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Expanding Benzotriazole Methodology

Permanent Link: http://ufdc.ufl.edu/UFE0024327/00001

Material Information

Title: Expanding Benzotriazole Methodology Difficult Peptides and Other Molecules of Biological Interest
Physical Description: 1 online resource (163 p.)
Language: english
Creator: Haase, Danniebelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, alkoxyimidoylation, amino, aminoacylbenzotriazole, aminoimidoylation, amyloid, arylthioimidoylation, auxiliary, benzotriazole, coupling, difficult, heterocyclic, microwave, peptides, phase, solid, synthesizer, thiocarbamoylation
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 1H-Benzotriazole has found wide applicability as a highly effective synthetic auxiliary in solution and solid phase reactions. The aim of this study was to develop practical synthetic routes of potential importance for the construction of biologically active structural motifs and compounds of biological interest. This thesis is divided into eight parts. Chapter 1 gives an overview of 1H-benzotriazole methodology, highlighting recent applications in synthetic organic chemistry. Chapter 2 describes the C-thiocarbamoylation and C-aminoimidoylation of ester enolates and other nucleophiles. Chapter 3 extends this work to the syntheses of novel O-aryl-benzotriazole-1-carbothioates, S-aryl-benzotriazole carbodithioates, benzotriazole-1-carboximidates and explores their potential to C-alkoxylate- and C-arylthioimidoylate nucleophiles. Chapter 4 describes the preparation of N-Fmoc-(alpha-aminoacyl)benzotriazoles and Chapter 5 their application in the microwave-assisted solid phase peptide synthesis (SPPS) of oligopeptides. Chapter 6 extends the utility of benzotriazole-mediated syntheses to the assembly of ?difficult? peptides. In Chapter 7, potential applications of benzotriazole methodology in the syntheses of azole-based peptides and water-soluble coupling reagents for peptide synthesis are presented. Finally, Chapter 8 provides conclusions, a summary of achievements and future directions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Danniebelle Haase.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024327:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024327/00001

Material Information

Title: Expanding Benzotriazole Methodology Difficult Peptides and Other Molecules of Biological Interest
Physical Description: 1 online resource (163 p.)
Language: english
Creator: Haase, Danniebelle
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, alkoxyimidoylation, amino, aminoacylbenzotriazole, aminoimidoylation, amyloid, arylthioimidoylation, auxiliary, benzotriazole, coupling, difficult, heterocyclic, microwave, peptides, phase, solid, synthesizer, thiocarbamoylation
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 1H-Benzotriazole has found wide applicability as a highly effective synthetic auxiliary in solution and solid phase reactions. The aim of this study was to develop practical synthetic routes of potential importance for the construction of biologically active structural motifs and compounds of biological interest. This thesis is divided into eight parts. Chapter 1 gives an overview of 1H-benzotriazole methodology, highlighting recent applications in synthetic organic chemistry. Chapter 2 describes the C-thiocarbamoylation and C-aminoimidoylation of ester enolates and other nucleophiles. Chapter 3 extends this work to the syntheses of novel O-aryl-benzotriazole-1-carbothioates, S-aryl-benzotriazole carbodithioates, benzotriazole-1-carboximidates and explores their potential to C-alkoxylate- and C-arylthioimidoylate nucleophiles. Chapter 4 describes the preparation of N-Fmoc-(alpha-aminoacyl)benzotriazoles and Chapter 5 their application in the microwave-assisted solid phase peptide synthesis (SPPS) of oligopeptides. Chapter 6 extends the utility of benzotriazole-mediated syntheses to the assembly of ?difficult? peptides. In Chapter 7, potential applications of benzotriazole methodology in the syntheses of azole-based peptides and water-soluble coupling reagents for peptide synthesis are presented. Finally, Chapter 8 provides conclusions, a summary of achievements and future directions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Danniebelle Haase.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024327:00001


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1 EXPANDING BENZOTRIAZOLE METHODOLOGY: DIFFICULT PEPTIDES AND OTHER MOLECULES OF BIOLOGICAL INTEREST By DANNIEBELLE NICKESHA HAASE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULF ILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Danniebelle Nickesha H aase

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3 To my parents, Kingsley and Beverley Haase T o my brother, Kevin Haase T o my fam ily and dear friends for their continued love and support

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Professor Alan R. Katritzky for the opportunity to study in his laboratory and for his supervision during the period. I thank current and form er members of the Katritzky group. I would like to thank Dr. Hall, Mr. Zouquan Wang, Mrs. Megumi Yoshioka Tarver, Dr. Srinivasa Tala, Dr. Sevil Oz c an, Dr. Anamika Singh, Dr. Adam Vincek, Ms. Janet Cusido and Dr. N iveen Khashab. Special thanks to my current labmate Ms. Claudia El Nachef and former undergraduate researchers Mr. Robert Johnson and Ms. Darcie Thomas for sharing their life as well as the lab space with me. I also thank Mrs. Lori Clark, Mrs. Elisabeth Sheppard, Ms. Gwendolyn McCann and Dr Sure yya Hanci I thank my collaborators Mr. Alfred Chung from Proteomics, Interdisciplinary Center for Biotechnology Research (ICBR) at UF and Dr. Jodie Johnson of the Mass Spectrometry Facility in the Department of Chemistry at UF for their assistance in data acqui sition and their patience with me I also want to express my gratitude to Mr. Alfred Chung for sharing his breath of knowledge of peptide chemistry and for sharing his great thoughts on life I would also like to thank my committee members, Professor s Lisa McElwee White, Kenneth Sloan, Sukwon Hong and Dr. Ion Ghiviriga for their valuable advice. Special thanks to Professor Helen Jacobs of the University of the West Indies for inspiring me in so many ways. I am forever indebted to her Very special tha nks to my dear friends Dr. Denise Simpson, Mr. Gilles Seburyamo, Ms. Cidya Grant and family, Ms. Sharon Hooper and Ms. Monique Blake for the huge impact you have had on my life gros bisous I thank all my friends around the world, especially in Jamaica. F inally, my foremost thanks go to my parents Kingsley and Beverley, my brother Kevin and my entire family for their love and encouragement throughout the years Words are in sufficient to express my heartfelt gratitude to them

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 LIST OF SCHEMES .......................................................................................................................... 14 LIST OF ABBREVIATIONS ............................................................................................................ 16 ABSTRACT ........................................................................................................................................ 20 CHAPTER 1 BENZOTRIAZOLE: A FASCINATING MOLECULE .......................................................... 22 1.1 Introduction ........................................................................................................................... 22 1.2 1H Benzotriazole as a Synthetic Auxiliary ......................................................................... 22 1.3 Recent Developments in Benzotriazole Methodology ....................................................... 26 Benzotriazolylamides ........................................................................ 26 1.3.2 1H Benzotriazole and its Derivatives in Cross Coupling Reactions ...................... 27 1.3.3 Uses of 1H Benzotriazole and its Derivatives in Materials Chemistry .................. 32 1.3.3.1 Benzotriazole derivatives as corrosion inhibitors .......................................... 32 1.3.3.2 Benzotriazole as a component of conjugated polymers ................................ 33 1.3.4 Aspects of Benzotriazole-mediated Syntheses ......................................................... 34 1.3.4.1 Benzotriazole assisted imidoylations ............................................................. 34 1.3.4.2 Benzotriazole assisted N acylations ............................................................... 35 1.4 Concluding Remarks ............................................................................................................. 35 2 C-AMINOIMIDOYLATION AND C THIOCARBAMOYLATION OF ESTERS .............. 37 2.1 Background ........................................................................................................................... 37 2.2 Introduction ........................................................................................................................... 40 2.3 Results and Discussion ......................................................................................................... 42 2.3.1 Preparation of 1 (Alkyl/aryl thiocarbamoyl)benzotriazoles 2.6a d and Benzotriazole 1 -carboxamidines 2.7a c. ....................................................................... 42 2.3.2 C -Aminoimidoylation and C Thiocarbamoylation of Doubly Activated Esters .... 42 2.3.3 C -Aminoimidoylation and C Thiocarbamoylation of Unactivated Esters and Other Activated Compounds ........................................................................................... 47 2.3.3.1 Reactions with unactivated esters ................................................................... 47 2.3.3.2 Reactions with sulfones and cyano compounds ............................................ 50 2.3.3.3 Reactions with nitro compounds ..................................................................... 50 2.4 Conclusions and Outlook ..................................................................................................... 51 2.5 Experimental Section ............................................................................................................ 52

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6 2.5.1 General ........................................................................................................................ 52 2.5.2 General Procedure for the Preparation of 2.20a -c, 2.21a,b, 2.23a,b, 2.24a d, 2.25a,b ............................................................................................................................... 52 3 C-ALKOXYIMIDOYLATION AND C -ARYLTHIOIMIDOYLATION .............................. 62 3.1 Introduction ........................................................................................................................... 62 3.2 Results and Discussion ......................................................................................................... 64 3.3 Conclusion ............................................................................................................................. 65 3.4 Experimental Section ............................................................................................................ 65 3.4.1 General Procedure for the Preparation of O -Arylbenzotriazole Carbothioate 3.9 or S -Arylbenzotriazole Carbodithioate 3.10a ........................................................... 65 3.4.2 General Procedure for the Preparation of Benzotriazole 1 carboximidates (3.12) ................................................................................................................................. 67 3.4.3 General Procedure for the Preparation of 3.14 ......................................................... 68 4 N(FMOC -AMINOACYL)BENZOTRIAZOLES: VERSATILE SYNTHETIC REAGENTS FROM PROTEINOGENIC AMINO ACIDS .................................................... 69 4.1 Introduction ........................................................................................................................... 69 4.2 Results and Discussion ......................................................................................................... 70 4.2.1 Preparation of N (Fmo c aminoacyl)benzotriazoles 4.1a r ................................... 70 (N -Fmoc amino)acid Amides 4.3a,b and 4.4a,b .......................... 71 4.3 Conclusion ............................................................................................................................. 73 4.4 Experimental Section ............................................................................................................ 73 4.4.1 General Procedure for the Preparation of 4.2bl, q, r ............................................... 74 4.4.2 General Procedure for the Preparation of 4.3a,b, 4.4a,b, (4.3a+4.4a) and (4.3b+4.4b) ....................................................................................................................... 79 5 MICROWAVE -ASSISTED SOLID PHASE PEPTIDE SYNTHESIS UTILIZING N FMOC -PROT ECTED( -AMINOACYL)BENZOTRIAZOLES ........................................... 83 5.1 Introduction ........................................................................................................................... 83 5.2 Results and Discussion ......................................................................................................... 84 5.2.1 Preparation of N -Fmoc ( aminoacyl)benzotriazoles (5.2a g) ............................... 84 5.2.2 Peptide Syntheses ....................................................................................................... 85 5.3 Conclusion ............................................................................................................................. 86 5.4 Experimental Section ............................................................................................................ 87 6 BENZOTRIAZOLE -ASSISTED SOLID -PHASE ASSEMBLY OF LEU ENKEPHALIN, AMYLOID SEGMENT 34 42, AND OTHER DIFFICULT PEPTIDE SEQUENCES ............................................................................................................ 92 6.1 Introduction ........................................................................................................................... 92 6.2 Results and Discussion ......................................................................................................... 93 6.3 Preparation of Leu En kephalin (9) and Amyloid42) (10) ....................... 99 6.4 Conclusions and Directions ................................................................................................ 100

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7 6.4.1 Conclusions ............................................................................................................... 100 6.4.2 Directions .................................................................................................................. 101 6.5 Experimental Section .......................................................................................................... 101 6.5.1 Solid Phase Protocol for the Preparation of Peptides 3 10 .................................... 102 6.5.2 Parameters for Microwave Reactions ..................................................................... 103 6.5.3 Peptide Analysis ....................................................................................................... 104 6.5.4 Characterization of Polymorphic Compounds 2c,d,j and 3 10 ............................. 104 7 SYNTHESES OF AZOLE -BASED AMINO ACIDS AND PEPTIDES, AND WATER SOLUBLE COUPLING R EAGENTS .................................................................................... 132 7.1 Azole based Amino Acids and Peptides ........................................................................... 132 7.1.1 Introduction............................................................................................................... 132 7.1.2 Proposed Synthetic Route to Azolo-based Amino Acids and Dipeptides ............ 133 7.1.3 Preparation of N aminoacyl)benzotriazoles 7.1................................. 134 7.1.4 Preparation of Amidrazones 7.2 .............................................................................. 135 7.1.5 Preparative routes to Amidoximes 7.3, Thiohydrazides 7.4 and Semicarbazides 7.5 ........................................................................................................ 136 7.1.6 Summary and Future Prospect ................................................................................. 136 7.1.7 Experimental Section ............................................................................................... 137 7.1.7.1 General procedure for the preparation of N aminoacyl)benzotriazoles 7.1 ................................................................................ 137 7.1.7.2 General procedure for the preparation of amidrazone 7.2b and dihydrotetrazine 7.17a,b ......................................................................................... 137 7.2 Synthesis of Water -soluble Coupling Reagents ................................................................ 138 7.2.1 Introduction............................................................................................................... 138 7.2.2. Pr eparative Routes to Water -soluble Coupling Reagents 7.22 7.23 .................... 141 7.2.2.1 Retrosynthetic analysis for 7.22.................................................................... 141 7.2.2.3 Retrosynt hetic analysis for 7.23.................................................................... 143 7.2.2.4 General procedure for the preparation of 1 H benzotriazole 6 -sulfonic acid 7.29 .................................................................................................................. 144 7.2.2.5. Future work ................................................................................................... 149 8 CONCLUSIONS, SUMMARY OF ACHIEVEMENTS AND FUTURE OUTLOOK ....... 150 LIST OF REFERENCES ................................................................................................................. 153 BIOGRAPHICAL SKETCH ........................................................................................................... 163

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8 LIST OF TABLES Table page 2 1 C-Aminoimidoylation and C -thiocarbamoylation of esters 2.19a c to give 2.20a -c 2.21a,b .................................................................................................................................... 43 2 2 C-Aminoimidoylation and C -thiocarbamoylation of esters 2.19d -f to give 2.23a,b 2.24a -c .................................................................................................................................... 48 4 1 Conversions of the 18 N -Fmoc amino acids 4.1a-r into N (Fmoc aminoacyl) benzotriazoles 4.2a -r ............................................................................................................. 71 4 2 Preparation of N (acylamino)amides 4.3a,b and 4.4a,b from N (Fmoc aminoacyl) benzo triazoles 4.2b,g and L or D PhCH(Me)NH2 (4.5 or 4.6 ) .......................................... 72 5 1 N-Fmoc -protected aminoacyl)benzotriazoles 5.2a -g utilized for peptide synthesis ..... 85 5 2 Synthesis of peptides 5.3 5.4 ................................................................................................. 86 6 1 Preparation of N -Fmoc aminoacyl)benzotriazoles ( 6.2 ) from the corresponding Fmoc -protected amino acids ( 6.1 ) ..................................................................................... 94 6 2 Analytical data of peptides 6.3 6.10 ..................................................................................... 97

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9 LIST OF FIGURES Figure page 1 1 Tautomeric equilibrium of benzotriazole ............................................................................. 22 1 2 Cycle of 1 H -benzotriazole assisted syntheses ...................................................................... 23 1 3 Multiple activating influences of the benzotriazoly l group ................................................. 25 1 4 Comparison of 1H -benzotriazole with other activating groups .......................................... 26 1 5 Literature methods for the preparation of -be nzotriazolylamides 1.2 .............................. 26 1 6 Synthesis of 1.4 from the CuI catalyzed N arylation of imidazole and benzimidazole with aryl halides ..................................................................................................................... 28 1 7 Synthesis of 1.5 from the Cu -catalyzed coupling of alkylthiols with aryl bromides ......... 28 1 8 Cu(I) mediated tandem synthesis of indolo and pyrrolo[2,1 a]isoquinolines 1.8 ............ 29 1 9 Cu -catalyzed N arylation of 1 H -benzotriazole 1.1 .............................................................. 31 1 10 Pd -catalyzed indolization of N aroylbenzotriazoles 1.20 .................................................... 31 1 11 Structures of benzotriazole derivatives 1.25 and 1.26 ......................................................... 33 2 1 General structure of isothiocyanates and carbodiimides ..................................................... 37 2 2 Formation of heterocycles from isothiocyanates ................................................................. 37 2 3 Biologically active heterocycles synthesized from carbodiimides ..................................... 38 2 4 Possible tautomeric forms of 2.20 ......................................................................................... 44 2 5 Relevant 1H and 13C chemical shifts in compounds 2.21a,b ............................................... 44 2 6 Relevant 1H and 13C chemical shifts in CDCl3 of keto -enamine tautomeric structures 2.20a -c .................................................................................................................................... 45 2 7 Trans and cis forms of thioamides ........................................................................................ 49 2 8 1H NMR spectrum of the reaction of 2.6f with ethyl nitroacetate ...................................... 51 2 9 IR spectrum of 2.20c .............................................................................................................. 57 2 1 0 IR spectrum of 2.23b ............................................................................................................. 57 2 11 High Resolution Mass Spectrum of 2.23b ............................................................................ 58

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10 2 12 1H NMR spectrum of 2.24d ................................................................................................... 59 2 13 13C NMR spectrum of 2.24d .................................................................................................. 59 2 14 1H NMR spectrum of 2.25a ................................................................................................... 60 2 15 13C NMR spectrum of 2.25a .................................................................................................. 60 2 16 1H NMR spectrum of 2.25b ................................................................................................... 61 2 17 13C NMR spectrum of 2.25b .................................................................................................. 61 3 1 General Structure of isothioureas, isoureas, ketene O N and S N acetals ......................... 62 3 2 Specific examples of isothioureas applied in medicine and agriculture ............................. 62 3 3 Trehazolin ( 3.3 ), a natural cyclic isourea ............................................................................. 63 3 4 Examples of ketene O N and S N acetals .............................................................................. 63 5 1 General Peptide Fragmentation ............................................................................................. 88 5 2 The HPLC profile of peptide 5.3 (Pro Trp -Met Trp NH2) ................................................. 89 5 3 Expected pr oduct ions from the (+)ESI MSn of the m/z 618 [M+H]+ ion. ........................ 89 5 4 The HPLC profile of peptide 5.4 (Leu -Met Gly -Phe -Ala NH2) ......................................... 90 5 5 Expected product ions from the (+)ESI MSn of the m/z 537 [M+H]+ ion. ........................ 90 5 6 The profile of peptide 5.5 (Pro -Leu -Met Gly -Phe -Ala -NH2) ............................................. 91 5 7 Expected product ions from the (+)ESI MSn of the m/z 634 [M+H]+ ion......................... 91 6 1 HPLC profiles of a ) crude and b ) pure peptide 6.3 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage. ..................................................................................... 96 6 2 HPLC profiles of a ) crude and b ) pure peptide 6.9 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage. ................................................................................... 100 6 3 1H NMR spectrum of 6.2c in CDCl3 ................................................................................... 106 6 4 13C NMR spectrum of 6.2c in CDCl3.................................................................................. 106 6 5 1 6.7) of 6.2d in CDCl3 ................................................................ 107 6 6 1 1.1) of 6.2d in CDCl3 ................................................................ 107 6 7 13C NMR spectrum of 6.2 d in CDCl3 ................................................................................. 108

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11 6 8 1H NMR spectrum of 6.2j in CDCl3 ................................................................................... 109 6 9 13C NMR spectrum of 6.2j in CDCl3 .................................................................................. 109 6 10 High Resolution Mass Spectrum of 6.2j ............................................................................. 110 6 11 Expected product ions from the (+)ESI MSn of crude 6.3 (m/z 501 [M+H]+ ions) ........ 111 6 12 Fragmentation of crude 6.3 .................................................................................................. 111 6 13 High Resolution Mass Spectrum of crude 6.3 .................................................................... 112 6 14 HPLC profiles of a ) crude and b ) pure 6.3 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage .................................................................................... 113 6 15 Expected product ions from the (+)ESI MSn of crude 6.3 (m/z 501 [M+H]+ ion s) ........ 113 6 16 HPLC profile of crude 6.4 ................................................................................................... 114 6 17 Expected product ions from the (+)ESI MSn dissociation of crude 6.4 (m/z 600 [M+H ]+ ion) .......................................................................................................................... 114 6 18 High Resolution Mass Spectrum of crude 6.4 .................................................................... 115 6 19. HPLC profiles of a ) crude and b ) pure 6.5 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage ...................................................................................................... 116 6 20 Expected product ions from the (+)ESI MSn collision induced dissociation (CID) of crude 6.5 (m/z 499 [M+H]+ ion) ......................................................................................... 116 6 21 Expected product ions from the (+)ESI MSn CID of pure 6.5 (m/z 500 [M+H]+ ion) ... 117 6 22 High Resolution Mass Spectrum of pure 6.5 ...................................................................... 117 6 23 HPLC profiles of a ) crude and b ) pure 6.6 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage .................................................................................... 118 6 24 Product ions from the (+)ESI -MSn dissociation of crude 6.6 (m/z 618 [M+H]+ ion). .... 118 6 25 High Resolution Mass Spectrum of pure 6.6 ...................................................................... 119 6 26 HPL C profiles of a ) crude and b ) pure 6.7 obtained after SPPS using 5% piperazine DMF for Fmoc cleavage ...................................................................................................... 120 6 27 Expected product ions from the (+)ESI MSn of crude 6.7 (m/z 693 [M+H]+ ion) ......... 120 6 28 High Resolution Mass Spectrum of crude 6.7 .................................................................... 122

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12 6 29 HPLC profiles of a ) crude and b ) pure 6.8 obtained after SPPS using 5% pi perazine DMF for Fmoc cleavage ...................................................................................................... 123 6 30 Expected product ions from the (+)ESI CID -MS/MS of crude 6.8 (m/z 735 [M+H]+ ion) ........................................................................................................................................ 123 6 31 High Resolution Mass Spectrum of crude 6.8 .................................................................... 124 6 32 Expected product ions according to classical cleavage along the amide backbone to create b a -, y and z ions ................................................................................................. 125 6 33 High Resolution Mass Spectrum of pure 6.9 ...................................................................... 125 6 34 Gradient analysis .................................................................................................................. 126 6 35 T he (+)ESI -MS spectrum of peptide 6.9 ............................................................................ 127 6 36 The (+)ESI -MS/MS of 6.9 ................................................................................................... 128 6 37 HPLC profiles of a ) crude and b ) pure 6.10 obtain ed after SPPS using 20% piperidine DMF for Fmoc cleavage .................................................................................... 129 6 38 High Resolution Mass Spectrum of pure 6.10 ................................................................... 129 6 39 The (+)ESI -MS of p eptide 6. 10. .......................................................................................... 130 6 40 T he traditional amide cleavage of 6.10 during (+)ESI -MS and MSn ............................. 131 7 1 Synthesis of 1,2,4 triazo lo 1,2,4 -oxadiazolo 1,3,4 thiadiazolo amino acid derivatives 7.6 7.7 ................................................................................................................ 133 7 2 Synthesis of 1,2,4 triazolo 1,2,4 -oxadiazolo 1,3,4 thiadiazolo dipeptide derivatives 7.117.14 ............................................................................................................................... 134 7 3 Structures of some of water soluble N protecting groups ................................................. 139 7 4 Structures of water soluble activating groups .................................................................... 140 7 5 Potential water -soluble coupling reagents 7.22 and 7.23 .................................................. 140 7 6 Retrosynthetic analysis of 7.22 ........................................................................................... 141 7 7 Proposed synthetic route to 7.23 ......................................................................................... 143 7 8 1H NMR spectrum of 7.29 in D2O ...................................................................................... 145 7 9 13C NMR spectrum of 7.29 in D2O ..................................................................................... 145

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13 7 10 The m/z 200 [M+H]+ ion of 7.29 (top) plus the primary and secondary product ions of dissociation (middle and bottom) ................................................................................... 146 7 11 The m/z 198 [M H]ion of 7.29, self adduct ions (top) plus primary and secondary product ions of dissociation (bottom) ................................................................................. 147

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14 LIST OF SCHEME S Scheme page 1 1 A plausible mechanism for the Cu -catalyzed N arylation of imidazoles and S arylation of thiols to produce 1.4 and 1.5 respectively ........................................................ 29 1 2 A possible mec hanism for the Cu -mediated tandem synthesis of indolo and pyrrolo[2,1 a]isoquinolines 1.8 [09AGE1138] .................................................................... 30 1 3 Mechanism for the formation of polysubstituted indoles 1.19 from N aroylbenzotriaz oles 1.20 and disubstituted alkynes 1.21 ..................................................... 32 1 4 Preparation of guanidines from guanylating reagents 1.29 and 1.30 .................................. 34 1 5 Prepara tion of N acylbenzotriazoles 1.31 ............................................................................. 35 2 1 Representative reactions of 2.6 and 2.7 with nucleophiles .................................................. 39 2 2 Reactions of 1 (alkyl/ arylthiocarbamoyl)benzotriazoles 2.6 .............................................. 40 2 3 N -Aminoimidoylation with benzotriazole 1 -carboxamidines 2.7 ...................................... 40 2 4 Preparation of C aminoimidoylation product 2.16 .............................................................. 41 2 5 Preparation of C -thiocarbamoylation product 2.17 .............................................................. 41 2 7 Preparation from esters 2.19a -c of the C aminoimidoylation products 2.20a c and the C-thiocarbamoylation products 2.21a,b ............................................................................... 43 2 8 Four possible rotameric conformations of 2.20a -c .............................................................. 46 2 9 Literature methods for the preparation of ketene aminals 2.22 ........................................... 47 2 10 Preparation from esters 2.19d -f of the C aminoimidoylation products 2.23a,b and the C-thiocarbamoyl ation products 2.24a -c ............................................................................... 47 3 1 Preparation of O aryl -benzotriazole carbothioate ( 3.9 ), S aryl -benzotriazole carbodithioate ( 3. 10) and benzotriazole 1 carboximidates ( 3.12) ...................................... 65 3 2 Synthesis of product 3.14 ...................................................................................................... 65 4 1 Amide bond formation ........................................................................................................... 69 4 2 Preparation of N -Fmoc aminoacyl)benzotriazoles 4.2a -r from the corresponding Namino acids 4.1a -r ......................................................................................... 70 4 3 Preparation of N (acylamino)amides 4.3a,b and 4.4a,b from N (Fmoc aminoacyl) benzotriaz oles 4.2b,g and L or D PhCH(Me)NH2 (4.5 or 4.6 ) .......................................... 72

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15 5 1 Preparation of N -Fmoc aminoacyl)benzotriazoles 5.2a g ............................. 85 6 1 Preparation of N -Fmoc aminoacyl)benzotriazoles ......................................................... 93 6 2 SPPS approach using the Rink amide MBHA resin and N Fmoc aminoacyl)benzotriazoles ...................................................................................................... 94 6 3 Possible (+)ESI -MSn dissociation pathway for the formation of m/z 449 and m/z 336 product ions from the m/z 693 [M+H]+ ion of 6.7 ............................................................ 121 7 1 Preparation of N -protected a minoacyl)benzotriazoles 7.1 ........................................... 134 7 2 Preparation of amidrazones 7.2 and dihydrotetrazines 7.17 .............................................. 135 7 3 Preparation of amidoximes 7.3 ............................................................................................ 136 7 4 Preparation of thiohydrazides 7.4 and semicarbazides 7.5 ................................................ 136 7 5 Preparation of 1 H -benzotriazole 6 -sulfonic acid 7.2 9 ....................................................... 141 7 6 Preparation of 7.24 from 7.25.............................................................................................. 142 7 7 Proposed route to 7.22 ......................................................................................................... 142 7 8 Proposed preparation of 7.23 from 7.35 ............................................................................. 143 7 9 Probable ( )ESI -MSn dissociation of the m/z 198 [M -H]ion of 7.29 ............................. 148 7 10 Proposed SPPS using potential water -soluble reagents 7.22 or 7.23 ................................ 149

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16 LIST OF ABBREVIATIONS alpha locant specific rotation [ expressed without units ; the units, (deg mL)/(g dm) are understood] Ala alanine Al k alkyl Ar aryl Asp aspartic acid beta locant br broad (spectral) Bt benzotriazol 1 yl BtH 1 H benzotraizole C carbon oC degree Celcius calcd calculated Cbz benzyloxycarbonyl CDCl3 deuterated chloroform Cu copper Cys cysteine chemical shift in parts per million downfield from tetramethylsilane d doublet D (10 point) dextrorotary (right) DCC N, N -dicyclohexylcarbodiimide DCM dichloromethane DMF dimethylformamide DMSO dimethylsulfoxide

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17 DTT 1,4 Dithiothreitol EDT 1,2 Ethanedithiol eq uiv equivalent(s) Et eth yl et al. and others Et3N triethylamine Fe iron Fmoc 9 -fluorenylmethoxycarbonyl g gram(s) Gly glycine Glu glutami c acid Gln glutamine h hour H hydrogen HRMS high resolution mass spectrometry Hz hertz i iso (as in i -Pr; never i -propyl) Ile isoleucine i -Pr i sopropyl IR infrared J coupling constant (in NMR spectroscopy) L (10 point) levorotary (left) Leu leucine lit literature Lys lysine

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18 m multiplet (spectral); metre(s); milli Me methyl Met methionine min minute(s) mol mole(s) mp melting point m / z mass-to -char ge ratio N nitrogen NMR nuclear magnetic resonance o ortho locant O oxygen OEt ethoxy OMe methoxy Oxone potassium peroxymonosulfate p para locant Pd palladium Pg protecting group Ph phenyl Phe phenylalanine ppm parts per million Pro proline Pz pyrazine q quartet R rectus (right) (naming groups around a central carbon) (opposite of S ) ref. reference

PAGE 19

19 rt room temperature s singlet (spectral) S sinister (left) (naming groups around a central carbon) (opposite of R ) Ser serine SOCl2 thionyl chloride t triplet (spectral) t tertiary TFA trifluoroacetic acid Thr threonine TIPS triisopropylsilane TLC thin layer chromatography TMS trimethylsilane Tr (triphenylmethane) trityl Trp tryptophan Tyr tyrosine Val valine W watt(s)

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20 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXPANDING BENZOTRIAZOLE METHODOLOGY: DIFFICULT PEPTIDES AND OTHER MOLECULES OF BIOLOGICAL INTEREST By Danniebell e Nickesha Haase May 2009 Chair: Alan R. Katritzky Major: Chemistry 1 H Benzotriazole has found wide applicability as a highly effective synthetic auxiliary in solution and solid phase reaction s The aim of this study was to develop practical synthetic r out es of potential importance for the construction of biologically active structural motifs and compounds of biological interest This thesis is divided into eight parts. Chapter 1 gives an overview of 1 H -benzotriazole methodology, highlighting recent appl ications in synthetic organic chemistry. Chapter 2 describes the C thiocarbamoylation and C aminoimidoylation of ester enolates and other nucleophiles Chapter 3 extends this work to the synthes e s of novel O aryl benzotriazole 1 -carbothioates S aryl -benzo triazole carbodithioate s benzotriazole 1 carboximidates and explores their potential to C alkoxylate and C arylthioimidoylate nucleophiles Chapter 4 describe s the preparation of N Fmoc aminoacyl )benzotriazoles and Chapter 5 their application in the microwave assisted solid phase peptide synthesis (SPPS) of oligopeptides. Chapter 6 extends the utility of b enzotriazole-mediated syntheses to the assembly of difficult pept i des. In C hapt er 7 potential applications of benzotriazole methodology in the syntheses of azole -based peptides and water -soluble coupling reagents for peptide synthesis are

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21 presented Finally, Chapter 8 provides conclusions, a summary of achievements and future direct ion s

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22 CHAPTER 1 BENZOTRIAZOLE: A FASCINATING MOLECULE 1.1 Introduction Ben zazoles, in particular, 1 H -benzotriazole (1.1 ) play a unique role in heterocyclic chemistry (Figure 1 1) [09MRC142] 1 H B enzotriazole is a n intriguing molecule that possesses inter esting chemical and biological activities. [54SCI989, 95EJMC77, 91T2683] T he chemistry of 1 H -benzotriazole has been extensively studied by Katritzky and coworkers over the last t wo decades and is a mature field however, new discoveries are constantly being made. C urrently there is a resurgence of interest in 1 H -benzotriazole and it s derivatives due to wide applicability in fields such as medicine, environmental science, technology polymer science and synthetic organic chemis try. [09MRC142] Figure 1 1. Tautomeric equilibrium of benzotriazole 1.2 1 H -Benzotriazole as a S ynthetic A uxiliary 1 H Benzotriazole, a benzofused heterocycle possessing an azole ring, has three contiguous nitrogen atoms (Figure 1 1) The presence of concatenated nitrogen atoms in 1 H -b enzotriazole is responsible for its innate properties such as a p Ka of 8.2, indicating a high N H acidity and a p KaH 0 for proton addition. In other words, the 1 H -benzotriazole ring is able to accept or donate electrons. [91T2683]

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23 A synthetic auxiliary i s a compound that is temporarily incorporated during the synthesis of a chemical entity. N umerous neutral and charged heterocyclic spec ies, for example 1 H benzotriazole can act as efficient synthetic auxiliarie s (Figure 1 2 ). Figure 1 2. Cycle of 1 H be nzotriazole assisted syntheses As a n ideal synthetic auxiliary, 1 H -benzotriazole is: Economically favored, that is, it is inexpensive and readily available Readily attached to the substrate by substitution, addition or three c omponent condensation reaction s A good leaving group and thus is e asily displaced for example, by hydrolysis, reduction, or Pd -cataly zed substitution Readily separated from the product usually with minimal effort Easily recovered and reused E xhibits interesting and diverse reactivity patterns

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24 The use of 1 H benzotriazole as a versatile synthetic auxiliary, for both solution and solid phase syntheses, was previously established by Katritzky and coworkers. [91T2683, 98CR409, 03CEJ4586] The benzotriazolyl group is an excellent leaving group and can replace halogens in many reactions. [98CR409] Additionally, t he benzotriazolyl moiety is known to activate carbon atoms to which it is attached, thereby promoting common synthetic transformations. [91T2683, 98CR409, 03CEJ4586] T he benzotriazolyl moiety conveys it s activation of carbon atoms in several ways for example as (i) a proton activator, (ii) a cation stabilizer, (iii) an ambident anion directing group, (iv) a radical precursor and (v) an anion precursor (Figure 1 3 ). The leaving group ability, CH activation towards proton loss and electron donor properties of 1 H -benzotriazole ha ve been compared with other activating groups, such as the cyano and phenyl groups. [91T2683, 98CR409, 03CEJ4586] It was determined that 1 H -benzotriazole is compar able to cyano and phenylsulfonyl with respect to leaving group ability and in its ability to enabl e deprotonation (Figure 1 4). [ 98CR409] However, when compared with the electron donor properties of the phenyl and vinyl groups, 1 H -benzotriazole exhibits ad vantages. Examples of activating groups that possess all three aforementioned attributes, that is, they are good leaving groups, electron donors and activators of CH proton loss, are rare. 1 H -benzotriazole and the phenylthio group are examples of activatin g groups possessing these three properties, however when compared the former has considerable advantages (Figure 1 4). [ 91T2683, 98CR409, 03CEJ4586] In general, benzotriazole chemistry is straightforward and easy to comprehend. The benzotriazole derivativ es, many of which are odorless and stable solids, are usually prepared in high yields and are capable of a plethora of reactions. Now, an overview of the numerous

PAGE 25

25 synthetic possibilities of benzotriazole derivatives and recent developments in the syntheses of these derivatives are provided in Section 1.2 Figure 1 3 Multiple activating influences of the benzotriazolyl group

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26 Figure 1 4 Comparison of 1H -benzotriazole with other activating groups 1. 3 Recent Developments in Benzotriazole Methodology 1 3 .1 Synthesis of Benzotriazolylamides B enzotriazolylamides 1.2 are biologically active and are synthetic precursors to pyrid 2 ones. [88F A 29, 97JOC6210, 09OL995] In the literature, the synthesis of -benzotriazolylamides has been achieved by (i) the r eaction of sodium benzotriazolate with halides in average yields of 74% [ 97JOC6210] and (ii) an Ugi -Smiles/deallylation / diazotization sequence in average yields of 65% [09OL995] (Figure 1 5 ). Figure 1 5 Literature methods for the preparation of -b enzo triazolylamides 1.2

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27 In addition to being a powerful addition to the available synthesis of benzotriazolylamides 1.2 the Ugi Smiles/deallylation sequence is useful for the synthes e s of other privileged structures, for example, benzimidazoles 1.3 [09OL995 ] 1. 3 .2 1 H -Benzotriazole and its Derivatives in Cross Coupling Reactions Over the last twenty years cross coupling reactions ha ve emerged as a n integral tool for the synthes e s of natural products and polymers. [06EJO 3283] The Cu -mediated C N, C -S and C O bond forming reactions ha ve attracted much attention as a result of its economy and efficiency when compared to Pd-catalyzed cross coupling reaction s [07TL4207] However, the benefits of Cu -catalyzed bond formation reactions are often masked by the lack of highly efficient ligands and alternate copper sources [03A G E5400] The use of 1 H benzotriazole and its derivatives as ligands in transition metal mediated cross coupling reactions is relatively unexplored. A search of the literature revealed few examples in which 1 H -benzotriazole and its derivatives were used as ligands. Recently, Verma and coworkers assessed the potential of 1 H benzotriazole as a ligand for Cu -catalyzed C -N and C-S bond formation. [07TL4207, 07TL7199] 1 H benzotriazole is moisture insensi tive, inert to air and has excellent coordinating capabilities so Verma and coworkers considered it a potential ligand in Cu -mediated bond formations [07TL4207] As a result of it s coordinating abilities it was postulated that 1 H benzotriazole would be u seful in stabilizing catalytic species and in that way promot e catalytic cycles under mild conditions In a study on the preparation of N arylimidazoles 1.4 [07TL4207] (Figure 1 6 ) and aryl sulfides 1.5 [07TL7199] (Figure 1 7 ) via Cu -mediation, 1 H benzotri azole was found to be an exceptional ligand and afforded 1.4 and 1.5 in good to excellent yields.

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28 Figure 1 6 Synthesis of 1.4 from the CuI catalyzed N arylation of imidazole and benzimidazole with aryl halides Figure 1 7 Synthesis of 1.5 from the C u -catalyzed coupling of alkylthiols with aryl bromides A possible mechanism for the formation of N arylimidazoles 1.4 and aryl sulfides 1.5 is shown in Scheme 1 1. [07TL4207, 07TL7199] It is postulated that chelation of Cu(I) with 1 H benzotriazole results in the formation of species L1 Reactive species L1 readily participates in the oxidative addition reaction with aryl halides (ArX) to form species L2 Intermediate L3 is then generated by reaction of L2 with thiols or imidazoles in the presence of base. S ubsequent reductive elimination releases the product 1.4 or 1.5 and regenerates the active Cu(I) species. The described activation mode affords c ompounds 1.4 and 1.5 which are significan t components of numerous bioactive compounds. [07TL4207, 07TL7199] Ver ma and coworkers also utilized benzotriazol 1 ylmethanol 1.7 as a ligand in the Cu catalyzed tandem synthesis of multiring heterocycles. [09AGE1138] Multiring heterocyclic indolo and pyrrolo[2,1 a]isoquinolines 1. 8 were synthesized in good yields with exc ellent regioselectivity from indoles 1.9 and ortho haloarylalkynes 1.10 in the presence of catalytic CuI, base and hydroxymethyl benzotriazole 1.7 (Figure 1 8). [09AGE1138] The proposed mechanistic pathways are illustrated in Scheme 1 2. [09AGE1138]

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29 Sche me 1 1. A plausible mechanism for the Cu -catalyzed N arylation of imidazoles and S arylation of thiols to produce 1.4 and 1.5 respectively In one pathway, copper complex 1. 11 is generated from CuI and 1.7 Oxidative addition and ensuing complexation wit h the ortho haloarylalkyne produces intermediate 1.1 2 Intermolecular and an intramolecular nucleophilic attack on 1.1 2, followed by elimination of HBr results in the formation of Cu complex 1.1 5 via intermediates 1.1 3 and 1.14 respectively Product 1.8 is then generated from the reductive elimination of 1.1 5 Figure 1 8 Cu(I) mediated tandem synthesis of indoloand pyrrolo[2,1 a]isoquinolines 1.8

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30 Scheme 1 2. A possible mechanism for the Cu -mediated tandem synthesis of indoloand pyrrolo[2,1 a]isoquinolines 1.8 [09A G E1138] Contrary to its use as a ligand in Cu -mediated cross coupling reactions, substituted benzotriazoles have also been the synthetic target s [08SL3068] The Cu -catalyzed N arylation of 1 H benzotriazole 1.1 with aromatic and heteroarom atic chlorid es 1.17 provided the corresponding aryl -functionalized benzotriazole derivatives 1.18 in good to excellent yields (Figure 1 9 ). [08S L3068]

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31 Figure 1 9 Cu -catalyzed N arylation of 1 H benzotriazole 1.1 More recently, Nakamura and coworkers [09 OL 1055] demonstrated that benzotriazole derivatives are synthetic equivalents of 2 -haloanilides in Pd -mediated reactions. Nakamura and coworkers reported the construct ion of polysubstituted indole derivatives 1.19 via the Pd catalyzed denitrogenative indol ization of N aroylbenzotriazoles 1. 20 and disubstituted alkynes 1.2 1 (Figure 1 10). [09OL 1055] Figure 1 10. Pd -catalyzed indol iz ation of N aroylbenzotriazoles 1.20 The mechanism proposed by the authors [09OL 1055] (Scheme 1 3) involves insertion of Pd(0) into the C N bond of the diazonium species 1.22 to produce intermediate 1.23. Subsequent insertion of the alkyne 1.21 into the C -Pd bond of 1.23 generates the palladacycle species 1.24. Reductive elimination of Pd(0) from 1.2 4 provides the polysubstituted indole derivatives 1.19. [09OL 1055]

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32 Scheme 1 3. Mechanism for the formation of polysubstituted indoles 1.19 from N aroylbenzotriazoles 1.20 and disubstituted alkynes 1.21 1. 3 .3 Use s of 1 H -Benzotriazole and its Derivatives in Materials Chemistry 1. 3 .3.1 Benzotriazole derivatives as corrosion inhibitors Triazole containing compounds, such as 1H -benzotriazole are known to be efficient corrosion inhibitors. [09JAE269, 09JMPT1729, 09ME367] 1H benzotriazole reduces the rate of decay of metals or alloys such as Cu and Fe. [09JAE269] Recently b enzotriazole derivatives, 1 (2 thienylcarbonyl)benzotriazole 1.25 and 1 (2 pyrrolecarbonyl)benzotriazole 1.26 have been examined for their potential efficacy as corrosion inhibitors (Figure 1 1 1 ). [09JAE269]

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33 Figure 1 1 1 Structures of benzotriazole derivatives 1.25 and 1.26 1. 3 .3.2 Benzotriazole as a component of conjugated polymers Conjugated polymers have diverse applications, especially in sensors, LEDs, electrochromic devices and solar cells. [08CM7510] The efficac y of e lectrochromic materials depend s on its ability to reversibly change colors on modification of the redox state of the material [08CM7510] Despite major advances in materials chemistry, there still exists a demand for optically responsive materials wi th electrochr o mic properties for u se in data storage and display devices. [0 8 CM7510] Accordingly, Toppare and coworkers synthesized a novel donor acceptor type polymer poly(4,7-bis(2,3 -dihydrothieno[3,4 -b][1,4]dioxin5 yl) 2 -dodecyl 2 H benzo[1,2,3]triazole ) (PBEBT) 1.27 and investigated its electrochromic properties. Their results indicated that t he spectroelectrochemistry of PBEBT was nearly identical to the well known poly(ethylenedioxythiophene) (PEDOT) 1.28 with a maximum absorption of 618 nm (Figure 1 1 2 ). Also, 1.27 displayed advantages over 1.28 with respect to optical contrast, switching time and coloration efficiency Moreover 1.27 is capable of p and n -type doping, thus it is superior to 1.28 and a better option for use in electrochromic display d evices. Figure 1 1 2 Structures of conjugated polymers PBEBT 1.27 and PEDOT 1.28

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34 1. 3 .4 Aspects of Benzotriazole mediated S yntheses Benzotriazole -mediated acylation and imidoylation are examples of synthetically useful reactions that have been extensively studied. [03CEJ4586] In many instances the benzotriazole assisted strategy represents the most efficient routes to synthetic intermediates and target molecules [91T2683, 98CR409, 03CEJ4586] 1. 3 .4.1 Benzotriazole -assist ed i midoylation s Imidoylation reacti ons have received much attention by numerous researchers. Recently, Katritzky and coworkers developed g uanylating reagents d i(benzotriazolyl)methanimine 1.29 and benzotriazolylcarboximidoyl chlorides 1.30. As illustrated in Scheme 1 -4, the reaction of 1 H b enzotriazole and cyanogen bromide affords 1.29, while 1.30 is easily prepared from the reaction of isonit riles and 1 -chlorobenzotriazole Guanylating reagents 1.29 and 1.30 were then utilized in the preparation of tri and tetra -substituted guanidines (Sch emes 1 4) [03CEJ4586] Scheme 1 4. Preparation of guanidines from guanylating reagents 1.29 and 1.30 Chapter 1 of this study introduces novel C thiocarbamoylating reagents and describes their application in the preparation of C aminoimidoylating reagent s. Subsequently, reactions of the Caminoimidoylating and C thiocarbamoylating reagents with nucleophiles were explored. Chapter 2, an extension of Chapter 1, describes the preparation of C alkoxyimidoylating reagents.

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35 1. 3 .4.2 Benzotriazole -assisted N acyl ation s N-A cylbenzotriazoles can be prepared from (i) acyl halides and benzotriazole in the presence of a base, (ii) carboxylic acids and sulfonylbenzotriazole in the presence of Et3N [00JOC8210] and (iii) carboxylic acids treated with thionyl chloride and excess benzotriazole [03S2795] (Scheme 1 5 ). Scheme 1 5 Preparation of N acylbenzotriazoles 1. 31 It is of particular importance that N acylbenzotriazoles can be prepared from N protected amino acids and that these N -protected aminoacyl)benzotriazoles are chemically stable, crystalline and chirally pure. Chapters 4 7 will discuss aspects of the preparation of N -Fmoc aminoacyl)benzotriazoles and their use in microwave -mediated peptide chain extension. 1. 4 Conclud ing Remarks The exciting discoveries within the last few years demonstrate the incredible potential of 1 H benzotriazole methodology. This study contributes to the major advances in 1H benzotriazole chemistry by expanding the scope of 1 H benzotriazole metho dology to the syntheses of biologically important structural motifs and other molecules of interest. The aim of this study was to (i) discover and/or optimize synthetic routes to interesting compound classes, and (ii) assess the potential limitations of th is methodology.

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36 Herein the application of 1 H -benzotriazole methodology in the syntheses of precursors to heterocycles and small biomolecules is reported.

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37 CHAPTER 2 C-AMINOIMIDOYLATION AND C THIOCARBAMOYLATION O F ESTERS 2.1 Background Recently there has be en widespread focus on isothiocyanates (Figure 2 1) as a result of their usefulness in pharmacology, medic ine and industry. [06JPCA13195] Isothiocyanates, phytochemicals that frequently occur in cruciferous plants and vegetables, form one of several classe s of organic compounds possessing cumulat ive double bonds. [06JPCA13195, 91CR1, 05EJO 1184] Additional sources of isothiocyanates are blue -green algae, fungi and marine organisms. [05EJO 1184] Figure 2 1. General structure of isothiocyanates and carbodiim ides Isothiocyanates possess a range of activity, but are widely exploited for their in vitro and in vivo ant icancer activity. [06JPCA13195] In addition to their biological activity, they act as valuable synthetic intermediates for the preparation of biologically active heterocycles, for example, thiohydantoins ( 2.1 ), thiopyrimidone derivatives ( 2 .2 ) and pyridopyrimidinethiones (2.3 ) (Figure 2 2). [91CR1 ] Fi gure 2 2 Formation of heterocycles from isothiocyanates

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38 Similarly, carbodiimides are heterocumelenes that are useful for the construction of biologically heterocycles such as imidazolidinones ( 2.4 ) and quinazoline s ( 2.5 ) (Figure 2 3). [81CR589] In syntheses, carbodiimides are frequently employed as dehydrating agents in the preparation of pept ides and nucleotides. [81CR589] In contrast to isothiocyanates, carbodiimides are not naturally occurring and multiple methods have been devised for the syntheses of carbodiimides many of whi ch utilize thioureas [81CR589] Figure 2 3. Biologically active heter ocycles synthesized from carbodiimides While isothiocyanates and carbodiimides are valuable reagents, their utility has been somewhat limited by their great reactivity, moisture sensitivity, and the need for careful handling and storage. Therefore reaction s involving the use of these compound classes can be quite tedious. In preceding work, Katritzky and coworkers synthesized 1 (alkyl/arylthiocarbamoyl) benzotriazoles ( 2.6 ) [04JOC2976] and benzotriazole 1 carboxamidines ( 2.7 ) [05HCA1664], which are synthet ic equivalents of isothiocyanates and carbodiimides respectively. 1 (Alkyl/arylthiocarbamoyl)benzotriazoles ( 2.6 ) and benzotriazole 1 carboxamidines ( 2.7 ) are mainly stable solids that are moisture insensitive. [ 04JOC2976, 05HCA1664 ] It is postulated that reactions involving 2.6 and 2.7 occur via i) an E1cB mechanism with a concomitant in situ generation of the corresponding isothiocyanates and carbodiimides or ii) an addition -elimination mechanism (Scheme 2 1).

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39 Scheme 2 1. Representative reactions of 2. 6 and 2.7 with nucleophiles Recently, the utility of 1 (alkyl/arylthiocarbamoyl)benzotriazole thiocarbamoylating reagents ( 2.6 ) was reported in the synthes e s of di and trisubstituted thioureas ( 2.8 and 2.9 ) [04JOC2976], N-hydroxythioureas ( 2.10) and thios emicarbazides ( 2.11) [06ARK226] (Scheme 2 2). B enzotriazole 1 -carboxamidine N aminoimidoylating reagents ( 2.7 ) were also reported for the synthesis of 1,2,3-trisubstituted guanidines ( 2.12) [05HCA1664], Nhydroxy and N-amino guanidines ( 2. 13 and 2. 14) [06JOC6753] (Scheme 2 3). To expand the efficacy of 2.6 and 2.7 the reaction of 1 (alkyl/arylthiocarbamoyl) benzotriazole thiocarbamoylating reagents ( 2.6 ) and benzotriazole 1 -carboxamidine N aminoimidoylating reagents ( 2.7 ) with carbon nuc l eophiles was exam ined

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40 Scheme 2 2. Reactions of 1 (alkyl/arylthiocarbamoyl)benzotriazoles 2.6 Scheme 2 3. N -Aminoimidoylation with benzotriazole 1 -carboxamidines 2.7 2.2 Introduction The wide availability of acylating and imidoylating reagents reflects the wide atten tion given to C acylation [ 47JACS119, 03JOC1443, 59JACS4882] and C -imidoylation [97TL6771, 99OL977, 02JOC4667]. By contrast, C aminoimidoylation and C thiocarbamoylation are both

PAGE 41

41 relatively unexplored synthetically. In the literature examples of compounds that could conceptually have been made by C aminoimidoylation and C -thiocarbamoylation have generally been obtained via multiple steps. [ 04JOC188, 00JO C1583, 79S343, 83LA 290] A search of the literature disclosed no examples of the direct C aminoimidoylati on or C thiocarbamoylation of esters. Examples of the C aminoimidoylation products 2.16 were obtained by the nucleophilic attack of amines on isoxazolones 2.15 leading to ring opening and concomitant loss of CO2 (Scheme 2 4).[ 81JOC4068] Scheme 2 4. Prep aration of C aminoimidoylation product 2.16 Similarly potential C thiocarbamoylation products 2.17 were prepared from isothiocyanates, alcohols and carboxylic acids (Scheme 2 5). [04JOC188, 00JOC1583, 79S343, 83LA 290] Scheme 2 5. Preparation of C thioca rbamoylation product 2.17 Therefore a general straightforward approach to the synthesis of C aminoimidoylation and C-thiocarbamoylation products would be useful. Now the C aminoimidoylation and C -

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42 thiocarbamoylation of esters via 1 (alkyl/arylthiocarbamoyl) benzotriazoles ( 2.6a -c) and benzotriazole 1 -carboxamidines ( 2.7a b ) is reported 2.3 Results and Discussion 2.3.1 Preparation of 1-( A lkyl/aryl -thiocarbamoyl)benzotriazoles 2.6a d and Benzotriazole 1 -carboxamidines 2. 7a c. Bis(benzotriazolyl)methanethione [78JOC337] 2.18 and amines afforded 1 (alkyl thiocarbamoyl)benzotriazoles 2.6a f,h k or 1 (arylthiocarbamoyl)benzotriazoles 2.6g, which were converted in turn by iminophosphoranes into the benzotriazole -1 -carboxamidines 2.7a i (Scheme 2 6). Scheme 2 6. Pre paration of reagents 2.6 and 2.7 2.3.2 C -Aminoimidoylation a nd C -Thiocarbamoylation o f D oubly A ctivated Esters Reactions of 2.0 equiv. of ester enolates (from ester 2.1 9 a -c with 2.5 equiv potassium tert butoxide) with 1.0 equiv of benzotriazole 1 carboxami dines 2.7a c or 1 (alkylthiocarbamoyl)benzotriazoles 2.6b,d afforded 2. 20a c 2.2 1 a,b respectively; after limited optimization, yields of 20 51% were obtained (Scheme 2 7, Table 2 1). Elemental analysis HRMS and NMR spectral data support the structural as signments ( See 2. 5 Experimental Section ).

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43 Scheme 2 7. Preparation from esters 2.1 9 a -c of the C aminoimidoylation products 2. 20a -c and the C -thiocarbamoylation products 2.2 1 a,b Table 2 1 C -Aminoimidoylation and C thiocarbamoylation of esters 2.1 9 a c to give 2. 20a -c 2.2 1 a,b Ester R1 R2 Reagent R3 R Product Yield, % 2.1 9 a CO 2 Et Et 2.7a CH 3 (CH)Ph m CNC 6 H 4 2. 20 a 24 2.1 9 b CO 2 Me Me 2.7b CH 2 CH(CH 3 )CH 2 CH 3 p Tol 2. 20 b 51 2.1 9 c CN Et 2.7c Allyl m CNC 6 H 4 2. 20 c 20 2.1 9 b CO 2 Me Me 2.6b CH 3 (CH)Ph 2.2 1 a 27 2.1 9 a CO 2 Et Et 2.6d (CH 2 ) 2 Ph 2.2 1 b 49 Different tautomeric structures are possible for the ester products. The determination of the precise tautomeric structure of the compounds was of interest due to the influence of tautomeric structures on the physical properties [for example, boiling point, solubility] and chemical properties [for example : acid ity /bas icity, reactivity] of compounds. X ray data suggests that the compounds 2.2 1 a,b would exist as the keto ( CH ) rather than enol ( OH) tautomer in th e solid st ate (Figure 2 4 ).[ 07JOC6742] Conceptually, in solution three tautomeric forms of 2.2 1 can be present that is, t he keto ( 2.2 1 ), enol ( 2.2 1 ) and the enethiol (2.2 1 ) forms may coexist in a dynamic equilibrium (Figure 2 4 ). [05TEC284] Also it is known tha t the extent of the keto -enol -enethiol tautomerism of compounds is dependent on the compound structure and the nature of the solvent. [05TEC284] Compounds 2. 2 1 a ,b were examined in chloroform d at 25 C and 2D NMR verified the existence of a single form, th e keto (CH) tautomer, under the conditions examined (Figure 2 5 ).

PAGE 44

44 Figure 2 4 Possible tautomeric forms of 2.20 Figure 2 5 Relevant 1H and 13C chemical shifts in compounds 2.2 1 a,b Compounds 2. 20a -c were examined in chloroform d at 25 C to determine whether they ex ist in the enol or keto forms. On examination in chloroform d at 25 C, the NMR spectra indicated the absence of keto tautomers. Coupling of one of the NH protons with the alpha protons on R3 allowed assignment of the NH protons, and suppor ted the conclusion that 2. 20a -c are present in solution as the keto -enamine taut omers (2. 20) and not the corresponding amidine enol isomers (2. 20 ), as depicted in Figure 2 6 For 2. 20c two conformers are possible in solution however, the NMR spectra displayed signals from either a single stereoisomer (E

PAGE 45

45 isomer) or an average structure as the rotation about the C=C is quite rapid when compared wi th the NMR timescale [04JOC188] Additionally, there are four probable C N rotameric conformations for 2 20a -c These are the syn syn (ss), syn anti (sa ), anti syn (as ) and anti anti (aa) conformations (Scheme 2 8) [04JOC188] A survey of the literature di sclosed that 2 20a,b should adopt the unusual sterically demanding aa conformation as the two stabilizing intramolecular hydrogen bonds compensate for the steric hindrance between R and R3. On the other hand, 2 20c should preferentially adopt the more favo red sa or as conformation but may assume the aa conformation although it is capable of a single stabilizing hydrogen bond. [4JOC188] Figure 2 6 Relevant 1H and 13C chemical shifts in CDCl3 of keto -enamine tautomeric structures 2. 20a -c

PAGE 46

46 Scheme 2 8. F our possible rotameric conformations of 2 20a -c For 2. 20a -c t he formation of the keto enamine tautomer was further corroborated by IR spectroscopy. The IR spectra of 2. 20c indicated the presence of a hydrogen bonded NH at 3246 cm1, while t he carbonyl and alkene stretching frequencies were observed at 1655 and 1605 cm1 respectively. The carbonyl frequency was characteristic for a conjugation effect of the aminal group and intramolecular hydrogen bonding of the aminal NH with the carbonyl group. [04JOC188] In the literature c ompounds 2. 20a -c are known as N N -disubstituted ketene aminals, important compounds for the construction of heterocyclic systems.[04JOC188] Also, the N, N disubstituted k etene aminals, referred to as 1,1 -enediamines and -dioxoketene aminals, are considered bioisosteres of urea and are an important class of compounds in the fields of agriculture and medicine. [04JOC188] The heterocyclic analogues to N, N -disubstituted k etene aminals heterocyclic ketene aminals (HKAs) h ave also garnered much attention in recent decades [07S L 761] Literature methods for the preparation of N, N -disubstituted ketene aminals include the reaction of (i) activated methylene compounds and isothiocyanates, [04JOC188] ii) oxoketene N, S acetals an d lithiated secondary amines or aniline [00JOC1583], or (iii) tris(dimethylamino)ethoxymethane and simple ketones [79S343, 83LA 290] Methods i iii (Scheme 2 9 ) each consist of multi ple steps with average overall yields of ca 30%, whereas the

PAGE 47

47 current C ami noimidoylation method provides a direct access to these compound classes in comparable yields. Scheme 2 9 Literature methods for the preparation of ketene aminals 2.2 2 2.3. 3 C -Aminoimidoylation and C -Thiocarbamoylation of U nactivated Esters and O ther A ctivated Compounds 2.3.3.1 Reactions with unactivated esters Reactions of 2.0 equiv of ester enolates (from ester 2.1 9 d -f with 2.5 equiv potassium tert butoxide) with 1.0 equiv of benzotriazole 1 carboxamidines 2.7 d,e or 1 -(alkylthiocarbamoyl) benzotriazo les 2.6b, e,f afforded 2. 2 3 a ,b 2.2 4 a -d respectively; after limited optimization, yields of 2 8 49% were obtained (Scheme 2 10, Table 2 2 ). Elemental analysis HRMS and NMR spectral data support the structural assignments (See 2. 5 Experimental Section ). Sc heme 2 10. Preparation from esters 2.1 9 d -f of the C aminoimidoylation products 2.2 3 a,b and the C -thiocarbamoylation products 2.2 4 a -c

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48 Table 2 2. C -Aminoimidoylation and C thiocarbamoylation of esters 2.1 9 d -f to give 2. 2 3 a ,b 2.2 4 a -c Ester R1 R2 Reagent R3 R Product Yield, % 2.1 9 d H i Pr 2.7 d i Pr p Tol 2.2 3 a 33 2.1 9 d H i Pr 2.7 e i Pr p CO 2 i PrC 6 H 4 2.2 3 b 49 2.1 9 d H i Pr 2. 6 f Bn 2.2 4 a 2 8 2.1 9 e H Me 2.6 b CH(CH 3 )Ph 2.2 4 b 40 2.1 9 e H Me 2.6 e i Pr 2.2 4 c 71 2.1 9 f Cyclopropyl Et 2.6e i Pr 2.2 4 d 4 5 Ex amination of the C aminoimidoylation and C thiocarbamoylation of unactivated esters yielded O alkyl isoureas 2.2 3 a ,b and O alkyl thiocarbamates 2.2 4 a d and not the expected C aminoimidoylated and C thiocarbamoylated ester products. Although potassium t -but oxide is a bulky base, t he bulkiness of the t -butyl group did not hinder the nucleophilic addition of the potassium t -butoxide to the carbonyl group of the ester 2.1 9 d -f. [06JACS14268] The subsequent reaction of the alkoxide of the ester 2.1 9 d -f and benzot riazole 1 carboxamidines 2.7 d,e or 1 (alkyl thiocarbamoyl)benzotriazoles 2.6b, e,f afforded the products 2.2 3 a,b and 2.2 4 a -d In the case of benzotriazole 1 -carboxamidine 2.7e transe s terification of the aromatic ester substituent also occurred. As expected t he 1H NMR spectra of O alkyl thiocarbamates 2.2 4 a d and O alkyl isoureas 2.2 3 a ,b indicated the presence of rotamers in a ratio of approximately 1:1. In the solid state i t is well established that thioamides for example 2.24a -d are stable and exist in a s ingle form. H owever, in solution thioamides exist as a dynamic equilibrium of the cis and trans amide forms due to the relatively low energy barrier to rotation of the amino group in thioamides (Figure 2 7 ). [88BKCS236] While the energy barrier to rotation around the C -N bond of a thioamide is relatively low, it is somewhat larger than the barrier to rotation about the C -N bond of an amide (usually 1620 kcal mol1 in magnitude). Thus t he electron delocalization in amides and

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49 thioamides probably results in a higher rotational barrier around the C N bond when compared to a normal C N bond. The presence of conformational isomers in the O alkyl isoureas 2.2 3 a,b can also be rationalized by moderately low rotational barriers Figure 2 7 Trans and cis forms of thioamides The formation of O alkyl isoureas was further corroborated by IR spectroscopy. Compound 2.2 3 b displayed strong IR absorption bands for a non -hydrogen bonded NH a carbonyl group and amidine C=N at 3373, 1701 and 1641 cm1 respectively Mostly, the C aminoimidoylation and C -thiocarbamoylation of unactivated esters proceeded by reaction of the alkoxides of the unactivated esters however reactions of benzyl acetate with 2.6j,k and 2.7 e with methyl 2 (napthalen 1 -yl)acetate did not yield products In addition to O alkyl isoureas 2.2 3 t he hydrolysis of C aminoimidoylating reagents 2.7f,h yielded ureas 2.2 5 (Scheme 2 11). Scheme 2 11. Preparation of ureas 2.2 5 a, b fro m benzotriazole 1 -carboxamidines 2.7f,h The functions of O alkyl isoureas 2.2 3 O a lkyl thiocarbamates 2.2 4 and ureas 2.2 5 are diverse. O -A lkyl thiocarbamates and O alkyl isoureas find potential use as anti HIV drugs [93PR1076] and synthetic intermediates in the preparation of primary alkyl bromides [08S3565]

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50 respectively. Versatile u rea s are often used as neutral and extremely directional binding units in anion receptors. [06CCR3200] 2.3.3.2 Reactions with sulfones and cyano compounds C-A minoimidoylation of carbon nucleophiles derived from sulfones was examined. The reaction of C aminoim idoylating reagent 2.7 f with ethyl phenyl sulfone (2.0 equiv.) in the presence of potassium t -butoxide (2.5 equiv.) failed to generate the desired product. Therefore the reaction was repeated using 2.7 a and 2.7g and benzyl phenyl sulfone (2.0 equiv.) in t he presence of n BuLi (2.0 equiv.) The product of the reaction of n BuLi with 2.7g was the replacement of the benzotriazo yl group with the butyl group. Next, the more sterically demanding LDA was reacted with 2.7a but again no product w as formed. Likewis e, the reaction of (i) 2.6b,e with malononitrile in the presence of potassium t butoxide and (ii) 2.6b and malononitrile in the presence of NaH did not afford the corresponding C-thio carbamoylation products. C thiocarbamoylating reagents 2.6c,i,f were reacted with ethyl cyanoacetate in the presence of potassium t butoxide, but again, no products were formed. 2.3.3.3 Reactions with nitro compounds C-A minoimidoylation and C -thiocarbamoylation of nitro compounds were examined in the presence of 2.0 equiv. of t he nitronate anion (made from the n itro compound and 2.5 equiv. of potassium t -butoxide). In most cases, with the exception of 2.6f the desired C thiocarbamoylation and C aminoimidoylation product s w ere not obtained. Changing the solvent to DMSO and the base to NaH did not alter the outcome of the reaction. The reaction of 2.6 f with ethyl nitroacetate and nitromethane produced the corresponding C-thiocarbamoylation products, however, these compounds were unstable and decomposed subsequent to the acquisitio n of the 1H NMR (For example of product 1H NMR see Figure 2 8 ).

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51 Figure 2 8 1H NMR spectrum of the reaction of 2.6f with ethyl nitroacetate 2.4 Conclusion s and Outlook Nontraditional approaches to the preparation of C t hiocarba moylated ester products 2. 21, N, N -disubstituted ketene aminals 2. 20, O alkyl isoureas 2.2 3 and O alkyl thiocarbamates 2.2 4 were outlined. Compounds 2. 20 and 2. 21 were prepared via t he successful C aminoimidoylation and C thiocarbamoylation of este rs, in 34% average yield under mild reaction conditions. While optimization of the reaction conditions is required, t his method still provides an easy access to interesting classes of compounds that may be used for further synthetic transformations. C-A min oimidoylating reagents 2.7 possess the amidine functional group. Amidines are important in organic chemistry and are often employed as bases. [08ARK153] The presence of the electon donor benzotriazolyl group may introduce interesting reactivity to 2.7 and may warrant investigation

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52 To summarize, C -thiocarbamoylating reagents 2.6 and C aminoimidoylating reagents 2.7 hold great promise in synthetic organic chemistry. The chemical reactivity of 2.6 and 2.7 require greater examination in order for the full pote ntial of these reagents to be evaluated. 2. 5 Experimental Section 2.5.1 General Column chromatography was conducted on flash silica gel (200425 mesh). Visualization of TLC plates was via UV and phosphomolybdic acid staining. Melting points were determined on a hot -stage apparatus and are uncorrected. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were determined in CDCl3 with TMS as the internal standard. 2. 5 2 General Procedure for the Preparation of 2. 20a -c, 2.2 1 a,b 2.2 3 a,b, 2.2 4 a -d 2.2 5 a,b To a solutio n of the desired ester 2.1 9 (2.0 mmol) in THF (15 mL), was added potassium t butoxide (2.5 mmol). After st irring the mixture for 30 min, 1.0 mmol of the desired reagent 2.6 or 2.7 were added to the reaction mixture. The progress of the reaction was monitor ed by TLC. Upon completion, water (20 mL) was added to quench the reaction followed by extraction with dichloromethane (3 x 30 mL). The combined extracts were dried over magnesium sulfate and the solvent removed under vacuum. The crude mixture was purified by gradient column chromatography over silica gel (EtOAc hexanes) to give the desired products in moderate yields. Diethyl -2 -(3 -cyanoanilino)[1-phenethyl)amino]methylene}malonate ( 2. 20 a). Recrystallized from EtOAc h exanes to give white microcrystals (24%) ; mp 99.6100.6 oC; 1H NMR (CDCl3 J = 6.0 Hz, 1H), 7.367.34 (m, 2H), 7.247.23 (m, 5H), 6.876.84 (m, 2H), 4.264.12 (m, 5H), 1.40 (d, J = 6.0 Hz, 3H), 1.32 (t, J = 15.0 Hz, 6H); 13C NMR (CDCl3, 125.3, 118.1, 113.2, 103.4, 81.2, 60.2, 54.7, 24.5,14.3. HRMS calcd for C23H25N3O4 [M + H]+ 408.1918, found 408.1917.

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53 Dimethyl -2 -[[(2 -methylbutyl)amino](4 -methyltoluidino)methylene]malonate ( 2. 20b) Yellow oil (51%); 1H NMR (CDCl3 s, 1H), 7.11 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 6.0 Hz, 2H), 3.74 (s, 6H), 2.972.58 (m, 2H), 2.31 (s, 3H), 1.561.44 (m, 1H), 1.31 1.21 (m, 1H), 1.06 1.04 (m, 1H), 0.84 (d, J = 9.0 Hz, 3H), 0.77 (t, J = 9.0 Hz, 6.0 Hz, 3H); 13C NMR (CDCl337.2, 134.1, 129.6, 122.5, 77.8, 51.1, 35.1, 26.6, 20.8, 17.1, 11.1. Anal. Calcd for C18H26N2O4: C, 64.65; H, 7.84; N, 8.38. Found: C, 65.03; H, 8.17; N, 8.50. Ethyl -3 -(allylamino) -2 -cyano -3 -[(3 -cyanophenyl)imino]propionate (2. 20c). Yellow oil (20%); 1H NM R (CDCl3) 10.76 (br s, 1H), 9.36 (br s, 1H), 7.507.33 (m, 4H), 5.765.70 (m, 1H), 5.24 (m, 2H), 4.24 (quartet, J = 9.0 Hz, 2H), 3.57 (t, J = 6.0 Hz, 2H), 1.34 (t, J = 9.0 Hz, 6.0 Hz, 3H) ; 13C NMR (CDCl3) 169.9, 162.5, 140.4, 132.3, 130.9, 128.4, 125.6, 124.4, 119.4, 118.6, 118.4, 114.1, 61.0, 47.8, 30.1, 14.9. HRMS calcd for C16H16N4O2 [M+ H]+ 297.1346, found 297.1341. Dimethyl 2 -{[(1 -phenylethyl)amino]carbothioyl}malonate (2.2 1 a). Yellow oil (27%); 1H NMR (CDCl3) 9.43 (br s, 1H), 7.39 7.26 (m, 5H), 5.66 (quint et, J = 9.0 Hz, 1H), 5.03 (s, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 1.63 (d, J = 9.0 Hz, 3H); 13C NMR (CDCl3) 187.3, 165.6, 165.4, 140.7, 128.3, 127.2, 125.8, 65.1, 54.7,53.2, 53.1, 20.1. Anal. Calcd for C14H17NO4S: C, 56.93; H, 5.80; N, 4. 74. Found: C, 57.30; H, 6.02; N, 4.65. Diethyl 2 -{[(1 -phenethyl)amino]carbothioyl}malonate (2.2 1 b). Yellow oil (49%); 1H NMR (CDCl3) 9.23 (br s, 1H), 7.337.23 (m, 5H), 4.96 (s, 1H), 4.20 ( q uartet J = 6.0 Hz, 4 H), 3.95 ( q uartet J = 6.0 Hz, 2H), 2.99 (t, J = 9.0 Hz, 6.0 Hz, 2H), 1.25 (t, J = 9.0 Hz, 6.0 Hz, 6H); 13C NMR (CDCl3) 189.2, 165.2, 137.9, 128.5, 128.4, 126.5, 65.6, 62.6, 47.3, 33.3, 13.6. Anal. Calcd for C16H21NO4S: C, 59.42; H, 6.54; N, 4. 33. Found: C, 59.36; H, 6.67; N, 4.36.

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54 1 -{[(Z)-I sopropoxy(isopropylami no)methylidene]amino} -4 methylbenzene (2.2 3 a). Yellow oil (33%); 1H NMR (CDCl3) 7.08 (d, J = 7.8 Hz, 2H), 6.76 (d, J = 8.1 Hz, 2H), 5.19 (quintet, J = 14.7, 6.0 Hz, 1H), 3.79 3.6 9 (overlapped m, 1 H), 3.68 (overlapped br s, 1H) 2.30 (s, 3H), 1.33 (d, J = 6.3 Hz, 6H), 1.04 (d, J = 6.3, 6H); 13C NMR (CDCl3) 152.9, 146.8, 131.6, 130.5, 123.1, 68.6, 43.6, 23.9, 22.4, 21.2. Anal. Calcd for C14H22N2O: C, 71.76; H, 9.46; N, 11.95. Found: C, 71.50; H, 9.93; N, 11.59. Isopropyl 4 -{[(Z)-isopropoxy(phenethylamino) methylidene]amino}benzoate (2.2 3 b). Colorless oil (49%); 1H NMR (CDCl3) 7.90 ( d J = 8. 7 Hz, 2H), 7. 31 7. 18 (m, 3 H) 7. 10 (d J = 6.9 Hz, 2H ), 6.80 (d, J = 8.7 Hz, 2H), 5.2 7 5.1 0 (m 2H), 3.92 (br s, 1H), 3.31 (quartet, J = 5.9 Hz, 2H), 2.72 (t, J = 6.9 2 H), 1.3 7 1.3 1 (m, 12H); 13C NMR (CDCl3) 166.2, 153.8, 152.1, 138.8, 131.1, 128.8, 128.6, 126.5, 124.5, 122.6, 69.1, 67.8, 42.8, 36.7, 22.0. HRMS calcd for C22H28N2O3 [M+ H]+ 369.2173, found 369.2180. O -I sopropyl N -benzylcarbamothioate (2.2 4 a) Colorless o il (28%); 1H NMR (CDCl3) (ca. 1:1 mixture of rotamers) 7. 40 7. 19 (m, 5H), 7.09 (br s, 1H), 6.42 (br s, 1H), 5.62 5. 48 (m, 1H), 4.74 (d, J = 5.4 Hz, 1 H), 4.4 0 (d, J = 5.7 Hz, 1H), 1.3 2 (d, J = 6. 3 Hz, 6H); 13C NMR (CDCl3) 189.8, 189.0, 136. 8 136.7, 128. 7, 128.7, 127.9, 127.8, 127.7, 127.6, 76.0, 74 .0, 49.0, 4 7.0 21.8, 21.7. Anal. Calcd for C6H15NO S 0.2H2O : C, 62.05; H, 7.29; N, 6.58. Found: C, 62.41; H, 7. 55; N, 6.7 8. O -M ethyl N (1 -phenylethyl)carbamothioate (2.2 4 b). Yellow oil (40%); 1H NMR (CDCl3) (ca 1:1 mixture of rotamers) 7.3 8 7.2 3 (m, 10H), 7.01 (br s, 1H), 6.49 (br s, 1H), 5.45 (quintet, J = 7.5 Hz, 1H), 4.97 (quintet, J = 7. 1 Hz, 1H), 4.00 (s, 3H), 3.97 (s, 3H), 1.57 (d, J = 6.9 Hz, 3H), 1.50 (t, J = 6. 8 Hz, 3H) ; 13C NMR (CDCl3) 190.3, 190.1, 142.2, 142.0, 128.6,

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55 127.5, 127.4, 126.2, 126.0, 125.7, 58.3, 56.9, 54.4, 53.0, 22 .2, 21.0 Anal. Calcd for 2( C10H13NOS) : C, 61.50; H, 6.71; N, 7.17. Found: C, 61.65; H, 6.92; N, 6.98 O -M ethyl N isopropylcarbamothioate (2.24 c). Colorless oil ( 71%); 1H NMR (CDCl3) (ca. 1:1 mixtur e of rotamers) 7.03 (br s, 1H), 6.30 (br s, 1H), 4.46 4. 28 (m, 1H), 4.07 (overlapped s 3H), 4.063.96 ( overlapped m, 1H), 3.96 ( overlapped s, 3H), 1.24 (d, J = 6.6 Hz, 6H), 1.18 (d, J = 6.5 Hz, 6H); 13C NMR (CDCl3) 189.5, 189.4, 57.9, 56.4, 47.0, 45.3, 22.1, 21.7. Anal. Calcd for C5H11NO S : C, 45.08; H, 8.32; N, 10.51. Found: C, 45.06; H, 8.55; N, 10.34. O -Ethyl N isopropylcarbamothioate (2.24 d) Colorless oil (4 5 %); 1H NMR (CDCl3) ( ca. 1:1 mixture of rotamers) 6.71 (br s, 1H), 6.11 (br s, 1H), 4.56 (q, J = 7. 1 Hz, 2H), 4.45 (overlapped q, J = 7.0 Hz, 2H), 4. 41 4.31 (overlapped m, 1H), 4.0 9 3.94 ( overlapped m, 1H), 1.37 (t, J = 7. 1 3H), 1.31 (t, J = 7.2 Hz, 3H), 1.24 (d J = 6.6 Hz, 6H), 1.18 (d, J = 6.6 Hz, 6H) ; 13C NMR (CDCl3) 189.4 68.1, 66. 3 47. 3 45.6, 22. 7, 22.3, 14.6. Anal. Calcd for C6H13NO S : C, 48.94; H, 8. 90; N, 9 .51 Found: C, 4 9 72; H, 9 37; N, 9 29. 1 -Butyl -3 -(3 -cyanophenyl)urea (2.2 5 a). Colorless oil (40%); 1H NMR (CDCl3) 7.78 (s, 1H), 7.62 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.30 (overlapped t, J = 8.0 Hz, 1H), 7.24 (overlapped t, J = 7.4 Hz, 1H), 5.64 (t, J = 5.4 Hz, 1H), 3.23 (q, J = 6.6 Hz, 2H), 1.53 1.41 (m, 2H), 1.401.24 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) 155.9, 140.1, 129.8, 125.9, 123.4, 122.1, 118.8, 112.5, 40.0, 32.0, 20.0, 13.7. Anal. Calcd for C12H15N3O: C, 66.34; H, 6.96; N, 1 9. 34. Found: C, 66.58; H, 6.37; N, 18.86. 1 -Phenyl -3 -p -tolylurea (2.25 b). White solid (65%); 1H NMR (CDCl3) 7.44 7.29 (m, 5H), 7.26 (d, J = 4.5 Hz, 2H), 7.07 (d, J = 8.1 Hz, 2H), 3.72 (s, 2H), 2.28 (s, 3H) ; 13C NMR

PAGE 56

56 (CDCl3) 169.4, 135.4, 134.9, 134.5, 130.0, 129.8, 129.6, 128.0, 120.3, 21.2. HRMS calcd for C14H14N2O [M+ H]+ 226. 1242, found 226. 1106.

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57 Figure 2 9 IR spectrum of 2. 20c Figure 2 10. IR spectrum of 2.2 3 b

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58 Figure 2 1 1 High Resolution Mass Spectrum of 2.2 3 b

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59 Figure 2 12. 1H NMR spectrum of 2.2 4 d Figure 2 13. 13C NMR spectrum of 2.2 4 d

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60 Figure 2 14. 1H NMR spectrum of 2.2 5 a Figure 2 1 5 13C NMR spe ctrum of 2.2 5 a

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61 Figure 2 1 6 1H NMR spectrum of 2.2 5 b Figure 2 17. 13C NMR spectrum of 2.2 5 b

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62 CHAPTER 3 C-ALKOXYIMIDOYLATION AND C -ARYLTHIOIMIDOYLATION 3 .1 Introduction Isothioureas [96TL2619, 08JOC2003, 05TL7597], isoureas [ 95RCR929, 04T61], keten e O Nand S N acetals [90S195] are all useful building blocks in the construction of many heterocycles [96TL2619, 08 JOC2003, 04T61, 95RCR929 90S195] and frequently form part of the structure of biologically active compounds (For examples, see Figure s 3 -2 3 3 and 34 ). Figure 3 1. General Structure of isothioureas, isoureas, ketene O N and S N acetals The applications of isothioureas are diverse and they are widely employe d especially in the fields of medicine and ag riculture. Examples include 3 1 [08JOC2003] an isothiourea that possesses potent herbicidal activity, and 3. 2 [05TL7597], a commercial drug known as Clobenpropit that is an anticonvulsant (Figure 3 2) Figure 3 2. Specific examples of isothioureas applied in medicine and agriculture Isothioureas are widely use d for example, (i) as solubility modulators [04JOC1571], (ii) potent inhibitors of nitric oxide synthases (NOSs) [96TL2619, 96EJP341, 96PBB179], (iii) to treat acute kidney failure, septic shock, and to prevent organ rejection after transplant surgery [96TL2619, 96EJP341, 96PBB179]. Also, isothioureas are postulated to be potent agonists of

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63 neurodegenerative diseases, such as Alzheimers disease (AD) [96TL2619] In synthesis, i sothioureas are va luable intermediates in the construction of heterocycles as well as guanidines [05TL7597, 07JOM 545] Isoureas, frequently used as synthetic intermediates in carbodiimide -based peptide coupling, are known appetite suppressants, agricultural agents, inhibito rs of pheromone synthesis and antihypertensive agents [04T61, 02JC A 293, 08BMC8210]. Trehazolin ( 3. 3 ), the first na tural cyclic isourea derivative of carbohydrates, exhibits potent activity as a t rehalase inhibitor (Figure 3 3). [04T61] Trehalase is a glyco side hydrolase enzyme found in most animals and is responsible for the conversion of trehalose to glucose. Figure 3 3. Trehazolin ( 3.3 ), a natural cyclic isourea Ketene O Nand S N acetals serve as versatile building blocks in the preparation of functi onalized heterocycles. K etene O Nand S N acetals for example t rifluoroacetylketene O,N and S,N -acetals ( 3. 4 ) and ( 3. 5 ) respectively are easily accessed and potential ly useful medicinal and agricultural agents (Figure 3 4). [90S195] Figure 3 4 Exam ples of ketene O N and S N acetals

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64 Multiple methods are available for the synthesis of these four compound classes ; h owever, the ir use as potential drugs is limited due to the lack of general synthetic routes. Previously, Katritzky and coworkers synthesize d (i) 1,2,3 trisubstituted guanidines [05HCA1664], Nhydroxy and N-amino guanidines [04JOC2976] (ii) di and trisubstituted thioureas [06JOC6753], N-hydroxythiourea s and thiosemicarbazides [06ARK2 26] using novel 1 (alkyl/arylthiocarbamoyl)benzotriazole th iocarbamoylating reagents. Now the syntheses of novel benzotriazole 1 -carboximidates 3. 12 and benzo triazole 1 carboximidothioates 3. 13 are describe d It will be demonstrate d that esters, ketones and amines can be C alkoxyimidoylated by benzotriazole 1 -car boximidates 3. 12 and C -aryl thioimidoylated by benzotriazole 1 -carboximidothioates 3. 13 to provide isothioureas, isoureas, ketene O N and S Nacetals. This simple procedure comprises nucleophilic substitution of the benzotriazolyl group in benzotriazole 1 -c arboximidates 3.12 and benzotriazole 1 -carboximidothioates 3.13 by the appropriate carbon and nitrogen nucleophiles. 3.2 Results and Discussion Bis(benzotriazolyl)methanethione 3. 6 [78JOC337] reacted with phenols ( 3. 7 ) to afford O aryl -benzotriazole carbot hioate s 3. 9 Similarly, the reaction of 3. 6 with p toluenethiol 3. 8 afforded S aryl -benzotriazole carbodithioate 3. 10. The synthesis of benzotriazole 1 carboximidates 3. 12 was achieved by reacting 3. 9 with iminophosphoranes 3. 11 (Scheme 3 1). Reaction of 2 .0 equiv of ester enolates (from ester 3.14 with 2.5 equiv potassiu m tert butoxide) with 1.0 equiv of benzotriazole 1 carboximidates 3. 12a, afforded 3. 15; after limited optimization, a yield of 54% was obtained (Scheme 3 2).

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65 Scheme 3 1. Preparation of O aryl -benzotriazole carbothioate ( 3. 9 ), S aryl benzotriazole carbodithioate ( 3. 10) and benzotriazole 1 carboximidates ( 3.12) Scheme 3 2. Synthesis of product 3.1 4 3. 3 Conclusion A facile route to the synthesis of C alkoxyimidoylated ester products has b een developed. Optimization of the procedure is still required and further experimental work is necessary to determine the scope of these reactions. 3.4 Experimental Section Melting points were determined on a hot -stage apparatus and are uncorrected. 1H (3 00 MHz, with TMS as the internal standard) and 13C NMR (75 MHz) NMR spectra were recorded in CDCl3. Elemental analysis was carried out in an Eager 200 CHN analyzer. 3.4.1 General Proc edure for the Preparation of O A ryl benzotriazole C arbothioate 3. 9 or S A r ylbenzotriazole C arbodithioate 3. 10a Bis -b enzotriazol 1 -yl -methanethione 3. 6 (1 mmol) was dissolved in methylene chloride (10 mL). In another flask, the appropriate phenol 3. 7 or thiol 3. 8 (1 mmol) and sodium hydride

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66 (5 mmol) were combined in methylene chl oride (10 mL) and stirred for 15 min. The sodium salt of the former was added to the bis -bezotriazol 1 -yl -methanethione solution and the resulting mixtu re stirred at room temperature. The progress of the reaction was monitored by TLC. On completion of the reaction, water (20 mL) was added to the reaction mixture, which was then extracted with m ethylene chloride (3 x 30 mL). The combined organic extracts was washed with 10% sodium carbonate solution (3 x 30 mL), dried over magnesium sulfate and the solvent removed in vacuo. The crude mixture was purified by gradient column chromatography over silica gel ( E thyl acetate/hexanes) to give O aryl -benzotriazole 1 -carbothioates 3. 9a-c or S a ryl b enzotriazole 1 -carbodithioates 3. 10a in 3 4 61% yield. O -(4 -Ethylphenyl) 1 H -1,2,3 -benzotriazole -1 -carbothioate ( 3. 9a). Recrystallized from EtOAc h exanes to give p ale yellow crystals (34%); mp 91 7 94.1 oC; 1H NMR 8.49 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 7. 8 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 2.74 (q, J = 7. 6 Hz, 2H), 1.30 (t, J = 7.5 Hz); 13C NMR 182.4, 150.6, 146.6, 143.3, 131.6, 130.7, 129.2, 126.3, 121.7, 120.8, 115.1, 28.3, 15. 3 Anal. Calcd for C15H13N3O: C, 63.58; H, 4.62; N, 14.83. Found: C, 63.77; H, 4.54 ; N, 14.88. O -(2,6 -D imethylphenyl) 1 H -1,2,3 -benzotriazole -1 -carbothioate ( 3. 9b) Yello w oil (35%) 1H NMR 8.53 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H) 7.72 (t, J = 7.8 Hz, 1H), 7.56 (t, J = 7.7 Hz 1H), 7.227.16 ( m 3H), 2.27 (s, 6H); 13C NMR 180.1, 150.0, 146.7, 131.7, 130.8, 130.4, 129.1, 127.1, 126.3, 120.9, 115.1, 16.3. HRMS calcd for C15H13N3O S [M+ H]+ 284.0852, fou nd 284.0837. O -(3,5 -D imethylphenyl) 1 H -1,2,3 -benzotriazole -1 -carbothioate ( 3. 9c). Recrystallized from EtOAc h exanes to give yellow c rystals (37%) ; mp 127.7 129.8 oC; 1H NMR 8.48 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 7. 7 Hz, 1H), 7.5 5 (t, J = 7.8 Hz, 1H), 7.03 (s,

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67 1H), 6.90 (s, 2H), 2.40 (s, 6H); 13C NMR 182.3, 152.6, 146.6, 139.9, 131.6, 130.7, 128.9, 126.2, 120.8, 119.4, 115.1, 21.3. Anal. Calcd for C15H13N3O: C, 63.58; H, 4.62; N, 14.83. Found: C, 63.70; H, 4.53; N, 14.78. 4 -M eth ylphenyl 1 H -1,2,3 -benzotriazole -carbodithioate ( 3. 10a ). Recrystallized from EtOAc h exanes to give yellow c rystals (61%) ; mp 86. 6 88. 4 oC; 1H NMR 8.71 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.6 6 (t, J = 7. 8 Hz, 1H), 7.53 (t, J = 7. 5 Hz, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 2.47 (s, 3H); 13C NMR 198.4, 146.9, 141.0, 135.8, 132.0, 130.7, 130.2, 126.2, 125.4, 120.3, 115.3, 21.2 Anal. Calcd for C14H11N3S2: C, 58.92; H, 3.88; N, 14. 72. Found: C, 58.98; H, 3.75; N, 14. 69. 3.4.2 Gen eral P rocedure for the P reparation of Benzotriazole -1 -carboximidates ( 3. 12) To a solution of O aryl -benzotriazole1 -carbothioates 3. 9 (1 mmol) in toluene (12 mL), was added the i minophosphorane 3.11 (1 mmol). The mixture was stirred for 5 min then refluxed for 12 h. The solvent was removed in vacuo and the crude product was chromatographed over silica gel using a gradient of ethyl acetate/hexanes. Further purification by crystallization from ethyl acetate/hexanes yielded pure benzotriazole 1 -carboximidates 3. 12a and 3.12b in 13 and 58% respectively. 3,5 -D imethylphenyl N -(4 methylphenyl) -1 H -1,2,3 -benzotriazole -1 -carboximidoate (3. 12a). Recrystallized from EtOAc h exanes to give white crystals ( 13%); mp 1 39.6142.1 oC;1H NMR 8.36 (br s, 1H), 8.12 (d, J = 8 1 Hz, 1H), 7.65 (t, J = 7 7 Hz, 1H), 7.49 (t, J = 7 7 6.0 Hz, 1H), 7.227.0 3 (m, 4 H), 6 .74 6.56 (m, 3H), 2.31 (s, 3H), 2.20 (s, 6H); 13C NMR 170.0, 154.1, 146.2,140.5, 139.7, 135.1, 131.9, 129.5, 129.4, 126.2, 125.4, 122.9, 120.2, 115.0, 114.1, 21.2, 20.9. Anal. Calcd for C22H20N4O: C, 74.14; H, 5.66; N, 15.72. Found: C, 74.40; H, 5.62; N, 15.58. Anal. Calcd for C22H20N4O: C, 74.14; H, 5.66; N, 15.58. Found: C, 74.40; H, 5.62; N, 15.58.

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68 4 -Ethylphenyl N -(4 -chlorophenyl) -1 H -1,2,3 -benzotriazole -1 -carboximidoate (3. 12b). Recrystallized from EtOAc h exanes to give white crystals (58%); 1H NMR 8.2 5 (s, 1H), 8.1 1 (d, J = 8 1 Hz, 1H), 7.6 4 (t, J = 7.5 Hz, 1H), 7. 48 (t, J = 7. 7 Hz, 1H), 7.2 1 (d, J = 8. 4 Hz, 2H), 7.0 6 (d, J = 7 2 Hz, 3H), 6.97 (s, 2H), 2.56 (quartet, J = 7. 2 Hz, 2H), 1.17 (t, J = 7.5 Hz); 13C NMR 152.2, 146.5, 142.3, 141.4, 141. 3, 132.2, 130.9, 130.1, 130.0, 129.6, 129.5, 129.4, 129.4, 126.1, 126.0, 124.4, 120.8, 118.2, 114.2, 28.5, 15.9. Anal. Calcd for C21H17Cl N4O : C, 66.93; H, 4.55; N, 14.87 Found: C, 6 6 9 0 ; H, 4.5 4 ; N, 14. 67. 3.4.3 General Procedure for the P reparation of 3. 1 4 To a solution of potassium tert -butoxide (2.5 eq uiv ) in THF (15 mL) was added d imethylmalonate ( 3. 1 3 ) (2.0 eq uiv ). After the solution was stirred for 30 min, 3,5 dimethylphenyl N (4 -methylphenyl) 1 H 1,2,3 benzotriazole 1 -carboximidoate ( 3. 12a ) (1 .0 eq ui v ) was added and the resulting mixture stirred at room temperature. The progress of the reaction was monitored by TLC. On completion of the reaction, water (15 mL) was added to the reaction mixture, which was then extracted with d ichloromethane (3 x 30 m L). The combined extracts were dried over magnesium sulfate and the solvent removed in vacuo. The crude mixture was purified by gradient column chromatography over silica gel (ethyl acetate/hexanes) to give a yellow oil, tetramethyl 2 (p tolylamino)prop 1 -ene1,1,3,3 -tetracarboxylate ( 3. 1 4 ) (0.03 g, 54 %). Tetramethyl 2 -(p -tolylamino)prop -1 -ene -1,1,3,3 -tetracarboxylate (3.1 4 ). Yellow oil (54%) ; 1H NMR 11.72 (br s, 1H), 7.17 (d, J = 8.1 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 4.76 (s, 1H), 3 .76 (s, 3H), 3.71 (s, 6H), 3.68 (s, 3H), 2.36 (s, 3H) ; 13C NMR 170.2, 167.8, 165.2, 158.5, 138.0, 134.3, 130.3, 126.8, 103.5, 93.9, 53.1, 52.3, 51.7, 21.1. HRMS calcd for C18H21NO8 [M+ H]+ 380.1340, found 380.1341.

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69 CHAPTER 4 N(FMOC -AMINOACYL)BENZOTRIAZOLES: VERSATILE SYNT HETIC REAGENTS FROM PROTEINOGENIC AMINO ACIDS 4.1 Introduction Namino acids need activation of the carboxylic acid function to facilitate peptide bond formation. Usually, activation of the carboxylic acid moiety is achieved by reaction with an appropriate peptide coupling reagent. [04T2447] Subsequent reaction of the activated amino acid with the amino group of a second amino acid generates the amide bond. Scheme 4 1. Amide bond formation Activation methods can be in situ with no isolable intermediates as exemplified by the use of carbodiimides, EDC, DCC and DIC, in combination with additives such as HOBt and HOAt. [04T2447]. Alternatively, isolated activated intermediates include N -protected amino acid halides and azides. N-Acylbenzotriazoles have been utilized in (i) N acylation for the preparation of primary, secondary and tertiary amides [00JOC2810, 06S3231, 08OBC2400] and N acylsulfonamides, [04ARK14] (ii) S acylation for the synthesis of thiol esters [04S1806] and (iii) C acylation for the preparation of ketones, [06JOC9861] diketones, [00JOC3679] ketosulfones, [03JOC1443] enaminones [00S2029] and C acylated pyrroles, indoles, [04CCA175] 2 -methylfurans and thiophenes [04CCA175] P reviously the N (Boc Fmoc and Cbz aminoacyl)benzotriazoles of some amino acids were prepared by Katritzky and coworkers [05S397, 02ARK134,

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70 07CBDD465] Now s table, crystalline N -Fmoc ( aminoacyl)benzotriazoles derivatives of 18 proteinogenic amino acids an ( N-Fmoc amino)acid amides are reported 4.2 Results and Discussion 4.2.1 Preparation of N -(Fmoc aminoacyl)benzotriazoles 4.1a -r Eighteen of the twenty natural, N -Fmoc amino acids 4. 1a -r ( purchased from P eptides International, Louisville, KY, USA and used without further purification) when treated with 1 H benzotriazole and thionyl chloride in THF at 20 oC for 2 hours [0 5S397] (Scheme 4 2 Table 4 1), afforded crystalline N (Fmoc aminoacyl)benzotriazoles 4.2a -r in 6 9 90% yields. Novel 4.2b -l q -r were characterized by 1H and 13C NMR spectroscopy, elemental analysis and high resolution mass spectrometry (HRMS) ; known 4.2a and m p were verified by comparison of the melting points and spectroscopic data with that of the literature. [07CBDD45, 06S4135, 06S411] The spectra of 4.2b l q -r displayed the expected 13C NMR chemical shifts at ca. 120, and 114 ppm, and that of the amide and carbamate carbonyl carbons at ca. ppm, respectively. [02ARK134] Scheme 4 2 Preparation of N Fmoc ( aminoacyl)benzotriazoles 4.2a-r from the corresponding Namino acids 4 .1a -r The N -Fmoc -( aminoacyl)benzotriazoles of arginine and aspargine were not obtained under these conditions; they appeared to be formed but were rapidly hydrolyzed before they could be isolated.

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71 Table 4 1. Conversions of the 18 N Fmoc amino acids 4. 1a -r into N (Fmoc aminoacyl) benzotriazoles 4.2a -r Reactant Product Yield a (%) mp (C) (Lit. mp) D Fmoc L Ile OH ( 4.1a ) Fmoc L Ile Bt ( 4.2a ) 78 165.4 167.25 c (168.8 170.0) 32.8 Fmoc L Val OH ( 4.1b ) Fmoc L Val Bt ( 4.2b ) 84 148.3 149.8 40.4 Fmoc L Thr( t Bu) OH ( 4. 1c) Fmoc L Thr( t Bu) Bt ( 4.2c ) 80 62.2 65.0 30.0 Fmoc L Lys(Boc) OH ( 4.1d ) Fmoc L Lys(Boc) Bt ( 4.2d ) 75 138.4 140.6 33.3 Fmoc L Glu(O t Bu) OH (4.1e ) Fmoc L Glu(O t Bu) Bt (4.2e ) 81 65.5 67.6 21.2 Fmoc L Ser( t Bu) OH ( 4.1f ) Fmoc L Ser( t Bu) B t ( 4.2f ) 70 91.7 92.4 14.8 Fmoc L Tyr( t Bu) OH ( 4.1g ) Fmoc L Tyr( t Bu) Bt ( 4.2g ) 83 138.4 139.3 +15.0 Fmoc L Gln(Trt) OH ( 4.1h ) Fmoc L Gln(Trt) Bt ( 4.2h ) 69 167.0 168.0 16.3 Fmoc L Asp(O t Bu) OH ( 4.1i ) Fmoc L Asp(O t Bu) Bt ( 4.2i ) 73 102.0 104.0 11.1 Fmo c L Cys(Trt) OH (4.1j ) Fmoc L Cys(Trt) Bt ( 4.2j ) 88 96.0 98.0 11.0 Fmoc L His(Trt) OH ( 4.1k ) Fmoc L His(Trt) Bt ( 4.2k ) 73 137.4 139.5 +13.0 Fmoc L Leu OH ( 4.1l ) Fmoc L Leu Bt ( 4.2l ) 80 121.3 123.2 +53.1 Fmoc L Trp OH ( 4.1m ) Fmoc L Trp Bt ( 4.2m ) 90 92.5 93.6 c ; 192.4195.2b (88.0 90.0) e +9.0 Fmoc L Phe OH ( 4.1n ) Fmoc L Phe Bt ( 4.2n ) 85 159.1 160.2 c (136.5 137.4) d +3.4 Fmoc L Met OH ( 4.1o ) Fmoc L Met Bt ( 4.2o ) 82 122.7 123.35c (98.0100.0)e 44.7 Fmoc L Ala OH ( 4.1p ) Fmoc L Ala Bt ( 4.2p ) 72 160.0 160.3 c (160.7 161.3) d 60.8 Fmoc L Pro OH ( 4.1q ) Fmoc L Pro Bt ( 4.2q ) 89 163.5 165.4 c 60.5 Fmoc Gly OH ( 4.1r ) Fmoc Gly Bt ( 4.2r ) 88 161.5 161.9 c Non chiral a Isolated yield, b mp of polymorph, c 07CBDD465, d 06S4135, e 06S411 4.2.1 -(N -Fmoc -a mino)acid Amides 4.3a,b and 4.4a,b Methylbenzylamides of N -protected amino acids provide criteria for optical purity and stability towards racemization. N Fmoc ( aminoacyl)benzotriazoles 4.2b,g were separately reacted with L-methylbenzylamine 4.5 and D-methylbenzylamine 4.6 in THF at 20 oC to

PAGE 72

72 afford amides 4.3a,b and 4.4a,b in 66 7 4 % yields (average 7 2 %, Scheme 4 3 4.4 Experimental Section). (N -Fmoc amino)acid amides 4.3a,b and 4.4a,b were determined by 1H NMR and HPLC. A comparison of the 1H NMR spectra obtained from the derivatization of 4.2b with D/Land L-methylbenzylamine respectively demonstrated the chirality of 4.3a and thus the conversion of the 4.1b to 4.2b also occurred with retention of chiral ity. HPLC analyses of 4.3a 4.4a and corresponding diastereomeric mixtures provided further evidence for the smooth conversion of chiral 4.1b,g to chiral 4.2b,g (Table 4 2). Scheme 4 3 Preparation of N (acylamino)amides 4.3a,b and 4.4a,b from N (Fmoc aminoacyl) benzotriazoles 4.2b,g and Lor DPhCH(Me)NH2 (4.5 or 4.6 ) Table 4 2. Preparation of N (acylamino)amides 4. 3a,b and 4. 4a,b from N (Fmoc aminoacyl) benzotriazoles 4. 2b,g and Lor D-PhCH(Me)NH2 (4. 5 or 4. 6 ) Reactant Product Yielda (%) Retenti on time t R (min) Fmoc -L-Val -Bt ( 4.2b ) Fmoc L Val L NHCH(Me)Ph ( 4.3a ) 73 5.42 Fmoc -L-Val -Bt ( 4.2b ) Fmoc L Val D/L NHCH(Me)Ph (4.3a+4.4a ) 74 5.41, 8.00 Fmoc L Tyr(tBu) Bt ( 4.2g ) Fmoc L Tyr( t Bu) D NHCH(Me)Ph ( 4.3b ) 74 1.91 Fmoc L Tyr(tBu) Bt (4.2g) Fmoc L Tyr( t Bu) D/L NHCH(Me)Ph ( 4.3b+4.4b ) 66 1.73, 1.91 a Isolated yield. b For condition s, see 4. 4 Experimental Section HOBt based aminium and phosphonium derivatives (PyBOP, HBTU, HATU, etc),1 N hydroxysuccinimide esters, [64JACS1839] and p -nitrophenyl est ers [02TL7717] are widely used

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73 in peptide synthesis but the preparative routes require multiple steps. N -Fmoc ( aminoacyl)benzotriazoles are easily prepared peptide coupling reagents whose generality has been demonstrated in the solution phase syntheses o f sterically hindered peptides [07JOC5794] or pept ide conjugates [08JOC511] and the solid phase preparation of simple oligopeptides [07CBDD465] Additionally, N -Fmoc ( aminoacyl)benzotriazoles are fully amenable to microwave assisted syntheses. [07CBDD465, 07JOC5794] 4.3 Conclusion In summary, the convenient, cost effective preparation of N (Fmoc aminoacyl)benzotriazoles 4.2a -r (6 9 90%) was describe d. N (Fmoc aminoacyl)benzotriazoles 4.2a -r are storable at 20 oC for months without special handling. 1H NMR and HPLC analyses of N(Fmoc aminoacyl)amides 4.3a,b and 4.4a,b easily prepared in high yields, demonstrated that the chirality is maintained during amide bond formation. 4.4 Experimental Section Reagents were obtained as follows: N Fmoc -Lamino aci ds from Peptides International, Louisville, KY, USA; 1 H benzotriazole, dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EtOAc), hexanes, magnesium sulfate (MgSO4) and sodium carbonate (Na2CO3) from Fischer Scientific, Fair Lawn, NJ, USA. Meltin g points were determined on a hot -stage apparatus and are uncorrected. 1H (300 MHz, with TMS as the internal standard) and 13C NMR (75 MHz) NMR spectra were recorded in CDCl3. Optical rotations were recorded on Perkin Elmer 241 polarimeter. HPLC analyses w ere performed on a Shimadzu instrument using a Zorbax Rx -C18 reverse phase column (4.6 x 150 mm) with UV detection at 210 nm, a flow rate of 1.0 mL/min and MeOH:H2O as the eluting solvent. High resolution mass spectrometry was performed in the ESI (electro spray ionization) mode on an Agilent 6210 LC TOF (liquid

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74 chromatography-time of flight) instrument. Elemental analysis was carried out in an Eager 200 CHN analyzer. 4.4.1 General Procedure for the Preparation of 4. 2b -l, q, r Thionyl chloride (5 mmol) was a dded dropwise to a solution of 1H -benzotriazole (20 mmol) in THF (50 mL). After stirring at room temperature for 30 min N -Fmoc amino acid (5 mmol) was added in one p ortion. After stirring for 2 h at room temperature, the solvent was evaporated in vacuo T he crude mixture obtained was dissolved in EtOAc (30 mL) and the organic layer washed with saturated Na2CO3 solution (30 mL x 3) and dried over MgSO4. Concentration under reduced pressure gave the desired product, which was precipitated from dichloromethan e -hexanes. S -(9H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -3 methyl -1 -oxobutan -2 ylcarbamate (Fmoc -LVal -Bt, 4. 2b). White microcrystals (84%), mp 148.3149.8 o24 D = 40.4 (c = 1.5, CHCl3); 1J = 8.1 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H ), 7.76 (d, J = 7.5 Hz, 2H), 7.69 (t, J = 7.8 Hz, 1H) 7.61 (d, J = 7.2 Hz, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 5.6 (dd, J = 9.5, 5.0 Hz, 1H), 5.63 (d, J = 9.3 Hz, 1H), 4.44 (d, J = 6.3 Hz, 2H), 4.24 (t, J = 7. 2, 6.3 Hz, 1H), 2.582.42 (m, 1H), 1.13 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H); 13 128.1, 127.5, 127.0, 125.4, 120.8, 120.4, 114.8, 67.6, 59.8, 47.6, 32.1, 20.1, 17.5. Anal. Calcd for C26H24N4O3: C, 70.89; H, 5.49; N, 12.72; Found: C, 71.25; H, 5.57; N, 12.82. S -(9H -F luoren -9 -yl)methyl(2 S ,3 R) 1 -(1 H -benzotriazol -1 -yl) -3 -tert -butoxy -1 oxobutan -2 ylcarbamate (Fmoc -L-Thr( t Bu) -Bt, 4. 2c). White microcrystals (80%), mp 62.2 65.0 oC: [ ]24 D = 30.0 (c = 1.5, CHCl3); 1H NMR 8.28 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.78 (d J = 7.5 Hz, 2H) 7.727.64 (m, 3H), 7.54 (t, J = 7.8 Hz, 1H), 7.457.31 (m, 4H), 5.94 (d, J = 9.6 Hz, 1H), 5.67 (dd, J = 9.6, 1.5 Hz, 1H), 4.624.51 (m, 1H), 4.43 (t, J = 6.6 Hz, 2H),

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75 4.30 (t, J = 7.2 Hz, 1H), 1.43 (d, J = 6.0 Hz, 3H), 0.92 (s, 9H); 13C N MR 169.9, 156.8, 145.8, 143.9, 143.7, 141.3, 131.1, 130.8, 127.7, 127.1, 126.5, 125.2, 125.2, 120.3, 120.0, 114.2, 74.3, 68.0, 67.4, 60.6, 47.1, 28.0, 27.8, 21.1. Anal. Calcd for C29H30N4O4: C, 69.86; H, 6.06; N, 11.24; Found: C, 70.04; H, 6.23; N, 11.14. S -(9H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -6 -(tert-butoxycarbonylamino) -1 oxohexan -2 ylcarbamate (Fmoc -L-Lys(Boc)-Bt, 4. 2 d). White microcrystals (75%), mp 138.4 140.6 oC: [ ]24 D = 30.0 (c = 1.5, CHCl3); 1H NMR 8.27 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.5 Hz, 2H), 7.68 (overlapped t, J = 7.5 Hz, 1H), 7.65-7.60 (m, 2H) 7.54 (t, J = 7.8 Hz, 1H), 7.40 (t, J = 7.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 5.87 (d, J = 7.8 Hz, 1H), 5.82 5.72 (m, 1H), 4.62 (br s, 1H), 4.494.37 (m, 2H), 4.24 (t, J = 6.9 Hz, 1H), 3.203.00 (m, 2H), 2.201.90 (m, 2H), 1.60 1.50 (m, 4H), 1.43 (s, 9H); 13C NMR 171.7, 156.2, 146.0, 143.8, 143.6, 141.2, 131.1, 130.7, 127.7, 127.1, 126.5, 125.1, 120.3, 120.0,114.4, 79.3, 67.2, 54.5, 47.1, 39.6, 32.2, 29.6, 28.4, 22.5. Anal. Calcd for C32H35N5O5: C, 67.47; H, 6.19; N, 12.29; Found: C, 67.38; H, 6.22; N, 11.90. S -tert -butyl 4 -(((9H -F luoren -9 -yl)methoxy)carbonylamino) -5 -(1 H -benzotriazol -1 yl) 5 -oxopentanoate (Fmoc -L-Glu(O t Bu) -Bt, 4. 2 e). White microcrystals (81%), mp 65.567.6 C, [ ]20 D = 21.2 (c = 2.4, CHCl3); 1H NMR 8.19 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 8.1 Hz 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.65 7.39 (m, 4H), 7.33 (t, J = 6.9 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H), 7.00 (br s, 1H), 5.91 (d, J = 8.1 Hz, 1H), 5.78 5.68 (m, 1H), 4.424.27 (m, 2H), 4.16 (t, J = 6.9 Hz, 1H), 2.412.28 (m, 2H), 2.25 2.10 (m, 2H), 1.36 (s 9H); 13C NMR 172.0, 171.2, 156.1, 146.0, 143.8, 143.6, 141.2, 131.1, 130.8, 127.7, 127.0, 126.6, 125.1, 120.3, 119.9, 114.4, 81.2, 67.2, 54.5, 47.1, 31.6, 28.0, 27.5. HRMS calcd for C30H30N4O5 [M+Na]+ 549.2108, found 549.2071.

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76 S -(9H -F luoren -9 -yl)methyl 1 -(1 H -benzotri azol -1 yl) -3 -tert -butoxy -1 -oxopropan -2 ylcarbamate (Fmoc -L-Ser( t Bu) -Bt, 4. 2 f). White microcrystals (70%), mp 91.792.4 C, [ ]20 D = 14.8 (c = 2.4, CHCl3); 1H NMR 8.30 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 7.5 Hz, 2H), 7.72 7.63 (m 2H), 7.587.52 (m, 2H), 7.447.26 (m, 4H), 6.02 (d, J = 9.0 Hz, 1H), 5.885.86 (m, 1H), 4.474.35 (m, 2H), 4.314.22 (m, 2H), 3.92 (dd, J = 9.0, 3.2 Hz, 1H), 1.03 (s, 9H); 13C NMR 169.5, 156.2, 143.9, 143.7, 141.3, 131.2, 131.0, 127.8, 127.1, 126.5, 126.1, 125.2, 120.3, 120.0, 114.4, 74.0, 67.5, 62.9, 55.9, 47.1, 27.1. HRMS calcd for C28H28N4O4 [M+Na]+ 507.2003, found 507.1986. S -(9H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -3 -(4 -tert -butoxyphenyl) -1 oxopropan -2 -ylcarbamate (Fmoc -L-Tyr( t Bu) -Bt, 4. 2 g) White microcrystals (83%), mp 138.4139.3 C, [ ]20 D = +15.0 (c = 1.9, CHCl3); 1H NMR 8.19 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.2 Hz, 2H), 7.62 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 4.5 Hz, 2H), 7.48 (t, J = 7.5 Hz, 1H), 7.387.23 (m, 4H), 7.03 (d, J = 8.1 Hz, 2H), 6.83 (d, J = 7.8 Hz, 2H), 6.13 6.06 (m, 1H), 5.83 (d, J = 8.4 Hz, 1H), 4.38 (t, J = 6.6 Hz, 2H), 4.204.16 (m, 1H), 3.39 (dd, J = 13.5, 5.4 Hz, 1H), 3.21 (dd, J = 13.5, 8.0 Hz, 1H), 1.24 (s, 9H); 13C NMR 170.9, 155.7, 154.4, 145.8, 143.6, 143.5, 141.1, 130.8, 130.6, 129.7, 129.6, 127.6, 126.9, 126.4, 124.9, 124.2, 120.2, 119.8, 114.1, 78.4, 67.1, 55.6, 46.9, 38.2, 28.6. Anal. Calcd for C34H32N4O4: C, 72.84; H, 5.75; N, 9.99. Found: C, 72.84; H, 6.00; N, 9.70. S -(9 H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -1,5 -diox o -5 -(tritylamino)pentan 2 -ylcarbamate (Fmoc -L-Gln(Trt)Bt, 4. 2 h). White microcrystals (69%), mp 167.0168.0 C, [ ]20 D = 16.3 (c = 1.4, CHCl3); 1H NMR 8.26 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 6.9 Hz, 2H), 7.68 (t, J = 7.2 Hz, 1H), 7.59 7.51 (m, 2H), 7.447.38 (m, 2H), 7.327.26 (m, 12H), 7.23 7.20 (m, 6H), 6.81 (s, 1H), 6.14 (d, J = 7.2 Hz, 1H), 5.83 5.22 (m, 1H),

PAGE 77

77 4.524.45 (m, 1H), 4.36 (t J = 6.6 Hz, 1H), 4.23 (t J = 6.3 Hz, 1H), 2.57 (t, J = 6.6 Hz, 2H), 2.44 (br s 1H), 2.27 (br s 1H); 13C NMR 171.1, 170.8, 156.5, 146.1, 144.5, 143.9, 143.7, 141.3, 131.1, 130.8, 128.7, 128.1, 127.8, 127.2, 126.6, 125.2, 120.4, 120.0, 114.5, 70.9, 67.3, 54.7, 47.2, 35.6, 27.7. Anal. Calcd for C45H37N5O4: C, 75.93; H, 5.24; N, 9.84. Found: C, 75.82; H, 5.46; N, 9.89. S -tert -butyl 3 -(((9 H -F luoren -9 -yl)methoxy)carbonylamino) -4 -(1 H -benzotriazol -1 yl) 4 -oxo butanoate (Fmoc -L-Asp(O t Bu) -Bt, 4. 2 i). White microcrystals (73%), mp 102.0104.0 C, [ ]20 D = 11.1 (c = 2.5, CHCl3); 1H NMR 8.29 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 7.2 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H), 7.60 7.51 (m, 3H), 7.427.28 (m, 4H), 6.15 (d, J = 6.9 Hz, 1H), 6.00 5.86 (m 1H), 4.44 4.40 (m, 2H), 4.27 4.25 (m, 1H), 3.26 (dd, J = 15.5, 5.6 Hz, 1H), 3.14 (dd, J = 18.6, 5.4 Hz, 1H), 1.38 (s, 9H); 13C NMR 169.5, 169.1, 155.7, 145.9, 143.7, 143.5, 141.2, 131.1, 131.8, 127.7, 127.0, 126.6, 125.1, 120.3, 120.0, 114.3, 82.3, 67.3, 51.9, 47.0, 38.5, 27.9. Anal. Cal cd for C29H28N4O5: C, 67.96; H, 5.51; N, 10.93. Found: C, 67.98; H, 5.81; N, 10.96. R-(9 H -F luoren -9 yl)methyl 1 -(1 H -benzotriazol -1 -yl) -1 -oxo -3 -(tritylthio)-propan-2 ylcarbamate (Fmoc -L-Cys(Trt) -Bt, 4. 2 j). White microcrystals (88%), mp 96.098.0 C, [ ]20 D = 11.0 (c = 2.0, CHCl3); 1H NMR 8.24 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.787.76 (m, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.64 7.56 (m, 3H), 7.41 7.38 (m, 2H), 7.337.30 (m, 7H), 7.197.11 (m, 11H), 5.78 5.70 (m, 1H), 5.52 (d, J = 7.2 Hz, 1H), 4. 41 (d, J = 6.9 Hz, 2H), 4.24 (t, J = 6.9 Hz, 1H), 3.13. (dd, J = 14.1, 5.7 Hz, 1H), 2.92 (dd, J = 12.6, 6.2 Hz, 1H); 13C NMR 169.1, 155.6, 146.0, 144.0, 143.5, 141.3, 131.1, 130.8, 129.4, 128.0, 127.8, 127.1, 127.0, 126.7, 125.2, 120.4, 120.0, 114.5, 67. 5, 67.3, 53.9, 47.1, 34.1. Anal. Calcd for C43H34N4O3S: C, 75.20; H, 4.99; N, 8.16. Found: C, 75.09; H, 5.28; N, 7.82.

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78 S -(9 H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -1 -oxo -3 -(1 -trityl -1H -imidazol -4 yl)propan-2 -ylcarbamate (Fmoc -L-His(Trt)-Bt, 4. 2 k). Ye llow microcrystals (73%), mp 137.4139.5 oC, [ ]24 D = 60.5 (c = 1.5, CHCl3); 1H NMR (300 Hz, CDCl3) 8.19 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.667.57 (m, 3H), 7.427.44 (m, 3H), 7.387.31 (m, 4H), 7.267.24 (m, 15H), 6.45 (s, 1H), 6.085.80 (m, 1H), 4.404.20 ( m, 3H), 3.50 3.38 (m, 2H); 13C NMR (75Hz, CDCl3) 170.5, 156.3, 145.8, 143.8, 142.0, 141.1, 139.0, 135.5, 130.5, 129.6, 129.6, 128.0, 127.6, 127.0, 126.3, 125.2, 120.2, 119.8, 114.3, 103.3, 77.2, 70.0, 60.4, 55.4, 30.3. HRMS calcd for C46H36N6O3 [M+H]+ 721.2922, found 721.2919. S -(9 H -F luoren -9 -yl)methyl 1 -(1 H -benzotriazol -1 yl) -4 methyl -1 -oxopentan -2 ylcarbamate (Fmoc -L-Leu -Bt, 4. 2 l). White microcrystals (80%), mp 121.3123.2 oC: [ ]24 D = + 53.1 (c = 1.5, DMF); 1H NMR 8.27 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.3 Hz, 2H), 7.69 (overlapped t, J = 7.1 Hz, 1H), 7.62 7.40 (m, 2H), 7.54 (overlapped t, J = 7.8 Hz, 1H), 7.41 (t, J = 7.0 Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 5.85 (t, J = 7.8 Hz, 1H), 5.54 5.44 (m, 1H), 4.45 (d, J = 7.0 Hz, 2H) 4.25 (t, J = 6.6 Hz, 1H), 1.88 (m, 2H), 1.821.71 (m, 1H), 1.11 (d, J = 4.9 Hz, 3H), 0.99 (d, J = 5.4 Hz, 3H); 13C NMR 172.4, 156.1, 146.0, 143.8, 143.6, 141.3, 131.1, 130.7, 127.7, 127.0, 126.5, 125.0, 120.3, 120.0, 114.4, 67.1, 53.0, 47.1, 41.9, 25.2, 23.2, 21.3. Anal. Calcd for C27H26N4O3: C, 71.35; H, 5.77; N, 12.33; Found: C, 71.19; H, 6.06; N, 12.21. S -(9 H -F luoren -9 -yl)methyl 2 -(1 H -benzotriazole -1 -carbonyl)pyrrolidine 1 -carboxylate (Fmoc -L-Pro -Bt, ca. 1:1 mixture of rotamers, 4. 2 q). White microcrystals (89%), mp 163.5165.4 oC, [ ]24 D = 60.5 (c = 1.5, DMF); 1H NMR 8.29 (d, J = 8.2 Hz, 0.5H), 8.20 (d, J = 8.2 Hz, 0.5H), 8.14 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 7.5 Hz, 2H), 7.747.28 (m, 6H), 7.21 (t, J = 6.0 Hz, 1.5H), 7.09 (t, J = 6.7 Hz, 0.5H), 6.896.78 (m, 1H), 5.89 (d, J = 4.2 Hz, 0.5H), 5.86 (d, J =

PAGE 79

79 4.2 Hz, 0.5H), 5.44 (d, J = 3.3 Hz, 0.5H), 5.41 (d, J = 3.9 Hz 0.5H), 4.614.53 (m, 1H), 4.524.43 (m, 0.5H), 4.404.26 (m, 0.5H), 4.02 (t, J = 5.0 Hz, 0.5H), 3. 90 3.81 (m, 0.5H), 3.773.57 (m, 1.5H), 2.71 2.57 (m, 0.5H), 2.56 2.42 (m, 0.5H), 2.312.19 (m, 1.5H), 2.182.00 (m, 1H), 1.991.88 (m, 1.5H); 13C NMR 171.0, 170.6, 154.9, 154.1, 146.0, 144.0, 143.8, 143.5, 141.3, 141.0, 140.8, 131.2, 131.2, 130.5, 130.5, 127.7, 127.4, 127.1, 126.9, 126.8, 126.5, 126.4, 126.4, 125.2, 125.1, 124.1, 124.0, 120.2, 120.2, 120.0, 119.7, 119.4, 114.6, 114.5, 67.7, 66.5, 60.0, 59.2, 47.2, 47.0, 46.9, 31.6, 30.7, 24.5, 23.2. Anal. Calcd for C26H22N4O3: C, 71.22; H, 5.06; N, 12.78; Found: C, 71.16; H, 5.03; N, 13.12. (9 H -F luoren -9 -yl)methyl 2 -(1H -benzotriazol -1 -yl) -2 -oxoethylcarbamate (Fmoc -Gly Bt, 4. 2 r). White microcrystals (88 %), mp 161.5161.9 oC: 1H NMR 8.25 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.4Hz, 1H), 7.77 (d, J = 7.4Hz, 2H), 7.717.68 (overlapped t, J = 7.8 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.54 (t J = 7.7 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.14 (br s, 1H), 5.59 (t, J = 5 .4 Hz, 1H), 5.10 (d, J = 5.7 Hz, 2H), 4.49 (d, J = 6.9 Hz, 2H), 4.28 (t, J = 6.9 Hz, 1H); 13C NMR 168.4, 156.5, 146.0, 143.7, 141.3, 130.9, 130.8, 127.7, 127.1, 126.6, 125.1, 120.4, 120.0, 114.0, 67.4, 47.0, 44.8. Anal. Calcd for C23H18N4O3: C, 69.34; H, 4.55; N, 14.03; Found: C, 69.40; H, 4.36; N, 14.08. 4.4.2 General Procedure for t he Preparation of 4. 3a,b, 4. 4a,b, ( 4. 3a+ 4. 4a) and ( 4. 3b+ 4. 4b) N(Fmoc aminoacyl)benzotriazoles 4. 2b,g (1 mmol) was dissolved in THF and Lmethylbenzylamine 4. 5 D-methy lbenzylamine 4. 6 -methylbenzylamine ( 4. 5 + 4. 6 ) (1 mmol) was added to the solution. The mixture was stirred at room temperature and monitored by TLC. On completion of the reaction the solvent was evaporated in vacuo. The resulting solid was dissolved in EtOAc (30 mL) and washed with saturated Na2CO3 (30 mL x 3) and dried with MgSO4. The solution was reduced to dryness in vacuo to yield 4. 3a,b 4. 4a,b ( 4. 3a + 4. 4a ) and (4. 3b + 4. 4b ).

PAGE 80

80 (9 H -F luoren -9 -yl)methyl S -3 -methyl -1 -oxo -1 -((R)-1 -phenylethylamino)butan -2 y lcarbamate ( 4. 3a). White powder (73%), mp 205.7206.2 o25 D = 32.6 (c = 2.4, CHCl3); 1J = 7.8 Hz, 2H), 7.63 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz, 2H), 7.38 7.32 (m, 7H), 6.34 (d, J = 6.3 Hz, 1H), 5.57 (d, J = 8.1 Hz, 1H), 5.17 (quintet, J = 7.2 Hz, 15.6 Hz, 1H), 4.504.35 (m, 2H), 4. 284.20 (m, 1H), 4.01 (t, J = 5.6 Hz, 1H), 2.182.06 (m, 1H), 1.75 (s, 1H), 1.52 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 6.0 Hz, 6H); 13C NMR 170.3, 156.5, 143.7, 142.8, 141.3, 128.6, 127.7, 127.4, 127.1, 126.1, 125.0, 120.0, 67.0, 60.5, 48.9, 47.1, 31.3, 21.7, 19.2, 17.9. Anal. Calcd for C28H30N2O3: C, 75.99; H, 6.83; N, 6.33; Found: C, 75.90; H, 6.99; N, 6.09. (9 H -F luoren -9 -yl)methyl S -3 -(4 -tert -butoxyphenyl) -1 -oxo -1 -((R)-1 phenylethylamino)propan-2 ylcarbamate ( 4. 3b). White microcrystals (74%), mp 180.1 181.1, [ ]24 D = +8.6 (c = 1.0, CHCl3); 1H NMR 7.81 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 6.9 Hz, 2H), 7.387.26 (m, 5H), 7.21 7.15 (m, 4H), 6.98 (d, J = 7.8 Hz, 2H), 5.76 (d, J = 5.1 H z, 1H), 5.53 (d, J = 5.5 Hz, 1H), 5.04 (quintet, J = 6 .9 Hz, 1H), 4.45 4.41 (m, 3H), 4.24 (t, J = 6.6 Hz, 1H), 3.18 (br s, 1H), 2.99 2.96 (m, 1H), 1.421.31 (m, 12H); 13C NMR 169.6, 154.5, 143.8, 143.7, 142.8, 141.3, 129.9, 128.7, 127.8, 127.5, 127.1, 126.0, 125.1, 124.4, 120.0, 78.5, 77.3, 67.0, 56.6, 49.0, 47.1, 38.5, 28.8, 21.6. Anal. Calcd for C36H40N2O5 .H2O: C, 74.46; H, 6.94; N, 4.82; Found: C, 74.49; H, 7.07; N, 4.58. (9 H -F luoren -9 -yl)methyl S -3 -methyl -1 -oxo -1 -((S )-1 phenylethylamino)butan-2 ylcarbamate ( 4. 4a). White powder (77%), mp 166.3168.8 oC, [ ]25 D = +22.1 (c = 2.2, CHCl3); 1H NMR 7.75 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.34 7.27 (m, 7H), 6.24 (d, J = 7.8 Hz, 1H), 5.44 (d, J = 8.7 Hz, 1H), 5.185.06 (m, 1H), 4.434.23 (m, 2H), 4.224.13 (m, 1H), 4.003.90 (m, 1H), 2.212.06 (m, 1H), 1.48 (d, J = 6.6 Hz, 3H ), 0.97 ( 2 overlapped d, J = 7.7 Hz, 6H); 13C NMR 170.1, 169.9, 143.8, 142.6, 141.3, 128.7, 127.7, 127.4,

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81 127.1, 126.1, 125.0, 119.9, 67.0, 60.5, 48.9, 47.1, 31.1, 21.7, 19.2, 18.0. Anal. Calcd for C28H30N2O3: C, 75.99; H, 6.83; N, 6.33; Found: C, 76.10; H, 7.01; N, 6.51. (9 H -fluoren -9 -yl)methyl S -3 -(4 -tert -butoxyphenyl) -1 -oxo -1 -((S)-1 phenylethylamino)propan-2 ylcarbamate ( 4. 4b). White powder (79%), mp 196.3197.7 oC, [ ]24 D = 6.2 (c = 1.0, CHCl3); 1H NMR 7.76 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.33 7.22 (m, 5H) 7.12 (d, J = 7.2 Hz, 2H), 7.006.97 (m, 2H), 6.83 (d, J = 8.1 Hz, 2H), 5.88 (br s, 1H), 5.42 (br s, 1H), 5.065.01 (m, 1H), 4.45 4.35 (m, 2H), 4.23 4.13 (m, 1H), 3.113.00 (m, 1H), 2.982.86 (m, 1H), 1.911.82 (m, 1H), 1.39 (d, J = 6.9 Hz, 3H), 1.31 (s, 9H); 13C NMR 169.9, 154.6, 143.9, 142.6, 141.5, 130.0, 128.8, 127.9, 127.6, 127.3, 126.3, 125.2, 124.5, 120.2, 78.6, 77.4, 67.2, 56.6, 49.2, 47.3, 38.3, 29.0, 21.7. Anal. Calcd for 2(C72H78N4O9)H2O: C, 75.63; H, 6.88; N, 4.90; Found: C, 75.59; H, 6.82; N, 4.77. (9 H -fluoren -9 -yl)methyl S -3 methyl -1 -oxo -1 -(1 -phenylethylamino)butan -2 ylcarbamate ( 4. 3a+ 4. 4a). Yellow oil (74%); 1H NMR 7.69 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.2 Hz, 2H), 7.43 (d, J = 7.2 Hz, 1H), 7.34 7.12 (m, 21H), 6.01 (s, 0.46H), 5.155.05 (m, 1.58H), 3.94 (t, J = 6.5 Hz, 1H), 3.74 (q, J = 6.0 Hz, 1H), 3.16 (t, J = 3.0 Hz, 2H), 2.96 (dd, J = 12.0, 6.2 Hz, 1H), 2.82 (dd, J = 12.2, 6.9 Hz, 1H), 2.342.16 (m, 2H), 1.42 (d, J = 6.9 Hz, 6H), 1.24 (d, J = 6.6 Hz, 3H), 0.91 (t, J = 7.5 Hz, 6H), 0.78 (d, J = 6.9 Hz, 6H), 0.68 (d, J = 6.6 Hz, 3H); 13C NMR 173.8, 173.7, 146.4, 146.4, 146.0, 144.0, 143.8, 141.6, 141.6, 129.1, 129.0, 129.0, 128.8, 127.6, 127.6, 127.5, 127.4, 127.2, 127.1, 126.6, 126.5, 125.0, 125.0, 121.4, 120.3, 120.2, 120.1, 60.4, 60.4, 58.6, 51.1, 48.5, 48.4. HRMS calcd for C28H30N2O3 [M+H CO2]+ 399.2436, found 399.2393 (9 H -fluoren -9 -yl)methyl S -3 -(4 -tert -butoxyphenyl) -1 -oxo -1 -(1 phenylethylamino)propan-2 ylcarbamate ( 4. 3b+ 4. 4b). Off-white powder (66%), mp 170.8-

PAGE 82

82 176.8 oC; 1H NMR 7.76 (d, J = 6.9 Hz, 4H), 7.55 (d, J = 6.9 Hz, 4H), 7.40 (t, J = 7.0 Hz, 4H), 7.347.20 (m, 12H), 7.187.06 (m, 6H), 6.92 (d, J = 7.8 Hz, 2H), 6.83 (d, J = 8.1, 2H), 5.94 (br s, 1H), 5.74 (br s ,1H), 5.50 (br s, 2H), 5.30 4.92 (m, 2H), 4.464.26 (m, 6H), 4.224.12 (m, 2H), 3.222.78 (m, 4H), 1.33 (s, 18H), 1.32 (overlapped d, J = 6.3 Hz, 6H); 13C NMR 169.8, 169.6, 154.4, 154.4, 14 3.7, 143.7, 142.4, 142.4, 141.3, 129.9, 129.8, 128.6, 128.6, 127.7, 127.4, 127.4, 127.1, 126.1, 126.0, 125.0, 124.4, 124.3, 120.0, 78.5, 78.4, 77.2, 67.0, 56.6, 48.9, 47.1, 38.5, 38.1, 28.8, 21.5. HRMS calcd for C36H38N2O4 [M+H]+ 563.2910, found 563.2904.

PAGE 83

83 CHAPTER 5 MICROWAVE -ASSISTED SOLID PHASE PEPTIDE SYNTHESIS UTILIZING N -FMOC PROTECTED( -AMINOACYL)BENZOTRIAZOLES 5.1 Introduction Peptides and proteins are ubiquitous and essential to all cellular processes (for example, cell division, biochemical control, storage and transport, etc.). [97MI1 02MI2 ] To solve biological problems, it is of interest to understand the function of peptides a nd proteins in such processes. Moreover peptides and peptide -related drugs are widely used in the medicine, hence the c onstant demand for new and improved approaches directed toward peptide and protein synthes e s. [ 97MI1 02MI2 ] Peptides and proteins are constructed via the sequential coupling of amino acid residues, with peptides containing fewer amino acid residues whe n c ompared to proteins. The ground breaking synthesis of the first peptides was achieved by Fischer and Curtius over a century ago However in the last thirty years, Bruce Merrifield further revolutionized peptide synthesis by the invention of the solid phase peptide synthesis (SPPS) technique. [97MI1 02MI2 ] D espite major advances in peptide synthesis the simple and efficient preparation of many peptides, especially the so called difficult sequences remains a challenge. [97MI1 02MI2 ] Compared with classic al solution synthesis, SPPS, a simple and rapid technique, offers greater ease of separation of the products from the reagents, with the elimination of inherent product losses associated with conventional chemistries ( filtration, recrystallization, etc. ); SPPS also utilizes an excess of reagents that promotes the rapid completion of the coupling reaction. The increased efficiency of SPPS when compared to conventional solution phase methodologies has resulted in its near -exclusive use for the preparation of peptides and in turn led to better coupling reagents, shorter coupling time s and better yields over the last three decades. [95JACS5401, 06CEJC285, 04JMC5662, 05JMC3060]

PAGE 84

84 M icrowave heating can enhance the rate of a variety of reactions including the solid phase assembly of peptides. Although this area is still relatively unexplored, a search of the current literature disclosed faster coupling times and improved peptide yields with the incorporation of microwave heating. [02S1592, 05OL1521, 92JOC4781, 06JOC3 051] Acylbenzotriazoles are easily prepared, chirally stable nonhydroscopic synthetic equivalents of acid halides [02ARK134, 05ARK116, 07JOC407, 06S411] Recently, Katritzky and coworkers reported that the solution phase peptide coupling reactions of N pr otected ( aminoacyl) benzotriazoles with unprotected amino acids proceed with minimal epimerization in partially aqueous media under mild conditions [05S397] Additionally, the preparation of di -, tri and tetrapeptides, [06S411, 05S397, 04S2645] the C acy l ation of activated heterocycles, [05JOC4993] as well as the O aminoacylation of hydroxysteroids [06ST660 ] and t erpenes [06S4135] can be achieved using N -protected ( aminoacyl) benzotriazoles Now the convenient microwave -enhanced solid phase syntheses of simple tetra penta and hexa peptides using N Fmoc ( aminoacyl ) benzotriazoles [07CBDD465] is reported. 5 2 Result s and Discussion 5.2.1 Preparation of N -Fmoc -( aminoacyl)benzotriazoles (5.2a-g) N-Fmoc ( aminoacyl ) benzotriazoles 5. 2a -g were prepared i n yields of 6090% from the reaction of NFmoc protected amino acids 5. 1a -g ( purchased from Peptides International and used without further purification) with precomplexed 1 H -benzotriazole (4.0 equiv ) and SOCl2 (1.0 equiv ) in THF at 20C for 2 h following the publishe d procedure (Scheme 5 1, Table 5 1 ). The original chirality was preserved in all cases (>95% as evidenced by NMR comparison of the diastereomers and the corresponding diastereomeric mixture ). [ 05S397, 05JOC4993, 09ARK47]

PAGE 85

85 Scheme 5 1 Prepara tion of N Fmoc aminoacyl)benzotriazoles 5.2a g Table 5 1 N Fmoc -protected aminoacyl)benzotriazoles 5. 2a -g utilized for peptide synthesis Entry Compound 2 mp ( o C) Lit. mp ( o C) [ref.] D 1 Fmoc L Ala Bt ( 5. 2a ) 160.0 160.3 160 161 [ 06S4135 ] 60.8 b 2 Fmoc L Trp Bt ( 5. 2b ) 92.5 93.6 88 90 [ 06S411 ] +9.0 b 3 Fmoc L Met Bt ( 5. 2c ) 122.7 123.3 98 100 [ 06S411 ] 44.7 b 4 Fmoc L Pro Bt ( 5. 2d ) 163.0 165.0 163.5 165.4 [ 09ARK47 ] 60.0 b 5 Fmoc L Phe Bt ( 5. 2e ) 159.1 160.2 136 137 [ 06S4135 ] +3.4 b 6 Fmoc Gly Bt ( 5 2f ) 160.9 161.5 161.5 161.9 [ 09ARK47 ] 7 Fmoc L Leu Bt ( 5. 2g ) 121.0 122.8 121.3 123.2 [ 09ARK47 ] +50.2 aIsolated yields; bLit. Optical rotation 5. 2a [06S4135] D 23 = 96.8 ( c 1.6, DMF), 5. 2b [06S411]D 25 = +12.7 ( c 1.5, DMF), 5. 2c [06S411]D 25 = 75.1 ( c 1.5, DMF), 5.2d [06S4135]D 24 = 60.5 ( c 1.5, DMF), 5. 2e [06S4135]D 23 = +35.6 ( c 1.6, DMF), 5. 2g [06S4135]D 24 = +53.1 ( c 1.5, DMF) 5. 2.2 Peptide Syntheses Standard Fmoc SPPS was performed manually in a 25 mL Discover SPS (solid ph ase synthesis) reaction vessel. The peptides 5. 1 5. 3 were prepared on Rink amide 4 methylbenzhydryl amine (MBHA) resin (0.1 mmol). Following the swelling of the resin in DCM (4 mL, 0.5 h) and treatment with 20% piperidine DMF for 20 min. to afford the free -base amide resin, a DMF DCM (5:1 ca 3 mL) solution of the appropriate N -Fmoc ( aminoacyl) benzotriazole (0.5 mmol) and the resin were combined and coupled using microwave irradiation (75 oC, 80 W, 15 min) T he completion of the coupling reaction was monitored b y the ninhydrin (Kaiser) test. Successive N -Fmoc aminoacyl)benzotriaz oles were coupled to the growing peptide in this manner. Deprotection of the N Fmoc aminoacyl)benzotriazole was ach ieved with 20% piperidine DMF. Finally, the resulting peptidyl resin was cleaved with cleavage cocktail B (88% TFA/ 5 % phenol/ 5% water/ 2% TIPS), K (82.5% TFA/ 5% phenol/ 5% water/

PAGE 86

86 5% thioanisole/ 5% EDT) or L (88% TFA/ 5% DTT/ 5% water/ 2% TIPS) for 1.5 2.0 h to afford the crude peptides 5. 3 5.5 Table 5 2. Synthesis of peptides 5.3 5.4 Entry Structure (C N terminus) Crude After HPLC sep aration Retention time, tR(min) HRMS [M+H]+ Yield (%) a Yield (%) c Purity (%) d Purity (%) b 1 NH 2 Trp Met Trp Pro ( 5.3 ) 73 39 >95 82 14.79 618.2502 2 NH 2 Ala Phe Gly Met Leu ( 5.4 ) 68 24 >99 92 11.67 537.2626 3 NH 2 Ala Phe Gly Met Leu Pro ( 5.5 ) 73 31 >99 54 12.42 634.3678 a Samples were weighed after precipitation of the cleaving mixture, b Estimated from the crude peptide HPLC trace, c Samples were weighed after HPLC purification, d Purity after HPLC purification. The preparation of peptides 5.3 5. 5 was straightforward. In the literature the thioether side chain of Met has reportedly been accompanied by alkylation and oxidation side reactions, either during the synthetic process or during subsequent handlin g of the Met -containing peptides. [00 MI3 ] Ho wever, n o oxidation product was observed during the preparation of Met containing peptides 5.3 5.5 although other works reported relatively easy partial oxidation of Met to its sulphone upon prolonged exposure to air. [87JACS620] Characterization of the pu rified compounds 5.3 5.5 by HPLC revealed compl ete retention of configuration (see 5.4 Experimental Section). 5.3 Conclusion A plethora of coupling reagents is widely available for SPPS. Often, when the more conventional carbodiimide -based methods are empl oyed long coupling times are required. Onium and aminium -based coupling reagents react rapidly, but are costly. N-Fmoc aminoacyl)benzotriazoles are easily accessed, cheap, chirally pure reagents and were demonstrated to be useful alternatives for both solid and solution phase peptide synthes e s. As

PAGE 87

87 summarized in Table 5 2, application of benzotriazole methodology in SPPS afforded peptides 5.3 5.5 in crude yields of 68 73%. 5.4 Experimental Section Analytical reversed -phase HPLC was performed on a Rainin HPXL system with a Vydac C18 (5 m. 2.1 x 250 mm) silica column at a 1m L /min flow rate. Peptides were eluted using a 1080% gradient of solvent B (0.1% TFA in acetonitrile) v s. solvent A (0.1% TFA in water) and the peaks were detected at 214 nm. The identification of the products was a chieved by matrixassisted laser desorption/ionization time -of -flight mass spectrometry (MALDI TOF, ABI 4700 Proteomics Analyzer) with a -cyano 4 hydroxy cinnamic acid matrix. MS/MS peptide fragmentation was obtained on the crude peptides by way of low re solution MS and tandem mass spectrometry (MSn) data obtained using an Agilent (Palo Alto, CA) 1100 series HPLC equipped with a Phenomenex Synergi 4u HydroRP 80A (2 x 150 mm, 4 m) column plus a C18 guard column (2 mm x 4 mm). Mass Analysis was performed u sing a ThermoFinnigan LCQ ion trap mass spectrometer (San Jose, CA) in electrospray ionization (ESI) mode. The peptides yielded abundant [M+H]+ and [M+Na]+ ions under the ( +)ESI -MS conditions used here. With (+)ESI -MS/MS and MSn of each peptides [M+H]+ io n, dissociation of the peptide proceeds, in general, due to fragmentation at the amide bonds as indicated below to yield a series of y and b ions which then form z and a ions, respectively.

PAGE 88

88 Figure 5 1. General Peptide Fragmentation N H 2 N H O R 1 R 2 N H N H N H O O O R 4 R 5 O O H R 3 b 5 b 4 b 3 b 2 b 1 y 1 y 2 y 3 y 4 N H 3 + R 5 O O H y 1 N H 2 N H O R 1 R 2 N H N H O O O + R 4 R 3 b 4 N H 2 N H O R 1 R 2 N H N H + O O R 4 R 3 a 4 CO NH 3 or H 2 O dependent on R grp z 1 C H + R 5 O O H + H + [M+H] +

PAGE 89

89 Figure 5 2. The HPLC profile of peptide 5.3 (Pro Trp -Met Trp NH2): a ) after purification and b ) crude Figure 5 3. Expected product ions from the (+)ESI -MSn of the m/z 618 [M+H]+ ion. The shaded ions were observed in the spectra. a b

PAGE 90

90 Figure 5 4 The HPLC profile of peptide 5 .4 (Leu -Met Gly -Phe -Ala NH2): a ) after purification and b ) crude Figure 5 5. Expected product ions from the (+)ESI -MSn of the m/z 537 [M+H]+ ion. The shaded io ns were observed in the spectra. L M G F A(NH 2 ) MW = 536.3 [M+H] + = 537.3 a1 Ions (loss of CO) 86.1 217.1 274.1 4 21.2 492.3 b ions N term 114.1 245.1 302.1 449.2 520.3 C term Residue H L M G F A NH 2 Residue mass 1.0 113.1 131.0 57.0 147.1 71.0 16.0 y ions 537.3 424.2 293.2 236.2 89.1 Loss of NH 3 520.3 407.2 276.2 219.2 72.1 a b

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91 Figure 5 6 The profile of peptide 5.5 (Pro Leu -Met Gly Phe -Ala NH2): a ) after purification b ) crude Figure 5 7. Expected product ions from the (+)ESI -MSn of the m/z 634 [M+H]+ ion. The shaded ions were observed in the spectra. P L M G F A(NH 2 ) MW = 633.3 [M+H] + = 634.4 a1 Ions (loss of CO) 70.1 183.1 314.2 371.2 518.3 589.3 b ions N term 98.1 211.1 342.2 399.2 546.3 617.3 C term Residue H P L M G F A NH 2 R esidue mass 1.0 97.1 113.1 131.0 57.0 147.1 71.0 16.0 y ions 634.4 537.3 424.2 293.2 236.2 89.1 Loss of NH 3 617.4 520.3 407.2 276.2 219.2 72.1 a b

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92 CHAPTER 6 BENZOTRIAZOLE -ASSISTED SOLID -PHASE ASSEMBLY OF LEU ENKEPHALIN, AMYLOID SEGMENT 34 42, AND OTHER DIFFICULT PEPTIDE SEQUENCES 6.1 I ntroduction Solution phase and especially the solid phase peptide syn thesis (SPPS) of potentially bioactive peptides is of great interest. [00MI3 97MI1 ] While SPPS has enabled major advances in scope, yields, purities, and reaction times, [ 00MI3 88JCS2895] SPPS [ 63JACS2149, 86S341] has also encountered difficult peptide sequences when incomplete aminoacylation and/or deprotection reactions at various stages in the synthetic scheme [ 88ARB957, 90JACS6039, 94IJPPR431, 95JACS12058] have resulted in low yields and purities [ 00MI3 ]. Such difficulties can arise in attempting to form a peptide link due (i) to steric effects when both amino acid units possess -branched side chains (e.g. valine, isoleucine and threonine) [ 90JACS6039] and (ii) as a result of the formation of secondary structures by intra and interchain hydrogen -bo nded associations [ 88ARB957, 90JACS6039, 94IJPPR431, 95JACS12058]. In such cases, the synthesized peptides can be partly racemized and/or adulterated with deletion sequences or form aspartimides and related side products [ 00MI3 03JPS518] Some of the probl ems associated with these difficult sequences in Fmoc-based SPPS have been alleviated by the use of (i) bases such as DBU and piperazine (less nucleophilic than the conventional piperidine) to suppress racemization and to reduce aspartimide formation [03J PS518, 07JPS143]; (ii) chemical ligation techniques, for example the O -Acyl isopeptide method which can significantly reduce isomerization of the peptide backbone [ 04CC124, 06TL3013]; (iii) microwave acceleration of the deprotection and coupling steps [ 0 7JPS143, 07CBDD465], which decreases racemization as the growing peptide has less time available for carbon epimerization.

PAGE 93

93 The formation of aspartimides and related side products remains a problem in SPPS. Backbone protection using the 2,4 -dimethoxybenzyl (Dmb), 2 -hydroxy4 -methoxybenzyl (Hmb), 2,4,6 -trimethoxybenzyl (Tmb) or 2 -nitrob enzyl (Nbzl) groups [ 95TL7523, 95JACS11656, 00JOC5460] can help, but requires additional steps. Recently, N -Fmoc aminoacyl)benzotriazoles of proteinogenic amino acids have been utilized in the syntheses of tri to heptapeptides in crude yields of 65 77% on the Rink amide MBHA solid support. [07CBDD465] T he microwave assisted syntheses of six short difficult peptide sequences is now described in attempts to examine the extent of racemization, incomplete aminoacylation/ deprotection reactions and the f ormation of aspartimides when N Fmoc ( aminoacyl)benzotriazoles are used as activating reagents for SPPS. 6.2 R esults and Discussion N-Fmoc ( aminoacyl)benzotriazoles 6. 2 a -l (76 91%) were prepared as previously described [09ARK47] by treatment of purchas ed Fmoc -L-protected amino acids 6. 1 a l with 4 equivalents of benzotriazole and 1 equivalent of SOCl2 in THF at room temperature for 2 hours (Scheme 6 1, Table 6 1). Scheme 6 1. Preparation of N Fmoc aminoacyl)benzotriazoles A standard SPPS approach w as employed in the syntheses of the difficult peptides ( 6.3 6.10), in which the appropriate N Fmoc ( aminoacyl)benzotriazole ( 2 ) was coupled in turn to the growing peptide (Scheme 6 2). Subsequent cleavage [TBAB ] from the Rink amide MBHA resin and purif ication of the crude peptide provided the desired product.

PAGE 94

94 Table 6 1. Preparation of N -Fmoc aminoacyl)benzotriazoles ( 6. 2 ) from the corresponding Fmoc -protected amino acids ( 6. 1 ) Entry Compound Yield a (%) mp (oC) Lit mp (oC) Reference 1 Fmoc L Val B t ( 6.2a ) 84 151.9 152.6 148.3 149.8 09ARK47 2 Fmoc L Thr( t Bu) Bt ( 6. 2b ) 80 64.6 66.8 62.2 65.0 09ARK47 3 Fmoc L Ser(tBu) Bt ( 6. 2c ) 78 63.8 65.4 91.7 92.4 b 4 Fmoc L Tyr( t Bu) Bt ( 6. 2d ) 84 99.0 100.5 138.4 139.3 b 5 Fmoc L Ile Bt ( 2e ) 78 165.4 167.2 168. 8 170.0 09ARK47 6 Fmoc L Lys(Boc) Bt ( 6. 2f ) 78 138.2 141.7 138.4 140.6 09ARK47 7 Fmoc Gly Bt ( 6. 2g ) 76 161.8 163.3 161.5 161.8 09ARK47 8 Fmoc L Asp(OtBu) Bt (6. 2h ) 81 102.1104.3 102.0104.0 09ARK47 9 Fmoc L Phe Bt ( 6. 2i ) 78 157.0 158.3 159.1 160.2 09A RK47 10 Fmoc L Leu Bt ( 6. 2j ) 87 75.3 78.6 121.3 123.2 b 11 Fmoc L Met Bt ( 6. 2k ) 91 129.1 131.8 122.7 123.3 09ARK47 12 Fmoc L Ala Bt ( 6. 2l ) 88 160.5 161.8 160.0 160.3 09ARK47 a Isolated b See 6. 5 Experimental Section for characterization of the polymor phs 6. 2c d j Scheme 6 2. SPPS approach using the Rink amide MBHA resin and N -Fmoc aminoacyl)benzotriazoles Hydroxy amino acids, such as serine, 3 -hydroxyproline, threonine, and certain analogues (for example, -hydroxyphenylalanine and hydroxytyrosine) are widely distributed as components of biologically active natural pr oducts. [ 06JOC7106, 07TA1667, 00JOC7663, 97JACS11734] The synthesis of peptides containing hydroxyamino acids can be challenging due i) to the risk of racemization of such residues during both the stepwise and convergent approaches to Fmoc SPPS [ 06TL3013, 98TL8529] and ii) because of aggregation,

PAGE 95

95 which may commence as early as the addition of the fifth amino acid residue in certain hydroxy amino acid containing sequences [ 06TL3013, 98TL8529]. Hydrophobic and branched chain amino acids (BCAAs), such as valine, isoleucine and leucine, promote aggregation during peptide synthesis and purification, particularly when a large percentage of such hydrophobic residues is present. In the literature the effect of the amino acid hydrophobicity is evident in the DIPCDI HOBt (1,3 diisopropylcarbodiimide hydroxybenzotriazole) stepwise SPPS of H -Val -Val -Ser Val -Val -NH2 (3 ) [04CC124, 06TL3013] where the undesired N -protected peptide amide, Fmoc Val -Val -Ser -Val -Val NH2 was produced as the major compound (1.1 fold high er than the desired peptide 6. 3 as evidenced by HPLC [ 04CC124]). In the presently reported work, using N Fmoc ( aminoacyl)benzotriazoles, peptide 6. 3 was obtained as the major product (Table 6 2, Figure 6 1; Figure 6 13, 6. 5 Experimental Section ) with no evidence of the undesired Fmoc -Val Val -Ser -Val -Val NH2 pe ptide or any racemized product. A possible explanation for the absence of micro aggregates in the benzotriazole assisted synthesis of 6. 3 when compared to the DIPCDI HOBt route [ 04CC124, 06TL3013] co uld be the impact of microwave irradiation on the environment of the growing peptide. In the present work, the alteration of the microenvironment by microwave irradiation may hinder the formation of insoluble micro aggregates and facilitate the removal of the Fmoc groups from the resin bound peptide 3 [ 07JPS143] Additionally, our microwave assisted stepwise protocol reduced the total coupling time for the synthesis of 6. 3 from 10 hours [ 04CC124] to 2.5 hours (open vessel; 1.5 hours when closed vessel condi tions are applied) and impr oved the overall yield from 1.4 % [ 04CC124] to 7%. While using the O acyl isopeptide chemical ligation technique to synthesize 6. 3 may provide a peak overall yield of 28%

PAGE 96

96 (extrapolated from the yield of the O acyl isopeptide of 6. 3 ), this requires a coupling time of 40 h. [ 06TL3013] Figure 6 1. HPLC profiles of a ) crude and b ) pure peptide 6. 3 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage. A synthesis analogous to that of 6. 3 gave hydrophobic peptide 6. 4 as the major product (20 mg of 60% purity, comparing to a yield of pure 6. 4 of 34%), which is further evidence of the utility of N -Fmoc -( aminoacyl)benzotriazoles in the synthesis of difficult peptides containing hydroxy amino acid residues. The HPLC and HRMS spectra of crude 6. 4 showed no peaks for the analogous Fmoc -protected peptide amide or for any racemized product (Figures 6 1 6 a nd 6 1 8 6. 5 Experimental Section ). A literature search for 6. 4 gave no details of the synthesis or yield. Peptide amides, such as 6. 3 and 6. 4 contain valine, a naturally occurring bra nched chain amino acid (BCAA). BCAAs constitute an important class of n atural products and occur frequently in the cores of proteins and are found in skeletal muscle. Such BCAAs are important in determining the three dimensional structure of globular proteins and play a pivotal role in the interactions of membrane proteins wi th phospholipid bilayers. [ 07MM129] T he absence of any peaks for the Fmoc protected peptide s in both the HPLC and HRMS spectra of 6. 3 and 6. 4 suggests that interference by aggregation was negligible in these two examples of our present work. a b

PAGE 97

97 Table 6 2. An alytical data of peptides 6. 3 6. 10 Sequence N C terminus Retention time, tR (min) Crude Purity (%) Crude Yieldb (%) After 2nd HPLC Purity (%) After 2nd HPLC Yield (%) [M+H]+ d Found H Val Val Ser Val Val NH 2 ( 6. 3 ) 7.57 60 63 >99 7 501.3385 H Val Val Ser Val Val Val NH 2 ( 6. 4 ) 9.79 60 34 n.d. c n.d. c 600.4058 H Val Ile Val Ile Gly OH a ( 6. 5 ) 10.82 70 40 >99 2 4 500.3643 H Thr Val Thr Val Thr -Val -NH 2 (6. 6 ) 8.70 50 35 89 16 618.3864 H Val Lys Asp Gly Tyr Ile NH 2 ( 6. 7 ) 8.31 30 68 >99 24 693.3916 H Val Lys As p Val Tyr Ile -NH 2 (6. 8 ) 9.10 37 64 >99 29 735.4419 H Tyr Gly Gly Phe Leu NH 2 ( 6. 9 ) 11.54 80 86 >99 37 555.2948 H Leu Met Val Gly Gly -Val -Val -Ile -Ala NH2 (6. 10) 13.82 28 42 89 22 857.5277 a On purification the peptide amide hydrolyzed to the corresponding acid b Yields quoted are calculated from the amount of product obtained multiplied by % purity from the HPLC. c n .d not determined. d For calculated [M+H]+ values see 6. 5 Experimental Section N ext synthesized peptide 6. 5 (Table 6 2; Figures 6 1 9 and 6 20, 6. 5 Experimental Section ) possessing 80% BCAAs and with a hydrophobicity similar to 6. 3 was isolated in 28% yield with no evidence of interference by aggregation ( Figure s 6 1 9 and 6 22, 6. 5 Experimental Section ; during purification the peptide amide was hydrolyzed to the corresponding acid ). Previously, the Boc -based SPPS strategy along with the DCC/HOBt active ester coupling method was applied in the synthesis of 6. 5 while our present work utilized the milder Fmoc based strategy along with N (Fmo c -protected aminoacyl)benzotriazoles. A comparison of the yield of 6. 5 via the literature Boc based SPPS with our yield by the Fmoc -based benzotriazole assisted strategy could not be calculated from the information provided by the previous authors. [01JPS641]

PAGE 98

98 Hexape ptide 6. 6 c ontains only -branched side chains and is less hydrophobic than 6. 3 and 6. 4 Peptide 6. 6 (Table 6 2; Figures 6 23, 6 24 and 6 25, 6. 5 Experimental Section ) was prepared by six successive coupling with (i) Fmoc -Val -Bt, (ii) Fmoc Thr( t Bu) -Bt, (iii) Fmoc -Val Bt, (iv) Fmoc Thr( t Bu) Bt, ( v) Fmoc -Val Bt (vi) Fmoc Thr( t Bu) Bt. The linear geometry of 6. 6 can be attributed to destabilizing steric effects and the restricted rotational freedom of peptides containing only -branched amino acids. [ 94B12022] Peptides, such as 6. 6 could be useful building blocks in tests for the stabilizing or destabilizing effect of BCAAs in peptide and protein -helix formation. [ 94B12022, 04JOC8804] The nearest literature comparison appears to be the synthesis by Jolliffe e t al. [ 04JOC8804] of the TBS -protected linear peptide amide (Val Thr)3 as an intermediate in the formation of cyclo (Val Thr)3; again no direct comparison of yields is possible because of lack of data in the literature reference. [ 04JOC 8804] In the SPPS of peptides containing asparagine or aspartic acid, aspartimides are frequently formed. [ 00MI3 03JPS518, 00LIPS107] Aspartimide formation can occur under both acidic and basic conditions. [00MI1] In base -catalyzed aspartimide formation the proportion of asp artimide side products attained depends on the base used for removal of the Fmoc group [ 03JPS518 ], the nature of the preceding amino acid residue [ 03JPS518, 00LIPS107] and to a lesser extent, the protecting group on the aspartyl residue [ 03JPS518, 00LIPS107 ]. Next hexapeptide 6. 7 [03JPS518, 06TL4121] was examined ; structure 6. 7 is the peptide fragment 16 of toxin II from the scorpion Androctonus australis Hector [ 87T5961]) and contains the Asp Gly fragment. Syntheses of compound 6. 7 (Table 6 2; Figures 6 2 6 and 6 27, 6. 5 Experimental Section ) and analogue 6. 8 [03JPS518] (Table 6 2; Figures 6 29 and 6 3 0 6. 5 Experimental Section ) containing Asp -Val serve to test the tendency for aspartimide formation using N -Fmo c aminoacyl)benzotriazoles. As anticipated, t his synthesis of peptide amide 6. 7 (m/z 735.4419, tR

PAGE 99

99 8.31 min; Figures 6 26 and 6 2 8 6. 5 Experimental Section ) did produce the corresponding aspartimide ( m/z 675.3808, tR 8.11 min; Figures 6 26 and 6 28, 6. 5 Experimental Section ) as a by product. Howeve r, the presently described synthesis of peptide amide 6. 8 (Table 6 2; Figures 6 29 and 6 3 1 6. 5 Experimental Section ) proceeded without any of the analogous aspartimide by product, the formation of which was evidently suppressed by replacement of t he glyc ine residue with valine. For both 6. 7 and 6. 8 5% piperazine DMF (and not 20% piperidine -DMF) was used for the removal of the Fmoc group and this eliminated ring opening of the aspartimides to the corresponding piperidides. Although 7 was used as a test pep tide by four sets of authors [ 03JPS518, 00LIPS107, 06TL4121, 87T5961], no comparison of the yield from this benzotriazole assisted synthesis with the literature can be made because each literature case reported only product purity and no yield was provided [ 03JPS518, 00LIPS107, 06TL4121, 87T5961] Again, for 6. 8 only the product purity was provided in the literature [ 03JPS518], making a yield comparison impossible. 6.3 Preparation of Leu -Enkephalin ( 9 ) a nd Amyloid -42) ( 10) Two biologically im portant difficult peptides 6. 9 [00OL1815] and 6. 10 [04PPL377] were prepared by the currently described microwave -enhanced and benzotriazole -mediated methodology. Leu -enkephalin ( 6. 9 ) (Table 6 2, Figure 6 2) is a natural peptide neurotransmit ter and a pow erful painkiller. For 6. 9 no direct yield comparison with the literature [00OL1815] can be made; as no literature yield is provided. Alzheimers disease (AD) is slowly progressive and is characterized by dementia. Intracellular amyloid aggregates are regularly associated with Alzheimers disease (AD). Formation of these aggregates heralds the formation of insoluble plaque, the principal biological marker indicating the development and progression of AD. The formation of the agg regates observed in an AD afflicted individual is due to the hydrophobic interaction between the core

PAGE 100

100 hydrophobic amino acid residues in the polypeptide. The preparation of hydrophobic segment (34 42) ( 6. 10) of amyloid (Table 6 2) was previously de scribe d by Halverson [90B2639] and coworkers who used Kaiser oxime resin with the alanine residue already attached; they then coupled N Boc -protected amino acids eight times to assemble 6. 10, stating that the solubilization was extremely difficult and recovery subsequent to HPLC analysis was extremely sensitive but claiming a 40% yield of material that was quite susceptible to oxidation. Figure 6 2 HPLC profiles of a ) crude and b ) pure peptide 6. 9 obtained after SPPS using 20% piperidine DMF for Fmo c cleavage. 6.4 C onclusions and Directions 6.4 .1 Conclusions As summarized in Table 6 2, benzotriazole assisted solid phase assembly affords difficult peptides in crude yields of 3486%. These benzotriazole assisted syntheses, in tandem with microwave acceleration have the following advantages over the previously used carbodiimide based coupling methods: (i) no base is required for the coupling reactions, (ii) the conditions are comparatively mild, (iii) the reactions are more rapid and (iv) importantly the products are chirally homogeneous. 1 Hydroxybenzotriazole hydrate (HOBt) is a widely used coupling additive in peptide synthesis that suppresses racemization when used in combination with car bodiimides such as b a

PAGE 101

101 DCC. Other 1 -hydroxybenzotriazole derivatives for example, 1 hydroxy7 -azabenzotriazole (HOAt), 6 -chloro 1 -hydroxybenzotriazole (6 Cl -HOBt), phosphonium and aminium salts of hydroxybenzotriazol es are also used as additives. Recently, the availability of HOBt has decreased due to the propensity for a n explosion during transportation. [05JHM1]Thus, this shows the efficacy of N Fmoc aminoacyl)benzotriazoles in peptide synthesis. There is a present and growing need for efficient synthetic methods for the assembly of proteins and peptides needed to study diverse systems in the body and/or develop a cure for neurodeg enerative diseases such as AD. N Fmoc aminoacyl)benzotriazoles were demonstrated to be useful for the solid phase assembly of difficult peptides, and represent viable alternatives as SPP S reagents. 6.4 .2 Directions In order to access longer chain peptides with the benzotriazole -mediated strategy aggregation of the growing peptide must be controlled. One approach is to apply backbone amide protecting groups such as 2 -hydroxy 4 -methoxybenz yl (Hmb) to disrupt hydrogen bonded associations. This would permit better s o lvation of the peptide chain and lead to more efficient coupling and deprotection steps. Investigations into the preparation of N Hmb, N -Fmoc dipeptidoylbenzotriazole derivatives for use in SPPS are underway. 6.5 Experimental Section Reagents were obtained as follows: N -fluorenylmethoxycarbonyl amino acids, and the Rink amide MBHA resin (substitution 0.43 meq/g) from Peptides International, Louisville, KY, USA; the Ninhydrin test kit from AnaSpec, San Jose, CA, USA; N, N -dimethylformamide (DMF), dichloromethane (DCM) and trifluoroacetic acid (TFA) from Fischer Scientific, Fair Lawn, NJ, USA; triisopropylsilane (TIS) and piperidine from Sigma Aldrich St. Louis, MO; piperazin e from Acros Organics, NJ, USA. A Discover Benchmate, upgraded for peptide

PAGE 102

102 synthesis, 10 mL vials and 25 mL Discover solid phase synthesis (SPS) reaction vessels from CEM Corporation, Matthews, NC, USA were used for manual SPS. For SPS, the set temperature was monitored using an internal fiber optic probe. Melting points were determined on a hot -stage apparatus and are uncorrected. NMR spectra were recorded in CDCl3 using TMS as the internal standard for 1H NMR (300 MHz) and the solvent, CDCl3, as the internal s tandard for 13C NMR (75 MHz). Analytical reversed -phase HPLC was performed on a Rainin HPXL system, equipped with a Vydac C were eluted using a 10 80% gradient of solvent B (0.1% TFA in acetonitrile) versus solvent A (0.1% TFA in water) and peaks were detected at 214 nm. The identification of the products was achieved by matrixassisted laser desorption/ionization time -of -flight mass spectrometry (MALDI -cyano 4 -hydroxy cinnamic acid as the matrix. MS/MS peptide fra gmentation was obtained on the peptides Tandem mass spectrometry (MSn) via HPLC -UV/(+)ESI -MS and -MSn was acquired using a ThermoFinnigan (San Jose, CA) LCQ ion trap mass spectrometer in electrospray ionization (ESI) mode at a wavelength of 220 nm. High resolution mass spectrometry (HRMS) via flow injection positive [(+)ESI] time of flight (TOF) was obtained on an Agilent 1200 series spectrometer. 6. 5 .1 S olid Phase Protocol for the Preparation of Pepti des 3 -10 Standard stepwise solid phase synthesis was performed manually in a 25 mL Discover SPS reaction vessel. Peptides 6. 3 6. 10, were each prep ared on Rink amide MBHA resin. After swelling the resin (0.1 mM) in DCM (4 mL, 0.5 h) and treatment with 20% p iperidine DMF ( ca 3 mL) for 20 min, the free -base amide resin was filtered off, washed with DMF (5 mL x 3) and DCM (5 mL x 3), dried and a DMF DCM (5:1 ca 3 mL) solution of the appropriate N -Fmoc -

PAGE 103

103 aminoacyl)ben zotriazole (0.5 mM) was added. The coupling was induced using microwave irradiation (70 75 oC, 70 80 W, 1030 min) and the completion of the coupling reaction was assessed by a negative n inhydrin (Kaiser) test. Successive N Fmoc a minoacyl)benzotriazole were similarly coupled to the growing peptide. Deprotection of the N -Fmoc aminoacyl)benzotriazole was achieved at each stage with 20% piperidine DMF or 5% piperazine DMF. Finally, the resulting peptidyl resin was cleaved with cle avage cocktail B [TBAB] (88% TFA/ 5 % phenol/ 5% water/2% TIPS), K [TBAB] (82.5% TFA/ 5% phenol/ 5% water/ 5% th ioanisole/ 5% EDT) or L [TBAB ] (88% TFA/ 5% DTT/ 5% water/ 2% TIPS) for 1.5 2.0 h. Following cleavage, the peptide was precipitated with cold di ethyl ether, the ether peptide mixture incubated for 24 h at 4 oC and lyophilized to afford the crude peptides 6. 3 6. 10. 6. 5 .2 Parameters for Microwave Reactions Microwave experiments were performed using single mode irradiation in pulsed tempe rature contr ol mode (SPS mode). Open vessel reactions were performed in 25 mL Discover SPS reaction vessels as previously described (See Experimental). In addition to open vessel conditions, closed vessel syntheses were performed in a capped 10 mL vial for 3 9 Simil ar results were obtained under both conditions, however, closed vessel reaction were marginally faster than the corre sponding open vessel reaction. Average coupling times per N -Fmoc aminoacyl)benzotriazole under open vessel conditions were 12 15 min, when compared to closed vessel reactions wh ich were complete in ~ 10 min. Most N Fmoc aminoacylbenzotriazole produced a negative ninhydrin (Kaiser) test after the 10 15 min coupling time; however, double coupling was frequently required for Fmoc -L-Val -Bt ( 2a ). The characterization data presented for peptides 3 10 are for open vessel SPS, unless indicated.

PAGE 104

104 6. 5 3 Peptide Analysis Analyses of the peptides 6. 3 6. 10 (Table 2) were ca rried out on an Agilent (Palo Alto, CA) 1100 series HPLC equipped with a Phenomenex Synergi 4u Hydrocolumn plus a C18 guard column (2 mm x 4 mm). Mass Analysis was performed using a ThermoFinnigan LCQ ion trap mass spectrometer ( San Jose, CA) in electrospray ionization (ESI) mode. 6. 5 4 Characterization of Polymorphic Compounds 2c,d,j and 3 -10 Full characterization data for N -Fmoc ( aminoacyl ) benzotriazoles 2a,b,e -i,k,l are reported in the literature. [0 9ARK47 ] S (9 H F luoren 9 yl )methyl 1 (1 H benzotriazol 1 yl) 3 tertbutoxy 1 oxopropan 2 ylcarbamate (Fmoc -LSer( t Bu) Bt, 6. 2c) Recrystallized from EtOA c -hexanes to give white crystals (78%); mp 63.8 65.4 oC; 1H NMR 8.31 (d, J = 8.1 Hz, 1 H), 8.16 (d, J = 8.1 Hz, 1 H), 7.78 (d, J = 7.8 Hz, 2H), 7.727.64 (m, 3H), 7.55 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7. 4 Hz, 2H) 5.99 (d, J = 9.0 Hz, 1H), 5.905.84 (m, 1H), 4.504.48 (m, 2H), 4.31 4.26 (overlapped m, 1H), 4.2 4 (overlapped dd J = 10.0 Hz, 3.0 Hz, 1H), 3.9 1 (dd, J = 9. 8 Hz, 3. 2 Hz, 1H), 1.03 (s, 9H) ; 13C NMR 169.7, 156.6, 146.2, 144. 3, 144.1, 141.7, 131.6, 131.2, 128.1, 127.5, 126.9, 125.6, 125.5, 120.7, 120.4, 114.8, 74.4, 67.8, 63. 2, 56.2, 47.5, 37.8, 27.7, 27.5. Anal. Calcd. for C28H28N4O4: C, 69.40; H, 5.82 ; N, 11. 56. Found: C, 69.00; H, 5.86 ; N, 11. 33. S (9 H F luoren 9 yl)methyl 1 (1 H benzotriazol 1 yl) 3 (4 tertbutoxyphenyl) 1 oxopropan 2 ylcarbamate (Fmoc -LTyr( t Bu) Bt, 6. 2d) Recrystallized from EtOA c h exanes to give white crystals ( 84 %) ; mp 99.0100.5 oC; 1H NMR 8.19 (d, J = 8.1 Hz, 1 H), 8.10 (d, J = 8.4 Hz, 1 H), 7.72 (d, J = 7.5 Hz, 2H), 7.62 (overlapped t, J = 7. 7 H z 1H), 7.58 7.54 (m, 2H), 7.48 (overlapped t, J = 7. 7 Hz, 1H), 7.360(t, J = 7.4 Hz, 2H), 7.27 (t, J = 7.2 Hz, 2H) 7.02 (d, J = 7.8 Hz, 2H), 6.83 (2H, J = 7.8 Hz, 2H), 6.106 .08 (m, 1 H), 5.79 (d, J = 7.5 Hz, 1H), 4.434.35 (m, 2H), 4.18 (t, J = 6. 8 Hz, 1H),

PAGE 105

105 3. 38 (dd, J = 10.4 Hz, 5.6 Hz, 1H), 3.21 (dd, J = 13.8 Hz, 7.5 Hz, 1H) 1.24 (s, 9H); 13C NMR 170.9, 155.6, 154.5, 145.8, 143.6, 143.5, 141.1, 130.8, 130.6, 129.7, 129.6, 127.6, 126.9, 126.4, 124.9, 124.2, 120.2, 119.8, 114.1, 78.4, 67.0, 55.6, 46.9, 38.3, 28.6. Anal. Calcd. for C34H32N4O4: C, 72.84; H, 5.75; N, 9.99. Found: C, 72.84; H, 6.00; N, 9.70. S (9 H F luoren 9 yl)methyl 1(1 H benzotriazol 1 yl) 4 methyl 1 oxopentan 2 ylcarbamate ( Fmoc -LLeu Bt, 6. 2 j ) Recrystallized from EtOA c -hexanes to give white crystals (87%); mp 75.3 78.6 oC; 1H NMR 8.20 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H) 7.69 (d, J = 7.2 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.58 7.50 (m, 2H), 7.46 (t, J = 7.7 Hz, 1H) 7.33 (t, J = 7.1 Hz, 2H), 7.25 (t, J = 7.0 Hz, 2H), 5.825.72 (m, 1H), 5.43 (d, J = 8.7 Hz, 1H), 4.37 (d, J = 6.9 Hz, 2H), 4.17 (t, J = 6.5 Hz, 1H), 1.881.6 4 (m, 3H), 1.04 (d, J = 4.8 Hz, 3H), 0.91 (d, J = 5.1 Hz, 3H) ; 13C NMR 172.4, 156.1, 146.0, 143.8, 143.6, 141.3, 131.1, 130.7, 127.7, 127.0, 126.5, 125.1, 120.3, 120.0, 114.4, 67.1, 53.4, 47.1, 41.9, 25.2, 23.2, 21.3. HRMS calcd for C27H26N4O3 [M+Na]+ 477.1897, found 477.1898.

PAGE 106

106 Figure 6 3 1H NMR s pectr um of 6. 2c in CDC l3 Figure 6 4 13C NMR s pectr um of 6. 2c in CDCl3

PAGE 107

107 Figure 6 5 1H NMR s pectr um ( 8.4 6.7) of 6. 2 d in CDCl3 Figure 6 6 1H NMR s pectr um ( 6.2 1.1) of 6. 2 d in CDCl3

PAGE 108

108 Figure 6 7 13C NMR s pectr um of 6. 2 d in CDCl3

PAGE 109

109 Figure 6 8 1H NMR s pectr um of 6. 2 j in CDCl3 Figure 6 9 13C NMR s pectr um of 6. 2 j in CDCl3

PAGE 110

110 Figure 6 10. High Resolution Mass Spectr um of 6. 2 j

PAGE 111

111 Figure 6 11. Expected product ions from the (+)ESI -MSn of crude 6.3 (m/z 501 [M+H]+ ions). The shaded ions were observed. Figure 6 12. Fragmentation of crude 6. 3 H V V S V V NH 2 MW = 500.4 [M+H] + = 501.4 b ions H 2 O 268.2 367.2 466.3 a ions (loss of CO) 72.1 171.1 258.2 357.2 456.3 b ions N term 100.1 199.1 286.2 385.2 484.3 C term Residue H V V S V V NH 2 Residue mass 1.0 99.07 99.07 87.03 99.07 99.0 7 16.0 y ions 501.4 402.3 303.2 216.2 117.1 Loss of NH 3 484.4 385.3 286.2 199.2 100.1

PAGE 112

112 Figure 6 13. High Resolution Mass Spectrum of crude 6. 3

PAGE 113

113 Figure 6 14. HPLC profiles of a ) crude and b ) pure 6. 3 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage. In this instance 6.3 was synthesized using closed vessel conditions H V V S V V NH 2 MW = 500.4 [M+H] + = 501.4 Loss of H 2 O 240.2 339.2 438.3 b ions H 2 O 268.2 367.2 466.3 a ions (loss of CO) 72.1 171.1 258.2 357.2 456.3 b ions N term 100.1 199.1 286.2 385.2 484.3 C term Residue H V V S V V NH 2 Residue mass 1.0 99.07 99.07 87.03 99.07 99.07 16.0 y ions 501.4 402.3 303.2 216.2 117.1 Loss of NH 3 484.4 385.3 286.2 199.2 100.1 Loss of H 2 O 466.4 367.2 268.2 Figure 6 15. Expected product ions from the (+)ESI -MSn of crude 6.3 (m/z 501 [M+H]+ ions). There was a series of ions resulting from the loss of the serine (Ser) hydroxyl group as loss of H2O from the b ions and other product ions. The shaded ions were observed. In this instance 6.3 was synthesized using closed vessel conditions a b

PAGE 114

114 Figure 6 16. HPLC profile of crude 6. 4 Figure 6 17. E xpected product ions from the (+)ESI -MSn dissociation of crude 6.4 (m/z 600 [M+H]+ ion). The shaded ions were observed.

PAGE 115

115 Figure 6 18. High Resolution Mass Spectrum of crude 6. 4 ( a ) zoomed in -view, b ) full) b a

PAGE 116

116 Figure 6 19. HPLC profiles of a ) crude and b ) pure 6. 5 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage H V I V I G NH 2 MW = 498.4 [M+H] + = 499.4 a ions (loss of CO) 72.1 185.2 284.2 397.3 454.3 b ions N ter m 100.1 213.2 312.2 425.3 482.3 C term Residue H V I V I G NH 2 Residue mass 1.0 99.07 113.08 99.07 113.08 57.02 16.0 y ions 499.4 400.3 287.2 188.2 75.1 Loss of NH 3 482.4 383.3 270.2 171.2 58.1 Figure 6 20. Expected product ions from the (+ )ESI -MSn collision induced dissociation (CID) of crude 6.5 (m/z 499 [M+H]+ ion) with the product ions resulting from traditional cleava ge along the peptide backbone. The shaded product ions were observed. b a

PAGE 117

117 H V I V I G OH MW = 499.3 [M+H] + = 500.3 a NH 3 28 u 140.2 239.2 352.3 409.3 a NH 3 55.1 168.2 267.2 380.3 437.3 a ions (b CO, 28 u) 72.1 185.2 284.2 397.3 454.3 b ions 100.1 213.2 312.2 425.3 482.3 500.3 Residue H V I V I G OH Residue mass 1.0 99.1 113.1 99.1 113.1 57.0 17.0 y -ions N Ter m 500.3 401.3 288.2 189.1 76.0 C Term z ions (y NH 3 ) 483.3 384.3 271.2 172.1 59.0 z CO 455.3 356.3 243.2 144.1 31.0 Figure 6 21. Expected product ions from the (+)ESI -MSn CID of pure 6. 5 (m/z 500 [M+H]+ ion) with the product ions resulting from tr aditional cleavage along the peptide backbone. The shaded product ions were observed. Figure 6 22. High Resolution Mass Spectrum of pure 6. 5

PAGE 118

118 Figure 6 23. HPLC profiles of a ) crude and b ) pure 6. 6 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage H T V T V T V NH 2 MW = 617.4 [M+H] + = 618.4 a 1 Ions (loss of CO) 74.1 173.1 274.2 373.2 474.3 573.4 b ions N term 102.1 201.1 302.2 401.2 502.3 601.4 C term Residue H T V T V T V NH 2 Residue mass 1.0 101 .1 99.1 101.1 99.1 101.1 99.1 16.0 y ions 618.4 517.4 418.3 317.2 218.2 117.1 Loss of NH 3 601.4 500.4 401.3 300.2 201.2 100.1 b ions 302 401 502 601 H 2 O 284 383 484 583 H 2 O 266 365 466 565 H 2 O 448 547 Figure 6 24. Product ions from the (+)ESI MSn dissociation of crude 6.6 (m/z 618 [M+H]+ ion). In addition to the traditional b and y ions, the b ions yielded a number of ions due to successive losses of H2O (from Thr, threonine). a b

PAGE 119

119 Figure 6 25. High Resolution Mass Spectrum of pure 6. 6

PAGE 120

1 20 Figure 6 26. HPLC profiles of a ) crude and b ) pure 6. 7 obtained after SPPS using 5% piperazine DMF for Fmoc cleavage H V K D G Y I NH 2 MW = 692.4 [M+H] + = 693.4 b ions H 2 O 325.2 382.2 54 5.3 658.3 a -ions (loss of CO) 72.1 200.2 315.2 372.2 535.3 648.4 b ions N term 100.1 228.2 343.2 400.2 563.3 676.4 C term Residue H V K D G Y I NH 2 Residue mass 1.0 99.07 128.0 9 115.0 3 57.02 163.06 113.0 8 16.0 y ions 693.4 594.3 466.2 351.2 294.2 131.1 Loss of NH 3 676.4 577.3 449.2 334.2 277.2 114.1 Figure 6 27. Expected product ions from the (+)ESI -MSn of crude 6. 7 (m/z 693 [M+H]+ ion) The shaded ions were observed. b a

PAGE 121

121 Scheme 6 3. Possible (+)ESI MSn di ssociation pathway for the formation of m/z 449 and m/z 336 product ions from the m/z 693 [M+H]+ ion of 6.7 N H 2 1 N H 2 N H 2 + 3 N H 4 N H 5 N H 6 N H 2 O O O O O O H C H 3 C H 3 N H 2 O O H C H 3 C H 3 O H N H 3 + 3 N H 4 N H 5 N H 6 N H 2 O O O O H O O H C H 3 C H 3 O H C H + 3 N H 4 N H 5 N H 6 N H 2 O O O O H O O H C H 3 C H 3 O H C H + 3 N H 4 N H 5 N H 2 O O O H O O H O H m/z 693 [M+H] + m/z 466 m/z 449 m/z 336

PAGE 122

122 Figure 6 28. High Resolution Mass Spectrum of crude 6. 7 H N H N N H N H O O O O O N H2O H N H2O H 6 7

PAGE 123

123 Figure 6 29. HPLC profiles of a ) crude and b ) pure 6. 8 obtained after SPPS using 5% piperazine DMF for Fmoc cleavage H V K D V Y I NH 2 MW = 734.4 [M+H] + = 735.5 b ions H 2 O 325.2 424.3 587.3 700.4 a ions (loss of CO) 72.1 200.2 315.2 414.3 577.3 690.4 b ions N term 100.1 228.2 343.2 442.3 6 05.3 718.4 C term Residue H V K D V Y I NH 2 Residue mass 1.0 99.07 128.09 115.03 99.07 163.06 113.08 16.0 y ions 735.5 636.4 508.3 393.3 294.2 131.1 Loss of NH 3 718.5 619.4 491.3 376.3 277.2 114.1 Figure 6 3 0 Expected product ions from the (+)ESI CID -MS/MS of crude 6.8 (m/z 735 [M+H]+ ion). The shaded product ions were observed. a b

PAGE 124

124 Figure 6 3 1 High Resolution Mass Spectrum of crude 6. 8

PAGE 125

125 H Y G G F L NH 2 MW = 554.3 [M+H] + = 555.3 a NH 3 28u 91.1 148.1 205.1 352.2 465.3 a NH 3 119.1 1 76.1 233.1 380.2 493.3 a ions (b CO, 28 u) 136.1 193.1 250.1 397.2 510.3 b ions 164.1 221.1 278.1 425.2 538.3 555.3 Residue H Y G G F L NH 2 Residue mass 1.0 163.1 57.0 57.0 147.1 113.1 16.0 y ions N Term 555.3 392.2 335.2 278.2 131.1 C Term z io ns (y NH3) 538.3 375.2 318.2 261.2 114.1 z CO 510.3 347.2 290.2 233.2 86.1 Figure 6 3 2 Expected product ions according to classical cleavage along the amide backbone to create b a -, y and z ions. The shaded ions were observed for pure 6.9 Figure 6 3 3 High Resolution Mass Spectrum of pure 6. 9

PAGE 126

126 Figure 6 3 4 Gradient analysis: The major compound was the expected peptide 6.9 ( MW 554, shaded peaks). There was also a minor compound with a MW 555 eluting shortly after 6.9

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127 F:\!-LCQ-Data-To-Archive\...\SEQ-9584-01 03/19/2008 12:24:25 PM DH/V / LeuEnk; 10 uL Atlantis dC18;0.15;100:0(0)>70:30(10)>5:95(55-65)/254 nm/(+)ESI SEQ-9584-01 # 978-1045 RT: 28.62-29.55 AV: 11 NL: 4.14E8 F: + c ESI Full ms [ 200.00-1600.00] 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance (+)ESI-MS, no SCID 1109.2 555.2 1110.3 538.1 556.2 425.0 397.1 1111.3 510.2 577.4 278.0 426.1 220.8 375.0 1112.2 578.3 851.7 492.9 1108.3 261.9 612.2 346.9 1166.2 482.9 306.8 x5 SEQ-9584-02 # 553-731 RT: 6.82-8.34 AV: 26 NL: 1.15E8 F: + c ESI sid=25.00 Full ms [ 50.00-600.00] 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance (+)ESI-MS, with 25% SCID 555.1 538.1 577.3 278.0 510.2 425.0 397.0 347.1 261.8 120.1 375.0 539.1 493.0 335.4 220.9 578.3 511.2 136.0 260.9 277.2 557.1 279.0 233.0 426.0 380.0 465.2 176.9 398.1 540.1 204.3 318.9 348.0 494.1 297.1 579.3 537.3 164.9 91.0 238.0 107.1 558.1 155.0 x5 Figure 6 3 5 The (+)ESI MS spectrum of peptide 6.9 produced m/z 555 [M+H]+, m/z 577 [M+Na]+ and m/z 1109 [M+H+M]+ ions Collision induced dissoc i ation in the source region of the mass spectrometer (SCID) was used to produce some characteristic fragment ions. Some of these fragment ions were then chosen for MSn scans.

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128 F:\!-LCQ-Data-To-Archive\...\SEQ-9584-01 03/19/2008 12:24:25 PM DH/V / LeuEnk; 10 uL Atlantis dC18;0.15;100:0(0)>70:30(10)>5:95(55-65)/254 nm/(+)ESI SEQ-9584-01 # 972-1048 RT: 28.55-29.58 AV: 12 NL: 2.77E8 F: + c ESI sid=2.00 Full ms2 555.30@37.50 [ 150.00-750.00] 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 538.1 510.2 539.2 425.0 493.1 375.1 397.1 511.3 426.1 494.2 261.9 347.1 520.2 465.1 380.1 278.1 398.0 492.4 424.4 SEQ-9584-01 # 975-1039 RT: 28.57-29.50 AV: 11 NL: 6.65E7 T: + c sid=2.00 d Full ms3 555.30@37.50 538.12@37.50 [ 165.00-1085.00] 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 510.2 493.1 375.0 511.2 425.0 494.1 465.1 380.0 347.1 397.1 520.2 261.9 426.0 492.2 466.2 509.5 348.0 381.1 346.2 330.0 398.1 278.1 419.2 436.1 288.9 216.9 232.8 SEQ-9584-01 # 979-1030 RT: 28.59-29.34 AV: 9 NL: 3.45E7 T: + c sid=3.00 d Full ms3 555.30@37.50 510.27@37.50 [ 155.00-1030.00] 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 493.1 465.1 380.0 351.1 346.1 494.1 466.2 397.0 375.0 425.0 289.1 492.3 436.1 352.1 381.1 261.2 398.1 188.1 278.1 464.3 289.9 204.1 238.1 329.9 Figure 6 3 6 The (+)ESI MS/MS of 6. 9 ( m/z 555 [M+H]+ ion ) (top) and (+)ESI -MS/MS/MS of m/z 538 (middle) and m/z 510 (bottom) primary product ions of 6. 9

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129 Figure 6 3 7 HPL C profiles of a ) crude and b ) pure 6. 10 obtained after SPPS using 20% piperidine DMF for Fmoc cleavage Figure 6 3 8 High Resolution Mass Spectrum of pure 6. 10 b a

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130 F:\!-LCQ-Data-To-Archive\...\SEQ-9420-02 02/23/2008 04:11:58 AM AIVVGGVML(NH2); in glass vial; 10 uL Atlantis dC18;0.15;95:5(0)>5:95(45-65)/210 nm/(+)ESI SEQ-9420-02 # 1183-1205 RT: 36.23-36.55 AV: 4 SB: 2 35.95-36.05 37.08-37.13 NL: 9.34E8 F: + c ESI Full ms [ 200.00-1800.00] 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance MW 856 m/z 857 = [M+H]+ m/z 879 = [M+Na]+ m/z 1713>1715 = [M+H+M]+ 857.4 858.4 879.6 1714.3 880.6 1715.2 656.3 840.4 769.3 557.2 1305.3 529.2 928.3 288.1 398.0 SEQ-9420-02 # 1179-1212 RT: 36.16-36.69 AV: 6 NL: 3.35E8 F: + c ESI sid=2.00 Full ms2 857.50@37.50 [ 235.00-900.00] 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 840.3 769.4 841.3 741.4 727.4 656.4 770.4 742.4 812.3 822.5 709.4 795.3 628.3 670.3 557.2 823.4 610.3 796.3 497.2 724.4 751.4 638.4 682.3 696.2 529.3 596.4 558.3 426.2 444.1 539.3 512.2 783.4 458.2 313.0 483.2 381.2 412.1 842.2 SEQ-9420-02 # 1183-1205 RT: 36.28-36.50 AV: 3 NL: 6.37E7 T: + c sid=2.00 d Full ms3 857.50@37.50 840.35@37.50 [ 220.00-1690.00] 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 m/z 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 741.3 727.3 822.4 742.3 709.2 628.1 823.4 795.2 812.2 796.2 710.3 610.3 582.3 497.1 656.3 682.1 634.2 528.4 747.1 426.1 480.2 696.1 769.1 557.2 450.2 511.0 412.2 397.9 794.4 627.4 343.8 284.7 313.0 383.8 568.1 369.8 327.1 266.8 Figure 6 39. The (+)ESI MS of p eptide 6. 10 predominantly produced its m/z 857 [M+H]+ ion (top). The m/z 857 [M+H]+ ion then underwent collision -induced dissociation (CID) to form m/z 840 and numerous other primary product ions (middle). The m/z 840 product ion was further dissociated to yield m/z 741 and other secondary product ions (bottom).

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131 Figure 6 4 0 T he traditional amide cleavage of 6.10 during (+)ESI MS and MSn produced t he expected a, b, y, and yNH3 ions. In addition, there were abundant ions due to loss of NH3 from the a ions The shaded ions were detected. MW = 856.5 [M+H] + = 857.5 b 1 b 2 b 3 b 4 b 5 b 6 b 7 b 8 b 9 a-ions -NH3 69.1 200.1 299.2 356.2 413.2 512.3 611.4 724.4 795.5 a-ions (loss of CO) 86.1 217.1 316.2 373.2 430.2 529.3 628.4 741.5 812.5 C-term b-ions N-term 114.1 245.1 344.2 401.2 458.2 557.3 656.4 769.5 840.5 857.5 Residue H L M V G G V V I A NH2 Residue mass 1.007825 113.08 131.04 99.07 57.02 57.02 99.07 99.07 113.08 71.04 16.01905 y-ions 857.5 744.4 613.4 514.3 457.3 400.3 301.2 202.2 89.1 18.0 Loss of NH3 840.5 727.4 596.4 497.3 440.3 383.3 284.2 185.2 72.1 y9 y8 y7 y6 y5 y4 y3 y2 y1 H L M V G G V V I A NH 2

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132 CHAPTER 7 SYNT HESES OF AZOLE -BASED AMINO ACIDS AND PEPTIDES, AND WATER SOLUBLE COUPLING REAGENTS 7.1 Azole -based Amino Acids and Peptides 7.1.1 Introduction Recently there has been considerable interest in the synthes e s of modified peptides and novel amino acids posse ssing a heterocyclic moiety due to their promising biological activity. [00JCS(PT 1)2311, 07OL2381, 08S2462] Amino acid and peptide conjugates with 1, 3,4 oxadiazole moieties display various properties including but not limited to antidepressa nt, antimicrobi al, hypoglycemic and analgesic effects [08TL4746] Furthermore, 1,3,4 -oxadiazoles are known to be bioisosteres of amides and esters. [08TL4746] Compounds with 1,2,4 -triazole and 1,3,4 thiadiazole components also display a considerable range of biological a ctivity including antimycotic, antibacterial, antidepressant, HIV inhibitory and antimycobacterial effects [93T165] In nature, a vast array of biologically active peptides is present. Despite the abundance of bioactive peptides and proteins, p eptide drugs are rare due to the prevalence of human peptidases. -Amino acids and peptides containing such amino acid residues occur less frequently in nature and are more stable to human peptidase, thus may be useful in peptidomimetics [07OBC2884] The attention of many research groups has focused on the synthe s e s of hybrid amino acids and peptides for use in peptidomimetics [07OL2381, 08TL4746, 07OBC2884] Hamz et al. [03JOC7316] reported the synthesis of various 3 -substituted 1,2,4 oxadiazole -containing chiral 3and amino acids from Fmoc protected asparti c acid Recently, Katritzky and coworkers de scribed the synthesis of chiral 1,2,4 oxadiazoles using N aminoacyl)benzotriazoles of some amino a cids in 7094% yield. [05ARK36] A search of the literature disclose d very few

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133 reported synthes e s of 1, 2,4 -triazolo or 1,3,4 -thiadiazolo -substi tuted amino acids or peptides. [07MOL103, 07HAC316] Prompted by the potential biological activities of these compounds and i n continu ation of the work on the versatility of N aminoacyl)benzotriazoles, th e proposed synthetic route to 1,2,4 -oxadiazolo -substituted dipeptides, 1,2,4 triazolo -substituted amino acids and peptides and 1,3,4 thiadiazolo -substituted amino acids and peptides is now described 7.1.2 Proposed Synthetic Route to Azolo -b ased Amino Acid s and Dipeptides A practical method for the synthesis of 1,2,4-triazolo -, 1,2,4 oxadiazolo 1,3,4 thiadiazolo amino acid derivatives involves the reaction of N -protected aminoacyl)benzotriazoles 7.1 with amidrazones 7.2 amidoximes 7.3 and thiohydrazides /thiosemicarbazides 7.4 / 7.5 respectively ( Figure 7 1). Figure 7 1. Synthesis of 1,2,4 triazolo 1,2,4 -oxadiazolo 1,3,4 -thiadiazolo amino acid derivative s 7.6 7.7

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134 Similarly azolo containing dipeptides can be made from dipeptidoylbenzotriazoles 7. 10 and amidrazones 7.2 amidoximes 7.3 and thiohydrazides /thiosemicarbazides 7.4 / 7.5 ( Figure 7 2). Figure 7 2. Synthesis of 1,2,4 triazolo 1,2,4 -oxadiazolo 1 ,3,4 -thiadiazolo dipeptide derivatives 7. 117. 14 7.1.3 Preparation of N aminoacyl)benzotriazoles 7.1 N-Protected aminoacyl)benzotriazoles 7.1 were prepared from commercial N protected -Lamino acids according to the literature (Scheme 7 1 ). [09ARK47] Scheme 7 1. Preparation of N protected aminoacyl)benzotr iazoles 7.1

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135 7.1.4 Preparation of Amidrazones 7.2 A midrazones 7.2 also referred to as hydrozonamides, can be obtaine d by several routes. This includes the reaction of hydrazines with (i) nitriles [70CR151] (ii) imidates and their salts [92JCS(PK2)671, 68J OC1679], (iii) imidoyl halides [70CR151], (iv) amides or thioamides in the presence of POCl3 [50JACS2783, 58JOC1931], (v) ketenimines [65JOC3718], and (vi) imidoyl benzotriazoles [06JOC9051] Although the reaction of nitriles with hydrazines appears to be simple, i t is known that the reaction of nitriles with hydrazine can lead the formation of dihydrotetrazines and subsequently tetrazines by oxidation [70CR151], however the reaction outcome is largely controlled by the nature of the nitrile. [06JOC9051] In itially, the reaction of nitriles 7.15 with hydrazine 7.16 yielded the corresponding dihydrotetrazines 7.17 rather the amidrazone as the major product (Scheme 7 2). For nitrile 7.1 5 a a trace amount of the amidrazone was present as evidenced in the high res olution mass spectrum (See 7.3 Experimental Section ). Subsequently, the reaction of amidine 7.18 with hydrazine 7.16 afforded the smooth conversion to amidrazone 7.2b Scheme 7 2. Preparation of amidrazones 7.2 and dihydrotetrazines 7.17

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136 7.1.5 Preparat i ve routes to Amidoximes 7.3 Thiohydrazides 7.4 and Semicarbazides 7.5 Amidoximes 7.3 can be readily prepared following the protocol of Hamz and coworkers [03JOC7316]. Us ing this procedure amidoximes 7.3 will be synthesized by reacting nitriles 7.15 and hydroxylamines 7.19 (Scheme 7 3). Scheme 7 3. Preparation of amidoximes 7.3 The key intermediate in the preparation of t hiohydrazides 7.4 and semicarbazides 7.5 is hydrazine 7.16. T hiohydrazides 7.4 will be readily available via the reaction of various thiocarbonylbenzotriazoles 7.20 with hydrazine 7.16 (Scheme 7 4) [05JOC7866] Similarly, semicarbazides 7.5 can be obtained by reacting thiocarbamoylbenzotriazoles 7.2 1 with hydrazine 7.16 (Scheme 7 4 ). [07JOC 6742] Scheme 7 4. Preparation of thi ohydrazi des 7.4 and semicarbazides 7.5 7.1.6 Summary and Future Prospect Precursors to azole based amino acids and peptides such as n ovel N -Cbz ( aminoacyl)benzotriazoles 7.1a and amidrazone 7.2b were s ynthesized and will be utilized in subsequent coupling reactions. Previously, N -p aminoacyl)benzotriazoles 7.1 and amidoximes were coupled by refluxing in ethanol in the presence of trieth ylamine. [05ARK36] Following the syntheses of a vast array of intermediates 7. 2 -7.5 investigations into the optimum

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137 conditions for the microwave assisted couplings of N aminoacyl)benzotriazoles 7.1 with amidrazones 7.2 amidoximes 7.3 and thio hydrazides /thiosemicarbazides 7.4 / 7.5 will be explored. 7 .1.7 E xperimental Section Melting points were determined on a hot -stage apparatus and are uncorrected. 1H (300 MHz, with TMS as the internal standard) and 13C NMR (75 MHz) NMR spectra were recorded i n CDCl3 or DMSO d6. Elemental analysis was carried out in an Eager 200 CHN analyzer. 7.1.7.1 General p rocedure for the p reparation of N aminoacyl)benzotriazoles 7.1 Naminoacyl)benzotriazoles 7.1 were prepared using established protocol. [09ARK47]. Preparative details and characterization data for 7.1b,c were described in 4.4 Experimental Section. Benzyl N -[(1 S )-1 -(1 H -1,2,3 -benzotriazol -1 -ylcarbonyl) -3 methylbutyl]carbamate (7.1a). Recrystallized from EtOAc -hexanes to give white crystals ( 86%); mp 61. 7 63. 9 oC; 1H NMR (CDCl3) 8.23 (d, J = 8.4 Hz 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.39 7.26 (m, 5H), 7.09 (br s, 1H), 5.885.78 (m, 1H), 5.51 (d, J = 8.7 Hz, 1H), 5.11 (s, 2H), 1.90 1.80 (m, 2H), 1.801.64 (m, 1H), 1.08 (d, J = 5.1 Hz, 3H), 0.95 (d, J = 6.0 Hz, 3H); 13C NMR (CDCl3) 172.4, 146.0, 130.7, 128.5, 128.2, 128.1, 126.4, 120.3, 114.4, 67.3, 53.4, 42.0, 25.2, 23.2, 21.3. Anal. Calcd for C20H22N4O3: C, 65.56; H, 6.05; N, 15.29. Found: C, 65.66; H, 6.43; N, 14.96. 7.1.7.2 General procedure for the preparation of amidrazone 7. 2b and dihydrotetrazine 7.17a,b To an e thanolic solution of benzamidine hydrochloride ( 8 mmol ) was added hydrazine ( 8 m mol). The mixture was refluxed for 12 h, and then allowed to cool to room temperature. On

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138 cooling, an o ff-white solid precipitated. This solid was filtered and washed with cold ethanol to afford (Z ) -benzohydrazonamide 7.2b as an off -white powder that turned pink on standing. Similarly, dihydrotetrazines 7.17 were prepared by adding hydrazine ( 8 m mol) to an ethanolic solution of 4 -methoxyphenylacetonitrile 7.15a (8 mmol) or p -tol unitrile 7.15b (1 mol) The mixture was refluxed for 12 h, and then allowed to cool to room temperature. On cooling, an off-white solid precipitated. This solid was filtered and washed with cold ethanol to afford dihydrotetrazines 7.17a,b (Z )-Benzohydrazo namide (7.2b). Recrystallized from isopropanol to give an off -white powder (6 5 %); mp 183.6184.6 oC; 1H NMR ( DMSO d6) 7.877.81 (m, 1H), 7.507.40 (overlapped m, 4H), 7.35 (br s, 4H); 13C NMR ( DMSO d6) 148.0, 130.3, 128.6, 126.0. HRMS calcd for C7H9N3 [M+H]+ 136.0865, found 136.0869. 3,6 -D i -p -tolyl -1,2 -dihydro -1,2,4,5 -tetrazine (7.17 a ). Recrystallized from ethanol to give pink crystals (71%); mp 218.4219.6 oC; 1H NMR (CDCl3) 7.55 (d, J = 8.1 Hz, 4H), 7.28 (d, J = 6.9 Hz, 4H), 2.42 (s, 6H); 13C NMR (CDCl3) 143.8, 132.2, 130.0, 129.7, 126.0, 22.0. HRMS calcd for C16H16N4 [M+H]+ 265.1403, found 265.1453. 3,6 -Bis(4 -methoxybenzyl) -1,2 -dihydro -1,2,4,5 -tetrazine (7.17b). Recrystallized from ethanol to give pink crystals ( 60%); mp 192.2196. 9 oC; 1H NMR (CD Cl3) 7.16 (d, J = 8.7 Hz, 4H), 6.82 (d, J = 8.7 Hz, 4H), 4.09 (s, 6H), 3.77 (s, 4H) ; 13C NMR (CDCl3) 158.7, 154.3, 129.7, 127.5, 114.4, 55.4, 30.2. Anal. Calcd for C18H20N4O2: C, 6 6 56; H, 6.21; N, 17.27. Found: C, 66.33; H, 6.19; N, 17.32. 7. 2 Synthe sis of Water -soluble Coupling Reagents 7. 2 .1 Intro duction Solid phase peptide synthesis (SPPS) is a simple and rapid technique that offers ease of separation of products from reagents, while eliminating inherent product losses associated with

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139 conventional solution phase methodologies (filtration, recr ystallization, etc.). [97 MI1 ] When c ompared with conventional solution phase methodologies, the increased efficiency of SPPS has resulted in its near -exclusive use for the preparation of peptides over the last thirty years [73JACS4501, 06CEJC285] However, there is concern regarding the environmentally friendly yet safe disposal of large amounts of organic solvents requi red for SPPS. [01JPS615, 04TL9293, 06PPL189] Reactions performed in aqueous media could reduc e the copious amount of solvents required for SPPS Published SPPS in aqueous media water has used water soluble N protected amino acids. In standard N C SPPS water soluble coupling reagents can be made by (i) protecting the N terminus with a polar group a nd (ii) activating the C -terminus with a polar group. To date (i) aqueous SPPS with water soluble N -pro tecting groups [75IJPPR295, 76C B3693, 78JOC4808, 06PPL189, 04CPB422], has been emphasized with little attention to (ii) water soluble N protected amino acids via the activation of the C terminus [04TL9293, 06PPL189, 79JACS3394]. Water soluble N -protecting groups utilized include the methylsulfonylethoxycarbonyl [75IJPPR 295], 2 (triphenylphos phino)ethoxycarbonyl [76CB3693], 9 (2 -sulfo)fluor enylmethoxycarbo nyl [78JOC4808], 2 [phenyl(methyl)sulfonio] ethoxycarbonyl (Pms) [06PPL189], 2 (4 -sulfophenyl) ethoxycarbonyl (Sps) [06PPL189] and N -ethanesulfonylethoxy carbonyl (Esc) [0 4CPB422] groups (Figure 7 3 ). Figure 7 3 Structures of some of water soluble N -prote cting groups

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140 Conversely, a search of the literature revealed a few methods involving SPPS using water soluble carbodiimides (WSCD) and water soluble active esters (Scheme 7 4). [06PPL189, 04TL9293, 79JACS3394] Figure 7 4 Structures of water soluble ac tivating groups Recently the preparation of N (Fmoc aminoacyl)benzotriazoles of 18 proteinogenic amino acids, their utility in the microwave accelerated synthesis of tri to heptapeptides in cru de yields of 6577% [09ARK47, 07CBDD465] and short difficul t peptides in crude yie lds of 3486% [09JOC2028] on the Rink amide MBHA solid support was reported N (Fmoc aminoacyl)benzotriazoles are sparingly soluble in water, therefore, novel water -soluble coupling reagents 7.22 and 7.23 we re designed with the a i m of performing SPPS in aqueous media (Figure 7 5 ). Herein preparative routes to benzotriazole 6 -sulfonic acid derivatives 7.22 and N (2 (2 pyridyl)ethoxycarbonyl) aminoacyl)benzotriazoles 7.23 as well as their potential for application in SPPS is descri bed Figure 7 5 Potential water -soluble coupling reagents 7.22 and 7.23

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141 7. 2 .2. Pr eparative Routes to Water -soluble Coupling Reagents 7.22-7.23 7. 2 .2.1 Retrosynthetic analysis for 7.22 The retrosynthetic pathway leading to title compound 7.22 is shown in Figure 7 6 The water -soluble C activating group, 1 H -benzotriazole 6 -sulfon ic acid 7.2 9 was readily obtained via nitro group reduction and subsequent cyclization of commercially available sodium 4 amino 3 nitrosulfonate 7.30 (Scheme 7 5 ). Figure 7 6 Retrosynthetic analysis of 7.22 Scheme 7 5 Preparation of 1 H -benzotriazole 6 -sulfonic acid 7.2 9

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142 The preparation of the N protecting group is more involved and is now described. To commence, t he reaction of 4 -mercaptophenol with bromoethanol in the p resence of NaOH will yield 4(2 hydroxyethylsulfanyl)phenol 7.25. [03OPRD418] Oxidation of 7.25 with oxone 7.3 3 and subsequent reaction with bis(benzotriazo 1 -yl) methanone 7.26 in the presence of Et3N should afford 7.3 2 (Scheme 7 6 ). [62USP3068278] Reaction of 7.3 2 with sultone and further coupling with free Lamino acids 7.28 should furnish 7.24 (Scheme 7 6 ). Once 7.24 is obtained the coupling reaction with 1 H -benzotriazole 6 sulfon ic acid 7.24 can be performed (Scheme 7 7 ). Scheme 7 6 Preparation of 7. 24 from 7.25 Scheme 7 7 Proposed route to 7.22

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143 7.2.2.3 Retrosynthetic analysis for 7.23 The retrosynthesis for possible water -soluble N -(2 (4 -pyridyl)ethoxycarbonyl) aminoacyl)benzotriazoles 7.23 is shown in Figure 7 7 The reaction of 2 (pyrid in 4 yl ) ethanol with bis(benzotriazo1 -yl)methanone 7.26 in the presence of Et3N should afford 7.3 6 (Scheme 7 8) Further reaction of 7.3 6 with amino a cids should yield 7.3 4 which will be reacted with 1 H benzotriazole or 7 azabenzotriazole to afford 7.23 (Scheme 7 8) Figure 7 7 Proposed synthetic route to 7. 23 Scheme 7 8. Proposed preparation of 7.2 3 from 7.3 5

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144 7.2.2.4 General procedure for the preparation of 1 H -benzotriazole -6 -sulfonic acid 7.2 9 So dium 4 amino 3 -nitrosulfonate 7.30 (1.50 g, 6.25 mmol) was dissolved in water (25 mL) an d p alladium/carbon (50 % wet, 0.66 g, 0.312 mmol, 20 mol %) was slowly added to the solution. The mixture was sti rred under hydrogen for 3 h. After 3 h the mixture was filtered through a celite bed and t he red -brown filtrate lyophilized for 24 hours to afford a red brown liquid, sodium 3,4 diaminobenzenesulfonate 7.31 (1.19 g, 5.66 mmol, 90%) To an acidic solution o f the sodium 3,4 -diaminobenzenesulfonate 7.31 (0.50 g, 2.379 mmol) was slowly added NaNO2 solution (0.16 g, 2.379 mmol) at 0 oC. The solution was stirred at room temperature for 4 h then neutralized with 2N NaOH. The water was then evaporated to yield 1 H b enzotriazole 6 -sulfonic acid 7.29 (0.32 g, 1.447 mmol, 60%). The dissociation of MW 199 compound 7.29 is shown in Figure 7 10. In Figure 7 10 compound 7.29 formed a m/z 200 [M+H]+ ion (top spectrum) that dissociated to yield a number of product ions (middl e spectrum). Further dissociation of the m/z 136 product ion of 7.29 is also illustrated in Figure 7 10 (bottom spectrum) Additionally, the m/z 198 [M H]ion of 7.29, a number of self adduct ions (for example, m/z 397 [(M H)+M]-) and the product ions of dissociation are shown in Figure 7 11. Sodium 3,4 -diaminobenzenesulfonate (7.31). Red brown liquid (90%); 1H NMR (D2O) 7.08 (d, J = 2.1 Hz, 1H), 7.04 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H); 13C NMR (D2O) 137.2, 133.1, 133.0, 117.6, 115. 7, 113.6. HRMS calcd for C6H7N2O3S [M Na ]1 87.0181, found 187.0183. 1 H -Benzotriazole -6 -sulfonic acid (7.29). Brown wax (6 0 %); 1H NMR ( D2O ) 8.38 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H), 7.83 (dd, J = 8.7, 1.6 Hz, 1H); 13C NMR (D2O ) 138.9, 122. 2, 115.3, 113.6. HRMS calcd for C6H4N3O3S [M]197.9973, found 197.8068.

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145 Figure 7 8. 1H NMR spectrum of 7.29 in D2O Figure 7 9 13C NMR spectrum of 7.29 in D2O

PAGE 146

146 E:\0-Spec\0-Data\Seq-10152-01 06/11/08 01:12:01 PM DH / VI /010; 10 uL Atlantis dC18; 0.15;100:0(0)>5:95(45-65)/254 nm/(+)ESI SEQ-10152-01 # 334-359 RT: 8.28-8.71 AV: 4 SB: 2 6.55-6.85 NL: 8.08E5 T: + c ESI sid=2.00 Full ms [ 105.00-1000.00] 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 20 40 60 80 100Relative Abundance MW 199 200.2 842.6 685.9 657.7 864.8 464.8 881.5 454.4 482.6 794.5 638.7 916.7 808.2 201.0 714.8 120.8 621.8 743.3 298.7 321.7 999.1 358.8 226.9 149.1 944.8 567.1 390.5 536.1 SEQ-10152-01 # 288-354 RT: 7.19-8.59 AV: 11 NL: 6.82E4 T: + c sid=2.00 d Full ms2 200.10@45.00 [ 55.00-415.00] 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 m/z 0 20 40 60 80 100Relative Abundance 119.2 136.1 122.1 108.1 80.1 81.1 124.1 139.7 135.1 96.1 107.0 118.3 90.0 151.0 171.9 78.9 163.0 200.2 125.1 180.9 71.1 215.4 231.3 186.2 SEQ-10152-01 # 296-344 RT: 7.37-7.90 AV: 4 NL: 4.47E3 T: + c sid=2.00 d Full ms3 200.17@45.00 136.13@45.00 [ 25.00-280.00] 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 m/z 0 20 40 60 80 100Relative Abundance 108.1 67.9 107.2 52.2 81.3 108.9 Figure 7 10. The m/z 200 [M+H]+ ion of 7.29 (top) plus the primary and secondary product ions of disso ciation (middle and bottom )

PAGE 147

147 E:\0-Spec\0-Data\Seq-10152-03 06/11/08 03:34:52 PM DH / VI /010; 2 uL Atlantis dC18; 0.15;100:0(0) Isocratic/254 nm/(-)ESI SEQ-10152-03 # 403-484 RT: 8.62-9.64 AV: 21 NL: 2.17E6 F: c ESI sid=1.00 Full ms [ 105.00-1000.00] 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 20 40 60 80 100Relative Abundance 198.1 639.9 418.9 860.7 396.8 640.9 818.9 862.8 634.0 199.1 419.9 461.0 486.7 681.8 170.0 821.0 396.0 617.6 552.9 960.7 748.5 981.2 290.4 872.6 907.6 592.8 248.0 781.8 732.6 135.0 366.8 SEQ-10152-03 # 402-489 RT: 8.63-9.70 AV: 22 NL: 3.82E5 F: c ESI sid=1.00 Full ms2 198.10@37.50 [ 50.00-220.00] 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 m/z 0 20 40 60 80 100Relative Abundance 170.0 90.1 171.1 169.2 91.1 80.3 89.4 118.1 199.0 SEQ-10152-03 # 408-468 RT: 8.74-9.44 AV: 11 NL: 1.33E5 T: c sid=1.00 d Full ms3 198.10@37.50 170.08@37.50 [ 35.00-350.00] 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 m/z 0 20 40 60 80 100Relative Abundance 90.2 105.2 91.0 Figure 7 11. The m/z 198 [M H]ion of 7.29, self adduct ions (top) plus primary and secondary product ions of dissociation (bottom)

PAGE 148

148 N H N S O O O-N N H S O O OC-N H N-N S O O O H N N-S O O O H Nm/z 90 m/z 170 m/z 198 [M-H][SO3]-, m/z 80 N H O m/z 105 Scheme 7 9. Probable ( )ESI -MSn dissociation of the m/z 198 [M H]ion of 7.29

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149 7.2.2. 5. Future work Subsequent to the synthesis of the 1 H -benzotriazole 6 -sulfonic acid amino acid derivatives 7.22 and N -(2 (2 -pyridyl)ethoxycarbonyl) aminoacyl)benzotriazoles 7.23, w ater solubility studies will be conducted on 7.22 and 7.23. In anticipation of a favorable outcome, the synthes e s of Leu and Met -e nkephalin will be attempted with water -soluble coupling reagents 7.22 and 7.23 (Scheme 7 10) and a comparison of the results made with the previous synthesis of Leuenkephalin (See Section 6.2 .1 ). Scheme 7 10. Proposed SPPS using potential water -soluble reagents 7.22 or 7.23

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150 CHAPTER 8 CONCLUSIONS SUMMARY OF ACHIEVEMENTS AND FUTURE OUTLOOK Synthetic organic chemistry had it genesis in the study of natural products. In t he broadest sense, the development of new methodologies still finds its inspiration from nature which is a constant source of intellectual challenge. Unlocking the myster ies surrounding the biogenesis of heterocycles and other pertinent biological molecule s, as well as understanding their properties and functions in nature requires a focused, multidisciplinary approach and s ynthetic organic chemistry has been a valuable tool in this process Chapter 1 presented an overview 1 H -benzotriazole methodology and c urrent applications of 1 H -benzotriazole and its derivatives in varying fields of chemistry. This chapter illustrated the renewed interest of research groups in 1 H -benzotriazole chemistry From Chapter 1, it is apparent that there has been an explosion in r esearch into some of the less studied aspects of 1 H benzotriazole and its derivatives such as its use in cross coupling reactions. Chapter 2 of this study predominantly focused on C a minoimidoylation and C thiocarbamoylation of ester enol ates. Chapter 3 a n extension of the methodology applied in Chapter 2 covered the p reparation of C alkoxyimidoylating reagents. C -Amino and C alkoxy imidoylation, as well as C thiocarbamoylation are extremely useful reactions for the formation of precursors to heterocycles. These precursors to heterocycles were obtained in one step from the C aminoimidoylating and C -thiocarbamoylating reagents, whereas previous methodologies required multiple steps. Furthermore when compared with the previous methodologies, the present C am inoimidoylation and C thiocarbamoylation methodologies provided comparable product yields Although the discussions on C amino, C alkoxy and C arylthioimidoylation, as well as C thiocarbamoylation are by no means comprehensive, the present methodologies pr ovide viable access to a range of important compound classes It is

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151 likely that further optimization of the C amino, C alkoxy and C arylthioimidoylation, as well as C-thiocarbamoylation reactions will provide even more method generality. Thus these reactio ns warrant further research. Over the last four decades there has been an escalation in the demand for synthetic peptides and proteins. The search for more promising bioactive peptid es, vaccine development and elucidating the factors influencing the three dimensional structure of proteins are a few reasons for the increased demand of synthetic peptides. Traditional solution phase methodologies involve the elaboration of the peptide or protein one amino acid at a time. This process is slow and can be a limiting factor in some studies. Thus, SPPS evolved in response to the need for a rapid reliable and inexpensive method for peptide syntheses. Today, both the solution and solid phase techniques for peptide synthesis ha ve their niche in synthetic organic chemi stry. In response to the increasing need for methods to access synthetic peptide s Chapters 4 7 of t his study has expanded well -developed and reproducible benzotriazole based methods to the novel synthes e s of structural motifs frequently found in biologica lly relevant compounds, including peptides and proteins. N -Protected aminoacyl)benzotriazoles of 18 proteinogenic amino acids were prepared from precomplexed 1 H benzotriazole and thionyl chloride. In this study N -p rotected aminoacyl)benzotriazoles we re demonstrated to be highly efficient tools for the syntheses of peptides and peptide conjugates. The scope of this aminoacylation reaction is quite general and affords a rapid and easy access to peptides and peptide conjugates. Evidently the ability to synthesize peptides and proteins (Chapters 4 6) for example, amyloid can greatly expand our comprehension of the significant role that these biomolecules play in the progression of neurodegenerative diseases such as Parkinsons and Alzheimers Thus an

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152 understanding of the degeneration process in AD and Parkinson s disease will assist with finding cures Extensions of the benzotriazole -based strategy of peptide synthesis via N -protected aminoacyl)benzotriazoles was discussed in Chapter 7. I n Chapter 7 the synthes e s of azole-based peptides can provide access to compound classes that may serve as valuable tools for peptidomimetics. Although work on the water based synthesis of peptides (Chapter 7) using 1 H benzotriazole methodology is still in its infancy, the development of this aqueous -based methodology is expec ted to have a profound impact on SPPS and by extension solution syntheses. To summarize, in this study synthetically useful b enzotriazole assisted method s were applied to the syntheses of a variety of significant compounds ( N N -disubstituted ketene aminals, peptides etc.) Among the challenges for the future is t he development of 1 H benzotriazole -mediated aqueous synthetic methods. While the Katritzky group has e x plored many aspects of 1H -benzotriazole chemistry over the last t hree decades, the maximum potential of 1 H -benzotriazole as a synthetic auxiliary has not been attained and many novel and interesting chemical transformations and applications await discovery. Undoubtedly the renewed interest in the chemistry of 1 H benzotr iazole and its derivatives will lead to insights at the interface of a range of chemistry related disciplines.

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153 LIST OF REFERENCES The reference citation system employed throughout this dissertation is that from Advances in Heterocyclic Chemistry (vol. 9 6) Academic Press, 2008 (Ed. A. R. Katritzky). Each time a reference is cited a number letter code is designated to the corresponding reference with the first two, or four if before 1910, numbers indicating the year followed by the letter code of the journ al and the page number in the end. Additional notes to this reference system are as follows: (i) Each reference code is followed by the conventional literature citation as depicted in the Advances in Heterocyclic Chemistry instruction for authors (ii) Le ss commonly used books and journals are coded as MI for miscellaneous (iii) The list of references are arranged according to the designated code in the order of (a) year, (b) journal in alphabetical order, (c) page number 47JACS119 J. C. Shivers, M. L. Dillon, and C. R. Hauser, J Am. Chem. Soc. 69, 119 (1947). 50JACS2783 H. Rap oport and R. M. Bonner, J. Am. Chem. Soc ., 72, 2783 ( 1950). 54SCI989 D. Davis, Science, 120, 989 (1954). 58JOC1931 R. F. W. Ratz and H Schroeder, J. Org. Chem. 23, 1931 (1958). 59JACS4882 G. B. Bachman and T. Hokama, J Am. Chem. Soc. 81, 4882 ( 1959). 62USP3068278 J. Bernstein and E. R. Spitzmiller, U.S. Patent 3,068,278 (1962). 63JACS2149 R. B. Merrifield, J. Am. Chem. Soc ., 85, 2149 (1963). 64JACS1839 G. W. Anderson, J. E Zim merman, and F. M Callahan, J. Am. Chem. Soc. 86, 1839 (1964). 65JOC3718 M Stiles, U Burckhardt, and G Freund, J. Org. Chem. 32, 3718 (1965). 68JOC1679 L Weintraub, S R. Oles, and N Kalish, J. Org. Chem. 33, 1679 (1968). 70CR151 D. G. Neilson, R. Roger, J. W. M. Heatlie, and L. R Newlands, Chem. Rev. 70, 151 (1970).

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154 73JACS4501 M. A. Barton, R. U. Lemieux, and J. Y Savoie, J. Am. Chem. Soc ., 95, 4501 (1973). 75IJPPR295 C. Jacobsen, Int. J. Peptide Protein Res. 8 295 (1975). 76C B3693 H. Kunz, Chem Ber. 109, 3693 (1976). 78JOC337 C. Larsen, K. Steliou, and D. N. Harpp, J. Org. Chem. 43, 337 ( 1978). 78JOC4808 R. B. Me rrifield and A E Bach, J. Org. Chem. 43, 4808 (1978). 79JACS3394 G P. Royer and G. M Anantharmaiah, J. Am. Chem. Soc ., 101, 3394 (1979). 79S343 W. Kantlehner and W. W. Mergen, Synthesis 343 (1979). 81CR589 A. Williams and I. T. Ibrahim, Chem. Rev. 81, 589 (1981). 83LA290 W. Kantlehner, W. W. Mergen, and E. Haug, Liebigs Ann Chem. 2 290 (1983). 86S341 B. Merrifield, Science 232 341 ( 1986). 87JACS620 W. R. Erickson and S. J. Gould, J. Am. Chem. Soc. 109, 620 ( 1987). 87T5961 F. Albericio, E. Nicol s, J. Josa, A. Grandas, E. Pedroso, E. Giralt, C. Granier, and J. van Rietschoten, Tetrahedron, 43, 5961 (1987). 88ARB957 S. B. H. K ent, Annu. Rev. Biochem. 57, 957 ( 1988). 88BKCS236 S. C. Shim and S. J. Lee, Bull. Korean Chem. Soc. 9 236 (1988). 88FA29 F. Sparatore, M. I. La Rotonda, G. Caliendo, E. Novellino, C. Silipo, and A. Vittoria, Farmaco 43, 29 (1988). 88JCS(PK1)2895 L. R. Cameron, J. L. Holder, M. Meldal, and R. C. Sheppard, J. Chem. Soc. Perkin Trans. 1 14, 2895 ( 1988). 90B2639 K. Halverson, P. E. Fraser, D. A. Kirschner, and P. T. Lansbury, Biochemistry 29, 2639 (1990). 90JACS6039 R. C. De L. Milton, S. C. F. Milton, and P. A. Adams, J. Am. Chem. Soc ., 112, 6039 ( 1990). 90S195 M. Hojo, R. Masuda, E. Okada, H. Yamamoto, K. Morimoto, and K. Okada, Synthesis 195 (1990). 91CR1 A. K. Mukerjee and R. Ashare, Chem. Rev. 1 1 ( 1991).

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155 91T2683 A. R. Katritzky, S. Rachwal, and G. J. Hitchings, Tetrahedron, 47, 2683 (1991). 92JCS(PK2)671 M Haddad, F Dahan, J P Legros, L Lopez, M T Boisdon, and J. Barrans, J. Chem. Soc., Perkin Trans. 2 671 (1992). 92JOC4781 H. -M. Yu, S. T. Chen, and K. T. Wang, J. Org. Chem. 57, 4781 (1992). 93PR1076 R. G. Strickley, and B. D. Anderson, Pharm. Res. 10, 1076 (1993). 93T165 P J. Garratt, S N. Thorn, and R. Wrigglesworth, Tetrahedron, 49, 165 (1993). 94B12022 V. W. Cornish, M. I. Kaplan, D. L. Veenstra, P. A. Kollman, and P. G. Schultz, B iochemistry 33, 12022 (1994). 94IJPPR431 C. Hyde, T. Johnson, D. Owen, M. Quibell, and R. C. Sheppard, Int. J. Pept ide Protein Res. 43, 431 ( 1994). 95EJMC77 G. Caliendo, R. Di Carlo, G. Greco, R. Meli, E. Novellino, E. Perissutti, and V. Santagada, Eur. J. Med. Chem. 30, 77 (1995). 95JACS5401 L. A. Carpino and A. El -Faham, J. Am. Chem. Soc. 117, 5401 (1995). 95JACS11656 M. Quibell, L. C. Packman, and T. Johnson, J. Am. Chem. Soc 117, 11656 ( 1995). 95JACS12058 J. P. Tam, and Y. A. Lu, J. Am. Chem. Soc 117, 12058 (1995). 95RCR929 A. A. Ba kibaev and V. V Shtrykova, Russ. Chem. Rev. 64, 929 (1995). 95TL7523 L. C. Packman, Tetrahedron Lett. 36, 7523 ( 1995). 96EJP341 D. Jang, C. Szabo, and G. A. C. Murrell, Eur. J. Pharmacol. 312, 341 (1996). 96PBB179 R. L. C. Handy, P. Wallace, and P. K. Moore, Pharmacol Biochem Be 55, 179 (1996). 96TL2619 J. M. Mellor, and H. Rataj, Tetrahedron Lett. 37, 2619 (1996). 97JACS11734 T. Kimura, V. P. Vassilev, G. J. Shen, and C. H. Wong, J. Am. Chem. Soc ., 119, 11734 (1 997 ). 97JOC6210 S. A. Belyakov, A. E. Sorochinsky, S. A. Henderson, and J. Chen, J. Org. Chem 62, 6210 (1997). 97MI1 P. Lloyd Williams, F. Albericio, and E. Giralt, Chemical Approaches to the Synthesis of Peptides and Proteins CRC Press LLC, Florida, 19 97.

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156 97TL6771 S. Fustero, B. Pina, and A. Simn-Fuentes, Tetrahedron Lett. 38, 6771 (1997) 98CR409 A. R. Katritzky, X. Lan, J. Z.Yang, and O. V. Denisko, Chem. Rev. 98, 409 ( 2003). 98TL8529 A. Di Fenza, M. Tancredi, C. Galoppini, and P. Rovero, Tetrahedr on Lett. 39, 8529 (1998). 99OL977 M. Garcia de la Torre, A. Navarro, C. Ramrez de Arellano, and A. Simn, Org. Lett. 1 977 ( 1999). 00JCS(PT1)2311 R. M. Adlington, J E. Baldwin, D Catterick, G J. Pritchard, and L T Tang, J. Chem. Soc., Perkin Trans. 1 2311 (2000). 00JOC1583 O. Barun, H. Ila, and H. Junjappa, J. Org. Chem ., 65, 1583 (2000). 00JOC3679 A. R. Katritzky and A. Pastor, J. Org. Chem. 65, 3679 (2000). 00JOC5460 L. P. Miranda, W. D. F. Meutermans, M. L. Smythe, and P. F. Alewood, J. Org. Ch em. 65, 5460 ( 2000). 00JOC7663 F. A. Davis, V. Srirajan, D. L. Fanelli, and P. Portonovo, J. Org. Chem. 65, 7663 (2000). 00JOC8210 A. R. Katritzky, H. Y He, and K. Suzuki, J. Org. Chem. 65, 8210 (2000). 00LIPS107 J. D. Wade, M. N. Mathieu, M. Macris, a nd G. W. Tregear, Lett. Pept. Sci ., 7 107 (2000). 00MI3 W. C. Chan and P. D. White, in Basic Principles : Fmoc Solid Phase Peptide Synthesis; A Practical Approach Oxford University Press, New York, 2000, (W. C. Chan and P. D.White, eds), p. 32. 00MI3 F. Albericio, I. Annis, M. Royo, and G. Barany, in Preparation and handling of peptides containing methionine and cysteine: Fmoc Solid Phase Peptide Synthesis, A Practical Approach Oxford Oxford University Press Inc., New York, 2000 (W. C. Chan and P. D. Wh ite, ed.), p. 77. 00MI3 M. Quibell and T. Johnson, in Difficult Peptides: Fmoc Solid Phase Peptide Synthesis; A Practical Approach Oxford University Press, New York, 2000, (W. C. Chan and P. D.White, eds), p. 115. 00OL1815 N. Thieriet, F. Guib and F. A lbericio, Org. Lett. 2 1815 (2000). 00S2029 A. R. Katritzky, Y. Fang, A. Donkor, and J. Xu, Synthesis 2029 (2000). 01JPS615 K Hojo, M Maeda, and K Kawasaki, J. Peptide Sci. 7 615 (2001).

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157 01JPS641 P. K. Ajikumar, and K. S. Devaky, J. Pept. Sci. 7 641 (2001). 02ARK134 A. R. Katritzky M. Wang, H Yang, S. Zhang, and N. G. Akhmedov, Arkivoc (viii) 134 (2002). 02JCA293 C. Sowa, R. Miethchen, and H. Reinke, J. Carbohyd. Chem. 21, 293 (2002). 02JOC4667 S. Fustero, B. Pina, E. Salavert, A. Navarro, M. C. Ramrez de Arellano, and A. Simn Fuentes, J. Org. Chem. 67, 4667 ( 2002). 02MI2 J. Jones, Amino Acid and Peptide Synthesis 3rd Ed. Oxford University Press Inc., New York, 2002. 02S1592 M. Erdlyi and A. Gogoll, Synthesis 1592 ( 2002). 02TL7717 P. Ga gnon, X. Huang, E. Therrien, and J. W. Keillor Tetrahedron Lett. 43, 7717 (2002). 03AGE5400 S. V. Ley, and A. W. Thomas, Angew. Chem. Int. Ed. 42, 5400 (2003). 03CEJ4586 A. R. Katritzky, and B. V. Rogovoy, Chem. Eur. J. 9 4586 (1998). 03JOC1443 A. R. Katritzky A. A. A. Abdel Fattah, and M. Wang, J. Org. Chem. 68, 1443 ( 2003). 03JOC7316 A Hamze, J -F Hernandez, P Fulcrand, and J Martinez, J. Org. Chem. 68, 7316 (2003). 03JPS518 M. Mergler, F. Dick, B. Sax, C. St helin, and T. Vorherr, J. Peptide S ci. 9 518 (2003). 03OPRD418 M Scalone and P Waldmeier Org. Process Res. Dev. 7 418 (2003). 03S2795 A. R. Katritzky, Y. Zhang, S. K. Singh, Synthesis 2795 (2003). 04ARK14 A. R. Katritzky, S. Hoffmann, and K. Suzuki, Arkivoc (xii) 14 (2004). 04CC124 Y. Sohma, M. Sasaki, Y. Hayashi, T. Kimura, and Y. Kiso, Chem. Commun. 1 124 (2004). 04CCA175 A. R. Katritzky K. Suzuki, and S. K. Singh, Croat. Chem. Acta 77, 175 (2004). 04CPB422 K Hojo, M Maeda, T J. Smith, E Kita, F Yamaguchi, S Yamamoto, a nd K Kawasaki, Chem. Pharm. Bull. 52, 422 (2004).

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158 04JMC5662 A. Wilczynski, X. S. Wang, R. M. Bauzo, Z. Xiang, A. M. Shaw, W. J. Millard, N. G. Richards, A. S. Edison, and C. Haskell Luevano, J. Med. Chem. 47, 5662 (2004). 04JOC188 Y. Shi, J. Zhang, N. G razier, P. D. Stein, K. S. Atwal, and S. C. Traeger, J. Org. Chem. 67, 188 ( 2009) 04JOC1571 H. Sandin, M. L. Swanstein, and E. Wellner, J. Org. Chem. 69, 1571 (2004). 04JOC2976 A. R. Katritzky, S. Ledoux, R. M. Witek, and S. K. Nair, J. Org. Chem. 69, 2976 (2004). 04JOC8804 D. Skropeta, K. A. Jolliffe, and P. Turner, J. Org. Chem. 69, 8804 (2004). 04PPL377 A. K. Tickler, A. B. Clippingdale, and J. D. Wade, Protein Peptide Lett. 11, 377 ( 2004). 04S1806 A. R. Katritzky A. A. Shestopalov, and K. Suzuki, Synthesis 1806 (2004) 04S2645 A. R. Katritzky, K. Suzuki, and S. K. Singh, Synthesis 2645 (2004). 04T61 Lpez, I. Maya, J. Fuentes, and J. G. Fernndez Bolaos, Tetrahedron, 60, 61 (2004). 04T2447 S. Y Han and Y. -A Kim Tetrahedron, 60, 2447 (2004). 04TL9293 K Hojo, M Maeda, and K Kawasaki, Tetrahedron Lett. 4 5 9293 (2004). 05ARK116 A. R. Katritzky, A. S. Vincek, and K. Suzuki, Arkivoc (v), 116 (2005). 05ARK36 A R. Katritzky, A A. Shestopalov, and K. Su zuki, Arkivoc (vii) 36 (2005). 05EJO1 184 S. H.Von Reuss and W. A. Koenig, Eur. J. Org. Chem. 1184 (2005). 05HCA1664 A. R. Katritzky, N. M. Khashab, and S. Bobrov, Helv. Chim. Acta 88, 1664 (2005). 05JHM1 K. D. Wehrstedt, P. A. Wandrey, and D. Heitkamp, J. Hazard. Mater. A126, 1 (2005). 05J MC3060 A. Wilczynski, K. R.Wilson, J. W. Scott, A. S. Edison, and C. Haskell Luevano, J. Med. Chem. 48, 3060 (2005). 05JOC4993 A. R. Katritzky, R. Jiang, and K. Suzuki, J. Org. Chem. 70, 4993 (2005).

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159 05JOC7866 A R. Katritzky, R M. Witek, V Rodriguez G arcia, P P. Mohapatra, J W. Rogers, J Cusido, A. A. A. Abdel Fattah, and P J Steel, J. Org. Chem. 70, 7866 (2005). 05OL1521 B. C. Gorske, S. A. Jewell, E. J. Guerard, and H. E. Blackwell, Org. Lett. 7 1521 ( 2005). 05S397 A. R. Katritzky, P. Angrish D. Hr, and K. Suzuki, Synthesis 397 (2005). 05TEC284 N. N. Il'chenko, V. N. Britsun, and M. O. Lozinskii, Theor. Expt. Chem. 41, 284 (2005). 05TL7597 M. K. Denk and X. Ye, Tetrahedron Lett. 44, 7597 (2005). 06ARK226 A. R. Katritzky, N. M. Khashab, a nd A. V. Gromova, A rkivoc (iii) 226 (2006). 06CCR3200 M H. Filby, J. W. Steed Coord. Chem. Re v 250, 3200 (2006). 06CEJC285 C. Jia, W. Qi, Z. He, H. Yang, and B. Qiao, Central Eur. J. Chem. 4 285 (2006). 06EJO3283 M. Schnurch, R. Flasik, A. F. Khan, M. Spina, M. D. Mihovilovic, and P. Stanetty, Eur. J. Org. Chem. 3283 (2006). 06JACS14268 A. Tsubouchi, K. Onishi, and T. Takeda, J. Amer. Chem. Soc. 128, 14268 (2006). 06JOC3051 T. Matsushita, H. Hinou, M. Fumoto, M. Kurogochi, N. Fujitani, H. Shimizu, and S. I. Nishimura, J. Org. Chem. 71, 3051 (2006). 06JOC6753 A. R. Katritzky, N. M. Khashab, S. Bobrov, and M. Yoshioka, J. Org. Chem. 71, 6753 (2006). 06JOC7106 D. Crich and A. Banerjee, J. Org. Chem. 71, 7106 ( 2006). 06JOC9051 A R. Katritzky, N M. Khashab, N Kirichenko, and A Singh, J. Org. Chem. 71, 9051 (2006). 06JOC9861 A. R. Katritzky, K. N. B. Le, L. Khelashvili, and P. P. Mohapatra J. Org. Chem. 71, 9861 (2006). 06JPCA13195 A. Modelli, and D. Jones, J. Phys. Chem. A 110, 13195 (2006). 0 6PPL189 K Hojo, M Maeda, N Tanakamaru, K Mochida, and K Kawasaki, Protein Peptide Lett. 1 3 189 ( 2006). 06S411 A. R. Katritzky P. Angrish and K. Suzuki, Synthesis 411 (2006).

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160 06S3231 A. R. Katritzky S. R. Tala, and S. K. Singh, Synthesis 3231 (2006). 06S4135 A. R. Katritzky, and P. Angrish, Synthesis 4135 (2006). 06ST660 A. R. Katritzky and P. Angrish, Steroids 71, 660 (2006). 06TL3013 Y. Sohma, A. Taniguchi, M. Skwarczynski, T. Yoshiya, F. Fukao, T. Kimura, Y. Hayashi, and Y. Kiso, Tetrahed ron Lett. 47, 3013 (2006). 06TL4121 S. Zahariev, C. Guarnaccia, C. I. Pongor, L. Quaroni, Pongor, Tetrahedron Lett. 47, 4121 (2006). 07CBDD465 A. R. Katritzky, N. M. Khashab, M. Yoshioka, D. N. Haase, K. R. Wilson, J. V. Johnson, A. Chung, and C. Haskell Luevano, Chem. Biol. Drug Des. 70, 465 (2007). 07HAC316 T. Akhtar, S. Hameed, N. A. Al -Masoudi, and K. M. Khan, Heteratom Chem. 18, 316 (2007). 07JOC407 A. R. Katritzky, H. Tao, R. Jiang, Suzuki, and K. Kirichenko, J. Org. Chem. 72, 407 (2007). 07JOC5794 A. R. Katritzky, E. Todadze, P. Angrish, and B. Draghici J. Org. Che m. 72, 5794 (2007). 07JOC6742 A. R. Katritzky, N. M. Khashab, D. N. Haase, M. Yoshioka, I. Ghiviriga, and P. J. Steel, J Org. Chem. 72, 6742 ( 2007). 07JOM545 H. Ube, D. Uraguchi, and M. Terada, J. Organomet. Chem. 692, 545 (2007). 07JPS143 S. A. Palase k, Z. J. Cox, and J. M. Collins, J. Peptide Sci. 13, 143 (2007). 07MM129 M. P tek, in Branched-Chain Amino Acids : Amino Acid Biosynthesis Pathways, Regulation and Metabolic Engineering, Microbiology Monographs Springer -Verlag Berlin Heidelberg, New York 2007, (Wendisch, V. F., eds) Vol. 5, pp 129 07MOL103 O. Pintilie, L. Profire, V. Sunel, M. Popa, and A. Pui, Molecules 12, 103 (2007). 07OBC2884 M I. Aguilar, A W. Purcell, R Devi, R Lew, J Rossjohn, A. I Smith, and P Perlmutter, Org. Biomol. Che m. 5 2 884 (2007 ) 07OL2381 J K. Pokorski, L M. Miller Jenkins, H Feng, S R. Durell, Y Bai, and D H Appella, Org. Lett. 9 2381 ( 2007). 07SL761 T. Zhang, C. -Y. Yu, and Z.T. Huang, Synlett, 761 (2007).

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161 07TA1667 G. Cremonesi, P. Dalla Croce, F. Fonta na, A. Forni, and C. La Rosa, Tetrahedron: Asymmetry 18, 1667 (2007). 07TL4207 A. K. Verma, J. Singh, V. K. Sankar, R. Chaudhary, R. Chandra, Tetrahedron Lett. 48, 4207 (2007). 07TL7199 A. K. Verma, J. Singh, and R. Chaudhary, Tetrahedron Lett. 48, 7199 (2007). 08ARK153 A A. Aly and A M. Nour El -Din Arkivoc (i) 153 (2008). 08BMC8210 A. Goonan, A. Kahvedzic, F. Rodriguez, P. S. Nagle, T. McCabe, I. Rozas, A. M. Erdozain, J. J. Meana, and L. F. Callado, Bioorgan. Med. Chem. 16, 8210 (2008). 08CM7510 A. Balan, G. Gunbas, A. Durmus, and L. Toppare, Chem. Mater. 20, 7510 (2008). 08JOC511 A. R. Katritzky T. Narindoshvili, B. Draghici, and P. Angrish, J. Org. Chem. 73, 511 (2008) 08JOC2003 S. Caille, E. A. Bercot, S. Cui, and M. M. Faul, J. Org. Chem. 7 3 2003 (2008). 08OBC2400 A. R. Katritzky, Q. Y. Chen, and S. R. Tala Org. Biomol. Chem. 6 2400 (2008) 08S2462 A. Alizadeh, R Hosseinpour, and S Rostamnia, Synthesis 2462 ( 200 8). 08S3565 M. F. Moynihan, J. W. Tucker, and C. J. Abelt, Synthesis 35 65 (2008). 08SL3068 J. -P. Wan, Y. F. Chai, J. -M. Wu, and Y. J. Pan, Synlett, 3068 (2008). 08TL4746 J I. Gavrilyuk, A J. Lough, and R. A Batey, Tetrahedron Lett. 49, 4746 (2008). 09AGE1138 A. K. Ver m a, T. Kesharwani, J. Singh, V. Tandon, and R. C. Larock Angew. Chem. Int. Ed. 4 8 1138 (2009). 09ARK47 A. R. Katritzky, A. Singh, D. N. Haase, and M. Yoshioka, Arkivoc (viii) 47 (2009) 09JAE269 D. Gopi, K. M. Govindaraju, V. Collins Arun Prakash, V. Manivannan, and L. Kavitha, J. Appl. Electrochem. 39, 2 69 (2009). 09JMPT1729 H. Lee, B. Park, and H. Jeong, J. Mater. Process Tech. 209, 1729 (2009). 09JOC2028 A R. Katritzky, D N. Haase, J. V. Johnson, and A Chung, J. Org. Chem. 74, 2028 (2009).

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163 BIOGRAPHICAL SKETCH Danniebelle N. Haase was born in 1979, in St. Andrew, Jamaica West Indies She was the first of two children. She spent her formative years at the Herrick Basic and Dunrobin Primary scho ols and later attended the Immaculate Conception High School. Subsequently, she attended the University of the West Indies where she read for a Bachelor of Science in Chemistry and Management. Upon graduation in the summer of 2000, she taught science at the Merl Grove High S chool for girls. Once again, i n fall 2002 she matriculat ed as a student in the Master of Philosophy program in the Department of Chemistry at the University of the West Indies. She was awarded the M aster of Phil o sophy in the summer of 2005, after which she traveled to the USA to pursue doctoral studies, specializing in organic chemistry at the University of Florida. In the fall of 2005 she joined Professor Alan R. Katritzkys research group at the Florida Ce nter for Heterocyclic Chemistry