1460, 1340, 1263, 1111 cm-1; HRMS (CI pos) for C14H26NO3 [M+H]+: calcd 256.1913, found 256.1911. 2-17: Rf = 0.43 (EtOAc/hexane, 4:1); 1H NMR (CDCl3) 6.65 (s, 1H), 5.89 (dd, J = 10.6, 6.3 Hz, 1H), 5.81 (dd, J = 10.3, 7.2 Hz, 1H), 4.03-3.90 (m, 6H), 3.55-3.45 (m, 4H), 1.70-1.23 (m, 12H); 13C NMR (CDCl3) 169.95, 130.30, 130.01, 71.25, 70.67,

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99 70.38, 65.63, 35.42, 29.25, 28.45, 27.27, 27.16, 24.91, 24.61; HRMS (ESI-FTICR) for [2M+Na]+: calcd 533.3561, found 533.3580. Unsaturated Lactam 2-18 O HN O O Palladium on activated carbon (10% Pd ) (58 mg, 0.055 mmol) was added to a solution of 2-16 (507 mg, 1.99 mmol) and EtOAc (9 mL). The headspace was evacuated using a three way stop cock attached to a water aspirator. Hydrogen was admitted via a balloon also attached to the stop cock. Th e reaction mixture was stirred for 45 min and the catalyst removed by filtering through a sm all pipette column of Celite. The column was rinsed with EtOAc (3 x 5 mL) and the combined fractions concentrated under reduced pressure leaving crude 2-18 (500 mg, 98%) as a yellow-brown oil. The hydrogenated product was used in the next step without further purification. 2-18: Rf = 0.32 (EtOAc/hexane, 4:1); 1H NMR (CDCl3) 6.63 (s, 1H), 3.83 (s, 2H), 3.43-3.21 (m, 8H), 1.66-1.21 (m, 16H); 13C NMR (CDCl3) 170.00, 71.61, 70.23, 70.12, 38.05, 28.97, 28.85, 27.54, 27.38, 26.70, 26.61, 25.13, 24.70; IR (neat) 3420, 2932, 2857, 1681, 1530, 1446, 1340, 1261, 1120 cm-1; HRMS (CI pos) for C14H28NO3 [M+H]+: calcd 258.2069, found 258.2073. Lactam 2-8

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100 O N O O To a stirred solution of 2-18 (570 mg, 2.22 mmol) in THF (2.2 mL) under argon was added NaH (60% mass, 270 mg, 6.7 mmol) slowly in portions. The mixture was stirred for 30 min. TBAI (8.18 mg, 0.0222 mm ol) and allyl bromide (0.97 mL, 11 mmol) were added and the reaction was heated at reflux for 9 h. The reaction mixture was cooled to room temperature, neutralized with saturated ammonium chloride, and extracted with EtOAc. The organic layer wa s washed with saturated NaCl, dried with MgSO4, and concentrated under reduced pressu re. Purification by chromatography on silica gel with hexane/EtOAc (4:1) afforded 2-8 (0.41 g, 62%) as a slightly yellow oil. 2-8: Rf = 0.37 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 5.84-5.68 (m, 1H), 5.185.07 (m, 2H), 4.17-3.93 (m, 4H), 3.57-3.22 (m, 8H); 1.79-1.21 (m, 16H); 13C NMR (CDCl3) 169.89-169.26 (2 lines), 133.4-133.39 (2 lin es), 117.23-116.29 (2 lines), 71.67, 71.45, 71.41, 70.83, 70.21, 70.15, 70.03, 70.00, 48.97, 47.95, 47.32, 44.95, 29.51, 28.71, 28.67, 28.47, 28.03, 27.53, 27.35, 27.11, 26.71, 26.58, 25.87, 25.71, 25.08, 24.26, 24.22; IR (neat) 2931, 2858, 1651, 1459, 1352, 1282, 1232, 1118, 1038, 995, 922 cm-1; HRMS (CI pos) for C17H32 NO3 [M+H]+: calcd 298.2382, found 298.2391. Anal. calcd for C17H31NO3: C, 68.65; H, 10.51; N, 4.71. Found: C, 68.31; H, 10.73; N, 4.94% 6.2.2 General Procedures for Amino Acid Coupling To a cooled (0 C) solution of amino acid (7.7 mmol) and CH2Cl2 (13 mL) was added 1,3-diisopropylcarbodiimide (DIC) ( 15 mmol), 4-(dimethylamino)pyridine (DMAP) (1.5 mmol), and hydroxybenzotriazole (HOBt) (8.0 mmol). After stirring the

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101 mixture for 5 min, the alcohol (12 mmol) was slowly added. The mixture was allowed to warm to room temperature while stirring for a total of 3 h. All solids were filtered and the filtrate was concentrated under reduced pressure. Th e crude product was purified on a silica gel column, eluting with hexane:EtOAc to provide the amino acid derivative. t-Boc-allyl ester of phenylalanine 2-7 O O HN Boc Following amino acid coupling procedures, t-Boc-L-phenylalanine (2.04 g, 7.69 mmol), CH2Cl2 (13 mL), DIC (2.4 mL, 15 mmol ), DMAP (0.186 g, 1.52 mmol), HOBt (1.08 g, 7.99 mmol), and allyl alcoho l (0.85 mL, 12 mmol) afforded 2-7 (2.2 g, 92%) as a white solid, m.p. 71-72 C. 2-7: Rf = 0.35 (hexane/EtOAc, 4:1); [ ]25 D = -8.05 (c = 1.10 MeOH); 1H NMR (CDCl3) 7.08-7.32 (m, 5H), 5.84 (ddt, J = 17.2, 10.4, 5.2 Hz, 1H), 5.28 (dq, J = 17.1, 1.4 Hz, 1H), 5.22 (dq, J = 10.3, 1.3 Hz, 1H), 4.98 (d, J = 7.9 Hz, 1H), 4.63-4.54 (m, 3H), 3.11 (dd, J = 13.8, 6.3 Hz, 1H), 3.04 (dd, J = 13.8, 6.5 Hz, 1H), 1.40 (s, 9H); 13C NMR (CDCl3) 171.63, 155.14, 136.07, 131.59, 129.42, 128.58, 127.06, 118.94, 79.91, 65.97, 54.57, 38.39, 28.39; IR (neat) 3362, 3088, 2971, 1705, 1509, 1455, 1368, 1169, 1053; HRMS (CI pos) for C17H24NO4 [M+H]+: calcd 306.1705, found 306.1703. Anal. calcd for C17H23NO4: C, 66.86; H, 7.59; N, 4.59. Found: C, 66.65, H, 7.78; N, 4.52%. Spectral data are in agreement with literature.102 Lit102 [ ]29 D = -10.2 (c = 1.10 MeOH). CM product 2-6

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102 O O HN Boc O N O O To a stirred solution of 2-8 (140 mg, 0.469 mmol), amino acid 2-7 (290 mg, 0.950 mmol), and CHCl3 (0.50 mL), was added a solution of catalyst 1-4 (19.9 mg, 0.0234 mmol) and CHCl3 (0.40 mL). The solution was heated at reflux for 21 h while flushing the headspace with argon to remove evolved ethylene. The reaction was allowed to cool to room temperature and quenched with EVE (ca. 0.75 mL). The solution was stirred for 30 min and concentrated under reduced pressu re. Purification by chromatography with hexane/EtOAc (90:10-65:35) afforded CM product 2-6 (130 mg, 48%) as a colorless oil and homodimer 2-19 (120 mg, 45%) as a white solid, m.p. = 148-150 C. Starting materials 2-8 (40 mg, 29%) and 2-7 (71 mg, 25%) were recovered as well. 2-6: Rf = 0.26 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 7.09-7.32 (m, 5H), 5.795.53 (m, 2H), 4.99 (d, J = 8.5 Hz, 1H), 4.62-4.53 (m, 3H), 4.19-3.94 (m, 4H), 3.59-3.22 (m, 8H); 3.11 (dd, J = 13.4, 6.3 Hz, 1H), 3.03 (dd, J = 13.4, 6.5 Hz, 1H); 1.81-1.14 (m, 25H); 13C NMR (CDCl3) 171.75, 169.78, 169.38, 155.19, 136.07, 130.67, 130.50, 129.47, 129.43, 128.66, 127.15, 126.16, 125.36, 80.02, 71.80-70.04 (7 lines), 65.20-64.82 (2 lines), 54.55, 47.72-44.97 (4 lines), 38.46, 29.54-24.26 (13 lines); IR (neat) 3439, 2933, 2860, 2247, 1712, 1640, 1497, 1456, 1367, 1254, 1168, 1114, 910, 733 cm-1; [ ]25 D = +8.7 (c = 1.00, CHCl3); HRMS (CI pos) for C32H51N2O7 [M+H]+: calcd 575.3696, found 575.3680. Anal. calcd for C32H50N2O7: C, 66.87; H, 8.77; N, 4.87. Found: C, 66.56; H, 9.00; N, 4.70%.

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103 Homodimer t-Boc-allyl ester phenylalanine 2-19 O O N Boc H O O N Boc H 2-19: Rf = 0.67 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 7.34-7.09 (m, 10H), 5.765.64 (m, 2H), 4.99 (d, J = 7.9 Hz, 2H), 4.65-4.54 (m, 6H), 3.10 (dd, J = 13.6, 5.8 Hz, 2H), 3.03 (dd, J = 13.6, 5.8 Hz, 2H), 1.41 (s, 18H); 13C NMR (CDCl3) 171.76, 155.25, 136.07, 129.53, 128.76, 128.06, 127.26, 80.18, 64.70, 54.64, 38.54, 28.47; IR (neat) 3367, 2977, 1715, 1498, 1455, 1367, 1252, 1166, 1054, 1022 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 605.2833, found 605.2859. Anal. calcd for C32H42N2O8: C, 65.96; H, 7.27; N, 4.81. Found: C, 66.16, H, 7.53; N, 4.77%. 6.2.3 General Procedures for Ethylenolysis of 2-6 A flame dried 5 mL round bottom flask e quipped with a reflux condenser capped with a three-way stopcock was charged with 2-6 (110 mg, 0.191 mmol) and CH2Cl2 (95 l). To the stirred solution was added a solution of catalyst 1-4 (3.5 mg, 0.0041 mmol) in CH2Cl2 (95 l). The headspace was evacuated us ing a water aspirato r attached to the stopcock. Ethylene gas, used without pur ification, was admitted via a balloon also attached to the stopcock. The solution wa s heated at reflux for 2.5 h, cooled to room temperature, and quenched with EVE (ca. 0.5 mL). Concentration by reduced pressure and purification of the residue by chromat ography on silica gel with hexane/EtOAc (10:0-8:2) afforded 2-8 (20 mg) and 2-7 (22 mg) as a colorless oil. Starting material 2-1

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104 (30 mg) and amino acid dimer 2-19 (2.2 mg) were isolated as well. All spectral and TLC data were identical to data for 2-7, 2-6, 2-8, and 2-19 previously isolated. N-allyl dimer scaffold 3-3 N N O O NaH (60% mass, 4.0 g, 100 mmol) was adde d in portions to a stirred solution of glycine anhydride (3-10) (3.14 g, 27.5 mmol) and a nhydrous DMF (55 mL). After stirring for an additional 15 minutes, TBAI (1.60 g, 4.33 mmol) and allyl bromide (12 mL, 140 mmol) were added. The reaction mixt ure was maintained at room temperature for 3.5 h, then quenched with H2O. DMF and H2O were removed in vacuo, leaving behind an orange semi-solid. EtOAc was added to the residue and the sodium salts filtered. The filtrate was concentrated in vacuo. Column chromatography on silica gel with EtOAc/hexane (7:3) afforded 3-3 (4.1 g, 77%) as a white solid, m.p. = 97.5-99.0 C. 3-3: Rf = 0.24 (EtOAc); 1H NMR (CDCl3) 5.82-5.63 (ddt, J = 17.1, 10.2, 6.3 Hz, 2H), 5.29-5.17 (m, 4H), 4.02-3.97 (m, 8H); 13C NMR (CDCl3) 163.36, 130.94, 119.80, 49.27, 48.32; IR (KBr) 3079, 2914, 1656, 1487, 1441, 1415, 1336, 1294, 1193, 1142, 1074, 1011 cm-1; HRMS (EI pos) for C10H14N2O2 [M]+: calcd 194.1055, found 194.1047. Anal. calcd for C10H14N2O2: C, 61.84; H, 7.27; N, 14.42. Found C, 61.75; H, 7.42; N, 14.29%. H-(N-allyl)Gly-OEt 3-13 O O H N Following a procedure re ported in literature,49 ethyl bromoacetate (3-12) (11 mL, 99 mmol) in THF (160 mL) was added drop-wise to a cooled solution of allyl amine (311) (16 mL, 210 mmol) in THF (260 mL). The so lution was maintained at 0 C for 2 h,

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105 concentrated in vacuo, and suspended in Et2O. The suspension was filtered through a coarse filtration frit and washed with Et2O. The filtrate was concentrated in vacuo. Column chromatography on silica gel with Et2O afforded ester 3-13 (8.6 g, 60%) as an oil. 3-13: Rf = 0.29 (Et2O); 1H NMR (CDCl3) 5.83 (ddt, J = 17.3, 10.4, 6.0 Hz, 1H), 5.16 (dd, J = 17.4, 1.5 Hz, 1H), 5.08 (dd, J = 10.4, 1.3 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 3.36 (s, 2H), 3.23 (d, J = 6.1, 2H), 1.75 (s, 1H), 1.24 (t, J = 7.2, 3H); 13C NMR (CDCl3) 172.58, 136.21, 116.70, 60.85, 51.94, 50.08, 14.34. All spectral data are in agreement with literature.49 t-Boc-(N-allyl)Gly-OH 3-14 Boc N O HO Boc2O (2.33 g, 10.7 mmol) was added to a stirred solution of ester 3-13 (1.38 g, 9.65 mmol) in THF (40 mL). The solution was maintained for 5 h. The solvent was removed in vacuo to give the t-Bo c-protected ester as an oil; Rf = 0.44 (Et2O/hexane, 2:3). The oil was redissolved in MeOH (12 mL), followed by the addition of 4 N NaOH (3 mL). After stirring for 3 h, MeOH was removed in vacuo. Water (60 mL) was added and the aqueous layer washed with Et2O (2 x 50 mL). The aqueous layer was acidified with 1 N HCl (12 mL) and extracted with EtOAc (3 x 70 mL). The combined organic layers were washed with brine, dried with Na2SO4, and concentrated in vacuo to give tBoc-protected N-allyl glycine 3-14 (2.1 g, quantitative yiel d) as a colorless oil. Compound 3-14 was used in the next step wi thout further purification. 3-14: 1H NMR (CDCl3) 10.80 (s, 1H), 5.84-5.65 (m, 1H), 5.21-5.03 (m, 2H), 4.02-3.84 (m, 4H), 1.43 and 1.40 (s, 9H); 13C NMR (CDCl3) 175.09, 174.98, 155.92,

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106 155.33, 133.48, 133.30, 117.95, 117.19, 80.94, 50.87, 50.31, 47.71, 47.55, 28.32, 28.26 [2 rotamers detected]. All spectral data are in agreement with literature.49 6.2.4 General Procedures for EDCI Coupling t-Boc-(N-allyl)Gly-(N-allyl)Gly-OEt 3-15 Boc N O O N O A flame dried flask under argon was charged with acid 3-14 (4.26 g, 19.8 mmol), EDCI (5.70 g, 29.7 mmol), HOBt (4.02 g, 29.8 mmol), DMAP (187 mg, 1.53 mmol), DIPEA (10 mL, 57 mmol), and CH2Cl2 (157 mL). Ester 3-13 (2.70 g, 18.9 mmol), was added to the stirred solution and maintain ed for 18 h. The solvent was removed in vacuo, and the residue redissolved in EtOAc (290 mL ). The organic layer was washed with 1 N KHSO4 (3 x 150 mL), 1 N NaHCO3 (3 x 150 mL), water (150 mL), and brine (150 mL). The organic layer was then dried with Na2SO4 and concentrated in vacuo to give 3-15 (6.4 g, quantitative yield) as a viscous colorless oil. The oil was used in the next reaction without further purification. 3-15: Rf = 0.39 (hexane/EtOAc, 1:1). 1H NMR (CDCl3) 5.85-5.63 (m, 2H), 5.265.02 (m, 4H), 4.21-3.82 (m, 10H), 1.41 (s, 9H), 1. 23 (t, J = 6.8 Hz, 3H). Spectral data are in agreement with literature.49 6.2.5 General Procedures for Hydrolysis t-Boc-(N-allyl)Gly-(N-allyl)Gly-OH 3-16

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107 N O HO N O Boc NaOH (4 N, 8.6 mL) was added to a stirred solution of ester 3-15 (6.38 g, 18.7 mmol), and MeOH (23 mL). After 3 h, MeOH was removed in vacuo. Water (100 mL) was added and the aqueous layer washed with Et2O (2 x 90 mL). The aqueous layer was acidified with 1 N HCl (35 mL) and extracted with EtOAc (3 x 110 mL). The combined organic layers were washed with brine, dried with Na2SO4, and concentrated in vacuo to give 3-16 (5.8 g, 98%) as a viscous slightly yellow oil. Acid 3-16 was used in the next step without further purification. 3-16: 1H NMR (CDCl3) 5.85-5.64 (m, 2H), 5.22-5.08 (m, 4H), 4.10-3.85(m, 8H), 1.44 and 1.43 (s, 9H). t-Boc-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-OEt 3-17 Boc N O N N O O O Following general EDCI coupling procedures, acid 3-16 (1.19 g, 3.82 mmol), ester 3-13 (502 mg, 3.50 mmol), EDCI (1.00 g, 5.22 mmol), HOBt (713 mg, 5.28 mmol), DMAP (40 mg, 0.327 mmol), DIPEA (2.0 mL, 11.5 mmol), and CH2Cl2 (29 mL) yielded ester 3-17 (1.52 g, quantitative yield) as a viscous slightly yellow oil. The oil was used in the next step without fu rther purification. 3-17: Rf = 0.33 (hexane/EtOAc, 1:2); 1H NMR (CDCl3) 5.82.55 (m, 3H), 5.244.98 (m, 6H), 4.21-3.78 (m, 14H), 1.36 and 1.34 (s, 9H), 1.18 (t, J = 7.0 Hz, 3H); 13C

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108 NMR (CDCl3) 169.27-168.67 (4 lines), 155.67-155.41 (2 lines), 133.93-131.96 (7 lines), 118.50-116.43 (5 lines), 79.94-79.88 (2 lines), 61.67-61.16 (3 lines), 50.84-49.61 (5 lines), 48.30-46.35 (7 lin es), 28.29, 14.12; IR (neat) 3082, 2980, 2934, 1747, 1675, 1463, 1367, 1250, 1194 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 460.2418, found 460.2414. t-Boc-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-OEt 3-18 Boc N O N N O O HO Following general hydrolysis procedures, ester 3-17 (6.38 g, 18.7 mmol), 4 N NaOH (8.7 mL), MeOH (23 mL), and 1 N HCl (35 mL) yielded 3-18 (5.8 g, quantitative yield) as a viscous slightly yellow oil. Acid 3-18 was used in the next step without further purification. 3-18: 1H NMR (CDCl3) 10.56 (s, 1H), 5.89-5.57 (m, 3H), 5.31-4.97 (m, 6H), 4.30-3.68 (m, 12H), 1.39 and 1.37 (s, 9H); 13C NMR (CDCl3) 171.56-168.80 (8 lines), 156.14-155.57 (3 lines), 133.77-131.87 (7 lines ), 118.67-116.75 (5 lines), 80.60-80.48 (2 lines), 50.86-49.82 (6 lines), 47.96-46.74 (4 lines), 28.36; IR (neat) 3083, 2980, 2934 (region broader than ester 3-17), 1738, 1672, 1477, 1407, 1367, 1250, 1173 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 432.2105, found 432.2120. t-Boc protected 2,4,5-trichlorophenol ester 3-20 N O O O N N O O O Cl Cl Cl

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109 Following general EDCI coupling procedures, acid 4-14 (4.71g, 11.5 mmol), 2,4,5trichlorophenol (3-19) (2.06 g, 10.5 mmol), EDCI (3.06 g, 16.0 mmol), HOBt (2.12 g, 15.7 mmol), DMAP (0.130 g, 1.06 mmo l), DIPEA (5.5 mL, 32 mmol), and CH2Cl2 (87 mL) yielded ester 3-20 (3.9 g, 58%) as a white solid, m.p. = 81.5-82.5 C. 3-20: Rf = 0.34 (Et2O/hexane, 9:1); 1H NMR (CDCl3) 7.54 (s, 1H), 7.33 (s, 1H), 5.95-5.64 (m, 3H), 5.38-5.01 (m, 6H), 4.52-3.84 (m, 12H), 1.44 and 1.42 (s, 9H); 13C NMR (CDCl3) 169.62-169.02 (5 lines), 166.75, 155.96-155.64 (2 lines), 134.16-131.07 (8 lines), 126.10-125.42 (3 lines), 119.52116.73 (6 lines), 80.30, 51.34-49.89 (4 lines), 48.20-46.58 (7 lines); IR (KBr) 3073, 3007, 2978, 2932, 1792, 1698, 1659, 1460, 1408, 1348, 1310, 1249, 1222, 1176, 1122, 1080 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 610.1249; found 610.1290. Anal. calcd for C26H32N3O6Cl3: C, 53.03; H, 5.48; N, 7.14. Found: C, 53.42, H, 5.49; N, 7.06%. t-Boc-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-OEt 3-21 Boc N O N N O O N O O Following general EDCI coupling procedures, acid 3-18 (8.60 g, 21.0 mmol), ester 3-13 (2.73 g, 19.1 mmol), EDCI (5.15 g, 26. 9 mmol), HOBt (3.63 g, 26.9 mmol), DMAP (235 mg, 1.92 mmol), DIPE A (10 mL, 57 mmol), and CH2Cl2 (160 mL) gave a viscous slightly yellow oil. Purification by column chromatography on silica gel with EtOAc/hexane (5:5 to 7:3) yielded ester 3-21 (9.06 g, 87%) as a colorless oil. 3-21: Rf = 0.27 (EtOAc/hexane, 7:3); 1H NMR (CDCl3) 5.83-5.58 (m, 4H), 5.254.97 (m, 8H), 4.22-3.80 (m, 18H), 1.37 and 1.35 (s, 9 H), 1.24-1.16 (m, 3H); 13C NMR

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110 (CDCl3) 169.04, 168.52, 168.15, 155.65, 133.92-132.11 (5 lines), 118.58-116.38 (7 lines), 79.93, 61.75-61.21 (2 lines), 50.8248.24 (7 lines), 47.60-46 .50 (4 lines), 28.33, 14.17; IR (KBr) 3083, 2981, 1747, 1669, 1467, 1366, 1197, 1026 cm-1; HRMS for C27H43N4O7 [M]+: calcd 535.3132, found 535.3115. t-Boc-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-(N-allyl)Gly-OH 3-22 Boc N O N N O O N O HO Following general hydrolysis procedures, ester 3-21 (2.48 g, 4.64 mmol), 4 N NaOH (1.4 mL), MeOH (5.6 mL), and 1 N HCl (5.6 mL) yielded 3-22 (2.0 g, 85%) as a white foam. Acid 3-22 was used in the next step w ithout further purification. 3-22: 1H NMR (CDCl3) 11.00 (s, 1H), 5.82-5.58 (m, 4H), 5.23-4.97 (m, 8H), 4.26-3.70 (m, 16H), 1.36 and 1.35 (s, 9H); 13C NMR (CDCl3) 170.75, 169.63, 168.95, 168.68, 155.80, 155.43, 133.66-131.80 (6 lines), 118.50, 117.92, 117.23, 116.64, 80.30, 50.65, 50.34, 49.87, 47.12, 46.76, 28.29. t-Boc protected 2,4,5-trichlorophenol ester 3-23 Boc N O N N O O N O O Cl Cl Cl Following general EDCI coupling procedures, acid 3-22 (1.80 g, 3.55mmol), 2,4,5trichlorophenol (3-19) (0.636 g, mmol), EDCI (0.845 g, 4.41 mmol), HOBt (0.577 g, 4.27 mmol), DMAP (0.060 g, 0.49 mmol), DIPEA (1.7 mL, 9.7 mmol), and CH2Cl2 (30 mL)

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111 gave a viscous slightly yellow oil. Purifi cation by column chromatography on silica gel with Et2O/hexane (8:2 to 10:0) yielded 3-22 (1.2 g, 51%) as a white semi-solid. 3-22: Rf = 0.21 (Et2O); 1H NMR (CDCl3) 7.48-7.23 (m, 2H), 5.85-5.55 (m, 4H), 5.25-4.4.95 (m, 8H), 4.35-3.80 (m 16H), 1.38 (s, 9H); 13C NMR (CDCl3) 169.34, 168.97, 168.78, 166.54, 155.80, 145.46, 133.98-130.43 (9 lines), 125.97, 125.35, 119.48116.52 (6 lines), 80.15, 51.21-50.05 (4 lines), 47.67-46.71 (3 lines ), 28.45; IR (neat) 3085, 2979, 2931, 1784, 1673, 1460 1405, 1366, 1350, 1222, 1171, 1122, 1082 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 707.1778, found 707.1762. Tetramer scaffold 3-5 N N N N O O O O A solution of trifluoroacetic acid (4 mL) and CH2Cl2 (10 mL) was added drop-wise to a stirred solution of ester 3-23 (816 mg, 1.19 mmol) and CH2Cl2 (10 mL). After the solution was stirred for 45 min, the solvent and excess TFA were removed in vacuo, followed by neutralization with NaHCO3, and extraction with EtOAc. The organic layer was washed with brine and dried (Na2SO4) then concentrated to give a viscous oil. The oil was resuspended in anhydrous dioxane (3 00 mL) and pyridine (0.1 mL, 1.2 mmol), and the solution was heated at 100 C for 24 h. Concentration in vacuo and purification by column chromatography with EtOAc/hexane (5:5 to 9:1) afforded tetramer 3-5 (120 mg, 26%) as a white solid.

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112 3-5: Rf = 0.23 (EtOAc); 1H NMR (CDCl3) 5.81-5.67 (m, 4H), 5.31-5.22 (m, 8H), 4.04-3.96 (m, 16H); 13C NMR (CDCl3) 163.38, 130.94, 119.88, 49.34, 48.41; IR (KBr) 3294, 3081, 2932, 1658, 1487, 1415, 1338, 1297, 1195, 1162, 1128, 1074, 1011 cm-1; HRMS (CI pos) for C20H29N4O4 [M+H]+: calcd 389.2188; found 389.2198. Anal. calcd for C20H28N4O4: C, 61.84; H, 7.27; N, 14.42. Found C, 61.74; H, 7.40; N, 14.00%. t-Boc-allyl ester alanine 3-26b O O HN Boc Following amino acid coupling procedures, t-Boc-L-alanine 3-24b (1.73 g, 9.14 mmol), DIC (2.8 mL, 18 mmol), DMAP (0 .230 g, 1.89 mmol), HOBt (1.27 g, 9.40 mmol), allyl alcohol (1.0 mL, 15 mmol), and CH2Cl2 (15 mL) yielded 3-26b (2.0 g, 95%) as a colorless oil. 3-26b: Rf = 0.42 (hexane/EtOAc, 8:2); [ ]25 D = -35.0 (c = 1.04, MeOH); 1H NMR (CDCl3) 5.89 (ddt, J = 17.0, 10.2, 5.6 Hz, 1H), 5.37-5.19 (m, 2H), 5.11 (br s, 1H), 4.694.54 (m, 2H), 4.39-4.26 (m, 1H), 1.43 (s, 9H), 1.38 (d, J = 7.1, 3H); 13C NMR (CDCl3) 173.19, 155.23, 131.78, 118.72, 79.95, 65.94, 49.39, 28.47, 18.81; IR (neat) 3368, 2980, 2937, 1716, 1650, 1518, 1455, 1367, 1251, 1167, 1069; HRMS (ESI-FTICR) for [M+Na]+: calcd 252.1206, found 252.1227. Anal. calcd for C11H19NO4: C, 57.62; H, 8.35; N, 6.11. Found: C, 57.88; H, 8.77; N, 6.47. t-Boc-allyl ester proline 3-26c N C O O Boc Following amino acid coupling procedures, t-Boc-L-proline 3-24c (4.15 g, 19.3 mmol), DIC (6.0 mL, 39 mmol), DMAP (0.707 g, 5.79 mmol), HOBt (2.74 g, 20.3

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113 mmol), allyl alcohol (2.24 g, 38.6 mmol), and CH2Cl2 (32 mL) yielded 3-26c (4.7 g, 95%) as a clear oil. 3-26c: Rf = 0.25 (hexane/EtOAc, 4:1); [ ]25 D = -70.9 (c = 1.00, MeOH); 1H NMR (CDCl3) 5.89 (ddt, J = 16.7, 10.5, 5.7 Hz, 1H), 5.37-5.15 (m, 2H), 4.68-4.51 (m, 2H), 4.36-4.18 (m, 1H), 3.58-3.30 (m, 2H), 2.28-2.09 (m, 1H) 2.02-1.71 (m, 3H), 1.43 and 1.38 (s, 9H); 13C NMR (CDCl3) 172.99, 153.89, 131.96, 118.73-118.24 (2 lines), 80.0079.86 (2 lines), 65.58, 59.28-58.98 (s lines), 46.68-46.46 (2 lines) 31.05-30.08 (2 lines), 28.57-28.45 (2 lines), 24.46-23.77 (2 lines ); IR (KBr) 2978, 2882, 1749, 1702, 1397, 1258, 1162, 1122, 1089; HRMS (CI pos) for C13H21NO4 [M+H]+: calcd 256.1549, found 256.1541. t-Boc-allyl ester methionine 3-26d O O HN Boc S Following amino acid coupling procedures, t-Boc-L-methionine 3-24d (2.23 g, 8.94 mmol), DIC (2.8 mL, 18 mmol), DMAP (0.300 g, 2.46 mmol), HOBt (1.26 g, 9.33 mmol), allyl alcohol (0.9 0 mL, 13 mmol), and CH2Cl2 (15 mL) yielded 3-26d (2.5 g, 97%) as a colorless oil. 3-26d: Rf = 0.28 (hexane/EtOAc, 8:2); [ ]25 D = -32.4 (c = 1.04, MeOH); 1H NMR (CDCl3) 5.88 (ddt, J = 17.1, 10.4, 5.7 Hz, 1H), 5.35-5.16 (m, 3H), 4.67-4.55 (m, 2H), 4.44-4.34 (m, 1H), 2.51 (t, J = 7.5 Hz, 2H), 2.18-1.83 (m, 5H), 1.41 (s, 9H); 13C NMR (CDCl3) 172.11, 155.44, 131.67, 119.01, 80.07, 66.09, 52.95, 32.26, 30.08, 28.41, 15.56; IR (neat) 3362, 2977, 2920, 1716, 1650, 1511, 1447, 1367, 1251, 1167, 1050; HRMS (CI pos) for C13H24NO4S [M+H]+: calcd 290.1426, found 290.1421.

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114 Phenylalanine derivative 3-26e O O HN Boc Following amino acid coupling procedures, t-Boc-L-phenylalanine 3-24e (5.35 g, 20.2 mmol), DIC (5.2 mL, 33.6 mmol), DMAP (0 .48 g, 3.93 mmol), HOBt (2.96 g, 21.9), 3-buten-1-ol (2.7 mL, 31.4 mmol), and CH2Cl2 (40 mL) yielded 3-26e (5.8 g, 90%) as a white precipitate, m.p. = 79-80.5 C. 3-26e: Rf = 0.40 (hexane/EtOAc, 4:1); [ ]25 D = -9.0 (c = 1.00, MeOH); 1H NMR (CDCl3) 7.33-7.10 (m, 5H), 5.72 (ddt, J = 17.1, 10.6, 6.6 Hz, 1H), 5.14-5.03 (m, 2H), 4.99 (d, J = 8.2 Hz, 1H), 4.57 (dt, J = 8.2, 6.4 Hz, 1H), 4.14 (t, J = 6.8 Hz, 2H), 3.11 (dd, J = 13.6, 6.5 Hz, 1H), 3.03 (dd, J = 13.7, 6.6 Hz, 1H), 2.40-2.29 (m, 2H), 1.41 (s, 9H); 13C NMR (CDCl3) 172.06, 155.24, 136.21, 133.78, 129.50, 128.68, 127.15, 117.68, 80.01, 64.50, 54.58, 38.54, 33.01, 28.45; IR (KBr) 3355, 3077, 3030, 3006, 2973, 2930, 1735, 1708, 1645, 1516, 1455, 1391, 1365, 1288, 1220, 1187, 1086, 1054, 1020. Anal. calcd for C18H25NO4: C, 67.69; H, 7.89; N, 4.39. Found: C, 67.83; H, 8.07; N, 4.36%. Alanine derivative 3-26f O O HN Boc Following amino acid coupling procedures, t-Boc-L-alanine 3-24f (1.86 g, 9.83 mmol), DIC (2.5 mL, 16 mmol), DMAP (0 .240 g, 1.96 mmol), HOBt (1.45 g, 10.7 mmol), 3-buten-1-ol (1.3 mL, 16 mmol), and CH2Cl2 (20 mL) yielded 3-26f (2.4 g, 98%) as a colorless oil.

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115 3-26f: Rf = 0.35 (hexane/EtOAc, 4:1); [ ]25 D = -45.7 (c = 1.11, MeOH); 1H NMR (CDCl3) 5.75 (ddt, J = 17.1, 10.5, 6.8, 1H), 5.16-5.00 (m, 3H), 4.34-4.08 (m, 3H), 2.432.34 (m, 2H), 1.42 (s, 9H), 1.35 (d, J = 7.2 Hz, 3H); 13C NMR (CDCl3) 173.46, 155.21, 133.76, 117.61, 79.88, 64.32, 49.35, 33.15, 28.47, 18.87; IR (KBr) 3368, 2980, 1718, 1644, 1517, 1168, 1069; HRMS (CI pos) for C12H22NO4 [M+H]+: calcd 244.1549 found 244.1549. Proline derivative 3-26g N C O O Boc Following amino acid coupling procedures, t-Boc-L-proline 3-24g (4.06 g, 18.9 mmol), DIC (5.0 mL, 32 mmol), DMAP (0 .730 g, 5.98 mmol), HOBt (2.82 g, 20.9 mmol), 3-buten-1-ol (2.7 mL, 32 mmol), and CH2Cl2 (31 mL) yielded 3-26g (4.5 g, 89%) as a colorless oil. 3-26g: Rf = 0.41 (hexane/EtOAc, 7:3); [ ]25 D = -72.3 (c = 1.24, MeOH); 1H NMR (CDCl3) 5.71 (ddt, J = 17.0, 10.2, 6.8 Hz, 1H), 5.11-4.96 (m, 2H), 4.27-4.01 (m, 3H), 3.54-3.26 (m, 2H), 2.38-2.27 (m, 2H), 2.21-1.73 (m, 4H), 1.39 and 1.34 (s, 9H); 13C NMR (CDCl3) 173.21-172.95 (2 lines), 153.87, 134.05-133.83 (2 lines), 117.46-117.23 (2 lines), 79.89-79.76 (2 lines), 63.93, 59.28-58.98 (2 lines), 46.63-46.41 (2 lines), 33.20, 31.03-30.08 (2 lines), 28.54-28.45 (2 lines), 24.37-23.67 (2 lines); IR (KBr) 3482, 3080, 2977, 1699, 1395, 1160; HRMS (CI pos) for C14H23NO4 [M+H]+: calcd 270.1705, found 270.1701. Methionine derivative 3-26h

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116 O O HN Boc S Following amino acid coupling procedures, t-Boc-L-methionine 3-24h (5.59 g, 22.4 mmol), DIC (5.2 mL, 34 mmol), DMAP (0.577 g, 4.72 mmol), HOBt (3.35 g, 24.8 mmol), 3-buten-1-ol (2.9 mL, 34 mmol), and CH2Cl2 (40 mL) yielded 3-26h (6.0 g, 88 %) as a clear oil. 3-26h: Rf = 0.26 (hexane/EtOAc, 8:2); [ ]25 D = -23.8 (c = 1.10, MeOH); 1H NMR (CDCl3) 5.76 (ddt, J = 17.5, 10.3, 6.7 Hz, 1H), 5.16-5.05 (m, 3H), 4.44-4.33 (m, 1H), 4.26-4.13 (m, 2H,), 2.52 (t, J = 7.6 Hz, 2H), 2.40 (qt, J = 6.7, 1.2, 2H), 2.18-1.86 (m, 5H); 1.44 (s, 9H); 13C NMR (CDCl3) 172.40, 155.46, 133.75, 117.74, 80.13, 64.59, 53.02, 33.16, 32.49, 30.15, 28.50, 15.64; IR (KBr) 3362, 3079, 2978, 2919, 1716, 1643, 1509, 1446, 1391, 1367, 1251; HRMS (CI pos) for C14H26NO4S [M+H]+: calcd 304.1582 found 304.1570. Leucine derivative 3-26i O O HN Boc Following amino acid coupling procedures, t-Boc-L-leucine 4-24i (2.08 g, 8.99 mmol), DIC (2.0 mL, 13 mmol), DM AP (0.179 g, 1.47 mmol), HOBt (1.44 g, 10.6 mmol), 3-buten-1-ol (1.0 mL, 12 mmol), and CH2Cl2 (50 mL) yielded 4-26i (2.1 g, 83%) as a clear oil. 3-26i: Rf = 0.33 (hexane/EtOAc, 9:1); [ ]25 D = -39.2 (c = 1.42, MeOH); 1H NMR (CDCl3) 5.72 (ddt, J = 17.3, 10.1, 6.7 Hz, 1H), 5.10-4.92 (m, 3H), 4.28-4.05 (m, 3H),

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117 2.35 (qt, J = 6.7, 1.1 Hz, 2H), 1.71-1.39 (m, 12H), 0.90 (d, J = 1.2, 3H ), 0.88 (d, J = 1.3 Hz, 3 H); 13C NMR (CDCl3) 173.50, 155.48, 133.79, 117.48, 79.72, 64.17, 52.24, 41.97, 33.10, 28.40, 24.86, 22.87, 22.03; IR (neat) 3368, 3081, 2960, 2872, 1718, 1644, 1509, 1455, 1367, 1165, 1122, 1048, 1023. Anal. calcd for C15H27NO4: C, 67.69; H, 7.89; N, 4.39. Found: C, 67.83; H, 8.07; N, 4.36%. Fmoc phenylalanine derivative 3-27a O O H N O O Following amino acid coupling proc edures, Fmoc-L-phenylalanine 3-25a (1.00 g, 2.58 mmol), DIC (0.60 mL, 3.87 mmol), DMAP (60.0 mg, 0.491 mmol), HOBt (422 mg, 3.12 mmol), 3-buten-1-ol (0.33 mL, 3.86 mmol), and THF (5.0 mL) yielded 3-27a (1.1 g, 99 %) as a white solid, m.p. = 52-54 C. 3-27a: Rf = 0.44 (hexane/EtOAc, 7:3); [ ]25 D = -20.0 (c = 1.06, MeOH); 1H NMR (CDCl3) 7.85-7.16 (m, 13 H), 5.86-5.72 (m, 1H), 5.46-5.09 (m, 3H), 4.80-4.20 (m, 6H), 3.25-3.15 (m, 2H), 2.48-2.32 (m, 2H); 13C NMR (CDCl3) 171.61, 155.67, 143.98-143.88 (2 lines), 141.42, 135.93, 133.67, 129.47, 128.69, 127.83, 127.23-127.17 (2 lines), 125.25-125.18 (2 lines), 120.11, 117.70, 67.05, 64.60, 54.94, 47.26, 38.38, 32.94; IR (KBr) 3327, 3064, 2962, 1696, 1605, 1536, 1450, 1388, 1263, 1104, 1086, 1045; HRMS (CI pos) for C28H28NO4 [M+H]+: calcd 442.2018, found 442.2025. Anal. calcd for C28H27NO4: C, 76.17; H, 6.16; N, 3.17. Found: C, 75.81; H, 6.22; N, 3.16%. Fmoc proline derivative 3-27b

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118 O O N O O Following amino acid coupling procedures, Fmoc-L-proline 3-25b (1.04 g, 3.07mmol), DIC (0.71 mL, 4.6 mmol), DM AP (0.0749 g, 0.613 mmol), HOBt (0.502 g, 3.71 mmol), 3-buten-1-ol (0.40 mL, 4.7 mmol), and THF (7.0 mL) yielded 3-27b (1.1 g, 92%) as a colorless oil. 3-23b: Rf = 0.35 (hexane/EtOAc, 7:3); [ ]25 D = -49.4 (c = 1.25, MeOH); 1H NMR (CDCl3) 7.62-7.12 (m, 8H), 5.69-5.50 (m, 1H), 5.00-4.84 (m, 2H) 4.33-3.89 (m, 6H), 3.55-3.29 (m, 2H), 2.27-1.66 (m, 6H); 13C NMR (CDCl3) 172.43-172.36 (2 lines), 154.67-154.27 (2 lines), 144.10-143.68 (4 lin es), 141.18-141.13 (2 lines), 133.79-133.60 (2 lines)127.58, 126.94, 125.09-124.86 (3 lines ), 119.86, 117.31-117.17 (2 lines), 67.31, 63.85, 59.20-58.75 (2 lines), 47.20-46.34 (4 lin es), 32.96-32.91 (2 lines) 30.96-29.80 (2 lines) 24.19, 23.17; IR (KBr) 3068, 2957, 2884, 1745, 1705, 1451, 1417, 1349, 1194, 1120, 1089; HRMS (ESI-FTICR) for [M+Na]+: calcd 414.1676, found 414.1669. Anal. calcd for C24H25NO4: C, 73.64; H, 6.44; N, 3.58. Found: C, 73.28; H, 6.61; N, 3.54%. Fmoc glycine derivative 3-27c O O H N O O Following amino acid coupling procedures, Fmoc-glycine 3-25c (1.90 g, 6.39 mmol), DIC (1.5 mL, 9.7 mmol), DMAP (0.318 g, 2.60 mmol), HOBt (1.38 g, 10.2

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119 mmol), 3-buten-1-ol (0.85 mL, 9.9 mmol), and THF (15 mL) yielded 3-27c (2.2 g, 85 %) as a white solid, m.p. = 78.5-80 C. 3-27c: Rf = 0.40 (hexane/EtOAc, 7:3); 1H NMR (CDCl3) 7.81-7.30 (m, 8 H), 5.79 (ddt, J = 17.2, 10.2, 6.7 Hz, 1H), 5.49-5.41 (m, 1H), 5.18-5.08 (m, 2H), 4.43 (d, J = 7.0 Hz), 4.28-4.20 (m, 3H), 4.00 (d, J = 5.6 Hz, 2H), 2.42 (qt, J = 6.8, 1.3 Hz, 2H); 13C NMR (CDCl3) 170.15, 156.43, 143.93, 141.40, 133.63, 127.83, 127.19, 125.20, 120.10, 117.71, 67.28, 64.54, 47.21, 42.85, 33.02; IR (KBr) 3335, 3065, 3017, 2947, 1767, 1685, 1541, 1451, 1414, 1389, 1361, 1288, 1192, 1104, 1081, 1055; HRMS (CI pos) for C21H22NO4 [M+H]+: calcd 352.1549, found 352.1556. Anal. calcd for C21H21NO4: C, 71.78; H, 6.02; N, 3.99. Found: C, 71.62; H, 6.06; N, 3.98%. 6.2.6 General Procedures for CM of Dimer 3-3 with an Amino Acid CM product t-Boc-allyl ester phenylalanine-dimer 3-28a N N O O O O O N H O N Boc Boc H A solution of catalyst 1-4 (46 mg, 0.054 mmol) and CHCl3 (0.50 mL) was added to a stirred solution of dimer 3-3 (105 mg, 0.541 mmol), amino acid 3-26a (825 mg, 2.70 mmol), and CHCl3 (0.50 mL). The reaction was heated at reflux for 10 h while flushing the headspace with argon to remove evolved ethylene. The reaction was allowed to cool to room temperature and quenched with EVE (ca. 0.5 mL). The solution was stirred for 30 min and concentrated under reduced pressure. Purification by column

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120 chromatography on silica gel with hexane/EtOAc (9:1-4:6) yielded 3-28a as an oil (150 mg, 37%) and homodimer 3-30a (301 mg, 38%) as a white solid. 3-28a: Rf = 0.33 (EtOAc); 1H NMR (CDCl3) 7.35-7.09 (m, 10H), 5.78-5.57 (m, 4H), 5.03 (d, J = 7.9 Hz, 2H), 4.66-4.51 (m, 6H), 4.07-3.90 (m, 8H), 3.16-2.98 (m, 4H), 1.41 (s, 18H); 13C NMR (CDCl3) 171.70, 163.08, 155.14, 135.99, 129.39, 128.80, 128.65, 127.40, 127.13, 80.07, 64.56, 54.57, 49.34, 47.03, 38.41, 28.40; IR (KBr) 3324, 2978, 1666, 1498, 1366, 1169, 1022 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 771.3576, found 771.3544. CM product t-Boc-allyl ester alanine-dimer 3-28b N N O OO O H N Boc O O N H Boc Following general CM procedures, dimer 3-3 (104 mg, 0.533 mmol), amino acid 426b (600 mg, 2.62 mmol), Grubbs catalyst 1-4 (46 mg, 0.054 mmol), and CHCl3 (0.5 mL) gave a brown residue. Purification by column chromatogra phy on silica gel with hexane/EtOAc (9:1 to 6:4, 2:8 to 0:10) yielded 3-28b (130 mg, 40%) as an oil and homodimer 3-30b (180 mg, 42%) as a white solid, m.p. = 96-97 C. 3-28b: Rf = 0.21 (EtOAc); 1H NMR (CDCl3) 5.85-5.60 (m, 4H), 5.10 (d, J =7.2 Hz, 2H), 4.65-4.55 (m, 4H), 4.35-3.89 (m, 10H), 1.40 (s, 18H), 1.35 (d, J = 7.3 Hz, 6H); 13C NMR (CDCl3) 173.13, 163.14, 155.17, 128.93, 127.24, 79.95, 64.49, 51.52, 49.30, 47.03, 28.41, 18.61; IR (KBr) 3330, 2980, 2935, 2361, 2250, 1666, 1520, 1478, 1366, 1252, 1165, 1070, 1023 cm-1; HRMS (EI pos) for C28H44N4O4 [M]+: calcd 619.2950, found 619.2946. Homodimer t-Boc-allyl ester alanine 3-30b

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121 O O H N Boc O O N H Boc 3-30b: Rf = 0.34 (hexane/EtOAc, 6:4); 1H NMR (CDCl3) 5.87-5.78 (m, 2H) 5.11 (d, J = 7.2 Hz, 2H), 4.64-4.57 (m, 4H), 4.354.22 (m, 2H), 1.40 (s, 18H), 1.36 (d, J = 7.2 Hz, 6H); 13C NMR (CDCl3) 173.12, 155.19, 127.91, 79.95, 64.60, 49.32, 28.43, 18.67; IR (KBr) 3370, 2983, 2938, 1737, 1685, 1522, 1456, 1369, 1274, 1163, 1085, 1024 cm-1; HRMS (CI pos) for C20H35N2O8 [M+H]+: calcd 431.2393, found 431.2379. CM product t-Boc-allyl ester proline-dimer 3-28c N N O OO O N Boc O O N Boc Following general CM procedures, dimer 3-3 (95.5 mg, 0.492 mmol), amino acid 3-26c (726 mg, 2.51 mmol), Grubbs catalyst 1-4 (42.0 mg, 0.0495 mmol), and CHCl3 (1.0 mL) gave a brown residue. Purification by column chromatography on silica gel with hexane/EtOAc (9:1 to 7:3, 2:8 to 0:10) yielded 3-28c (135 mg, 42% NMR yield) as an oil and homodimer 3-30c (188 mg, 31%) as an oil. 3-26c: Rf = 0.28 (EtOAc); 1H NMR (CDCl3) 5.88-5.65 (m, 4H), 4.66-4.58 (m, 4H), 4.36-4.20 (m, 2H), 4.07-3.92 (m, 8H), 3. 60-3.35 (m, 4H), 2.30-2.13 (m, 2H), 2.021.82 (m, 6H), 1.46 and 1.41 (s, 18H). Homodimer t-Boc-allyl ester proline 3-30c O O N Boc O O N Boc 3-30c: Rf = 0.37 (hexane/EtOAc, 5:5); 1H NMR (CDCl3) 5.94-5.60 (m, 2H), 4.764.52 (m, 4H), 4.36-4.18 (m, 2H), 3.58-3.32 (m, 4H), 2.29-2.10 (m, 2H), 2.02-1.77 (m,

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122 6H), 1.44 and 1.38 (s, 18H); 13C NMR (CDCl3) 172.92, 172.67, 154.48, 153.83, 128.46-127.61 (3 lines), 80.00, 64.41, 64.27, 59.22, 58.92, 46.68, 46.46, 31.04, 30.06, 28.55, 28.45, 24.47, 23.78 cm-1; IR (neat) 3482, 2974, 1950, 1747, 1698, 1399, 1259, 1170 cm-1. HRMS (CI pos) for C24H39N2O8 [M+H]+: calcd 483.2706, found 483.2724. Anal. calcd for C24H38N2O8: C, 59.73; H, 7.94; N, 5.81. Found: C, 60.05; H, 8.20; N, 5.70% CM product t-Boc-homoallyl ester phenylalanine-dimer 3-28e N N O O O O N Boc H O O N Boc H Following general CM procedures, dimer 3-3 (103 mg, 0.530 mmol), amino acid 326e (908 mg, 2.84 mmol), catalyst 1-4 (45.9 mg, 0.0541 mmol) and CHCl3 (1.0 mL) gave a brown residue. Purification by chromat ography on silica gel with hexane/EtOAc (100:0 0:100) afforded 3-28e (180 mg, 44%) as a silver foam and homodimer 3-30e (440 mg, 50%) as a gray solid, m.p. = 142-144 C. 3-28e: Rf = 0.27 (EtOAc); 1H NMR (CDCl3) 7.38-7.08 (m, 10H), 5.58 (dt, J = 15.2, 6.3 Hz, 2H), 5.42 (dt, J = 15.3, 6.3 Hz 2H), 5.10 (d, J = 8.0 Hz, 2H), 4.63-4.47 (m, 2H), 4.18-3.83 (m, 12H), 3.15-2.96 (m, 4H ), 2.39-2.25 (m, 4H), 1.41 (s, 18H); 13C NMR (CDCl3) 172.01, 163.28, 155.21, 136.22, 131.46, 129.39, 128.63, 127.09, 125.68, 79.92, 64.11, 54.62, 49.11, 47.40, 38.41, 31.56, 28.40; IR (KBr) 3328, 2976, 2249, 1713, 1664, 1498, 1366, 1170, 1052 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 799.3889; found 799.3879. Homodimer t-Boc-homoallyl ester phenylalanine 3-30e

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123 O O N Boc H O O N Boc H 3-30e: Rf = 0.30 (hexane:EtOAc, 7:3); 1H NMR (CDCl3) 7.35-7.10 (m, 10H); 5.47-5.35 (m, 2H), 5.09 (d, J = 7.2 Hz, 2H), 4. 56 (dt, J = 7.6, 7.0 Hz, 2H) 4.08 (t, J = 6.8 Hz, 4H), 3.09 (dd, J = 13.6, 6.5 Hz, 2H), 3.02 (dd, J = 13.6, 6.2 Hz, 2H), 2.37-2.20 (m, 4H), 1.40 (s, 18H); 13C NMR (CDCl3) 171.90, 155.13, 136.17, 129.34, 128.53, 128.24, 126.99, 79.80, 64.55, 54.52, 38.39, 31.81, 28.33; IR (KBr) 3365, 3003, 2971, 2931, 1709, 1517, 1456, 1391, 1365, 1222, 1184, 1087, 1053, 1019 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 633.3146, found 633.3156. CM product t-Boc-homoallyl ester alanine-dimer 3-28f N N O O O O N Boc H O O N Boc H Following general CM procedures, dimer 3-3 (200 mg, 1.03 mmol), amino acid 326f (1200 mg, 4.94 mmol), Grubbs catalyst 1-4 (84.0 mg, 0.099 mmol) and CHCl3 (3.0 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (100:0 0:100) afforded 3-28f (250 mg, 39%) as a silver foam and homodimer 3-30f (750 mg, 66%) as an oil. 3-28f: Rf = 0.31 (EtOAc); 1H NMR (CDCl3) 5.65 (dt, J = 15.4, 6.8 Hz, 2H), 5.46 (dt, J = 15.3, 6.4 Hz, 2H), 5.16 (s, 2H), 4.28-4.07 (m, 6H), 4.01-3.90 (m, 8H), 2.44-2.37 (m, 4H), 1.43 (s, 18H), 1.36 (d, J = 7.7, 6H); 13C NMR (CDCl3) 173.20, 163.22, 155.10, 131.37, 125.49, 79.45, 63.73, 49.13, 48.87 47.15, 31.51, 28.24, 22.81, 18.20; IR (neat)

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124 3327, 2979, 2934, 1666, 1521, 1478, 1391, 1367, 1335, 1250, 1166, 1115, 1068, 1023 cm-1; HRMS (CI pos) for C30H49N4O10 [M+H]+: calcd 625.3449, found 625.3450. Homodimer t-Boc-homoallyl ester alanine 3-30f O O N Boc H O O N Boc H 3-30f: Rf = 0.39 (hexane/EtOAc, 7:3); 1H NMR (CDCl3) 5.47-5.39 (m, 2H) 5.15 (s, 2H), 4.28-4.02 (m, 6H), 2.38-2.24 (m, 4H ), 1.37 (s, 18H), 1.30 (d, J = 7.3 Hz, 6H); 13C NMR (CDCl3) 173.41, 155.19, 131.70, 128.29, 127.39 (cis), 79.81, 64.47, 49.31, 32.01, 28.43, 18.73; IR 3366, 2979, 1715, 1518, 1455, 1392, 1367, 1251, 1166, 1069, 1025 cm1; HRMS (CI pos) for C22H39N2O8 [M+H]+: calcd 459.2706, found 459.2705. CM product t-Boc-homoallyl ester proline-dimer 3-28g N N O O O O O N O N Boc Boc Following general CM procedures, dimer 3-3 (90.0 mg, 0.463 mmol), amino acid 3-26g (624 mg, 2.32 mmol), Grubbs catalyst 1-4 (41.2 mg, 0.048 mmol) and CHCl3 (1.0 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (100:0 0:100) afforded 3-28g (140 mg, 45%) and homodimer 3-30g (325 mg, 55%) as an oil. 3-28g: Rf = 0.28 (EtOAc); 1H NMR (CDCl3) 5.68-5.31 (m, 4H), 4.24-3.75 (m, 14H), 3.52-3.24 (m, 4H), 2.44-1.73 (m, 12H), 1.39 and 1.34 (s, 18H); 13C NMR (CDCl3) 173.13, 172.92, 163.24, 163.14, 154.36, 153.75, 131.75, 131.41, 125.60, 125.38, 79.83, 79.69, 63.62, 59.14, 58.81, 49.05, 47.35, 46.58, 46.34, 31.66, 30.93, 29.99, 28.46, 28.36,

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125 24.32, 23.64; IR (neat) 3494, 2975, 1745, 1670, 1477, 1399, 1279, 1162 cm-1; HRMS (CI pos) for C34H53N4O10 [M+H]+: calcd 677.3762, found 677.3751. Homodimer t-Boc-homoallyl ester proline 3-30g O O N Boc O O N Boc 3-30g: Rf = 0.33 (hexane/EtOAc, 7:3); 1H NMR (CDCl3) 5.50-5.32 (m, 2H), 4.26-3.92 (m, 6H), 3.54-3.20 (m, 4H), 2.36-1.74 (m, 12H), 1.39 and 1.34 (s, 18H); 13C NMR (CDCl3) 173.10, 172.82, 154.32, 153.73, 128.43, 128.22, 127.98, 79.71, 79.61, 64.07, 59.11, 58.81, 46.51, 46.28, 31.97, 30.89, 29.94, 28.40, 28.30, 26.86, 24.25, 23.55 IR (neat) 3522, 2976, 2882, 1747, 1700, 1478, 1455, 1397, 1258, 1162, 1122 cm-1; HRMS (CI pos) for C26H43N2O8 [M+H]+: calcd 511.3019, found 511.3017. Homodimer methionine t-Boc-homoallyl ester 3-30h O O HN Boc O O NH Boc S S Following general CM procedures, dimer 3-3 (86.0 mg, 0.443 mmol), amino acid 3-26h (690 mg, 2.27 mmol), Grubbs catalyst 1-4 (39.9 mg, 0.0470 mmol) and CHCl3 (1.0 mL) gave a brown residue. Purifi cation by chromatography on silica gel with hexane/EtOAc (9:1) afforded homodimer 3-30h (94 mg, 14%) as an oil. 3-30h: Rf = 0.31 (hexane/EtOAc, 7:3); 1H NMR (CDCl3) 5.78-5.39 (m, 2H), 5.19 (s, 2H), 4.44-4.30 (m, 2H), 4.22-4.07 (m, 4H), 2.56-2.29 (m, 8H), 2.17-1.83 (m, 10H), 1.42 (s, 18H); 13C NMR (CDCl3) 172.39, 155.48, 128.42-126.62 (3 lines), 80.09, 65.6364.19 (4 lines), 52.98, 32.38-31.69 (4 lines), 30.14-29.80 (2 lines), 28.45, 27.19-26.95 (2

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126 lines), 15.59; IR (n eat) 3357, 2976, 1715, 1515, 1366, 1252, 1166, 1051 cm-1; HRMS (CI pos) for C26H47N2O8S2 [M+H]+: calcd 579.2774, found 579.2768. CM product t-Boc-homoallyl ester leucine-dimer 3-28i N N O O O O O NH O HN Boc Boc Following general CM procedures, dimer 3-3 (95.0 mg, 0.489 mol), amino acid 326i (682 mg, 2.39 mmol), Grubbs catalyst 1-4 (43.5 mg, 0.0512 mmol) and CHCl3 (1.0 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (9:1 1:9) afforded 3-28i (103 mg, 30%) and homodimer 3-30i (381 mg, 59%) as an oil. 3-28i: Rf = 0.42 (EtOAc:hexane, 8:2); 1H NMR (CDCl3) 5.72-5.41 (m, 4H), 5.03 (d, J = 7.6 Hz, 2H), 4.31-3.90 (m, 14H), 2.53-2.35 (m, 4H), 1.86-1.38 (m, 24H), 0.94 (d, J = 6.72, 12H); 13C NMR (CDCl3) 173.58, 163.41, 155.60, 131.74, 125.74, 79.90, 64.01, 52.35, 49.21, 47.53, 41.85, 31.84, 28.54, 25.00, 23.04, 22.09; IR 3325, 2960, 1713, 1665, 1522, 1475, 1390, 1366, 1334, 1253, 1166, 1048, 1020 cm-1; HRMS (CI pos) for C36H60N4O10 [M+H]+: calcd 709.4388, found 709.4390. Homodimer t-Boc-homoallyl ester leucine 3-30i O O HN Boc O O NH Boc 3-30i: Rf =0.29 (hexane/EtOAc, 8:2); 1H NMR (CDCl3) 5.53-5.36 (m, 2H), 4.97 (d, J = 6.4 Hz, 2H), 4.30-4.00 (m, 6H), 2.41-2.26 (m, 4H), 1.73-1.37 (m, 24H), 0.90 (d, J = 6.3 Hz, 12H); 13C NMR (CDCl3) 173.49, 155.49, 128.33, 127.42 (cis) 79.77, 64.38,

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127 52.25, 41.95, 32.05, 28.45, 26.95-24.89 (2 lin es), 22.94-22.05 (2 lines); IR 3379, 2960, 1716, 1510, 1391, 1367, 1253, 1165, 1048 cm-1; HRMS (CI pos) for C28H50N2O8 [M+H]+ calcd 543.3645, found 543.3630; Anal. calcd for C28H50N2O8: C, 61.97; H, 9.29; N, 5.16. Found: C, 62.13; H, 9.62; N, 5.04 % CM product Fmoc-homoally l phenylalanine-dimer 3-29a O O H N Fmoc O O N H Fmoc N N O O Following general CM procedures, dimer 3-3 (61.0 mg, 0.314mol), amino acid 327a (614 mg, 1.39 mmol), Grubbs catalyst 1-4 (30.0 mg, 0.035 mmol) and CHCl3 (1.5 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (9:1 2:8) afforded 3-29a (136 mg, 42%) as brown foam, and homodimer 3-31a (285 mg, 48%) as a white solid, m.p. = 54-56 C. 3-29a: Rf = 0.35 (EtOAc/hexane, 8:2); 1H NMR (CDCl3) 7.70-7.01 (m, 26H), 5.53-5.26 (m, 6H), 4.59-3.74 (m, 18H), 3.09-2.92 (m, 4H), 2.34-2.17 (m, 4H); 13C NMR (CDCl3) 171.65, 163.35, 155.75, 143.87, 141.39, 136.02, 131.46, 129.42, 128.70, 127.82, 127.17, 125.73, 125.19, 120.09, 67.01, 64.28, 55.06, 49.10, 48.32, 47.30, 38.31, 31.63; IR 3304, 2956, 1721, 1662, 1478, 1451, 1334, 1260 cm-1; HRMS (ESI-FTICR) for [M+Na]+: calcd 1043.4202, found 1043.4214. Homodimer Fmoc-homoallyl phenylalanine 3-31a O O H N Fmoc O O N H Fmoc

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128 3-31a: Rf = 0.30 (hexane/EtOAc, 7:3); 1H NMR (CDCl3) 7.72-6.98 (m, 26H), 5.55-5.20 (m, 4H), 4.64-3.92 (m, 12H), 3.10-2.75 (m, 4H), 2.29-2.12 (m, 4H); 13C NMR (CDCl3) 171.64, 155.73, 144.01-143.90 (2 lines), 141.46, 135.96, 129.49, 128.74, 128.38, 127.87, 127.29-127.21 (2 lines), 125.29125.21 ( 2 lines), 120.15, 67.10, 64-8764.77 (2 lines), 54.99, 47.30, 38.44, 31.93; IR 3343, 2953, 1728, 1521, 1450, 1211, 1051 cm-1; HRMS (ESI-FTICR) for [M+Na]+ calcd 877.3459, found 877.3485. Anal. calcd for C54H50N2O8: C, 75.86; H, 5.89; N, 3.28. Found: C, 75.46; H, 5.99; N, 3.26 %. CM product Fmoc-homoallyl proline-dimer 3-29b O O N Fmoc O O N Fmoc N N O O Following general CM procedures, dimer 3-3 (63.0 mg, 0.324mol), amino acid 327b (558 mg, 1.42 mmol), Grubbs catalyst 1-4 (30.0 mg, 0.0353 mmol) and CHCl3 (1.5 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (9:1 2:8) afforded 3-29b (140 mg, 46% NMR yi eld) and homodimer 331b (280 mg, 52% NMR yield) as a brown semi-solid. 3-29b: Rf = 0.32 (EtOAc); 1H NMR (CDCl3) 7.67-7.18 (m, 16H), 5.58-5.27 (m, 4H), 4.41-3.38 (m, 24H), 2.31-1.80 (m, 12H); 13C NMR (CDCl3) 172.56, 163.27163.15 (2 lines), 154.84-154.40 (2 lines), 144.18-143.77 (3 lines), 141.32, 131.53-131.28 (2 lines), 127.73, 127.12, 125.58-125.02 (4 lines), 120.02, 67.49, 63.84, 59.27-58.88 (2 lines), 49.06, 47.34-46.54 (4 lines), 31.6431.59 (2 lines), 31.15, 30.00, 24.42, 23.42; HRMS (ESI-FTIR-MS) for [M+Na]+: calcd 943.3899, found 943.3900. Homodimer Fmoc-homoallyl proline 3-31b

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129 O O N Fmoc O O N Fmoc 3-31b: Rf = 0.37 (hexane/EtOAc, 1:1); 1H NMR (CDCl3) 7.73-7.15 (m, 16H), 5.43-5.27 (m, 2H), 4.40-3.88 (m, 12H), 3.62-3.37 (m, 4H), 2.32-1.73 (m, 12H); 13C NMR (CDCl3) 172.57, 154.91-154.49 (2 lines), 144.24-143.86 (2 lines), 141.37, 128.44128.11 (3 lines), 127.77, 127.13, 125.28-125.07 (3 lines), 120.06, 67.57, 64.39, 59.3858.97 (2 lines), 47.43-46.58 (4 lines) 32.05, 31.22, 30.06, 24.46, 23.45; HRMS (ESIFTIR) for [M+Na]+ calcd 777.3148, found 777.3129. CM product Fmoc-homoallyl glycine-dimer 3-29c O O H N Fmoc O O N H Fmoc N N O O Following general CM procedures, dimer 3-3 (94.0 mg, 0.484 mol), amino acid 327c (854 mg, 2.43 mmol), Grubbs catalyst 1-4 (67.5 mg, 0.0795 mmol) and CHCl3 (2.0 mL) gave a brown residue. Purifica tion by chromatography on silica gel with hexane/EtOAc (9:1 1:9) afforded 3-29c (120 mg, 30%) as a silv er solid, m.p. = 52-54 C, and homodimer 3-31c (357 mg, 44%) as a white solid, m.p. = 121-123 C. 3-29c: Rf = 0.42 (EtOAc); 1H NMR (CDCl3) 7.67-7.15 (m, 16H), 5.86-5.20 (m, 6H), 4.32-3.72 (m, 22H), 2.37-2.18 (m, 4H); 13C NMR (CDCl3) 170.24, 163.54, 156.62, 143.93, 141.33, 131.84, 127.78, 127.13, 125.59, 125.23, 120.04, 67.12, 63.94, 48.89, 47.18, 42.77, 31.80; IR 3319, 3065, 2957, 1724, 1662, 1534, 1478, 1450, 1333, 1263, 1193, 1104, 1051, 1008 cm-1; HRMS (ESI-FTICR) for [M+H]+: calcd 841.3443, found 841.3432. Homodimer Fmoc-homoallyl glycine 3-31c

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130 O O H N Fmoc O O N H Fmoc 3-31c: Rf = 0.39 (hexane/EtOAc, 5:5); 1H NMR (CDCl3) 7.78-7.27 (m, 16H), 5.50-5.36 (m, 4H), 4.40 (d, J = 7.3 Hz, 4H), 4.26-4.14 (m, 6H), 3.98 (d, J = 5.7 Hz, 4H), 2.43-2.31 (m, 4H); 13C NMR (CDCl3) 170.24, 156.52, 143.99, 141.48, 128.48, 127.91, 127.26, 125.27, 120.18, 67.38, 67.38, 64.80, 47.28, 42.93, 32.03; IR 3319, 2950, 1758, 1691, 1541, 1450, 1411, 1361, 1287, 1191, 1105, 1082, 1053 cm-1; HRMS (ESI-FTICR) for [M+Na]+ calcd 697.2520, found 697.2528. Anal. calcd for C40H38N2O8: C, 71.20; H, 5.68; N, 4.15. Found: C, 70.83; H, 5.74; N, 4.12% Independent synthesis of cis 3-30a ONHBoc Ph O O O BocHN Ph A flame dried flask under argon was charged with acid 3-24a (2.07 g, 7.8 mmol), and CH2Cl2 (10 mL). The solution was cooled to 0 C, followed by the addition of EDCI (1.47 g, 7.67 mmol), HOBt (1.05 g, 7. 77 mmol), DMAP (0.095 g, 0.78 mmol), and DIPEA (1.8 mL, 10 mmol). After stirri ng for 20 min, (Z)-2-butene-1,4-diol (3-33) was added drop-wise to the solution and mainta ined for 6 h. The solvent was removed in vacuo, and the residue redissolved in EtOAc. The organic layer was washed with 1 N KHSO4, 1 N NaHCO3, water, and brine. The organic layer was then dried with Na2SO4 and concentrated in vacuo to give 3-30a (1.7 g, 97%) as a white solid. 3-30a: 1H NMR (CDCl3) 7.32-7.09 (m, 10H), 5.72-5.60 (m, 2H), 4.98 (d, J = 8.1 Hz, 2H), 4.72-4.54 (m, 6H), 3.15-2.96 (m, 4H), 1.39 (s, 18H); 13C NMR (CDCl3) 171.81, 136.09, 129.54, 128.76, 128.09, 127.27, 80.16, 60.74, 54.64, 38.57, 28.48. Hex-3-ene-1,6-diol (3-35)

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131 HO OH Following literature procedures,62 a solution of trans-hydromuconic acid 3-34 (1.00g, 6.94 mmol), concentrated sulfuric acid (0.34 mL), and absolute methanol (50 mL) were refluxed overnight under an atmosphere of argon. The solution was cooled to room temperature and the MeOH was removed by redu ced pressure. Extraction with ether, NaHCO3, H2O and brine, and drying with MgSO4 afforded the diester (1.0 g, 85%). A solution of the diester (1.0 g, 5.8 mmol) a nd THF (30 mL) was added to a reaction vessel containing LiAlH4 (925 mg, 24.4 mmol) and THF (12 mL ) by an addition funnel, and the reaction mixture stirred unde r argon at room temperature for 6 h. The reaction was quenched with EtOAc. The white precipitate that was formed was filtered off and washed with cold ether. The combined orga nic layers was passed through a pad of celite and concentrated under reduced pressure to give diol 3-35 (0.44 g, 65%). 3-35: 1H NMR (CDCl3) 5.48-5.36 (m, 2H), 3.78 (s, 2H) 3.53 (t, J = 7.0 Hz, 4H); 13C NMR (CDCl3); 129.424, 61.648, 35.979. Independent synthesis of trans 3-30e A flame dried flask under argon was charged with acid 3-30e (3.45 g, 13.0 mmol), and CH2Cl2 (40 mL). The solution was cooled to 0 C, followed by the addition of EDCI (2.90 g, 15.1 mmol), HOBt (2.21 g, 16. 4 mmol), DMAP (0.130g, 1.06 mmol), and DIPEA (4.0 mL, 23 mmol). Af ter stirring for 20 min, diol 3-35 was added drop-wise to the solution and maintained for 16 h. The solvent was removed in vacuo, and the residue redissolved in EtOAc. The orga nic layer was washed with 1 N KHSO4, 1 N NaHCO3, water, and brine. The organic layer was then dried with Na2SO4 and concentrated in

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132 vacuo to give 3-30e (1.2 g, 54%) as a white solid. Analytical data were identical to data from CM product 3-30e. 4-iodo benzoate (4-10) O O I Following literature procedures,119 thionyl chloride (1.8 mL, 25 mmol) was added drop-wise to a solution of 4-iodobenzoic acid (4-9) (2.90 g, 11.7 mmol ) in ethanol (30 mL). The solution was heated at 60 C for 4 h, then concentrated in vacuo. The residue was diluted with EtOAc and the organic layer washed with H2O and brine. After drying the organic layer (MgSO4) and concentrating under reduced pressure, the oil that resulted was purified by flash chromatography with hexane/CH2Cl2 (8:2) to afford pure ethyl 4iodobenzoate 4-10 (3.2 g, 98%) as a colorless o il. Proton and carbon NMR data are identical to those reported in SDBS.140 4-10: Rf = 0.31 (hexane/CH2Cl2, 7:3); 1H NMR (CDCl3) 7.78-7.68 (m, 4H), 4.35 (q, J = 7.1, 2H), 1.35 (t, J = 7.1, 3H); 13C NMR (CDCl3) 166.07, 137.71, 131.07, 130.01, 100.69, 61.27, 14.41. 4-allyl benzoate (4-11) O O Following literature procedures,119 isopropylmagnesium chloride (8.1 mL, 2 M THF solution) was added drop-wise to a solution of ethyl 4-iodobenzoate (4-10) (3.02 g, 10.9 mmol) in dry THF (115 mL) cooled to -40 C. The reaction was stirred for 1 h before adding a daily prepared CuCN, 2LiCl solution in dry THF. To prepare the solution, anhydrous LiCl (0.977 g, 23.1 mmol) wa s first dried in a flask heated with an oil

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133 bath at 130 C under vacuum for 2 h. Upon cooling to room temperature, CuCN (0.990 g, 11.1 mmol) and dry THF (37 mL) were adde d and the flask flushed with argon. After stirring for 15 min, the yellow-green soluti on was cooled to -40 C and added to the solution of ethyl 4-iodobenzoat e via a stainless steel cannula The reaction was stirred for 15 min, followed by the addition of allyl bromide (3.8 mL, 44 mmol). After stirring for 1 h, the reaction mixture was diluted with EtOAc and filtered over Celite. The filtrate was concentrated under reduced pressure a nd the residue redissol ved in EtOAc. The organic layer was washed with H2O and brine, dried (MgSO4) and concentrated in vacuo. Purification by flash chroma tography with hexane/CH2Cl2 (85:15) afforded pure ethyl 4allyl benzoate 4-11 (1.9 g, 89%) as a yellow oil. 4-11: Rf = 0.32 (hexane/ CH2Cl2, 7:3); 1H NMR (CDCl3) 7.95 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 5.99-5.85 (m, 1H), 5. 11-5.03 (m, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.39 (d, J = 6.7 Hz, 2H), 1.36 (t, J = 7.13 Hz, 3H); 13C NMR (CDCl3) 166.41, 145.28, 136.40, 129.69, 128.71, 128.52, 116.45, 60.69, 40.08, 14.30. 4-allyl benzoic acid (4-5) O HO An aqueous solution of sodium hydroxide (2 M, 40 mL) was added to an ethanolic solution of 4-allyl benzoate 4-11 (55 mM, 80 mL). The solu tion was stirred at room temperature for 7 h, then quenched with HCl (1 M, 80 mL). Ethanol was removed in vacuo and the aqueous layer was extracted 3x with EtOAc. The organic layer was washed with brine, dried (MgSO4), and concentrated under reduced pressure. Recrystallizing with hexane/Et2O afforded 4-allybenzoic acid (4-5) as a white solid (0.67 g, 94%). Proton and carbon NMR data are id entical to those repor ted in literature.119

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134 4-5: Rf = 0.25 (CH2Cl2/MeOH, 95:5); 1H NMR (CDCl3) 8.04 (d, J = 8.32, 2H), 7.28 (d, J = 8.31, 2H), 6.02-5.88 (m, 1H), 5.15-5.05 (m, 2H), 3.45 (d, J = 6.66, 2H); 13C NMR (CDCl3) 172.76, 146.79, 136.43, 130.64, 128.95, 127.48, 116.94, 40.42. 6.2.7 General Procedures for Removing Boc Protecting Group with TFA Deprotected homoallyl phenylalanine 4-12 NH2O O A solution of trifluoroacetic acid (TFA) (4.0 mL) and CH2Cl2 (10 mL) was added drop-wise to a stirred solution of amino acid 3-26e (2.35 g, 7.36 mmol) and CH2Cl2 (10 mL). After stirring for 3.5 h, the solvent and excess TFA were removed in vacuo, followed by neutralization with NaHCO3 and extraction with Et OAc. The organic layer was washed with brine and dried (Na2SO4). Removal of the solvent followed by recrystallization in Et2O/hexane afforded a white solid (quantitative yield), m.p. = 77-78 C. 4-12: Rf = 0.34 (EtOAc); 1H NMR (CDCl3) 7.99 (s, 2H), 7.48-7.32 (m, 5H), 5.75 (ddt, J = 18.0, 10.2, 6.7 Hz, 1H), 5.23-5.14 (m, 2H), 4.40-4.18 (m, 3H), 3.42 (dd, J = 14.2, 6.5 Hz, 1H), 3.34 (dd, J = 14.7, 7.2 Hz, 1H), 2.43-2.35 (m, 2H); 13C NMR (CDCl3) 169.24, 133.94, 133.25, 129.51, 129.14, 127.94, 117.86, 65.66, 54.33, 36.62, 32.62 Compound 4-13

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135 N O H N O O O O A flame dried flask under argon was charged with acid 3-24c (1.69 g, 7.83 mmol) and CH2Cl2 (18 mL). The reaction flask was cooled to 0 C and EDCI (1.75 g, 9.13 mmol), HOBt (1.15 g, 8.49 mmol), DMAP (0.591 g, 0.484 mmol), and DIPEA (2.1mL, 12 mmol) were added to the stirred soluti on. After stirring for 30 min, amino acid 3-24c (1.43 g, 6.52 mmol) was added and the reaction maintained for 4 h at room temperature. The solvent was removed in vacuo, and the residue redissolved in EtOAc. The organic layer was washed with KHSO4 (1 N), NaHCO3 (1 N), water, and brine. The organic layer was then dried with Na2SO4 and concentrated in vacuo. Purification by column chromatography with hexane/Et2O (7:3) afforded 4-13 (2.4 g, 85%). 4-13: Rf = 0.29 (Et2O/hexane, 6:4); 1H NMR (CDCl3) 7.24-7.02 (m, 6H), 5.67 (ddt, J = 17.4, 10.3 6.7 Hz, 1H), 5.08-4.96 (m 2H), 4.77 (s, 1H), 4.24-4.02 (m, 3H), 3.342.89 (m, 4H), 2.34-1.67 (m, 6H), 1.35 (s 9H); IR (neat) 3315, 3065, 2977, 1743, 1698, 1521, 1394, 1165, 1122, 1088 cm-1. Compound 4-6 HN O H N O O Following general deprotection procedures using TFA, amino acid derivative (1.99 g, 4.78 mmol), TFA (4.0 mL), and CH2Cl2 (22 mL) gave a yellow oil. Column

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136 chromatography on silica gel with CH2Cl2/MeOH (97:3) yielded 4-6 as a white solid (1.5 g, 98%), m.p. = 88-90 C. 4-6: Rf = 0.31 (CH2Cl2/MeOH, 95:5); [ ]25 D = -27.7 (c = 1.16, MeOH);1H NMR (CDCl3) 8.18-8.08 (m, 2H), 7.27-7.11 (m, 5H), 5.70 (ddt, J = 17.0, 10.3, 6.7 Hz, 1H), 5.13-5.03 (m, 2H), 4.75-4.47 (m, 2H), 4.19-4.06 (m, 2H), 3.35-2.97 (m, 4H), 2.38-1.82 (m, 6H), ; 13C NMR (CDCl3) 171.12, 169.13, 136.33, 133.69, 129.23, 128.70, 127.20, 117.68, 64.76, 59.50, 54.52, 46.43, 37.26, 32.91, 30.10, 24.43; IR (KBr) 3347, 3286, 3091, 2985, 1739, 1714, 1667, 1573, 1556, 1499, 1457, 1427, 1299, 1181, 1137, 1084, 1031 cm-1; HRMS (ESI-FT-ICR-MS) for [M+H]+: calcd 317.1860; found 317.1880. Compound 4-4 N O NH O O O Following EDCI coupling procedures, acid 4-5 (336 mg, 2.07 mmol), 4-6 (243 mg, 0.768 mmol), EDCI (480 mg, 2.50 mmol), HO Bt (338 mg, 2.50 mmol), DMAP (101 mg, 0.827 mmol), DIPEA (0.55 mL, 320 mmol), and CH2Cl2 (6 mL) yielded 4-4 (244 mg, 69%) as a white solid, m.p. = 84.5-86 C. 4-4: Rf = 0.33 (hexane/EtOAc, 1:1); [ ]25 D = 39.4 (c = 1.10, MeOH); 1H NMR (CDCl3) 7.43-7.08 (m, 10H), 6.03-5.65 (m, 2H), 5.14-4.74 (m, 6H), 4.21-4.11 (m, 2H), 3.47-2.97 (m, 6H), 2.42-1.72 (m, 6H); 13C NMR (CDCl3) 171.43, 171.15, 171.00, 142.67, 136.79, 136.30, 133.99, 133.77, 129.48, 128.60, 128.54, 127.61, 126.99, 117.60, 116.53, 64.52, 59.91, 53.65, 50.49, 40.13, 38.08, 32.98, 27.43, 25.46zz; IR (KBr) 3258, 3078, 2978, 1733, 1633, 1539, 1423, 1351, 1324, 1272, 1228, 1178, 1118, 1083, 1043

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137 cm-1; HRMS (ESI-FT-ICR-MS) for [M+Na]+: calcd 483.2254, found 483.2258; Anal. calcd for C28H32N2O4: C, 73.02; H, 7.00; N, 6.08. Found C, 72.76; H, 7.15; N, 6.00% Dimer 4-7a 4-7a: 1H NMR (Acetone d6) 7.39-7.18 (m, 9H), 6.62 (d, J = 7.2 Hz, 1H), 5.84 (dtt, J = 15.3, 6.0, 1.1 Hz, 1H), 5.04 (dddt, J = 14.3, 7.9, 6.2, 1.6 Hz, 1H), 4.55 (dd, J = 8.3, 3.5 Hz, 1H), 4.28 (q, J = 7.3 Hz, 1H), 4.27 (m, 1H), 3.82 (m, 1H), 3.62 (m, 2H), 3.38 (dd, J = 15.1, 6.6 Hz, 1H), 3.22 (dd, J = 14.5, 6.2 Hz, 1H), 3.02 (dd, J = 14.1, 7.6 Hz, 1H), 2.66 (dd, J = 13.7, 7.7 Hz, 1H), 2.25 (m, 2H), 2.18 (m, 1H), 1.84 (m, 2H), 1.76 (m, 1H); 13C NMR (CDCl3) 171.3, 169.1, 169.8, 142.6, 138.1, 132.7, 129.8, 128.4, 128.2, 126.7, 63.5, 62.1, 55.2, 47.4, 37.9, 37.3, 32.4, 32.2, 22.9; HRMS (ESI-FT-ICR-MS) for [M]+: calcd 865.4171, found 865.4174. 5,6-nitrofluorescein (5-21) O O O OH HO N O O Following literature procedures,135 resorcinol (5-19) (26.7 g, 243 mmol) and 4nitrophthalic acid (5-20) (25.5 g, 121 mmol) were thoroughl y mixed in a flask and heated on an oil bath at 190-200 C until the black-b rown liquid dried to a glass-like mass (ca.12 h). After cooling to room temperature, th e melt was chipped from the flask, ground in a mortar, and suspended in 0.6 N HCl (400 mL). The mixture was boiled for 1 h, filtered hot in a 350 mL coarse fritted filter, washed with hot 0.6 N HCl (3 x 75 mL), followed with 1.5 L of boiling water. The brown mass wa s dried in the oven at 112 C to give 5,6nitrofluorescein (5-21) (42 g, 94%), m.p. = 338-340 C. Rf = 0.41 (benzene/EtOH, 4:1).

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138 5,6-nitrofluorescein diacetate (5-22) O O O O O N O O O O To a stirred solution of 5,6-nitrofluorescein (5-21) (35.5 g, 76.9 mmol) in pyridine (136 mL) at room temperature was added glac ial acetic anhydride (53 mL). Acetylation occurred immediately and no changes in TLC were observed after 10 minutes. Three volumes of toluene were added to the so lution, which was stirred for 5 min, and concentrated under reduced pressure to form a black-brown viscous gum. After repeating this step two more times, the viscous mass was vacuum dried to form 5,6-nitrofluorescein diacetate 5-22, 100 C decomposition. Rf = 0.70 (benzene/EtOH, 4:1). 5-nitrofluorescein diacetate (5-23) O O O O O N O O O O Following literature procedures,135 5,6-nitrofluorescein diacetate 5-22 was redissolved in 100 mL refluxing acetic anhydride, cooled to room temperature, seeded with 5-nitrofluorescein diacetate crystal prev iously obtained to indu ce crystallization, and set overnight. Recrystallization continued fo r another day at 4 C. The creamy yellowwhite crystals formed were co llected in a Buchner funnel, and washed twice with 35 mL of acetic anhydride (6.3 g, 14%, fraction 1). The filtrate from fraction 1 was concentrated to half the volume under reduced pressure seeded with crystals, and allowed to crystallize at 4 C for 48 h to obtain additional 5-nitrofluorescein diacetate. The crystals

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139 were collected in a Buchner funnel and rinsed twice with 35 mL of acetic anhydride (9.3 g, 21%, fraction 2). To purify the 5-nitroflu orescein diacetate, fr actions 1 and 2 were combined, dissolved with 50 mL refluxi ng acetic anhydride, and filtered hot on a Buchner funnel. The filtrate was cooled to 4 C for 24 h. The white crystals formed were collected in a Buchner funnel and rinsed twice with 20 mL ace tic anhydride to give purified 5-nitrofl uorescein diacetate 5-23 (9.0 g, 21%). To increase the yield, the filtrate from the purification of fraction 1 and 2 wa s concentrated under reduced pressure, and cooled at 4C for 24 h. The crystals formed in the flask were isolated and purified as described above (1.1 g, 3%, fraction 3). To tal yield of all fractions combined after purification was 10.1 g (24%), m.p. = 223-224 C. 5-23: Rf = 0.71 (benzene/EtOH, 4:1); 1H NMR (CDCl3) 8.88 (d, J = 1.9 Hz, 1H), 8.55 (dd, J = 2.0, 8.4 Hz, 2H), 7.40 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 1.9 Hz, 2H), 6.906.79 (m, 4H), 2.34 (s, 6H) ; 13C NMR (CDCl3) 168.93, 166.78, 157.87, 152.67, 151.54, 149.67, 130.29, 128.80, 127.71, 125.75, 121.20, 118.34, 114.98, 110.96, 82.22, 21.25; HRMS for C20H25NO9 [M]+, calcd 461.0747, found 461.0747. Melting point is in agreement with literature.132 6-nitrofluorescein diacetate (5-24) O O O O O N O O O O The filtrate from fraction 2 of 5-23 was concentrated under reduced pressure to form a dark brown viscous gum. The mass was redissolved in 180 mL of benzene and recrystallized overnight at room temperature. The yellow-white crystals were collected in a Buchner funnel and washed with 40 mL of benzene (5.1 g, 12%), m.p. = 195 C. If

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140 higher yields were required, the filtrate was concentrated under vacuum and recrystallized at 4 C in benzene. 5-24: Rf = 0.69 (benzene/EtOH ,4:1); 1H NMR (CDCl3) 8.49 (dd, J = 1.8, 8.5 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 1.7Hz, 1H), 7.15 (d, J = 2.1Hz, 2H), 6.90-6.79 (m, 4H), 2.33 (s, 6H); 13C (CDCl3) 168.87, 166.97, 154.26, 152.69, 151.65, 130.77, 128.75, 125.50, 126.90, 125.76, 119.84, 118.37, 114.98, 110.99, 82.22, 21.28. Melting point is in agreement with known literature.132 5-nitrofluorescein (5-25) O O O OH HO N O O 5-Nitrofluorescein diacetate 5-23 (4.75 g, 10.3 mmol) was dissolved in hot filtered methanol saturated with NaOH (95 mL), and st irred at 50 C. The dark-red solution was filtered on a Buchner funnel, poured into 4 vol umes of water, and acidified with 12.1 N HCl (2 mL). The orange mixture was allowed to sit at room temperature for 4 h. The orange precipitate was collected in a Buchne r funnel, washed with water (480 mL), and vacuum dried to give 5-25 (3.8 g, 97 %), m.p. above 350 C. 5-25: Rf = 0.40 (benzene/EtOH, 4:1); 1H NMR (DMSO-d6) 10.22 (s, 2H), 8.66 (d, J = 2.0Hz, 1H), 8.56 (dd, J = 2.2, 8.4 Hz, 1H), 7.57 (d, 8.4 Hz, 1H), 6.72-6.52 (m, 6H); 13C NMR (DMSO-d6) 166.74, 159.88, 157.40, 151.83, 149.10, 130.40, 129.41, 127.74, 125.93, 120.35, 112.78, 108.29, 102.38, 83.77; IR (KBr) 3064.97, 1599.09, 1528.31, 1459.81, 1353.42, 1311.36, 1240.52 1212.21, 1172.09, 1120.48 cm-1; HRMS for

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141 C20H11NO7 [M]+, calcd 377.0536, found 377.0531; Melti ng point is in agreement with known literature.132 IR is in agreement with SDBS.140 5-aminofluorescein salt (5-26) O O O OH HO N H H H To a stirred solution of s odium sulfide nonahydrate, Na2SH2O, (4.35 g, 18.1 mmol), in H2O (75 mL) was added 5-nitrofluorescein (5-25) (1.89 g, 5.01 mmol). After dissolution of 5-25, sodium hydrosulfide, NaHS (2.04 g, 36.4 mmol), was introduced and the solution was refluxed for 24 h at 125 C The solution was cooled to room temperature and acidified with glacial acetic acid (3.0 mL). The dark red precipitate was collected in a Buchner funnel, dissolved in refluxing 6% HCl (150 mL), and filtered hot in a Buchner funnel to remove elemental su lfur. The filtrate was cooled to room temperature then placed at 4 C for 48 h to al low crystallization. Th e red-orange crystals were collected in a Buchner funnel, redi ssolved in refluxing 6% HCl (50 mL), and filtered hot in a Buchner funnel to remove any additional sulfur. Crystallization at 4 C for 48 h afforded 5-aminofluorescein salt (5-26) (0.88 g, 46%), 204 C decomposition. 5-26: Rf = 0.26 (benzene/EtOH, 4:1); 1H NMR (DMSO-d6) 7.56 (s, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.77-6.55 (m, 6H). 5-aminofluorescein (5-27)

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142 O O O OH HO N H H 5-Nitrofluorescein salt (5-26) (425 mg, 1.11 mmol) in NaOH (88 mL) was precipitated with glacia l acetic acid (2.5 mL). The so lid was collected in a Buchner funnel, and dried under vacuum to give 5-27 (270 mg, 67%), m.p. = 222 C. 5-27: Rf = 0.58 (benzene/EtOH, 3:2); 1H NMR (DMSO-d6) 10.97 (s, 2H), 7.006.82 (m, 3H), 6.67-6.50 (m, 6H), 5.75 (s, 2H); 13C NMR (DMSO-d6) 169.55, 159.35, 152.05, 150.58, 139.72, 129.12, 127.58, 124.27, 121.77, 112.50, 110.77, 106.24, 102.19, 82.96; HRMS for C20H13NO5 [M]+, calcd 347.0794, found 347.0793. Data are consistent with NMR spectra of a sample of 5-amin ofluorescein purchased from Aldrich and SDBS.140 N-(5-fluoresceinyl)maleamic acid (5-29) O O O OH HO N H O O OH Following literature procedures, maleic anhydride (5-28) (49.6 mg, 0.506 mmol) was added to a stirred solution of 5-aminofluorescein (5-27) (174 mg, 0.500 mmol) in glacial acetic acid (50 mL). The mixture was stirred for 4 h at room temperature. The bright canary yellow precipitate was collected on a filtered frit, washed with EtOAc (100 mL), dried overnight, and used in the next step without purificati on (190 mg, 85%), m.p. above 350 C.

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143 5-29: Rf = 0.10 (benzene/EtOH, 60:40,); 1H NMR (DMSO-d6) 12.93 (br, 1H), 10.77 (s, 2H), 10.13 (s, 2H), 8.32 (s, 1H), 7.84 (dd, J = 1.8, 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 6.51-6.68 (m, 7H), 6.34 (d, J = 12.0 Hz 1H). All data are in agreement with known literature.130 N-(5-fluoresceinyl)maleimide (5-14) O O O OH HO N O O To a stirred solution of maleic acid 5-29 (161 mg, 0.25 mmol) in anhydrous DMF (1.5 mL) was added distilled benzene (149 mL) and ZnCl2 (68.2 mg, 0.500 mmol). Freshly distilled HMDS (211 ul) was added drop-wise, and the mixture refluxed for 2.5 h. After cooling to room temperature, the mixture was filtered and the orange-yellow filtrate collected. The solvent was remove d under reduced pressure, leaving behind DMF and the maleimide. The residual solution was poured into an ice-water bath (50 mL), and the aqueous phase acidified with 0.1 N HCl to a pH of 6. Th e orange-yellow precipitate was collected on a filtered frit and dried in a dessicator under vacuum in the dark to give maleimide 5-13 (97 mg, 91%), m.p. above 350 C. 5-14: Rf = 0.64 (benzene/EtOH, 3:2); 1H NMR (DMSO-d6) 10.18 (s, 2H), 7.98 (d, J = 1.2 Hz, 1H), 7.79 (dd, J = 1.7, 8.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.28 (s, 2H), 6.55-6.71 (m, 6H); 13C (NMR) (DMSO-d6) 169.65, 168.02, 159.62, 151.83, 151.10, 134.96, 133.50, 133.20, 129.14, 126.73, 124.72, 122.02, 112.76, 109.17, 102.32, 83.34; HRMS for C24H13NO7 [M]+: calcd 427.0692 found 427.0685. All data are in agreement with known literature.130

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144 Mono-norbornene ester (5-fl uoresceinyl)maleimide (5-13) and di-norborn ene ester (5fluoresceinyl)maleimide (5-15) O O O OH O N O O O O O O O O N O O O O Under argon, oxalyl chloride (510 l, 5.80 mmol ) was adde d drop-wise to a stirred solution of 5-norbornene-2-carboxylic acid (5-9) (98%, mixture of endo and exo, 160 mg, 1.16 mmol) and CH2Cl2 (1.2 mL). A catalytic amount of DMF was added and the mixture was stirred until no additional gas was released. Excess oxalyl chloride was removed under reduced pressure. To remove any oxalyl chloride residue, the acid chloride was redissolved in CH2Cl2 and concentrated under reduced pressure. The crude acid chloride was redissolved in CH2Cl2 (7.0 mL) once more, and added drop-wise to a stirred solution of maleimide 5-13 (472 mg, 1.10 mmol) in tr iethylamine (200 ul, 1.44 mmol) and CH2Cl2 (34 mL) at 0 C. The reaction was allowed to warm to room temperature and stirred for 3 h. The ye llow orange mixture was diluted with CH2Cl2. The organic layer was then extr acted with brine and dried (Na2SO4). Concentration of the organic layer by reduced pressure and pur ification by flash chromatography on silica gel with CH2Cl2/MeOH (100:0 to 98:2) afforded 5-13 (mixture of endo and exo) as a yellow-orange solid (130 mg, 22%), 183 C decomposition, and di-acylated 5-15 (mixture of endo and exo) (40 mg, 6%) as a white yellow precipitate, m.p. = 205.5-207 C.

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145 5-13: Rf = 0.38 (CH2Cl2/MeOH, 96:4); 1H NMR (CDCl3) 8.05 (d, J = 1.6 Hz, 1H), 7.67 (dd, J = 1.5, 8.3 Hz, 1H), 7.22-6.43 (m, 9H), 6.28 (dt, J = 2.8, 5.6 Hz, 1H), 6.07 (td, J = 2.8, 5.9Hz, 1H), 3.39 (s, 1H), 3.24 (dt, J = 4.5, 8.9Hz, 1H), 2.98 (s, 1H), 2.02 (ddd, J = 4.1, 9.2, 12.4 Hz, 1H), 1.65-1.22 (m, 3H); 13C NMR (CDCl3) 173.60, 169.08, 168.86, 158.76, 152.40, 152.26, 152.00, 151.83, 147.41, 138.69, 134.69, 133.15, 132.53, 132.16, 129.38, 127.61, 125.09, 122.20, 117.63, 116.07, 112.95, 110.66, 109.92, 103.28, 83.35, 49.98, 47.05, 46.21, 43.86, 42.87, 36.98, 29.55; IR (KBr) 3423.45, 2976.13, 1761.01, 1719.81, 1611.55, 1496.32, 1426.63, 1388.56, 1251.64, 1149.70; HRMS for C32H21NO8 [M]+: calcd 547.1267, found 547.1254. 5-15: Rf = 0.76 (CH2Cl2/MeOH, 96:4); 1H NMR (CDCl3) 8.00 (d, J = 2.1 Hz, 1H), 7.63 (dd, J = 2.1, 8.3 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.96 (dd, J = 3.4, 2.2 Hz, 2H), 6.86 (s, 2H), 6.81-6.76 (m, 2H), 6.73-6.68 (m, 2H), 6.20 (dt, J = 3.0, 6.0 Hz, 2H), 5.98 (td, J=2.8, 6.0 Hz, 2H), 3.31 (s, 2H), 3.18-3.11 (m, 2H), 2.91 (s, 2H), 1.99-1.90 (m, 2H), 1.54-1.26 (m, 6H); 13C NMR (CDCl3) 174.44, 172.85, 168.96, 168.19, 152.62, 151.63, 147.37, 138.56, 135.70, 134.66, 133.31, 132.43, 132.14, 130.28, 129.13, 127.29, 124.98, 122.20, 118.01, 115.78, 110.56, 82.02, 49.91, 46.98, 46.52, 46.12, 43.80, 43.48, 42.82, 41.90, 30.73, 29.50; IR (KBr) 3481.93, 3061.02, 1975.28, 2873.30, 1766.18, 1720.31, 1610.77, 1580.32, 1495.13, 1421.42, 1386.41, 1335.54, 1244.58, 1154.72, 1015.77, 903.43, 832.28, 710.82, 690.26, 607.50; HRMS for C40H29NO9 [M]+: calcd 667.1842, found 667.1857. N-phenyl-2-(phenylthio)succinimide (5-32)

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146 N OO S To a stirred solution of N-phenylmaleimide (5-30) (3.0 g, 17.3 mmol) in dry benzene (30 mL) and triethylamine (0.17 mL) was added drop-wise over 25 min a solution of benzenethiol (5-31) (1.8 mL, 17.5 mmol) in benz ene (30 mL). Within 15 min a white precipitate was formed. The creamy white mixture was diluted with benzene (20 mL) and stirred overnight. The solvent wa s removed under reduced pressure, and the resulting residue was recrystallized in petroleum/EtOAc (5:1) to give 5-32 (4.9 g, 90%) m.p. = 140-142 C. 5-32: Rf = 0.30 (Et2O/hexane, 3:2); 1H NMR (CDCl3) 7.60-7.55 (m, 2H), 7.457.30 (m, 6H), 7.05-7.00 (m, 2H), 4.13 (dd, J = 3.9, 9.3 Hz, 1H), 3.32 (dd, J = 9.3,19.1 Hz, 1H), 2.88 (J = 3.8, Hz, 1H); 13C NMR (CDCl3) 174.68, 173.69, 135.24, 131.64, 129.83, 129.66, 129.29, 128.95, 126.47, 44.24, 35.51. Melting point and 1H NMR data are in agreement with known literature values.136 N-phenyl-2-(dimethylamino-ethyl sulfanyl)succinimide (5-34) N OO S N Dimethylaminoethanethiol salt (5-33) (2.01 g, 14.1 mmol) in Et2O (8.0 mL) was stirred with aqueous sodium carbonate at r oom temperature for 20 min. The free thiol

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147 was extracted with Et2O several times (3 x 8 mL) and dried over sodium sulfate. The solution was added drop-wise for a peri od of 20 min to a stirred solution of N-phenylmaleimide (5-30) (810 mg, 4.70 mmol) in benzene (5.0 mL) and triethylamine (65 l) at room temperature. The reaction was monitored by TLC, and after 2 h, the solvent was removed under reduced pressure. The pi nk white residue was recrystallized in CH2Cl2/hexane (1:2) to give purified 5-34 (1.1 g, 87%), m.p. = 79.5-81 C; 5-34: Rf = 0.43 (CH2Cl2/MeOH, 85:15); 1H NMR (CDCl3) 7.53-7.28 (m, 5H), 4.02 (J = 3.7, 9.2 Hz, 1H), 3.32 (J = 9.2, 18.7 Hz, 1H), 3.15 (J = 6.6, 13.0 Hz, 1H), 2.98 (J = 6.6, 13.0 Hz, 1H), 2.74-2.59 (m, 3H), 2.28 (s, 6H); 13C NMR (CDCl3) 175.98, 173.93, 131.82, 129.41, 128.97, 126.61, 58.84, 45.38, 39.24, 36.25, 29.95; IR (KBr) 2946.68, 2758.14, 1707.63, 1501.56, 1455.19, 1398.60, 1300.80, 1210.37, 1187.06, 749.49, 698.93 cm-1; HRMS for C14H18N2O2S [M+H]+: calcd 278.1089, found 279.1090; N-phenylmaleimide (5-30) N OO Metachloroperbenzoic acid (925mg, 5.36 mmol) in CHCl3 (13 mL) was added to a stirred solution of 5-32 (1.11g, 3.92 mmol) in chloroform (20 mL) at 0 C. The solution slowly warmed to room temperature and stirred for 1 h. The mixture was extracted with CH2Cl2 and washed with NaHCO3 (1 N), H2O, HCl (0.5 N), H2O, and brine. The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure leaving behind a white solid. To the solid was adde d distilled toluene and the mixture refluxed for 1 h. The yellow solution was cooled to room temperature a nd the solvent removed

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148 under reduced pressure. The yellow residue was purified by flash chromatography with silica gel (Et2O/hexane, 2:8 to 3:7), to give maleimide 5-30 (590 mg, 87%), m.p. = 86 C. 5-30: Rf = 0.39 (Et2O/hexane, 3:2); 1H NMR (CDCl3) 7.48-7.26 (m, 5H), 6.74 (s, 2H); 13C NMR (CDCl3) 169.65, 134.32, 131.35, 129.26, 128.08, 16.21. All data is in agreement with 5-30 purchased from Aldrich.

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149 APPENDIX A HPLC DATA The HPLC spectra of selected compounds from Chapter 4 are illustrated in this appendix.

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150 Figure A-1. HPLC spectrum of e xperiment 1 reaction mixture (CH2Cl2, 5 mM, room temperature, 4 days, no template) Figure A-2. HPLC spectrum of e xperiment 2 reaction mixture (CH2Cl2, 36 mM, room temperature, 4 days, no template)

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151 Figure A-3. HPLC spectrum of e xperiment 3 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, no template) Figure A-4. HPLC spectrum of e xperiment 4 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, no template)

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152 Figure A-5. HPLC spectrum of e xperiment 5 reaction mixture (CH2Cl2, 5 mM, reflux 19 hours, no template) Figure A-6. HPLC spectrum of expe riment 7 reaction mixture (CHCl3, 5 mM, reflux 15 hours, no template)

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153 Figure A-7. HPLC spectrum of e xperiment 10 reaction mixture (CH2Cl2, 5 mM, room temperature 4 days, LiClO4) Figure A-8. HPLC spectrum of e xperiment 11 reaction mixture (CH2Cl2, 36 mM, room temperature 4 days, LiClO4)

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154 Figure A-9. HPLC spectrum of e xperiment 12 reaction mixture (CH2Cl2, 5 mM, reflux 13 hours, LiI) Figure A-10. HPLC spectrum of e xperiment 14 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, LiI)

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155 Figure A-11. HPLC spectrum of e xperiment 15 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, LiI) Figure A-12. HPLC spectrum of e xperiment 16 reaction mixture (CH2Cl2, 5 mM, reflux 15 hours, LiI)

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156 Figure A-13. HPLC spectrum of e xperiment 17 reaction mixture (CHCl3, 5 mM, reflux 13 hours, LiI)

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157 APPENDIX B SELECTED NMR SPECTRAL DATA The 1H and 13C spectra of selected compounds from Chapters 2-5 are illustrated in this appendix. The spectra along with the proposed structure are shown.

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186 BIOGRAPHICAL SKETCH Tammy K. C. Low was born in Taiwan (ROC ). As the daughter of a career Air Force non commissioned officer, she grew up in California, Nebraska, North Carolina, Taiwan, and Okinawa. After graduating fr om Eastern Wayne High School, Goldsboro, North Carolina, in 1993, she accepted an Ai r Force ROTC scholarship and attended North Carolina State University. Tammy was awarded a Bachelor of Science degree in biochemistry and her commission as a second li eutenant in the Air Force in May 1997. The United States Air Force Academy (U SAFA) selected Tammy to pursue her Master of Science degree in chemistry. She attended the University of Illinois at UrbanaChampaign and graduated in August 1998. Tammy was then assigned to Patrick Air Force Base (AFB), Florida, where she he ld the position of Biotechnology Research Officer, involved with treaty monitoring and counter proliferation of nuclear, chemical, and biological weapons. Her ne xt assignment took her to the Department of Chemistry at the USAFA, Colorado Springs, Colorado, wh ere she taught general, organic, and biochemistry, lectured in chemistry of w eapons, and researched on ionic liquids and nucleic acid derivatives. She discovered her joy of teaching at the Academy, and to continue her teaching aspirations, Tammy accepted the Air Force Ph.D. program. She moved to Gainesville, Florid a, in August 2003, to study organic chemistry under Dr. Eric Enholm at the University of Florida.


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Title: Olefin Metathesis in Peptidomimetics, Dynamic Combinatorial Chemistry, and Molecular Imprinting
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Copyright Date: 2008

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Permanent Link: http://ufdc.ufl.edu/UFE0015040/00001

Material Information

Title: Olefin Metathesis in Peptidomimetics, Dynamic Combinatorial Chemistry, and Molecular Imprinting
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
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OLEFIN METATHESIS IN PEPTIDOMIMETICS, DYNAMIC COMBINATORIAL
CHEMISTRY, AND MOLECULAR IMPRINTING















By

TAMMY KARRIE CHENG LOW


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006
































The views expressed in this dissertation are those of the author and do not reflect the
official policy or position of the United States Air Force, Department of Defense, or the
U.S. Government.
































This dissertation is dedicated to my parents for their unconditional love and support.















ACKNOWLEDGEMENTS

My sincere gratitude goes out to the many individuals who have supported me

throughout the years. I first like to thank the Air Force Academy for giving me the

opportunity to pursue my Ph.D. I look forward to returning and working in a wonderful

teaching environment. Special thanks go to Lt Col Ron Furstenau, a mentor and role

model, who taught me a lot about being a good instructor and inspired me to teach.

I would like to extend my sincere appreciation to my research advisor, Dr. Eric

Enholm, for his support, patience, understanding, and invaluable help. He provided me

all the necessary guidance to complete my dissertation, and allowed me the research

freedom to develop my own ideas. Most importantly, he is a caring person who was

always concerned for my family, especially during the monstrous hurricane season of

2004 in Florida. He has been a great advisor and I will never forget his encouragement

and kindness.

I would like to thank my committee members for their constructive feedback and

advice. Special thanks go to Dr. William Dolbier. He is one the most sincere and helpful

professors I have ever met, who shows true concern and interest toward his students. I

thank him for allowing me to drop by his office anytime to discuss mechanisms and

research, and for building my confidence in chemistry. I would also like to thank Dr.

Ronald Castellano. His excellent teaching style and well organized lectures gave me a

great start to the Ph.D. program. I am very appreciative for his advice on my research

and also for the generous use of the HPLC. I sincerely thank Dr. Ion Ghiviriga for









helping with the elucidation of the structure of my organic compounds, and for sharing

his vast NMR expertise, more than I thought I could ever learn about NMR. I also

appreciate Dr. Kenneth Sloan for being on my committee and providing valuable

feedback during my oral qualifier and the preparation of this dissertation. I truly have

been fortunate to have these individuals on my committee.

Graduate school would not have been enjoyable without my fellow Enholm group

members Jed Hastings, Sophie Klein, Kalyan Mondal, and Ryan Martin. It has been a

blessing to work in a cooperative environment, where laboratory discussions are open

and free, and everyone is so helpful and genuinely friendly. I especially like to thank Jed,

for his patience in helping with my lab experiments early on, for exchanging knowledge,

and for providing feedback as I prepared for my oral qualifier, final defense, and the

writing of my dissertation. It has been a pleasure working with Sophie. Her relaxed,

cheerful nature makes any working environment enjoyable. I also thank Kalyan for all

his help and assistance. Not only has it been ajoy working with these individuals, I also

appreciate their friendship outside of lab.

I extend my thanks to all of my supportive friends while in graduate school. I

would like to thank my thoughtful and caring friend Heshan Illangkoon, for our many

chemistry discussions, the proofreading of my work, and his willingness to help anyone

in need. It has also been a pleasure to share my AFIT Ph.D. experience with Lt Col John

Peak. I thank him for his encouraging words, assistance, and his support. I would also

like to acknowledge Dr. Rico Del Sesto and Andy Lampkins for taking the time to read

my dissertation and providing me their honest and constructive comments. I am also

very grateful to Dr. Joe Cradlebaugh, who provided tremendous advice for the









preparation of my oral and final defense and the reviewing and formatting of this

dissertation. Most importantly, I thank him for his wonderful friendship, especially my

last year in graduate school.

Finally, my most heartfelt acknowledgement must go to my parents, sister, and

brother for their continuous support, encouragement, and kindness. I especially thank my

parents for their inspiration, infinite love, and faith. They have made me a better person

by being my role models and instilling me with strong values. They taught me

determination and responsibility at an early age, and provided me the foundation to

achieve my life endeavors. I would not have been in the position to write this dissertation

without my parents. Words alone cannot express my gratitude, especially for their

tremendous love and belief in me during the Ph.D. period.

I am fortunate to have many family and friends who have supported and

encouraged me throughout these years in school and my Air Force career. With

heartfelt and sincere appreciation, I thank everyone.
















TABLE OF CONTENTS



A C K N O W L E D G E M E N T S ............................................................................................... iv

LIST OF TA BLES .................................................................... ............ .. ix

LIST OF FIGURES ............................... ... ...... ... ................. .x

LIST OF SCHEM ES ............... .............................. ............. ........... xiii

CHAPTER

1 H ISTORICAL BA CK GROUN D ....................................................... ....................

1.1 O lefin M etathesis ............................ ..... .. ........ .............. .. ........... 1
1.1.1 Development of Olefin M etathesis and Catalysts....................................... 1
1.1.2 Mechanism of Olefin Metathesis............... ...... ...............5
1.1.3 Important Types of Metathesis Reactions and Applications........................6
1.2 Peptidom im ethics .................. .................................... ... .. ............ 11
1.3 Dynamic Combinatorial Chemistry ......................................... ...............14
1.4 M olecular Imprinted Polymers............................. ........... .. ............. .. 18
1.5 C o n clu sio n s.................................................. ................ 2 3

2 USE OF CROSS-METATHESIS TO COUPLE L-PHENYLALANINE TO A
M ACROCYCLIC LACTAM ............................................................................... 24

2.1 Introduction........................................................................ ....... ...... 24
2.2 R results and D discussion ................................................... ........... ............... 28
2.2.1 Synthesis of Com pound 2-6 ......................... ............................... .... 28
2.2.2 Examining the Reversibility of the CM reaction in Model 2-6..................32
2 .3 C o n clu sio n s.................................................. ................ 3 3

3 OLEFIN METATHEIS OF AMINO ACID DERIVATIVES WITH CYCLIC
S C A F F O L D S .................................................... ................ 34

3.1 Introduction........................................................................ ....... ...... 34
3.2 R results and D discussion ................................................... ........... ............... 4 1
3.2.1 Synthesis of Cyclic Scaffolds ...................................... ...............42
3.2.2 Synthesis of Amino Acid Derivatives .............. ..................... ................45
3.2.3 CM Reactivity of Dimer with One Amino Acid .............. ... .............47









3.2.4 Generation of Small Libraries and Template Effects.............................53
3 .3 C o n clu sio n s.................................................. ................ 5 8

4 DYNAMIC COMBINATORIAL LIBRARIES EMPLOYING
PEPTIDOM IM ETIC DIENES ............................................................................59

4.1 Introduction ............... ......... ................. ...... .............. ......... 59
4 .2 R results and D iscu ssion ........................................ ...........................................63
4 .3 C o n clu sio n s.................................................. ................ 7 7

5 MODEL STUDY MOLECULAR IMPRINTING OF NERVE GASES .................78

5 .1 In tro du ctio n ...................................... ............................ ................ 7 8
5.2 R results and D discussion ....................................................... ........................... 85
5.2.1 Synthesis of N-(5-fluoresceinyl)maleimide 5-7 .......................................85
5.2.2 Synthesis of Compounds 5-13 and 5-15................... .......................... 88
5 .2 .3 M o d el S tu d y .................................................................... .................... 8 9
5 .3 C o n c lu sio n s ..................................................................................................... 9 3

6 E X PE R IM E N T A L S ....................................................................... ......................94

6.1 General Method and Instrumentation..... ...................... .............94
6.2 Experim mental Procedures and D ata.................................... ....................... 95
6.2.1 General Procedures for RCM ........................... ..... ....................... 98
6.2.2 General Procedures for Amino Acid Coupling .......................................100
6.2.3 General Procedures for Ethylenolysis of 2-6 .........................................103
6.2.4 General Procedures for EDCI Coupling............................106
6.2.5 General Procedures for Hydrolysis ........................ ......................... 106
6.2.6 General Procedures for CM of Dimer 3-3 with an Amino Acid..............19
6.2.7 General Procedures for Removing Boc Protecting Group with TFA.......134

APPENDIX

A HPLC DATA ........... ................................................. ............. 149

B SELECTED NMR SPECTRAL DATA..... .................. ...............157

LIST OF REFERENCES ......... ...................................... ........ .. ............... 178

B IO G R A PH ICA L SK ETCH ............ .................................................... .....................186
















LIST OF TABLES


Table page

1-1. Differences between traditional and dynamic combinatorial libraries.....................15

2-1. R CM to obtain 2-16 and 2-17.......................................................... ............... 30

3-1. Yields, melting points and optical rotations of amino acid derivatives....................46

3-2. Yields, melting points, and optical rotations of amino acid derivatives without use
of H O B t during synthesis ........... ................................................... ............... 47

3-3. Yields of cyclic and amino acid dim ers.................................. ........................ 49

3-4. Expected products from CM reaction of dimer scaffold and two or three amino
acid d eriv ativ es ................................................. ................ 5 6

3-5. CM conditions with and without lithium template.........................................57

4-1. Series of C M reaction s ....................................................................... ..................65

4-2. Proton chemical shifts and J values............. ........ .. ..................... ... ........... 75
















LIST OF FIGURES


Figure page

1-1. Schrock's alkoxy imido molybdenum-based catalyst 1-1................. ...............4

1-2. R uthenium catalysts........ ..................................................................................... .5

1-3. Hydrogen-bonding leads to bent conformation.............. ............. ............... 13

1-4. Design of putative azasugar peptidomimetic.............................14

1-5. A dynamic combinatorial library and its free energy landscape .............................15

1-6. Casting and molding process in DCC .... ......................................16

1-7. Dynamic combinatorial chemistry (top) versus virtual combinatorial libraries
(bottom ) ............................................................... ..... ..... ......... 17

1-8. Mass spectrometric analysis of the vancomycin dimer mixture.............................18

1-9. A comparison between the Lock and Key model and the MIP model .....................20

1-10. Schem atic of m olecular im printing process ............................ .............................21

2-1. Oxazole-based m acrocyclic lactam ........................................ ........................ 24

2-2. Phenylalanine on a m acrocyclic lactam .................................. ........................ 26

2-3. Macrocyclic lactam with anchors for CM with amino acids............................. 27

3-1. E xam ples of scaffolds............................ ........... .. ........................... ............... 34

3-2. Dynamic combinatorial libraries of peptidomimetics ............................................. 35

3-3. Schematic of a DCL and a model of amino acids linked to a cyclic scaffold by
o lefi n C M ......................................................................... 3 6

3-4. Glycine-based dimer, trimer and tetramer scaffolds ...............................................37

3-5. N natural cyclic tetrapeptide............................................................... .....................37

3-6. Small dynamic library of cyclic dipeptidomimetics............................................38









3-7. A dynamic library of cyclic dipeptidomimetics including cis/trans isomers............39

3-8. Dynamic library of cyclic tripeptidomimetics.....................................39

3-9. Dynamic library of cyclic tripeptidomimetics including cis/trans isomers ...............40

3-10. Dynamic library of cyclic tetrapeptidomimetics ............................................... 40

3-11. Dynamic library of cyclic tetrapeptidomimetics including cis/trans isomers..........41

3-12. A m ino acids used in the library ........................................ .......................... 47

3-13. Satellites of the alkene protons from cis homodimer 3-30a...................... 52

3-14. Satellites of the alkene protons from trans homodimer 3-30e ..............................53

4-1. F orm action of [2]catenane ................................................................. ..................... 62

4-2. Com prison of experim ents 1 and 10 ....................................................................... 67

4-3. Comparison of experiments 2 and 11 .....................................................................68

4-4. Comparison of experiments 4 and 14 ....................................................................... 69

4-5. Com prison of experim ents 7 and 17 ....................................................................... 70

4-6. HPLC spectrum of isolated compound from experiment 3.....................................72

4-7. Structures of dimers 4-7a, 4-7b, and catenane 4-14................................................73

4-8. 13C NMR chemical shifts and the corresponding protons of isolated compound ......74

4-9. M odel of catenane 4-14 ...................................................................... .................. 76

4-10 M odel of dim er 4-7a ................................................................................ ........ 76

4-11. HPLC spectrum of experiment 3 reaction mixture................................................77

5-1. Structures of com m on nerve agents....................................... ......................... 79

5-2. Mechanism of acetylcholine in the transmission of nerve impulses ........................79

A-1. HPLC spectrum of experiment 1 reaction mixture......................................150

A-2. HPLC spectrum of experiment 2 reaction mixture...............................150

A-3. HPLC spectrum of experiment 3 reaction mixture...............................151

A-4. HPLC spectrum of experiment 4 reaction mixture...............................151









A-5. HPLC spectrum of experiment 5 reaction mixture............................152

A-6. HPLC spectrum of experiment 7 reaction mixture............................152

A-7. HPLC spectrum of experiment 10 reaction mixture............... ...............153

A-8. HPLC spectrum of experiment 11 reaction mixture...............................................153

A-9. HPLC spectrum of experiment 12 reaction mixture...............................................154

A-10. HPLC spectrum of experiment 14 reaction mixture......................................154

A-11. HPLC spectrum of experiment 15 reaction mixture......................................155

A-12. HPLC spectrum of experiment 16 reaction mixture......................................155

A-13. HPLC spectrum of experiment 17 reaction mixture......................................156













LIST OF SCHEMES

Scheme age

1-1. Olefin metathesis .................................. ... ...... .. ....................

1-2. Proposed interim ediates for olefin m etathesis.................................... .....................3

1-3. Metallacyclobutane intermediate proposed by Chauvin ...........................................3

1-4. Dissociative substitution of ruthenium catalysts ................... ......................... 5

1-5. M echanism of olefin m etathesis ........................................... ............................ 6

1-6. Types of olefin m etathesis ........................................ ................................. 8

1-7. Utilizing RCM to synthesize coumarins......................................... 8

1-8. Em playing ROM P to create new m materials ........................................ .....................9

1-9. CM of asymmetric internal olefins............... .... .................................. ........ ... 10

1-10. Primary and secondary CM metathesis reactions................................. ...............11

1-11. Synthesis of C-glycosyl asparagines via CM ..................................................11

1-12. Cyclization of a linear peptide using coupling agents............................................13

1-13. Use of RCM toward the synthesis of P-turn mimetics .................. .. ............. 14

1-14. Dimerization of monomeric vancomycin derivatives with terminal olefins by
m etathesis .............................. .. ............................ ... ...... ........ 18

1-15. An example of the pre-organized approach via covalent interactions....................21

2-1. Synthesis of an analogue of Trienomycin A utilizing RCM ....................................25

2-2. Synthesis of a m acrocyclic inhibitor ............................................... .....................25

2-3. Retrosynthethic analysis of 2-6 ............. .......................................... ............... .....28

2-4 Synthesis of 2-10 and 2-12 ........................................................................................28

2-5. Synthesis of diene 2-15 ...................................................................... ...................29









2-6. R C M of 2-15 ............................................... ............................ 29

2-7 Synthesis of lactam 2-8 ....................................................................... ..................30

2-8. CM reaction to obtain 2-6 and 2-19....................................... ......................... 31

2-9. CM reaction with ethylene gas ............................................. ...................33

3-1. Combinatorial synthesis of piperazine-2,5-dione derivatives ........................ 37

3-2. Synthesis of dim er scaffold ............................................... ............................. 42

3-3. Synthesis of trim er scaffold .......................................................................... .... ... 44

3-4. Synthesis of tetram er scaffold ............................................................................. 45

3-5. Synthesis of am ino acid derivatives ........................................ ........ ............... 46

3-6. Olefin CM of dimer scaffold with an amino acid derivative............... ............... 48

3-7. Synthesis of cis hom odim er 3-30a .............. .................................. ...... ................51

3-8. Synthesis of trans homodimer 3-30e .................................................52

3-9. CM of dimer scaffold with two amino acid derivatives .............. ................ 55

4-1. Comparison of building blocks 4-1 and 4-4 and their library constituents ...............60

4-2. Retrosynthesis of dipeptide 4-4 ............................................................................ 61

4-3. Library of dimers, tetramers, hexamers, oligomers, linear compounds and
c ate n an e s ................................................................6 2

4-4. Synthesis of 4-allylbenzoic acid (4-5).................................... ...................63

4-5. Synthesis of dipeptide 4-4 ................................................ .............................. 64

5-1. Degradation products of VX, following hydrolysis .................................................80

5-2. Form ation of hydrogen-bonded complexes............................................................. 81

5-3. F orm action of the M IP ......... ................. ..................................... ............................82

5-4. Mechanism of the ROMP polymerization..... ............................................. ... ...........82

5-5. Coupling of norbornene moiety to fluorescein maleimide .............. .....................84

5-6. Synthesis of cross-linking m onomer 5-11 ...................................... ............... 85

5-7. Synthesis of fluorescein amino hydrochloride 5-26............................................86


xiv









5-8. Synthesis of N-(5-fluoresceinyl)maleimide (5-7).....................................................87

5-9. Synthesis of com pounds 5-13 and 5-15.................................... ....... ............... 89

5-10. Synthesis of succinim ide 5-32........................ .. ............................. ............... 89

5-11. Synthesis of succinim ide 5-34........................ .. ............................. ............... 90

5-12. Oxidation and elim nation reactions................................... .......................... 91

5-13: Hydrogenation of fluorescein maleimide 5-7.............. ........ .................. 92

5-14: Attempted oxidation of fluorescein 5-37........................ ...................... 93















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

OLEFIN METATHESIS IN PEPTIDOMIMETICS, DYNAMIC COMBINATORIAL
CHEMISTRY, AND MOLECULAR IMPRINTING

By

Tammy Karrie Cheng Low

August 2006

Chair: Eric J. Enholm
Major Department: Chemistry

Catalysis based olefin metathesis is a very valuable and useful tool in synthetic

organic chemistry. Our research goals consisted of employing olefin metathesis in the

synthesis of peptidomimetics, and studying the feasibility of this method in dynamic

combinatorial chemistry and molecular imprinting of bioactive molecules.

One of the approaches to developing peptidomimetics is attaching biologically

significant molecules, such as amino acid chains, to a scaffold. Grubbs' second

generation ruthenium catalyst was used to couple phenylalanine to a 17-membered

lactam using cross-metathesis in 48% yield with an E:Z ratio of 1.2:1. The cross-

metathesis product of two phenylalanine amino acids was isolated in 45% yield and

found to be predominantly trans. The 17-membered lactam was constructed in 6 steps,

including the fundamental ring-closing metathesis reaction. The reversibility of the

cross-metathesis of this macrocyclic system was demonstrated, which is essential to the

development of a dynamic combinatorial library.









Olefin metathesis in dynamic combinatorial chemistry is of interest as a method in

generating peptidomimetic libraries. The olefin cross-metathesis reactivity and

selectivity of amino acid derivatives with a cyclic scaffold to generate diketopiperazine

peptide derivatives were investigated. Product yields were dependent on the amino acid

R groups, and whether the amino acid possessed an allyl or homoallyl moiety at the

carboxylate side. Stereoselectivity of the dipeptide derivatives was found to be

predominantly trans. Larger sized cyclic scaffolds were also synthesized to create a more

diversified library. In addition, several peptide derivatives possessing diene functionality

were examined and subjected to Grubbs' second generation ruthenium catalyst. Various

conditions, including substrate and catalyst concentrations, as well as diverse metal

templates, were explored.

Also described is a model study to investigate the feasibility of using ring-opening

metathesis polymerization in molecular imprinting technology capable of detecting

bioactive molecules, namely nerve agents. The project involved the synthesis of a

fluoresceinyl dye to be used as the detector and the investigation of thiols as templates in

the molecular imprinted system.














CHAPTER 1
HISTORICAL BACKGROUND

1.1 Olefin Metathesis

Olefin metathesis is a powerful synthetic tool that has found its way into a vast

array of applications, ranging from the development of small molecule drug candidates to

the industrial scale synthesis of petrochemicals.17 This catalytic organic reaction is

unlike other carbon-carbon bond forming strategies due to the versatility of synthetic

transformations it promotes, such as the synthesis of various sized cycloalkenes from

dienes and specialized polymers by the ring-opening of cyclic molecules. Olefin

metathesis has opened efficient synthetic routes to complex natural products, drug

molecules, and new materials as demonstrated by the explosion of metathesis related

applications found in the literature during the past decade. In 2005, the value of this

organic reaction was prestigiously recognized by the award of the Nobel Prize in

Chemistry to the major contributors of olefin metathesis Yves Chauvin, Robert H.

Grubbs, and Richard R. Schrock.

1.1.1 Development of Olefin Metathesis and Catalysts

Olefin metathesis was discovered accidentally by researchers in petrochemical

companies in the 1950s when they were searching for heterogeneous catalysts to convert

olefins to high-octane gasoline.7'8 Instead of the expected products, the chemists

observed newly formed olefins. It was not until the 1960s when researchers at Goodyear

Tire & Rubber determined that these new products were the result of an exchange of









substituents on different olefins, which they referred to as olefinn metathesis"9 as shown

in Scheme 1-1.

R1

R2 Catalyst R R2
SR3 R 4

R4

Scheme 1-1. Olefin metathesis

For years, chemists attempted to explain the mechanism behind the novel reaction

for the skeletal transformation of olefins. Calderon et al.,10 Lewandos and Pettit,11 and

Grubbs and Brunck12 initially suggested cyclobutane, tetramethylene complex, and

rearranging metallacyclopentane intermediates as part of the mechanism, respectively,

but all proposals later proved to be incorrect (Scheme 1-2).8 It was in 1971 when

Chauvin and Herisson proposed a metal-carbene mechanism which involves the

formation of a metallacyclobutane intermediate (Scheme 1-3).8,13 The debate over the

mechanism continued for years until Katz, Schrock, and Tebbe independently conducted

experiments, which supported Chauvin's proposal.1'8

During the debate over the olefin metathesis mechanism, several groups continued

to develop transition metal carbene complexes, including Fischer carbenes (low oxidation

state metals and electron poor carbon centers) and Schrock carbenes (high oxidation state

metals and electron poor metal centers).1'8 The Fischer carbenes showed little activity for

olefin metathesis, and Schrock's early tantalum and niobium complexes proved

unsuccessful as well.1'8 These initial studies however paved the road to improved

alkylidene complexes which eventually demonstrated improved reactivity for olefin

metathesis.










R1 R2 R1 R2 R1 R2 R3 R1

LM M
R3 pR4 R3 M R4
4M R3 R4 R4 R2


cyclobutane tetramethylene rearranging
intermediate complex metallacyclopentane

Scheme 1-2. Proposed intermediates for olefin metathesis

R
LnM--/ R
LnM

R1
R1

Scheme 1-3. Metallacyclobutane intermediate proposed by Chauvin

Despite early advances in catalyst development, olefin metathesis was not a

practical synthetic methodology due to the catalyst's low reactivity, instability, and lack

of tolerance towards functional groups. It was not until the 1990s when Schrock lab

introduced the well-defined alkoxy imido molybdenum-based catalyst 1-1, which made

olefin metathesis a useful tool (Figure 1-1).14,15 In contrast to many of the early catalytic

systems of the 1970s and 1980s, which are often referred to as "classical" or "ill-defined"

catalysts because the propagating species can not be observed, isolated, or structurally

characterized, the molybdenum alkylidene complex is highly reactive and leads to desired

products in high yields, even with sterically hindered alkenes.1,16 However, the downfall

is the catalyst's relatively limited tolerance toward polar functional groups such as

alcohols and carboxylic acids, and its sensitivity to air and moisture.5











iPr iPr
N Ph
(F3C)2 MeCO" 'Mo C Me
4/ Me
(F3C)2MeCO

Shrock's catalyst
1-1

Figure 1-1. Schrock's alkoxy imido molybdenum-based catalyst 1-1

In an effort to improve tolerance for functional groups and moisture, Grubbs group

examined ruthenium catalysts, which have an oxidation state lower than Schrock's

metallaolefins but higher than the Fischer carbenes. 117 Despite the development of the

ruthenium catalyst [(PPh3)2Cl2Ru=CHCH=C(Ph)2] (1-2) in 1992, which was stable in

protic and aqueous solvents, the catalyst exhibited limited reactivity compared to

Schrock's carbene complexes (Figure 1-2).1'18'19 Modifications of the ruthenium catalyst

were conducted throughout the years. Eventually in 1996, "Grubbs' first generation

catalyst" 1-3 was introduced, which not only displayed functional group tolerance, but

also up to 20-10,000 times greater activity than ruthenium catalyst 1-2 (Figure 1-2).20 In

1999, based on Herrmann and coworkers' studies on N-heterocyclic carbenes,21 Grubbs

group substituted one of the tricyclohexyl phosphine (PCy3) ligands from 1-3 with a

mesityl N-heterocyclic ligand to afford the more stable ruthenium complex 1-4, which is

now referred to as "Grubbs' second generation catalyst" and is far superior to other

catalysts because of its tolerance to air, moisture, and a wide variety of functional groups

(Figure 1-2).1,6,22,23 As a result of these enhanced properties, our research efforts focused

on olefin metathesis utilizing Grubbs' second generation catalyst 1-4.










PCy3
ClI I
KRu- Ph
Cl" -
PCy3 Ph



1-2


PCy3
Rum P
CI'"I Ph
PCy3

Grubbs' first
generation catalyst
1-3


Mes-N N'Mes
Cl1Y
c,.Ru=\
Cl Ph
PCy3

Grubbs' second
generation catalyst
1-4


(Cy = cyclohexyl, Mes = 2,4,6-trimethylphenyl)

Figure 1-2. Ruthenium catalysts

1.1.2 Mechanism of Olefin Metathesis

Commercial availability of ruthenium catalysts 1-3 and 1-4 has made them a

practical, standard organic tool; thus the synthesis of the metal alkylidene complexes will

not be discussed. To better apply olefin metathesis towards the synthesis of target

compounds and polymers, it is helpful to examine the mechanism that was first

introduced by Chauvin. When utilizing Grubbs' catalysts 1-3 and 1-4, the first step of the

mechanism involves the dissociation of the PCy3 ligand, followed by the binding of the

alkene to the carbene (Scheme 1-4).24,25 The next step is a [2+2] cycloaddition with the

metal catalyst to form the metallacyclobutane intermediate, which can then undergo a

cycloreversion to produce a new metal alkylidene (Scheme 1-5).4,25 The mechanism

proceeds as a catalytic cycle where the metal alkylidene undergoes another [2+2]

cycloaddition with a second alkene, followed by the cycloreversion leaving the newly

formed olefin with R1 and R2 groups and the metal alkylidene for further catalytic use.

L L L
Cl,, R -PCy3 Cl/,, R + olefin R
Ru-, R Ru/ Cl2-Ru
1 +PCy3 C olefin
PCy3 R

Scheme 1-4. Dissociative substitution of ruthenium catalysts









R1

R2 LnM=
+
[2+2]
cycloreversion R1

R2 LnM

LnM RI R1
LnM71 LnMF-R1

[2+2 ---R2 cycloreversion
[2+2 -
+- H2C=CH2

R1

Scheme 1-5. Mechanism of olefin metathesis

Since ethylene gas is released as a byproduct,6 it is possible to shift the equilibrium

toward the desired products by deliberately flushing the headspace with inert gas to

remove the evolved ethylene.26 The cycle continues until the reaction is quenched, for

example, with ethyl vinyl ether (EVE), which reacts with the ruthenium catalyst and

forms the Fischer carbene L(PCy3)(Cl)2Ru=CHOEt.24 The formation of the Fischer

carbene is virtually irreversible and the electron rich carbene complex is significantly less

reactive than the ruthenium alkylidenes.24,27

1.1.3 Important Types of Metathesis Reactions and Applications

As highlighted many times, olefin metathesis is a versatile technique which

includes ring-closing metathesis (RCM), ring-opening metathesis (ROM), cross-

metathesis (CM), ring-opening metathesis polymerization (ROMP) and acyclic diene

metathesis (ADMET) (Scheme 1-6).6 Discussed in greater detail are the three main

metathesis reactions of interest in our studies, RCM, CM and ROMP.









RCM is the cyclization of a diene to generate various sized cycloalkenes, ranging

from small 5-membered rings to macrocycles.16 The stereochemistry of the cycloalkene

products is dependent on the substrates; for example, small and medium sized rings

formed from RCM are in a less strained cis conformation while in contrast, the

stereochemistry of non-rigid RCM derived macrocyclic compounds is difficult to predict

and can encompass a mixture of cis and trans stereoisomers.28

RCM reactions are conducted under highly dilute conditions to prevent ADMET

polymerization. In addition, heat is often employed to improve ring closures due to the

entropy of activation required to bring the two ends of the chain together.29 However,

higher temperatures can cause the catalyst to decompose; thus a greater catalyst loading

is required.5 Despite this requirement, RCM has provided a shorter, more efficient

synthetic route to natural products, drug molecules, and new materials, compared to

conventional methods. An example is shown in Scheme 1-7 in which Van et al. utilized

RCM to synthesize coumarins in excellent yields, while other methods reported in

literature had disadvantages and required harsher conditions.30

The reverse reaction of a RCM is ROM, where the cycloalkene breaks open to form

two terminal dienes, which can be followed by a CM reaction with other acyclic alkenes

to form new products.5 Similar to RCM, ROM requires dilute conditions due to the

resulting dienes undergoing polymerization, referred to as ROMP. The polymerization is

quite practical in the synthesis of specialized polymers and is more widely used than

ROM. Highly strained cycloalkenes, such as norbornene, cyclopentene and cyclooctene

favor ROMP.16 By reducing ring strain, the reaction is enthalpically driven forward and

is not reversible.











-C2H4
RCM
ROM
+C2H4


ADMET
- n C2H4


CM
R1 + -C2H4
/ 7:----------

R2

Scheme 1-6. Types of olefin metathesis


O


ROMP


R R R


Heterodimer Homodimers
Heterodimer Homodimers


O
k^


1-4 (5 mol%)
CH2C12, reflux, 4 h


1-5a R1 = OMe; R2 = R3 = R4 =H 1-6a 81%
1-6a 81%
1-5b R1 = R2 = R4 = H;R3 = OMe 1-6b 90%
1-5c R1 = R4 = H; R2 + R3 = O-CH2-O 1-6c 83%
1-5d R1 = R4 = OMe; R3 = R2 =H 1-6d 72%
1-6e 70%
1-5e R1 = R4 = R3 = R2 = H

Scheme 1-7. Utilizing RCM to synthesize coumarins

Grubbs' catalyst 1-4 has high functional group tolerance and has been

demonstrated in ROMP to generate functionalized, telechelic and trisubstituted

polymers.31 ROMP has been responsible for the synthesis of a variety of new materials,

from the development of nonlinear optics to biologically relevant polymers.32 A recent









application of this polymerization is shown in Scheme 1-8, where a polymer was

synthesized to create biomaterials that can undergo a [2+2] cycloaddition when irradiated

with UV light.33



/ 0 <- Ph ROMP OPh
O -.Ph 1-3, CH2Cl2 n P

O 79% O
1-7 1-8

Scheme 1-8. Employing ROMP to create new materials

RCM and ROMP started as the most popular types of metathesis reactions, but due

to recent studies and a better understanding of the selectivity and stereoselectivity of CM,

it has become a more useful and versatile synthetic technique. The concerns over

selectivity arose from the mixture of heterodimers, homodimers, and cis/trans

stereoisomers that can be generated from CM reactions. In addition, employing

asymmetric internal olefins in CM can also lead to a greater number of product mixtures

(Scheme 1-9). Factors such as sterics and electronic effects may also affect CM

reactivity and selectivity, and must be considered when planning reactions. For example,

olefins possessing electron withdrawing or bulky substituents often lead to little or no

homodimerization because of the poor reactivity with the catalyst. However, steric

effects can favor trans selectivity.34









R1 R1 R2 R2
R2 + R+ + Heterodimers

R1 CM
R R1 R2 R3 4

R3 + R+ R+ Homodimers
R1 R 3 R4


Scheme 1-9. CM of asymmetric internal olefins

Fortunately, new models and methodology were developed to improve selective

CM. For instance, Grubbs group categorized olefins as Type I, II, III, and IV based on

their reactivity to form homodimers by CM with catalyst 1-3 and 1-4. Primary allylic

alcohols, protected amines, and esters are examples of Type I alkenes (sterically

unhindered, electron-rich) because they readily form homodimers by CM and also

undergo secondary metathesis reactions.26'34'35 The more sterically hindered Type II

alkenes (i.e., secondary alcohols and vinyl ketones) are less reactive, and Type III alkenes

are nonreactive (i.e., tertiary allylic carbons). Type IV alkenes (i.e., protected

trisubstituted allyl alcohols) are spectators and do not participate in the CM reaction. The

examples given above are based on the utilization of catalyst 1-4. One strategy toward

selective CM involves a two step procedure in which homodimers of Type I alkenes are

generated, followed by a secondary metathesis reaction with Type II/III alkenes to

preferentially form the heterodimer product, which can favor the trans isomer in the

presence of selected functional groups (Scheme 1-10).34 CM is more widely used now,

and an example of a recent application of CM is shown in Scheme 1-11, where Nolen and

coworkers were able to synthesize C-glycosyl asparagines in good yields with

predominantly trans selectivity.36










RI- R2
SCat. R1. R 1 Cat
R2- +
R1-

Scheme 1-10. Primary and secondary CM metathesis reactions

AcA A5 H\ + 5 CO2Me 1-4 (20 mol%) Ac 0.\
^Acc AcO < NHCbz CH2C12, C Ac OCO2Me
reflux, 12h NHCbz
1-9 1-10 1-11 82%
predominantly E

Scheme 1-11. Synthesis of C-glycosyl asparagines via CM

1.2 Peptidomimetics

Olefin metathesis is now a common synthetic tool for the organic chemist. As

shown by the examples in the previous section, it is versatile and has been used to make

new materials and analogues of natural products. Other areas of science that have a

particular interest in metathesis are peptide and medicinal chemistry. Peptides, made of

amino acid building blocks, are vital biological molecules with vast functionality. For

example, they can serve as antibiotics, analgesics, and building blocks of important

proteins, such as enzymes. Therefore, peptides have gained significant interest as drug

candidates. Unfortunately, natural peptides typically do not make good pharmaceuticals

because of their lack of bioavailability and susceptibility to premature hydrolysis in-

vivo.37,38 A solution to this downfall is peptidomimetics, which are synthetic molecules

that can mimic the peptide's topography and functionality, but possess improved

pharmaceutical properties.39'40

One strategy to enhance these properties is by synthesizing cyclic molecules that

mimic peptides, which are referred as cyclic peptidomimetics. Frequent discovery of









natural cyclic peptides possessing antibiotic, antiviral, antitumor, and therapeutic

properties has made them an active area of research.41'42 In addition, cyclic peptides are

more stable than linear peptides because of their constrained conformation, which

improves receptor selectivity.37 Therefore, there is great interest in the synthesis of

mimetics of cyclic peptides for use in medicinal chemistry and drug development.

Peptidomimetics can be designed to be less susceptible to proteolysis, and biostable with

improved ability for absorption in the body.43'44

Several methods exist to synthesize cyclic amino acids and peptidomimetics. One

strategy involves cyclization of a linear peptide by conventional coupling agents to form

a new amide bond (Scheme 1-12).37,45 Some common reagents used to perform this task

are dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and expensive

reagents such as HATU or PyBOP.45,46 Racemization of the chiral center is of great

concern, and often times racemization suppressants such as 1-hydroxy-7-aza-

benzotriazole (HOAt) and 1-hydroxybenzotriazole (HOBt) must be employed.45

Cyclization can sometimes be complicated due to difficulties in bringing the two terminal

ends together;42 thus peptidomimetics are often designed where hydrogen-bonds can

assist in the ring closure by inducing the linear peptide to turn (Figure 1-3).37,39

It is also possible to synthesize cyclic peptidomimetics by attaching biologically

significant molecules, such as amino acids, to a scaffold.43'44'47'48 The scaffold can be

designed where the functional groups are properly oriented to their corresponding

binding sites. For example, Chery and Murphy synthesized potential HIV protease

inhibitors by grafting pharmacophoric groups on a azasugar scaffold (Figure 1-4),48 but a

rather lengthy synthetic scheme starting from D-fructose and involving selective









deprotection was required. Other types of scaffolds have been generated including

conformationally constrained bicyclic amino acid motifs39 and pyridine derivatives.44


H2N H
HN coupling reagents N

0 -H20 0


linear peptide cyclic peptide

Scheme 1-12. Cyclization of a linear peptide using coupling agents

O R
R O

HN H
O-----H-N

NH 0



Figure 1-3. Hydrogen-bonding leads to bent conformation

Above is a brief overview of peptidomimetics and some strategies used to generate

cyclic mimetics. Methodologies used to synthesize scaffolds and cyclic peptides often

involve intricate synthetic procedures. Recently, RCM has been utilized as an alternative

method towards generation of cyclic peptidomimetics.37,49'50 An example is shown in

Scheme 1-13, where Gmeiner group employed RCM to synthesize P-turn mimetics in

excellent yields.5









Charged amine can hydrogen
bond with the carbonyl group
of HIV-protease amide
backbone


H OH < Binds to aspartic acids


S)OH Oriented into enzyme subsites


1-12

Figure 1-4. Design of putative azasugar peptidomimetic

0 OMe O Me
1-4 (7 mol%) c- -
-N CH2C12, 40 OC
0 NHBoc 88%
O NHBoc

1-13 1-14

Scheme 1-13. Use of RCM toward the synthesis of 3-turn mimetics

1.3 Dynamic Combinatorial Chemistry

In addition to applications in peptidomimetics, olefin metathesis in dynamic

combinatorial chemistry (DCC) is very promising. DCC has gained interest in recent

years as a powerful methodology for exploring molecular recognition systems, thereby

leading to the discovery of new biologically active molecules, drugs, receptors, and

catalysts.52'53 A dynamic combinatorial library (DCL) is made from molecular building

blocks connected via reversible linkages which interconvert in a thermodynamic

equilibrium.52,53 A template, such as a biological molecule, is added to the system which

can shift the equilibrium to form one major constituent in the library (Figure 1-5).54

Unlike traditional combinatorial techniques, where molecules are created en masse and









tested for desired properties, DCC combines the synthesis and selectivity of library

constituents in one pot. Key differences between DCC and traditional combinatorial

chemistry are shown in Table 1-1. DCC is not only valuable in discovering new

properties, but also as a learning tool towards a better understanding of molecular

recognition, self-assembly, and supramolecular chemistry.






f. To'Tlate,





Reprinted from Publication: Curr. Opin. Chem. Bio.; Vol 6; Otto, S.; Furlan, R. L. E.; Sanders, J. K. M.;
Recent Developments in Dynamic Combinatorial Chemistry; 321-327; Copyright 2002, with permission
from Elsevier.

Figure 1-5. A dynamic combinatorial library and its free energy landscape

Table 1-1. Differences between traditional and dynamic combinatorial libraries
COMBINATORIAL LIBRARY DYNAMIC COMBINATORIAL LIBRARY
Real set Virtual set
Collection of molecules Collection of components
Covalent Covalent or non-covalent
Non-reversible Reversible
Systematic Recognition-directed
Preformed by synthesis Self-assembled
In absence of target In presence of target

There are two types of dynamic combinatorial recognition described by biological

aspects of receptors and substrates (Figure 1-6).53,55 In the casting process, the target

receptor (TR) influences the organization of the building blocks into the substrate with

specific binding affinity to the TR. In the molding process, the target molecule is a

substrate (Ts) which results in the molding of the receptor.









The examples in Figure 1-6 demonstrate the addition of the target molecule to the

reaction mixture after the library of building blocks have assembled. The presence of the

target can then shift the equilibrium where only the constituents with the highest affinity

for the target will be amplified. This is considered a true dynamic combinatorial

system.56 The other method, which is sometimes referred as the generation of a virtual

combinatorial library, involves the addition of the target molecule without formation of a

library of interchangeable species, as depicted by the lock and key metaphor shown in

Figure 1-7.56

TR
LC EA) TR



Casting











Molding

Figure 1-6. Casting and molding process in DCC

There are different types of reversible reactions that can potentially be used in

DCC, such as covalent bond formation, non-covalent interactions and intramolecular

processes.56 Some reversible reactions that have already been studied for use in DCC

include disulfide exchange,5 metal-ligand coordination,58 exchange of oximes,59

hydrazones,60 and olefin metathesis.61'62














generation election


j hairy of
SInterhanging


u <^ uidirhdg-- Receptor q




SveclionOf
Receptor best binder




Nature Reviews I Drug Discovery
Reprinted from Publication: Nature Rev. Drug Discov.; Vol 1, Ramstrom, 0.; Lehn, J. M.; Drug Discovery
by Dynamic Combinatorial Libraries; 26-36; Copyright 2002, with permission from Nature Review Drug
Discovery, MacMillan Magazines Ltd.

Figure 1-7. Dynamic combinatorial chemistry (top) versus virtual combinatorial libraries
(bottom)

Even with the expanding amount of dynamic combinatorial related research found

in literature, very few studies have examined olefin metathesis in DCC. One of the most

interesting developments came from Nicolaou group in 2000, where they proved the

applicability and significance of DCC via olefin metathesis by synthesizing dimers of

vancomycin derivatives (Scheme 1-14).63 The building blocks possessed various olefin

chain lengths and different amino acid R groups, while the amino acid target chosen had

a high affinity to vancomycin.63 As they expected, the dimers with the shorter tethers

were amplified upon addition of the target (Figure 1-8).63 Despite Nicolaou's important

studies on DCL of vancomycin derivatives, there is still more to learn about the








18



reactivity, influence of functional groups, and stereochemistry of olefin metathesis


reactions for use in DCC.


Olefin Metathesis


Dimers


Scheme 1-14. Dimerization of monomeric vancomycin derivatives with terminal olefins
by metathesis


'10 shorter tethered
d im er q I


IL ^UO.IC2
50 tLNelC
4LSUNM 'C;


'V


13-(LuNflM)C2- .(L4MN)C4
. + 13-(LuNeM)Ce- (LaNlMWaJC3


I fLK4Me|C1
ML.tHY eCI


S


iLcuLM. Cj
iLuY~C


- I


.1 ~IBl~iF lkC
iii ~OS L~ITF



r'-~~1~t


J
I',


'VPi* I'M


Reprinted from Publication: Angew. Chem. Int. Ed.; Vol 39; Nicolaou, K. C.; Hughes, R.; Cho, S. Y.;
Winssinger, N.; Smethurst, C.; Labischinski, H.; Endermann, R.; Target-Accelerated Combinatorial
Synthesis and Discovery of Highly Potent Antibiotics Effective Against Vancomycin-Resistant Bacteria;
3823-3828; Copyright 2000, with permission from Wiley.


Figure 1-8. Mass spectrometric analysis of the vancomycin dimer mixture. (A) Absence
of a target and (B) in the presence of a target


1.4 Molecular Imprinted Polymers


There has been a great interest in the Enholm group to employ ROMP in molecular


imprinted technology, which has only been reported once in the literature by Steinke


A


I


% f N W.%


:i i II
f~' i
*









lab.64 Molecular imprinted polymers (MIPs) are useful as mimics of biological receptors,

enzymes, and antibodies,6570 and can be designed as drug delivery or drug separation

systems.67 In addition, research has already shown that MIPs can have enhanced

catalytic activities compared to corresponding natural catalytic antibodies.70 In addition

to a plethora of studies found in the literature related to biological applications, this

technology has also been used for chemical sensors71'72 and environmental analysis.71

The general principle behind molecularly imprinted technology involves the

synthesis of a polymer that possesses a 3-dimensional molecular memory or "imprint" for

a target. For example, in the lock and key metaphor by Emil Fischer, an enzyme ("lock")

has active sites specific for a particular substrate ("key") (Figure 1-9). In MIPs, the

polymer acts as an enzyme by having a cavity with functional groups that can interact

covalently or non-covalently with the template (Figure 1-9). Like enzymes, the polymer

has a specific binding affinity for the template.

The general process of molecularly imprinted technology is shown in Figure 1-10,

in which a template and functional monomers, which include a polymerizable moiety,

interact to form a stable template-functional monomer assembly.69 A cross-linker is

added and polymerization proceeds resulting in a rigid template-cross-linked polymer

complex. The template is then extracted to give the MIP.

There are two main ways a template can interact with the functional monomers -

the self-assembly approach via non-covalent (i.e. electrostatic, hydrogen bonding, and

hydrophobic) or metal coordination interactions, and the pre-organized approach via

reversible covalent interactions,69 where both methods have their advantages and

disadvantages. With the pre-organized approach, the template is fixed in its proper









orientation during polymerization resulting in a pronounced imprint.65 However, only

selected reversible covalent reactions, such as the formation and hydrolysis of boronate

esters, are suitable for MIP.68,72 An example of the pre-organized approach is shown in

Scheme 1-15, in which Wang et al. built fluorescent sensors using boronate esters.73

A: "Lock and Key"

SS +. Enzyme S Enzyme

Active Sites
S = Substrate


B: MIP



S + Active S





\ Polymer

Figure 1-9. A comparison between the Lock and Key model and the MIP model

The non-covalent approach is the most widely used due to the ease of template

removal by breaking non-covalent interactions.67 However, more functional monomers

are required to fix the template in place prior to polymerization. If the template is not

properly set, then the resulting molecular imprint could have a reduction in the specificity

for the template. A solution to both methods is a semi-covalent approach, in which the

template is covalently bound during imprinting and non-covalently bound during

rebinding.72 For example, Vulfson group designed a molecular imprint of a tripeptide

using this semi-approach (Scheme 1-16).74














Template
Template


Functional Monomers


CPolross-Linking
Polymerization Monomer


Template Extraction





0


Polymer


Molecular Imprinted Polymer

Figure 1-10. Schematic of molecular imprinting process


Scheme 1-15. An example of the pre-organized approach via covalent interactions
Radical polymerization is the standard method for the synthesis of MIPs, where the

functional and cross-linking monomers chosen for MIP often possess vinyl or acrylic

groups, such as methyl methacrylate (MMA) and ethylene glycol dimethacrylate









(EDMA). A large variety of these monomers are readily available and have functional

groups exhibiting hydrogen bonding, hydrophobicity, and other interactions with the

template.66 The radical polymerization can proceed via thermal initiators (azo-bis-

isobutyronitrile (AIBN) is most commonly used), or photoinitiators.72



0N
HN 0 HN O



S N CO2H divinylbenze, N C 02
H n AIBN H H'


Scheme 1-16. An example of a semi-covalent approach to MIPs

In 2003, Steinke group used ROMP rather than radical polymerization to

synthesize enantioselective MIPs to thermodynamically control the polymerization

reaction.64 The goal was to prevent the formation of MIPs polyclonal cavities, which can

decrease selectivity for the template. This was the first time ROMP was demonstrated in









the generation of MIPs. During that same time period, Allias performed a side by side

comparison of radical polymerization and ROMP employing Grubbs' 1-4 catalyst, and

discovered that ROMP required shorter reaction times and easier work-up conditions.75

Preliminary studies also showed that there was an increase in the affinity for the template

using the ROMP strategy, while the MIP cavity generated by radical polymerization

showed poor selectivity for the template. Another benefit of employing ROMP is that

heat or photo labile templates which are not acceptable in radical polymerization can now

be used in the system. Based on these initial studies, it appears promising to examine

olefin metathesis in molecularly imprinted technology.

1.5 Conclusions

Olefin metathesis is a powerful organic synthetic tool, attested by the large volume

of metathesis related research found in literature. Grubbs' second generation catalyst 1-4

and its tolerance for functional groups have made this methodology even more useful.

However, there are still areas of olefin metathesis that require more studies in the field of

peptidomimetics and MIPs. The work presented here will examine the use of olefin

metathesis in the 1) attachment of an amino acid on a macrocyclic scaffold by CM; 2)

synthesis of dipeptide mimetics by CM and the examination of the reactivity,

stereochemistry, and feasibility of CM in DCC; 3) generation of a DCL of macrocyclic

compounds employing peptidomimetic dienes and 4) development of chemical sensors

using ROMP in molecularly imprinted technology to detect nerve agents.














CHAPTER 2
USE OF CROSS-METATHESIS TO COUPLE L-PHENYLALANINE TO A
MACROCYCLIC LACTAM

2.1 Introduction

Peptidomimetic research is of paramount importance to the field of medicinal

chemistry. One approach toward the synthesis of peptidomimetics is to use molecular

templates or scaffolds to which biologically significant functional groups, such as amino

acid chains, are covalently anchored.26'30'33'62 These molecules have an excellent

potential for chiral discrimination and display stabilization of functional groups.

Macrocyclic lactams are excellent choices as scaffolds because many naturally

occurring macrocycles possess important biological and medicinal properties.76'77 In

addition, the ability to attach multiple functional groups on a large ring can increase

diversity, essential to drug discovery.7 One example of a macrocyclic scaffold used for

drug discovery is shown in Figure 2-1, in which side chains are attached to a rigid

macrocyclic lactam in a well-defined orientation toward receptor sites.79

AllO2C


NH HN
N 0 /NHBoc

MeO2C 0 N
/ \ NH HN

0 0

2-1 NHCbz
2-1Figure 2-1. Oxazole-based macrocyclic lactam
Figure 2-1. Oxazole-based macrocyclic lactam









Ring-closing metathesis (RCM) has recently become a useful organic reaction to

synthesize macrocylic scaffolds as demonstrated in Scheme 2-1.77 In this example, Peng

and Blagg utilized Grubbs' first generation catalyst 1-3 to generate an analogue of

Trienomycin A, an antibiotic which also possesses antitumor activity.77

R R


NH 1-3 (12 mol%) NH

S 0 CH2C12
r.t. 24 h
trans:cis, 3.5:1
45%
2-2 2-3

Scheme 2-1. Synthesis of an analogue of Trienomycin A utilizing RCM

Another example of metathesis derived macrocyclic lactams is shown in Scheme 2-

2, in which Hu and associates discovered that 15-17 membered rings provided the best

inhibitor properties against an enzyme involved in bacterial biosynthesis.76


H H
H NNH, 1-3 (7 mol0/o HNN N N
H N N N
OBn H ) CH2Cl2 OBn H 0 n
( n reflux 20 h n


2-4 2-5

Scheme 2-2. Synthesis of a macrocyclic inhibitor

The examples above demonstrate the importance of macrocyclic lactams as

scaffolds in drug development. As part of our continuing interest in cyclic

peptidomimetics, we envisioned RCM as a means to generate macrocyclic scaffolds and

cross-metathesis (CM) as a way to anchor amino acids to the scaffolds. Model system 2-









6 is ideally suited for a CM reaction1'34'80'81 since it has a dumbbell shape with two halves

connected together by an E-alkene tether (Figure 2-2). We believed that Grubbs' well-

defined second generation catalyst 1-4 would be an ideal choice to facilitate this reaction

due to its high tolerance of functional groups.8284


H 0 O
t-BOC


O0
2-6

Figure 2-2. Phenylalanine on a macrocyclic lactam

In addition to examining RCM and CM as a synthetic approach toward

peptidomimetics, we also wanted to test the reversibility of the CM reaction in this model

system for potential use in dynamic combinatorial chemistry (DCC) of cyclic

peptidomimetics.85'86 We envisioned a macrocyclic scaffold possessing several

anchoring points where functional groups could be attached by a CM reaction, as shown

in Figure 2-3. The CM reaction must be reversible such that functional groups can attach

and detach onto the scaffold in a dynamic equilibrium. Olefin metathesis is known to be

reversible, though only Miller lab has systematically examined the reactivity and

selectivity of CM and the effects of remote functionality for potential use in DCC.62

However, the work conducted by Miller group62 and other researchers61'63 focused on the

homodimerization of their substrates. In contrast, our focus is on anchoring amino acid

derivatives on molecular scaffolds. Dynamic combinatorial libraries (DCL) of cyclic

peptidomimetics will be discussed further in Chapter 3. The main focal point of this









chapter will be on the synthesis of model compound 2-6 by RCM and CM, followed by

examination of the reversibility of the CM reaction.

Sites for CM reactions



R1
R4
ON O R2

o N 0
N N R3



R = various amino acids

Figure 2-3. Macrocyclic lactam with anchors for CM with amino acids

In examining the viability of our approach to the synthesis of 2-6, we decided that

anchoring the amino acid to the nitrogen atom of a large-ring lactam would function well

(Scheme 2-3). Compound 2-6 could be prepared from protected L-phenylalanine 2-7

bearing an allyl ester and allyl lactam 2-8. Each of these halves of the molecule contains

a terminal alkene to be used in the CM reaction. Precursor 2-8 was to be prepared by

RCM and the alkene would later be removed by hydrogenolysis. Eventually we

envisioned 2-8 as emanating from the SN2 coupling of bromo-amide 2-9 and alcohol 2-

10.










H O
2-6 = > t-BOC.N_. O + 0N



2-7 2-8

IH Br OH


0
2-9 2-10

Scheme 2-3. Retrosynthethic analysis of 2-6

2.2 Results and Discussion

2.2.1 Synthesis of Compound 2-6

We began our synthesis with a Williamson etherification using commercially

available 1,8-octanediol (2-11) and allyl bromide (0.9 eq), as shown in Scheme 2-4. The

desired allyl ether 2-10 was obtained in 70% yield, while the minor product, double

Williamson ether 2-12, was isolated in 25% yield. The use oftetrabutylammonium

iodide (TBAI) was essential in obtaining high yields.87

NaH, THF, TBAI O
allyl bromide
2-11 2-10: Y = H, 70%
2-12: Y = allyl, 25%

Scheme 2-4. Synthesis of 2-10 and 2-12

Next, N-Allyl-2-bromoacetamide (2-9) was readily synthesized in 73% yield from

allyl amine (2-13) and dibromide 2-14 followed by recrystallization in hexane/ether,88 as

shown in Scheme 2-5. Deprotonation of 2-10 with NaH, addition of TBAI, and

nucleophilic substitution of allyl-bromoacetamide 2-9 gave a 54% yield of the terminal

diene 2-15.










NH Br Br CH2Cl 0 OC, 3 h H Br
N 73% N

2-13 2-14 2-9



NaH, THF, TBAI, 2-10H H

54% 0 O
0
2-15

Scheme 2-5. Synthesis of diene 2-15

Diene 2-15 was reacted with Grubbs' catalyst 1-4 under various conditions

(Scheme 2-6 and Table 2-1) to give two 17-membered RCM lactams, 2-16 and 2-17, as

geometric isomers.3 All reactions were refluxed in CH2C12 and monitored by thin layer

chromatography (TLC). In every entry, 2-15 was never fully consumed. We were

pleased with the excellent trans selectivity of our macrolactam; however, the next

synthetic procedure required the hydrogenation of the alkene making stereoselectivity a

non-issue. None the less, the E-isomer 2-16 was isolated as the major product, which

was separated from Z-isomer 2-17 by chromatography over silica gel. For entries 6-8, the

cis-isomer was not isolated due to the small scale of the reaction.



HN
1-4
2-15 1 HN
CH2C12 reflux
61%
61 2-16 (E) : 2-17 (Z)

20: 1


Scheme 2-6. RCM of 2-15









After RCM, cycloalkenes 2-16 and 2-17 were hydrogenated using Pd on activated

C (10% Pd) as the catalyst to give the corresponding saturated lactam 2-18 in high yields

(Scheme 2-7). According to NMR spectra and TLC, the compound was pure enough for

use in the next step. Addition of NaH, TBAI, and nucleophilic substitution of allyl

bromide, gave 2-8 in 62% yield. Two different conformations of 2-8, due to the rotation

about the lactam C-N bond, were present in roughly equal amounts at ambient

temperature and readily detected by 1H NMR and 13C NMR by the doubling of peaks.

Table 2-1. RCM to obtain 2-16 and 2-17
Conc 1-4 Time Yield E:Za
Entry (M) mol% (h) % 2-16:2-17
1 0.001 10 25 59 93:7

2 0.0014 10 21 45 98:2
3 0.0016 10 24 46 92:8
4 0.0016 10 16 61 96:4
5 0.0018 8 19 60 93:7
6 0.003 14 3.5 48 NA
7 0.007 8 2 35 NA
8 0.013 5 20 18 NA
a The reported E/Z ratio is based on isolated compounds


H2, Pd on C, 98% NaH, THF, TBAI
2-16 IN allyl bromide, 62% N


0 0
2-18 2-8

Scheme 2-7. Synthesis of lactam 2-8

The next step involved the CM ofN-allyl lactam 2-8 with the allyl ester of t-Boc-

phenylalanine 2-7 (Scheme 2-8). To synthesize the amino acid derivative, commercially

available t-Boc-L-phenylalanine was treated with 1,3-diisopropylcarbodiimide (DIC),

hydroxybenzotriazole (HOBt) and allyl alcohol to give 2-7 in 95% yield. We were









pleased that the CM of phenylalanine derivative 2-7 (2 equiv.) and N-allyl lactam 2-8 was

observed with 5 mol% of catalyst 1-4. The reaction was heated at 55 C in CHC13 for 21

h while continuously flushing out ethylene with argon to drive the reaction forward.

Additional CHC13 was added as needed to keep the concentration to ca. 1 M. After

quenching the reaction with ethyl vinyl ether (EVE), purification by column

chromatography gave the desired product 2-6 in 48% yield with an E:Z ratio of 1.2:1.

The stereoisomers were not separable by column chromatography and the reported

cis/trans ratio was determined by 1H NMR of the isolated compound. Similar to the 13C

NMR of N-allyl lactam 2-8, we also observed the doubling of peaks for 2-6 due to the

rotation about the lactam C-N bond. Column chromatography of the crude reaction

mixture also afforded amino acid dimer 2-19 in 45% yield with a predominantly trans

configuration.


H 0 y
t-BocN oO N

2-6
E:Z, 1.2:1 0
H O (48% yield)
t-Boc' 0N '' 1-4, 2-8

2-7 CHC13 H i
t-Boc O Or N t-BOC
S 2-19 O H
(45% yield)

Scheme 2-8. CM reaction to obtain 2-6 and 2-19

The moderate yields and lack of CM stereoselectivity in the preparation of the

heterodimer 2-6 were expected since both amino acid 2-7 and lactam 2-8 were considered

Type I olefins with fast homodimerization reactivity,34 as discussed in Chapter 1.









Interestingly, the dimer ofN-allyl lactam 2-8 was not observed. A 2:1 ratio of amino acid

2-6 and lactam 2-8 in the CM reaction should result in a statistical heterodimer selectivity

of 66%, which is slightly higher than the 52% selectivity we obtained. In regards to the

stereoselectivity, the trans homodimers were predominantly formed, which is most likely

due to secondary CM reactions to produce the more thermodynamically favored isomer.34

However for heterodimer 2-6, we believed that the bulky lactam may prevent cis/trans

isomerization by secondary metathesis reactions; thus resulting in the lack of

stereoselectivity.34

2.2.2 Examining the Reversibility of the CM reaction in Model 2-6

We examined the reversibility of CM in this model system for potential use in DCC

using compound 2-6. It is known that CM is reversible, and was tested in our

system.52,85,86 We were able to confirm that 2-6 can be converted back to allyl lactam 2-8

and amino acid derivative 2-7 (Scheme 2-9). Ethylene gas, used without purification,

was admitted via a balloon to a stirred solution of compound 2-6, catalyst 1-4 (2 mol%),

and CH2C12. The solution was first maintained at room temperature overnight. TLC

showed the formation ofN-allyl lactam 2-8 and allyl ester 2-7. In an attempt to drive the

reaction further, the solution was refluxed in CH2C12 for 2.5 h under an atmosphere of

ethylene gas, then quenched with EVE upon cooling. Purification by chromatography

afforded N-allyl lactam 2-8 in 36% yield and allyl ester 2-7 in 37% yield. Both the amino

acid dimer 2-19 and unreacted starting material 2-6 were recovered as well. These

studies demonstrated the potential of using allyl lactams and allyl amino acids as building

blocks for DCL of cyclic peptidomimetics.










"0 HQ
2-6 1-4 CN + -BocN O
CH Cl2
ethylene
0
2-8 2-7

Scheme 2-9. CM reaction with ethylene gas

2.3 Conclusions

In summary, Grubbs' second generation ruthenium catalyst was used to couple the

amino acid phenylalanine to a 17-membered lactam using CM in 48% yield with an E:Z

ratio of 1.2:1. The CM product of two phenylalanine amino acids was isolated in 45%

yield and found to be predominantly trans. The 17-membered lactam was constructed in

6 steps, including the fundamental RCM reaction. The reversibility of the CM of this

macrocyclic system was demonstrated, which is essential to the development of a DCL.

Based on the results of our model study, the next goal was to generate a small library of

cyclic peptidomimetics to determine the shift in dynamic equilibrium upon addition of a

template.














CHAPTER 3
OLEFIN METATHEIS OF AMINO ACID DERIVATIVES WITH CYCLIC
SCAFFOLDS

3.1 Introduction

As discussed in Chapter 2, one of the approaches to the development of

peptidomimetics is to attach biologically significant functional groups to a scaffold. An

example of a scaffold used for drug development is shown in Figure 3-1, where amino

acids are linked to a carbohydrate backbone.47 Another important type of scaffold is one

that is peptide based. For example, glycine based scaffolds are of interest because it has

been shown that they can potentially act as host molecules.89

/NH
BnO NR2

BnO' O I R2N O

BnO -0 O -N-NR
0 N

NH2 R = CH2CH2OBn

Glucose-based nonpeptide scaffold Glycine-based scaffold

Figure 3-1. Examples of scaffolds

By having terminal olefin moieties attached to peptide based scaffolds, there is

potential to create a dynamic combinatorial library (DCL) of peptidomimetics via olefin

cross-metathesis (CM). Numerous studies have shown the advantages of dynamic

combinatorial chemistry (DCC) over traditional combinatorial chemistry. Employing

olefin metathesis in DCC can lead to the efficient synthesis and isolation of biologically

significant peptidomimetics. There have been no reports in literature where cyclic









scaffolds and amino acid building blocks were used to create a dynamic library of

peptidomimetics.

As part of our ongoing study directed toward the attachment of amino acid

derivatives to cyclic scaffolds by olefin CM,90 our research group is interested in studying

the viability of reversible olefin CM for DCC of cyclic peptidomimetics. The

constituents of our library are made by CM of amino acid precursors with a cyclic

scaffold (Figure 3-2). Both the amino acid precursor and the cyclic molecule must

possess a terminal alkene. We are examining the reactivity of various types of amino

acid precursors and cyclic scaffolds. In addition, several templates are also being tested

on the reaction to select for the best constituent in the library.










Building Blocks j


Dynamic Library

Figure 3-2. Dynamic combinatorial libraries of peptidomimetics

Amino acids can be combinatorially arranged around a glycine based lactam by a

reversible CM reaction, as shown in Figure 3-3, to prepare DCL 3-1 on a cyclic scaffold.

To examine the viability of the approach, model compound 3-2 was utilized in anchoring

amino acid derivatives to the nitrogen atoms of a lactam.









aa7 reversible
aa6 aa2 CM linkage
aa4 aal n\ N, k)n
aa8 aa5 aal n
aa3
3-1 3-2
dynamic combinatorial model
library

Figure 3-3. Schematic of a DCL and a model of amino acids linked to a cyclic scaffold by
olefin CM

Miller group recently demonstrated the importance of remote functionality of olefin

CM using Grubbs' first generation catalyst 1-3 for potential use in DCC.62 Here we

report product yields and distributions of CM reactions of allyl and homoallyl ester

amino acid derivatives with a rigid cyclic scaffold using Grubbs' second generation

catalyst 1-4.

Several cyclic scaffolds are in consideration for these studies dimer 3-3, trimer 3-

4, and tetramer 3-5 (Figure 3-4). Dimer 3-3 is of interest because cyclic dipeptides

possess significant medicinal and biological characteristics.41,91-93 In comparison to

linear peptides, diketopiperazines are conformationally constrained and more stable

toward hydrolysis, which is critical in drug design.9496 In addition, the diketopiperazine

peptide derivatives could also be used as building blocks for the synthesis of larger or

more complex cyclic peptides.93 There has also been great interest in combinatorial

synthesis of cyclic dipeptide derivatives.93'97 For example, Loughlin et al. demonstrated

the solution-phase combinatorial synthesis and evaluation of piperazine-2,5-dione

derivatives for cytotoxic activities (Scheme 3-1).97












N INTO ONO
1N 0N N\ N N
3-3 3-4

3-3 3-43-


Figure 3-4. Glycine-based dimer, trimer and tetramer scaffolds

KtBuO
Ac O Ac O DMF
O RI O N 1) R2 tBuOH
N R1 H 0 NRH 24 h
N DM KtBuO N 2) Acetic Acid
Ac DMFBuOH R Ac
Ac tBuOH R1 Ac


3-6


HR2
O N

R N 0
R1 H


24 h


Scheme 3-1. Combinatorial synthesis of piperazine-2,5-dione derivatives

We are also interested in synthesizing triallyl-cyclo-triglycine 3-4 to serve as the

trimer scaffold. Similar to diketopiperazines, cyclic tripeptides possess a rigid

conformation and display important pharmaceutical properties.50 In addition to cyclic

dipeptides and tripeptides, the ability to create a DCL of molecules that mimic cyclic

tetrapeptides would be important since they can also exhibit phytotoxic, cytotoxic,98 and

medicinal properties.42 An example of a natural cyclic tetrapeptide is shown in Figure 3-

5. Comparable to the structures of the dimer and trimer scaffolds, tetrallyl-cyclo-

tetraglycine 3-5 can serve as a tetramer scaffold.



O N HN,
NH 1aOH HNCyclo(Proline-Valine-Proline-Tyrosine)

N 0 Tyrosinase inhibitor

3-9

Figure 3-5. Natural cyclic tetrapeptide









To generate a library of peptidomimetics, various amino acids possessing a

terminal olefin moiety can couple with these cyclic scaffolds by olefin CM. For example,

the metathesis of two amino acid derivatives with N-allyl glycine scaffold 3-3 can lead to

three amino acid dimers and three cyclic CM products (Figure 3-6). The number of

amino acid dimers generated by metathesis is given by N*(N+1)/2 excluding E/Z isomers,

where Nis the number of unequivalent amino acid derivatives in the pool. When

cis/trans isomers are considered, then there are 16 total possible products (Figure 3-7).

Products from oligomerization of the dimer scaffold or olefin isomerization are excluded

from these projected numbers.99'100 There is great interest in synthesizing cyclic

diketopiperazine peptide derivatives. However, these simple systems for use in DCC are

not ideal since at most, only two different amino acids could be attached to the dimer

scaffold. This methodology would be more suitable towards a traditional combinatorial

library. The main interest in using the dimer scaffold in these experiments is to better

understand CM reactivity, yield, and stereochemistry of the expected products in a simple

dynamic combinatorial system.







Amino Acid Dimers
Cyclic CM Products
Building Blocks Dynamic Library


Figure 3-6. Small dynamic library of cyclic dipeptidomimetics










IE Z Z E Z IZ IE Z 1E Z Z Z


IE I/EI U E Z I E ?E IE Z Z Z

Building Blocks Dynamic Library

Figure 3-7. A dynamic library of cyclic dipeptidomimetics including cis/trans isomers

Employing the trimer scaffold 3-4 can lead to a larger library. If three amino acid

derivatives react with the trimer scaffold in CM coupling, then the library will be

composed often unique cyclic CM molecules and six amino acid dimers (Figure 3-8).

This excludes any olefin isomerization products or oligomerization of the cyclic scaffold.

If stereoisomers are considered, then there are 48 isomers of cyclic CM molecules and 12

isomers of amino acid dimers (Figure 3-9). CM of tetramer scaffold 3-5 with four

different amino acid derivatives would result in a larger library, where there can

potentially be 55 cyclic peptidomimetics (652 including E/Z isomers) and ten amino acid

dimers (20 including E/Z isomers) (Figures 3-10 and 3-11).







Building Blocks ET g 4 l

r Dynamic Library


Figure 3-8. Dynamic library of cyclic tripeptidomimetics






Cyclic Molecules



YYT Y Y0 H 1 B

00^ i00 =3 Mjj LIU| 930 ML DO


Homodimers
Z F Z F


F Z F Z


F Z F


E
Figure 3-9. Dynamic library of cyclic tripeptidomimetics including cis/trans isomers


40 4_
+3 13
L+
- o L-Q-
13 La L


Building Blocks Dynamic Library


Figure 3-10. Dynamic library of cyclic tetrapeptidomimetics









Cyclic Molecules




EH EH EM"


o u o[BBJ


6 E/Z isomer
each/ 24 total
10 E/Z isomers
each/ 120 total
10 E/Z isomers
each/ 60 total

8 E/Z isomers each/
48 total


16 E/Z isomers
each/ 192 total
12 E/Z isomers
each/ 144 total


16 E/Z isomers
each/ 64 total
652 E/Z isomers of
cyclic molecules total


Amino Acid Dimers
LO LO


O U LO U L


2 E/Z isomers
each/ 20 dimers
total


672 isomers


Figure 3-11. Dynamic library of cyclic tetrapeptidomimetics including cis/trans isomers

3.2 Results and Discussion

In order to study the viability of olefin metathesis for DCC, we first needed to

synthesize the various monomers the cyclic scaffolds and amino acid derivatives. It

was then necessary to react the monomers with Grubbs' second generation catalyst 1-4 to

study the olefin CM selectivity and reactivity. The generation of a small library and


template effects were also examined.


03 = a- 1'


a a raay

[3 MI BM









3.2.1 Synthesis of Cyclic Scaffolds

To obtain the N-allyl dimer scaffold 3-3, NaH was added to a solution of

commercially available glycine anhydride (3-10), tetrabutylammonium iodide (TBAI),

and DMF (Scheme 3-2). Excess allyl bromide was added and the reaction was stirred at

70 C to give the desired product in 4 h. However, workup of the reaction proved to be

cumbersome. Due to the polarity, some of dimer 3-3 remained in DMF during the

aqueous workup even after numerous extractions with organic solvents. The best method

we found to obtain good yields was to quench the reaction with H20, then remove the

DMF and water in vacuo with heat. The residue was redissolved in EtOAc, leaving

behind the sodium salts which were filtered. Concentration in vacuo and purification by

chromatography gave the desire product as a white solid in good yields.

O T N H NaH, DMF, TBAI, 0 N

H N O BN O
3-10 77% 3-3


Scheme 3-2. Synthesis of dimer scaffold

Triallyl-cyclo-triglycine 3-4 was prepared following literature procedures,49'89 with

modifications as described in the experimental section (Scheme 3-3). Procedures

consisted mainly of hydrolysis and typical amino acid coupling. Reichwein and Liskamp

used benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as

the coupling reagent.49 We chose N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride (EDCI) as our coupling reagent simply because it was less expensive and

we were able to obtain high yields. In addition to EDCI, 4-dimethylaminopyridine

(DMAP), diisopropylethylamine (DIPEA, Hunig's base), and hydroxybenzotriazole









(HOBt), were used with the coupling reagent. All of the above hydrolysis and

condensation reactions gave high yields comparable with literature, and typically did not

require purification by column chromatography.

Trichlorophenol 3-19 was coupled to acid 3-16 to afford compound 3-20.101 After

removing the t-Boc protecting group from ester 3-20 with trifluoroacetic acid (TFA), we

attempted to cyclize the compound. The reaction was run in DMF under very dilute

conditions (4 mM) to preferentially form the cyclic trimer rather than the cyclic hexamer.

Closure to form a nine member ring is known to be difficult.29 Hioki et al. reported a

11% yield for the cyclic trimer, with the cyclic hexamer as the major product.89 Upon

purification by column chromatography, we isolated a white solid appearing to be either

the trimer or the hexamer, according to H NMR and 13C NMR. High resolution mass

spectroscopy (HRMS) indicated the presence of trimer 3-4 and an unknown compound.

A pure sample of trimer could not be isolated even after numerous recrystallizations in

various solvent systems, or by column chromatography.

Tetramer scaffold 3-5 was synthesized through simple hydrolysis and condensation

starting from acid 3-18 (Scheme 3-4). The t-Boc protecting group oftrichloro ester 3-23,

was removed by TFA. Using similar procedures for the cyclization of the trimer, the

ester was heated for 24 h in dioxane (4 mM) and pyridine (1.1 eq). Purification by

column chromatography afforded tetramer 3-5 in 26% yield. Identical results were

obtained using DMF, but this is a less desirable solvent because of the difficulty of

removing DMF during workup.










0
10 ,./Br


-.NH2 +

3-11


O Boc
HO N3-
3-14


o
HO 'N -., -Boc
O 0

3-16


THF
60%


3-12


3-13


>90%


3-15


1) Boc2O, THF
2) NaOH/MeOH

>90%


1) NaOH/MeOH
2) HC1
>90%


3-13. EDCI. DMAP.,
HOBt, DIPEA
>90%


1) NaOH/MeOH
2) HC1
>90%


0
HO- N- N- NN Boc

0 0

3-18


3-19


EDCI, DMAP, HOBt, DIPEA
58%


1) TFA, CH2C12
2) Py, DMF, 100 OC


3-20


+ Unknown Compound


Scheme 3-3. Synthesis oftrimer scaffold


N0=
0


S H


O
N O0










9 S o o?
HO N N N-Boc 3-_13 FDpT MAP, O .N N N -Boc
O I HOBt, DIPEA 0 O
S87% O O 1
3-18 |1 1| 3-21


1) NaOH/MeOH
2)HC1
>90%


HO
HONAI N -NN N '-NBoc
0 o
3-22 II


3-19, EDCI DMAP,.
HOBt, DIPEA
51%


1) TFA, CH2C2 N C1
-Boc 2) Py, Dioxane, 100C OC
62 %O i


Cl 0 o0 O 05
3-23 3-5

Scheme 3-4. Synthesis of tetramer scaffold

3.2.2 Synthesis of Amino Acid Derivatives

Different amino acid derivatives were synthesized to determine the effects of

protecting groups, side chains, and alkene moieties in olefin CM. Miller group had

examined the effect of remote functionality but with allyl and homoallylamides.62

Herein, we investigated the allyl and homoallyl olefin moiety of the carboxylate end of

the amino acid.

A series of amino acid derivatives were synthesized by coupling t-Boc or Fmoc

protected amino acids with allyl alcohol or 3-buten-l-ol using DIC, DMAP, and HOBt

(Scheme 3-5). The starting material was typically consumed within 20 minutes as

indicated on TLC. The urea byproduct was filtered and purification of this filtrate by

column chromatography gave the amino acid products in high yields (Table 3-1 and

Figure 3-12). MeOH was used as the solvent to determine the optical rotation of the

samples.


w









PG f PG 0O
HNi O DIC, DMAP, DCML H R1
S OH HOBt, R1OH 0
R R
3-24, PG = Boc 3-26, PG = Boc
3-25, PG = Fmoc 3-27, PG = Fmoc


0
Boc u

0
Fmoc= \^ O"


Scheme 3-5. Synthesis of amino acid derivatives

Table 3-1. Yields, melting points and optical rotations of amino acid derivatives
Entry N-PG-AA R Producta Yield mp [a]D(0) (C, c)
(%) (OC) MeOH
1 Boc-Phe Allyl 3-26a >95 71-72 -8.05 (25, c = 1.10)
3-24a
2 Boc-Ala Allyl 3-26b >95 oil -35.0 (25, c = 1.04)
3-24b
3 Boc-Pro Allyl 3-26c 95 oil -70.9 (25, c = 1.00)
3-24c
4 Boc-Met Allyl 3-26d >95 oil -32.4 (25, c = 1.04)
3-24d
5 Boc-Phe Homoallyl 3-26e 90 79- -9.01 (25, c = 1.00)
3-24e 80.5
6 Boc-Ala Homoallyl 3-26f 98 oil -45.7 (25, c= 1.11)
3-24f
7 Boc-Pro Homoallyl 3-26g 89 oil -72.3 (25, c = 1.24)
3-24g
8 Boc-Met Homoallyl 3-26h 88 oil -23.8 (25, c = 1.10)
3-24h
9 Boc-Leu Homoallyl 3-26i 86 oil -39.2 (25, c = 1.42)
3-24i
10 Fmoc-Phe Homoallyl 3-27a >95 52-54 -20.0 (25, c = 1.06)
3-25a_
11 Fmoc-Pro Homoallyl 3-27b 92 oil -49.4 (25, c = 1.25)
3-25b
12 Fmoc-Gly Homoallyl 3-27c 85 78.5- N/A
3-25c 80


a Allyl Boc-Phe was first discussed in chapter 2 and referred as compound 2-7. For
clarity and simplicity in the numbering of our libraries, the compound will be labeled as
3-26a.









O 0 0
II II II
HzN-CH-C-OH HzN-CH-C-OH C-OH
I I
CH2 CH3
HN



Phenylalanine (Phe) Alanine (Ala) Proline (Pro)

o 0 o
II II II
H2N-CH-C-OH H2N-CH-C-OH HzN-CH-C-OH
CH2 CH2 H
CH2 CH-CH3
S CH3
CH3
Methionine (Met) Leucine (Leu) Glycine (Gly)

Figure 3-12. Amino acids used in the library.

As shown in Table 3-2, HOBt was required to prevent racemization of the chiral

center. The 1H NMR and optical rotation data of allyl ester phenylalanine 3-26a102 and

alanine 3-26b103 were similar to those reported in literature. Full characterization of the

other amino acid derivatives is found in the experimental section of Chapter 6.

Table 3-2. Yields, melting points, and optical rotations of amino acid derivatives without
use of HOBt during synthesis
Entry N-PG-AA R1 Product Yield mp [a]D(0) (C, c)
(%) (OC) MeOH
1 Boc-Phe Allyl 3-26a 87 70-71 -2.31 (25, c = 1.00)
2 Boc-Ala Allyl 3-26b 88 oil -13.6 (25, c= 1.08)
3 Boc-Phe Homoallyl 3-26e 56 78-80 -2.20 (25, c = 1.00)
4 Boc-Ala Homoallyl 3-26f 80 oil -33.4 (25, c= 1.00)

3.2.3 CM Reactivity of Dimer with One Amino Acid

The CM reactivity of dimer scaffold 3-3 with amino acid derivatives possessing

different protecting groups and olefin moieties was examined. Dimer scaffold 3-3 was









allowed to react with an amino acid derivative (5 eq.) using 10 mol% Grubbs' second

generation catalyst 1-4 (Scheme 3-6). The reaction was stirred at reflux in CHC13 for 10

h while flushing the headspace with argon to remove evolved ethylene. The reaction was

quenched with EVE and purification by column chromatography gave the desired

products in moderate yield (Table 3-3).

We attempted to isolate compound 3-32, the CM product of one amino acid

derivative with dimer scaffold 3-3, by column chromatography. Separation and

purification of 3-32 from other byproducts was unsuccessful, but the mass spectrometer

data did indicate the presence of compound 3-32. The low yields of 3-32 were expected

due to excess amount of amino acid derivatives which would favor the homodimer or

heterodimer products. In addition, these CM reactions were run on a small scale.

PG O
HN OH: 3-26, PG = Boc
0~ n 3-27, PG = Fmoc
N^ R

0 CHC13, 1-4
3-3
R PG O R
H PG O nO -PG HNNH
pG'Ny 0 R 0 PG
R Cyclic CM Products Amino Acid Dimers
3-28, PG = Boc 3-30, PG = Boc
3-29, PG = Fmoc 3-31, PG = Fmoc


H 0 O "N "
+ PG O N, O
R
3-32
Not Isolated

Scheme 3-6. Olefin CM of dimer scaffold with an amino acid derivative










Table 3-3. Yields of cyclic and amino acid dimers
Entry Starting N-PG-AA R1, n Cyclic Yielda Amino Yield
Material CM % Acid %
Product Dimer
s
1 3-26a Boc-Phe Allyl, n=l 3-28a 37 3-30a 38

2 3-26b Boc-Ala Allyl, n=l 3-28b 40 3-30b 42

3 3-26c Boc-Pro Allyl, n=l 3-28c 42 3-30c 31

4 3-26d Boc-Met Allyl, n=l 3-28d 0 3-30d 0

5 3-26e Boc-Phe Homoallyl, 3-28e 44 3-30e 50
n=2
6 3-26f Boc-Ala Homoallyl, 3-28f 39 3-30f 66
n=2
7 3-26g Boc-Pro Homoallyl, 3-28g 45 3-30g 55
n=2
8 3-26h Boc-Met Homoallyl, 3-28h 0 3-30h 14
n=2
9 3-26i Boc-Leu Homoallyl, 3-28i 30 3-30i 59
n=2
10 3-27a Fmoc-Phe Homoallyl, 3-29a 42 3-31a 48
n=2
11 3-27b Fmoc-Pro Homoallyl, 3-29b 46 3-31b 52
n=2__
12 3-27c Fmoc-Gly Homoallyl, 3-29c 30 3-31c 44
n=2
a Isolated yields except 3-31b, which is based on NMR

Similarly, the cyclic CM products 3-28 and 3-29 were difficult to purify by column

chromatography due to the polarity of the compounds. Based on TLC, these compounds

have Rf values similar to dimer 3-3 and compound 3-32. Fortunately, we were able to

separate the cyclic CM products from the other compounds for characterization. For all

of the CM reactions, the amino acid starting material was not fully consumed. The amino

acid dimers had Rf values close to the amino acid starting material, but we were able to

obtain pure samples by column chromatography.









The yields for the cyclic CM products 3-28 and 3-29 were comparable, whether an

allyl or homoallyl olefin moiety was attached to the amino acid. There was also little

difference between the yields of Fmoc protected 3-29a and Boc protected 3-28a and 3-

28e. However, the yields improved for the amino acid dimers 3-30 e-h and 3-31 a-b,

which possess the homoallyl olefin chain, in comparison to dimerized products 3-31 a-d

with the allyl chain. A possible reason for the higher yield is that the ruthenium catalyst

is less sterically hindered by the amino acid moiety and has better access to the longer

chain terminal olefin.26 Again we saw little difference between the Fmoc 3-31 a-b and

Boc protected 3-30e and 3-30g. In all cases, CM of methionine derivatives resulted in

zero or low yields, which was a surprise since Grubbs and Mioskowski groups

demonstrated the CM of sulfur containing compounds in good yields.23'104 TLC analysis

of the reaction mixture showed that mostly starting material 3-26d and dimer 3-3 were

present even after 2 days of reflux.

The stereochemistry of the cyclic CM products was found to be predominantly

trans by 1H NMR of the isolated compounds. We examined the 1H NMR of the crude

reaction mixture to determine the cis/trans ratio of amino acid dimers 3-30 and 3-31. The

chemical shift of the cis isomers was expected to be further downfield than the trans

isomers, but the large number of library constituents in the crude reaction mixture made

the NMR data rather complex. We therefore isolated the amino acid dimers by column

chromatography. Based on the NMR spectra, most of the isolated compounds appeared

to be a pure isomer, rather than a mixture of cis and trans isomers. We expected them to

be mostly trans based on Grubbs work.34 However, experiments conducted by Miller lab









showed a 3:1 ratio of cis/trans but using Grubbs' first generation catalyst 1-3 instead of

catalyst 1-4.62

To ensure we properly assigned the cis/trans ratio of amino acid dimers, we

examined the satellites of the alkene protons using a 500 MHz spectrometer with

deuterated acetone as the solvent. These weak satellites were formed from protons

attached directly to the 13C (1% natural abundance), rather than protons attached to the

more abundant 12C isotope.105 The satellites were located 80 Hz to the right and left side

of the of the stronger proton signal. We expected the alkene protons of the trans isomers

to have a larger coupling constant (J= 15-17 Hz) than the cis isomers (J= 9-11 Hz).106

We independently synthesized two authentic homodimers, where the

stereochemistry was known, to confirm the predicted J coupling values of the weak

satellites. We first synthesized the cis allyl homodimer 3-30a by coupling (Z)-2-butene-

1,4-diol (3-33) with N-(tert-butoxycarbonyl)-phenylalanine (3-24a, 3 eq) using DMAP,

HOBt, DIPEA, and EDCI in 95% yield (Scheme 3-7). Homodimer 3-30a was also

synthesized using DIC as the coupling agent. However, removal of the urea byproduct

was cumbersome, and the yield was 88%. NMR analysis of the weak satellites of the cis

alkene protons indicated a pattern of a doublet of a triplet with J= 11 and 6 Hz (Figure 3-

13).

0
J MRTT o BocHN O O NHBoc
HOOH + HO NHBoc EDCI, HOBt, B0 NHB
HO--OH + Ph
Ph DMAP, DIPEA, DCM Ph 0 Ph
95%
3-33 3-24a 3-30a

Scheme 3-7. Synthesis of cis homodimer 3-30a












i I









.. ~~ ~~~- - ----- I----.- ,'" ,

Figure 3-13. Satellites of the alkene protons from cis homodimer 3-30a

We also independently synthesized the trans homoallyl dimer by employing the

same method as above, but starting from the less expensive trans-3-hexenedioic acid 3-

34 rather than diol 3-35 (Scheme 3-8). Acid 3-34 was converted to the diester, which

was then reduced to the diol by LiAlH.62 Amino acid coupling with Boc-protected L-

phenylalanine 3-24a afforded the trans homodimer product 3-30e. NMR analysis of

trans 3-30e and the weak satellites of the alkene protons indicated a doublet of a triplet

pattern with J= 16 and 7 Hz (Figure 3-14).

O
OOH 1) MeOH, H2SO4 85%
HO OH
HO OH 2) LiAlH 65% Ho
3-34 0 3-35

0 /Ph
EDCI, HOBt, DMAP BocHN OO NHBoc
DIPEA, DCM, 3-24a N
54% Ph' 3-30e

Scheme 3-8. Synthesis of trans homodimer 3-30e

NMR analysis of our homodimer samples from the CM reactions indicated the

presence of trans isomers, as determined by the J coupling of the weak satellites since we

observed a doublet of a triplet pattern with J = 16 and 7 Hz.













I i














5.70 5.65 .O 5. 5 0 5.- 5 5.40 5.35 5.30 5.25 pp

Figure 3-14. Satellites of the alkene protons from trans homodimer 3-30e

3.2.4 Generation of Small Libraries and Template Effects

After examining the CM reactivity of the amino acid derivatives with the dimer

scaffold, we were interested in generating a small library and examining the template

effects. As a preliminary study, dimer 3-3 was allowed to react with two or three

different amino acid derivatives (2-3 eq) in a cross-coupling reaction at room temperature

or at reflux using 10-18 mol% of catalyst 1-4. See Scheme 3-9 for a representative

reaction. For this particular example, in which two different amino acids were used, we

would expect to see three different amino acid dimers and cyclic CM products, and

possibly the mono-coupled compounds (Table 3-4). The resulting equilibrium mixture

was frozen upon addition of EVE, which significantly reduced the activity of the catalyst.

The catalyst was removed by washing with water soluble tris(hydroxymethyl)phosphine

[THP, P(CH20H)3]107 or through column chromatography. Isolation of each CM product

by column chromatography was not feasible due to the number of possible CM products









as well as any unreactive starting materials. We then analyzed the samples by high

performance liquid chromatography (HPLC).

Several groups have studied lithium salts and their effect on the conformation of

peptides.108s109 Sanders group used hydrazone chemistry to prepare a DCL of cyclic

peptidomimetics.60 They observed the formation of one major 42-membered macrocyclic

compound upon addition of lithium salts. This was a surprising result since Li is a small

cation that would not be expected to coordinate with a large cyclic compound. In our

system, the diketopiperazine CM product had a linear configuration where the amino acid

derivatives were separated by the cyclic scaffold. Because of this configuration, we

would not expect a small ion to influence the dynamic equilibrium very much.

Nevertheless, we were interested in knowing the effects, if any at all, of lithium salts on

our small dynamic library. Several reaction conditions were examined to determine the

effects of the lithium ion template (Table 3-5).

In experiments 1 and 2, the reaction mixture was refluxed for 11 h without the

addition of a template (Table 3-5). These reactions were compared to experiment 3, in

which the dimer, amino acid derivatives, and catalyst were refluxed for 3 h followed by

the addition of the LiC104. The reaction mixture was then refluxed for another 8 h.

Based on HPLC data, there were no significant changes when the template was added to

the reaction.

We then examined the influence of a proline derivative, which possesses a rigid

structure compared to other amino acids. For experiments 3-7, the reaction was refluxed

in its respective solvent as listed in Table 3-5. There were some changes to the peak

patterns and area % of the HPLC traces of samples with and without LiC104. However,









there was no major increase in concentration of one or two peaks after the addition of

LiC104 to conclude that the template influenced the equilibrium in a significant capacity.

PG O PG O
H O (3 eq) and (3 eq)
O N- N R1 R2
N -O 3-26a, PG = Boc 3-26b, PG = Boc
3-3


1-4, CH2C12 or CHC13

R1
N OyON O NPG
H 0 0 H

R 3-30a

H o OYH. HoY PG
PG.N NoON o 0
R2 3-30b
R2
NH O0 N o NPG
PG'H-O ", N o O H
R1 3-31a


H O R1
oPGN'-, 0" -N--PG
S 3-30a 0 H

H R2
.PG O : -PG
PG O N
R2 3-30b O H


H O O N-

R1
3-32a



H 0 0O N
PG N O N OI-


H R2 R2
pG'-H-T^o O@ N-P
P R1 O H
3-34a

Scheme 3-9. CM of dimer scaffold with two amino acid derivatives


3-32b









Table 3-4. Expected products from CM reaction of dimer scaffold and two or three amino
acid derivatives
Entry Starting Possible Productsa
Material
Scaffold Coupled Scaffold Amino Acid
w/ 2 Amino Acids Coupled w/ 1 Dimers
Amino Acid
1 Dimer 3-3 3-28a (Phe-D-Phe) 3-32a (D-Phe) 3-30a (Phe-Phe)
Boc-Allyl Phe 3-28b (Ala-D-Ala) 3-32b (D-Ala) 3-30b (Ala-Ala)
3-26a 3-33a (Phe-D-Ala) 3-34a (Phe-Ala)
Boc-Allyl Ala
3-26b
2 Dimer 3-3 3-28e (Phe-D-Phe) 3-32c (D-Phe) 3-30e (Phe-Phe)
Boc-Homoallyl 3-28g (Pro-D-Pro) 3-32d (D-Pro) 3-30g (Pro-Pro)
Phe 3-26c 3-33b (Phe-D-Pro) 3-34b (Phe-Pro)
Boc-Homoallyl
Pro 3-26g
3 Dimer 3-3 3-29a (Phe-D-Phe) 3-32e (D-Phe) 3-31a (Phe-Phe)
Fmoc- 3-29b (Pro-D-Pro) 3-32f (D-Pro) 3-31b (Pro-Pro)
Homoallyl Phe 3-33c (Phe-D-Pro) 3-32c (D-Gly) 3-31c (Gly-Gly)
3-27a 3-33d (Phe-D-Gly) 3-34c (Phe-Pro)
Fmoc- 3-33e (Gly-D-Pro) 3-34d (Phe-Gly)
Homoallyl Pro 3-34e (Pro-Gly)
3-27b
Fmoc-
Homoallyl Gly
3-27c
a The dimer scaffold is abbreviated as "D."

In experiment 8, the system was analyzed using Fmoc amino acid derivatives,

which were also easier to observe by HPLC because of the aromatic rings (Table 3-5).

Grubbs' catalyst 1-4 (13 mol%) was added to a solution of the dimer 3-3, Fmoc-L-Phe 3-

27a, Fmoc-L-Pro 3-27b, Fmoc Gly 3-27c, and CHC13 at room temperature and stirred for

24 h to ensure equilibration. LiCl (3 eq) was added and the reaction stirred for 1 h,

followed by another addition of catalyst 1-4 (7 mol%). After stirring at room temperature

for 21 h, additional LiCl (4 eq), catalyst 1-4 (7 mol%), and CHC13 (0.5 mL) were added.

The reaction was maintained for another 23 h. In order to observe the influence of heat,

catalyst 1-4 (4%) and CHC13 (1 mL) were added and reaction heated at 40 OC for 23 h.









Aliquots of the reaction mixture were taken at numerous time points to monitor the

changes by HPLC. Similar to previous experiments, minor changes were observed but

there was no dramatic shift in the equilibrium.

Table 3-5. CM conditions with and without lithium template
Experiment Starting Material (equivalent) Solvent Catalyst Template
(Molarity) 1-4 (equivalent)
(mol%)
1 Dimer 3-3 (1 eq) CHC13 10% None
Boc-Allyl Phe 3-26a (2 eq) (0.5 M)
Boc-Allyl Ala 3-26b (2 eq)
2 Dimer 3-3 (1 eq) CHC13 10% None
Boc-Allyl Phe 3-26a (3 eq) (0.5 M)
Boc-Allyl Ala 3-26b (3 eq)
3 Dimer 3-3 (1 eq) CHC13 10% LiC104
Boc-Allyl Phe 3-26a (3 eq) (0.5 M) (1 eq)
Boc-Allyl Ala 3-26b(3 eq)
4 Dimer 3-3 (1 eq) CH2C12 10% LiC104
Boc-Allyl Phe 3-26a (2 eq) (0.7 M) (1.4 eq)
Boc-Allyl Pro 3-26g(2 eq)
5 Dimer 3-3 (1 eq) CHC13 16% None
Boc-Homoallyl Phe 3-26e (2 (0.4 M)
eq)
Boc-Homoallyl Pro 3-26g (2 eq)
6 Dimer 3-3 (1 eq) CHC13 13% LiC104
Boc-Homoallyl Phe 3-26e (3 (0.2 M) (1.4 eq)
eq)
Boc-Homoallyl Pro 3-26g (3 eq)
7 Dimer 3-3 (1 eq) CHC13 16% LiC104
Boc-Homoallyl Phe 3-26e (3 (0.3 M) (1.6 eq)
eq)
Boc-Homoallyl Pro 3-26g (3 eq)
8 Dimer 3-3 (1 eq) CHC13 18% LiCl
Fmoc-Homoallyl Phe (0.2 M) (7 eq)
3-27a (3 eq)
Fmoc-Homoallyl Pro
3-27b (3 eq)
Fmoc-Homoallyl Gly
3-27c (3 eq)









3.3 Conclusions

Our main objective in this project was to examine the olefin CM reactivity and

selectivity of amino acid derivatives with cyclic scaffolds, and its potential for use in

dynamic combinatorial chemistry. Having the olefin moiety further from the amino acid

functional groups increased the yields of the amino acid dimers, but there were little

differences in yields for the diketopiperazine products. Altering protecting groups on the

amino acids from Boc to Fmoc resulted in little changes to the yields as well. The

stereochemistry of the isolated cyclic and amino acid dimers was found to be

predominantly trans.














CHAPTER 4
DYNAMIC COMBINATORIAL LIBRARIES EMPLOYING PEPTIDOMIMETIC
DIENES

4.1 Introduction

In our continuing studies of cyclic peptidomimetics, we were interested in

generating a dynamic combinatorial library (DCL) 52,54,56,110 of macrocyclic compounds

by olefin metathesis. Several groups have reported the use of reversible chemical

reactions, such as disulfide exchange,5 metal-ligand coordination,58 exchange of

oximes59 and hydrazones,60 formal metathesis111 and olefin metathesis in dynamic

combinatorial chemistry (DCC).61-63 However, few have examined olefin metathesis as a

means to generate a library of cyclic peptidomimetics. The chemistry involves amino

acid building blocks possessing two terminal olefin moieties. Cyclic molecules can be

formed from these building blocks by ring-closing metathesis (RCM), or by cross-

metathesis (CM) followed by RCM. A large number of library constituents, such as

cyclic and linear compounds, as well as oligomers can be generated from this method.

Upon addition of a template with specific binding properties to the library constituents,

the equilibrium can shift to amplify one or two major products in good yields.

Sanders group used building block 4-1 consisting of L-proline, L-phenylalanine

and an aromatic linker which can engage in non-covalent interactions, hydrogen bonding,

Lewis acid-base, pi-pi and cation-pi interactions.54'60'112 Hydrazone formation was used

to create these libraries of linear compounds, oligomers, and cyclic molecules. They

were able to show amplification of one of his library constituents, trimer 4-2, by adding a










lithium metal ion as a template (Scheme 4-1).54 They employed other metals such as KI,

RbI and CsI as templates but did not observe the same shift toward the trimer.60

However, under the same hydrazone based library, Sanders group observed the

amplification of the trimer using alkylammonium salts.110'113

We were curious to see if similar results could be obtained using a comparable

building block but with olefin metathesis rather than hydrazone exchange. A trimer made

up of dipeptide 4-4 would consist of a 51 membered ring (Scheme 4-1). A 48 membered

macrocyclic could also be made by replacing the homoallyl moiety on building block 4-4

with an allyl ester; however, previous studies in our lab have shown that allyl groups are

more sluggish toward metathesis than longer chain olefins.

R1 H 0 R1 HNiN'

NRN H 0 N? ,
N 0 Library of Dimer, Li eN -O O HN Work done by Sij bren Otto,
Trimer, Hexamer, ~ [ Ricardo LE Furlan, and
Setc. 42-membered O Jeremy KM Sanders

R2 N'-
HN
4-1 R' =CH2Ph O 4-2
R2 CH(OMe)2 R1 NH


RHO -N
R1


N0 R H 0
51-membered N 0N
versus 0 Building Block


0 0 O T I/

R 4-3 4-4


Scheme 4-1. Comparison of building blocks 4-1 and 4-4 and their library constituents









The proposed building block 4-4 would consist of phenylalanine, proline, an

aromatic linker, and terminal alkene moieties for olefin CM and/or RCM. The

retrosynthesis of dipeptide 4-4 is shown in Scheme 4-2. The molecule can easily be

synthesized by the coupling of allyl benzoic acid 4-5 and peptide 4-6, which is made

from proline 3-24c and phenylalanine 3-26e.

0

/Y OH
0 Boc
NH 0 H H 0N O 3-24c
^ NH H
YHOV H 0


BoCN Oe

4-4 4-5 4-6 H
3-26e

Scheme 4-2. Retrosynthesis of dipeptide 4-4

The CM and/or RCM reactions of dipeptide 4-4 can give a library of linear

compounds, cyclic dimers 4-7, trimers 4-8, hexamers and oligomers (Scheme 4-3). The

regioselectivity and stereochemistry of the metathesis reactions can also increase the

number and types of library constituents as well. For example, the coupling of two

molecules can occur in a head to tail or a tail to tail fashion, to form dimers 4-7a and 4-7b

respectively. In addition, CM and RCM reactions can give cis or trans alkenes.

Catenanes, interlocked molecular rings, can also be generated by intramolecular

RCM mediated syntheses under thermodynamic control.114 Two molecules can become

intertwined and the two-fold RCM can result in the formation of rings covalently linked

(Figure 4-1). Several groups have also demonstrated the template or hydrogen bond

directed synthesis of catenanes in good yields using olefin metathesis.115-118










Dimers 4-7


Head-Tail Head-Head
4-7a 4-7b

(E/E, E/Z, Z/Z)

Trimers 4-8







Head-Tail Head-Head
4-8a 4-8b

(EIEIE, EIZIE, ZIEIZ, Z/Z/Z)

Tetramers, Hexamers, Oligomers, Linear Compounds


Scheme 4-3. Library of dimers, tetramers, hexamers, oligomers, linear compounds and
catenanes





2


Figure 4-1. Formation of [2]catenane









4.2 Results and Discussion

In order to study olefin CM and RCM in DCL of cyclic peptidomimetics, we first

needed to synthesize dipeptide 4-4. The molecule can be synthesized by the coupling of

4-allyl benzoic acid (4-5) with peptide 4-6. Allylbenzoic acid 4-5 was synthesized

following literature procedures,119'120 starting from commercially available 4-iodobenzoic

acid (4-9) (Scheme 4-4). The synthesis involves esterification of acid 4-9, followed by

the conversion of the aryl iodide to the Grignard reagent. Treatment of the magnesium

compound with allyl bromide and CuCN-2LiCl afforded benzoate 4-11, which was then

hydrolyzed to give 4-allyl benzoic acid (4-5) in excellent yield. 121

O O O
HO SOC12 1) iPrMgC1
SEtOH 2) CuCN.2LiC1,
I 98% I allyl bromide
4-9 4-10 89% 4-11


0
NaOH, 1
N HO
94%0 HO
4-5


Scheme 4-4. Synthesis of 4-allylbenzoic acid (4-5)

Dipeptide 4-4 was synthesized by a series of N-(3-Dimethylaminopropyl)-N'-

ethylcarbodiimide hydrochloride (EDCI) amino acid coupling reactions (Scheme 4-5).

Boc-homoallyl ester phenylalanine 3-26e was first deprotected with TFA, and then

coupled with Boc-L-proline 3-24c to afford compound 4-13. The Boc protecting group

was removed with TFA once again, followed by another EDCI coupling reaction with

allylbenzoic acid 4-5 to produce dipeptide 4-4 in good yield.










NH0 TFA, DCM NH2 EDC HOBt

0 87%

3-26e 4-12


o0
H 0 H,'; ) FDrT HORt
0'- N TFA, DCM NH DIPEA, 4-5
0 O 0 73%
0
4-13 4-6





HON HO


4-4


Scheme 4-5. Synthesis of dipeptide 4-4

Upon the synthesis of dipeptide 4-4, a series of CM experiments were conducted in

various conditions (Table 4-1) to generate a library of peptidomimetics. We were

interested in seeing the major components of the reaction when dipeptide 4-4 was

allowed to react with catalyst 1-4, with and without a template. A representative

procedure for the CM reactions listed in Table 4-1 (experiment 12) is as follows: under

an atmosphere of argon, a solution of LiI (1 eq) and THF (2 mL) was added to a solution

of dipeptide 4-4 and CH2C2 (20 mL). After stirring for 30 minutes, a solution of Grubbs'

catalyst 1-4 and CH2C12 was added drop-wise and the reaction refluxed for 13 h. Upon

cooling, the reaction was quenched with EVE. The work-up included an aqueous

extraction with tris(hydroxymethyl)phosphine (THP) to remove the ruthenium catalyst.107









Table 4-1. Series of CM reactions
Experiment Solvent Catalyst 1-4 Template Conditions
(mM) mol% (equivalent)
1 CH2C12 (5) 5 None Monitored reaction for 4
days at r.t.
2 CH2C12 5 None Monitored reaction for 4
(36) days at r.t.
3 CH2C12 (5) 5 None 15 h at reflux

4 CH2C12 (5) 5 None 15 h at reflux

5 CH2C12 (5) 5 None 19 h at reflux

6 CHC13 (5) 5 None 19 h at reflux

7 CHC13 (5) 5 None 15 h at reflux

8 THF (5) 5 None Monitored reaction for 3
days at r.t.
9 THF (36) 5 None Monitored reaction for
days at r.t.
10 CH2C12 (5) 5 LiC104 (1.5) Monitored reaction for 4
days at r.t.
11 CH2C12 5 LiC104 (1.5) Monitored reaction for 4
(36) days at r.t.
12 CH2C12 (5) 5 Lil (1 eq) 13 h at reflux

13 CH2C12 (5) 5 Lil (1 eq) 15 h at reflux

14 CH2C12 (5) 5 Lil (1 eq) 15 h at reflux

15 CH2C12 (5) 5 Lil (1 eq) 15 h at reflux

16 CH2C12 (5) 5 Lil (1 eq) 15 h at reflux

17 CHC13 (5) 5 Lil (1 eq) 13 h at reflux

18 CHC13 (5) 5 Lil (1 eq) 15 h at reflux


The samples were analyzed on a Shimadzu high performance liquid

chromatography (HPLC) using a reverse phase column. The HPLC conditions consisted

of an isocratic (65% acetonitrile, 35% water) flow rate of 0.5 mL/min and UV detection









at 254 nm. HPLC spectra of selected experiments can be found in appendix A. To

ensure accurate comparison of the HPLC traces, aliquots of the reaction mixture from

each experiment were co-injected into the HPLC. The peaks were also labeled for ease

of comparison and do not necessarily represent known compounds, unless otherwise

indicated.

For experiments 1-2, the reaction mixture was stirred at room temperature in

CH2C12 for 4 days (Table 4-1). There were only minor changes in the HPLC trace

whether the concentration was 5 mM or 36 mM. After four days the major component

was the starting material. We also examined THF as a solvent because it was later used

to dissolve the templates and we wanted to make sure THF was not a contributor to the

equilibrium shift. Similarly to experiments 1-2, starting material was the major

component when THF was employed both at concentrations of 5 mM and 36 mM

(experiments 8 and 9, respectively).

We then studied the effects of LiC104 and Lil as templates in various conditions.

As shown in Figure 4-2, there was a decrease in peak F and an increase in peak J when

LiC104 was employed and the reaction was maintained at room temperature (Table 4-1,

experiments 1 and 10). However, the starting material (peak D) was predominantly

present in the reaction mixture. No major shift in the equilibrium was detected between

experiments 2 and 11, when the reaction was maintained at a higher concentration

(Figure 4-3). Minimal changes to the equilibrium when LiC104 was employed may be

due to reaction conditions at room temperature, rather than lack of template effects. The

minor product formation was most likely due to the entropy of activation required to







67


bring two terminal alkene moieties together for CM or ring closure.29 Therefore, we next

conducted experiments where heat was employed.


E


A B G H


LAI
I_.._.~ _. ___~,_....--- ---- --_.


Figure 4-2. Comparison of experiments 1 and 10. A) HPLC spectrum of experiment 1
reaction mixture (CH2C2, 5 mM, room temperature, 4 days, no template). B)
HPLC spectrum of experiment 10 reaction mixture (CH2C12, 5 mM, room
temperature 4 days, LiC104)


(14 t m 29 ^ 2* r6 ^ iC 3 3 II 3 u
hlinili







68





D




-F

A B G





A




D
A


B CE
SB










Figure 4-3. Comparison of experiments 2 and 11. A) HPLC spectrum of experiment 2
reaction mixture (CH2C12, 36 mM, room temperature, 4 days, no template).
B) HPLC spectrum of experiment 11 reaction mixture (CH2C12, 36 mM, room
temperature 4 days, LiC104)

In experiments 3-5, a solution of dipeptide 4-4, Grubbs' catalyst 1-4, and CH2C12

was refluxed for 15 h and for 19 h (Table 4-1). There were negligible differences in the

HPLC traces between the two reaction times. However, we observed new products

compared to reactions maintained at room temperature. There was more of a dramatic

change in the HPLC trace when Lil was used as a template and the reaction was refluxed

in CH2C12 (5 mM) (Figure 4-4). We observed a large decrease of the starting material







69


(peak D), along with an increase in some of the larger molecules (peaks G, H, and I). Lil

was chosen as the template rather than LiC104 for these set of experiments to better

replicate Sanders' work. The CM reactions, with and without lithium, were repeated

several times as shown in Table 4-1, and for all cases, similar results were obtained.

HPLC samples from the olefin metathesis reactions were also co-injected with the

starting material to ensure we had properly labeled the peaks.


G H I


G H


Figure 4-4. Comparison of experiments 4 and 14. A) HPLC spectrum of experiment 4
reaction mixture (CH2C12, 5 mM, reflux 15 hours, no template). B) HPLC
spectrum of experiment 14 reaction mixture (CH2C12, 5 mM, reflux 15 hours,
LiI)


a
I :
+*30


LIW







70


We next examined conditions in which a solution of dipeptide 4-4, Grubbs' catalyst

1-4, and CHC13 was refluxed for 13 h and 15 h without the addition of a template (Table

4-1, experiments 6-7). In contrast to experiments conducted in refluxing CH2C12, there

was little amount of starting material present after 13 h or 15 h (Figure 4-5).


















D

B


A






E



B
D F"

O A J C


00 25 *0 7 5 T0 25 ISO 5 no 225 MD D a25 350 W 00 45 *50 175 WO
B

Figure 4-5. Comparison of experiments 7 and 17. A) HPLC spectrum of experiment 7
reaction mixture (CHC13, 5 mM, reflux 15 hours, no template). B) HPLC
spectrum of experiment 17 reaction mixture (CHC13, 5 mM, reflux 13 hours,
LiI)









We then explored the effects of LiI as a template. As shown by the HPLC traces in

Figure 4-5, there was little change to the starting material dipeptide 4-4. However, we

did observe a decrease in peak E and an increase in peaks B and F.

A series of reactions were also monitored by HPLC where the dipeptide was

allowed to react with catalyst 1-4 for a period of several hours to 24 hours at room

temperature to allow equilibration. Afterwards, different lithium templates (Lil or

LiC104) were added and the reaction maintained for another 24 h. We continued to

monitor the reaction after refluxing for another 24 h. For some experiments, additional 5

mol% of the catalyst was introduced every 24 h to ensure the catalyst was not degraded.

However, we did not observe any significant changes in the HPLC data when the lithium

template was added before the catalyst to allow pre-coordination of the amino acids with

the metal.

Even though we did not see a significant shift in the equilibrium before and after

the addition of the template as was seen in Sanders' work, we felt it was of interest to

identify the major peaks. Attempts were made to isolate the major products by column

chromatography. We were able to recover the starting material, dipeptide 4-4. However,

obtaining pure CM products was more difficult than anticipated. We did isolate a

compound that appeared to be a dimer made up of two molecules of dipeptide 4-4 based

on high resolution mass spectrometry (HRMS) and 2D NMR [(acetone d6), COSY

(Correlated Spectroscopy), HMBC (Heteronuclear Multiple Bond Correlation), HMQC

(Heteronuclear Multiple Quantum Correlation), NOESY (Nuclear Overhauser and

Exchange Spectroscopy)]. The 1H and 13C spectra were obtained on an Inova

spectrometer operating at 500 MHz for proton and 100 MHz for carbon. Two









dimensional NMR experiments were conducted on the same spectrometer, and samples

were dissolved in deuterated acetone. The HPLC trace of the isolated compound is

shown in Figure 4-6.

HRMS data of the isolated compound revealed three possible structures as shown

in Figure 4-6. The dimer of dipeptide 4-4 can be formed by the CM of two molecules in

a head to head or head to tail fashion, followed by a RCM to give 4-7a and 4-7b,

respectively. In addition, the HRMS data also corresponded with the structure of

[2]catenane 4-14, in which two rings of dipeptide 4-4 are interlocked within each other

(Figure 4-7).



















Figure 4-6. HPLC spectrum of isolated compound from experiment 3










Theoretical m/z: C52H56N408 [M] = 865.4171
Found: C52H56N408 [M]+ =865.4174


Head to Tail Head to Head
4-7a 4-7b


[2]Catenane
4-14

Figure 4-7. Structures of dimers 4-7a, 4-7b, and catenane 4-14

The NMR chemical shifts and coupling values of the isolated compound are shown

in Figure 4-8 and Table 4-2. NMR data proved that two dipeptide building blocks were

attached in a head to tail fashion because the alkene protons had two different chemical

shifts (5.04 and 5.84 ppm), which ruled out structure 4-7b. If the building blocks were

linked in a head to head fashion, then the chemical shifts would be identical.









(128.2)


(63.5) (37.9)
q J/k b m/n
(32.4) (132.7)
(142.6)



(126.7) (138.1) e (169.1)
NH
(62.1) N
b c o/p h (47.4)
(128.4) (129.8) (37.3) 1
O (171.3)

r/t s
(32.2) (22.9)


Numbers in parenthesis refer to 13C chemical shifts

Figure 4-8. 13C NMR chemical shifts and the corresponding protons of isolated
compound

To determine if the isolated product was structure 4-7a or catenane 4-14, we

examined the NOESY spectrum. Molecular models of the most stable conformation of

catenane 4-14 were generated using the HyperChem program (HyperCube Inc., Version

7). The most likely conformation of catenane 4-14 is shown in Figure 4-9, where the

aromatic ring of one molecule is perpendicular to the alkene of the second ring. Because

of the shielding effects of the aromatic ring on the olefinic protons, we would expect the

chemical shifts to be approximately 2-3 ppm further upfield.106 However, we did not

observe the shielding effects on the olefinic protons. Therefore, catenane 4-14 was ruled

out based on the models, leaving the most likely structure to be dimer 4-7a (Figure 4-10).

The large Jvalue (15 Hz) also indicated the presence of a trans alkene.









Table 4-2. Proton chemical shifts and J values
Proton Chemical Shift (ppm) Spin Coupling (Jvalue in Hz)a

a 7.39 m
b 7.26 m
c 7.22 m
d 7.18 m
e 6.62 d (7.2)
f 5.84 d,t,t(15.3, 6.0, 1.1)
g 5.04 dddt (14.3, 7.9, 6.2, 1.6)
h 4.55 dd (8.3, 3.5)
i 4.28 q (7.3)
j 4.27 m
k 3.82 m
1 3.62 m
m 3.38 dd (15.1, 6.6)
n 3.22 dd (14.5, 6.2)
o 3.02 dd (14.1, 7.6)
p 2.66 dd (13.7, 7.7)
q 2.25 m
r 2.18 m
s 1.84 m
t 1.76 m
a 1 1 ,/s 1 1, s,*1 /s


doublet (a), multiple (m), triplet (t), q (quartet)

In addition to dimer 4-7a, we also attempted to isolate some other major CM

products by column chromatography. We obtained a compound that corresponded to

peak X of the HPLC trace shown in Figure 4-11. HRMS of the compound showed the

presence of a dimer made up of two dipeptide 4-4 molecules. However, H NMR of the

isolated compound appeared oligomeric and 2D NMR confirmed the presence of terminal

alkenes. Therefore, we could only conclude that there was a mixture of CM products

corresponding to peak X.




































Figure 4-9. Model of catenane 4-14


Figure 4-10. Model of dimer 4-7a







77



HPLC of crude reaction mixture (Experiment 3]



Starting Material
Dipeptide 4-4

Peak X



Dimer 4-7a





I





Figure 4-11. HPLC spectrum of experiment 3 reaction mixture (CH2C12, 5mM, reflux 15
hours, no template)

4.3 Conclusions

A series of CM reactions were conducted to generate a library of peptidomimetics.

The building block utilized was a dipepetide molecule consisting of a phenylalanine,

proline, aromatic linker, and two terminal olefin moieties. Lithium templates were

introduced into the system to determine the influence on the equilibrium. There was no

dramatic shift in the equilibrium where one major product was formed as demonstrated

by Sanders group. However, the largest change occurred in our system when the reaction

mixture was refluxed in CH2C12 (5 mM) for 15 h and Lil was used as the template. We

have isolated and characterized dimer 4-7a, which was formed by the CM and RCM of

dipeptide 4-4 in a head to tail arrangement with the trans alkene favored. The research

group is continuing the project to isolate and purify other peptide molecules.














CHAPTER 5
MODEL STUDY MOLECULAR IMPRINTING OF NERVE GASES

5.1 Introduction

Molecular imprinted polymers (MIPs) are useful mimics of biological receptors,

enzymes and antibodies.71'72 In addition, their application in chemical sensors is

imperative due to recent national concerns over chemical warfare. This current threat

involves the use of chemical agents that can incapacitate or kill those who are exposed by

affecting the functions of the body, such as the nervous system, lungs, blood, or skin. 122

Chemical weapons were first used during World War I by the Germans. During an

attack against the Allied troops, the release of chlorine gas resulted in an estimated 5,000

dead and 10,000 disabled.123 Chemical weapons are not just a concern for the military,

but also civilians. In 1981, the Iraqis attacked a Kurdish town with mustard gas, a blister

agent that can cause nausea, vomiting, pain, and death.123 Due to recent terrorist attacks

on U.S. soil, the ability to detect these deadly agents quickly and accurately has been a

priority for national defense.

Our group is interested in using molecular imprinted technology as a sensor for

chemical agents such as the "nerve gases," a family of organophosphates that interfere

with the nervous system and can cause injury or death (Figure 5-1).122,123 In a healthy

body, a neurotransmitter called acetylcholine is released from the presynaptic neuron and

crosses over the synaptic cleft (Figure 5-2). Upon attachment to a receptor on the

postsynaptic neuron, a nerve signal is transmitted.124 Acetylcholine is then released from

the receptor, and the enzyme acetylcholinesterase hydrolyzes the neurotransmitter to









prevent build up of the chemical in the synapse and over "firing" of the nerve. Nerve

agents are toxic because they have the ability to phosphorylate acetylcholinesterase; thus

inactivating the enzyme and causing an accumulation of acetylcholine, which

continuously binds to the receptors.122 This essentially means the nerve is constantly in

the "on" position and can lead to a variety of effects, such as vomiting, coma, paralysis of

the muscles and respiratory arrest.123 Of the four chemical agents shown below in Figure

5-1, VX (5-1) is the most toxic.122


YO~


0
O-P-F
I


VX Sarin

5-1 5-2

Figure 5-1. Structures of common nerve agents


I1
0-P-F

Soman

5-3


i 0
O-P-F
/N\
Tabun

5-4


18
Aw


Acetate P \Acetylcholinesterase

Choline E


Presynaptic Neuron




Synaptic Cleft



Acetylcholine Receptor

Postsynaptic Neuron


Figure 5-2. Mechanism of acetylcholine in the transmission of nerve impulses









Due to VX toxicity, we have chosen 2-(diisopropylamino)ethanethiol (5-5) as the

template. This thiol is a degradation product of VX and less hazardous to handle.125

Thiol 5-5 can be synthesized from thiourea and 2-(diisopropylamino)ethyl chloride

following literature procedures.126




0 _11 fTSH-
"N S + HO-P-O


5-1 5-5 5-6

Scheme 5-1. Degradation products of VX, following hydrolysis

Development of our proposed MIP involves the Michael addition of thiol template

5-5 to the fluorescein maleimide 5-7, which serves as the detector (Scheme 5-2).

Fluorescein maleimide is a widely used fluorescent green dye selective toward thiols.127

Compound 5-8 is then coupled to 5-norbomene-2-carboxylic acid (5-9, mixture of endo

and exo), which is required for polymerization. Carboxylic acid 5-9 serves as the

functional monomer and is allowed to form hydrogen-bonded complexes with 5-10.

Ring Opening Metathesis Polymerization (ROMP) with the complex and cross-linking

monomer (5-11) using Grubbs' second generation catalyst 1-4 results in the rigid network

(Schemes 5-2 and 5-3). The mechanism of the ROMP with the monomers and dye is

shown in Scheme 5-4. Removal of the template and the regeneration of the maleimide

leave the desired imprint. In other words, a molecular memory is introduced in the MIP,

in which the recognition sites possess the correct shape and orientation of the functional

groups to make it selective for thiol template 5-5.










H O

O N


X^yO
5-7


Functional Monomers
5-9


Hydrogen-bonded Complexes


/IN Oi

5-9




O~O


-OO
S 5-10

N


O O
0
ROMP Cross-Linking Monomer
5-11
Grubbs' 2nd Generation Catalyst 1-4

Cross-linked polymer (See Scheme 5-3)

Scheme 5-2. Formation of hydrogen-bonded complexes


'~ N SH


Template
5-5
























Cross-Linked
Polymer


Scheme 5-3. Formation of the MIP



/=MLn R MLn
R( ) 12+2] 12+21


5-11


5-9: X = OH

5-10: X = Fluoresceinyl thiol ether


Repeat with 5-9 or 5-11
------------
-------------


Cross-Linked Polyme


Scheme 5-4. Mechanism of the ROMP polymerization









Fluorescein maleimide 5-7 was chosen as the dye because it has been extensively

used as a thiol reactive dye.127-129 It possesses many useful photophysical properties and

is very reactive and selective toward thiol groups.127 130 It absorbs light of mean

wavelength 427 nm and emits visible green light at 515 nm.127 One research group

demonstrated the increase in fluorescence emission when glutathionine, a tripeptide

containing a thiol group, was allowed to react with fluorescein maleimide.131 We plan on

examining the reaction of our thiol substrates with the dye and determining the effects on

the fluorescence emission. Fluorescein maleimide was also selected as the dye because

the norbornene moiety, which is required for ROMP, can be coupled to either one or two

of the alcohol groups in one step without modification to the dye (Scheme 5-5). With

only one norbornene molecule attached, the fluorescein molecule can exist in two

tautomeric forms.

Cross-linking monomers 5-11 have been made previously in our group, starting

from commercially available 5-norbomene-2-carboxaldehyde (5-16).75 The aldehyde can

be reduced to alcohol 5-17, which can then react with adipoyl chloride (5-18) to form

diester 5-11 (Scheme 5-6).

Ratios of the template/monomer/cross-linking agent will be tested to determine the

best polymerization conditions. Binding properties of the MIP will be evaluated using

gas chromatography and mass spectrometry methods to ensure whether it is specific to

only thiol 5-5.