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Cysteine based PNA (CPNA)

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Title:
Cysteine based PNA (CPNA) design, synthesis and application
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Book
Language:
English
Creator:
Yi, Sung Wook
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects

Subjects / Keywords:
Cell permeable biomolecules
Antisense therapy
Solid phase synthesis
Hydrazines and hydrazides
Regio-selective acylation
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: This report mainly discusses the development of the cysteine based PNA (CPNA), which is an analogue of PNAs. Peptide nucleic acids (PNA), a pseudopeptide DNA mimic, was discovered by Nielsen and his coworker in 1991. PNA is proved to sequence-specifically form a very stable duplex with complementary DNA and RNA strands through Watson-Crick base paring, and it is also capable of binding to duplex DNA by helix invasion. These intriguing properties of PNA implicated great potential for medical and biotechnical applications. Therefore, PNA has attracted many scientists in the fields of chemistry, biology, medicine including drug discovery and genetic diagnostics, molecular recognition. Due to its acyclic, achiral and neutral nature of the backbone, PNA has shown problems such as its poor aqueous solubility, poor cell permeability and instability of PNA-DNA duplexes and triplexes.Accordingly, many synthetic approaches have been directed toward developing modified backbones of PNA. Among those PNA analogs, only few examples including lysine-based monomers, guanidine-based peptide nucleic acids (GPNA) and the aminoethylprolyl PNA (aep-PNA) showed noticeable enhancements with regards to the daunting challenges mentioned above. Reported herein is the summary of our research endeavor to develop the CPNA oligomers with the great water-solubility and cell permeability. Chapter one briefly summarizs the background and history of the PNA as the front-runner of the antisense therapeutic agents. Chapter two discusses the novel protocols that enabled synthesis of the various versions of CPNA monomers for both Fmoc and Boc solid phase synthesis strategies. Chapter three includes the experimental procedures for solution phase preparation of the CPNA monomers. Chapter four starts with the introduction of solid phase synthesis strategy.After the brief review, our efforts on solid phase based synthesis of CPNA oligomers are discussed. Detailed procedures for the solid phase synthesis are summarized in Chapter five. Disclosed In the final chapter is a methodology which enables regioselective mono-acylation of hydrazines. Remarkably, this new protocol gives the mono-acylation on the less-reactive nitrogens of the hydrazines. Carbon disulfide takes the key role for this unique transformation. At the end of the dissertaion, selected NMR and Mass spectra are attached.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Sung Wook Yi.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 220 pages.
General Note:
Includes vita.

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aleph - 002007393
oclc - 403799916
usfldc doi - E14-SFE0002346
usfldc handle - e14.2346
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ABSTRACT: This report mainly discusses the development of the cysteine based PNA (CPNA), which is an analogue of PNAs. Peptide nucleic acids (PNA), a pseudopeptide DNA mimic, was discovered by Nielsen and his coworker in 1991. PNA is proved to sequence-specifically form a very stable duplex with complementary DNA and RNA strands through Watson-Crick base paring, and it is also capable of binding to duplex DNA by helix invasion. These intriguing properties of PNA implicated great potential for medical and biotechnical applications. Therefore, PNA has attracted many scientists in the fields of chemistry, biology, medicine including drug discovery and genetic diagnostics, molecular recognition. Due to its acyclic, achiral and neutral nature of the backbone, PNA has shown problems such as its poor aqueous solubility, poor cell permeability and instability of PNA-DNA duplexes and triplexes.Accordingly, many synthetic approaches have been directed toward developing modified backbones of PNA. Among those PNA analogs, only few examples including lysine-based monomers, guanidine-based peptide nucleic acids (GPNA) and the aminoethylprolyl PNA (aep-PNA) showed noticeable enhancements with regards to the daunting challenges mentioned above. Reported herein is the summary of our research endeavor to develop the CPNA oligomers with the great water-solubility and cell permeability. Chapter one briefly summarizs the background and history of the PNA as the front-runner of the antisense therapeutic agents. Chapter two discusses the novel protocols that enabled synthesis of the various versions of CPNA monomers for both Fmoc and Boc solid phase synthesis strategies. Chapter three includes the experimental procedures for solution phase preparation of the CPNA monomers. Chapter four starts with the introduction of solid phase synthesis strategy.After the brief review, our efforts on solid phase based synthesis of CPNA oligomers are discussed. Detailed procedures for the solid phase synthesis are summarized in Chapter five. Disclosed In the final chapter is a methodology which enables regioselective mono-acylation of hydrazines. Remarkably, this new protocol gives the mono-acylation on the less-reactive nitrogens of the hydrazines. Carbon disulfide takes the key role for this unique transformation. At the end of the dissertaion, selected NMR and Mass spectra are attached.
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Regio-selective acylation
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Cysteine Based PNA (CPNA): Design, Synthesis and Application by Sung Wook Yi A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Mark L. Mclaughlin, Ph.D. Edward Turos, Ph.D. Bill J. Baker, Ph.D. Kirpal S. Bisht, Ph.D. Date of Approval: April 2, 2008 Keywords: cell permeable biomolecules, antisense therapy, solid phase synthesis, hydrazines and hydrazides, regio-selective acylation Copyright 2008, Sung Wook Yi

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Dedicated to My entire family and friends, Especially to my mom who lives in my heart.

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Acknowledgements I would like to thank my major professor, Dr. McLaughlin, for his support. He has been paving the way for me as advisor and given me the opportunity to work on the great projects. It has been a great experience to work in his lab and I have l earned so much from him. I just dont think I can express my gratitude enough in words for his favor. I must thank Dr. Turos, Dr. Baker and Dr. Bi sht for their support and care as my committee throughout the course. They have been parenting me as well from the beginning of my graduate program. I thank Priyesh, Mingzhou, Shrida, Laura, Missi, David and Mehul who shared time with me in the McLaughlin lab. I also would like to thank Tanagi, Umut, Kiran, Stephanie, Rao, Gabriel for their time and discussion. I owe special thanks to Drs, Advait Nagle, David Flanigan and Young Chun Jung who have been always supporting and standing by me as my se nior students. I thank Chiliu(John) Chen, Matt Cross, Robert, and other old members who spent their valuable time with me. I also would like to thank Dr. Jung and Dr. Yoon for their support as well. I would like to thank Dr. Gauthi er, Dr. Rivera, Dr. Larsen, Dr. Antilla and their group members for discussion and supports for the analytical instruments. I would like to thank all the people in the Chemistry Department who have been sharing the idea and time with me. Finally, I would like to thank my family and friends for everything.

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i Table of Contents List of Tables................................................................................................................. ............... vi List of Figures................................................................................................................ ............... vii List of Schemes................................................................................................................ ............ ix Abstract........................................................................................................................................ xi 1. Chapter One: An Introduction to the Properties a nd Applications of PNA and why CPNA: Cysteine Based PNAs are Important New Advances. .................................................................. 1 1.1. Intr oduc tion.............................................................................................................. ............... 1 1.2. DNA: structure and its prope rties. ........................................................................................ .. 2 1.2.1. Struct ure of DNA. ....................................................................................................... ....... 2 1.2.2. Functi on of DNA. ........................................................................................................ ....... 4 1.3. Antisens e Ther apy. ........................................................................................................ ........ 6 1.3.1. Introduction to antisense therapy...................................................................................... 6 1.3.2. DNA and RNA oligonucleotides as antisense t herapeuti c agents.................................... 7 1.3.3. The endeavor to overcome the inconvenient truths of antisense therape utic agents. ..... 8 1.4. PNA: structure, property and appli cat ion............................................................................. 10 1.4.1. Structure and pr operties of PNA. .................................................................................... 10 1.4.2 PNA ap plic ation.......................................................................................................... ..... 14 1.5. PNA analogues and deriva tives. .......................................................................................... 14 1.5.1. Rationales for modif ication of PNA................................................................................. 15 1.5.2. Structural modific ation of PN A backbone....................................................................... 15 1.5.3. Structural modific ation of nucleobases........................................................................... 20 1.6. Cysteine Ba s ed PNA( CPNA)............................................................................................... 22

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ii 1.7. Refe rences................................................................................................................ ........... 23 2. Chapter Two: Synthesis of Cystein e based PNA Monomers. ................................................... 29 2.1. Intr oduc tion.............................................................................................................. ............. 29 2.2. CPNA sub-monomers with acetoni de protec ting on N, Stermini....................................... 30 2.3. Nucleo base coupli ng on the submonomers....................................................................... 33 2.4. CPNA monomers with Boc and tr ityl groups on the N, S-termini ......................................... 36 2.5. Synthesis of CPNA monomers with the alkyl groups on the S-terminus. ............................ 39 2.6. Inversed CPNA: new CPNA backbone. ............................................................................... 45 2.7. Conclusion................................................................................................................ ............ 47 2.8. Refe rences ................................................................................................................ ........... 48 3. Chapter Three: Experimental Procedures for Chapter Two. ..................................................... 49 3.1. Intr oduc tion.............................................................................................................. ............. 49 3.2. Experimental procedures for c ompounds in Sc hemes 2 and 3........................................... 49 3.2.1. Preparat ion of 1. ....................................................................................................... ...... 49 3.2.2. Preparat ion of 2. ....................................................................................................... ...... 50 3.2.3. Preparat ion of 3. ....................................................................................................... ...... 50 3.2.4. Preparat ion of 4. ....................................................................................................... ...... 51 3.2.5. Preparat ion of 5. ....................................................................................................... ...... 51 3.2.6. Preparat ion of 6. ....................................................................................................... ...... 51 3.2.7. Preparat ion of 7. ....................................................................................................... ...... 52 3.2.8. Preparat ion of 8. ....................................................................................................... ...... 52 3.2.9. Preparat ion of 9. ....................................................................................................... ...... 53 3.2.10. Prepara tion of 10. ..................................................................................................... .... 53 3.2.11. Prepara tion of 11. ..................................................................................................... .... 54 3.2.12. Prepara tion of 12. ..................................................................................................... .... 54 3.3. Experimental procedures for compounds in Sche mes 3, 4 and 5....................................... 55 3.3.1. Preparat ion of 17. ...................................................................................................... ..... 55

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iii 3.3.2. Preparat ion of 14....................................................................................................... ..... 55 3.3.3. Preparat ion of 18. ...................................................................................................... ..... 56 3.3.4. Preparat ion of 15. ...................................................................................................... ..... 56 3.3.5. Preparat ion of 19. ...................................................................................................... ..... 57 3.3.6. Preparat ion of 16. ...................................................................................................... ..... 57 3.3.7. Preparat ion of 20. ...................................................................................................... ..... 57 3.3.8. Preparation of 21, 24 and 25. ......................................................................................... 58 3.3.9. Preparat ion of 35. ...................................................................................................... ..... 59 3.3.10. Prepara tion of 36. ..................................................................................................... .... 59 3.4. Experimental procedures for c ompounds in Sc hemes 6 and 7........................................... 60 3.4.1. Preparat ion of 37. ...................................................................................................... ..... 60 3.4.2. Preparat ion of 38a. ..................................................................................................... .... 60 3.4.3. Preparat ion of 38b. ..................................................................................................... .... 60 3.4.4. Preparat ion of 39. ...................................................................................................... ..... 61 3.4.5. Preparation of 40, 41 and 42. ......................................................................................... 62 3.5. Experimental procedures for compo unds in Schemes 8, 9, 10, 11, 12 and 13. .................. 63 3.5.1. Preparat ion of 47. ...................................................................................................... ..... 63 3.5.2. Preparat ion of 48. ...................................................................................................... ..... 63 3.5.3. Preparat ion of 49. ...................................................................................................... ..... 64 3.5.4. Preparat ion of 50. ...................................................................................................... ..... 64 3.5.5. Preparat ion of 51. ...................................................................................................... ..... 65 3.5.6. Preparat ion of 52. ...................................................................................................... ..... 65 3.5.7. Preparat ion of 53. ...................................................................................................... ..... 66 3.5.8. Preparat ion of 54. ...................................................................................................... ..... 66 3.5.9. Preparat ion of 55. ...................................................................................................... ..... 67 3.5.10. Prepara tion of 56. ..................................................................................................... .... 67 3.5.11. Prepara tion of 57. ..................................................................................................... .... 68 3.5.12. Prepara tion of 58. ..................................................................................................... .... 68 3.5.13. Prepara tion of 67. ..................................................................................................... .... 69 3.6. Experimental procedures for c ompounds in Schemes 14................................................... 69 3.6.1. Preparat ion of 72. ...................................................................................................... ..... 69 3.6.2. Preparat ion of 73. ...................................................................................................... ..... 70 3.6.3. Preparat ion of 84. ...................................................................................................... ..... 70 3.6.4. Preparat ion of 85. ...................................................................................................... ..... 71 3.6.5. Preparat ion of 86. ...................................................................................................... ..... 71

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iv 3.6.7. Preparat ion of 87....................................................................................................... ..... 72 4. Chapter Four: Synthesis of Cysteine ba se d PNA Oligomers: Solid Phase Synthesis. ............. 73 4.1. Intr oduc tion.............................................................................................................. ............. 73 4.1.1. Historical introduction to solid phase sy nth esis: R. Bruce Merrifield and the solid-phase synthesis................................................................................................................................... 73 4.2. Strategies of so lid phas e synthesis...................................................................................... 74 4.3. Solid supports for soli d phase meth odologi es...................................................................... 78 4.3.1. Essential factor for the s olid supports............................................................................. 78 4.3.2. Representative Solid s upports for SPPS........................................................................ 79 4.3.3. Ananyltical methods for SPPS. ....................................................................................... 87 4.3.4. Peptide co upling methods .............................................................................................. 87 4.4. Synthesis of CPNA oligomers .............................................................................................. 93 4.4.1. Preparation for soli d phase synthesi s............................................................................. 93 4.4.2. Solid phase sy nthesi s using native chemical ligation..................................................... 97 4.4.3. Solid phase synthesis usi ng S-alkylat ed monome rs.................................................... 100 4.5. Conclusion................................................................................................................ .......... 109 4.6. Refe rences ................................................................................................................ ......... 109 5. Chapter Five: Procedures and Experimental Data for Chapter Four. ...................................... 111 5.1. Intr oduc tion.............................................................................................................. ........... 111 5.2. General procedures and data for the solution-phase deprotect ion of the Monomers........ 111 5.2.1. General pr ocedure for deprotec tion of Boc protected monomers................................ 111 5.2.2. General pr ocedure for deprotec tion of Fmoc protect ed monomers............................. 114 5.2.3. Procedures for solution bas ed preparation of CPNA Dimers ....................................... 115 5.3. General procedures for the s olid-phase synthesis............................................................. 116 5.3.1. Selecti on of Solvents ................................................................................................... 116 5.3.2. Deprotecting N-term inal protec ti ng group..................................................................... 117 5.3.3. Coupling of a monomer or spacer using HA T U activation........................................... 117 5.3.4. Cleavage fr om the res in................................................................................................ 117 5.3.5. Post cl eavage work -up. ................................................................................................ 119

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v 5.4. Analytical procedures for solid phase synthesis................................................................ 119 5.5.1. The procedure for Kais er Test...................................................................................... 119 5.5.2. The procedur e for Fmoc Test....................................................................................... 120 6. Chapter Six: Regioselective Mono Acylation of the Electro nically Less Reactive Nitrogen of Aryl Hydrazines via Temporary Protection with Carbon Disulfide. .................................................. 121 6.1. Intr oduc tion.............................................................................................................. ........... 121 6.2. Diverse biological pro perties of hydrazines........................................................................ 122 6.3. Practice of the hydrazines and their derivatives in the synthesis....................................... 123 6.4. Regioselective mono acylation of the electr oni cally less reactive nitrogen of aryl hydrazines via temporary protection with carbon disulfide................................................................... 125 6.4.1. Developing story of our new protoc ol........................................................................... 125 6.4.2. Optimization of the protocol.......................................................................................... 126 6.4.3. Scope of the protocol. .................................................................................................. 128 6.4.4. Experimental proce dure of the protoc ol........................................................................ 131 6.5. Conclusion................................................................................................................ .......... 131 6.6. Refe rences ................................................................................................................ ......... 132 Appendix ...................................................................................................................................... 133 About the Author End Page

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vi List of Tables Table 1: Comparison of the Boc and Fm oc SPPS strategies........................................................ 77 Table 2: Characteristics of an effici ent resin................................................................................. 78 Table 3: Class of Res ins................................................................................................................ 79 Table 4: Functiona lized PEG-PS res ins......................................................................................... 85 Table 5: Optimization of the protoc ol.......................................................................................... 127 Table 6: Solvent effect s on this reaction...................................................................................... 127 Table 7: Scope of the pheny l hydraz ine substrates..................................................................... 128

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vii List of Figures Figure 1: Structur e of DNA and PNA............................................................................................. .. 1 Figure 2: Chemical s tructure of DNA............................................................................................ ... 2 Figure 3: Structures A, B and Z DNAs. .......................................................................................... .. 3 Figure 4: The basic principles of the antis ense approach............................................................... 7 Figure 5: Analogues of ODNs .................................................................................................... ...... 8 Figure 6: Modified ba c kbones of RNA............................................................................................ 9 Figure 7: Structures of PNA and DNA backbones ......................................................................... 11 Figure 8: PNA binding modes for targeti ng double stranded DNA................................................ 12 Figure 9: Structures of various PNA complexes............................................................................ 13 Figure 10: The first genera t ion of modi fied PNA............................................................................ 16 Figure 11: Second Genera t ion of modi fied PNA............................................................................ 17 Figure 12: PNAs with c hiral backbones......................................................................................... 17 Figure 13: More analogue s of the PN A backbone. ........................................................................ 18 Figure 14: The backb ones of aeg-PNA and other a nalogues........................................................ 19 Figure 15: Structure of the Main Nuc leobases.............................................................................. 20 Figure 16: The non-stan dard nucleobases .................................................................................... 21 Figure 17: Analysis of the cys teine based PNA backbone............................................................ 22 Figure 18: Comparison of the uni ts for P eptide, PNA and CPNA.................................................. 29 Figure 19: Possible pathway s for CPNA backbones..................................................................... 39 Figure 20: Comparison of the CP NA and Invers ed CPNA (I -CPNA)............................................. 45 Figure 21: General scheme of SPPS............................................................................................. 75 Figure 22: General scheme for the conv ergent SPPS. .................................................................. 76 Figure 23: Preparation of common PS resins. ............................................................................... 81

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viii Figure 24: Preparation of comm on PS resins B............................................................................ 82 Figure 25: Preparation of common PS re sins C. ........................................................................... 83 Figure 26: Preparation of common PS re sins D. ........................................................................... 84 Figure 27: Polyethyl ene glycol resins. ......................................................................................... .. 86 Figure 28: coupli ng reagents A................................................................................................. ..... 89 Figure 29: Coupli ng reagents B................................................................................................. .... 90 Figure 30: Coupling reagents C.TPFT IS incorrect........................................................................ 91 Figure 31: Commonly us ed bas es for SPPS................................................................................. 92 Figure 32: Additives fo r the amide c oupling................................................................................... 92 Figure 33: First generation of CPNA mo nomers. ........................................................................... 93 Figure 34: Second Generation of the CP NA monom ers & the IC PNA (T) monomer.................... 94 Figure 35: Comparison of the two mass spectr a (MALDI-TOF) from PEG before (top) and after (bottom) the coupling of the linker.............................................................................. 106 Figure 36: Derivative s of hydrazines........................................................................................... 121 Figure 37: Mechanism of the trans for mations............................................................................. 126 Figure 38: Comparison of the two pr otocols ................................................................................ 130

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ix List of Schemes Scheme 1: Boc protec ted s ub-monomers...................................................................................... 30 Scheme 2: Synthesis of Fmoc sub-monomers 11 an d 12 ............................................................. 31 Scheme 3: Synthesis of acetic ac i d derivatives of nucleo bases 14, 15 and 16 ........................... 33 Scheme 4: Optimization of the coupli ng condition for t he CPNA monomers................................ 34 Scheme 5: Cbz protecte d A and C deri vatives .............................................................................. 35 Scheme 6: Two different routes toward the trityl and Boc pr otected su b-monom er...................... 36 Scheme 7: Preparing the N-Bo c, S-trityl Monom ers...................................................................... 38 Scheme 8: A SN2 protocol (Route C)............................................................................................. 40 Scheme 9: Preparation of the charged al kyl bromides for S-al kylation......................................... 41 Scheme 10: Preparation of R1-alkylated Fmoc Monomers............................................................ 42 Scheme 11: Preparation of R2-alkylated Fmoc Monomers............................................................ 43 Scheme 12: Preparation of Smethyl Fmoc Monomers................................................................. 43 Scheme 13: Alkylated Boc monomers with R1 and methyl groups................................................ 44 Scheme 14: Synthesis of i nversed-CPN A monomer..................................................................... 46 Scheme 15: Deprotection of Boc monome rs for Boc-solid phase synthesis................................. 94 Scheme 16: Deprotection of Fmoc monome rs for Fmoc-solid phase synthesis........................... 95 Scheme 17: Preliminary study befor e the s olid phase synthesis.................................................. 96 Scheme 18: Early trials on s olid-phase synthesis......................................................................... 98 Scheme 19: The attempt of a solid phase synthe si s using sequential native chemical ligation.... 99 Scheme 20: SPS with the S-alkylated monomers ....................................................................... 100 Scheme 21: Optimizing the solid phas e s ynthesis for CPNA oligomers..................................... 101 Scheme 22: Employing the s pacer on the resin.......................................................................... 102 Scheme 23: Fmoc-SPS of the CPNA 15 -mer on a pepti de s ynthesizer...................................... 103

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x Scheme 24: Manual solid phase synthesis with G unit................................................................ 104 Scheme 25: Liquid phase synthesis and silica su pported sy nthesis........................................... 105 Scheme 26: Optimization of the SPS pr otoc ol............................................................................. 107 Scheme 27: Cbz prot ected G m onom ers..................................................................................... 108 Scheme 28: CPNA 18-mer synt hes is (G: Cbz protected)............................................................ 108 Scheme 29: Representative r eac tions of hydrazines.................................................................. 123 Scheme 30: Hydrazines as synthetic intermedia te s for the synthesis of indomethacin and its derivativ es................................................................................................................ 124

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xi Cysteine Based PNA (CPNA): Design, Synthesis and Application Sung Wook Yi Abstract This report mainly discusses the development of the cysteine based PNA (CPNA), which is an analogue of PNAs. Peptide nucleic acid s (PNA), a pseudopeptide DNA mimic, was discovered by Nielsen and his cowo rker in 1991. PNA is proved to sequence-specifically form a very stable duplex with complementary DNA and RNA strands through Watson-Crick base paring, and it is also capable of binding to duplex DNA by helix invasion. These intriguing properties of PNA implicated great potential for medical and bi otechnical applications. Therefore, PNA has attracted many scientists in the fields of chem istry, biology, medicine including drug discovery and genetic diagnostics, molecular recognition. Due to its acyclic, achiral and neutral nature of the backbone, PNA has shown problems such as its poor aqueous solubility, poor cell permeability and instability of PNA-DNA duplexes and triplexes. Accordingly, many synthetic approaches have been directed toward developing modified backbones of PNA. Among those PNA analogs, only few examples including lysine-based monomers, guanidine-based peptide nucleic acids (GPNA) and the aminoethylprolyl PNA (aep-PNA) showed noticeable enhancements with regards to the daunting challe nges mentioned above. Reported herein is the summary of our research endeavor to develop the CPNA oligomers with the great water-solubility and cell permeability. Chapter one briefly summari zs the background and history of the PNA as the front-runner of the antisense therapeutic agents. Chapter tw o discusses the novel protocols

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xii that enabled synthesis of the various versions of CPNA monomers for both Fmoc and Boc solid phase synthesis strategies. Chapter three includes the experimental procedures for solution phase preparation of the CPNA monomers. Chapter four starts with the introduction of solid phase synthesis strategy. After the brief review our efforts on solid phase based synthesis of CPNA oligomers are discussed. Detailed procedures for the solid phase synthesis are summarized in Chapter five. Disclosed In the final chapter is a methodology which enables regioselective mono-acylation of hydrazines. Remarkably, this new protocol gives the monoacylation on the less-reactive nitrogens of the hydrazines. Carbon disulfide takes the key role for this unique transformation. At the end of the dissertaion, selected NMR and Mass spectra are attached.

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1 1. Chapter One: An Introduction to the Properties and Applications of PNA and why CPNA: Cysteine Based PNAs are Important New Advances. 1.1. Introduction. Peptide nucleic acids (PNA), first discov ered in 1991 by Nielsen, Egholm, Berg and Buchardt, is a pseudopeptide DNA mimic, which re places the phosphate ribose ring backbone of DNA with a N-(2-aminoethyl) glycine polyamide backbone1. In spite of the fact that PNAs have a very different structure, they have shown the capa bility of sequence specific binding to DNA as well as RNA. H2N N A O O H N N C O O H N N G O O H N N T O NH2 O DNAO O P O O O O O P O O O O O P O O O O O T G C A HO P O O OH PNA Figure 1: Structure of DNA and PNA. Moreover, their complexes with complementary DNA or RNA have proven to be extremely stable2. Due to their extraordinary binding properties and stability, PNA is considered as a promising reagent for antigene and antisense therapies as well as gene diagnostics.3 In this chapter, the

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2properties of PNA as well as its applications are detailed as to put chapters 2 and 4 in context. In addition, the limitations that PNAs have faced and the approaches to overcome those barriers are discussed in the later stage of this chapter. At the end of this chapter, the backbone of the cysteine based PNA will be disclosed with our rationale for the design of the PNA. 1.2. DNA: structure and its properties. 1.2.1. Structure of DNA. Since PNA was designed as a DNA mimic, and it s applications are directly related to the structure and function of DNA, it is neces sary to understand the nature of DNA. Figure 2: Chemical structure of DNA4 Deoxyribonucleic acid, DNA, is a long polymer made from repeating units called nucleotides.5 The backbone of the DNA strand consists of alternating phosphate and 2deoxyribose residues6 and a base is attached to the sugar in its unit. In DNA, four bases are

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3found and they are called adenine (often abbrev iated as A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate backbone to form the complete nucleotide. James Watson and Francis Crick revealed the double helix molecular structure of the DNA In 1953, and nine years later, they shared the Nobel Prize in Physiology or Medicine with Maurice Wilkins for solving one of the most important of all biological riddles7. In living organisms, DNA normally exists in t he shape of a double helix. The DNA double helix is stabilized by hydrogen bonds between the bases, hydrophobic effects and pi stacking.8 Unlike the complementary base paring, the latter fact ors stabilizing the double helix are not influenced by the sequence of the DNA. Figure 3: Structures of A, B and Z DNAs.4 Due to the fact that two complementary bas es are tied with the weak hydrogen bonding, the two DNA strands can be pulled apart and rejoined relatively easily. Indeed, this reversible interaction between the complementary base pairs is essential in DNA replication and critical for its other functions9.

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41.2.2. Function of DNA. DNA normally occurs as linear chromosomes in eukaryote cells, and circular chromosomes in prokaryote cells. The set of chromosomes in a cell makes up its genome and the human genome has about 3 billion base pairs of DNA arranged into 46 chromosomes10. DNA carries the information held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is accomplished vi a complementary base pairing. For example, in transcription, when a cell utilizes the information in a gene, the sequence of the DNA is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then us ed to make a matching protein sequence in a process called translation, which can also be desc ribed as protein biosynthesis. Alternatively, a cell may simply copy its genetic information in a process called DNA replication. RNAs and their properties will be discussed with more details in a section as part of this chapter. 1.2.2.1. Transcription of DNA. Transcription is the proces s that synthesizes RNA under the direction of DNA. The DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA)11. One notable differenc e between RNA and DNA sequences is the presence of uracyl (U) in RNA in stead of the thymine (T) of DNA. In the case of protein-encoding DNA, transcription is the first step that usually leads to the expression of the genes, by the production of the mRNA intermediate which is a transcript of the gene's proteinbuilding instruction. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit that is translated into protein contains sequences that direct and regulate protein synthesis in addition to coding the sequence that is translated into protein. The regulatory sequence that is before, or 5' of the coding sequence, is called the 5' untranslated (5'UTR) sequence, and the sequence found following or 3' of the coding sequence, is called the 3' untranslated (3'UTR) sequence. Transcription has some proofreading mechanisms, but they

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5are fewer and less effective than the controls for copying DNA; theref ore, transcription has a lower copying fidelity than DNA replication11. As in DNA replication, transcription proceeds in the 5' to 3' direction. Only one of the two DNA strands is transcribed and this strand is called the template strand, because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The DNA template strand is read 3' to 5' by RNA polymerase and the new RNA strand is synthesized in the 5' to 3' direction. RNA polymerase binds to the 5' end of a gene (promoter) on the DNA template strand and travels toward the 3' end. Except for the fact that thymines in DNA are represented as uracils in RNA, the newly synthesized RNA strand will have the same sequence as the coding (non-template) strand of the DNA. For this reason, scientists usually refer to the DNA coding st rand that has the same sequence as the resulting RNA when referring to the directionality of genes on DNA, not the template strand. Transcription is divided into 3 stages: initiation, elongation and terminat ion. These three steps will not be discussed in detail. Once the DNA is transcribed to mRNA the next step is to translate the genetic information from the mRNA into the proteins using the nut rients and fuel provided. The translating process will be briefly discussed la ter in this chapter. 1.2.2.2. DNA replication. The process of copying a double-stranded DNA molecule is called DNA replication and it is essential in all living organisms. Generally, the mechanisms of DNA replications in prokaryotic and eukaryotic organisms are somewhat differen t, and some antibiotics take advantage of those different mechanisms. The basic mechanism of DNA replication became clearer when the complementary, double-stranded structure of DNA was recogni zed by Watson and Crick in 19537,5. Although the replication of the DNA is one of the most important biological processes, it is beyond the scope of this dissertation to discuss in detail.

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61.2.2.3. Structure of RNA and translation. Like DNA, RNA is a nucleic acid made from nucleotide units. This long chain of nucleotides has a nitrogenous base, a ribose su gar, and a phosphate in its unit. It structurally differs from DNA. RNA is usually single stranded in the cell in contrast to the double stranded DNA. Of course, the hydroxyl group in ribose is the functional group that makes the difference. In addition, RNA nucleotides contain uracils instead of thymines, which are found in DNA.5,11 There are many types of RNA f ound in the cell, but it is beyond the scope of this section to discuss all of them and their functions in this report. Among all the RNAs, the messenger RNA (mRNA) is the one that carries the specific information from DNA to the ribosome for production of a protein in a process called translation. Translation can be simply called protein biosynthesis and this important process is the part of the gene regul ation that is proceeded by transcription. The genetic information from chromosomes is carried by the mRNA, and the information is recorded as a ribonucleotide sequence, which is read by the translational machinery in a sequence of nucleotide triplets. These triplets of nucleotid es are called codons and many of them have unique functions. For example, a codon codes for a specific amino acid and a codon also can be a specific order for an action in translation.12 1.3. Antisense Therapy. 1.3.1. Introduction to antisense therapy. Antisense refers to short sequenced oligonucleotides that have a complementary sequence to a mRNA. At present, antisense therapy is an emerging trend in biotechnology. This trend was initiated after the understanding of t he mechanism of gene expression and is powered by the Human Genome Project (HGP), which was completed in 2003 when the approximately 25,000 genes in human DNA were sequenced.13

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7 translation transcription Antigene strategy e.g DNA aptamers, triple helix formation oligonucleotides Antimessage strategy e.g. Antisense oligonucleotides, ribozymes, peptide nucleic acids, RNA aptamers Traditional drug inhibition Figure 4: The basic principles of the antisense approach. The idea of antisense therapy is outlined in Figure 4. About 20 to 25 bases containing nucl eotides of DNA or RNA can bind to a specific messenger RNA (mRNA), which can subsequently prevent protein synthesis.14 Likewise, other molecules that sequence specifically bind to mRNA can target gene related diseases by preventing expression of the targeted protein. Summarized here in Figure 5 is an overview of families of antisense agents.15 1.3.2. DNA and RNA oligonucleotides as antisense therapeutic agents. As discussed briefly before, DNA and RNA oligonucleotides can be great therapeutic agents. However, a couple of studies reported the stability problems of these oligonucleotides in biological system.16 The unmodified phosphodiester backbone of antisense oligodeoxynucleotides (ODNs) is rapidly degrade d in biological systems. Like DNA, unmodified RNA has also shown its instability in the biological environments.17 Moreover, the cellular uptake of antisense DNA and RNA oligonucleotides in cultured cells is very poor. These combined

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8inconvenient truths could not di minish scientists passion for the idea of the antisense therapy. The various strategies to overcome these pr oblems are summarized in the next section. 1.3.3. The endeavor to overcome the inconvenient truths of antisense therapeutic agents. 1.3.3.1 Modification of the backbones of the oligonucleotides. As it is discussed before, the DNA and RNA oligonucleotides have poor bioavailability and delivery problems as antisense therapeutic agents. To increase the stability for the therapeutic applications the backbone of the oligonucleotides has been extensively modified. O O P O O O O P O O O O O O P O O O B B B O DNA B N O O HN N O B O HN N O B O HN HN PNA O O P O O O O P O O S S O O P O O S B B B O S-DNA N P O O Morpholino N O B N O B O P O N N O B O O O P O O O O P O O O O O O P O O O B B B O O O P O O O O P O O O O O O P O O O B B B O O O O O O O O O O 2'-Methoxyethyl ODN LNA Figure 5: Analogues of ODNs. First, the phosphorothioate (PS) ODNs18 were widely studied as the representative of first generation modified oligonucleotides. Peptide nucleic acids (PNAs), 19 2-methoxyethyl based ODNs,20 morpholino-based ODNs21 and locked nucleic acids (LNA) are considered as second

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9generation in this category. The representations of the structural variation of modified versions of ODNs are illustrated in Figure 5. Similar to DNA, since unmodified RNA showed instability, various backbone modifications have been done to increase the stability of ribozymes while retaining its catalytic activity.22 2-O-methyl ribonucleotides,23 chimeric RNA-phosphorothioate DNA,24 and 3-3inverted thymidines at the 3 termini25 are the representative analogues that are partially replaced in the ribozyme to preserve catalytic activity.26 The structures of modified backbones of the RNA are represented in Figure 6. B O OH O O unmodified RNA B O OMe O O B O O O B O NH2 O O T O O O 2'O-methyl ribonucleotides 2'-amino RNA 2'-C-allyl RNA 3'-3'-inverted thymidine Figure 6: Modified backbones of RNA. In summary, the endeavor to enhance the plasma stability of the oligonucleotides by modifying the backbones has show n some positive improvements, but in terms of improving their cellular uptake, the available modifications hav e solved the intracellular delivery issues. More recently, the phenomenon of RNAi has gai ned substantial attention from the world of antisense therapy after Andrew Fire and Craig Mello introduced double-stranded (ds) RNA to inhibit targeted unc-22 RNA gene, producing severe twitching movements in the worm.27 In 2006, Fire and Mello won the Nobel Prize for their wo rk which began in 1998. The mechanism of RNA

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10interference is conceptually similar to antisense but the RNA appears to play the key role for the process is double stranded RNA (dsRNA) or siRNA, so it is technically a little different than the mechanism of traditional antisense mechanisms. In contrast to the approaches that directly modify the DNA/RNA nucleotides, there are indirect ways to overcome the barriers that possible antisense agents must overcome. An alternate method is the conjugation of the antisense oligomers with other molecules as aids for the delivery. Among other possible methods, conjugating the oligomers with lipids,28 dendrimers,29 biodegradable polymers30 or cell penetrating peptides31 are the strategies that are commonly used to deliver the ant isense agents to the targets. 1.4. PNA: structure, property and application. 1.4.1. Structure and properties of PNA. According to the article, which disclosed PN A for the first time in 1991, it was designed by computer-assisted modeling by building a normal TAT triplex.1 The deoxyribose phosphate backbone of the third (the T) strand was remov ed and a polyamide backbone was built to replace the original backbone. Surprisingly, the resulting polyamide nucleic acids proved to be a good structural mimic of the ribose phosphate ba ckbone of nucleic acids. Consequently, PNA immediately attracted scientists with wide in terests because it had great potential as a gene therapeutic agent or as a t ool for genetic diagnostics.

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11H2N N A O O H N N C O O H N N G O O H N N T O NH2 O DNAO O P O O O O O P O O O O O P O O O O O T G C A HO P O O OH PNA Figure 7: Structures of PNA and DNA backbones. The structure of the PNA backbone is completely changed so it looks little like a ODN and more like a peptide. It certainly has polyamide backbone, however technica lly it is not peptide because the basic unit is no longer an amino acid. As early as the 1970s, De Koning and Pandit disclosed a class of nucleopeptides derived from lysine,32 and Buttrey et al. prepared the polymers of thyminyl-alanine.33 However, these oligomers of nucleobase amino acids did not hybridize to polyA efficiently; subsequently t he application towards the antisense technology could not be extended further.34 Nonetheless, the aminoethyl gl ycine based PNA presented great binding properties to DNA as well as RNA, and this result revived the world of antisense therapeutics one more time.35

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12 a) Triplex b) Triplex invasion c) Duplex Invasion d) Double Duplex Invasion Figure 8: PNA binding modes for ta rgeting double stranded DNA. (Bold structure signifies the 10mer PNA and Bl ue line represents Watson-Crick base paring). PNAs have shown that a mimi c of DNA/RNA do not have to re ly on the backbones of the DNA and/or RNAs. According to a study which was reported by Nielsen,35 the reason why the PNA is a good mimic of DNA can be explained by the favorable geometry of the backbone combined with a structure of constrained flexibility. This allows the oligomer to adapt to the helical structure preferred by DNA or RNA without losing the entire gain in binding enthalpy in decreased entropy upon formation of the much more rigid dupl ex or triplex structures. His explanation could be supported by the study that was reported in 1996.36 In that report, they removed the rigidity imposed by the planar amide by reducing the amide linker and the DNA binding properties of the PNA with the reduced backbone was detrimental. T herefore, a certain degr ee of the rigidity in PNA backbone is important for the DNA/RNA bindi ng. More structural studies with regards to their binding properties shall be discussed in t he section that addresses the analogues of PNA.

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13 Originally, PNA was designed with the expectation that it could bind to DNA to make a triplex; however, it was observed that PNA binds to DNA duplex by triplex invasion.37 A single PNA can bind to the DNA duplex by duplex invasion38 although with less efficiency. In addition, when alternative pseudo complementary nucl eobases (such as the 2,6-diaminopurine-2thirouracil) were used, PNA can make very stable complexes by double duplex invasion.38 In Figure 8, binding modes of PNA to DNA duplex are illustrated. Figure 9: Structures of various PNA complexes. At present, NMR and X-ray crystallography hav e determined four three-dimensional (3-D) structures of PNA complexes. As shown in Figure 9, the structures illustrate d in the top panel are the representation of views from the side as compared to the views from the top, which are in lower panel. The PNA-RNA39 and PNA-DNA40 duplex structures were solved by NMR methods while the PNA2DNA triplex41 and PNA-PNA42 duplex structures were determined by X-ray crystallography. The studies have shown that PNA is able to adapt to its partner relatively well. In terms of sugar puckering, the PNA-RNA dupl ex structure is close to DNAs natural A-

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14conformation while the PNA-DNA duplex adopts clos e to B-conformation. Interestingly, PNA in other structures prefers a uni que helix form, the P-form, which can be seen in the PNA-PNA duplex while PNA2DNA triplex structure is a unique-form. 1.4.2 PNA application. ` The fascinating DNA and RNA hybridizat ion properties of PNA have gained great attention because it has such a potential for variou s medical purposes. As it is discussed earlier, it is a contender for antisense t herapy due to its excellent binding properties although exploitation of PNA to the antisense world is hampered by it s inefficient cellular uptake in most eukaryotic cells.43 Its sequence specific DNA/RNA binding properties can be readily applied to gene diagnostic technologies such as in situ hybridization,44 pre-Southern blotting,45 array hybridization,46 modulation of PCR analyses47 and tools for genome mapping.48 It has been reported that PNA could arrest transcription sequence specifically by binding the template strand,49 which suggests that it may also be used as an antigene therapeutic agent. 1.5. PNA analogues and derivatives. The structure of the original PNA monomer has been subjected to rational modification with the expectation of understandi ng the structure activity relations hips in this class of antisense and/or antigene agents as well as with the aim of improving the properties of oligomers to overcome the problems of intracellular delivery. The major problems include low aqueous solubility, ambiguous DNA binding orientation and poor membrane permeability.50

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151.5.1. Rationales for modification of PNA. To solve the problems associated with using standard PNA mentioned earlier, PNA could be structurally modified in different ways. It coul d be the ethylendiamine or glycine sector that undergoes transformation. The linker to the nucle obases can also be modified. In fact, the nucleobase itself can be a factor. Strategically, the common rationale for the modification of PNA can be categorized as follows. The strategic rationales behind the structural PNA modification are listed. First, introducing chirality to the backbone of PNA to gain the orientational selectivity in complementary DNA binding. Secondly, increasing the rigidity of the backbone to entropically drive PNA to the duplex formation. Third, introduction of cationic functional groups directly in the PNA backbone somehow to potentially increase intracellular delivery. Fourth, modulating base pairing by changing the linker to either the nucleobase or the nucleobase itself. Lastly, conjugating the PNA with a molecules that could deliver it into the ta rgeted cells. These changes are necessary to use PNAs for mainly therapeutic purposes. In addition, PNA has been modified to optimize its diagnostic applications. 1.5.2. Structural modifi cation of PNA backbon e. The early attempts to modify the PNA involv e the simple extension of the original PNA backbone, which is the aminoethyl glycine unit or the base linker. The earliest and the simplest modified PNA backbones are grouped as the first generation and illustrated in Figure 10. I repres ents the classic backbone re ported in 1991. Then, simply extending parts of the backbone gave II51, III52 and IV52 respectively, however all of these attempts resulted in a significantly lowering of melting points (Tm) of the derived PNA-DNA hybrids. When replacing the carbonyl group of the tertiary amide it gives the tertiary amine in the backbone, which is V ,53 and this flexible or less rigid and cationic backbone result ed in destabilization of the PNA-DNA hybrid.

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16This result suggested that a certain degree of the rigidity in the backbone is necessary for hybridization properties of PNA. Further modifi cations of the PNA backbone were directed in a way to gain the rigidification of the backbone by introducing alkyl substi tuents on the backbone. These attempts are summarized in Figure 11. N N H O O B N N H O B O N H N O O B N N H O O B NH N H O B N O O O B II I III IV V VI Figure 10: The first generation of modified PNA VII54 shows the template that can substitute the al kyl group on either side of the backbone or on the both sides. A number of the modifications, wh ich replaced the glycine component with other amino acids, consequently leads to chiral PNA VII when R1 is hydrogen. The PNAs with R2 group include both hydrophobic and hydrophilic group, and even charged alpha substituted groups54C have been reported. PNA oligomers with the chir al monomers showed the retention of the hybridization properties with a little less efficiency. Small and medium substituents at the alpha position of the glycine were reported to exhibit tolerance. However, among other replacements, only those derived from D-lysine showed DNA bindi ng properties as good as that of classical PNA.54 Even a cyclohexyl ring is corporated into t he backbone with different approaches to give VIII55 with the 1,2-cyclohexylamino group and IX56 with the spirocyclic hexyl group. These trials though did not improve PNAs DNA binding properties.

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17 N N H O O B R1 R2 *N N H O O B N N H O O B N H N O B O N N H O O B O N N H O O B O N O B H N VII VIII IX X XI XII Figure 11: Second Generation of modified PNA. N H* N H B O B O N H O B N H H N O B O N H H N O O B N H H N O O B N H H N O O O B XIII XIV XV XVI XVII XVIII XIX Figure 12: PNAs with the chiral backbones. By interchanging of various carbonyl and NH groups on the peptide linkages, different types of PNAs( X ,57 XI58 and XII59) have been generated. All these systems exhibited a lower potency for duplex formation with the complem entary DNA/RNA implying that in addition to the

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18geometric factors, other subtle changes or factors may influence the PNA-DNA or PNA/RNA hybridization. N H O B n O R B HN HO HO O NH O B O N H B O O N P O B O O R N H N P O B O O R N H N O B R O P O O XX XXI XXII XXIII XXIV XXV XXVI Figure 13: More analogues of the PNA backbone. XIII60 represents the early strategy that using alpha-amino acids carrying nucleobase building blocks that reported even bef ore PNA was introduced. These nucleopeptides did not have significant interaction with polynucleotides. A study to incorporate XIII and XIV in one oligomer was reported, but the result was not encouraging.61 Adenylic analogues of PNA derived from XV, which were interspersed with the glycine, serine, threonine or tyro sine exhibited strong binding properties with poly (dT) and poly (U) to form triplexes.62 PNA oligomers derived from alternating monomers of XVI XVII XVIII and XIX with the type XIII and other amino acids have been reported without any enhanced properties.63 Although with much weaker affinity, PNAs derived from XX with glycine and alanine linker showed very high sequence specificity.64 Glucopyranosy PNA (GNA)65 derived from XXI have

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19shown its RNA binding affinity equivalent to that of DNA-RNA hybridization but with much better specificity. Tetramers of XXII which have a aromatic moiety in the backbone showed favorable bas stacking interactions66 while the olefin PNAs67 derived from XXIII had less affinity toward DNA which indicates that the importance of amide bond for its electrostatic effects. PNA analogues based on XXIV, XXV and XXVI which, replace the amide to phosphonic acid,68 phosphoamide69 and phosproroamide70 respectively, have shown interesting DNA duplexation properties with the excellent water-solubility due to the negative charges on their backbones. N N H N H N H O B O O B NH2 N H N H B S *H N COOH H2N 2 4 * O H N O B N H N O B O R N H N O B * O O O B P O O 2 4 2 4 3 * O O B P O O 2O XXVII XXVIII XXIX XXX XXXI XXXII XXXIII XXXIV Figure 14: The backbones of aep-PNA and other analogues. The chimerical PNAs, which are derived from alternating monomers of PNA and DNG ( XXVII ) have been reported71. These PNA-DNG oligomers have show n great binding properties with

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20DNA and RNA. Similarly, PNAs with the XXVIII72 as the alternating monomer have also been reported. PNAs from monomers derived from XXIX73 also have shown relatively positive results with its flexibility to interchange the position of the nucleobase and backbone among the different nitrogens. Three monomers XXX,73 XXXI74 and XXXII75 have cyclic ring systems on the backbone. While oligomers of XXXI have shown negative results due to the extreme rigidity of the tertiary amide with the ring system, PNAs derived from XXX and XXXII have shown promising results. Especially the latter one, the N-(2-aminoethyl) prolyl (aep) PNA, is considered as one of the most successful analogues because it has shown an un precedented affinity to wards DNA strand with the great discriminating power and at least 25 times better aqueous solubility than the original PNAs. Oligomers from XXXIII and XXXIV have been reported76 with some good results. However, these backbones are closer to that of DNA than PNA. 1.5.3. Structural modifi cation of nucleobases. N N N H N NH2 NH N N H N O NH2 N N H NH2 O NH N H O O N N H NH2 O N N N N NH2 O OH OH HO N N N N NH2 O OH OH O P -O OO Nucleobases :AdenineGuanine Thymine Cytosine Uracil Adenosine Adenosine monophosphate ( AMP) N N N N NH2 O OH O P -O OO Deoxyadenosine monophosphate (dAMP) Purines PyrimidinesA nucleoside Nucleotides Figure 15: Structure of Main Nucleobases.

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21 There has been a steady stream of reports in modulating and expanding the recognition motif of standard base pairs to better understand the recognition process in the world of nucleotides chemsitry.50 Compared to the efforts of bac kbone modification, not many nonstandard nucleobases has been employed in the PNA or oligonucleotides scaffolds. The limited studies on the modification of the nucleoba ses have reported some interesting results.77 The structures of the modified the nucleobases have been summarized in Figure 16 along with the stand ard nucleobases ( Figure 15) for the comparison. In short, these approaches could provide very useful i nformation concerning the binding properties of DNA analogues as well as the applications in diagnostic and nanomaterial chemistry. N N N N NH2 H2N HN N H N N O O N N N N H2N HN N N N S H2N N NH N N HN N HNN O NH2 O NH O S O NHCOPh XXXV XXXVI XXXVII XXXVIII XXXIX XL XLI XLII Figure 16: The non-standard nucleobases.

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221.6. Cysteine Based PNA(CPNA). Improving the bioavailability of potential drugs is the one of the major challenges for drug discovery and PNAs have been facing these co mmon problems. Despite all the modifications from different angles, the development of PNAs with the therapeutic applications is still being hampered by cellular uptake and delivery problems. PNAs have been successful so far in gene diagnostics, however, it is too early to be sati sfied with the progress that PNAs have made. H N* N O S O (B) O R H N N O O (B) O Chirality Rigidity Cationic charge ? ? PNA CPNA Figure 17: Analysis of the cysteine based PNA backbone. By carefully analyzing the data from the ear lier studies, a new backbone of the PNA was designed and developed. As it is shown in Figure 17, this backbone consists of a cysteine and an aminoethyl g roup. Cysteine is a chiral amino acid that has a highly nucleophilic thiol group. Most of time, this functional group has to be protec ted when one needs to do chemical transformations

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23on the other functional groups. This explains the reason why the cysteine has rarely been involved with the modification of PNA by other groups. We have envisioned that the thiol group can add many positive aspects as part of the PNA backbone in many ways. The aminoethylcysteinyl backbone ideally meets many of the criteria t hat have been applied for modifying the backbone of PNA. First, as a chiral amino acid, cysteine can provide the PNA the chirality so the complementary DNA/RNA binding could have selective orientation. Second, Its additional alkyl group moderately increases the ri gidity of the backbone compared to that of classical PNA. It could provide the cationic functional group. In fa ct, by utilizing the nucleophilicity of thiol, we can put a library of other functional gr oups in that position. In addition, the flexibility in terms of modulating the size of the alkyl group and the type of alkyl group is unprecedented. Furthermore, when designing the alkyl groups that are going to be attached to the backbone, the rationales have been adapted to increase the aqu eous solubility of the oligomers as well to increase the cell permeability. The rationales for the design were developed after studying the peptides that are reported to penetrate the cells78, which are very inspiring. Our synthetic endeavor to complete the ba ckbone as well as the oligomer synthesis using solid phase chemistry is discussed in the following chapters. 1.7. References. 1 Nielsen, P. E., Egholm, M., Berg, R.H. and Buchardt, O. Science, 254 1497-1500 (1991). 2 (a) Egholm, M; Nielsen, P.E. Nature 1993 365, 566. (b) Nielsen, P.E. Peptide Nucleic Acids: Protocols and Applications 2nd ed.; Nielsen, P.E., Ed.; Horizon Bioscience: Wymondham, Norfolk, 2004. 3 Nielsen, P.E. Quarterly Reviews of biophysics 2005 38, 345. 4 Permission was granted to copy this image, distribute and/or modify this document under the terms of the GNU Free Documentation license

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24 5 Devlin, T.M. Textbook of biochemistry with clinical correlations 5th ed.; Wiley-Liss: New York, NY, 2002. 6 Ghosh, A; Bensal, M Acta Crystallogr D Biol Crystallogr 2003 59, 620. 7 Watson, J; Crick, F Nature 1953 171, 4356. 8 Ponnuswamy, P; Gromiha, M J Theor Biol 1994 169, 419. 9 Johnson, A; Lewis, J; Raff, M; Roberts, K; Walters, P Molecular Biology of the Cell; Fourth Edition 4th ed.; Alberts, B, Ed.; Garland Science: New York, NY, 2002. 10 Venter, J. et al. Science 2001 291, 1304. 11 Berg, J. M.; Tymoczko, J. L.; Stryer, L Biochemistry, 6th ed.; W. H. Freeman: San Francisco, CA, 2006. 12 Champe, P.C.; Harvey, R.A.; Ferrier, D.R. Lippicott's Illustrated Reviews: Biochemistry 3rd ed.; Williams & Wilkins: NY, New York, 2005. 13 http://www.ornl.gov/sci/techresources/Hum an_Genome/home.shtml (March 06,2008) 14 Fire, A.; Xu, S.; Montgomery, M.K.; Kostas, S.A.; Drive, S.E.; Mello, C.C. Nature 1998 391, 806. 15 Jansen, B.; Zangemeister-Wittke, U. The Lancet oncology 2002 3, 672. 16 (a) Wickstorm, E. J. Biochem. Biophys. Methods 1986 13, 97. (b) Akhtar, S. et al. Life Sci. 1991 49, 1793. 17 Jarvis, T.C. et al. Antisense Nucleic Acid Drug Dev. 2000 10, 11. 18 (a) Zon, G. Pharm. Res. 1988 5, 1988. (b) Kashihara, N. et al. Exp. Nephrol. 1988 6, 84. 19 (a) Hamilton, S.E. et al. Chem. Biol. 1999 6, 343. (b) Shammas, M.A. et al. Oncogene 1999 18, 6191. 20 (a) Khatsenko, O. et al. Antisense Nucleic Acid Drug Dev. 2000 10, 35. (b) Knight, D.A. et al. Transplantation 2000 3, 417. 21 (a) Arora, V. et al. J. Pharmacol. Exp. Ther. 2000 292, 921. (b) Qin, G.Z. et al. Antisense Nucleic Acid Drug Dev. 2000 10, 11. 22 (a) Gerwitz, A.M. Curr. Opin. Mol. Ther. 1999 1, 297. (b) Reding, M.T Expert Opin. Ther. Pat. 2000 10, 1201.

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25 23 Prasmickaite, L. et al. Nucleic Acids Res. 1998 26, 4241. 24 Konopka, K. et al. Int. J. Pharm. 1996 136, 23. 25 Fell, P. et al. Antisense Nucleic Acid Drug Dev. 1997 7, 319. 26 Hertel, K.J. et al. Nucleic Acids Res. 1992 20, 3252. 27 Fire, A.; Xu, S.; Montgomery, M .; Kostas, S.; Driver, S.; Mello, C Science 1998 391, 806. 28 (a) Maurer, N. et al. Mol. Membr. Biol. 1999 16, 129. (b) Hughes, J. et al. Methods Enzymol. 2000 313, 372. (c) Tari, A.M. Methods Enzymol. 2000 313, 372. (d) Roh, H. et al. Cancer Res. 2000 60, 560. 29 Yoo, H.; Juliano, R.L. Nucleic Acids Res. 2000 28, 4225. 30 Troy, C.M. et al. J. Drug Targeting 1998 5, 291. 31 Fischer, R.; Fotis-Mleczek, M.; Hufnagel, H.; Brock, R. Chembiochem 2005 6, 287. 32 De Koning, H.; Pandit, U.K. Recl. Trav. Chim. Pays-Bas 1971 90, 1069. 33 Buttrey, J.D.; Jones, A.S.; Walker, R.T. Tetrahedron 1975 31, 73. 34 Uhlmann, E.; Peyman, A. Chem. Rev. 1990 90, 563. 35 Nielsen, P.E. Acc. Chem. Res. 1999 32, 624. 36 Hyrup, B.; Egholm, M.; Buchardt, O.; Nielsen, P.E. Bioorg. Med. Chem. Lett. 1996 6, 1083. 37 Bentin, T.; Nielsen, P.E. Triple Helix Forming Oligonucleotides. ; Malvy, C., Ed.; Kluwer Academic Publishers: Barel, Bellan, 1999. 38 Lohse, J.; Dahl, O.; Nielsen, P.E. Proc. Natl. Acad. Sci. USA 1999 96, 11804. 39 Brown, S.C.; Thomson, S.A.; Veal, J.M.; Davis, D.G. Science 1994 265, 777. 40 Eriksson, M.; Nielsen, P.E. Nature Structural Biology 1996 3, 410. 41 Betts, L.; Josey, J.A.; Veal, J.M.; Jordan, S.R. Science 1995 270, 1838. 42 Rasmussen, H.; Kastrup, J.S.; Niels en, J.N.; Nielsen, J.M.; Nielsen, P.E. Nature sturctural biology 1997 4, 98. 43 (a) Hanvey, J.C. et al. Science 1992 258, 1481. (b) Knudsen, H.; Nielsen, P.E. Nucleic Acids Res. 1996 24, 494. (c) Bonham, M. A. et al. Nucleic Acids Res. 1995 23, 1197. (d) Gambacorti, C. et al. Peptide Nucleic Acid(PNA) Blood 1996 88, 1411.

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26 44 Lansdorp, P.M.; Verwoerd, N.P.; Rijke van de, F. M.; Dragowska, V.; Little, M.-T.; Dirks, R.W.; Raap, A.K.; Tanke, H.J. Hum. Mol. Genet. 1996 5, 685. 45 Perry-O'Keefe, H.; Yao, X.-W.; Co ull, J.; Fuchs, M.; Egholm, M. Proc. Natl. Acad. Sci. U.S.A. 1996 93, 14670. 46 Weiler, J.; Gausepohl, H.; Hauser N.; Jensen, O.N.; Hoheisel, J.D. Nucleic Acids Res. 1997 25, 2792. 47 Qrum, H.; Nielsen, P.E.; Egholm, M.; Berg, R.H.; buchardt, O. Nucleic Acids Res. 1993 21, 5332. 48 Bukanov, N.O.; Demidov, V.V.; Niel sen, P.E.; Frank-Kamenetskii, M.D. Proc. Natl. Acad. Sci. U.S.A. 1998 95, 5516. 49 Nielsen, P.E.; Egholm, M.; Buchardt, O. Gene 1994 149, 139. 50 Ganesh, K.N.; Nielsen, P.E. Current Organic Chemistry 2000 4, 931. 51 Hyrup, B.; Egholm, M.; Rolland, M.; Nielsen, P.E.; Berg, R.H.; Buchardt, O. J. Chem. Soc. Chem. Commun. 1993 10, 518. 52 Hyrup, B.; Egholm, M.; Nielsen, P.E. ; Wittung, P.; Norden, B.; Buchardt, O. J. Am. Chem. Soc. 1994 116, 7964. 53 Hyrup, B.; Egholm, M.; Buchardt, O.; Nielsen, P.E. Bioorg. Med. Chem. Lett. 1996 6, 1083. 54 (a) Dueholm, K.L.; Nielsen, P.E. New J. Chem. 1997 21, 19. (b) Dueholm, K. et al. Bioorg. Med. chem. Lett. 1994 4, 1077. (c) Haaima, G. et al. Angew. Chem. Int. Ed. Eng. 1996 35, 1939. (d) Puschl, A. et al. Tetrahedron Lett. 1998 39, 4707. 55 Lagriffoule, P. et al. Chem. Eur. J. 1997 3, 912. 56 Maison, W. et al. J .Bioorg. Med. Chem. Lett. 1999 9, 581. 57 Krotz, A.H. et al Bioorg. Med. Chem. 1998 6, 1983. 58 Almarison, O. et al. Proc. Natl. Acad. Sci. USA 1993 90, 9542. 59 Lagriffoul, P.-H. et al. Bioorg. Med. Chem. Lett. 1994 4, 1081. 60 (a) De Koning, H. et al. Recl. Trav. Chim. Pays-Bas 1971 90, 1069. (b) Lenzi, A. et al. Tetrahedron Lett. 1995 36, 1717. 61 Savithri, D.; Leumann, C.; Scheffold, R. Hlvet. Chim. Acta 1996 79, 288.

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27 62 (a) Shah, V.J.; Cerpa, R.; Kuntz, I.D.; kenyon, G.L. Bioorg. chem. 1996 24, 201.(b) Bergmeier, S.C.; Fundy, S.L. Bioorg. med. Chem. Lett. 1997 7, 3135. 63 (a) Howarth, N.M.; Wakelin, L.P.G. J. Org. Chem. 1998 36, 1716. (b) Lenzi, A. Tetrahedron Lett. 1998 36, 1716. 64 Kuwahara, M.; Arimitsu, M.; Sisido, M. J. Am. Chem. Soc. 1999 121, 256. 65 Goodnow, R.A. Jr.; Tam, S.; Pruess, D.L.; McComas, W.W. Tetrahedron Lett. 1997 38, 3199. 66 Tsantrizos, Y.S.; Lunetta, J.F.; Bo yd, M.; Fader, L.D.; Wilson, M.-C J. Org. Chem. 1997 62, 5451. 67 Schultz, R.; Cantin, M.; Roberts, Cl; Greiner, b.; Uhlmann, E.; leumann, C. Angew. chem. Int. Ed. Eng. 2000 39, 1250. 68 (a) Peyman, A. et al. Angew. Chem. Int. Ed. Eng. 1996 35, 2632. (b) Uhlmann, E. et al. Nucleosides & Nucleotides 1997 16, 603. 69 Efimov, V.A. et al. Nucleosides & Nucleotides 1999 18, 1393. 70 van der Laan, A.C; van Amsterdam, I.; Tess er, G.I.; van Boom, J.H.; Kuyl-Yehskiely, E. Nucleosides & Nucleotides 1998 17, 219. 71 Barawkar, B.A.; Bruice, T.C. J. Am. Chem. Soc. 1999 121, 10418. 72 Dallaire, C.; Arya, P. Tetrahedron Lett. 1998 39, 5129. 73 (a) Jordan, S. et al. Bioorg. med. Chem. Lett. 1997 7, 681. (b) Gangamani, B.P. et al. Tetrahedron 1996 52, 15017. 74 Lowe, G.; Vilaivan, T. J. Chem. Soc. Perkintrans. 1 1997 547. 75 Decosta, M.; Kumar, V.A.; ganesh, K.N. Organic Lett. 1999 1513. 76 Verheijen, J.C. et al. Nucleosides & Nucleotides 1999 18, 493. 77 (a) Haaima, G. et al. Acc. Chem. Res. 1997 25, 4639. (b) Egholm, M. et al. Acc. Chem. Res. 1995 23, 217. (c) Gangamani, B.P. et al. JCS. Chem. Commun. 1997 240, 778. (d) Dldrup, A.B.; Dahl, O.; Nielsen, P.E. J. Am. Chem. Soc. 1997 119, 11116. (e) Timar, Z. et al. Nucleosides & Nucleotides 1999 18, 1131. 78 (a) Fischer, R.; Fotis-Mleczek, M.; Hufnagel, H.; Brock, R. Chembiochem 2005 6, 287.(b) Koppelhus, U.; Awastahi, S.K.; Zachar, V .; Holst, H.U.; Ebbesen, P.; Nielsen, P.E. Antisense &

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28 Nucleic Acid Drug Development 2002 12, 51.(c) Shiraishi, T.; Pankratova, S.; Nielsen, P.E. Chemistry & Biology 2005 12, 923. (d) Wadia, J.S.; Stan, R.V.; Dowdy, S.F. Nature Medicine 2004 10, 310.

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29 2. Chapter Two: Synthesis of Cysteine based PNA Monomers. 2.1. Introduction. H2N N O HS O B OH H2N H2N N R O O B OH R O OH C-terminus C-terminus C-terminus N-terminus N-terminus N-terminus S-terminus U n i t o f P e p t i d e : A m i n o a c i d s U n i t o f P N A : U n i t o f C P N A Figure 18: Comparison of the units for Peptide, PNA and CPNA. PNA oligomers are polyamide compounds but they are not peptides because their building blocks are not amino acids79. However, since they have polyamide bonds in their backbones, PNA oligomers are cons tructed by methods similar to those of peptides. In most cases, application of solid phase chemistry is necessary for their construction and their monomers are prepared with orthogonal prot ecting groups on both the C and N termini. Discussed herein is our work to prepare the cysteine based PNA monomers with different protecting groups. The synthesis of PNA oligomers will be discussed in chapter three. Notably, since the newly designed backbone contains cysteine residues, synthesis protocols of the monomers has to allow 3 sites to be protected befor e each termini is reacted. In other words, our cysteine based PNA (CPNA) have C, N and S termin i in one building block that require protection. Therefore the preparation of the monomers was not trivial. Figure 18 shows differences in their stru ctures between peptides, regular PNAs and our cysteine based PNAs.

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30 2.2. CPNA sub-monomers with acetonide protecting on N, Stermini. When we began this project, our main goal was to improve the physical properties of PNA oligomers by adding charged alkyl groups on the S-termini. Moreover, it was also anticipated that the native peptide ligation effect80 of the thiol group could well be adapted for the solid phase synthesis of the resulting CPNAs. So we developed a scheme for the monomers that are protected with acetonide for the N, S-termini and methyl ester for the C-termini. Scheme 1 pre sents the protocol to develop the Boc protected sub-monomers. Scheme 1: Boc protected sub-monomers. Synthesis of the first sub-monomer for the project started out with HCl salt of L-cysteine. The first reaction in the procedure t hat was reported in 1958 provided us 1 with excellent yield. The semi-protected compound 1 was then protected with Boc anhydride to give compound 2 The yield of the second step was normally repo rted to give poor yield. Among the precedented L-cysteine HCl S H N OH O HCl S N OH O Boc S N N O Boc O S N H O Boc S N N H Boc CO2Me (a) acetone, reflux 3hr, 92%. (b) (Boc)2O, DIPEA, acetonitrile, 3 day, rt, 40 %. (c) N,O-dimethylhydroxylamine hydrochloride, DCC, TEA, EtOAc, 70 %. (d) DIBAL-H, Et2O, 0oC, 2 h, 92 %. (e) glycine methyl ester hydrochloride, MgSO4, NMM, NaBH3CN, 75 %. b c d e 1 2 3 4 5 a

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31procedures, the best reported yield for this reac tion was 60 %. Several attempts from our laboratory to reproduce or match the best yield werent satisfactory and our best result was 40 % which matches with the recently reported work from other groups81,82. The next step was a Weinreb amide83 formation to give 3 with 70 percent yield. A reduction of 3 using DIBAL-H gave aldehyde 4 which was then directly subjected to a reductive amination to give 5 The combined yield for the last two steps was 69 percent. So the first sub-monomer for the CPNA project was Scheme 2: Synthesis of Fmoc sub-monomers 11, 12. obtained after the 5-step procedure with overall yields of 17.8 %. Although it was manageable to get grams of 5 we werent so enthusiastic on this protocol. Thereafter, we decided to try Fmoc group instead of the Boc group for the protection of the N-terminus. The change of the protecting group gave us exciting results as shown in Scheme 2. The esterification of the starting material using thionyl chloride in MeOH gave the 6 which was then semi-protected with acetonide to give L-Cysteine methyl ester.HCl L-Cysteine.HCl H N S CO2Me N S CO2Me Fmoc N S Fmoc OH N S CHO Fmoc N S Fmoc N H CO2Me (a) SOCl2, MeOH, reflux 3 hr, 95%. (b) acetone, TEA, reflux, 12hr 85%. (c) Fmoc-Cl, NaHCO3, DCM, rt, 83%. (d) DIBAL-H, THF, 0 oC, 91%. (e) Dess Martin Periodinane, DCM, 0 oC, 85%. (f) glycine methyl ester hydrochloride,TEA, MgSO4, NaBH3CN, THF, rt, 80%. (g) glycine t-butyl ester acetic acid TEA, MgSO4, NaBH3CN, THF, rt, 75%. N S Fmoc N H CO2 t-Bu (a) (b) (c) (d) (e) (f) (g) 6 7 8 9 10 11 12

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32784. The yields of the first two steps were 95% and 85%, respectively. The base-labile Fmoc group was then introduced to 7 to give 8 with 83% yield. First three steps gave us 8 with combined yield of 67% which was considerably better than the 37% overall yield of compound 2 (see the Scheme 1). The next step was to reduce the methyl ester to the corresponding aldehyde 10. Even though there are known proc edures to directly reduce 8 using DIBAL-H at -78 C, it was the practical decision for us to follow the 2-step protocol which gave us consistent results with a good combined yield. Reduction of 8 using DIBAL-H at 0 C gave us the primary alcohol 9 with 91% yield. Oxidation of 9 with Dess-Martin Periodinane (DMP) afforded us 10 with a good yield. The somewhat unstable aldehyde 10 was directly subjected to a reductive amination reaction with the two different glycine esters. With the me thyl glycine ester, the reaction gave us 11 with 80% which was slightly better than that of 12.

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332.3. Nucleo base coupling on the sub-monomers. Scheme 3: Synthesis of acetic acid derivatives of nucleo bases 14, 15 and 16. (a) K2CO3, benzyl bromoacetate, DMF, 70% (b) Na, benzyl alcohol 80% (c) ethyl bromoacetate, NaH, DMF, 55% (d) NaOH, H2O (e) KOt-Bu, benzyl bromoacetate, DMF, 85% N NH O O OH O Thymine-1-acetic acids ( commercially available ) 13 N N N N O NH2 HO O 14 N N N N NH2 O HO 15 NN O NH2 HO O 16 N N N N H NH2 Cl N N N N NH2 Cl O O 14 N N N N H NH2 HNN O NH2 N N N N NH2 O O NN O NH2 O O 15 16 (a) (b) (c) (d) (e) (d) 17 18 19 84% 90%

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34Scheme 4: Optimization of the coupling condition for the CPNA monomers. With the sub monomers in hand, we needed to couple the nucleo bases to the backbone. As it was shown in Chapter One, nucleo bases are connected to the PNA backbone through an acetyl linker. Only thymine acetic acid is commerc ially available and other nucleo bases have to S N N H Boc CO2Me 5 N S Fmoc N H CO2Me N S Fmoc N H CO2 t-Bu 11 12 a, 13 a, 14 S N N Boc CO2Me O (T) S N N Boc CO2Me O (G) a, 13 a, 13 5 20 21 N S Fmoc N CO2Me O (T) 24 N S Fmoc N CO2 t-Bu O (T) 25 86% 80% 93% 70% a, 15 5 No reaction a, 16 5 No reaction DCC, 13 bases Solvents No reaction or poor yields 5 (a) HATU, TEA, DMF

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35be manually prepared to have the linker and the proper protecting groups. Scheme 3 shows our first gen eration of the bases that are prepared fo r the coupling onto the CPNA backbones. In fact, the compounds 15 16 and 17 are not fully protected because we thought that the amines on the bases are not as nucleophilic as ordinary amines on our backbones so it wouldnt be necessary for the coupling steps to fully protect the bases. The simple procedures to make acetic acid derivatives of nucleo bases are known85,86 and shown in Scheme 3. Scheme 5: Cbz protected A and C derivatives. Scheme 4 briefly presents the conditions we tried to cou ple our sub-monomers and the acetic acid derivatives of the nucleo acids. Presumably due to the structural nature of our submonomers, ordinary coupling procedures with 13 using DCC and EDC had given us unsatisfactory results. However, a trial of the amide formation using HATU in DMF with TEA gave us the desired monomer 20 with a quantitative yield. We then successfully coupled the benzyl protected guanine derivative onto the backbone. Unfortunately, the coupling reactions with 15, 16 N N H N N N N H NH2 O N N H NH-Cbz O N N NH-Cbz O O Ot-Bu N N NH-Cbz O OH O NH2 N N N N H NH-Cbz N N N N NH-Cbz O t-BuO N N N N NH-Cbz O HO a b c d e f (a) Cbz-Cl, DMAP, pyridine (b) BrCH2CO2C(CH3)3, K2CO3, Cs2CO3, DMF (c) HCl/ 1,4-dioxane, CH2Cl2(d) NaH, Cbz-Cl, DMF (e) BrCH2CO2C(CH3)3, K2CO3, Cs2CO3, DMF (f) TFA, CH2Cl2, Et3SiH. 27 28 29 30 31 32 33 by Mehul by Priyesh Jain 26

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36didnt give the desired monomers. The problem with coupling the unprotected adenine and cytosine derivatives was the insolubilities of the derivatives in variours organic solvents. To solve the problem, we decided to protect the adenine (A) and cytosine (C) derivatives with the Cbz group. The Cbz protected adenine and cytosine derivatives were prepared by the reported procedure86 which is described in the Scheme 5. 2.4. CPNA monomers with Boc and trityl groups on the N, S-termini. Scheme 6: Two different routes toward the trityl and Boc protected sub-monomer. BocHN S N H CO2Me Ph Ph Ph BocHN CO2H S Ph Ph Ph BocHN S N H CO2Me Ph Ph Ph O BocHN S N OMe Ph Ph Ph O Me BocHN S Ph Ph Ph O H 93 % 92 % 40 % (55 % after recovering SM) a b c d e 34 35 36 37 38a (from 36) 38b (from 37 ) 60 % for 2 steps (a) EDC.HCl, DMAP, DCM, rt 4 hr (b) DIBAL-H, Et2O, 0 oC, 3 hr (c) Glysine methyl ester.HCl, MgSO4, NMM, NaBH3CN, THF, rt, 6 hr. (d) Glysine methyl ester.HCl, EDC.HCl, DMAP, DCM, rt, 4 hr (e) BH3 .THF, THF, 60 oC 20 hr With the monomers prepared above, we have tr ied solution based and also solid phase coupling of monomers to get dimers and oligomers. The studies towards synthesis of the oligomers are

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37discussed in chapter four. After analyzing the data from those studies, we concluded that utilizing the acetonide as the co-protecting group of N, S-termini in the continuous solid phase synthesis sequence is not feasible. Therefore, we decid ed to use different protecting groups which can be deprotected in milder conditions before the solid phase synthesis. Consequently, the new protocols had to be developed. After examining thoroughly, the trityl group was selected to be the protecting group for the thiol. Summarized in the Scheme 6 is the protocol to prepare th e trityl protected monomers. The commercially available 34 was used to give 38a by the protocol we previously used. On the other hand, we have developed another protocol wh ich utilizes the borane reduction. The reason why we have sought the other route for the same sub-monomer was not only did we want to improve the overall yield but also we wanted to prepare the optically pure monomers. Like peptides, PNAs oligomers also require optically pure monomers since they usually need to have certain lengths for their sequence specific activity. So, the newly developed route begins with an amide formation of 34 with glycine methyl ester to give 37 which is then subjected to the key step, the borane reduction. The mild borane reduction is reportedly very likely to cause the epimerization of the alpha stereogenic center.87 After the reduction, we were able to isolate the desired 38b However, along with 38, significant amounts of byproducts were observed. The major byproduct was identified as the alcohol compound which is the result of the reduction of the ester group. After numerous attempts, the best yield we obtained was 40 %. When we recover the starting material, the yield improves slightly to 55 %. Nevertheless, it is still important to make sure which route gives less epimerization. Theoret ically, we know that the reductive amination route has to go through the aldehyde stage so it is very difficult to avoid the epimerization with the protocol using reductive amination. To prove this matter experimentally, we first coupled both 38a and 38b with an optically pure Fmoc-proline to perform an NMR analysis of the ratio of potential diastereomers. Although this NMR study convincingly showed the difference of the spectra formed by the two different routes, yet we were not able to quantitatively analyze the data. Thereafter, we have decided to use HPLC with a chiral column. After finding good resolution conditions for the racemic 38, both 38a and 38b were tested and it turned out to give the result

PAGE 53

38that we were anticipating. The reductive ami nation route which had been conducted with extreme care to avoid the epimerization gave about 10% of the minor diastereomer while the other route gave no epimerization at all. The ee (enantiomeric excess) values for 38a and 38b were 80 % and 99.9% respectively. This result technically was exceedingly important for us because the purity of the single unit for oligomers is critical. For example, if one wants to make a peptide or an oligomer with the 10 units in the sequence, and if the optical purity of the single unit used is only 90%, one could get the desired product with the maximum yield of 60% with a possible 1022 diastereomers and the other enantiomer. As the oligomer gets longer, the efficiency drops precipitously. Therefore, although the second st ep has a productivity issue, the borane route still is valuable. Scheme 7: Preparing the N-Boc, S-trityl Monomers. BocHN S N CO2Me Ph Ph Ph O N HN O O 38b 39 >95 % b BocHN S N CO2Me Ph Ph Ph O N 40 >95 % a N N N NH2 BnO BocHN S N CO2Me Ph Ph Ph O BocHN S N CO2Me Ph Ph Ph O N N N O NHCbz N N N CbzHN 41 42 (a) HATU, 13, TEA, DMF, rt, 2 hr. (b) HATU, 14, TEA, DMF, rt, 2 hr. (c) HATU, 29 TEA, DMF, rt, 2hr. (d) HATU, 33 TEA, DMF, rt, 2 hr. 38b >95 % d >95 % c

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39 The optically pure 38b was then coupled with the four different nucleo bases. As shown in Scheme 7, all four reactions under the same conditions gave the desired 39, 40, 41 a nd 42 with quantitative yields. 2.5. Synthesis of CPNA monomers with the alkyl groups on the S-terminus. After the preliminary study on the solid phase synthesis, which is discussed in chapter four, weve learned that it is better to reduce the nu mbers of steps in the solid phase synthesis if it is possible. Therefore, we decided to introduce the side chains that we designed before the solid phase synthesis. As a sequence, our protocol again had to be totally revised. FmocHN CO2H S Ph Ph Ph FmocHN S Ph Ph Ph N H FmocHN S Ph Ph Ph X FmocHN S Ph Ph Ph H O O FmocHN S Ph Ph Ph NH2 CO2R Route A Route C Route B Route D FmocHN S Ph Ph Ph N H CO2R FmocHN S Ph Ph Ph N H CO2R FmocHN S Ph Ph Ph N H CO2R FmocHN S Ph Ph Ph N H CO2R Figure 19: Possible pathways for CPNA backbones. To come up with a new protocol, we needed to carefully look at all the termini and their possible protecting groups in the big picture. We decided to use Fmoc group for N-terminus

PAGE 55

40because it is a good function that can be utilized in the solid phase synthesis when one needs to quantitatively analyze the coupling. Matters regarded with the solid phase synthesis will be discussed in detail in chapter four. After fixing the N-terminus protecting group as Fmoc group, the C-terminus should have an orthogonal protection. Therefore, the acid labile tert-butyl ester was selected as the protecting group. The remaining S-terminus must have a protecting group which can be cleaved selectively from both of the ot her termini. After considering it thoroughly, we chose the trityl group. With all the protective gr oups set, we had to go over all the possible ways to carry out our synthesis. Illustrated in Figure 19 are the possible pathways including the new protocol to get the Fmoc p rotected backbone of our CPNA monomer s. Route A is one of the most conventional ways to construct the PNA backbone. However, we now have learned that this route can cause epimerization. Route B proved to have no epimerization, but this route also has a problem which is the poor yield of the key step, borane reduction. So, we had to look for the alternatives. Route C, elaborated in Scheme 8, is a procedure that is designed to use a SN2 reaction to couple Scheme 8: A SN2 protocol (Route C). FmocHN CO2H S Ph Ph Ph Borane FmocHN S Ph Ph Ph OH FmocHN S Ph Ph Ph Br PBr3CCl4THF 85 % 44 45 43 75 % H2N CO2t-Bu bases Solvents XFmocHN S Ph Ph Ph N H CO2t-Bu

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41Scheme 9: Preparation of the charged alkyl bromides for S-alkylation. H2N NH2 CbzHN NHBoc CbzHN N H O Br CbzHN NH2 (46) 48 49 50 (a) (Boc)2O, CHCl3, 0 oC. (b) Cbz-Cl, THF, 0 oC. (c) TFA, DCM (d) bromoacetyl bromide, Na2CO3,DCM, -10 oC (e) 1,3-bis(benzyloxycarbonyl) -2-methyl -2-pseudothiourea, HgCl2, TEA, DCM, rt, 3 hr H2N NHBoc 47 N H NHBoc CbzHN NCbz N H NH2 CbzHN NCbz N H N H CbzHN NCbz 51 52 53 a b c d e c d (R1Br) (R2Br) O Br by Laura Anderson by Mellisa Topper our cysteine and the glycine ester. A mild borane reduction of 43 gave 44 with a good yield and the bromo compound 45 was obtained when 44 was treated with the phosphorus tribromide in CCl4. The next step was the SN2 reaction that would complete the backbone, but all the conditions we tried gave no desired product or Fm oc-deprotected by-product. As an alternative to route C, a totally new procedure was proposed. The protocol was examined to be promising both in terms of productivity and optical purity. Using this procedure shown in Scheme 10, the charged alkyl chain s are successfully induced on to the S-te rmini. Two different alkyl chains were initially designed and synthesized by the sequences that were conducted by Melissa Topper and Laura Anderson.

PAGE 57

42Scheme 10: Preparation of R1-alkylated Fmoc Monomers. FmocHN S Ph Ph Ph NH2 FmocHN S Ph Ph Ph N H CO2t-Bu FmocHN CONH2 S Ph Ph Ph FmocHN S N CO2t-Bu N H O CbzHN O (Base) FmocHN S N H CO2t-Bu N H O CbzHN 58 (T), 59 (G), 60 (C), 61 (A) DCC, HOBT, NH4OH THF BH3 .THF THF BrCH2CO2t-Bu, DIPEA THF i)TFA/DCM, Et3SiH ii) R1Br ( 50 ), NaOH, EtOH, 90 % HATU, DIPEA, 13, 14, 29 or 33 DMF, 90 ~ 98 % 92 % 65 % 92 % FmocHN CO2H S Ph Ph Ph 43 54 55 56 57 The procedure to synthesize the R1-attached Fmoc monomers begins with the commercially available 43 The simple amide formation using DCC in the presence of HOBt was well precedented88 and gave us 54 with 92% yield. The borane reduction which is also used for route B earlier gave us the reduced amine 55 with 65 % yield. This time, because the substrate for the borane reduction didnt have any other bor ane labile functional group, the yield of the reduction was improved. The next st ep was the key step to couple 55 and the bromo acetate in the presence of a bulky amine base. The SN2 reaction gave 56 with 92% yield. Trityl protected 56 then had to be deprotected selectively. 15% TFA solution in DCM was able to cleave the trityl group from the thiol without deprotecting the tert-butyl ester.

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43Scheme 11: Preparation of R2-alkylated Fmoc Monomers. FmocHN S Ph Ph Ph N H CO2t-Bu 56 i) TFA/DCM, Et3SiH ii) R2Br, NaOH, EtOH, TBAI, 75 % FmocHN S N H CO2t-Bu N H O N H CbzHN CbzN FmocHN S N CO2t-Bu N H O N H CbzHN CbzN O (Base) 13, 14, 29 or 33 HATU, DIPEA DMF 63 (T), 64 (G), 65 (C), 66 (A) 90~98 % 62 Trityl cation was quenched by triethylsilane89 and the progress of the reaction can be monitored by the color of the solution because the trityl cation in the solution displays a distinctive yellow color. The naked thiol compound was then direct ly subjected to the next step without further purification. Scheme 12: Preparation of S-methyl Fmoc Monomers. FmocHN S Ph Ph Ph N H CO2t-Bu 56 i)TFA/DCM, Et3SiH ii)MeI, NaOH, EtOH FmocHN MeS N H CO2t-Bu 67 91% 13, 14, 29 or 33 HATU, TEA DMF FmocHN MeS N CO2t-Bu O (Base) 68 (T), 69 (G), 70 (C), 71 (A) The alkylation of the crude thiol with 50 in the presence of the sodium hydroxide in EtOH yielded the sub-monomer 57 with a 90% yield for the 2 step sequence. 57 was then coupled with acetic

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44acid derivatives of the nucleo bases usi ng the HATU protocol to give monomers 58, 59, 60 and 61 with a quantitative yields. With the protocol developed in Scheme 10, R2-alkylated Fmoc monomers are also prepared as shown in Scheme 11. To co ntrol the degree of charge on the CPNA oligomer, we needed to have a neutral backbone as well. Therefore, a neutral alkyl chain had to be designed or selected. The simple methyl group was chosen as the neutral alkyl group due to its availability and the simplicity. The procedure to synthesize the neutral monomers with all the bases is summarized in Scheme 12 and the proto col is quite similar to that of charged monomers. Scheme 13: Alkylated Boc monomers with R1 and methyl groups. BocHN CO2H S Ph Ph Ph BocHN S Ph Ph Ph NH2 BocHN CONH2 S Ph Ph Ph DCC, HOBT, NH4OH THF BH3 .THF THF 98% THF Methyl bromoacetate, DIPEA, DMAP(Cat.) BocHN S Ph Ph Ph N H CO2Me BocHN RS N H CO2Me 92% i)TFA/DCM, Et3SiH ii) NaOH, R1Br, MeOH, TBAI 87 % or MeI, NaOH, MeOH, 91% BocHN RS N CO2Me O (Base) 70 % 13, 14, 29 or 33 HATU, TEA DMF 34 72 73 38c 74 R = Me 75 R = R176 R= Me and T 77 R= Me and G 78 R= Me and C 79 R= Me and A 80 R= R1 and T 81 R= R1 and G 82 R= R1 and C 83 R= R1 and A 96~99 % Many of reported PNA oligomers were synthesized with Boc-solid phase synthesis. As summarized in Scheme 13. We have also prepared our alkylated CPNA monomers with Boc prote cting group. The protocol adopted the pr ocedure that was developed for alkylated Fmoc

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45CPNA monomers. Overall, the yields for the Bo c-protocol were slightly better than the Fmoc protocol. 2.6. Inversed CPNA: new CPNA backbone. H2N N O HS O B OH C-terminus N-terminus S-terminus U n i t o f C P N A N HS NH2 O HO O B U n i t o f i n v e r s e d C P N A C-terminus N-terminus S-terminus Figure 20: Comparison of the CPNA and Inversed CPNA (I-CPNA). A totally different cysteine based PNA backbone was designed when we were developing the protocols for CPNA monomers. As it can be seen in Figure 20, this new backbone is simply an inverte d version of the CPNA backbone. C and N termini of the new backbone are located on the other side of the backbone as compared to the normal CPNA. Moreover, the nucleo bases are connected to the nitrogen which comes from the original cysteine while in the normal CPNA, the nitrogen from the cysteine becomes the N-term inus. We believe this design can probably help the solid phase synthesis of its oligomers becau se the N-terminus of this new monomer is a primary amine with less steric hindrance for coupling to make the amide bond that connects the monomer units. The steric nature of the monomers can well be crucial factor for their coupling efficiency. On the other hand, coupling of the nucleo base derivatives could be tougher since the secondary amine is sterically hindered as compared to that of the normal CPNA. The protocol to synthesize the inversed-CPNA monomer is shown in Scheme 14. This protocol shares the same

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46starting materials with normal CPNA monomers that are shown in Scheme 13. When the synthe sis reached to 73 the procedure becomes different. The primary amine, 73 gets protected with the Fmoc group to give 84 with a good yield. The Fmoc protected amine is now the Nterminus for the monomer. So it does not get deprotected until it reaches the solid phase synthesis stage. The other Boc protected amine on 84 however, needs to be deprotected as well as the S-terminus that is protected with the trityl group. Since both Boc and trityl groups are acid labile group, we needed to be careful when we conduct the selective deprotection of 84. After trying a couple of preliminary experiments, it was figured that the both N and S termini can be selectively deprotected. Scheme 14: synthesis of inversed-CPNA monomer. BocHN S Ph Ph Ph NHFmoc N S NHFmoc t-BuO2C O Fmoc-Cl, DIPEA, DMAP(Cat.) THF i)TFA/DCM, Et3SiH ii) NaOH, R1Br, EtOH, TBAI, 87% (2 steps) ii)BrCH2CO2t-Bu, DIPEA, DMF, 60% for ( 2 steps) 85% 50% (95% after SM recovery) BocHN S Ph Ph Ph NH2 73 84 BocHN S NHFmoc H N O 85 H N S NHFmoc H N O 86 t-BuO2C i) HCl(g), 1,4-dioxane H N O CbzHN CbzHN CbzHN (T) Cl O (T) 87

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47 To selectively deprotect the thiol of 84 we can treat 10 % TFA solution in DCM at room temperature in the presence of the triethylsilane as a scavenger of the trityl cation. For selective Boc deprotecton, simply stirring it in the 50% TFA solution in DCM for about an hour and concentration of the reaction solution would give the TFA salt of the deprotected amine. By the way, we deprotected the thiol selectively and put the alkyl group to get 85 with 87% yield for 2 steps. Then the Boc group was deprotected with the HCl gas instead of TFA solution because the HCl salt of the amine gave the better yield for the next alkylation step. So, the crude HCl salt from the 85 was subjected to the alkylation with the bromo t-butyl acetate in the presence of DIPEA in DMF. The overall yield for the two steps to get 86 was 60%. The subsequent step was to couple the nucleo base derivatives on 86. Surprisingly, the condition that gave us always good yield for nucleo base coupling didnt work for this substr ate. DCC and EDC based coupling conditions and other coupling conditions didnt work for 86 either. Luckily, when we treated the acid chloride version of the nucleo base, it gave us the desired product 87 with about 50% yield and we were able to recover most of the starting material unreacted. The thymine acetic acid chloride was freshly generated before the coupling by refluxing 13 with thionyl chloride. 2.7. Conclusion. Variou s versions of CPNA monomers have been synthesized. Both Boc and Fmoc protected protocols have been developed to provide the wider options for the solid phase synthesis. Notably, the unique protocols that utilize the bor ane reduction are free of epimerization. This astonishing result can contribute to other PNA pr ojects or peptide chemistry. As reported in the latter part of this chapter, the inverse-CPNA protocol has also revealed a unique synthetic strategy and it may have a structural advantage for solid phase synthesis. In short, demonstrated throughout this chapter are synthetic strategies that successfully synt hesized monomers of the CPNA with the charged alkyl chains whic h could enhance the water-solubility and cell permeability of the CPNA oligomers.

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482.8. References. 79 Nielsen, P.E. Peptide Nucleic Acids: Protocols and Applications, 2nd ed.; Horizon Bioscience: Wymondham, Norfolk, 2004. 80 Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B.H. Science 1994 266, 776. 81 Kemp, D.S.; Carey, R.I. J. Org. Chem. 1989 54, 3646. 82 Nicolaou, K.C.; Nevalainen, M.; Safina, B.S.; Zak, M.; Bulat, S. Angew. Chem. Int. ed. 2002 41, 1941. 83 Nahm, S.; Weinreb, S. M. Tetrahedron lett. 1981 22, 3815. 84 Lee, H.S.; Kim, D.H. Bull. Korean Chem. Soc. 2002 23, 593. 85 Kofoed, T.; Hansen, H.F.; Qrum, H.; Koch, T. J. Peptide Science 2001 7, 402. 86 Thomson, S. A. et al. Tetrahedron 1995 50, 6179. 87 Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D.G. J. Org. Chem. 2001 66, 874. 88 Nakamura, Y.; Okumura, K.; Kojima, M.; Takeuchi, S. Tetrahedron Lett. 2005 47, 239. 89 Schwarz, M.K.; Tumelty, D.; Gallop, M.A. J. Org. Chem. 1999 64, 2219.

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49 3. Chapter Three: Experimental Procedures for Chapter Two. 3.1. Introduction. All the experiments w ere carried out under ar gon or nitrogen atmosphere using oven dried glassware. Most of chemicals were purchased from Aldrich Chemical Co. and Acros Organics. When the solvents must be anhydrous, CH2Cl2, THF and diethyl ether were distilled over calcium hydride or proper metals. 1H(250 MHz) and 13C(62.5 MHz) nuclear magnetic resonance (NMR) data were recorded at room temperature in CDCl3 using a Bruker DPX 250 spectrometer unless otherwise noted. High Resolu tion Mass Spectrometry (HRMS) spectra were recorded using an Agilent 1100 Series in the ESI-TOF mode. Selected NMR and Mass spectra are available in the Appendix section. 3.2. Experimental procedures for compounds in Schemes 2 and 3. 3.2.1. Preparation of 1. L-cysteine HCl S H N OH O HCl 1 reflux acetone L-Cysteine HCl monohydrate (20 g) was refluxed in 1 L of acetone for 3 hr and allowed to cool to RT. The resulting white solid was filtered and washed with ice-cold acetone twice and dried under vacuum to yield 23 g of 1 as a white solid in a 92% yield.

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503.2.2. Preparation of 2. S H N OH O HCl S N OH O Boc 1 2 (Boc)2O, DIEA Acetonitrile 1 (10 g) was suspended into 200 mL of anhydr ous acetonitrile and 15.5 g (1.4 eq) of (Boc)2O and 8.8 mL (1.05 eq) of DIPEA was added to make a so lution and the mixture was stirred at RT for 3 days. The solvent was evaporated under vacuum a nd the residue was taken up in diethyl ether. The white precipitate was removed by filtration th rough Celite and the filtrate was washed with 0.1 N HCl twice and once with water and brine. The organic layer was concentrated to an oil (sometimes solid) and crystallized (or recrystallized) from hexanes to give 5 g of 2 as a white solid in a 40% yield. 3.2.3. Preparation of 3. S N OH O Boc S N N O Boc O 2 3 ETOAc N,O-dimethylhydroxylamine, DCC, TEA 2 (3.8 g) was dissolved in 100 mL of EtOAc and 3.2 g (1.1 eq) of DCC, 1.52 g (1.1 eq) of N,Odimethylhydroxylamine HCl, and 1.55 mL of DIPEA were added into the soluti on. After stirring at rt for 4 hours, the solution was concentrated and subjected to flash column chromatography which yielded 3 g of 3 as a white solid in a 70% yield. (Rf = 0.55, Hexane/EtOAc = 1/1)

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513.2.4. Preparation of 4. S N N O Boc O S N H O Boc 3 4 DIBAL-H Et2O 3 (2.0 g) was dissolved in 100 mL of diethyl ether and cooled to 0 C and 10 mL of 1M DIBAL-H solution in hexanes (1.5 eq) was slowly added. The mixture was stirred for 3 hr at 0 C and then carefully quenched with methanol. After stirring for 30 min, the aluminum complex was filtered through the Celite and washed with EtOAc. The organic filtrate was concentrated to give 1.45 g of 4 as a white solid in a 92% yield. 3.2.5. Preparation of 5. S N H O Boc 4 S N N H Boc CO2Me 5 ClH.H2N CO2Me, MgSO4, NMM, NaBH3CN 4 (1.11 g) was dissolved in 100 mL of anhydrous THF and 3 g of MgSO4, 570 mg (1 eq) of glycine methyl ester HCl, and 500 mL (1.05 eq) of N-methylmorpholine were added. After stirring for 10 min, 600 mg (2 eq) of NaBH3CN was added and the mixture was stirred for 4 hr. The concentrated mixture was subjected to the fl ash column chromatography to give 1 g of 5 as a white solid in a 75% yield. (Rf = 0.39, Hexane/EtOAc = 1/1) 3.2.6. Preparation of 6. L-Cysteine methyl ester.HCl L-Cysteine.HCl 6 MeOH SOCl2, Reflux

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526) L-cysteine HCl monohydrate (30 g) was dissolved in 300 mL of methanol and the solution was cooled to 0 C. 14.5 mL (1.05 eq) of thionyl chloride was carefully added dropwise and the mixture was refluxed for 3 hr. After concentration of the solution, the white solid residue was recrystallized with MeOH and EtOAc to yield 30 g of 6 in a 95% yield. 3.2.7. Preparation of 7. L-Cysteine methyl ester.HCl H N S CO2Me 6 7 TEA, Acetone, reflux 6 (5.0 g) was dissolved in 100 mL of acetone and 3.09 g (1.05 eq) of TEA was added. The solution was refluxed under Argon for 12 hr and the solution was concentrated and subjected to the column chromatography to give 4.1 g of 7 in an 85% yield. (Rf = 0. 33, Hexane/EtOAc = 1/1) 3.2.8. Preparation of 8. H N S CO2Me 7 N S CO2Me Fmoc 8 Fmoc-Cl, NaHCO3DCM 7 (1.0 g) was dissolved in 60 mL of DCM and 1.5 g of Fmoc-Cl and 3 g of NaHCO3 was suspended in the solution. The mixture was vigorously stirred for 3 hr and the filtrate was concentrated. Flash column chromatography gave 1.8 g of 8 in an 83% yield. (Rf = 0.45, Hexane/EtOAc = 2/1 )

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533.2.9. Preparation of 9. N S Fmoc OH 9 N S CO2Me Fmoc 8 DIBAL-H THF 8 (1.37 g) was dissolved in 60 mL of anhydrous THF and the solution was cooled to 0 C and 17 mL of 1 M DIBAL-H solution in hexanes was added under Argon. The solution was stirred at 0 C for 3 hr and quenched carefully with methanol. After 30 min of stirring, the aluminum complex was filtered and washed with EtOAc thoroughly. The combined organic layer was concentrated and subjected to flash column chromatography to yield 1.15 g of 9 in a 91% yield. (Rf = 0.31, Hexane/EtOAc = 2/1) 3.2.10. Preparation of 10. N S CHO Fmoc 10 N S Fmoc OH 9 DCM Dess-Martin Periodinane 9 (400 mg) was dissolved in 20 mL of anhydrous DCM and the solution was cooled to 0 C. Dess-Martin Periodinane (2.3 mL, 15% in DCM) was added and stirred for an hr. As soon as TLC detects the full conversion of the starting material, the reaction was quenched with saturated NaHCO3 solution and 10% sodium bisulfate solution. The organic later was washed with brine, dried over sodium sulfate, concentrated, and subjec ted to the next step without further purification. (Rf = 0.6, Hexane/EtOAc = 2/1)

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543.2.11. Preparation of 11. N S CHO Fmoc 10 N S Fmoc N H CO2Me 11 Glycine methyl ester Hydrochloride, TEA, MgSO4, NaBH3CN THF 10 (700 mg) was dissolved in THF and 1.5 g of MgSO4 was suspended. Glycine methyl ester (240 mg, 1 eq) and 202 mg (1.05 eq) of NMM were added with vigorous stirring. After 10 min, 250 mg of NaBH3CN was added and stirred for 4 hr. The mixture was filtered and the filtrate was concentrated and subjected to flash column chromatography to yield 660 mg of 11 in an 80% yield. (Rf = 0.38, Hexane/EtOAc = 1/1) 3.2.12. Preparation of 12. N S CHO Fmoc 10 N S Fmoc N H CO2 tBu 12 glycine t-butyl ester. acetic acid, TEA, MgSO4, NaBH3CN THF The same reaction conditions with glycine t-butyl ester acetic acid ( 11 ) salt gave 12 in a 75% yield.

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553.3. Experimental procedures for compounds in Schemes 3, 4 and 5. 3.3.1. Preparation of 17. N N N N H NH2 Cl N N N N NH2 Cl O O 17 K2CO3, benzyl bromoacetate DMF 2-Amino-6-chloro-purine (2.0 g) and 3.26 g K2CO3 were placed in 50 mL of DMF and the mixture was stirred for 1 hr and then 2.1 mL of benzyl bromoacetate was added in one portion. The reaction mixture was stirred for 2 hr at room te mperature and 2.5 gm of Celite was added and the mixture was filtered. The Celite was wa shed with 2 mL of DMF. The solution was concentrated to a volume of 80 mL and after cooling to 15 C the mixture was filtered and the filtrate was evaporated to dryness. The residue wa s added to a mixture of ethyl acetate and water (20 mL/4 mL), and stirred for 10 min. The crud e product was collected upon filtration, washed with cold ethanol and dried overnight in high vacuum to give 2.5 g of 17 in a 70% yield. 3.3.2. Preparation of 14. N N N N O NH2 HO O 14 N N N N NH2 Cl O O 17 Na, Benzyl alcohol Fresh cut pieces of sodium (304 mg) were added to 30 mL of benzyl alcohol. The reaction was left for 1 hr at rt until the sodium was completely reacted. 17 (1.5 g) was added in one portion and the mixture was stirred overnight. After additi on of water (30 mL) and washing with diethyl ether, the pH was adjusted to 2 using 2 M NaHSO4. The white solid was collected and dissolved

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56in 120 mL of ethanol:water (3:1). 14 (1.1 g) was obtained after cooling and filtration in an 80% yield. 3.3.3. Preparation of 18. N N N N H NH2 N N N N NH2 O O 18 Ethylbromoacetate,NaH DMF Adenine (5 g) was suspended in 75 mL of DMF. NaH (1.6 g, 60% suspension) was added in portions over 1 hr at 10 C with stirring. The mixture was stirred at RT for 1 hr. ethyl bromoacetate (12.3 g) was added dropwise over 3 hr. The mixture was stirred at RT for 20 hr. The solution was evaporated to dryness in vacuo and stirred for 2 hr with 50 mL of water. 18 (4.5 g, 55% yield) was collected by filtration, washed with water, and absolute ethanol. 3.3.4. Preparation of 15. N N N N NH2 O HO 15 N N N N NH2 O O 18 NaOH H2O 18 (3.75 g) was dissolved in 160 mL of water, followed by dropwise addition of 50 mL of 2 M NaOH solution at 0 C. After 30 min, the temperature was allowed to reach RT and the reaction mixture was stirred for 1 hr the pH was adjusted to 2.5 with 2 M NaHSO4. 15 (2.75 g), a gray solid, was obtained after filtration and washing with cold water for an 84% yield.

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573.3.5. Preparation of 19. HNN O NH2 NN O NH2 O O 19 KOt-Bu, benzyl bromoacetate DMF To 2.06 g of cytosine in 18 mL of DMF was added 2.32 g of potassium tert-butoxide and the mixture was heated to 100 C for 2 hr. The reaction was cooled to 10 C and 3.21 g of benzyl bromoacetate was added dropwise over 30 min. The reaction was allowed to warm to RT while stirring for 12 hr and quenched with 1.2 mL of acetic acid, concentrated in vacuum. The residue was re-suspended in 20 mL of water and stirred for 4 hr and filtered, washed with water, and dried under high vacuum to recover 3.8 g of a pinkish powder, 19, in an 85% yield. 3.3.6. Preparation of 16. NN O NH2 HO O 16 NaOH H2O NN O NH2 O O 19 The same procedure as with 15 was applied to give 2.5 g of compound 16 from 4 g of 19 in a 90% yield. 3.3.7. Preparation of 20. S N N H Boc CO2Me 5 S N N Boc CO2Me O (T) 20 13, HATU, TEA DMF

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58To a solution of 23 mg of 5 13 mg of 13 28 mg of HATU in 0.5 ml of DMF was added 8 mg of TEA and the reaction mixture was stirred overnight. The solution was concentrated in vacuo and the residue was diluted with EtOAc, washed with brine, dried over the sodium sulfate, concentrated, and subjected to column chromatography to yield 30 mg of compound 20 in an 86% yield. (Rf = 0.55, EtOAc) 3.3.8. Preparation of 21, 24 and 25. S N N Boc CO2Me O (G) 21 N S Fmoc N CO2Me O (T) 24 N S Fmoc N CO2 tBu O (T) 25 N S Fmoc N H CO2Me N S Fmoc N H CO2 tBu 11 12 S N N H Boc CO2Me 5 13, HATU, TEA DMF 13, HATU, TEA DMF 14, HATU, TEA DMF The same procedure as with 20 was applied to give 21 in an 80 %, 24 in a 93%, and 25 in a 70% yield with the respective protecting groups. (Rf = 0.4, 0.6, 0.65, EtOAc)

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593.3.9. Preparation of 35. BocHN CO2H S Ph Ph Ph BocHN S N OMe Ph Ph Ph O Me 93% a 34 35 Solution of 34( 2 g), EDC.HCl(910 mg), DMAP(630 mg) and N,O-dimethyl hydroxylamine HCl(463 mg) in DCM(20 ml) was stirred for 4hr. Reaction mixture was washed with water and brine, concentrated, and the crude product was subjected to a flash column chromatograph to give 2 g of white soild 35 in a 93% yield. (Rf = 0.55, EtOAc/Hexane = 1/1). 3.3.10. Preparation of 36. BocHN S N OMe Ph Ph Ph O Me BocHN S Ph Ph Ph O H 35 36 DIBAL-H, Et2O, 0 oC, 3hr 35 (410 mg) was dissolved in 10 mL of diethyl ether and cooled to 0 C and 1.05 mL of 1M DIBAL-H solution in hexanes (1.3 eq) was slowly added. The mixture was stirred for 3 hr at 0 C and then carefully quenched with methanol. After stirring for 30 min, the aluminum complex was filtered through the Celite and washed with EtOAc. The organic filtrate was concentrated to give 370 mg of oily product 36. The crude product was subjected directly to the next reductive amination step without further purification.

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603.4. Experimental procedures for compounds in Schemes 6 and 7. 3.4.1. Preparation of 37. BocHN CO2H S Ph Ph Ph 34 Glycine methyl ester.HCl EDC.HCl, DMAP, DCM rt 4hr BocHN S N H CO2Me Ph Ph Ph O 37 Solution of 2 g of 34, 910 mg of EDC HCl, 630 mg of DMAP and 600 mg of Glycine methyl esterHCl in 20 mL of DCM was stirred for 4 hrs. Reaction mixture was washed with water and brine, concentrated, and the crude product was subjected to a flash column chromatograph to give 2.1 g of white soild 37 in a 92% yield. (Rf = 0.55, EA/Hexane = 1/1). 3.4.2. Preparation of 38a. BocHN S N H CO2Me Ph Ph Ph 38a BocHN S Ph Ph Ph O H 36 Glycine methyl ester.HCl, MgSO4, NMM, NaBH3CN THF, rt, 6hr Crude 36 (260 mg) was dissolved in anhydrous THF (10 mL) and MgSO4 (450 mg), glycine methyl esterHCl (73 mg), and N-methylmopholine (67 mL) were added. After stirring for 5 min at room temperature, NaBH3CN (75 mg) was added and the mixture was stirred for 8 hr. The concentrated mixture was subjected to the flash column chromatography to give 177 mg of 38a as a white solid in a 60% yield (2 steps). ( Rf = 0.4, Hexane/EtOAc = 1/1) 3.4.3. Preparation of 38b.

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61BH3 .THF, THF, reflux, 20hr BocHN S N H CO2Me Ph Ph Ph O 37 BocHN S N H CO2Me Ph Ph Ph 38b To the solution of 37 (500 mg) dissolved in anhydrous THF (10 mL) in a flame dried 25 mL round bottom flask was slowly added 1 M BH3THF (2 mL) solution at room temperature. The reaction mixture was refluxed for 20 hrs and then treated with morpholine (0.5 mL). The reaction mixture was stirred for 2 hrs. Reaction mixture was d iluted with EtOAc and filtered through Celite. The filtrate was concentrated and subjected to a flash chromatography to give 38b (150 mg) in a 40% (55 % after recovering 130 mg of starting material). 3.4.4. Preparation of 39. BocHN S N CO2Me Ph Ph Ph O N HN O O BocHN S N H CO2Me Ph Ph Ph 38b >95% HATU, 13, TEA, DMF rt, 2hr 39 To a solution of 15 mg of 38b 10 mg of 13, 20 mg of HATU in 0.3 mL of DMF was added 5 mg of TEA and the reaction mixture was stirred for 15 hr. The solution was concentrated in vacuo and the residue was diluted with EtOAc, washed with brine, dried over the sodium sulfate, concentrated, and subjected to column chromatography to yield 19.5 mg of 39 in a 99% yield. (Rf = 0.5, EtOAc)

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623.4.5. Preparation of 40, 41 and 42. BocHN S N CO2Me Ph Ph Ph O N BocHN S N H CO2Me Ph Ph Ph 38b 40 N N N NH2 BnO BocHN S N CO2Me Ph Ph Ph O BocHN S N H CO2Me Ph Ph Ph 38b BocHN S N CO2Me Ph Ph Ph O BocHN S N H CO2Me Ph Ph Ph 38b N N N O NHCbz N N N CbzHN 41 42 >95% HATU, 14 TEA, DMF rt, 2hr >95% HATU, 43 TEA, DMF rt, 2hr >95% HATU, 47, TEA, DMF rt, 2hr The same procedure as with 61 was applied to give, 62, 63 and 64 from 55b in 95~99% yield for each compound.

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633.5. Experimental procedures for compounds in Schemes 8, 9, 10, 11, 12 and 13. 3.5.1. Preparation of 47. (Boc)2O, CHCl3, 0 oC H2N NH2 (46) H2N NHBoc 47 To 1,3-propanediamine (150 mL, 1.8 mol) in CHCl3 (300 mL) at 0 C was added (Boc)2O (6.42 g, 32 mmol) dissolved in CHCl3 (40 mL) dropwise during 0.5 hr. The reaction mixture was allowed to warm to ambient temperature and stirred for 3 hr. After filtering the mixture, the filtrate was concentrated in vacuo and the oily residue was dissolved in EtOAc (150 mL). The solution was washed with brine and dried over Na2SO4. Evaporation of the solvent in vacuo gave 47 (4.92 g) as a clear oil. Yield 89%. 3.5.2. Preparation of 48. CbzHN NHBoc 48 H2N NHBoc 47 Cbz-Cl, THF, 0 0C To a solution of 47 (4.92 g, 28.3 mmol) in THF (150 mL) at 0 C was added benzyl chloroformate (5.23 mL, 37 mmol) in NaOH (3 N, 30 mL). The reaction mixture was allowed to warm to ambient temperature and stirred for 3 hr. The solution was ac idified with HCl (6 N), extracted with EtOAc. The organic layer was dried over Na2SO4, concentrated in vacuo to give the crude product which was flash chromatographed on silica gel (EtOAc/Hexane, 1:1) to provide 48 (7.47 g, 24 mmol) as a clear oil. Yield 86%

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643.5.3. Preparation of 49. CbzHN NH2 49 CbzHN NHBoc 48 TFA, DCM A solution of 48 (4.53 g, 15 mmol) in DCM (10 mL) was added TFA (10 mL). The reaction was stirred at room temperature for 0.5 hr. The solvent s were removed in vacuo. To the yellow, oily residue, DCM (15 mL) was added and removed in vacuo. This step was repeated four more times. The solvent was removed in vacuo to give the crude product which was flash chromatographed on silica gel (DCM/MeOH, 4:1) to provide 49 (4.62 g, 14 mmol). Yield 96%. 3.5.4. Preparation of 50. CbzHN N H O Br 50 CbzHN NH2 49 Bromoacetyl bromide, Na2CO3, DCM, -10 oC To compound 49 (4g, 12.3 mmol),40 mL of DCM and 20 mL of saturated aqueous Na2CO3 were added. The mixture was vigorously stirred with a mechanical stirrer and cooled to -10 C before the addition of bromoacetyl bromide (2.49 gm, 12.3 mmol). Stirring was continued for 30 min at 0 C. The reaction mixture was warmed to ambient temperature and then stirred overnight. After 18h, the solvents were removed in vacuo. To the resulting residue, EtOAc (150 mL) and water (100 mL) were added. The two phases were separated and the organic layer was washed with 5% NaHCO3 (100 mL), 15 % citric acid solution (100 mL), and brine (3 x 100 mL). Drying over Na2SO4 and removal of the solvent under reduced pressure afforded the crude product as a white solid. Purification by flash chroma tography (EtOAc/hexane 1:1) gave 50 as a white solid (3.6 g, 1.09 mmol). Yield 89%

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653.5.5. Preparation of 51. H2N NHBoc 47 N H NHBoc CbzHN NCbz 51 1,3-bis(benzyloxycarbonyl) -2-methyl -2-pseudothiourea HgCl2, TEA, DCM, rt, 3h Compound 47 (2.6 g, 14.92 mmol) was dissolved in anhydrous DCM and 7.15 mL of triethylamine was added. HgCl2 (6.07 g, 22.38 mmol) was added to this mixture followed by the portioned addition of 1,3-bis(benzyloxycarbonyl)-2-methyl-2-ps eudothiourea, (6.15 g, 17.2 mmol). The flask flushed with Ar and reaction was stirred at r oom temperature for 3 hours. After reaction was completed (3 hr), the mixture was filtered th rough Celite under vacuum and the residue was washed with methanol. The filtrate was then concentrated under reduced pressure and the crude was purified by column chromatography (1:1 EtOAc/hexane) to give 6.5 g of 51 as a pale, thick oil in a 90% yield. 3.5.6. Preparation of 52. N H NHBoc CbzHN NCbz 51 N H NH2 CbzHN NCbz 52 TFA, DCM Compound 51 was dissolved in a 1:1 mixture of DCM and Trifluoroacetic acid. After strring at room temparature for 1h, the solvent was evaporated under reduced pressure and the crude compound 52 was obtained and was subjected to the next reaction without purification. For Analyzing purpose, crude compound was purifi ed by column chromatography (89:10:1, DCM: MeOH: NH4OH).

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663.5.7. Preparation of 53. N H NH2 CbzHN NCbz 52 N H N H CbzHN NCbz 53 O Br Bromoacetyl bromide, Na2CO3DCM, -10oC Compound 52 as the trifluoroacetate salt (5.6 g) was dissolved in 40 mL of CH2Cl2 and 40 mL of sat. aqueous sodium carbonate. This mixture was stirred and cooled to -10 C. Bromoacetyl bromide (0.8 mL) was added and stirring continued for 0.5 hr at 0 C before a second portion of 2 (0.4 mL) was added. The reaction was left stirring for 3 hrs. The reaction mixture was poured into 100 mL EtOAc and 80 mL of water. The phases were separated and the organic layer was washed with 5% NaHCO3 (100 mL), 1 M HCl (100 mL), and brine (100 mL x 2). Crude was dried over Na2SO4, concentrated under vacuo and subjected to column chromatography (6:4 = EtOAc: hexane) to give 1.65 g of 53 as white solid in a 55% yield for 2 steps. 3.5.8. Preparation of 54. FmocHN CO2H S Ph Ph Ph FmocHN CONH2 S Ph Ph Ph DCC, HOBT, NH4OH THF 43 54 Synthesis of (R)-(9H-fluoren-9yl)methyl 1-amino-1-oxo-3-(tri tylthio)propan-2-ylcarbamate, 54. To a solution of 43 (2 g, 3.41 mmol), HOBT (549 mg, 4.1 mmol) and DCC (832 mg, 4.1 mmol) in THF (10 mL) was added 28% NH4OH (715 mL, 5.1 mmol) at 0 C. After 2 hr of stirring at 0 C, the reaction mixture was filtered through CeliteTM and filtrate was concentrated, diluted with EtOAc, and washed with water and brine. The organic layer was dried over sodium sulfate, concentrated and subjected to flash column chromatography to give 54 (1.85 g, 92%) as a white solid.

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673.5.9. Preparation of 55. FmocHN S Ph Ph Ph NH2 FmocHN CONH2 S Ph Ph Ph BH3 .THF THF 54 55 Synthesis of (R)-(9H-fluoren-9-yl)methyl 1amino-3-(tritylthio)propan-2-ylcarbamate, 55. To solution of 54 (430 mg, 0.74 mmol) in THF (3 mL) was added 1 M BoraneTHF complex (2.2 mL, 2.2 mmol) at 0 C and after stirring at 0 C for 3 hr, the reaction mixture was heated to 70 C and stirred for 12 hrs. The reaction mixture was cooled to 0 C and quenched by MeOH, the reaction mixture was concentrated and diluted with MeOH and concentrated again in vacuo. The concentrated oil was subjected to flash column chromatograph to afford 55 (256 mg) in a 61% yield. 3.5.10. Preparation of 56. FmocHN S Ph Ph Ph N H CO2t-Bu FmocHN S Ph Ph Ph NH2 BrCH2CO2t-Bu, DIPEA THF 55 56 Synthesis of 56. To a solution of 55 (123 mg, 0.21 mmol) in THF (3 mL) was added tert-butyl bromoacetate (41.5 mg, 0.21 mmol) and DIPEA (0.040 mL, 0.23 mmol) at 0 C. The reaction mixture was slowly allowed to warm to r.t. T he reaction mixture was co ncentrated in vacuo, diluted with EtOAc and washed with brine. The organic layer was dried over sodium sulfate, concentrated in vacuo, and subjected to a flash column chromatography to give 56 (133.5 mg, 90%).

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683.5.11. Preparation of 57. FmocHN S N H CO2t-Bu N H O CbzHN FmocHN S Ph Ph Ph N H CO2t-Bu i) TFA/DCM, Et3SiH ii) R1Br, NaOH, EtOH 56 57 Synthesis of (R)-tert-butyl 13-(((9H-fluoren-9 -yl)methoxy)carbonylamino)-3,9-dioxo-1-phenyl-2oxa-11-thia-4,8,15-triazaheptadecan-17-oate, 57. To a solution of 56 (50 mg, 0.086 mmol) and in DCM (5 mg) stirring at 0 C was added TFA (2 mL). The reaction mixture was treated with triethylsilane (8.5 mg, 0.086 mmol). The reaction mixture was stirred for 30 min at 0 C and concentrated in vacuo. The crude product was dissolved in EtOH (2 mL) and the solution was cooled to 0 C. Aqueous NaOH (0.2M, 0.8 mL) was added slowly to the solution followed by addition of the alkyl bromide, (25 mg). The reaction was stirred for 4 hrs at 0 C. After the removal of organic solvents in vacuo, the residue was diluted with EtOAc and washed with water and brine. The organic layer was dried over sodium su lfate, concentrated in vacuo, and subjected to flash column chromatography to give 57 (46.3 mg, 92%). 3.5.12. Preparation of 58. FmocHN S N CO2t-Bu N H O CbzHN O N HN O O FmocHN S N H CO2t-Bu N H O CbzHN HATU, TEA, Thymine acetic acid DMF 58 57 General procedure for the nucleobase coupling : Synthesis of (R)-tertbutyl 13-(((9H-fluoren9-yl)methoxy)carbonylamino)-15-(2-( 5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)-3,9dioxo-1-phenyl-2-oxa-11-thia-4,8,15-triazaheptadecan-17-oate, 58 To a stirring solution of 57 (22 mg, 0.032 mmol), HATU (18 mg, 0.048 mmol) and Thymine acetic acid (9 mg, 0.048 mmol) in DMF was added TEA (5 mg) at r.t. for 4 hr.

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69The reaction mixture was diluted with EtOAc, washed with cold water and brine, dried over sodium sulfate, concentrated in vacuo and subjected to flash column chromatography to give 58 (25.8 mg, 95%). 3.5.13. Preparation of 67. FmocHN MeS N H CO2t-Bu FmocHN S Ph Ph Ph N H CO2t-Bu i)TFA/DCM, Et3SiH ii)MeI, NaOH, EtOH 57 67 Synthesis of 67. To a solution of 57 (1.12 gm, 1.6 mmol) in DCM (40 mL) stirring at 0 C was added TFA (4 mL). The reaction mixture was then trea ted with triethylsilane (0.187 gm, 1.6 mmol). The reaction mixture was stirred for 30 min at 0 C and concentrated in vacuo. The crude product was kept on vacuum for 12 hours and dissolved in EtOH (75 mL). After cooling the solution to 0 C, aqueous NaOH (2M, 2.42 mL) was added slowly, followed by addition of iodomethane, (1.0 mL). The reaction was stirred fo r 10 min at 0 C. After the removal of organic solvents in vacuo, the residue was diluted with EtOAc and washed with water and brine. The organic layer was dried over sodium sulfate, concentrated in vacuo and subjected to flash column chromatography to give 67 (0.67 g, 90%). 3.6. Experimental procedures for compounds in Schemes 14. 3.6.1. Preparation of 72. CO2H BocHN S Ph Ph Ph CONH2 BocHN S Ph Ph Ph 34 72 DCC, HOBt, NH4OH THF Synthesis of (R)-tert-butyl 1-amino-1-oxo-3-(tritylthio)propan-2-ylcarbamate, 72. To a solution of 34 (2 g, 4.31 mmol), HOBT (700 mg, 5.17 mmol) and DCC (1.07 g, 5.18 mmol) in THF (10 mL)

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70was added 28% NH4OH (0.44 mL, 6.46 mmol) at 0 C. After 2 hr of stirring at 0 C, the reaction mixture was filtered through CeliteTM and the filtrate was concentrated, diluted with EtOAc, and washed with water and brine. The organic layer was then dried over sodium sulfate, concentrated and subjected to flash column chromatography to give 72 (1.95 g, 98%) as a white solid. 3.6.2. Preparation of 73. CONH2 BocHN S Ph Ph Ph 72 BocHN S Ph Ph Ph 73 NH2 BH3.THF THF Synthesis of (R)-tert-butyl 1-amino3-(tritylthio)propan-2-ylcarbamate, 73. To solution of 72(1 g, 2.16 mmol) in anhy THF (5 mL) was added 1 M BoraneTHF complex (5.4 mL, 5.4 mmol) at 0 C and after stirring at 0 C for 3 hrs, reaction mixture was heated to 60 C and refluxed for 12 hr. The reaction mixture was cooled to 0 C, quench ed by MeOH, concentrated, diluted with MeOH, and concentrated again. The concentrated o il was then subjected to water work-up pH = 8.5 followed by flash column chromatography to afford 73 (0.58 mg, 60%). 3.6.3. Preparation of 84. BocHN S Ph Ph Ph 73 NH2 BocHN S Ph Ph Ph 84 Fmoc-Cl, DMAP, DIPEA DCM NHFmoc Synthesis of 84. To a solution of 73 (0.1168 gm, 0.26 mmol) in DCM, Fmoc-Cl (70.7 mg, 0.273 mmol) was added followed by addition of DM AP (8 mg, 0.0654 mmol) and DIPEA (0.043 mL, 0.26 mmol) was added at 0 C and stirred for 3 h at room temperature. The reaction mixture was concentrated to give a crude oil. 84 was obtained as a solid by triturating the crude oil with ethyl acetate and hexane (0.147 gm, 85%).

PAGE 86

71 3.6.4. Preparation of 85. BocHN S Ph Ph Ph NHFmoc 84 TFA, TES DCM R1Br, NaOH, TBAI EtOH 85 BocHN R1S NHFmoc Synthesis of 85. To a solution of 84 (62.9 mg, 0.093 mmol) in DCM was added TES (12 mg, 0.103 mmol). The reaction mixture was cooled to 0 C. TFA (0.19 mg, 1.69 mmol) was added dropwise. The reaction mixture was concentrated and subjected to flash column chromatography affording the free thiol product which was subjected to the next alkylation without further purification (39.5 mg, 98.8 %). To a solution of crude thiol (31.2 mg, 0.0728 mmol) in ethanol was added TBAI (2.7 mg, 0.0073 mmol) at 0 C. 2N NaOH (2.9 mg,0.00728 mmol) was added dropwise followed by dropwise addition of alkylating agent R1Br (28 mg, 0.0872 mmol) in ethanol. The reaction mixture was stirred for 3 hr and concentrated followed by water work-up at pH = 8.5. The organic layer was dried over anhydrous sodi um sulfate and concentrated followed by flash column chromatography to give 85 (0.0435 mg, 89%). 3.6.5. Preparation of 86. 85 HCl Gas 1,4-Dioxane BrCH2CO2t-Bu, DIPEA DMF H N R1S NHFmoc 86 t-BuO2C BocHN R1S NHFmoc Synthesis of 86. To a solution of 85 (1.16 g, 1.71 mmol) in 1,4-dioxane at 0 C HCl gas was passed for 15 min and the reaction was stirred at room temperature for 30 min. The reaction mixture was concentrated and dried by applying higy vaccum to give the crude amine which is in the HCl salt form (0.988 g, 100%). To a suspention of crude amine HCl salt (0.988 g, 1.71 mmol) in DMF was added tert-butyl bromoacetate (0.351 g, 1.79 mmol) at 0 C followed by addition of DIPEA (0.443 g, 3.42 mmol) and tetrabutylammoni um iodide (63.3 mg, 0.171 mmol). The reaction

PAGE 87

72mixture was stirred for 3 hr. The reaction mixtur e was concentrated, subjected to water work-up at pH = 8.5 followed by drying with anhydrous sodi um sulfate, and flash column chromatography to obtain 86 (0.71 gm, 60%). 3.6.7. Preparation of 87. N R1S NHFmoc 87 t-BuO2C O (T) H N R1S NHFmoc 86 t-BuO2C DMF i) SOCl2, 13 Solution of 13 in thionyl chloride was refluxed for 2 hr, then the solution was concentrated with vaccum and the acid chloride was diluted with anhydrous DMF. 86 was added into the acid chloride solution followed by DIPEA. The reaction was stirred for an hour at room temperature and then heated up to 50 C. The reaction mixture was concentrated and subjected to flash column chromatography to give 87 in 50% yield. After the column, 45% of 86 was recovered.

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73 4. Chapter Four: Synthesis of Cysteine based PNA Oligomers: Solid Phase Synthesis. 4.1. Introduction. As discussed in Chapter 2, the CPNA monomers were prepared with the protecting groups designed to allow oligomerization of the monomers using a solid phase synthetic strategy. In the 1960s, the solid-phase methodology was first developed by R Bruce Merrifield90 and it has been developed at a revolutionary pace. The solid phase synthetic methodology is now one of the key components for modern bioscience and its impossible to imagine same areas of science would have evolved over the last half century withou t this discovery. It has been widely used for peptide chemistry and many applications in organic chemistry have also been reported. This chapter includes a brief history and the summary of the basic aspects for solid phase synthesis followed by the discussion of our experimental efforts to synthesize the CPNA oligomers with specific sequences. 4.1.1. Historical introduction to solid phase synthesis: R. Bruce Merrifield and the solidphase sy nthesis. The inventor of the solid phase synthesis methodology, R. Bruce Merrifield, was born in 1921 in Fort Worth, Texas, and grew up in California where he obtained his Bachelors and Ph.D. degrees from UCLA.91 It was 1949, when he moved to the eastern part of US with a one year postdoctoral research assistantship offer from Dr. Wooley who was a professor at the Rockefeller Institute for Medical Research. During his Post doctoral assistantship, he was involved with a project which studied a new bacterial growth factor, and was eventually introduced to a tedious

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74fragment synthesis of a pentapeptide. It took 11 m onths for him to complete the synthesis and the overall yield for the procedure was only 7 %. Th is bitter experience became a seed for the great invention he developed years later. He was appointed Assistant Professo r at The Rockefeller Institution and eventually was prom oted to associate professor in 1958. In 1959, Dr. Merrifield conceived the idea of a peptide synthesis on a solid support so that he would avoid the purification of the intermediates. It took about 4 years to overcome the challenges and publish a paper90 that unveiled the synthesis of first tetrapeptide by the solid-phase protocol. The seminal paper is now among the most cited papers in all of the scientific literature.91 He recognized the possibility of developing an automated peptide synthetic device, and later constructed the first automated peptide synthesizer using the solid-pha se protocol that he developed. In 1984, Dr. Merrifield won the Nobel Prize in Chemistry for his discovery and development of the solid phase methodology that impacted the direction of scientific research throughout the world. In 2006, Dr. Merrifield died of a long suffered illness after being recognized as one of the most influtential scientists of the past century. 4.2. Strategies of solid phase synthesis. Solid phase synthesis has several advantages over the traditional solution based method regardless of where it is a peptide or small organic molecule synthesis.94 First, it can be driven to completion by using excess amount of reagents while the solution based reaction often requires stoichiometric control of the reagents to simplif y isolation of the intermediates. Whereas with SPPS, the excess reagents can be removed by simple washing. Second, a solid supported reaction can minimize the loss of the desired product due to its simple work-up procedure. Third, the solid phase synthesis can be conducted in a aut omated system. In fact, most of peptides are.

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75 coupling = Solid support (Resin) = Handle (Linker) = Monomer (Amino acid) = Temporary protectiing = Permanent protecting group n n Cycles of Deprotection and Coupling n n coupling deprotection Desired Polymer(Peptide) deprotection deprtection of permanet protecting group & cleavage of the solid upport Selection of the resin Figure 21: General scheme of SPPS. prepared by using an automated peptide synthesizer these days. Fourth, since every single resin bead can be considered as a microscale reactors, it is possible to synthesize large number of compounds simultaneously by parallel or serial combinatorial approaches

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76 = Solid support (Resin) = Semipermanet Handle = Permanent Handle (Linker) = Monomer (Amino acid) = Temporary protectiing = Permanent protecting group segment segment 1 HO HO segment 2 O O segment 1 O O segment 2 O O H N O segment 1 segment 1 O OH segment 2 O OH H N O segment 2 O O H2N segment 3 O O H2N segment 2 O H N O segment 3 O O N H segment 1 segment 2 O H N O segment 3 O OH N H segment 1 H2N Figure 22: General scheme for the convergent SPPS. The basic concept of the stepwise solid phase peptide synthesis is illustrated in Figure 21.94 Generally two types of approache s are applied for SPPS. Stepwise SPPS92 is normally used for to prepare small size peptides and the convergent SPPS strategies94 is often used for large peptides with longer s equences. For c onvergent SPPS, the scheme is little different than the traditional one and it is briefly demonstrated in Figure 22. Generally, SPPS proceeds from the C terminus to N terminus. In other words, the N terminus is usually protected while the C terminus is being reacted. There have been only few cases reported where SPPS procedures allows C to N terminus synthesis.94 Among other protecting groups, Boc and Fm oc groups are the most widely used protecting groups for the N-terminus. Compared to the Boc method, the Fmoc strategy is relatively new. These two different methods hav e their own advantages over each other. One

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77must consider carefully which method would fit better for the oligomer synthesis depending on the composition of the sequence and the permanent protecting groups that are employed in the synthesis. Table 1: Comparison of the Boc and Fmoc SPPS strategies Boc-SPPS Fmoc-SPPS Notes N terminus Protecting group Boc group Fmoc group Deprotecting condition TFA Mildly bulky amine bases (usually piperidine) Repetitive TFA deprotecting steps often generates degradation of the peptides Common C terminus protecting groups Base labile ester (Methyl or Ethyl) Acid labile t-Butyl ester Other protecting groups can be applied as long as the deprotecting condition doesnt affect the other termini. i.e.: allylic ester. Cleavage condition HF or TFMSA TFA HF and TFMSA are extremely strong acids: There are safety concerns for the cleavage conditions in Boc SPPS. In Table 1, the two representative solid phase peptide s ynthesis (SPPS) strategies, Boc-SPPS and Fmoc-SPPS, are briefly compared. Many re view papers have compar ed these two methods and it seems that the Fmoc strategies are usually the preferred one compared to the other one in terms of overall productivity as well as the purity of the final compounds.93 The problem of the Boc strategy is believed to be the repetitive usage of the TFA. As a contrast to the SPPS which is cons idered as a quite established area, SPOC, the application of the solid-phase synthesis for org anic chemistry is still in its development. The SPOC is one of the most active subjects in the field, it seems that more organic chemists are getting interested in the field since the scope of the application is almost unlimited. Even though

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78SPOC is not discussed in detail due to the limit ed scope of this report, one should be aware of the fact that there are many reviews and report s on the SPOC with very interesting ways of mixing the solid supports and countless organic methodologies. 4.3. Solid supports for solid phase methodologies. It is somewhat a misleading term solid support used for the solid-phase peptide synthesis (SPPS) or solid phase or ganic chemistry (SPOC) becaus e the polymers that are used in the reactions are not quite solid solids but they are just insoluble polymers which could have reactions on the surface as well as inside their particles.94 Solid supports, commonly resins, can be classified depending on the matrices of the polymers; of course, the matrix dictates the properties of the resins. Resins can also be categorized by the external functional groups. Additionally it could also be divided by the strategies that the resin could support. Understanding the nature of the materials which support the SPPS or SPOC is definitely helpful when choosing the optimum support and to design the optimum synthetic schemes. 4.3.1. Essential factor for the solid supports. Table 2: Cha racteristics of an efficient resin.94 Questions Ideal Characteristics Is the size and shape of the resin OK? Or is the resin physically stable? Yes, so the resin can be manipulated in the process. It must go through the transferring and filtering and so on. Is the resin inert to the reagents or solvents Yes, of course. It must be inert. Does it swell in the solvents? Yes, most of time. It should swell extensively so it can allow all the reagents to access all the reactive sites of the resin.

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79Is it possible to modify/generate the functional group? Yes, It should be readily modified, so one can use it in a variety of ways. Is the loading capacity of resin high enough? It should be considered. High capacity resins are not always ideal. Before discussing the class of the resins, it s hould be pointed out that there are characteristics94 for the resins to act as good supports for the SPPS or SPOC and these are summarized in the Table 2. The resin should be physically stable and ch emically inert throughout the whole synthetic process. As briefly mentioned in the introduction, the more swelling in the solvents, the more reactive sites the resin can have. The resin should be compatible with the anchor and the monomers. 4.3.2. Representative Solid supports for SPPS. Conventionally, It is wise to have a resin t hat gets solvated well in the organic solvents so all the active sites of the solid support can be accessible from its own steric barrier. For this reason, the resins with the minimal level of cross-linking consistent with reasonable physical stabilities are generally considered as the best solid supports. Table 3: Class of Resins.94 Physical property Notes Matrix Notes Polystyrene(PS) Low cross-linked Polyacrylamide Hydrophilic alternative to PS PEG grafted (PEG-PS) Gel-type supports Most widely used supports. Good swelling, inert, flexible polymer network. PEG Sintered polyethylene Cellulose fibers Surface-type supports Hard surface, less (or) no swelling and fast reaction Highly Cross-linked PS Non-swelling, rapid reaction.

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80Pore glass, silica Composites and supported gets Increased mechanical stability Highly Cross-linked PS(exterior) + polyamide(interior) Brush polymers Linear components attached to a film or tubing PS grafted onto polyethylene As they are classified in Table 3, along with its hybrid forms, gel-type supports are the most widely used resins for SPPS and SPO C. They normal ly have adequate reaction rates due to their good swelling property and they are also physically st able. In contrast to gel type solid supports, resins with hard surfaces dont swell much in organic solvents. These types of resin can have higher reaction rates; however they often are ine fficient. Therefore these resins are often used for SPPS and SPOC. 4.3.2.1. Polystyrene based resins A suspension polymerization of styrene and divinylbenzene (DVB) provide cross-linked polystyrene as hydrophobic beads. The size of the bead can be controlled and virtually monodispersed sized beads are usually the product.94 There are two ways of functionalizing the resins: either by adding fuctionalized monomer for the polymerization or by functionalizing the polymer. The first method is not usually used because it is hard to control the polymerization this way. In addition, more of the functional groups may be located in positions that are sterically hindered so it would be difficult for them to re act with the substrates. Therefore, the direct derivatization has been most common strategy for solid phase methodology. Electrophilic aromatic substitution reactions of the styrene ring of the resin, especially Friedel-Crafts alkylations are the main approaches for initial fuctionalization. In Figure 23, Figure 24, Figure 25 and Figure 26, the simplified procedures for the preparation of common PS resins are summarized.94 These procedures are adapted from reference 94.

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81 Figure 23: Preparation of common PS resins.94 +Styrene Divinylbenzene(DVB) Polymerization Syntheis of copoly(styrene-DVB) resin. PS PS ClCH2OCH3/DCM, ZnCl2, 40 C PS Cl Chloromethyl resin (Merrifield resin) O (SnCl4) BCl3-CCl4, 0 C Cl + + P o l y m e r i z a t i o n PS PS PS NO2 NH2 HNO3SnCl2, 2H2O, HCl, DMF, 100 C PS Y1 Y2 COCl + PS O Y1 Y2 PS OH Y1 Y2 PS Cl PS NH2 aminomethylated resin amino resin Benzhydrylhydroxy resin NH3 Anhy, DCM AlCl3NaBH4 or LAH

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82 Figure 24: Preparation of common PS resins B .94 PS PS HgCl PS CO2H HgO,TFA,DCM n-BuLi, CO2 PS Carboxy resin PS Br O CH3CH(Br)COBr, DCM, AlCl32-Bromopropionyl resin ( Bromo Wang resin ) PS OH Y1 Y2 PS PS Br(or Cl) Y1 Y2 HX(g), DCM PS O Y1 Y2 i) HCO2NH4, HCO2H ii) HCl conc. PS NH2 Y1 Y2 NH3(g), DCM Benzhydrylhalo resin Benzhydrylamine resin PS Cl OH OCH3 H3CO O + ii) LiBH4, THF, reflux iii) methanol/CH3COCH3, 0 C PS O OCH3 OCH3 HO Rink acid resin NHFmoc OCH3 OCH3 O PS Rink amide resin

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83 PS Cl O O OH + i) CsOH, DMF, r.t ii) LiBH4,THF, 6h, reflux iii) FmocNH2, benzosulfonic acid DMF, 50 C PS C H2O O NHFmoc Sieber resin PS NH2 X OCH3 OH O HO O + DCC, DCM PS HN X O O OCH3 OH X = H, HMPB-BHA resin X = CH3, HMPB-MBHA resin PS Cl HO OH + NaOCH3 in DMA, 50 C PS CH2O OH Hydroxymethylphenoxymethyl resin ( Wang resin ) HO OH OCH3 NaOCH3 in DMA, 50 C PS CH2O OH + Sasrin resin OCH3 Figure 25: Preparation of common PS resins C.94

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84 PS + COCl NO2 AlCl3 O NO2 PS PS NOH NO2 NH2OH, HCl Pyridine Kaiser oxime resin PS i) nBuLi/TMEDA ii) PhCOPH( X R PS OH X R R = H, CH3, OCH3X = Cl, H AcCl Benzene PS OH X R Tritylalcohol resin Tritylchloride resin PS OH + O Cl DIPEA, DCM PS CH2O O Vinylcarbonyloxymethyl resin ( REM resin ) Figure 26: Preparation of common PS resins D.94 4.3.2.2. Polyethylene glycol based resins. Polyethylene glycol (PEG) is often used as a solid support for synthesis and also is used to modify the PS resins by grafting it on the func tional groups of the PS resins to improve the properties of the solid supports. The lengths of PEGs that get grafted onto PS resins can vary depending on the purposes. The lengthy grafted PEGs on PS resins are often considered as spacers between the polymer supports and the subs trates. The compositon of the PEG-PS resins can change the physical properties of the resin. Modifying the functional groups on the PEG-PS resins is also as common as that of PS based resin. Various types of linkers have been introduced to the PEG-PS solid supports and they are illustrated in Table 4.

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85Table 4: Functionalized PEG-PS resins. PS PEG OCH2CH2Br PS PEG NHCO OH PS PEG OCH2CHO PS PEG O OH PS PEG O NH2 O O PS PEG NHCO(CH2)4O OH O PS PEG OCH2CH2SH PS PEG OCH2CH2NH2 PS PEG OCH2CH2COOH PS PEG OCH2CH2OH PS PEG CO(CH2)2CO N O O PS PEG NHCOCH2O OH PS PEG NHCO OH PS PEG OH X X X = H, Cl PS PEG NHCO(CH2)nO O NHFmoc PS PEG NHCOCH2O NHFmoc O O

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86 (OCH2CH2)n.OCH2CH2OH PS (OCH2CH2)n.OCH2CH2X PS functionalization PEG-PS resins R R O O O R R NH O R R R R N O HN NH2 n PEGA resin O O O O O OH O O O R R POEPS resin POE-Spacer 34 34 34 O O O O O O O O OH O O O O 3 4 3 4 3 4 POEPOP resin CH2=CHCH2NH2CH2=C CH3 CO2(CH2)2NH2 CH2=C CH3 CO (OCH2CH2)9 OCOCH=CH2CH3 Precursors used for the synthesis of CLEAR Figure 27: Polyethylene glycol based resins. PEG based resins also can be utilized as solid phase supports. The PEG molecules have an amphipathic nature, so PEG based resins are well solvated in both polar and nonpolar solvents.95 There are mainly four types of PEG based resins; PEGA resins, polyoxyethylene-

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87polystryrene (POEPS) resins, Po lyoxyethylene-polyoxypropylene (POEPOP) resins and crosslinked ethoxylate acrylate resins (CLEAR). Their structures are shown in Figure 27. 4.3.3. Ananyltical methods for SPPS. There are several methods to determine whet her the coupling was efficient in SPPS. The most widely used qualitative method is the Kaiser Test. This method is also called the nynhydrin test. The Kaiser test indicates the whether free primary amines are present or not by the color development following the procedure. It should be noted that the Kaiser test sometimes give a false negative sign when the resins and substr ates are severely aggregated. There is another qualitative method known as the chlorine test. This test indicates the presence of secondary amines. When the solid phase synthesis is uses th e Fmoc group as protecting group, it can be quantitatively tested using the Fmoc test. This is a good method to monitor the solid phase synthesis. The authors have found the fact that there are at least too procedures available for monitoring Fmoc group. After using both Fmoc monitoring procedures, It was learned that the procedure from the catalog of the Novabiochem93 gave consistent result compared to the others reported. Therefore, the procedure from Nov abiochem is reported for our CPNA results. 4.3.4. Peptide coupling methods. The amide bond formation is the key step for the peptide synthesis and other organic synthetic projects as well. Therefore, t here are hundreds of coupling reagents that are commercially available these days. In addition, the ester formation may also share the protocol that are prepared for amide and vice versa. The coupling techniques and reagents have been rapidly developed in the last few decades. This c an be linked to the recent success of the peptide chemistry. Since peptide chemists now have ac cess to automated peptide synthesizers along with the other advanced analytical devices, large numbers of peptides and even longer peptides are becoming available. The amide formation step should proceed to essentially 100 % yield. The

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88epimerization of monomers and other side reactions in the process of using the certain activating reagents have been an issue. Nevertheless, the activating group or the coupling reagent must be chosen after a thorough examination in terms of the high coupling efficiency along with the potential side reactions. Fortunately, there are many activating reagents that have been developed, and they are extensively anal yzed, and compared in the reviews.93 There are generally 4 types of coupling strategies currently employed in solid phase synthesis: first, using carbodiimides as the coupling reagents; second, forming the symmetrical anhydride as an active species; third, using active esters as the coup ling partner; and forth, utilizing the uronium based coupling reagents. Carbodiimides are one of the most popular activation reagents of all time.92 However, it is well know that this family of reagents produce thee in soluble ureas in some of the solvents that are used in SPPS. Preformed sy mmetrical anhydrides have been widely used for Boc SPPS due to their high reactivity. They can be generated in situ by mixing two equivalents of the protected amino acid and one equivalent of the carbodimi de reagent. The urea can be easily filtered and the anhydrides are ready for the coupling. Freshly generated anhydrides should be used for satisfactory results. A drawback for this meth od is the waste of the monomers. Some of the activated esters have been extrem ely useful in SPPS. Obt este rs and OPfp este rs are great examples for this category.94 The most common method of all is using one of the in situ activating reagents which are based on phosphonium or aminium salts in the presence of a tertiary amine base. These reagents are known to give fast re actions generally without side reactions. The simple procedures are also one of the merits for these reagents. Among all the coupling reagents illustrated in Figure 28, Figure 29 and Figure 30, following reagents BOP, PyBOP, HBTU, TBTU, HATU, PyBrOP, TSTU, and TNTU are most commonly employed for routine SPPS. Common bases for the coupling reactions are also grouped in Figure 31. Often times, use of an additive accelerates the coupling and reduces racemiazation during the coupling. Commonly used additives are shown in Figure 32.

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89CN N CN N CN N NH Cl N N N 2BF4 N N N P N O N N P N O N N P N O N N P N N N PF6 -PF6 N N N N P N O N N PF6 N N N N P N O N N N N N O N N PF6 -PF6 PF6 -N N N N O N N PF6 N N N N O N N PF6 N N N O N N PF6 BF4 -N N N N O N N BF4 -N N N N O N N PF6 -N N N N O N N PF6 N N N O N N PF6 -N N N N O N N N N N O O N N N N N O O N N BF4 -DDC DIPCDI ECC, WSC Bates reagent BOP PyBOP AOP PyAOP HBTU HATU HAPyU TBTU TATU HAPipU HAMDU HBMDU, BOI HAMTU HDTU TPTU Figure 28: coupling reagents, part A.

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90PF6 -PF6 N N N O O N N BF4 N O N N BF4 O O N O N N BF4 O O O N N BF4 -S N O N O N N BF4 O O N N N O N N F3C N N O F F F F F PF6 O F F F F F TOPPipU TSTU TPhTU TMTU TNTU CF3-TBTU PfTU PfPyU, HPyOPfp PyNOP CF3-PyBOP, PyFOP N N N P N N N O2N PF6 N N N P N N N F3C PF6 -P N N N O N NN O PF6 -P N N N O F F F F F PyDOP PyPOP, PfOP, PyPfpOP P N N N O N PF6 -PF6 -P N O PF6 -N N N N N N S N N N N N N PF6 -S N N N N N N PF6 PyTOP AOMP HATTU HAPyTU Figure 29: Coupling reagents, part B.

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91PF6 -PF6 O F F F F F P N N N N N F PF6 -N N F F PF6 -P N Cl N N PF6 -P N Br N N PF6 -P N Br N N PF6 -Cl PF6 -P O Cl P O O P O Cl F F F F F P O N3 O O P O CN O O P O N N Cl O O O O P O CN N O O O P O N O O O O P O N O O N N O P O N O N N O O P O N O O O O P O O O O N N N S N O O N N NO2 S N O O N N NO2 N N N Cl OMe MeO N N N F F F F F F F F F F F F F F F HPySPfp TFFH BTFFH DFIH PyCloP PyBroP BroP CIP, DCIH Dpp-Cl Cpt-Cl FDPP DPPA DEPC BOP-Cl DEPBO DOPBO DOPBT DEPBT ENDPP BDP MSNT TPSNT CDMT TPft Figure 30: Coupling reagents, part C.

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92N N N N N N N N N N N N N O NO N O TEA DIPEA TMP, collidine DMAP NMI MPO NMM Pyridine 2,3,5,6-tetramethylpyridine 2,6,-Di-t-butyl-4methylpyridine DB(DMAP) Figure 31: Commonly used bases for SPPS. N N N OH N N N N OH N N N O OH N N N N OH N N N Cl OH N N N OH O EtO N H S O O N N N OH N N N OH N N OH NO2 S O HO O O HOBt HOAt, 7-HOAtHODhbt, HOOBt 4-HOAt HOCt 5-Chloro-1hydroxytriazole Polymeric N-benzyl1-hydroxybenzotriazole-6sulfonamide P-HOBt HOHppTDO Figure 32: Additives for the amide coupling.

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934.4. Synthesis of CPNA oligomers. 4.4.1. Preparation for solid phase synthesis. 4.4.1.1. Deprotecti on of the monomers. N S Fmoc N CO2Me O B N S Fmoc N CO2 t-Bu O B 25 (T) S N N Boc CO2Me O B BocHN S N CO2Me Ph Ph Ph O B 39 (T), 40 (G), 41 (C), 42 (A) 20 (T), 21 (G) 24 (T) Figure 33: First generation of CPNA monomers. A series of the PNA monomers were prepar ed as discussed in chapter 2. The first generation of monomers was prepared to test whet her the native chemical ligation method could be adapted to the solid phase synthesis of PNAs. Monomers prepared for this new approach are summarized in the Figure 33. These monomers are all in protect ed forms, so they need to be deprotected in a way that the other terminus could be still intact. Except for compound 24, all the other compounds are protected in the traditional ways, which are commonly used for ordinary peptide chemistry. We have found an interesting procedure96 that uses a mild trialkyltin hydroxide to hydrolyze the methyl ester while t he Fmoc group is still protected. Considering how vulnerable the Fmoc group is in basic conditions this methodology is a nice protocol because it could provide flexibility for designing peptide sy nthesis and organic synthesis. We tested the

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94N S NHFmoc t-BuO2C O H N O CbzHN B 87 (T) BocHN RS N CO2Me O B 76 R= Me and T 77 R= Me and G 78 R= Me and C 79 R= Me and A 80 R= R1 and T 81 R= R1 and G 82 R= R1 and C 83 R= R1 and A FmocHN MeS N CO2t-Bu O B 68 (T), 69 (G), 70 (C), 71 (A) FmocHN S N CO2t-Bu N H O N H CbzHN CbzN O B 63 (T), 64 (G), 65 (C), 66 (A) FmocHN S N CO2t-Bu N H O CbzHN O B 58 (T), 59 (G), 60 (C), 61 (A) Protected Fmoc(R1) Monomers Protected Fmoc(R2) Monomers Protected Fmoc (SMe) Monomers Protected Boc Monomers(R1 & SMe) Protected ICPNA T-monomer(R1) Figure 34: Second Generation of the CP NA monomers & the ICPNA (T) monomer. Scheme 15: Deprotection of Boc monomers for Boc-solid phase synthesis. BocHN S N CO2H Ph Ph Ph O B 90 (T), 91 (G), 92 (C), 93 (A) BocHN RS N CO2H O B 94 R= Me and T 95 R= Me and G 96 R= Me and C 97 R= Me and A 98 R= R1 and T 99 R= R1 and G 100 R= R1 and C 101 R= R1 and A Boc Protected Monomers: 20, 21, 39, 40, 41, 42, 76, 77, 78, 79, 80, 81, 82, 83 88 ~ 101 a) NaOH or LiOH, THF/H2O, rt. S N N Boc CO2H O B 88 (T), 89 (G)

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95protocol and it worked well for our monomer 24 as shown in Scheme 16. Before we began the solid ph ase synthesis, we had to decide which coupling procedure we would use for the solid phase synthesis and optimize the oligomer synthesis since PNA oligomer synthesis is not trivial. Scheme 16: Deprotection of Fmoc monomers for Fmoc-solid phase synthesis. N S Fmoc N CO2Me O B N S Fmoc N CO2H O B 102 (T) 24 (T) FmocHN MeS N CO2H O B 111 (T), 112 (G), 113 (C), 114 (A) FmocHN S N CO2H N H O N H CbzHN CbzN O B 107 (T), 108 (G), 109 (C), 110 (A) FmocHN S N CO2H N H O CbzHN O B 103 (T), 104 (G), 105 (C), 106 (A) N S NHFmoc HO2C O H N O CbzHN B 115 (T) Fmoc protected Monomers: 25, 58, 59, 60, 61, 63, 64, 65, 66, 68, 69, 70, 71, 87 b or c Fmoc methyl ester monomer, 24 a 102 ~ 115 102 (R1) Fmoc monomers (R2) Fmoc monomers (SMe) Fmoc monomers Inverted-(R1) Fmoc T-monomer a) Me3SnOH, DCE, 24 hr. 85% b) TFA/DCM, 1hr, quantatative c) HCl(g), 1,4-Dioxane, 0 C to rt. 3hr. quantatative

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964.4.1.2. Solution based coupling of monomers. Before conducting the solid phase synthesis, we needed to confirm the coupling of our monomers in solution. Therefore, we decided to check the coupling of our first generation monomers. Scheme 17: Preliminary study before the solid phase synthesis. S N N Boc CO2Me O T 20 HS H2N N CO2Me O T S H N N CO2Me O T 116 117 various conditions BocHN S N CO2Me Ph Ph Ph O T TFA H2N S N CO2Me Ph Ph Ph O T (DCM) TFA. H2N HS N CO2Me O T TFA.118 119 39 91(G) + 119(T) 90(T) + 118(T) HATU, TEA DMF HATU, TEA DMF BocHN S N Ph Ph Ph O T H N S N CO2Me Ph Ph Ph O T O 120 BocHN S N Ph Ph Ph O (BnO)G H N HS N CO2Me O T O 121 TFA, EDT

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97 In the process of deprotecti ng our Boc-thiazolidine monomer 20 we had figured that the deprotecting conditions to cleave the acetonide we re harsh, and many attempts to get the fully deprotected compound ended up giving poor yields. Even the step by step approach didnt give a satisfactory result while its first step was clean. That prompted us to change our protecting group for the thiol to the trityl group which can be cleaved with relatively milder conditions. After changing the protecting groups, we were able to deprotect our monomer 39 using TFA with an excess amount of EDT as a scavenger. Interestingly, the Boc group can be selectively in the absence of the EDT scavenger. Efficient coupling between 90 and 118 gave us the fully protected T-T dimer 120 with a good yield while showing the HATU and TEA protocol is good for the coupling of our monomers at least in the solution phase. Next, the production of 121 by coupling between 91 and 119 suggested that our hypothesis of usin g the native chemical ligation method is still worthy of further investigation. With this encouraging result, we decided to move on to solid phase synthesis stage. 4.4.2. Solid phase synthesis using native chemical ligation. After we fixed the coupling conditions from solution-based preliminary studies, we decided to start solid phase experiments with the Fmoc-solid phase strategy because we wanted to quantitatively monitor every step to conf irm the efficiency of the reaction. As discussed earlier, the Fmoc test is the mo st reliable way to quantitatively monitor the solid phase synthesis. With the starting materials ready, we had to choose the optimum resins as well. Through experimentation we learned the PNA oligomer synthesis should start with a resin that has a less loading capacity.

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98Scheme 18: Early trials on solid-phase synthesis. OCys(Fmoc,Trt) HMBA resin, subs = 0.741 mmol/g SPS # 1. OH 102 (T) OCys T SH Sub subs = 0.20, 0.18, 0.23 SPS # 2. HMBA-NovaGel resin Subs = 0.69 mmol/g Fmoc-Cys(Trt)-OH O Cys(Fmoc,Trt) HATU (PEG-PS) SPS # 3. OH HMBA-NovaGel resin Subs = 0.69 mmol/g (PEG-PS) DIPCDI DMAP TEA O Ala(Fmoc) DIPCDI DMAP Fmoc-Ala-OH Subs = 0.13 Subs = 0.09 SPS # 4. OH HMBA-MBHA resin Subs = 1.3 mmol/g PS (1% DVB) Fmoc-Cys(Trt)-OH DIPCDI DMAP O Cys(Fmoc,Trt) Subs = 0.12

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99Scheme 19: The attempt of a solid phase synthesis using sequential native chemical ligation. In Scheme 18, several solid phase syntheses were conducted to become familiar with the techni que. After getting used to the solid phase synthesis procedure, we obtained a good result on solid phase synthesis (SPS) experiment # 5 which is shown in Scheme 18. At that time, we have prepa red the newer monomer 90 which can be easily deprotected with TFA. Hence we wanted to see if we could employ the monomer and our charged alkyl chain simultaneously by taking advantage of the native chemical ligand effe ct of the thiol group. The experiment is simplified in the Scheme 19. In this procedure, The distinctive color of the trityl cation was used as the proof for the coupling of the T monomer. Unfortunately, after the third T-coupling we werent able to observe the distinctive yellow color that would have appeared if the trityl group was present. As a consequence, we decided to simplify our solid phase synthesis, so that we could minimize the complexity of the solid phase synthesis because it is hard to control in the solid phase. Thereafter, the entire scheme including the monomer synthesis had to be changed. As a result, our new solid phase synthesis scheme was simplified and became more like conventional SPPS. However, in contrast to the solid phase synthesis, the scheme for the monomer synthesis became more complex because we needed to introduce the charged alkyl chains on the thiol of the backbone. Thereafter, we developed a novel protocol which reduces the racemization of the cysteine as discussed in Chapter 2. We have prepared the second generation CPNA monomers with both Fmoc and Boc protec ting group. Therefore, we resumed the solid phase synthesis. SPS # 5. Ala(Boc) Boc-Ala-PAM-resin Subs = 0.44 mmol/g i) 90, HATUii) 90 iii) R1Br iv) 90 v) R1Br Ala-T T R1 T R1 ?

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100 Scheme 20: SPS with the S-alkylated monomers. 4.4.3. Solid phase synthesis using S-alkylated monomers. Schem e 20 Scheme 20 shows the ear ly experiments with the new monomers which now have the alkyl group attached already, so the procedures for the solid phase synthesis are simpler. SPS #6 shows our experiments on t he Rink-amide Am resin to see where we can put our monomers consistently. Rink NHFmoc SPS # 6. i) 103 ii) 103 iii) 103 Rink Amide AM resin Subs : 0.71 mmol/g coupling:HATU,TEA Rink T T T (Fmoc) Subs T : 0.18 mmol/g T-T: 0.17 mmol/g T-T-T: 0.05 mmol/g Sieber SPS # 7. i) 103 NovaSyn TG Sieber resin Subs : 0.142 mmol/g coupling:HATU,DIPEA Sieber T (Fmoc) Subs T : 0.14 mmol/g NHFmoc a) b) Sieber NovaSyn TG Sieber resin Subs : 0.142 mmol/g NHFmoc i) 103 coupling:HOBt, DIPCDI Sieber T (Fmoc) Subs T : 0.13 mmol/g

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101Scheme 21: Optimizing the solid phase synthesis for CPNA oligomers. SPS # 8. Sieber NovaSyn TG Sieber resin Subs : 0.142 mmol/g NHFmoc i) 113 ii) 113 iii) 113 coupling:HATU,DIPEA Sieber C C C (Fmoc) Subs C : 0.094 mmol/g C-C: 0.085 mmol/g C-C-C: 0.085 mmol/g SPS # 9 *Kaiser test : all negative Sieber NovaSyn TG Sieber resin Subs : 0.142 mmol/g NHFmoc i) 113 coupling:HATU,DIPEA *Kaiser test : negative a) b) Sieber C (Fmoc) Subs C: 0.075 mmol/g Sieber NHFmoc i) 113 coupling:HATU,DIPEA *Kaiser test : negative Sieber C (Fmoc) Subs C : 0.10 mmol/g d) C) capping Sieber C Sieber C (Fmoc) Subs T : 0.10 mmol/g : means capped i) 113 coupling:HATU,DIPEA *Kaiser test : negative C (Fmoc) Subs C-C : 0.87 mmol/g single C-C: 0.10 mmol/g double x2 Subs C: 0.075 mmol/g The first two attachments gave similar yields but t he yield for the third T attachment dramatically dropped. It is known that with high loaded resin, aggregation of the chain sometimes occur when the chain gets longer. So, we concluded that we need to use a resin with low loading capacity. SPS #7 in Scheme 20 employed a Sieber resin which has very low loading capacity. So we examined two different coupling conditions on this resin. The HATU and DIPEA condition gave slightly better yields on the first coupling of R1 alkylated monomer compared to the conditions employing DIPCDI with HOBt as additives. Neve rtheless, both conditions gave excellent coupling efficiency and we were very excited with the result. Thereafter, we wanted to see coupling efficiency of other monomers. As experiment SPS #9, the first coupling of the alkylated C monomer gave little lower yields than the T monom er, but the second and third coupling gave us

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102consistent results with no fluctuation. Again, this data shown in Scheme 21 was very encouraging. The T -T dimer was also successfully employed on the Sieber resin but this time the yield was a little lower. Scheme 22: Employing the spacer on the resin. PS PEG PAL-NHFmoc Fmoc-PAL-PEG-PS resin Subs : 0.19 mmol/g (Spacer) NHFmoc HO2C HATU,DIPEA X 3 PS PEG PAL-Spacer(Fmoc) Subs: 0.195 single 0.194 double 0.198 triple a) b) 111, HATU, DIPEA PS PEG PAL-Spacer T(Fmoc) X 2 Subs: 0.145 single 0.148 double SPS # 10 So we came up with the idea that if we put a little more space between the linker and the start of the PNA with a linear alkyl chain, the yield coul d be improved and this could possibly reduce the aggregation of the oligomers. So, we purchased a simple alkyl chain which contains six carbons. The experiment SPS #10 shows the result of the reaction. The spacer was employed with a quantitative yield which was expected. The result of the next coupling with T wasnt perfect but still its better than that of SPS #9. To briefly summarize, we now know that low loaded resin, such as the Sieber resin and PAL-PSPEG resin and HATU are the optimal conditions, and employing a spacer also increases the yield at least in the beginning. Therefore, we concluded that we were ready for a CPNA 15-mer synthesis. We had the peptide synthesizer set up and started the automated solid-phase synthesis using the instrument. The s equence of the 15-mer is shown in the Scheme 23. We actually dont know how much of the alkyl chain would give the best properties for our oligomer. We decided to put the alkyl chains on every other unit first. Eventually, we want at least 3 different compositions of the alkyl group with the same sequence so we can compare each other.

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103It took about 2 days from the day we set the experiment to figure out that the synthesis of PNA, especially with our bulky units, is not a trivia l task. It was convenient and time-efficient to utilize a automated peptide synthesizer. After the entire coup ling steps, we just cleaved the product from the resin and analyze it. Unfortunately, the MALDI-TOF spectrum of our crude product did not show any peaks corresponding to our desired product. Scheme 23 : Fmoc -SPS of the CPNA 15-mer on a peptide synthesizer. SPS # 11 : Using Peptide synthesizer. PS PEG PAL-NHFmoc SPACER,TGTGGCGCGCTGGGA B : SMe monomers B : R1 Monomers coupling: HATU, DIPEA ?MALDI-TOF Suggested No product Obtained. What did we get? C-terminus N-terminus After failing to obtain the CPNA 15mer with about 50 % of charged alkyl chain, we didnt have any other option but do the synthesis manually and check the coupling step by step to figure out where and what have caused the problem. A manual synthesis of the 15-mer initiated, However, after the second step, we monitored that the yield dramatically decreased. So we thought that the alkylated G monomer was giving us problem. The G monomer we used so far was the O-benzyl protected one which is actually in a partially protected form. After several control experiments, we figured that the G m onomer was giving us poor coupling consistently. Therefore, the acetic acid derivative of the nucleobase G had to be protected differently. We decided to use the conventional G protecting group which is the Cbz group. As shown in Scheme 27, the procedure for the Cbz protect ed nucleobase 122 was adapted from the precedented paper97 and the procedure for preparation of monomers using 122 was straightforward since the protocols were already well established from our lab. Meanwhile, we have tried the other

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104Scheme 24: manual solid phase synthesis with G unit. PS PEG PAL-Spacer(Fmoc) SPS # 12 : Manual SPS i) 111 ii) 104 Subs : 0.18 mmol/g HATU, DIPEA Subs T-G : 0. 082 mmol/g T-G : 0. 075 mmol/g(double G) a) c) PS PEG PAL-Spacer(Fmoc) i) 111 ii) 111 Subs : 0.18 mmol/g HATU, DIPEA x 2 + T-G(Fmoc) Subs T-T : 0. 143 mmol/g T-T(Fmoc) HATU, DIPEA d) 105 Subs T-T : 0. 06 mmol/g T-T-C(Fmoc) + b) PS PEG PAL-Spacer(Fmoc) i) 111 ii) 104 Subs : 0.18 mmol/g HATU, DIPEA Subs T-G : 0. 032 mmol/g + T-G(Fmoc) Microwave 40 W, 4min supporting material for our SPS such as a long PEG polymer and silca gel based resin as shown in Scheme 26. The silica gel supported resin was confirmed to be a poor supporting system for long sequences. However, we had a quite interesting result on the PEG polymer. Using this PEG polymer is actually completely different than the conventional solid-phase synthesis. It is actually a method that uses the unique solvation property of the certain lengths of PEG polymer. The PEG polymer we used has molecular weight around 5000. The polymer is soluble in MC but not in other organic solvents. So actually the reaction ta kes place in a solvent sy stem that dissolves all the reagents including the PEG polymer, and then by adding the solvents that could precipitate the polymer which now has a unit attached. This type of strategy is ca lled liquid phase syntheses. It is not widely used but it could be useful for a difficult peptide synthesis because the coupling reaction is taking place in a homogeneous system.

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105Scheme 25 : Liquid phase synthesis and silica supported synthesis. OMe -PEG -NH2M.W. : ~ 5000 SPS # 13 HATU, DIPEA Fmoc-Rink-Linker H NRink-Fmoc PEG MeO 111 a) Silca-NH2Subs : 1. 7 mmol/g HATU, DIPEA Fmoc-Rink-Linker Solvent c) HATU, DIPEA Solvent H NRink-Fmoc Silica Subs : 1. 7 mmol/g HATU, DIPEA Fmoc-Rink-Linker H NRink-Fmoc Silica b) Subs : 0.33 mmol/g Subs : 0.042 HBTU, DIEA NMP Fmoc-Ala-OH H NRink Silica Subs : 0.11 mmol/g Ala(Fmoc) H NRink PEG MeO T(Fmoc) Subs: 0.13 mmol/g Silca-NH2 Moreover, since the PEG polymer is linear, there shouldnt be an aggregation problem which can be observed in a SPPS. The coupling efficiency can be analyzed by Fmoc Test when the unit has the Fmoc protecting group. We figured that the effi ciency of the coupling in liquid phase synthesis could also well be observed by the MALDI-TOF spectroscopy.

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106 Figure 35: Comparison of the two mass spectr a(MALDI-TOF) from PEG before (top) and after (bottom)the coupling of the Rink linker.

PAGE 122

107Figure 35 shows the distinctive spectra obtained from the PEG before the coupling and after the coupling of the Rink linker. Unfortunately, we werent able to afford the amount of the monomers to do this experiment repetitively. Scheme 26: Optimization of the SPS protocol. PS PEG PAL-Spacer(Fmoc) i) 111 ii) 108 Subs : 0.18 mmol/g HATU, DIPEA Subs T: 0.13 mmol/g T-G : 0. 067mmol/g a) + T-G(Fmoc) Subs spacer-T : 0. 145 mmol/g Spacer-T(Fmoc) Subs T-T : 0. 126 mmol/g T-T(Fmoc) b) PS PEG Sieber(Fmoc) i) 111 x2 Subs : 0.23 mmol/gHATU, DIPEA Subs T: 0.11 mmol/g T: 0.11 mmol/g(double) T-(Fmoc) (R2) *different batch from previous experiment c) PS PEG Sieber(Fmoc) i) Spacer Subs : 0.23 mmol/gHATU, DIPEA Subs spacer : 0.23 mmol/g Spacer-(Fmoc) i) 111 HATU, DIPEA i) 111 HATU, DIPEA d) PS PEG Sieber-Spacer(Fmoc) Subs : 0.23 mmol/g Subs spacer-T : 0. 065 mmol/g Spacer-T-G(Fmoc) ii) 108 HATU, DIPEA + When the Cbz protected G monomers were ready, our target sequence was changed. The target sequence is shown in Scheme 28. This time we wanted to make the entire sequence with only the S-methyl monomers since we thought that it would be easier to get the desired 18-mer with the sterically less hindered m onomers. So the SPS was again tried using the peptide synthesizer. To be able to analyze the coupling at the final stage, we skipped the final Fmoc deprotection step. Hence, we were able to do the Fmoc test on the resin after all the couplings.

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108Scheme 27: Cbz protected G monomers. NH N N N O NHCbz O HO 122 N N N N NH2 O BnO Cl 17 FmocHN RS N CO2t-Bu O G (Cbz) FmocHN RS N H CO2t-Bu FmocHN RS N CO2H O G (Cbz) R = Me R1 (from Scheme 9) R2 (from Scheme 9) R = Me 123 R1 124 R2 125 R = Me 126 R1 127 R2 128 BocHN RS N CO2Me O G (Cbz) BocHN RS N H CO2Me BocHN RS N CO2H O G (Cbz) R = Me R1R = Me 129 R1 130 R = Me 131 R1 132 Scheme 28: CPNA 18-mer synthesis (G: Cbz protected). PS PEG Sieber(Fmoc) Subs : 0.20 mmol/g SPS # 13 : Automated synthesis Spacer, GGCTTGCGTGGGGCTCCG PS PEG Sieber-Spacer-GGCTTGCGTGGGGCTCCG(Fmoc) Subs : 0.046 mmol/g However, after the deprotection of Fmoc and cleavage no desired product was detected from the MALDI-TOF All SMe Monomers G: Cbz protected The substitution level turned out to be 0.046 mmol/g after 19 coupling steps. However, the desired peak from the MALDI spectrum could not be observed. The purities of our monomers were believed to be the biggest factor for the failure since we used some of the monomers without purification after the tertiary butyl ester deprotection using TFA or HCl gas. This

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109deprotection step is quite clean, however if it was not performed carefully or if the monomers have been stored for a long time we can actually see the monomers decomposing. The sensitive Fmoc group could cause the problem. We have purified some of the monomers with the recrystallization, but it should be noted that, to be able to make this type of long oligomer every single monomers should be perfectly clean. Even though several attempts to make the long oligomers have failed it is likely that we will get the oligomer soon because we now have collected sufficient amount of information to improve the Fm oc solid-phase synthesis of CPNA oligomers. The convergent solid phase strategy also can be considered for this project. 4.5. Conclusion. The solid p hase synthesis is one of the most us eful methodologies in history of chemistry or science. Peptide chemistry, polyamide chemistr y and normal organic methodologies are widely adapting this strategy as we extensively empl oyed the solid phase synthesis methodology for our CPNA project. We have manipulated the Fmoc soli d-phase synthesis strategy step by step to make CPNA oligomer which could be a water-soluble and cell permeable antisense therapeutic agent. Since we have figured out many important fa ctors to improve the solid-phase protocol for CPNA, its promising that we could get the CPNA oligomers in a short period of time. We are also investigating the Boc based solid phase strategies and we are getting exciting results from this protocol. The procedures for the solid phase synthesis includi ng the procedures for the analytical tests are discussed in the chapter five. 4.6. References. 90 Merrifield, R.B. Solid Phase Peptide Sy nthesis. I. The Synthesis of a Tetrapeptide,J. Am. Chem. Soc. 1963 85, 2149.

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110 91 Merrifield, R.B. Life During a Golden Age of Peptide Chemistry: The Concept and Development of Solid-Phase Peptide Synthesis.; Seemen, J.I., Ed.; American Chemical Society: Washington, DC, 1993. 92 Barany, G.; Merrifield, R.B. The Peptides: Analysis, Synthesis, Biology; Gross, E., meienhofer, J, Eds.; Academic Press: New York, NY, 1979; Vol. 2, 93 Novabiochem Catalog 2006/2007 EMD Biosciences, Inc: San Diego, Ca. 2006. 94 Kates, S. A.; Albericio, F.; Yokum, S.; Ba rany, G.; Forns, P.; Fields, G.B. et al. Solid-Phase Synthesis a Practical Guide; Kates, S.A., Albericio, F., Eds.; Ma rcel Dekker, Inc: New York.Basel, NY, 2000. 95 Meldal, M. Methods Enzymol 1997 289, 83. 96 Nicolaou, K.C.; Nevalainen, M.; Safina, B.S.; Zak, M.; Bulat, S. Angew. Chem. Int. ed. 2002 41, 1941. 97 Kofoed, T.; Hansen, H.F.; Qrum, H.; Koch, T. J. Peptide Science 2001 7, 402.

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111 5. Chapter Five: Procedures and Experimental Data for Chapter Four. 5.1. Introduction. All the experiments for sol id-phase synthesis were carried out under argon atmosphere using oven dried glassware. Most of chemicals were purchased from Aldrich Chemical Co., Acros Organics., EMD Biosciences Inc. When the solvents must be anhydrous, CH2Cl2, THF and diethyl ether were distilled over calcium hydride or proper metals. 1H (250 MHz) and 13C (62.5 MHz) nuclear magnetic resonance (NMR) data were recorded at room temperature in CDCl3 using a Bruker DPX 250 spectrometer unl ess otherwise noted. High Resolution Mass Spectrometry (HRMS) spectra were recorded usi ng an Agilent 1100 Series in the ESI-TOF mode. Selected NMR and Mass spectra are available in the Appendix section. For compounds with higher molecular weights, the mass spectra were obtained using a Bruker Autoflex series in the MALDI-TOF mode. 5.2. General procedures and data for the solution-phase deprotection of the Monomers. 5.2.1. General procedure for deprotection of Boc p rotected monomers.

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1125.2.1.1. C-termini deprotecti on of Boc protected monomers. 1.5 equivalents of NaOH or LiOH solution was added into a solution of protected monomer (1 equivalent) in 5 mL mixture of water and THF (1:1) at 0 C. The reaction mixture was carefully monitored by TLC and when the starting material was all consumed, 5 mL of EtOAc was added into the mixture to extract the unreacted starting material. The water layer was then treated with dilute HCl solution for neutralization. Adequat e amounts of EtOAc was added to extract the desired compound from the water layer. If needed, the extraction step was repeated a couple of times. The organic layer was washed with brine, dried over sodium sulfate, and concentrated to give the deprotected monomer with excellent yields. 5.2.1.2. N-termini deprotecti on of Boc protected monomers. S N N Boc CO2Me O (T) 20 S H.HCl N N CO2Me O (T) HCl, H2O/MeOH(1/ 3) rt, 2day 116 BocHN S N CO2H Ph Ph Ph O B 90 (T), 91 (G), 92 (C), 93 (A) BocHN RS N CO2H O B 94 R= Me and T 95 R= Me and G 96 R= Me and C 97 R= Me and A 98 R= R1 and T 99 R= R1 and G 100 R= R1 and C 101 R= R1 and A Boc Protected Monomers: 20, 21, 39, 40, 41, 42, 76, 77, 78, 79, 80, 81, 82, 83 88 ~ 101 a) NaOH or LiOH, THF/H2O, rt. S N N Boc CO2H O B 88 (T), 89 (G)

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113a) 20 (15 mg) was dissolved in 1.5 mL of methanolic HCl solution (conc. HCl/MeOH=3/10) and the solution was stirred for 2 days at room temper ature. The solution was concentrated and dried under vacuum to give 13 mg of 116 (crude product). BocHN S N CO2Me Ph Ph Ph O N HN O O 39 ClH.H2N S N CO2Me Ph Ph Ph O N HN O O 90% 118 HCl(g), Dioxane b) HCl(g) was bubbled into the solution of 39 (100 mg) in anhydrous dioxane (5 mL) at 0 C for 10 min with stirring, then the solution was allowed to slowly warm to room temperature. After 1 hr of stirring at room temperature, the solvent was removed in vacuo to give 90 mg of crude 118 as the HCl salt. The crude product was subjected to t he next reaction without further purification. BocHN S N CO2Me Ph Ph Ph O N HN O O 39 HS N CO2Me O N HN O O F3CCO2H.H2N TFA, H2O, Triethylsilane 119 c) 39 (100 mg) was stirred in 2 mL of TFA/H2O (9/1) solution for 30 min and then triethylsilane (1 eq) was slowly added and stirred until the yellowish solution became clear. TFA was removed under reduced pressure and the product was diluted in 5 mL of water. The solution was washed with hexane and concentrated to give 85 mg of crude 119.

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1145.2.2. General procedure for deprot ection of Fmoc pr otected monomers. 5.2.2.1. C-termini deprotection of Fmoc protected monomers (for t-Bu ester). FmocHN MeS N CO2H O B 111 (T), 112 (G), 113 (C), 114 (A) FmocHN S N CO2H N H O N H CbzHN CbzN O B 107 (T), 108 (G), 109 (C), 110 (A) FmocHN S N CO2H N H O CbzHN O B 103 (T), 104 (G), 105 (C), 106 (A) N S NHFmoc HO2C O H N O CbzHN B 115 (T) Fmoc protected Monomers: 25, 58, 59, 60, 61, 63, 64, 65, 66, 68, 69, 70, 71, 87 102 ~ 115 (R1) Fmoc monomers (R2) Fmoc monomers (SMe) Fmoc monomers Inverted-(R1) Fmoc T-monomer a) TFA/DCM, 1hr, quantatative b) HCl(g), 1,4-Dioxane, 0 C to rt. 3hr. quantatative a or b General procedure for deprotecting t-Bu esters: a) To a solution of a t-butyl protected monomer (0.018 mmol) in DCM (0.5 mL) was added TFA (1 mL). After stirring at room temperature for 2 h, the reaction solution was concentrated and diluted with EtOAc, washed with brine, dried over sodi um sulfate, and concentrated in vacuo to give a deprotected monomer in carboxylic acid form (quantitative yield). b) To a stirring solution of a t-butyl protected monomer (0.018 mmol) in 1,4-dioxane at 0 oC, HCl gas was bubbled in for 30 min. The solution was warmed to room temperature and stirred for 2 hr. The suspension was then concentrated over high vacuum to give the deprotected monomer.

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115 5.2.2.2. C-termini deprotection of Fmoc protected monomers (for methyl ester) N S Fmoc N CO2Me O B N S Fmoc N CO2H O B 102 (T) 24 (T) Fmoc methyl ester monomer, 24 a 102 a) Me3SnOH, DCE, 24 hr. 85% 24 (22 mg) was dissolved in 2 mL of DCE and 5 eq of Me3SnOH was added and the mixture was heated for a day. The mixture was concentrated and taken up in EtOAc. The organic layer was washed with brine and dried over sodium sulfate. Removal of the solvent in vacuo afforded 15 mg of the crude carboxylic acid, 102. 5.2.3. Procedures for solution based preparation of CPNA Dimers. 5.2.3.1. Procedures for Boc protected CPNA T-T dimer. 90(T) + 118(T) HATU, TEA DMF BocHN S N Ph Ph Ph O T H N S N CO2Me Ph Ph Ph O T O 120 TEA (50 mg) was added into the solution of 75 mg of 90, 90 mg of crude 118 and 60 mg of HATU in DMF (1 mL) with stirring. After 2 hr, the solvent was removed under reduced pressure. The residue was diluted in EtOAc and washed with water and brine. Organic layer was dried over sodium sulfate, concentrated and subjected to a flash column chromatography to give 110 mg of 120 in a 75 % yield (Rf = 0.3, MeOH/DCM = 1/9)

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1165.2.3.2. Procedures for Boc protected CPNA T-G dimer (Native chemical ligation). 91(G) + 119(T) HATU, TEA DMF BocHN S N Ph Ph Ph O (BnO)G H N HS N CO2Me O T O 121 The same procedure as that of 120 was applied to obtain 121 5.3. General procedures for the solid-phase synthesis. Generally, our solid phase synthesis wa s performed by the following steps: Swelling, washing, deprotecting the N-term inus, washing, coupling (double coupling as necessary), washing, analytical test as necessary (Kaiser test and/or Fmoc test), and repeating these step-wise cycles as many times as it is needed. Before the cleavage, deprotecting the Nterminus was performed. Finally cleavage from t he resin using appropriate conditions depends on the type of the resin. After the work-up, the pr oduct was subjected to analysis (usually mass spectroscopy in MALDI-TOF mode). Details of t he each step are described in this section. 5.3.1. Selection of Solvents. For our Fm oc protocol, we mostly used N-methylpyrrolidine (NMP). All of the resins we used showed great swelling properties in NMP. However, For the Boc strategy, we have used a mixture of DCM and NMP. For the washing step, DCM and NMP were alternatively used repeatedly.

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1175.3.2. Deprotecting N-terminal protecting group 5.3.2.1. Deprotecting N-terminal Fmoc group After making sure the resin was swelled for a sufficient time, 20 % piperidine solution in DMF was added for 10 min with bubbling under argon gas. And this was repeated one more time. After the deprotection, the washing step was done immediately. 5.3.2.2. Deprotecting N-terminal Boc group After swelling resin, 50 % TFA in DCM was added to the resin for 30 min. 5.3.3. Coupling of a monomer or spacer using HATU activation. Generally, 3.8 equivalent s of HATU and 8 equiva lents of the DIPEA were premixed in the minimum amount of the NMP, and the mixture was immediately added to the solution of monomer (4 equivalents) in NMP. The mixture was then agitated for a minute, then immediately added onto the resin. The reaction vessel was bubbled with argon for about 30 minutes at room temperature, and washed thoroughly after the co upling step. Double coupling was executed as necessary by repeating the coupling procedure without deprotecting the N-terminal protecting group. 5.3.4. Cleavage from the resin. 5.3.4.1. Clea vage procedure for the Sieber resin. Before the cleavage, removal of the N-terminal Fmoc group was performed. The cleavage procedure which adapted from Novabiochem catalog as described below: 1. Swell the resin (1 g) in DCM in a sealable sintered glass funnel and remove excess DCM. 2. Add 1 % TFA in dry DCM (10 mL). Seal funnel and shake for 2 min. Filter solution by applying pressure into a flask containing 10 % pyridine in methanol (2 mL).

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118 3. Repeat step 2 up to 10 times, wash the residual protected peptide from the resin with 3 x 30 mL DCM, 3 x 30 mL, MeOH, 2 x 30 mL DCM, 3 x 30 mL MeOH, and check filtrates by TLC or HPLC. 4. Combine filtrates which contain product and evaporate under reduced pressure to 5 % of the volume. Add water (40 mL) to the residue and cool mixture with ice to aid precipitation of the product. 5. Isolate product by filtration through a sintered glass funnel. Wash product three times with fresh water. Dry sample in a desiccator under vacuum over KOH. 5.3.4.2. Cleavage procedure for the PAM resin. 1. Place the resin (250 mg) in a round bottom flask with a stirring bar and cool in an ice bath to between 5 C and 0 C. 2. Add 250 L of m-cresol, 50 L EDT, 750 L DMS and 1.25 mL of TFA, add 250 L of TFMSA drop-wise to the reaction mixture with stirring to dissipate any heat produced. 3. Allow the mixture to react for 3 h, keeping the temperature between 0 C and 5 C. 4. Transfer the contents of the flask to a medium sintered glass funnel and wash the resin with several volumes Et2O and suction dry. 5. Dry the resin under vacuum for a minimum of 4 h over KOH. 6. Place the dried resin from above in a round bottom flask with a stirring bar and add 250 L of thioanisol and 75 L of EDT and stir for 5-10 min. 7. Cool to 5 C to 0 C using an ice bath and add 2.5 mL of TFA. Mix 5-10 min followed by addition of 250 L of TFMSA dropwise with mixing to dissipate heat. 8. Remove the flask from the ice bath and allow reaction to proceed at rt for 30 min with PAM resins for complete cleavage. 9. Remove the resin by filtration under reduced pressure. Wash the resin twice with TFA. Combine filtrates and add (dropwise) and 8-10 fold volume of cold ether. 10. Isolate the product as described in the section below.

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1195.3.5. Post cleavage work-up. The product work-up after the cleav age was executed by either precipitation or centrifugation. a) Precipitation: Filter the precipitated product through a filter paper in a Hirsch funnel under a light vacuum. Wash the precipitate further with cold ether, dissolve the peptide in a suitable aqueous buffer and lyophilize. b) Add a small volume of diethyl ether to the residue and triturate thoroughly until a free suspension is obtained. Transfer the suspension to a clean centrifuge tube, seal and centrifuge. Carefully decant the ether from t he tube. Repeat the ether wash as necessary. Dissolve the residual solid in a suitable aqueous buffer and lyophilize. 5.4. Analytical procedures for solid phase synthesis. 5.5.1. The procedure for Kaiser Test. The procedu re was adapted from Novabiochem catalog 2006/2007 version. a) Preparing the solution Dissolve 5 g of ninhydrin in 100 mL ethanol. Dissolve 80 g of phenol in 20 mL of ethanol. Add 2 mL of a 0.001 M aqueous solution of potassium cyanide to 98 mL pyridine. b) Testing sample a few resin beads and wash several times with ethanol. Transfer to a small glass tube and add 1 drop of each solution and leave for 5 min. Wash the beads with DMF to remove the red so lution. A positive test is indicated by red beads.

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1205.5.2. The procedure for Fmoc Test. The procedure was adapted from Novabiochem catalog 2006/2007 version. a) Preparing sample Take 3 x 10 mm matched silica UV cells. Weigh dry Fmoc amino acid-resin (approx. 5 mol with respect to Fmoc) into a 10 mL graduated flask. Add 2 mL of 2 % DBU in DMF. Agit ate gently for 30 min. Dilute solution to 10 mL with acetonitrile. Take 2 mL of this solution and dilute to 25 mL in a graduated flask. Prepare a reference solution as in step above, but without addition of the resin. Fill two cuvettes with 3 mL of test solution and one cuvette with 3 mL of reference solution. b) Testing the sample Place the cell in a spectrophotometer and record optical density at 304 nm. Obtain an estimate of first residue attachment from equation below. Substitution Level: mmole/g = (AbssampleAbsref) X 16.4 / mg of resin

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6. Chapter Six: Regioselective Mono Acylati on of the Electroni cally Less Reactive Nitrogen of Aryl Hydrazines via Temporary Protection with Carbon Disulfide. 6.1. Introduction. Hydrazines are an important class of compounds that contain the reactive nitrogennitrogen bond. Hydrazines and analogues such as hydrazides and hydrazones, are well known for their highly active biological profiles.98 For example, a substantial number of hydrazines and their analogues are known to have antineoplastic activity.98 Hydrazines are also frequently used in synthetic chemistry to make pyrazoles,99 oxadiazoles,100 triazoles101 and indoles.102 However, alkylating and acylating hydrazines can be cumber some especially when one needs to selectively acylate the electronically less reactive nitrogen of the hydrazine. The early part of this chapter discusses the general properties of hydrazines a nd several examples that utilize the hydrazines or hydrazine derivatives as synt hetic intermediates. The latter part of this chapter describes the developing story of a methodology which allows mono-acylation of arylhydrazines with good regioselectivities. Remarkably, our protocol facilitates mono-acylation particularly on the less nucleophilic nitrogen of aryl hydrazines. R H N NH2 H N NH2 R O R3H N N R2 R1 Hydrazines HydrazidesHydrazones Figure 36: Derivatives of Hydrazines

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1226.2. Diverse biological properties of hydrazines. Hydrazines are well-known for their diverse biological properties but especially for their toxicity. In some cases, it is believed that they are specif ically toxic to the liver or the eye. Therefore, some hydrazines were designated as live r or eye toxins by early researchers.103 According to the literature,98 most hydrazines are carcinogenic in experimental animals. Hydrazines are often carcinogenic with about 80 of 90 hydrazines having carcinogenic activity. Nonetheless, the point of discussing this data is to stress on the biological activity of hydrazines. In fact, it is well-known in cancer research that cancer induction and cancer inhibition often times occur simultaneously. The best examples to support this idea are the early chemotherapeutic agents, such as mustard gas and other alkylating compounds. Furthermore, some drugs that are presently used, such as bischloroethyl nitrosourea, cyclophosphamide, chlorambucil, etc. also are known to induce and inhibit cancer simultaneously.98 The original idea to use hydrazines as cancer chemotherapeutic agents was developed by Simon in 1952 in Germany where he used hydrazine and isonicotinic acid hydrazide, and a series of phenyl hydraz ine and thiosemicarbazide derivatives for the treatment of mouse ascites tumor.104 Since then, more than 400 hydrazine derivatives have been studied for antitumor activities in both humans and animals. Among those 400 compounds, a substantial number of compounds have been found to show anticancer activities for a variety of tumors.98 Interestingly, two hydrazine-containing na tural products both cultivated from edible mushrooms, Agaricus bisporus and Leninus edodes, have shown antineoplastic activities in animals. However, these compounds were not furt her investigated to see if they have cancerinhibiting properties due to their hydrazine content.98 The most important antineoplastic drug of the hydrazine class used in humans is N-isopropyl-(2-methylhydrazino)-p-toluamide HCl (procarbazine, Natulan). This drug was used in combination with other compounds and with irradiation for a variety of neoplasms including Hodgkins disease.105 A review paper which summarized those studies for over 400 hydrazine based compounds98 suggests that it is conceivable to look for a cure for cancer from the hydrazine class of chemicals. Later in this chapter, new aryl hydrazine derivatives are sy nthesized by a novel protocol that has been developed in our laboratory.

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1236.3. Practice of the hydrazines and their derivatives in the synthesis. Scheme 29: Representative reactions of hydrazines. R1 H R2 O S O O NH H2N CH3 H N N H Ts BuLi H2O R3X R1 R2 H R1 R2 R3 a) Shapiro reaction R1 H R2 N N H Ts NaOMe R1 R2 tosylhydrazones R1 R2 c) Fischer indole synthesis N H NH2 R2 O R1 + N H R1 R2 b) Bamford-Stevens reaction e) Four-component coupling reaction to make pyrazoles and Isoxazoles + + R H N NH2 CO+ OMe I d) Modified Fischer indole synthesis(Buchwald) Br N H2N R1 R2 + Pd(0) BINAP, KOt-Bu N H N R1 R2 N H R1 R2 PdCl2(PPh3)2rt, 1 atm N NH R OMe R NHNH2 O R1NCS THF, -22 oC R N H N H N R1 S O i) TsCl ii) pyridine THF, 65 oC N O N R NH R1 f) a protocol for 2 amino-1,3,4-oxadiazoles N2

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124Hydrazines readily react with ketones and aldehydes to give hydrazones and this allows one to detect or even quantitatively analyze the concentra tion of the ketones and aldehydes. Hydrazine and its derivatives including hydrazine and hydrazone are often used for the well-known organic procedures; such as the Shapiro reaction,106 the Bamford-Stevens reaction,107 the Wolff-Kishner reduction,108 the Fischer-indole synthesis102 and the Gabriel synthesis.109 Hydrazines are also frequently used to make heterocycli c systems; such as pyrazoles,99 oxadiazoles,100 triazoles101 and indoles.110 The hydrazine class of compounds very us eful intermediates for the synthesis of natural products.111 Some of the representative reactions are illustrated in the Scheme 29 to highlight the usefulness of hydrazines. Scheme 30: Hydrazines as synthetic intermediates fo r the synthesis of indomethacin and its derivatives. R1 H N NH2 CH3CHO toluene, 0 oC R H N N H Me R2 COCl pyridine, 0 oC R1 N N H Me O R2 HCl (g) toluene/ methanol 0 oC, 1hr R1 N NH2HCl O R2 N CO2H Me O R2 R1 O CO2H AcOH, 70 oC-80 oC Indomethacin and its derivatives Scheme 30 is a summarized procedure that enables the synthesi s of indomethacin and its derivatives. The synthesis uses the Fisc her indole synthesis. The synthesis involves protection-deprotection schemes of the hydrazine that it relevant to the new protocol that we have developed in our laboratory. Nevertheless, as br iefly shown above, hydrazines are frequently used to make other interesting classes of compounds.

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1256.4. Regioselective mono acylation of the electronically less reactive nitrogen of aryl hydrazines via temporary protection with carbon disulfide. As we discussed early in the chapter, hydraz ines are an important class of compounds that are often used as synthetic intermediates. Howe ver, alkylating and acylating hydrazines is cumbersome especially when one needs to selectively acylate the electronically less reactive nitrogen of the hydrazine. For that reason, in general, alpha-acylated aryl hydrazines are much less readily available than the beta-acylated aryl hydrazines. In this chapter, we disclose a simple one-pot protocol which allows mono-acylation of arylhydrazines with good regioselectivities. Remarkably, our protocol facilitates mono-acylat ion particularly on the less nucleophilic nitrogen of the aryl hydrazines. 6.4.1. Developing story of our new protocol. In 1962, Tarbell and Scharrer reported their study on the decomposition of mixed carboxylic dithiocarbamate anhydrides.112 They reported that this decomposition gives an amide and carbon disulfide as result of the decomposition and suggested a mechanism which involves a strained 4-membered ring transition state as shown in Figure 37. It requires additional energy su ch as heating or photoactivation to promote such a rearrangement. To the best of our knowledge, there have not been any reports that make use of their findings as a synthetic method. While we were revisiting their chemistry for one of our CPNA projects which is discussed in earlier chapters, we developed a hypothesis that the mixed carboxylic dithiocarbamate anhydrides of hydrazines would form a 5-member ed transition state for an intramolecular acyl transfer. The rearrangement would give mono-acyla ted products after the loss of carbon disulfide as shown in Figure 37.

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126N S S O R N S O R S N O R H N N H S S O R Ph NH H N S O R S N NH2 R O 4-membered ring transition state 5-membered ring transition state a) CS2CS2b) Tarbell et al. (1962). McLaughlin & Yi (2008) Figure 37: Mechanism of the transformations. 6.4.2. Optimization of the protocol. To test our hypothesis, our first attempt was carried out with phenyl hydrazine because it is simple and also it has distinctly different reactiviti es on the two nitrogens. It is obvious that without the extra base only half of the hydrazine would react with carbon disulfide to form dithiocarbamate and the other half would be the counter ion. Therefore, at least one equivalent of amine base is necessary to fully form the dithiocarbamate. As shown in Table 5, the first attempt to make the corresponding dithiocarbamate with one equivalent of carbon disulfide in the presence of the DIEA at room temperature follo wed by slow addition of the benzoyl chloride into the mixture at 0 oC gave the desired product but with poor yield ( Table 5, Entry 1). In fact, the major p roduct of the reaction was the di-acylated compound and more beta-acylated compound than alpha-acylated compound was formed.

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127Table 5: Optimization of the protocol. H N NH2 Base, CS2THF Ph Cl O N NH2 O Ph H N N H Ph O N N H O Ph Ph O A B C TaTb Entry Ta Tb Ratio of (A : B : C) 1 rt -0 oC Trace : 2 : 3 2 rt -78 oC 4 : 3 : 3 3 -78 oC -78 oC 10 : 1 : Trace Table 6: Solvent effects on this reaction. H N NH2 CS2, DIPEA Solvent CH3C(O)Cl N NH2 O Entry Solvents Yields (%) 1 THF 92 2 DCM 71 3 Ethyl Ether 40 4 Ethyl Acetate 60 Our next attempt was to add the benzoyl chloride at -78 oC and that gave us more of the desired alpha-acylated compound but still with significant amounts of the beta-acylated product and di-acylated byproduct (Table 5, Entry 2). At this point, we concluded that dithiocarbamate formation was certainly not as selective as we expected at ambient temperature. Thereafter, we decided to add the carbon disulfide at -78 oC and to slowly warm up the mixture to room

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128temperature to selectively form the dithiocarbamate on the more reactive nitrogen. The reaction mixture was then cooled again to -78 oC before adding benzoyl chloride to selectively form the intermediate which eventually transfers the acyl gr oup to the adjacent nitrogen by intramolecular acyl transfer while the reaction mixture was slowly allowed to warm to room temperature (Table 5, Entry 3). As we envisioned, the latter condition gave the desired compound with great selectivity. Careful observation via thin layer chromatography indicated that the intermediates, the carboxylic dithiocarbamate anhydrides, are unstable at temperatures above -70 oC. We then investigated solvent effects as summarized in Table 6. Anhydrous THF was the best solvent for our methodology while DCM and ethyl acetate also gave good results. 6.4.3. Scope of the protocol. Table 7: Scope of the phenyl hydrazine substrates. H N NH2 CS2, DIEA CH3C(O)Cl THF N NH2 R O R Entry Substrates Products Yields (%) 1 H N NH2 Ac N NH2 133 92 2a H N NH2 N NH2 O Ph 134 87 3 b H N NH2 Cl Cl Ac N NH2 Cl 135 80 4b H N NH2 Cl HCl Ac N NH2 Cl 136 72

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1295 b H N NH2 Br HCl Ac N NH2 Br 137 55 6 b H N NH2 Br HCl Ac N NH2 Br 138 40 7 b H N NH2 NO2 HCl Ac N NH2 NO2 139 75 8 b H N NH2 F F Cl Ac N NH2 F F 140 89 9 H N NH2 O2N NO2 Ac N NH2 O2N NO2 141 85 10 b H N NH2 MeO HCl Ac N NH2 MeO 142 9(85 c) aBenzoyl chloride was used instead of acetyl chloride. b1 more equivalent of DIPEA was used. cyield of the beta-acylated compound. With the optimized conditions, we have examined the scope of the reaction with various phenyl hydrazines and the results are summarized in the Table 7. Reactions with free hydrazines gene rally gave better yields than the substrates with HCl salt forms (Table 7, Entries 1, 2 and 9). Not su rprisingly, functional groups with electron withdrawing effect on the aryl groups such as nitro, fluoro, chloro, and bromo on the phenyl ring gave alpha acylated product as major product with good to excellent yields ( Table 7, Entries 3, 4, 5, 6, 7, 8 and 9) while 4-methoxy phenyl hydra zine ( Table 7, Entry 10) showed inverted selectivity most likely due to the electron-donating effect of the methoxy group. Indeed, we carri ed out a control experiment for the 4-methoxy substrate, and the major product of the reaction without carbon disulfide was the alpha acylated

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130product. These results support our assertion that this methodology provides selective acylation of the electronically less reactive nitrogen of aryl hydrazines. R1 H N NH2 CH3CHO toluene, 0 oC R H N N H Me R2 COCl pyridine, 0 oC R1 N N H Me O R2 HCl (g) toluene/ methanol 0 oC, 1hr R1 N NH2HCl O R2 A. R. Maguire et al. (2001) 15~32 % in 3 steps a) b) R1 H N NH2 CS2, DIPEA Cl O R2 THF R1 N NH2 O R2 McLaughlin & Yi (2008) One step, up to 92% N CO2H Me O R2 R1 O CO2H AcOH, 70 oC~ 80 oC Indomethacin and its derivatives Figure 38: Comparison of the two protocols. Figure 38 briefly illustrates an effort to synthesize Indomethacin and its analogues by utilizing aryl hydra zines.111 In the Maguire procedure, a three-step sequence was needed to obtain the alpha acylated hydrazines. These first three steps had moderate yields and the best yield for three steps overall was just 32 %. In c ontrast, our one-pot strategy gives maximum yields of 92 %. With our protocol it would just require two steps to synthesize this class of indomethacin analogues. Therefore, our one-pot protocol merits would avoid those cumbersome procedures and could provide the mono-acylated products with improved yields.

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1316.4.4. Experimental procedure of the protocol. 6.4.4.1. Detailed the procedure for the regioselective protocol. The arylhydrazines (2 mmol) were dissolved in anhydrous solvent with 2.1 mmols of DIEA (an extra equivalent of base was added when the hydrazine was in the HCl salt form) and the solution was cooled to -78 oC before adding 2 mmols of carbon disulfide. After 5 min, the solution was slowly warmed to room temperature over 30 min. After stirring an additional 30 min at room temperature, the solution was cooled to -78 oC before slow addition of the acylating reagent (2 mmols). After briefly stirring at -78 oC, the reaction was slowly allowed to warm to room temperature and left at RT for 2 hours. The solution was concentrated under vacuum and the resulting mixture was subjected to a flash column chromatography to give the product. 6.4.4.2. The analytical instruments used for the protocol. The proton nuclear magnetic resonance (1H NMR) spectra were obtained from a Bruker DPX250 (CDCl3 as main solvent, sometimes CD3OD were added to enhance the solubility of the product) and processed using ACD 1H NMR Manager (ACDLabs Software). High Resolution Mass Spectrometry (HRMS) spectra were recorded using Agilent 1100 Series the ESI-TOF mode. The NMR and HRMS spectra are attached in the appendix. 6.5. Conclusion. In conclusion, we have developed a novel pr otocol that facilitates mono-acylation of arylhydrazines with reversed selectivity. The cheap and readily available carbon disulfide used herein takes the important role of temporary protecting group and promotes acylation of the electronically less reactive nitrogen of aryl hydr azines. Although this mechanism is quite simple, we believe this protocol could be very useful fo r other hydrazines. In addition, it is conceivable

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132that the new compounds that are prepared by our protocol could possess interesting biological activity like other hydrazine compounds. 6.6. References. 98 Toth, B. In Vivo 1996 10, 65. 99 Ahmed, M.S.M.; Kobayashi, K.; Mori, A. Org. Lett. 2005 7, 4487. 100 Dolman, S.J.; Gosselin, F.; O'shea, P.D.; Davies, I.W. J. Org. Chem. 2006, 71, 9548. 101 Potts, K. T. Chem. Rev. 1960, 61, 87. 102 Fischer, E.; Jourdan, F. Ber 1883 16, 2241. 103 Curtius, T. Ber deutsch chem 1887 20, 1632. 104 Simon, vk. Z. Naturforch. 1952 76, 532. 105 Toth, B. Intern. J. Oncol. 1994 4, 231. 106 Shapiro, R.H. React. 1976 23, 405. 107 Bamford, W.R.; Stevens, T.S. J.Chem. Soc. 1952 4735. 108 (a) Kishner, N. J. Russ. Chem. Soc. 1911 43, 582. (b) Wolff, L. Justus Liebigs Annalen der Chemie 1912 394, 86. 109 Gabriel, S. Ber. 1887 20, 2224. 110 Wagaw, S.; Yang, B.H.; Buchwald, S. J. Am. Chem. Soc. 1998 120, 6621. 111 Maquire, A.R.; Plunkett, S.J.; Papot, S.; Clynes, M.; OConnor, R.; Touhey, S. Bioorg. Med. Chem. 2001 9, 745. 112 Tarbell, D. S.; Scharrer, R. P. F. J. Org. Chem. 1962 27, 1972.

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Appendix (Continued) 212 29 Jan 2008 Acquisition Time (sec) 6.3308 Comment spot above-1 PROTON Date 17 Jul 2007 01:33:52 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-6-71-2\W-6-71-2_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 25.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 3.273.05 1.08 1.00 No. (ppm) Value Absolute Value 1 [6.20 .. 6.50] 1.000 6.07396e+8 2 [6.80 .. 7.05] 3.053 1.85459e+9 3 [7.10 .. 7.37] 2.928 1.77868e+9 4 [7.37 .. 7.71] 3.271 1.98664e+9 5 [7.74 .. 7.89] 2.063 1.25276e+9 6 [7.92 .. 8.15] 1.083 6.58040e+8 H N N H Ph O N '-phenylbenzohydrazide Chemical Formula: C13H12N2O Exact Mass: 212.095 Molecular Weight: 212.2472

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Appendix (Continued) 213 30 Jan 2008 Acquisition Time (sec) 6.3308 Comment polar spot Date 13 Jan 2008 06:28:16 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-8-80-1\W-8-80-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 21.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 11.24 2.00 7.24 7.14 4.99 No. (ppm) (Hz) Height 1 4.99 1247.2 0.3336 2 7.14 1787.1 0.4240 3 7.24 1809.9 0.3166 No. (ppm) Value Absolute Value 1 [4.76 .. 5.17] 2.000 1.11203e+9 2 [6.77 .. 7.51] 11.240 6.24963e+9 N NH2 O Ph N -phenylbenzohydrazide Chemical Formula: C13H12N2O Exact Mass: 212.095 Molecular Weight: 212.2472

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Appendix (Continued) 214 30 Jan 2008 Acquisition Time (sec) 6.3308 Comment PROTON Date 25 Sep 2007 23:25:52 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-7-60-1\W-7-60-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 25.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 4.98 3.00 2.04 7.42 7.39 7.26 4.82 2.09 2.01 1.61 0.00 No. (ppm) (Hz) Height 1 -0.01 -1.6 0.1193 2 -0.00 -0.0 1.0000 3 1.61 403.7 0.4455 4 2.01 501.6 0.3918 5 2.09 521.7 0.1203 6 4.82 1205.5 0.1414 7 7.26 1816.8 0.7117 8 7.39 1848.4 0.3460 9 7.41 1854.4 0.2055 10 7.42 1855.9 0.2820 No. (ppm) Value Absolute Value 1 [1.83 .. 2.08] 3.000 1.65127e+9 2 [4.53 .. 5.03] 2.039 1.12221e+9 3 [7.29 .. 7.55] 4.985 2.74385e+9 N NH2 O N -phenylacetohydrazide

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Appendix (Continued) 215 30 Jan 2008 Acquisition Time (sec) 6.3308 Comment PROTON Date 17 Sep 2007 23:32:16 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-7-55-1\W-7-55-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 25.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 3.28 3.00 2.591.72 0.00 1.96 3.83 4.80 6.90 6.94 7.22 7.27 No. (ppm) (Hz) Height 1 0.00 0.1 0.3149 2 1.96 490.0 0.7946 3 3.83 958.0 1.0000 4 4.80 1201.0 0.1415 5 6.90 1726.2 0.2555 6 6.94 1735.2 0.3244 7 7.18 1796.3 0.2214 8 7.22 1805.2 0.1798 9 7.27 1818.0 0.2600 No. (ppm) Value Absolute Value 1 [1.86 .. 2.03] 3.000 2.02696e+9 2 [3.76 .. 3.97] 3.280 2.21592e+9 3 [4.59 .. 5.05] 1.715 1.15889e+9 4 [6.84 .. 7.07] 2.250 1.52045e+9 5 [7.09 .. 7.41] 2.586 1.74754e+9 N NH2 O MeO N -(4-methoxyphenyl)acetohydrazide

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Appendix (Continued) 216 30 Jan 2008 Acquisition Time (sec) 6.3308 Date 16 Jan 2008 01:44:32 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-86-2\W-86-2_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent MeOD Sweep Width (Hz) 5175.98 Temperature (degree C) 22.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 3.03 3.00 7.53 7.11 7.07 6.92 6.84 2.39 2.04 No. (ppm) (Hz) Height 1 2.04 510.4 0.7675 2 2.39 597.3 0.1431 3 6.84 1711.1 0.0461 4 6.92 1730.4 0.0574 5 7.07 1768.1 0.0209 6 7.11 1778.4 0.0142 7 7.53 1883.9 0.0120 No. (ppm) Value Absolute Value 1 [1.83 .. 2.48] 3.028 4.00729e+9 2 [6.67 .. 7.76] 3.000 3.97041e+9 N NH2 O F F N -(2,4-difluorophenyl)acetohydrazide

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Appendix (Continued) 217 30 Jan 2008 Acquisition Time (sec) 6.3308 Comment 4-bromo Date 18 Jan 2008 19:03:28 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-8-89-1\W-8-89-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 21.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 4.20 3.00 1.37 7.55 7.52 7.27 4.79 2.02 No. (ppm) (Hz) Height 1 2.02 504.0 0.5831 2 4.79 1197.2 0.1804 3 7.27 1818.0 0.6834 4 7.52 1880.5 0.5918 5 7.55 1889.2 0.4769 No. (ppm) Value Absolute Value 1 [1.79 .. 2.51] 3.000 2.65722e+9 2 [4.43 .. 5.02] 1.372 1.21549e+9 3 [6.88 .. 7.93] 4.201 3.72105e+9 N NH2 O Br N -(4-bromophenyl)acetohydrazide Chemical Formula: C8H9BrN2O Exact Mass: 227.9898 Molecular Weight: 229.0739

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Appendix (Continued) 218 30 Jan 2008 Acquisition Time (sec) 6.3308 Date 15 Jan 2008 07:25:52 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-8-85-1\W-8-85-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 22.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 4.28 3.00 1.56 7.40 7.37 7.27 7.26 4.79 2.01 0.00 No. (ppm) (Hz) Height 1 0.00 0.0 0.5434 2 0.00 0.7 0.4995 3 2.01 502.0 0.9379 4 4.79 1198.0 0.4215 5 7.26 1815.4 0.4711 6 7.27 1818.7 0.7425 7 7.37 1842.4 1.0000 8 7.40 1850.9 0.5670 No. (ppm) Value Absolute Value 1 [1.81 .. 2.48] 3.000 2.94014e+9 2 [4.43 .. 5.09] 1.558 1.52685e+9 3 [7.03 .. 7.79] 4.277 4.19159e+9 N NH2 O Cl N -(4-chlorophenyl)acetohydrazide Chemical Formula: C8H9ClN2O Exact Mass: 184.0403 Molecular Weight: 184.6229

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Appendix (Continued) 219 30 Jan 2008 Acquisition Time (sec) 6.3308 Date 22 Jan 2008 10:48:32 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-HZ-3nitro\W-HZ-3nitro_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 20.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 3.57 3.00 1.71 2.18 4.80 7.59 7.62 8.14 8.25 No. (ppm) (Hz) Height 1 2.18 544.5 0.6411 2 4.80 1200.8 0.0905 3 7.59 1898.5 0.2506 4 7.62 1906.6 0.1822 5 8.14 2035.0 0.1241 6 8.25 2062.5 0.1293 No. (ppm) Value Absolute Value 1 [1.82 .. 2.76] 3.000 3.06759e+9 2 [4.13 .. 5.14] 1.705 1.74381e+9 3 [7.42 .. 8.61] 3.566 3.64612e+9 N NH2 O NO2 N -(3-nitrophenyl)acetohydrazide Chemical Formula: C8H9N3O3Exact Mass: 195.0644 Molecular Weight: 195.1754

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Appendix (Continued) 220 30 Jan 2008 Acquisition Time (sec) 6.3308 Comment 3-Chloro Date 16 Jan 2008 01:36:00 File Name C:\Documents and Settings\Sung Wook Yi\Desktop\Hydrazine NMR\W-87-1\W-87-1_001000fid Frequency (MHz) 250.13 Nucleus 1H Number of Transients 16 Original Points Count 32768 Points Count 32768 Pulse Sequence zg30 Solvent CHLOROFORM-D Sweep Width (Hz) 5175.98 Temperature (degree C) 22.160 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) 4.27 3.00 1.74 7.35 7.32 4.79 2.05 No. (ppm) (Hz) Height 1 2.05 512.1 0.5858 2 4.79 1196.9 0.1983 3 7.32 1830.3 0.7235 4 7.35 1837.9 0.6934 No. (ppm) Value Absolute Value 1 [1.77 .. 2.53] 3.000 2.78144e+9 2 [4.36 .. 5.02] 1.737 1.61076e+9 3 [7.00 .. 7.57] 4.267 3.95576e+9 N NH2 O Cl N -(3-chlorophenyl)acetohydrazide Chemical Formula: C8H9ClN2O Exact Mass: 184.0403 Molecular Weight: 184.6229

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About the Author : Sung Wook Yi (Woogie) was born in 1975 in Kim-Chon, South Korea. He grew up in Kim-Chon and stayed there until 1993 when he finished his high early education from Kim-Chon high school. He then moved to the other part of the country for his college. He was a sophomore at Kangnung National University when he joined army for the mandatory service. After serving 3 years in the army, he returned to the college and graduated in 2000 with B.S degree in chemistry. He then moved to the U.S. to learn English and eventually started his graduate program at the University of South Florida in 2002. He has been involved in various synthetic projects including the CPNA, a HIV protease synthesis and the hydrazine proj ects as well as couple other methodology projects under Dr. McLaughlins supervision.