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Benzotriazole Intermediates for Heterocycles and Pharmaceuticals

Permanent Link: http://ufdc.ufl.edu/UFE0041025/00001

Material Information

Title: Benzotriazole Intermediates for Heterocycles and Pharmaceuticals
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Yoshioka Tarver, Megumi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: benzotriazole, c, fluorogenic, gfp, peptides, spps
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: Benzotriazole is a synthetic auxiliary and benzotriazole methodology has been utilized for a variety of organic syntheses. 1H-Benzotriazole (BtH) has useful properties such as its leaving group ability, its electron-donating charactor, or its activation of the CH bond toward proton loss. BtH behaves as a weak base as well as a weak acid, which allows reactions to occur in both acidic and basic media, thus BtH can be easily removed from reaction mixtures by a simple acid or base work-up during purification. The focus of this work is to expand the utility of BtH as an excellent leaving group in the synthesis of various pharmaceuticals and heterocycles both in solution and solid phase. The thesis is divided into six parts; Chapter 1: an overview of BtH in organic syntheses, Chapter 2: C-thiocarbamoylation and C-aminoimidoylation of sulfones and ketones, Chapter 3: segment condensation peptide synthesis using N?-Fmoc-protected-dipeptidoylbenzotriazoles, Chapter 4: coumarin lebeled peptide synthesis, Chapter 5: pH sensitive green fluorescent protein modified chromophore analogues, and finally, Chapter 6: summary of achievement.
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 Megumi Yoshioka Tarver.
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: UFE0041025:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041025/00001

Material Information

Title: Benzotriazole Intermediates for Heterocycles and Pharmaceuticals
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Yoshioka Tarver, Megumi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: benzotriazole, c, fluorogenic, gfp, peptides, spps
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: Benzotriazole is a synthetic auxiliary and benzotriazole methodology has been utilized for a variety of organic syntheses. 1H-Benzotriazole (BtH) has useful properties such as its leaving group ability, its electron-donating charactor, or its activation of the CH bond toward proton loss. BtH behaves as a weak base as well as a weak acid, which allows reactions to occur in both acidic and basic media, thus BtH can be easily removed from reaction mixtures by a simple acid or base work-up during purification. The focus of this work is to expand the utility of BtH as an excellent leaving group in the synthesis of various pharmaceuticals and heterocycles both in solution and solid phase. The thesis is divided into six parts; Chapter 1: an overview of BtH in organic syntheses, Chapter 2: C-thiocarbamoylation and C-aminoimidoylation of sulfones and ketones, Chapter 3: segment condensation peptide synthesis using N?-Fmoc-protected-dipeptidoylbenzotriazoles, Chapter 4: coumarin lebeled peptide synthesis, Chapter 5: pH sensitive green fluorescent protein modified chromophore analogues, and finally, Chapter 6: summary of achievement.
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 Megumi Yoshioka Tarver.
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: UFE0041025:00001


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1 BENZOTRIAZOLE INTERM EDIATES FOR HETEROCYCLES AND PHARMACEUTICALS By MEGUMI YOSHIOKATARVER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Megumi Yoshioka Tarver

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3 I dedicate this work to my father Hitoshi Yoshioka, my mother Kazuyo Yoshioka, my brother Yuta Yos h i oka, my sister Yukari Yoshioka, and finally my wonderful husband Dr. Matthew R. Tarver. I would have never accomplished any of this works without their love, support, and understanding.

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4 ACKNOWLEDGMENTS I greatly appreciated many people who have helped me in the preparation of this di ssertation First, I would like to thank my advisor, Dr. Alan R. Katritzky for the opportunity, great understanding, and support during my Ph.D. program. I also thank my committee members, Dr. Margaret O. James, Dr. So Hirata, Dr. Sukwon Hong, and Dr. Ion Ghiviriga, for their helpful suggestions and instructions. I also appreciate all of Katritzky group members; especially Dr. C. Dennis Hall, Dr. Niveen M. Khashab, Dr. Tamar i Narindoshvil i, Dr. Anamika Si n gh Dr. Danniebell e N. Haase, Dr. Geeta Meher, Mr. Bahaa El Dien M. El -Gendy Ms. Longchuan Huang Ms. Claudia El Nachef, Ms. Janet Cusido, and Ms. Judit Kovacs Special thanks go to Dr. Ben Smith, Dr. Tammy Davidson, Dr. Katsu Ogawa, Dr. Jodie Johnson, Dr. Peter Steel, Mr. Alfred Chung, Ms. Lori Clark, Ms. Elizabeth Shep p ard, Ms. Elizabeth Cox, Ms. Gwen McCann, and all my fellow Gators at the University of Florida. I also thank my parents, Hitoshi and Kazuyo Yoshioka, and my brother, Yuta Yoshioka, my sister, Yukari Yoshioka, my parents -in -law, Dr. Robert a nd Karen Tarver, for their support. Finally, I thank my husband, Dr. Matt hew R. Tarver, for supporting, understanding, and loving me continuously

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 LIST OF SCHEMES .......................................................................................................................... 11 LIST OF ABBREVIATIONS ............................................................................................................ 13 ABSTRACT ........................................................................................................................................ 17 CHAPTER 1 GENERAL INTRODUCTION .................................................................................................. 19 1.1 General Introduction of Benzotriazole ................................................................................ 19 1.2 General Methods ................................................................................................................... 24 2 C-AMINOIMIDOYLATION AND C THI OCARBAMOYLATION OF SULFONES AND KETONES ......................................................................................................................... 26 2 .1 Introduction ........................................................................................................................... 26 2.2 Results and Discussion ......................................................................................................... 28 2.2.1 Synthesis of 1 (Alkyl/arylthiocarbamoyl) benzotriazoles 2.4a -e and Benzotriazole 1 -carboxamidine 2.5a -c .......................................................................... 28 2.2.2 C -Aminoimidoylation and C Thiocarbamoylation of Sulfones .............................. 29 2.2.3 C -Aminoimidoylation and C Thiocarbamoylation of Ketones ............................... 29 2.2.4 Compound Characterization and Tautomeric Structures ......................................... 32 2.3 Conclusion ............................................................................................................................. 37 2.4 Experimental Section ............................................................................................................ 38 2.4. 1 General Procedure for the Preparation of Compounds 2.6 and 2.8 ......................... 38 2.4.2 General Procedure for the Preparation of Compounds 2.9a d and 2.11a d ......... 39 2.4.3 General Procedure for the Preparation of Compounds 2.12a c .............................. 41 3 PEPTIDE SYNTHESIS UTILIZING (N FMOC -PROTECTED -AMINOACYL) BENZOTRIAZOLES AND ( N FMOC -PROTECTED DIPEPTIDO YL)BENZOTRIAZOLES ..................................................................................... 43 3.1 Introduction ........................................................................................................................... 43 3.2 Results and Discussion ......................................................................................................... 45 3.2.1 Preparation of ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2 ......................... 45

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6 3.2.2 Preparation of (LL) Dipeptides 3.4a -f and Diastereomeric Mixtures (3.4a+3.4a ), (3.4b+3.4b ), ( 3.4c+3.4c ), ( 3.4e+3.4e ), and ( 3.4f+3.4f ) Using (N-Fmoc -protected aminoacyl)benzotriazoles 3.2a d ................................................. 46 3.2.3 Preparation of ( N-Fmoc -protected -dipeptidoyl)benzotriazoles 3.5a -f .................. 49 3.2.5 General Peptide Syntheses by Segment Condensation ............................................ 49 3.3 Conclusion ............................................................................................................................. 52 3.4 Experimental Section ............................................................................................................ 52 3.4.1 General Procedure for the Preparation of 3.1a -g ..................................................... 52 3.4.2 General Procedure for the Preparation of 3.4a -f, ( 3.4a+ 3 .4a ), ( 3.4b+3.4b ) (3.4c+3.4c ), ( 3.4e+3.4e ), and ( 3.4f+3.4f ) ................................................................... 54 3.4.3 General Procedure for the Preparation of 3.5a -f ...................................................... 58 3.4.5 HPLC Results of Pe ptide 3.6 3.10 ............................................................................ 62 4 COUMARIN LABELING OF PEPTIDES ON SOLID PHASE ............................................. 65 4.1 Introduction ........................................................................................................................... 65 4.2 Results and Discussion ......................................................................................................... 69 4.2.1 Preparation of N-Fmoc N[(7 -methoxycoumarin 4 -yl)acetyl] -L lysine ( NFmoc L -Lys(Mca) OH) 4.4 and Its Benzotriazole Derivative 4.6 ............................... 69 4.2.2 Preparation of N-Fmoc N(coumarin 3 -ylcarbonyl) L lysine Benzotriazolide (N-Fmoc L -Lys(Cc) Bt) 4.9 and N(Coumarin 3 -ylcarbonyl) N-Fmoc -L lysine Benzotriazolide ( N-Cc -L Lys(Fmo c) Bt) 4.11 ............................................................. 70 4.2.3 Solid Phase Fluorescent Labeling with 4.6 4.9 4.11 to Synthesize Labeled Peptides 4.12 4.17 ............................................................................................................ 70 4.2.4 Soli d Phase Fluorescent Labeling with 4.7 to Synthesize Labeled Dipeptide (Cc) -LLeu -LLeu NH2 4.18 and Labeling with 4.5 to Synthesize Labeled Dipeptide (Mca) -LLeu -LLeu NH2 4.19 ....................................................................... 72 4.2.5 Fluore scence Measurement s of Peptides 4.12 4.19 ................................................. 73 4.3 Conclusion ............................................................................................................................. 75 4.4 Experimental Section ............................................................................................................ 75 4.4.1 Preparation of ( S ) 2 (((9 H Fluoren 9 -yl)methoxy)carbonylamino) 6 (2 (7 methoxy2 -oxo 2 H -chromen 4 -yl)acetamido)pentanoic acid ( NFmoc -LLys(Mca) OH) 4.4 ........................................................................................................... 75 4.4.2 General Procedure for the Preparation of 4.5 4.6 4.9 4.11 ................................... 76 4.4.3 General Procedure of So lid Support Peptide Synthesis ........................................... 78 4.4.4 HPLC Profiles of Peptide 4.124.19 ......................................................................... 79 5 DESIGN AND SYNTHESIS OF PH SENSITIVE GFP CHROMOPH ORE ANALOGUES ............................................................................................................................. 84 5.1 Introduction ........................................................................................................................... 84 5.2 Results and Discussion ......................................................................................................... 87 5.2.1 Synthesis of Imidazolinone Chromophore s 5.9a -f and Their Fluorescent Activit y ............................................................................................................................. 87 5.2.2 Synthesis of Imidazolinone Chromophore 5.14a-b and Their Fluorescent Activity ............................................................................................................................. 93 5.2.3 Absorption and Emission Measurement of Chromophores 5.9b -f and 5.14a -b .... 95

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7 5.3 Conclusion ............................................................................................................................. 96 5.4 Experimental ......................................................................................................................... 96 5.4.1 General Synthesis for the Preparation of Azalactone 5.8 ........................................ 96 5.4.2 General Synthesis for the Preparation of Imidazolinone 5.9 ................................... 98 5.4.3 Synthesis of 2 (2 Naphthamido)acetic acid 5.10 ................................................... 100 5.4.4 Synthesis of N -(2 -Chl oroacetyl)benzamide 5.11 ................................................... 100 5.4.5 Synthesis of N -(2 -Azidoacetyl)benzamide 5.12 ..................................................... 101 5.4.6 Synthesis of 2 Phenyl 1 H -imidazol 5(4 H ) -one 5.13 ............................................. 101 5.4.7 General Procedure for the Preparation of Imidazolinone 5.14 .............................. 101 6 SU MMARY OF ACHIEVEMENT S ....................................................................................... 103 LIST OF REFERENCES ................................................................................................................. 105 BIOGRAPHICAL SKETCH ........................................................................................................... 112

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8 LIST OF TABLES Table page 2 1 1 (Alkyl/arylthiocarbamoyl)benzotriazoles 2.4a -e and benzotriazole 1 carboxamidine 2.5a -c ............................................................................................................. 29 2 2 C-Aminoimidoylation and C -thiocarbamoylation of ketones 2.10a d to give 2.9a d and 2.11a d ............................................................................................................................ 30 2 3 Formation of 1,3 -oxazol id ine 2 -thione 2.12......................................................................... 31 3 1 (N-Fmoc -protected aminoacyl)ben zotriazoles 3.2a -g ........................................................ 46 3 2 Preparation of (LL) dipeptides 3.4a f and diastereomeric mixtures ( 3.4a+3.4a ), and (3.4b+3.4b ), ( 3.4c+3.4c ), ( 3.4e+3.4e ), and ( 3.4f+3.4f ) from ( NFmoc -protected aminoacyl)benzotriazoles 3.2a d .......................................................................................... 48 3 3 Preparation of ( N-Fmoc -protected -dipeptidoyl)benzotriazole 3.5a f ................................ 49 3 4 Synthesized pe ptides 3.6 3.10 by solid phase segment condensation ................................ 51 3 5 MS/MS Sequence of peptide 3.9 ........................................................................................... 64 4 1 Preparation of fluorescent pepti des 4.124.17 ...................................................................... 72 4 2 Preparation of fluorescent peptides 4.18 and 4.19 ............................................................... 73 4 3 Absorption and f luorescence data of fluorescent la beled p eptides 4.124.19 .................... 74 4 4 MS/MS Sequence of peptide 4.16 ......................................................................................... 82 5 1 Synthesis of GFP modified fluorophore 5.9a f .................................................................... 88 5 2 Imidazolinone chromophore 5.14a -b .................................................................................... 93 5 3 Quantum yields and excitation coefficients of 5.9b -f and 5.14a -b ..................................... 95

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9 LIST OF FIGURES Figure page 1 1 Various functions of the benzotriazole groups ..................................................................... 19 1 2 Coumarin(carboxyl/acetyl) benzotriazole 1.16, 1.17 and coumarin labeled -Fmoc -L Lys Bt 1.181.20 .................................................................................................................... 23 1 3 GFP modified unnatural amino acids .................................................................................... 23 1 4 GFP modif ied chromophore analogues ................................................................................ 24 2 1 X ray structure of 2.12a ......................................................................................................... 31 2 2 Relevant 1H and 13C chemical shifts in compounds 2.6 and 2.8 ......................................... 32 2 3 1H and 13C Chemical shifts in the major and minor isomers of compound 2.9a ............... 34 2 4 Relevant 1H and 13C chemical shifts in co mpound 2.9 ........................................................ 34 2 5 1H and 13C Chemical shifts and the tautomers/rotamers for compound 2.11a................... 36 2 6 Rotamers of C aminoimidoylatio n product 2.9 and C -thiocarbamoylation product 2.11 .......................................................................................................................................... 36 2 7 1H and 13C Chemical shifts for the tautomers of compound 2.11c in CDCl3 and acetoned6 ............................................................................................................................... 37 3 1 Structure of coupling reagents ............................................................................................... 44 3 2 1H NMR of 3.4a and ( 3.4a+3.4a ). a) 1H NMR of 3.4a methyl group, b) 1H NMR of (3.4a+3.4a ), methyl groups .................................................................................................. 48 3 3 13C NMR of 3.4a and ( 3.4a+3.4a ).c) 13C NMR of 3.4a, carbonyl groups, d) 13C NMR of ( 3.4a+3.4a ), carbonyl groups ................................................................................ 49 3 4 HPLC Profile of peptid e 3.6 .................................................................................................. 62 3 5 HPLC Profile of peptide 3.7 .................................................................................................. 62 3 6 HPLC Profile of peptide 3.8 ................................................................................................. 63 3 7 HPLC Profile of peptide 3.9 .................................................................................................. 63 3 8 HPLC Profile of peptide 3.10. ............................................................................................... 64 4 1 Structure of Mca and Dnp moiety ......................................................................................... 67

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10 4 2 Absorption spectra of 4.12-4.17 ............................................................................................ 74 4 3 Absorption spectra of 4.18-4.19 ............................................................................................ 74 4 4 Emission spectra of 4.12, 4.17, 4.19 ..................................................................................... 75 4 5 HPLC Profile of peptide 4.12. ............................................................................................... 79 4 6 HPLC Profile of peptide 4.13 ................................................................................................ 80 4 7 HPLC Profile of peptide 4.14 ................................................................................................ 80 4 8 HPLC Profile of peptide 4.15 ................................................................................................ 81 4 9 HPLC Profile of peptide 4.16. ............................................................................................... 81 4 10 HPLC Profile of peptide ........................................................................................................ 82 4 11 HPLC Profile of peptide 4.18. .............................................................................................. 82 4 12 HPLC Profile of peptide 4.19 ................................................................................................ 83 5 1 T he proposed GFP -based lysin (Lys), asparagine (Asn), and glutamine (Gln) analogues 5.4 5.6 .................................................................................................................... 86 5 2 Prevention of photoisomerization of imidazolinonyl compound 5.9a................................ 89 5 3 Absorption ( left) and emission spectra ( right ) of 5.9b ........................................................ 91 5 4 Absorption ( left) and emission spectra ( right ) of 5.9c ......................................................... 92 5 5 Absorption (left) and emission spectra (right) of 5.9d ........................................................ 92 5 6 Absorption ( left) and emission spectra ( right) of 5.9f ......................................................... 92 5 7 15N NMR study of 5.9c .......................................................................................................... 93 5 8 Absorption (left) and emission spectra (right) of 5.14a....................................................... 94 5 9 Absorption ( left) and emission spectra ( right ) of 5.14b ...................................................... 95

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11 LIST OF SCHEMES Scheme page 1 1 Protonation equilibria ............................................................................................................. 20 1 2 Benzotriazole -mediated nucleophilic addition ..................................................................... 20 1 3 Displacement of benzotriazole derivatives by different nucleophile s ................................ 21 1 4 C-Aminoimidoylation and C -thiocarbamoylation of sulfones and ketones ....................... 21 1 5 Conversion of N-protected amino acid into N-protected(aminoacyl) -benzotriazole 1.12 and N-protected(benzopeptidoyl)benzotriazole 1.14 ................................................. 22 2 1 Literature method of the preparation of sulfonyl amidine 2.2 ............................................. 26 2 2 Literature methods for the preparation of ketene aminals 2.3 ............................................. 27 2 3 Preparation of mono N -hydroxy and N aminothiourea from 1 (alkyl/arylthio carbamoyl)benzotriazoles 2.4 ................................................................................................ 28 2 4 N-Aminoimidoylation with benzotriazole 1 carboxamidines 2.5 ....................................... 28 2 5 Synth esis of benzotriazole 1 carboxamidine 2.4a -e and 1 (alkyl/arylthio carbamoyl)benzotriazoles 2.5a -c .......................................................................................... 28 2 6 Preparation of C aminoimidoylation product 2.6 and C -thiocarbamoylation product 2.8 fr om sulfone 2.7 ............................................................................................................... 29 2 7 Preparation of the C aminoimidoylation products 2.9a d and C thiocarbamoylation products 2.11a d from ketones 2.10a d .............................................................................. 30 2 8 Formation of 1,3 -oxazolidine 2 -thione 2.12......................................................................... 31 2 9 Possible mechanism of 1,3 -oxazolidine 2 thione 2.12a -c formation ................................. 31 2 10 Mechanism of isomeric mixtures formation ......................................................................... 34 3 1 General procedure of segment condensation peptide synthesis .......................................... 43 3 2 Epimerization mechanism during segment condensation.................................................... 44 3 3 Preparation of ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2a -g ............................... 46 3 4 Preparation of N-Fmoc -protected -dipeptides 3.4a -f, ( 3.4a+3.4a ), and ( 3.4b+3.4b ), (3.4c+3.4c ), ( 3.4e+3.4e ), and ( 3.4f+3.4f ) ......................................................................... 47 3 5 Preparation of ( N-Fmoc -protected -dipeptidoyl)benzo triazoles 3.5a -f .............................. 49

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12 3 6 General procedure of peptide synthesis by segment condensation ..................................... 51 4 1 Mechanism of FRET .............................................................................................................. 66 4 2 Reported synthesis of N(fluoren 9 -ylmethoxycarbonyl) N[(7 -methoxycoumarin4 yl)acetyl -L lysine 4.4 ......................................................................................................... 68 4 3 Preparation of N-Fmoc L -Lys(Mca) Bt 4.6 ........................................................................ 69 4 4 Preparation of N-Fmoc L -Lys(Cc) Bt 4.9 and NCc -L Lys(Fmoc) Bt 4.11 ................... 70 4 5 Synthesis of coumarinlabeled dipeptide 4.12...................................................................... 71 4 6 Preparation of peptide 4.18 .................................................................................................... 73 5 1 Intramolecular biosynthesis of imidazolinonyl chromophore in wild -type GFP ............... 85 5 2 Lit erature examples ................................................................................................................ 86 5 3 Synthesis of GFP modified fluorophore 5.9a d ................................................................... 87 5 4 Synthesis of GFP modified fluorophore 5.9e -f .................................................................... 87 5 5 Prevention of photoisomerization of imidazolinonyl compound 5.9a by six membered ring intramolecular hydrogen bond .................................................................... 90 5 6 Expect ed protonation of GFP analogues 5.9b 5.9c 5.9e 5.9f ........................................... 91 5 7 Expected protonation of GFP analogues 5.9d ...................................................................... 91 5 8 Synthesis of imi dazolinone chlomophore 5.14a b ............................................................... 93 5 9 Possible mechanism of fluorophore 5.14a............................................................................ 94

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13 LIST OF ABBREVIATION S AcOH Acetic acid Ac2O Acetic anhydride Ala (A) Alani ne Anal Analytical aq Aqu e ous []D Optical rotation BAL 2, 3 D imercaproptopanol Bn Benzyl Boc tert-Butyl dicarbonyl br s Broad shinglet BtH 1 H Benzotriazole Bt Benzotriazo l 1 -yl Bu Buthyl Calcd Calculated Cbz Carboxybenzyl CDCl3 Deutrated chloroform CFP Cyan fluorescent prot ein C Celsius degree Chemical shift d Doublet D (11 point) Dextrorotary DCM Methylene chloride

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14 DMF Dimethyl formamide DMSO Dimethyl sulfoxide DMSO d6 Deutreted dimethyl sulfoxide Et Ethyl et al A nd others Et3N Triethyl amine EtOAc Ethyl acetate equiv Equivalent (s) Fmoc Fluorenylmethyloxycarbonyl g Gram(s) GFP Green fluorescent protein gHMBC Heteronuclear multiple bond c orrelation Gln Glutamine Gly (G) Glycine h Hour HCl Hydrochloric acid His Histidine HMBA H examethylene bisacetamide HPLC H igh purity liquid chromatography HRMS High resolution mass spectrometry Hz H ertz H2O Water J Coupling constants

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15 L (11 point) Levorotary Leu (L) Leucine Lit Litterature Lys (K) Lysine m M ultiple M Molar Me Methyl MeCN Acetonitrile MeO H Methanol Met (M) Methionine min Minute MgSO4 Magnesium sulfate mp Melting point Na2CO3 Sodium carbonate NaOH Sodium hydroxide NMR Nuclear magnetic resonance nOes Nuclear overhauser e ffect NOESY Nuclear o verhauser e ffect s pectroscopy OH Hydroxyl group p P ara Pg Protecting group Ph Phenyl Phe (F) Phenylalanin e

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16 Pro (P) Proline Proj Project q Quartet RT Room temperature s Singlet SOCl2 Thionyl chloride t Triplet t (tert) Tertiary t -BuOK P otassium tert butoxide TFA Trifluoroaceti c acid TIPS Triisopropylsilane THF Tetrahydrofuran TMS Tetramethylsilane Tol 4 Methyl -phenyl tR Retention time Trp (W) Tryptophane Tyr Tyrosine UV Ultraviolet W W att(s) YFP Yellow fluorescent protein

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17 Abstract of Dissertation Presented to the Gra duate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BENZOTRIAZOLE INTERMEDIATES FOR HETEROCYCLES AND PHARMACEUTICALS By Megumi Yoshioka Tarver December 2009 Chair: A lan R. Katritzky Major: Chemistry The focus of this work is to expand the utility of 1 H -benzotriazole (B tH) in organic synthes i s. Benzotriazole is a synthetic auxiliary and had been utilized in many reactions previously. T he application of BtH is descriv ed as an excellent leaving group in the synthes i s of various pharmaceuticals and heterocycles. Chapter 1 describes the properties of BtH and its previous application s In C hapter 2 C aminoimi doylation and C -thiocarbamoylation of s ulfones, and ketones fro m benzotriazole 1 -carboxamidines and 1 (alkyl -or arylthiocarbamoyl)benzotriazoles are reported in the formation of new C C bond respectively. Ch ir a l y pure NFmoc protected -dipeptides are readily synthesized in solution phase from commercially available N-Fmoc -protected amino acids In C hapter 3 and 4, small peptides were synthesized using solid phase technique and benzotriazole methodology. N-Fmoc protected (aminoacyl)benzotriazoles are converted into NFmoc protected( dipeptidoy l)benzotri azoles which are used under mild microwave irradiation in solid phase peptide segment condensations syntheses to give tri tetra penta hexa -, and he ptapeptides as isol ated pure peptides ( Chapter 3 ). In Chapter 4, N-Fmoc N[(7 -methoxycoumarin 4 -yl)acetyl] -Llysin e ( NFmoc -LLys(Mca) OH) is conveniently prepared by benzotriazole methodology. N -

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18 Acylbenzotriazoles: Mca Bt N-Fmoc -L Lys(Mca) -Bt, coumarin 3 -ylcarbonyl (Cc) Bt NFmoc -LL ys(Cc) Bt, and N(Cc) L -Lys(Fmoc) Bt enable efficient microwave-enhanced solidphase fluorescent labeling of peptides. Chapter 5 p resent s eight green fluorescent protein (GFP) chromophore -modified analogues which wer e designed to act as pH sensors. Syntheses of these analogues were carried out via two to four steps from commerciall y available compounds. The fluorescence and pH depen dency of analogues were studied Finally, a summ a ry of this work is presented in Chapter 6.

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19 CHAPTER 1 GENERAL INTRODUCTION 1.1 General Introduction of Benzotriazole Benzotriazole is a well studied synthetic auxiliary and benzotriazole methodology has been utilized for a wide variety of organic syntheses. 1 H Benzotriazole (BtH) has useful properties such as its leaving group ability 1.1 its electron -donating charact e r 1.2 or its activation of the CH bon d toward proton loss 1.3 (Figure 1 1). [98CR409] BtH is non-toxic, insensitive to moisture, and commercially available at low cost. Moreover, it has high solubility in common organic solvents such as MeOH b enzene, chloroform, toluene, and DMF. BtH behaves as a weak base (pKa = 1.6) as well as a weak acid (pKa = 8.3), [48JCS2240, 00JACS5849] which allows reactions to occur in both acidic and basic media (Scheme 1 1). Thus BtH can be easily removed from reaction mixtures by a simple acid or base work up duri ng purification My research has focused on the utilization of BtH as an activating reagent in the synthesis of several biological targets. Figure 1 1. Various functions of the benzotriazole groups

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20 Scheme 1 1. Protonation equilibria A recent review summarized the important uses of benzotriazolyl intermediates and the displacement of Bt by nucleophilic a ttack. [94CSR363] Because of its ability as a leaving group, benzotriazole can be easily displaced by the lone pair of a heteroatom, followed by react ion with a nucleophile (Scheme 1 2) Scheme 1 2. Benzotriazole -mediated nucleophilic addition Benzotriazole methodology has been used in our research group for more than two decades, and the utility of BtH has been exemplified in reactions such as al kyl ations, [94CSR363] acylation [03JOC4932, 03JOC5720, 05S1656] imine acylation, [00S2029] guanylation [06N GIR] and imidoylati o n [06N GIR] Displacement of benzotriazole derivatives have been utilized by different nucleophilic atoms such as C, S, N, or O (Sc heme 1 3). Benzotriazole methodology has also be en utilized in Mannich reaction s [94JHC917] Michael reaction s [01B CSJ2133] and Grignard reactions [07S3141]

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21 Scheme 1 3. Displacement of benzotriazole derivatives by different nucleophile s In Chapter 2, the C aminoimidoylation and C -thiocarbamoylation of sulfones and ketones using the 1(alkylthiocarbamoyl)benzotriazoles 1.5 and benzotriazole 1 -carboxamidine 1.6 were presented (Scheme 1 4 ). [07JOC6742] These compounds were prepared from bis(benzotriazoyl) methanethione 1.4 in two steps. Highly toxic and moisture sensitive thiophosgene was easily converted to bis(benzotriazoyl)methanethione 1.4 [78JOC337] which is nontoxic and moisture insensitive Scheme 1 4 C -Aminoimidoylation and C thiocarbamoylatio n of sulfones and ketones Using benzotriazole methodology also results in a facile preparation of peptides. Carboxylic acids are activated by BtH in the presence of SOCl2. Benzotriazole activated N -

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22 protected amino acids react with the -NH2 group of free am ino acids to form longer peptide chains without the necessity to protect C termini. [ 05JOC4993, 05S397, 09A47] My research utilized N Fmoc protected amino acid benzotriazole derivatives 1.12 and 1.14 in peptide synthesis both in solution phase and solid phase with retention of the original chirality (Chapter 3 Scheme 1 5 ). [07CBDD465, 08CBDD181] Compounds 1.12 and 1.14 couple on solid resin under microwave irradiation in 3 to 10 min. Because of C terminus activation by benzotriazole, the method does not re quire additional coupling reagents. In some sequences, peptide synthesis during segment condensation using 1.14 did not cause epimerization, which is known to be a major problem during segment condensation. [08CBDD181] Scheme 1 5 Conversion of N-protected amino acid into N-protected(aminoacyl) -benzotriazole 1. 1 2 and N-protected(benzopeptidoyl)benzotriazole 1. 1 4 Currently, t he focus is on fluorogenic peptide synthesis and f luorescent labeled peptides using benzotriazole methodology. Three couma rin labeled -Fmoc -LLys OH derivatives (Figure 1 2) were synthesized in an average yield of 52% in two steps and their benzotriazole derivatives 1.181.20 and coumarin(carboxyl/acetyl)benzotriazoles 1.16 and 1.17 were utilized in fluorogenic peptide synthes is [08OBC4582]

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23 Figure 1 2. Coumarin(carboxyl/acetyl)benzotriazole 1.16, 1.17 and coumarin labeled -Fmoc -LLys Bt 1.181.20 Recently, I designed green fluorescent peptide (GFP ) modified unnatural amino ac ids as potential pH sensors. [Proj# 1979] The GFP chromophores are subject to a photoisomerization which decreases their fluorescence activity. The project is designed to inhibit photoisomerization by intramolecular hydrogen bonding under acidic conditions. The designed chromophores are able to tag N-pr otect ed natural amino acids such as lysine, asparagine, or glutamine for the preparation of fluorogenic peptides (Figure 1 3). In order to optimize the fluorescent activity, I synthesized GFP chromophore analogues (Figure 1 4, Chapter 5 ) and reported their fluorescent activity under different pH conditions. Figure 1 3. GFP modified unnatural amino acids

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24 Figure 1 4. GFP modified chromophore analogues 1.2 General Method s Reagents were purchased from Peptides International Across or Aldrich and used w ithout further purification. Rink amide -MBHA resin (200 400 mesh, 0.35 meq/g) was purchased from Peptide International (Louisville, KY, USA). Melting points were det ermined on a capillary point apparatus equipped with a digital thermometer NMR spectra wer e recorded in CDCl3 or DMSO d6 with TMS for 1H (300 MHz) and 13C NMR (75 MHz) as an internal reference. Absorption and fluorescence measurements were recorded on Cary 100 UV -Vis and FluoroMax spectrophotometers respectively. Optical rotation values were me asured with the use of sodium D line. Column chromatography was performed on silica gel (200425 mesh) or basic alumina (60325 mesh) Elemental analyses were performed on a Carlo Erba 1106 instrument. MALDI analyses were performed on Bruker Reflex II TOF mass spectrometer retrofilled with delayed extraction. Analytical reversed -phase HPLC was performed on a Rainin HP XL system with a Vydac C18 (5 m, 2.1 250 mm) silica column at a 1ml/min flow rate. Peptides were eluted using a 10 80 % gradient of solvent B (0.1 % TFA in MeCN ) vs solvent A (0.1% TFA in H2O ) and peaks were detected at a wavelength of 214 nm. The identification of the products was achieved by matrix assisted laser desorption/ionization time -of -flight mass spectrometry (MALDI TOF, ABI 4700 Pr oteomics Analyzer) with -cyano 4 -hydroxy cinnamic acid as the matrix. Synthesis of the peptides was performed in a Discover BenchMate peptide synthesizer from CEM (Matthews,

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25 NC, USA). The conditions for a variety of coupling steps were optimized to increas e rate and eliminate epimerization. Single mode irradiation with monitoring of temperature, pressure, and irradiation power versus time was used throughout, making the procedure highly reproducible. MS/MS peptide fragmentation was obtained on the crude pep tides by way of low resolution MS and tandem mass spectrometry (MSn) data obtained via HPLC/UV/(+)ESI -MS and MSn on a ThermoFinnigan (San Jose, CA) LCQ Classic quadruple ion trap mass spectrometer in electrospray ionization (ESI) mode. High resolution mas s spectrometry (HRMS) via flow injection positive [(+)ESI] time of flight (TOF) was obtained on an Agilent 1200 series spectrometer.

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26 CHAPTER 2 C-AMINOIMIDOYLATION AND C THIOCARBAMOYLATION O F SULFONES AND KETONES1 2 .1 Introduction C-Acylation and C imidoyl ation are widely used for the preparation of biologically active compounds. Many acylating [47JACS119, 03JOC1443, 59JACS4882] and imidoylating reagents [97TL6771, 99OL977, 02JOC4667] have been reported to give C acylation and C imidoylation ; by comparison C aminoimidoylation and C thiocarbamoylation have both been relatively unexplored synthetically. Literature examples of compounds that could conceptually have been made by C aminoimidoylation and C -thiocarbamoylation have generally been accessed by multis tep synthesis. [79S343, 83LAC290, 00JOC1583, 04JOC188] A substructure search showed no example of C aminoimidoylation by C -C bond formation; one possible product of such a reaction ( 2.2 ) was reported by the reaction of an alkyl dichlorovinyl sulfone 2.1 with 4 -methoxyaniline (Scheme 2 1). [79ZOK2349] Scheme 2 1. Literature method of th e preparation of sulfonyl amidine 2.2 N, N Disubstituted ketene aminals 2.3 are used in many syntheses, especially in the construction of he terocyclic compounds. [04JOC188] Structurally, ketene aminals can be converted to the tautomers of C aminoimidoylated ketones. They have been prepared (Scheme 2 1Reproduced in part with permission from The Journal of Organic Chemistry 2007 72, 6742. Copyright 2009 American Chemical Society

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27 2) starting from (i) activated methylene compounds and isothiocyanates, [04JOC188] (ii) oxokete ne N, S acetals and lithiated secondary amines or aniline, [00JOC1583] or (iii) tris(dimethylamino)ethoxymethane and simple ketones. [79S343, 83LAC290] Methods (i) (iii) (Scheme 2 2) each consist of multi -steps with average overall yields of ca 30%. Methods (i) and (ii) are related to C aminoimidoylation but no such reaction of a ketone was located in a literature search. Scheme 2 2. Literature methods for the preparation of ketene aminals 2.3 Recently, our group synthesized a ) di and tri -substituted thi oureas, [04JOC2976] Nhydroxythioureas and thiosemicarbazides [06A226] using novel 1 (alkyl/arylthiocarbamoyl) benzotriazole thiocarbamoylating reagents 2.4 (Scheme 2 3) and b) 1,2,3 tri -substituted guanidines, [05HCA1664] N-hydroxy -, and N-amino -guanidi nes [06JOC6753] using novel benzotriazole 1 -carboxamidine -N aminoimidoylating reagents 2.5 (Scheme 2 4). I have demonstrated C aminoimidoylation and C thiocarbamoylation of sulfones and ketones using 1 (alkyl/arylthiocarbamoyl)benzotriazoles 2.4 a e and ben zotriazole 1 -carboxamidines 2.5 a c by a simple benzotriazole procedure.

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28 Scheme 2 3 Preparation of mono -N hydroxyand N aminothiourea from 1 (a lkyl/arylthio carbamoyl)benzotriazoles 2. 4 Scheme 2 4 N -Aminoimidoylation with benzotriazole 1 -carboxami dines 2. 5 2.2 Results and Discussion 2.2.1 Synthesis of 1 -( A lkyl/arylthiocarbamoyl) benzotriazoles 2.4a -e and Benzotriazole -1 carboxamidine 2.5 a -c Treatment of bis(benzotriazoyl)methanethione [78JOC337] with amines afforded 1 (alkyl/arylthiocarbamo -yl)benzotriazoles 2.4 a -e [04JOC2976] which were converted into benzotriazole 1 -carboxamidines 2. 5 a -c [05HCA1664] by treatment with triphenylphosphine ylides (Scheme 2 5 Table 2 1 ). Scheme 2 5. Synthesis of benzotriazole 1 -carboxamidine 2.4a -e and 1 (alkyl/a rylthio carbamoyl)benzotriazoles 2.5a -c

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29 Table 2 1. 1 (A lkyl/arylthiocarbamoyl)benzotriazoles 2.4 a -e and benzotriazole 1 carboxamidine 2.5 a -c Entry R 1 Yield (%) a R Yield (%) a 1 2.4a Cyclohexyl 95 2 2.4b n Bu 98 2.5a p Tol 92 3 2.5b p ClC 6 H 4 6 7 4 2.4c (CH 2 )Ph 85 2.5c Ph 87 5 2.4d Bn 98 6 2.4e t Bu 87 a Isolated yield 2.2.2 C -Aminoimidoylation and C -Thiocarbamoylation of Sulfones Reaction of 2.0 e quiv of sulfones 2. 7 with 2.5 equiv of potassium tert butoxide in THF at room temp erature for 0.5 h followed by the addition of the appropri ate benzotriazole reagent (2.4a or 2.5a ) afforded compounds 2.6 and 2.8 in yields of 30 and 40% respectively (Scheme 2 6). The reaction was also carried out with methyl phenyl sulfone or ethyl pheny l sulfone and compound 2.4 or 2.5 but the desired product did not form in a similar manner. Scheme 2 6. Preparation of C aminoimidoylation product 2.6 and C -thiocarbamoylation product 2.8 from sulfone 2.7 2.2.3 C -Aminoimidoylation and C -Thiocarbamoylat ion of Ketones Reaction of the enolates from ketones 2.10a d with 2.4 or 2.5 after 0.5 4 h gave the C aminoimidoylation and C thiocarbamoylation products 2.9a d and 2.11a d (27 65% yields, Table 2 2 ) (Scheme 2 7) as monitored by TLC. For compounds 2.9a an d 2.11a -d 2D NMR correlation experiments were carried out by Dr. Ion Ghiviriga to assign the 1H and 13C chemical shifts.

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30 Scheme 2 7. Preparation of the C aminoimidoylation products 2.9a d and C -thiocarbamoylation products 2.11a d from ketones 2.10a d T able 2 2 C -Aminoimidoylation and C thiocarbamoylation of ketones 2.10a d to give 2.9a d and 2.11a d Reagent R R1 Ketone R2 R3 Product Yield(%)a 2.5c Ph (CH 2 ) 2 Ph 2.10a Ph H 2.9a 32 2.5a p Tol n Bu 2.10b 2 Thienyl H 2.9b 27 2.5c Ph (CH 2 ) 2 Ph 2.10c Me Me 2.9c 27 2.5b p ClC 6 H 4 n Bu 2.10a Ph H 2.9d 31 2.4b n Bu 2.10a Ph H 2.11a 50 2.4c (CH 2 ) 2 Ph 2.10d Ph Ph 2.11b 65 2.4b n Bu 2.10b 2 Thienyl H 2.11c 51 2.4e t Bu 2.10c Me Me 2.11d 40 a Isolated yield Reaction of ketones 2.10a -c with 2.4 a (R1= Bn ) afforded the isomeric 1,3 -oxazolidine 2 thione s 2.12 instead of the expected C thiocarbamoylation product (Scheme 2 8 Table 2 3 ). Structures 2.12a c were verified by NMR and in the case of 2.12a by X ray crystallography (Figure 2 1). 1,3 O xazolidine 2 t hione were previously prepared by cycloaddition of -metalated alkyl isothiocyanates to carbonyl compounds. [76CB3047] Compounds 2.12a -c were assumed to form via a similar mechanism after isothiocyanate formation from 2.4a in the presence of excess potassium tert -butoxide (Scheme 2 9).

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31 Scheme 2 8. Forma tion of 1,3 -oxazolidine 2 thione 2.12 Table 2 3. Formation of 1,3 oxazolidine 2 thione 2.12 Entry Compound R 1 R 2 Yield (%) a 1 2.12a Me 2 Th ienyl 51 2 2.12b Me Ph 27 3 2.12c Me Et 40 a Isolated yield Figure 2 1. X ray structure of 2.12a Scheme 2 9 Possible mechanism of 1,3 oxazolidine 2 thione 2.12a -c formation

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32 Due to the weak basi c ity of p otassium tert butoxide it allows the deprotonation of NH group of 2.4a for isoth iocyanate formation and cycliza t i on to 1,3 oxazolidine 2 thione 2.12a -c This formation of 1,3 -oxazolidine 2 thione might not have occurr ed in the pres ence of stronger base to forbid to negate formation. 2.2.4 Compound Characterization and Tautomeric Structures The tautomeric structure of a heterocyclic [or indeed any] compound can profoundly influence its physical properties [e.g. boiling point, solubility] and chemical properties [e.g. acid/base, electron distribution, reactivity]. It was therefore of interest to investigate the tautomeric structures of the parent compounds, and in particular, to know whether they exist ed in the enol or keto forms. Figure 2 2. Relevant 1H and 13C chemical shifts in compounds 2.6 and 2.8 Compounds 2.6 and 2.8 exist in CDCl3 at 25 C solely as the imino and thiocarbonyl forms, respectively (Fig u re 2 2). The alpha protons of R3 phenyl group (5.20 ppm for 2.6 and 5.57 ppm for 2.8 ) couple with the NH proton (6.11 ppm for 2.6 and 9.08 ppm for 2.8 ) in both compounds. Compound 2.9a was present in CDCl3 as a mixture of two compounds in a 7:1 ratio both with the same hydrocarbon skeleton, as revealed by the gHMBC spectra (Figure 2 3). Exchange

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33 peaks in the NOESY spectrum between protons such as 5.43 ppm with 5.35 ppm, 7.88 ppm with 7.67 ppm, 7.03 ppm with 6.97 ppm, 7.13 ppm with 7.26 ppm, 2.89 ppm with 3. 00 ppm and 3.49 ppm with 3.60 ppm indicate d that these species were inter converting. The chemical shift of the carbonyl carbon in 2.9a is closer to that expected for an aromatic ketone than for an enol. Thus, the tautomeric structure of 2.9a differs from the compounds of structure 2.6 These chemical shifts suggest that both isomers of 2.9a are keto -enamine forms. Of the exchange cross -peaks at 11.77 ppm, the largest is 4.76 ppm, indicating that the two isomers differ in the amine hydrogen facing the carbo nyl group. In the major isomer of 2.9a nOes between 5.43 ppm and 3.49 ppm, and between 4.76 ppm and 7.03 ppm demonstrate that in this isomer the aniline NH is involved in the hydrogen bond and that the alkyl group on the other nitrogen is anti to the ani line nitrogen, as depicted in Figure 2 3. For the minor isomer, the nOes offered no information on the syn/anti geometry of the aniline group, because they were mainly transferred nOes. It is reasonable however to assume the same geometry as in the major isomer. Exchange peaks between 5.43 ppm, 5.35 ppm and all the four NH protons indicate that the keto tautomer is present in the equilibrium, although it was not detected in the proton spectra. Another transient species displays a signal at 13.26 ppm, in e xchange with all of the exchangeable protons in both isomers. The isomerization of 2.9a occurs due to the keto -enamine enol -imine tautomerization (Scheme 2 10). Compounds 2.9b and 2.9d displayed equilibria similar to that for 2.9a The species with the ani line NH involved in hydrogen bonding is ca. 7 times more abundant than the other isomer. Compound 2.9c is solely in the imidamide form in CDCl3 at 25 C as indicated by the coupling between the protons at 3.70 ppm and 1.13 ppm (Figure 2 4). The difference in tautomerism between 2.9c and 2.9a 2.9 b 2.9 d may be explained by the steric hindrance in the enol form of 2.9c where the two methyl groups are syn periplanar.

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34 Figure 2 3. 1H and 13C Chemical shifts in the major and minor isomers of compound 2.9a Scheme 2 1 0 Mechanism of isomeric mixtures form ation Figure 2 4. Relevant 1H and 13C chemical shifts in compound 2.9

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35 Compound 2.11a in CDCl3 at 25 C displays an equilibrium between three species, i n a ratio 44 : 5 : 1. The inter conversion of these s pecies was demonstrated by exchange peaks in the NOESY spectrum. The major compound is the keto tautomer, while the other two are enol tautomers, as demonstrated by the chemical shift of the carbon bearing the oxygen atom. The significant deshielding of th e NH proton in the keto form as compared to the enol forms suggests a hydrogen bond in the keto form, as in Figure 2 5. The spectra were repeated in acetone d6, a hydrogen bond acceptor that would compete with the carbonyl group of 2.11a for hydrogen bondi ng. The chemical shift of the NH proton in the enol moved downfield by 2 ppm, while for the keto form the change was only 0.07 ppm, demonstrating intramolecular hydrogen bonding in the ketone. The two enol species have to be the Z and E isomers resulting f rom rotation about the CN bond in the thioamide. Thioamides are known to prefer the Z configuration and this is the configuration in the intramolecular hydrogenbonded keto species. [96JMS45] The similarity of the proton chemical shifts at the NHCH2in the ketone form (3.71 ppm) and in the major enol form (3.67 ppm) indicates that the latter is also in the Z configuration. The E configuration is present in the enol tautomer and not in the ketone because it strengthens the resonance assisted hydrogen bonding (RAHB) in the enol. This is demonstrated by the higher chemical shift value of the proton involved in the RAHB in the minor E enol form (14.86 ppm) as compared to the Z enol (14.54 ppm). The rota mer s form of C thiocarbamoylation product 2. 11a can be ex plained with Newman projection shown in Figure 2 6. The 2.11ro t a mer 1 and 2.11-ro t a mer 2 were favored in chloroform due to less steric hin derance of the R group. H owever, the disappearance of 2.11 ro t a mer 2 can be explained because of favor e d intermolecu lar hydrogen bonding with acetone and NH group of 2.11 to t a mer 1

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36 Figure 2 5. 1H and 13C Chemical shifts and the tautomers/rotamers for compound 2.11a Figure 2 6. Rota mer s of C aminoimidoylation product 2. 9 and C thiocarbamoylation product 2.11 Comp ound 2.11c in CDCl3 at 25 C occurs solely as the keto tautomer. The chemical shift of the NH proton is 9.22 ppm, indicating an intramolecular hydrogen bond. In acetone d6 solution, both tautomers are present, and the keto : en ol ratio is 1 : 0.44 (Figure 2 7 ). For compound 2.11a also, the proportion of the enol was larger in acetone (keto : enol = 45 : 55) than in chloroform (keto : enol = 88 : 12). As with the case of 2.11a no E rotamer of the enol form was detected in acetone d6.

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37 Figure 2 7 1H and 13C Chemical shifts for the tautomers of compound 2.11c in CDCl3 an d a cetone d6 Compounds 2.11b and 2.11d in CDCl3 at 25 C display the keto tautomer only. The enol tautomer is higher in energy when R3 is not H, due to steric repulsion between R2 and R3 whi ch have to be syn periplanar in the enol. 2.3 Conclusion Successful C aminoimidoylation s and C thiocarbamoylation s of sulfones and ketones were achieved in yield s of 2765 % unde r mild reaction conditions. The method provides an easy access to interesting classes of compounds for further transformations. However, higher yields o f those products may be achiev ed using stronger bases such as sodium hydride (pKa = 42), n -but yl lithium (pKa = 48), or lithium diisopropylamide (pKa = 36) due to the weak acidity of ketones 2.10. Thus, incomplete deprotonation of ketones and formation of isothiocyanates can be explained by unexpected product s 1,3 oxazolidine 2 thione 2.12. In fu ture work, the reactions should be repeated using stronger base than potassium tert -butox ide for more efficient C aminoimidoylation and C thiocarbamoylation reaction, which may yield the desired C thiocarbamoyl product with compound 2.4a (R1 = Bn).

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38 2.4 Experimental Section 2.4.1 General Procedure for the Preparation of Compounds 2.6 and 2.8 To a solution of the desired ester or sulfone (2.0 mmol) in THF (15 mL), potassium tert butoxide (2.5 mmol) was added. After stirring for 30 min, the desired reagent 2.4 or 2.5 (1.0 mmol) (Scheme 2 6) was added to the reaction mixture. The progress of the reaction was monitored by TLC. Upon completion, water (20 mL) was added to quench the reaction followed by extraction with DCM (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. N -Butyl -N -(4 methylphenyl) -2 -phenyl -2 -(phenylsulfonyl)ethanimidamide 2.6 : White microcrystals (30 %); mp 117.6 C ; 1H NMR (CDCl3) 7.66 (t, J = 8.4 Hz, 3H), 7.51 (t, J = 7.1 Hz, 2H), 7.53 J = 7.8 Hz, 2H), 6.11 (s, 1H), 6.11 (d, J = 8.1 Hz, 2H), 5.20 (s, 1H), 3.39 1.73 1.00 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3) 148.5, 146.5, 137.4, 134.2, 131.8, 130.8, 130.1, 129.4, 129.1, 128.9, 128.8, 128.7, 121.8, 68.4, 41.4, 31.0, 20.8, 20.4, 13.9. Anal. Calcd for C25H28N2O2S: C, 71.40; H, 6.71; N, 6.66; found: C, 71.55; H, 6.96; N, 6.52. N -Cyclohexyl -2 -phenyl -2 -(phenylsulfonyl)ethanethioamide 2.8 : White microcrystals (40 %); mp 148.9 C ; 1H NMR (CDCl3) 9.08 (br s, 1H), 7.76 (m, 1H), 7.52 (m, 5H), 5.57 (s, 1H), 4.38 1.7813C NMR (CDCl3) 190.0, 137.2, 134.5, 129.7, 129.3, 129.2, 129.0, 128.7, 81.7, 54.8, 31.1, 30.8, 25.4, 24.4. Anal. Calcd for C20H23NO2S2: C, 64.31; H, 6.21; N, 3.75; found: C, 63.96; H, 6.25; N, 3.62.

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39 2.4.2 General Procedure for the Preparation of Compounds 2.9a d and 2.11a d To a solution of the desired ketone (2.0 mmol) in THF (20 mL), potassium tert -butoxide (2.5 mmol) was added, followed by the appropriate reagent 2.4 or 2.5 (1.0 mmol) (Scheme 2 7). The mixture was stirred at room temperature until full conversion of starting materials (0.5 4.0 h) was observed by TLC The crude reaction mixture was then evaporated under reduced pressure, washed with water (30mL x 3), and finally extracted with diethyl ether (30 mL x 3). Evaporation of the organic fraction followed by flash column chromatography on silica gel afforded 2.9a d or 2.11a d 3 -Oxo -N -phenethyl -N ',3 -diphenylpropanimidamide 2.9a : W hi te microcrystals (32 %); mp 156.2 C ; 1H NMR (CDCl3) 13.06 (s, 1H) 7.88 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.1 Hz, 2H), 7.41 (t, J = 7.1 Hz, 1H), 7.30 (t, J = 6.6 Hz, 2H), 7.30 (t, J = 7.6Hz, 2H), 7.25 (t, J = 7.6Hz, 1H), 7.13 (d, J = 7.6 Hz, 2H), 7.03 (d, J = 6.6 Hz, 2H), 5.43 (s, 1H), 4.76 (br s, 1H), 3.49 (q, J = 5.6 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H); 13C NMR (CDCl3) 185.6, 159.6, 141.8, 138.1, 136.5, 136.5, 130.2, 129.1, 129.0, 127.2, 126.9, 126.3, 125.4, 124.4, 76.0, 43.6, 35.3. Anal. Calcd for C23H24N2O2: C, 76.64; H, 6.71; N, 7.77; found: C, 76.61; H, 6.32; N, 7.64. N -Butyl -N (4 -methylphenyl) -3 -oxo -3 -(2 -thienyl)propanimidamide 2.9b : Yellow oil (27 %); 1H NMR (CDCl3) 11.32 (s, 1H), 7.53 (d, J = 3.7 Hz, 1H), 7.39 ( d, J = 4.9 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 8.1 Hz, 2H), 7.06 (t, J = 3.8 Hz, 1H), 5.32 (s, 1H), 4.67 (br s, 1H), 3.20 (q, J = 6.9 Hz, 2H), 2.36 (s, 3H), 1.591.52 (m, 2H), 1.36 (q, J = 7.1 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H ); 13C NMR (CDCl3) 178.0, 159.5, 148.2, 136.2, 133.4, 130.5, 128.2, 127.4, 125.3, 121.6, 74.9, 42.1, 31.2, 21.0, 20.0, 13.7. HRMS Calcd for [C18H22N2OS+ H]+, 315.1554; found 315.1564. 2 -Methyl -3 -oxo -N -phenethyl -N -phenylbutanimidamide 2.9c : Yellow oil (27 %); 1H NMR (CDCl3) 7.40 7.01 (t, J = 7.3 Hz, 1H), 6.76 (d, J = 7.8 Hz, 2H), 4.47 (br

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40 s, 1H), 3.70 (q, J = 7.0Hz, 1H), 3.61 (quintet, J = 6.9 Hz, 2H), 2.92 1.13 (d, J = 7.0 Hz, 3H); 13C NMR (CDCl3) 207.2, 154.7, 150.8, 139.2, 129.1, 128.8, 128.5, 126.4, 122.2, 122.0, 47.3, 41.9, 35.0, 29.0, 15.0. Anal. Calcd for C19H22N2O: C, 77.52; H, 7.53; N, 9.52; found: C, 77.12; H, 7.73; N, 9.22. (Z )-N -Butyl -N -(4 -chlorophenyl) -3 hydroxy -3 -phenyl -2 -propenimidamide 2.9d : Yellow oil (31 %); 1H NMR (CDCl3) 7.89 86 (m, 2H), 7.43 7.18 (m, 2H), 5.41 (s, 1H), 4.61 (br s, 1H), 3.23 (q, J = 5.5 Hz, 2H), 1.61 2H), 0.96 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) 185.6, 159.4, 141.4, 135.1, 130.1, 130.0, 128.1, 126.6, 126.5, 82.4, 75.8, 42.2, 31.1, 20.1, 13.7. HRMS Calcd for [C19H21ClN2O+ H]+, 329.1404; found 329.1404. N -Butyl -3 -oxo -3 -phenylpropanethioamide 2.11a : Brown oil (50 %); 1H NMR (CDCl3) 9.28 (br s, 1H), 8.04 (d, J = 7.3 Hz, 2H), 7.63 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 7 .5 Hz, 2H), 4.50 (s, 2H), 3.67 (m, 2H), 1.70 (m, 2H), 1.44 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) 197.2, 194.2, 136.1, 134.5, 129.2, 128.9, 52.7, 46.5, 30.1, 20.4, 14.0. Anal. Calcd for C13H17NOS: C, 66.35; H, 7.28; N, 5.95; found: C, 66.62; H, 7.41; N, 5.52. 3 -Oxo -N -phenethyl -2,3 -diphenylpropanethioamide 2.11b : White microcrystals (65 %); mp 110.8 C ; 1H NMR (CDCl3) 9.01 (br s, 1H), 7.98 7.46 J = 7.3 Hz, 2H), 6.33 (s, 1H), 4.01 3.0113C NMR (CDCl3) 197.7, 183.1, 135.9, 134.9, 134.0, 129.4, 129.0, 128.8, 128.7, 128.7, 128.4, 127.8, 126.6, 67.0, 47.5, 33.6. Anal. Calcd for C23H21NOS: C, 76.85; H, 5.89; N, 3.90; found: C, 77.05; H, 5. 89; N, 3.84. N -Butyl -3 -oxo -3 -(2 -thienyl)propanethioamide 2.11c : Yellow oil (51 %); 1H NMR (CDCl3) 9.36 (br s, 1H), 8.00 (d, J = 3.8 Hz, 1H), 7.57 (d, J = 4.9Hz, 1H), 7.25 (m, 1H), 4.39 (s,

PAGE 41

41 2H), 3.68 (m, 2H), 1.68 (q, J = 7.4 Hz, 2H), 1.43 (m, 2H), 0.94 ( t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) 194.9, 187.0, 144.0, 134.1, 128.7, 127.0, 55.7, 45.8, 30.1, 20.1, 0.94. Anal. Calcd. for C11H15NOS: C, 54.74; H, 6.26; N, 5.80; found: C, 55.06; H, 6.48; N, 5.60. N -(Tert-butyl) -2 methyl -3 -oxobutanethioamide 2.11d : White microcrystals (40 %); mp 108.3 C ; 1H NMR (CDCl3) 8.17 (br s, 1H), 3.96 (q, J = 7.1 Hz, 1H), 2.29 (s, 3H), 1.54 (s, 9H), 1.45 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3) 209.7, 198.7, 64.0, 55.6, 29.6, 27.3, 19.2. Anal. Calcd for C9H17NOS: C, 57.71; H, 9.15; N, 7.48; found: C, 57.60; H, 9.56; N, 7.41. 2.4.3 General Procedure for the Preparation of Compounds 2.12a c To a solution of the desired ketone (2.0 mmol) in THF (20 mL), potassium tert -butoxide (2.5 mmol) was added. After stirring the mixture for 30 min, 2.5a (1.0 mmol) (Scheme 2 8) was added and t he mixture was stirred at room temperature for 0.3 4 h. The reaction was stopped and the solvent evaporated under vacuum. The crude product was washed with water and then extracted with diethyl ether. Evaporation of the organic fraction followed by flash column chromatography on basic alumina afforded 2.12a -c in moderate yields. 5 -Methyl -4 -phenyl -5 -(2 -thienyl) -1,3 -oxazolidine -2 -thione 2.12a : White microcrystals (51 %); mp 157.3 C ; 1H NMR (CDCl3) 7.397.37 (m, 1H), 7.27 7.1513C NMR (CDCl3) 188.7, 145.9, 134.3, 129.4, 129.1, 127.2, 126.8, 126.1, 124.7, 91.7, 70.8, 23.8. Anal. Calcd for C14H13NOS2: C, 61.06; H, 4.76; N 5.09; found: C, 61.40; H, 4.78; N, 5.09. Crystal data for C14H13NOS2, MW 275.37, monoclinic, space group P2/n, a = 14.3023(4), b = 5.7750(2), c = 17.5354(5) = 112.690(1) o, V = 1336.26(7) 3, F(000) = 576, Z = 4, T = 180 oC, (MoK ) = 0.385 mm1, Dcalcd = 1.369 g.cm3, 2 max 55 o (CCD area detector, MoK radiation), GOF = 1.05, wR(F2) = 0.076 (all 3065 data), R = 0.028 (2981 data with I > 2 I).

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42 5 -Methyl -4,5 -diphenyloxazolidine -2 -thione 2.12b : White microcrystals (27 %); mp 178.3 C ; 1H NMR (CDCl3) 7.45 (s, 3H); 13C NMR (CDCl3) 189.0, 143.6, 135.1, 129.4, 129.2, 129.0, 128.4, 127.0, 124.1, 93.7, 70.8, 23.9. Anal. Calcd for C16H15NOS: C, 71.34; H, 5.61; N, 5.20; found: C, 71.13; H, 5.61; N, 5.15. 5 -Ethyl -5 -methyl -4 -phenyloxazolidine -2 -thione (mixture of stereo -isomers) 2.12c : White microcrystals (40 %); mp 119.0 C ; 1H NMR (CDCl3) 7.62 (br s, 1H), 7.49 7.42 (m, 3H), 7.32 ), 1.56 (m, 1H), 1.16 (t, J = 7.4 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3) 189.0, 135.7, 129.1, 129.0, 128.9, 126.9, 126.7, 94.3, 93.8, 69.6, 67.0, 33.9, 28.9, 24.3, 21.3, 7.8. Anal. Calcd for C24H30N2O2S2: C, 65.12; H, 6.83; N, 6.33; found: C, 65.50; H, 6.61; N, 6.36.

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43 CHAPTER 3 PEPTIDE SYNTHES IS UTILIZING (N FMOC -PROTECTED -AMINOACYL) BENZOTRIAZOLES AND ( N FMOC -PROTECTED DIPEPTIDOYL)BENZOTRI AZOLES1 3.1 Introduction Segment condensation (or co nvergent/fragment condensation) peptide synthesis [0 5FSPPS215] is the construction of a polypeptide target by the assembly of several intermediate segment s and is useful for the synthesi s of complex peptides and small proteins. Such convergent synthesis often allows flexibility in the choice of protecting groups and coupling methods. Usually, protected peptide fragments of up to 15 amino acids in length are used because they are simpler to purify by reverse phase -HPLC compared with the longer peptides (Scheme 3 1) Scheme 3 1. General procedure of segment condensation peptide synthesis A fundamental drawback of convergent synthesis is epimerization at the C terminal residue of an N -segment during the condensation reaction with the C -segment (Scheme 3 2 ). [99JACS1636, 05B238, 06JPS116] There are several pos sible mechanisms for the racemization/epimerization procedures during peptide synthesis as shown in Scheme 3 2; a) formation of an oxazolone and b) tautomerization [70JACS 5792] Poor solubility of large protected intermediate segments can also impede this approach. [05FSPPS215] The use of 1Reproduced in part with permission from Chemical Biology & Drug De sign, 2008, 72 181. Copyright 2009 Wiley Blackwell

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44 dicyclohexylcarbodiimide (DCC) coupl ing additives such as 1-hydroxy benzotriazole (HOBt), and reagents such as (2 (1 H -benzotriazole 1 -yl) 1,1,3,3 -tetramethyluronium hexafluoro phosphate) (HBTU) or (2 (7 aza 1 H -benzotri azol e 1 -yl) 1,1,3,3 tetramethyluronium hexafluoro phosphate) (HATU) can minimize these disadvantages (Figure 3 1). [77BCSJ1999, 84S572, 93JACS4397] Promising results in the suppression of racemization during peptide segm ent condensation in solution were obtained with new copper (II) complexes in conjunction with other additives [01P]. Recently disclosed segment condensations using the O acyl isopeptide method have provided racemization free syntheses of small (2 5 amino acid units) peptides. [06TL7905] Sche me 3 2 Epimerization mechanism during segment condensation Figure 3 1 Structure of coupling reagents

PAGE 45

45 Early attempts to synthesize oligomers with repeating units e.g. (Tyr -Ala Glu)n (n = 1 4) using Boc/benzyl protection strategy and polystyrene resin were plagued with failure sequences and low yields. [94IJPPR118] An attempted synthesis of the polymer (Tyr -Gly Glu)6 using Fmoc/ tert -butyl protection strategy also failed after the 10th amino acid was coupled to the growing chain. However McMurray et al [94IJPPR118] developed segment condensation on solid support to eliminate failure sequences caused by single amino acid deletions. Other tactics such as enzymatic condensation suffered problems of secondary hydrolysis during the synthesis and the instability of the enzyme in the condensation media. [93JACS7912, 93JPPS405, 01RJBC 306] (N-Protected aminoacyl)benzotriazoles have been widely used fo r the preparation of chiral di and tri peptides by stepwise and tetra peptides by segment condensation in solution phase. [05S397, 06CBDD326, 06S411] However, the benzotriazole methodology in so lution phase peptide synthesis does not allow f or the extension of tri -peptides or tetra -peptides Recently, ( N-p rotected aminoacyl)benzotriazoles were utilized as efficient coupling reagents for solid phase peptide synthesis. [07CBDD465] In this chapter segment condensation syntheses of five peptides from diverse ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2 and ( NFmoc protected -dipeptidoyl) -benzotriazoles 3.5 using mild microwave conditions are reported. The aim of this project wa s to minimize or pr event common epimerization problems during segment condensation using our benzotriazole methodology. 3.2 Results and Discussion Fmoc -protected -aminoacyl)benzotriazoles 3.2 I prepared ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2 a -g in 72 90% yield by the reaction of NFmoc protected amino acids 3.1a -g with 4 equiv of BtH and 1 equiv of SOCl2 in

PAGE 46

46 DCM at room temperature for 2 h following a published procedure (Scheme3 3 Table 3 1). [05S397, 06CBDD326, 06S411] Scheme 3 3 Prepa ration of ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2a -g Table 3 1. (NFmoc protected aminoacyl)benzotriazoles 3.2a -g Reagents Product s Yields (%)a mp (C) D 24 b Fmoc -LPhe OH 3.1a Fmoc -LPhe Bt 3.2a 84 159.1160.2 +5.7 c Fmoc L Met OH 3.1b Fmoc L Met Bt 3.2b 90 122.7 123.3 +74.4 d Fmoc -LTrp OH 3.1c Fmoc -LTrp Bt 3.2c 90 92.593.6 +15.0 e Fmoc -LLeu OH 3.1d Fmoc -LLeu Bt 3.2d 88 121.0122.8 +88.6 f Fmoc Gly OH 3.1e Fmoc Gly Bt 3.2e 85 160.9 161.5 g Fmoc L Ala OH 3.1f Fmoc L Ala Bt 3.2f 72 160.0 160.3 101.4 h Fmoc L Pro OH 3.1g Fmoc L Pro Bt 3.2g 88 163.0 165.0 100.9 i a Isolated yields, b c = 1.5 in DMF, c Lit. [ ] D 24 = +35.6 (c 1.6 in DMF) [05JOC4993], dLit. [ ]D 24 = +74.4 (c 1.5 in DMF) [05S397], eLit. [ ]D 24 = +12.7 (c 1. 5 in DMF) [05S397], fLit. [ ]D 24 = +53.1 (c 1.5 in DMF) [08A47], gNot chiral, hD 24 = 96.8 (c 1.6 in DMF)[05JOC4993], i Lit. [ ] D 24 = 60.5 (c 1.5 in DMF) [08A47 ] 3.2.2 Preparation of (LL )-Dipeptides 3.4 a -f and Diastereomeric Mixtures (3.4 a +3.4 a ), (3.4b+3.4b), (3.4c+3.4c), (3.4e +3.4 e ), and (3.4 f+3.4 f) Using (N-Fmoc -protected aminoacyl)benzotriazoles 3.2a -d I coupled readily avail able ( NFmoc -protected aminoacyl)benzotriazoles 3.2a -d [05A36, 05S397, 07CBDD465] with unprotected chiral amino acids L -Ala 3.3 a L-Met 3.3 b L-Phe 3.3 c and racemic mixtures DL-Ala ( 3.3 a + 3.3 a ) and DLMet ( 3.3 b + 3.3 b ) in aqueous Me CN (Me CH : H2O = 2 : 1) in the presence of Et3N at 20 C for 1 h to afford NFmoc -protected -dipeptides 3.4 a -f, ( 3.4 a + 3.4 a ), (3.4b+3.4b ), ( 3.4c+3.4c ), ( 3.4 e +3.4 e ), and ( 3.4 f+3.4 f) (77 93 %) isolated by simple recrystalization in EtOAc/ h exanes (Scheme 3 4 Table 3 -2).

PAGE 47

47 NMR an alysis of dipeptides 3.4a -f showed no detectable epimerization (<5 %). 1H NMR analysis for each LLdipeptide 3.4a, c e f derived from L-Ala 3.3a showed a clear doublet of methyl protons ranging from 1.28 1.34 ppm. However for the corresponding diastereomeri c mixture ( 3.4a + 3.4a ), (3.4c+3.4c ), ( 3.4 e +3.4 e ), and ( 3.4 f+3.4 f) the signal for the methyl protons was observed as two sets of doublets. Each of the LL-dipeptides 3.4b -d derived from LMet 3.3b showed singlets for the methyl group ( -S CH3) at 2.03, 2.04 and 2.02 ppm respectively, while each of the corresp onding diastereomeric mixtures (3.4b+3.4b ) showed two singlets. The 13C NMR spectra of diastereomeric mixtures (3.4 a + 3.4 a ), (3.4b+3.4b ), ( 3.4c+3.4c ), (3.4 e +3.4 e ), and ( 3.4 f+3.4 f) revealed duplicat ion of almost all aliphatic an d carbonyl carbon signals, but f or 3.4a -f, the 13C NMR spectra showed no such duplication of the carbon signals (Figure 3 2 3 3 ). The enantiopurity of the dipeptides 3.4a -f was supported by HPLC analysis using a Chirobiotic T column (detection at 220 nm, flow rate 0.7 mL / min, and MeOH as eluent). For each of the LL-dipeptides 3.4a -f, the HPLC results showed a single peak. In contrast, two peaks of equal intensity were observed for the diastereomeric mixtures ( 3.4a+3.4a ) and (3.4b+3.4b ) (Table 3 2). Scheme 3 4 Preparation of N-Fmoc -protect ed -dipeptides 3.4a -f ( 3.4a+3.4a ), and (3.4b+3.4b ), (3.4c+3.4c ), ( 3.4 e +3.4 e ), and ( 3.4 f+3.4 f)

PAGE 48

48 Table 3 2. Preparation of (LL)-dipeptides 3.4a -f and diastereomeric mi xtures ( 3.4a+3.4a ), and (3.4b+3.4b ), ( 3.4c+3.4c ), ( 3.4 e +3.4 e ), and (3.4 f+3.4 f) from ( NFmoc -protected aminoacyl)benzotriazoles 3.2a d Reactant Amino Acid Product Yield (%) a mp (o C) tR (min)b D 24 c Fmoc L Phe Bt 3.2a LAla 3.3a Fmoc L Phe L Ala OH 3.4a 85 208.7 210.6 5.4 16.9 Fmoc L Phe Bt 3.2a LMet 3.3b Fmoc L Phe L Met OH 3.4b 82 186.0 186.5 5.8 27.4 Fmoc L Met Bt 3.2b LAla 3.3a Fmoc L Met L Ala OH 3.4c 77 169.9 2.4 9.0d Fmoc L Met Bt 3.2b LPhe 3.3c Fmoc L Met L Phe OH 3.4d 89 186.0 5.7 6.3 Fmoc L Trp Bt 3.2c LAla 3.3a Fmoc L Trp L Ala OH 3.4e 83 119.0 121.0 5.7 15.8e Fmoc L Leu Bt 3.2d LAla 3.3a Fmoc L Leu L Ala OH 3.4f 87 179.0 179.8 2.3 10.9 Fmoc L Phe Bt 3.2a DL Ala (3.3a+3.3a) Fmoc L Phe DL Ala OH (3.4a+3.4a) 82 149.9 151.0 5.6, 6.0 21.8 Fmoc L Phe Bt 3.2a DL Met (3.3b+3.3b ) F moc L Phe DL Met OH (3.4b+3.4b) 74 156.9 5.4, 6.6 21.6 Fmoc L Met Bt 3.2b DL Ala (3.3a+3.3a) Fmoc L Met DL Ala OH (3.4c+3.4c) 93 115.0117.0 N/Af N/Af Fmoc L Trp Bt 3.2c DL Ala (3.3a+3.3a) Fmoc L Trp DL Ala OH (3.4e+3.4e) 78 146.3147.1 N/Af 20.4 Fmoc L Leu Bt 3.2d DL Ala (3.3a+3.3a) Fmoc L Leu DL Ala OH (3.4f+3.4f) 89 163.0164.0 N/Af 13.2 a Isolated yield, b t R = retention time, c c = 1.5 in DMF, d D 24 = 9.7 (c 1.5 in DMF) [05S397], e D 24 = 15.7 (c 1.5 in DMF) [05S397], f N ot determined Figure 3 2. 1H NMR of 3.4a and ( 3.4a+3.4a ). a) 1H NMR of 3.4a methyl group, b) 1H NMR of (3.4a+3.4a ), methyl groups

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49 Figure 3 3 13C NMR of 3.4a and ( 3.4a+3.4a ). c) 13C NMR of 3.4a carbonyl groups, d) 13C NMR of ( 3.4a+3.4a ), carbonyl groups 3.2.3 Preparation of ( N-Fmoc -protected dipeptidoyl)benzotriazoles 3.5a -f I treated N-Fmoc -protected dipeptides 3.4a f in DCM with 4 equiv of BtH and 1 equiv of SOCl2 at 15 C for 3 h to give the novel dipeptidoylbenzotriazoles 3.5a f (69 87 %) (Scheme 3 5 Table 3 3), fully characterized by 1H and 13C NMR spectroscopy and elemental analysis. Scheme 3 5 Preparation of ( N-Fmoc -protected -dipeptidoyl)benzotriazoles 3.5a -f Table 3 3. Preparation of ( NFmoc protected -dipeptidoyl)benzotriazole 3.5a -f Reactant Product Yield (% )a mp (o C) D 24 b Fmoc -LPhe -LAla OH 3.4a Fmoc -LPhe -LAla Bt 3.5a 83 155.2156.9 72.2 Fmoc -LPhe -LMet OH 3.4b Fmoc -LPhe -LMet Bt 3.5b 73 159.0161.0 53.8 Fmoc -LMet -LAla OH 3.4c Fmoc -LMet -LAla Bt 3.5c 70 147.2149.0 50.1 Fmoc -LMet -LPhe O H 3.4d Fmoc -LMet -LPhe Bt 3.5d 87 197.5199.0 52.5 Fmoc -LTrp -LAla OH 3.4e Fmoc -LTrp -LAla Bt 3.5e 69 153.8155.0 74.7 Fmoc -LLeu -LAla OH 3.4f Fmoc -LLeu -LAla Bt 3.5f 81 153.0154.7 60.9 a Isolated yield, b c 1.5 in DMF 3.2.5 General Peptide Syn theses by Segment Condensation I used ( NFmoc -protected -dipeptidoyl)benzotriazoles 3.5a -f and ( N-Fmoc -protected aminoacyl)benzotriazoles 3.2c -e for the synthesis of peptides 3.6 3.10 (Table 3.4) by solid phase segment condensation under mild microwave irradiation.

PAGE 50

50 Following standard Fmoc solid phase peptide synth esis (SPPS) strategy (Scheme 3 6 ), approximately 120 mg of N -Fmoc -protected Rink -Amide MBHA Resin (200 400 mesh, 0.35 meq/g), (0.05 mmol) was placed in a reaction vessel. The resin was allowed to swell for 1 h in DCM (5 mL) followed by the removal of Fmoc group using 20 % piperidine in DMF (5 mL x 2, 5 min and 10 min). The resinNH2 was coupled to 5.0 equiv of the benzotriazole derivative (3.2c -e or 3.5a -f) in 1.5 mL of solvent (DMF for 3.2c -e and DMSO for 3.5a -f) under microwave irradia tion (30 C 10 min, 30 W). When a negative Kaiser (ninhydrin) test verified completion of coupling (10 min), the solid resin was washed with DMF (5 mL x 3) and DCM (5 mL x 3). The Fmoc group was removed and another coupling was started. After the desired number of coupling steps, the peptide was deprotected and the desired peptide was cleaved from the resin u sing cleavage cocktails: i) TFA : anisole : thioanisole : BA L (90 : 2 : 3 : 5) (for peptide sequences i ncluding Trp or Met) or ii) TFA : water : TIPS (95 : 2.5 : 2.5) (for the other peptide sequences) at 20 C for 2 h. The resin was filtered off, and the crude mixture was concentrated; diethyl ether was added at 20 C to afford the crude peptide as a precipitate. Precipitates were filtered off and drie d to yield peptides 3. 6 3.1 0 in crude yields of 5090 %. The crude pe ptides were purified by reverse -phase HPLC to give the pure peptides 3. 6 3.1 0 isolated in yields of 20 to 68 % (Table 3 4). Eac h peptide was characterized by HRMS. HPLC analysis of crude tripeptide 3.6 and heptapeptide 3.10 revealed peaks for the desired products with no racemized by-product (Table 4 3). For the peptides 3.7 3.9 formation of one by -product in yields of 19 to 33 % derived from epimerization was detected. The HPLC profiles o f 3.6 3 10 are given in the experimental section.

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51 Scheme 3 6 General procedure of peptide synthesis by segment condensation Table 3 4. Synthesized peptides 3.6 3.10 by solid phase segment condensation Reactants coupled to Rink resin NH2 Product Stru cture (N to C terminus) Pure yielda/ Purityb (%) tR (min) HRMS [M+H]+ e 3. 2d + 3.5f 3.6 H -LLeu -LAla -LLeu NH2 68/99 8.49 315.2401 3.5 c + 3.5e 3.7 H -LTrp -LAla -LMet -LAla NH2 26c/99 9.38 477.2291 3.5f + 3.5b + 3.2c 3.8 H -LTrp -LPhe -LMet -LLeu -LAla NH2 21c/99 15.45 666.3432 3.5b + 3.5d + 3.5f 3.9 H L Leu L Ala L Met L Phe L Phe L Met NH 2 20d/72 16.43 758.3707 3.2e + 3.5a + 3.5c + 3.5f 3.10 H -LLeu -LAla -LMet -LAla -LPhe -LAla Gly NH2 30/99 11.29 679.3589 a Isolated yield after HPLC purification.; b Purity after HP LC purification.; c Isolated yield of the major peptide peak; dIsolated yield of the major peak and an impurity not derived from racemization (refer Figure 5. 4). ; eFor the calculated values, see the experimental section. During segment condensations, epim erization frequently occurs at the C -terminus residue because of the activation of the carboxylic acid function (Scheme 3 2a) [06TL7905] Gly and Pro are widely used as C -terminus residues to prevent epimerization. [05FSPPS215] P eptide 3.6 (H -LLeu -L-Ala -LLeu NH2) was synthesized using 3 .2d and 3 .5f which contains Ala at C terminus in the fragment .With DMF as solvent, at 70 C (60 W), for 3 min under m icrowave irradiation followed by reported procedure [07CBDD465] 50% of epimerization was observed. DMF i s generally avoided as a solvent due to higher possibility of epimerization during segment

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52 condensation. T he reactions were attempted in DMSO at 50 oC, and epimerization was again obser ved. The epimerization is more likely at higher temperature due to equi librium mechanism (Scheme 3 2a). C oupling in DMSO at 30 C (30 W) for 10 min provided 3.6 (13 min total coupling time) and also 3.10 (33 min total coupling time) without epimerization in isolated yields of 68 and 30 % respective ly after HPLC purification. Thus, from the sequences from the peptides synthesized, an L-Ala residue at the C terminus ( 3.5a, 3.5c 3.5f ) prevents epimerization by benzotriazole methodology. Although the epimerization site of peptide 3.73.9 is still unknown, partial epimerization wa s probably caused at 3.5e 3.5b or 3.5d for 3.7 3.9 under the same coupling conditions. After HPLC purification yields of isolated optically pure 3.7 3.9 were still 20 to 26 % 3.3 Conclusion In conclusion I have prepared tri tetra penta hexa and heptapeptides in average isolated yields of 33 % by solid phase segment condensation under microwave irradiation. Benzotr i azole intermediates, ( NFmoc protected dipeptidoyl)benzotriazoles 3.5a -f, are air and moisture insensitive acylation reagents enabling solid phase segment condensation without the use of other coupling reagents or additives. After modification of couplind conditions, epimerizat ion was not observed in some sequences (3.6 and 3.10), but partial epimerization was observed in other sequences (3.7 3.9 ). T e mperature during the c oupling is the crutial factor in prevent ing epimerization at the C terminus. Thus, low t emperature microwave synthesis may be able to solve epimerization problems and offer a short coupling time. 3.4 Experimental Section 3.4.1 General Procedure for the Preparation of 3.1a -g SOCl2 (1.0 mmol) was added to a solution of BtH (4.0 mmol) in DCM (16 mL), and the react ion mixture was stirred for 30 min. The appropriate Fmoc -protected amino acid 3.1a -g was

PAGE 53

53 added in one portion, and the reaction mixture was stirred at room temperature for 2 h. The solute was then washed with 5 % Na2CO3 aq, extracted by DCM, and dried over MgSO4. Evaporation of the solvent followed by recrystallization from DCM/hexanes to gave ( N-Fmoc protected aminoacyl)benzotriazoles 3.1a -g (Scheme 3 3 ). (S )-(9 H -Fluoren -9 -yl)methyl -1 -(1 H -benzo[d][1,2,3]triazol -1 -yl) -4 -methyl -1 oxopentan -2 ylcarbamate (Fmoc -L-Leu -Bt) 3.2d : White microcrystals (88 %); mp 121.0 1 22.8 C D 24 = +88.6 (c 1.5, D MF); 1H NMR (CDCl3) 8.27 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 7.3 Hz, 2H), 7.69 (t, J = 7.1 Hz, 1H), 7.61 J = 7.0 Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 5.85 (t, J = 7.8 Hz, 1H), 5.50 (br s, 1H), 4.45 (d, J = 7.0 Hz, 2H), 4.25 (t, J = 6.6 Hz, 1H), 1.88 (br s, 2H), 1.77 (t, J = 10.2 Hz, 1H), 1.61 (s, 2H), 1.11 (d, J = 4.9 Hz, 3H), 0.99 (d, J = 5.4 Hz, 3H); 13C NMR (CDCl3) 172.4, 156.1, 146.0, 143.8, 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 -Fluoren -9 -yl)methyl -2 -(1 H -benzo[d][1,2,3]triazole -1 -carbonyl)pyrrolidine -1 carboxylate (Fmoc -L-Pro -Bt, mixt ure of ro tomers) 3.2g: White microcrystals (88 %); mp 163.0 C ; [ ]D 24 = 100.9 (c 1.5, DMF); 1H NMR (CDCl3) 8.29 (d, J = 8.2Hz, 1H), 8.20 (d, J = 8.2Hz, 1H), 8.14 (d, J = 8.1Hz, 2H), 7.78 (d. J = 7.5Hz, 2H), 7.737.49 (m, 8H), 7.447.30 (m, 10H), 7.21 (t, J = 6.0Hz, 3H), 7.09 (t, J = 6.7Hz, 2H), 6.89 J = 4.1Hz, 1H), 5.43 (dd, J = 3.4Hz, 1H), 4.61 4.28 (m, 2H), 4.02 (t, J = 4.9Hz, 1H), 3.89 2.53 H), 2.28 2.20 (m, 1H), 2.18 2.02 (m, 4H), 1.99 13C NMR (CDCl3) 171.0, 170.6, 154.9, 154.1, 146.0, 144.0, 143.8, 143.5, 141.3, 141.0, 140.8,

PAGE 54

54 131.2, 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, 47.0, 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. 3.4.2 General Proce dure for the Prepara tion of 3.4a -f, (3.4a+ 3 .4a), (3.4b+3.4b) (3.4c+3.4c ), (3.4e+3.4e ), and (3.4f+3.4f ) (N -Fmoc aminoacyl)benzotriazoles 3.2a-d (0.5 mmol) were added at room temperatu re to a solution of unprotectedamino acid 3.3a -c ( 3.3a+3.3a ), or ( 3.3b+3.3b ) ( 0.5 mmol) in aqueous MeCN (MeCN : H2O = 7 mL : 3 mL) in the presence of Et3N (0.5 mmol). The r eaction mixture was then stirred at 20 C until 3.2 was consumed. Then 6M HCl aq (1 mL) was added, and the solution was concentrated under reduced pressure. The residue was extracted with EtOAc, washed with 6 M HCl aq, brine, and the organic layer was drie d over MgSO4. Evaporation of the solvent followed by recrystallization from DCM/hexanes gave the desired dipeptide s 3.4a -f, ( 3.4a+3.4a ), ( 3.4b+4.4b ), (3.4c+ 3 .4c ), (3.4e+3.4e ), (3.4f+3.4f ) (Scheme 3 4 ). (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonyla mino) -3 phenylpropanamido) propanoic acid (Fmoc -L-Phe -L-Ala -OH) 3.4a : White microcrystals (85 %); mp 208.7 C D 24 = 16.9 (c 1.5, DMF); 1H NMR (DMSO d6) 12.60 (s, 1H) 8.38 (d, J = 7.1Hz, 1H), 7.87 (d, J = 7.4Hz, 2H), 7.70 .14 (m, 10H), 4.46 (m, 3H), 3.10 J = 7.1Hz, 3H); 13C NMR (DMSO d6) 174.1, 171.5, 155.8, 143.8, 140.6, 138.2, 129.3, 128.0, 127.6, 127.1, 126.2, 125.4, 120.1, 65.6, 55.9, 47.5, 46.6, 37.4,17.2. Anal. Calcd for C27H26N2O5: C, 70.73; H, 5.72; N, 6.11; found: C, 70.45; H, 5.82; N, 5.93.

PAGE 55

55 2 -((S )-2 -(((9 H -Fluoren -9 -yl)methoxy)carbonylamino) -3 -phenylpropanamido) propanoic acid (Fmoc -L-Phe -DL-Ala -OH) (3.4a+3.4a): White microcrystals (82 %); mp 149.9 151.0 C ; [ ]D 24 = 21.8 (c 1.5, DMF); 1H NMR (DMSO d6) 12.64 (br s, 1H), 8.43 8.35 (m, 1H), 7.91 (d, J = 7.4Hz, 2H), 7.687.65 (m, 2H), 7.50 2H), 4.22J = 7.1 H z, 1.5 H), 1.26 (d, J = 7.1 Hz, 1.5 H); 13C NMR (DMSO -d6) 175.0, 174.9, 172.4, 172.1, 156.7, 156.6, 144.7, 144.7, 141.6, 139.2, 139.0, 130.2, 130.2, 129.0, 128.6,128.0, 127.2, 126.3, 121.0, 66.6, 56.8, 47.5, 38.9, 38.4, 18.4, 18.1. Anal. Calcd. for C27H2 6N2O5: C, 70.73; H, 5.72; N, 6.11; found: C, 70.39; H, 5.81; N, 5.95. (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonylamino) -3 phenylpropanamido) -4 (methylthio)butanoic acid (Fmoc -L-Phe -L-Met -OH) 3.4b : White microcrystals (82 %); mp 186.0 C ; [ ]D 24 = 27.4 (c 1.5, DMF); 1H NMR (DMSO d6) 12.78 (br s, 1H), 8.34 (d, J = 7.7Hz, 1H), 7.87 (d, J = 7.4Hz, 2H), 7.70 6H), 7.22 (m 1H), 2.59 2.56 (m, 2H), 2.03 (s, 3H), 2.00 13C NMR (DMSO d6) 173.2, 171.9, 156.8, 143.8, 140.7, 138.2, 129.3, 128.1, 127.6, 127.1, 126.3, 125.4, 120.1, 66.6, 56.0, 51.0, 46.6, 37.3, 30.8, 29.6, 14.6. Anal. Calcd for C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40; found: C, 67.07; H, 5.95; N, 5.31. 2 -{[(2 S )-2 -{[(9 H -Fluoren -9 ylmethoxy)carbonyl]amino} -3 -(1 H -indol -3 yl)propanoyl] amino}propanoic acid (Fmoc -L-Phe -DL-Met -OH) (3.4b+3.4b) : White microcrystals (74 %); mp 156.9 C ; [ ]D 24 = 21.6 (c 1.5, DMF); 1H NMR (DMSO d6) 12.77 (br s, 1H), 8.41 (d, J = 8.0Hz, 0.5H), 8.36 (d, J = 8.0Hz, 0.5H), 7.91 (d, J = 7.4Hz, 2H), 7.77 7.53 ),

PAGE 56

56 3.10 2.04 (s, 1.5H), 2.02 13C NMR (DMSO d6) 173.2, 171.8, 171.5, 155.8, 155.7, 143.8, 143.7, 140.7, 140.9, 138.2, 137.9, 129.3, 128.1, 127.6, 127.1, 126.3, 125.4, 125.3, 120.1, 65.7, 65.6, 56.0, 51.0, 50.9, 46.6, 38.1, 37.3, 30.8, 30.8, 29.6, 29.5, 14.6, 14.5. Anal. Calcd for C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40; found: C, 67.22; H, 6.04; N, 5.26. (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonylamino )-4 -(methylthio)butanamido) propanoic acid (Fmoc -L-Met -L-Ala -OH) 3.4c : White microcrystals (77 %); mp 169.9 C (Lit. 155 C ); [ ]D 24 = 9.0 (c 1.5, DMF); 1H NMR (DMSO d6) 12.58 (br s, 1H), 8.29J = 7.4Hz, 2H), 7.68 (m, 2H), 7.38 34 2H), 1.28 (d, J = 7.1Hz, 3H); 13C NMR (DMSO d6) 170.1, 171.3, 155.9, 143.9, 143.8, 140.7, 127.7, 127.1, 125.4, 120.1, 65.6, 53.5, 47.5, 46.6, 31.8, 29.5, 17.0, 14.6. Anal. Calcd for C23H26N2O5S: C, 62.42; H, 5.92; N, 6.33; found: C, 62.12; H, 6.02.; N, 6.27. 2 -((S )-2 -(((9 H -F luoren -9 -yl)methoxy)carbonylamino) -4 -(methylthio)butanamido) propanoic acid (Fmoc -L-Met -DL-Ala -OH) (3.4c+3.4c) : White microcrystals (93 %); mp 115.0 1H NMR (D MSO d6) 12.64 (br s, 1H), 8.24 (d, J = 7.4Hz, 0.5H), 8.18 (d, J = 7.4Hz, 0.5H), 7.91 (d, J = 7.4Hz, 2H), 7.77. 7.68 (m, 2H), 7.55 (t, J = 8.2Hz, 1H), 7.42 (t, J = 7.4Hz, 2H), 7.32 (t, J = 7.1Hz, 2H), 4.33 4.05 (m, 3H), 2.602.37 (m, 2H), 2.111.98 (m, 3H), 1.951.77 (m, 2H), 1.33 1.20 (m, 3H); 13C NMR (DMSO d6) 174.0, 173.9, 171.1, 155.9, 143.9, 143.7, 140.7, 127.6, 127.1, 125.3, 120.1, 65.6, 53.7, 53.5, 48.0, 46.6, 31.8, 29.5, 17.4, 17.0, 14.6,. Anal. Calcd for C23H26N2O5S: C, 62.42; H, 5.92; N, 6.33; found: C, 62.38; H, 5.95.; N, 6.19. (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonylamino) -4 -(methylthio)butanamido) 3 -phenylpropanoic acid (Fmoc -L-Met -L-Phe -OH) 3.4d : White microcrystals (86 %); mp

PAGE 57

57 186.0 C ; [ ]D 24 = 6.3 (c 1.5, DMF); 1H NMR (D MSO d6) 12.77 (br s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 7.7 Hz, 2H), 7.78 J = 8.1 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.26 4.15 0 J = 7.4 Hz, 2H), 2.02 (s, 3H), 1.9013C NMR (DMSO d6) 172.8, 171.4, 155.8, 143.9, 143.7, 140.7, 137.4, 129.1, 128.1, 127.6, 127.1, 126.4, 125.3, 120.1, 65.6, 53.7, 53.3, 46.6, 36.5, 31.8, 29.5, 14.6. Anal. Calcd for C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40; found: C: 67.20; H, 5.96; N, 5.38. (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonylamino) -3 -(1 H -indol -3 yl)propanamido)propanoic acid (Fmoc -L-Trp -L-Ala -OH) 3.4e : Yellow microcrystals (83 %); m p 119.0 121.0 C (Lit. 155.0 C ); [ ]D 24 = 15.8 (c 1.5, DMF); 1H NMR (DMSO d6) 12.58 (br s, 1H), 10.86 (s, 1H), 8.42 (d, J = 6.9Hz, 1H), 7.90 (d, J = 6.9Hz, 2H), 7.74 (d, J = 7.7Hz, 1H), 7.66 (t, J = 7.7Hz, 2H), 7.55 (d, J = 8.5Hz, 1H), 7.42 (d, J = 6.3Hz, 2H), 7.39 7.30J = 7.7Hz, 1H), 7.03 3.19J = 7.4Hz, 3H); 13C NMR (DMSO d6) 175.1, 172.9, 156.8, 144.7, 141.6, 128.6, 128.0, 126.3, 124.9, 121.8, 121.0, 119.6, 119.1, 112.2, 66.6, 56.1, 48.5, 47.5, 28.8, 18.1. Anal. Calcd for C29H27N3O5: C, 70.01; H, 5.47; N, 8.45; found: C, 69.68; H, 5.59; N, 8.14. 2 -{[(2 S )-2 -{[(9 H -Fluoren -9 ylmethoxy)carbonyl]amino} -3 -(1 H -indol -3 yl)pr opanoyl] amino}propanoic acid (Fmoc -L-Trp -DL-Ala -OH) (3.4e+3.4e) : White microcrystals (78 %); mp 146.3 147.1 C; [ ]D 24 = 20.4 (c 1.5, DMF); 1H NMR (DMSO d6) 12.63 (br s, 1H), 10.86 (s, 1H), 8.42 (d, J = 7.1Hz, 0.5H), 8.35 (d, J = 7.7Hz, 0.5H), 7.90 (d, J = 7.1Hz, 2H), 7.79 7.60 (m, 2H), 7.607.51 (m, 1H), 7.51 7.14 (m, 5H), 7.14 6.94 (m, 2H), 4.424. 07 (m, 4H), 3.203.05 (m, 2H), 3.052.82 (m, 2H), 1.35 (d, J = 7.1Hz, 1.5H), 1.24 (d, J = 7.1Hz, 1.5H) ; 13C NMR

PAGE 58

58 (DMSO d6) 174.1, 174.0, 171.9, 171.6, 155.8, 155.7, 143.8, 140.7, 136.1, 127.6, 127.3, 127.1, 125.4, 124.0, 120.8, 120.1, 118.7, 118.2, 111.3, 110.3, 110.2, 100.0, 65.7, 55.4, 55.2, 47.6, 46.6, 28.2, 27.8, 17.4, 17.2. Anal. Calcd for C29H27N3O5: C, 70.01; H, 5.47; N, 8.45; found: C, 69.72; H, 5.44; N, 8.29. (S )-2 -((S ) -2 -(((9 H -Fluoren -9 yl)methoxy)carbonylamino) -4 methylpentanamido) propanoic aci d (Fmoc -L-Leu -L-Ala -OH) 3.4f : White microcrystals (84 %); mp 179.0 C ; [ ]D 24 = 10.9 (c 1.5, DMF); 1H NMR (DMSO d6) 12.49 (s, 1H), 8.17 (d, J = 7.1 Hz, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.77 J = 8.8 Hz, 1H), 7.42 (t, J = 7.1 Hz, 2H), 7.37 .32 2H), 1.27 (d, J = 7.4 Hz, 3H), 0.98 13C NMR (DMSO d6) 174.0, 172.2, 155.9, 144.0, 140.7, 127.6, 127.0, 125.3, 120.1, 65.5, 52.7, 47.4, 46.7, 24.1, 23.2, 21.4, 17.1. Anal. Calcd for C24H28N2O5: C, 67.91; H, 6.65; N, 6.60; found: C, 68.06; H, 6.77; N, 6.51. 2 -((S )-2 -(((9 H -F luoren -9 -yl)methoxy)carbonylamino) -4 -methylpentanamido) propanoic acid (Fmoc -L-Leu -DL-Ala -OH) (3.4f+3.4f): White microcrystals (89 %); mp 163.0 164.0 C; [ ]D 24 = 13.2 (c 1.5, DMF); 1H NMR (DMSO d6) 12.56 (br s, 1H), 8.238.19 (m, 1H), 7.92 (d, J =7.4Hz, 2H), 7.827.70 (m, 1H), 7.57 7.40 (m, 3H), 7.40 7.29 (m, 2H), 4.484.07 (m, 5H), 1.77 1.59 (m, 1H), 1.59 1.38 (m, 2H), 1.321.28 (m, 3H), 0.940.89 (m 6H); 13C NMR (DMSO d6) 174.1, 174.0, 172.2, 172.2, 155.9, 155.9, 144.0, 143.8, 140.7, 127.7, 127.1, 125.4, 120.1, 65.6, 52.9, 52.7, 47.5, 46.7,24.2, 24.1, 23.2, 24.4, 17.4, 17.1. Anal. Calcd for C24H28N2O5: C, 67.91; H, 6.65; N, 6.60; found: C, 67.78; H, 6.80; N, 6.52. 3.4.3 General Procedure for the Preparation of 3.5a -f SOCl2 (1.0 mmol) was added to a solution of BtH (4.0 mmol) in DCM (16 mL), and the reaction mixture was stirred for 30 min, then cooled to 15 C The appropriate dipeptide 3.4a -f was added in one portion, and the mixture was stirred at 15 C for 3 h. The reaction mixture

PAGE 59

59 was diluted with DCM, washed with 5 % Na2CO3 aq, and dried with MgSO4. Evaporation of the solvent followed by recrystallization from DCM/hexanes afforded the desired benzotriazole derivatives 3.5a -f (Scheme 3 5 ). (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -1 oxopropan -2 ylamino) -1 -oxo -3 -phenylpropan -2 -ylcarbamate (Fmoc -L-Phe -L-Ala -Bt) 3.5a : White microcrystals (83 %); mp 155.2 C D 24 = 72.2 (c 1.5, DMF); 1H NMR (CDCl3) 8.23 (d, J = 7.4 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 7.0 Hz, 2H), 7.69 (t, J = 7.1 Hz, 1H), 7.60J = 7.4 Hz, 3H), 7.24 (m, 2H), 6.64 (br s, 1H ), 5.88 (quin, J = 7.1 Hz, 1H), 5.43 (m 1H), 4.58 J = 7.0 Hz, 1H), 3.20 J = 7.0Hz, 3H); 13C NMR (CDCl3) 171.1, 170.5, 156.0, 146.0, 143.6, 141.3, 136.1, 131.1, 130.8, 129.3, 128.7, 127.7, 127.1, 126.6, 125.0, 120.4, 120.0, 114.3, 67.1, 56.0, 49.1, 47.0, 38.5, 18.7. Anal. Calcd for C33H29N5O4: C, 70.83; H, 5.22; N, 12.51; found: C, 70.77; H, 5.23; N, 12.44. (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -4 -(methylthio) 1 -oxobutan -2 ylamino) -1 -oxo -3 -phenylpropan -2 -ylcarbamate (Fmoc -L-Phe -L-Met -Bt) 3.5b : White microsrystals (73 %); mp 159.0 C ; [ ]D 24 = 53.8 (c 1.5, DMF); 1H NMR (DMSO d6) 8.96 (d, J = 6.5 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 7.7 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.76 4 (m, 9H), 5.88 1H), 4.48 (m, 2H), 2.05 (s, 3H); 13C NMR (DMSO d6) 172.4, 171.1, 155.9, 145.4, 143.8, 140.7, 138.0, 131.1, 130.7, 129.2, 128.1, 127.6, 127.1, 126.8, 126.3, 125.3, 120.2, 120.1, 114.1, 65.7, 55.7, 52.0, 46.5, 37.3, 30.0, 29.5, 14.4. Anal. Calcd for C35H33N5O4S: C, 67.83; H, 5.37; N, 11.30; found: C, 67.99; H, 5.44; N, 10.98.

PAGE 60

60 (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -1 oxoprop an -2 ylamino) -4 -(methylthio) -1 -oxobutan -2 -ylcarbamate (Fmoc -L-Met -L-Ala -Bt) 3.5c : White microcrystals (70 %); mp 147.2 C ; [ ]D 24 = 50.1 (c 1.5, DMF); 1H NMR (CDCl3) 8.25 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.3 Hz, 2H), 7.68 (t, J = 7.3 Hz, 1H), 7.60 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.00 (d, J = 6.6 Hz, 1H), 5.95 (t, J = 7.1 Hz, 1H), 5.59 (d, J = 7.7 Hz, 1H), 4.58J = 6.9 Hz, 2H), 4.23 (t, J = 6.7 Hz, 1H), 2.67 (t, J = 6.5 Hz, 2H), 2.15 (s, 3H), 2.13 1.71 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3) 171.5, 171.0, 156.1, 146.0, 143.7, 143.6, 141.3, 131.1, 130.8, 127.7, 127.1, 126.6, 125.0, 120.4, 120.0, 114.4, 67.2, 53.3, 49.2, 47.1, 31.5, 29.9, 18.5, 15.1. Anal. Calcd for C29H29N5O4S: C, 64.07; H, 5.38; N, 12.88; found: C, 64.16; H, 5.43; N, 12.69. (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -1 oxo -3 phenylpropan-2 ylamino) -4 -(methylthio) -1 -oxobutan -2 -ylcarbamate (Fmoc -L-Met -L-Phe Bt) 3.5d : White microcrystals (87 %); mp 197.5 C ; [ ]D 24 = 52.5 (c 1.5, DMF); 1H NMR (CDCl3) 8 .23 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.4 Hz, 3H), 7.41 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.20 (t, J = 7.3 Hz, 3H ), 7.16 J = 8.2 Hz, 1H), 4.50 J = 6.7 Hz, 1H), 3.54 2.6313C NMR (CDCl3) 171.0, 170.2, 155.9, 146.0, 143.7, 143.7, 141.3, 134.8, 131.0, 130.9, 129.2, 128.8, 127.7, 127.5, 127.1, 126.6, 125.0, 120.4, 120.0, 114.3, 100.2, 67.1, 54.2, 53.2, 47.0, 38.3, 31.2, 29.9, 15.0. Anal. Calcd for C33H35N5O4S: C, 67.83; H, 5.37; N, 11.30; found: C, 68.21; H, 5.69; N, 10.96.

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61 (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -1 oxopropan -2 ylamino) -3 -(1 H -indol -3 -yl) -1 -oxopropan -2 ylcarbamate (Fmoc -L-Trp -L-Ala -Bt) 3.5e : Yel low microcrystals (69 %); mp 153.8 155.0 C ; [ ]D 24 = 74.7 (c 1.5, DMF); 1H NMR (DMSO d6) 10.90 (s, 1H), 9.01 (d, J = 5.5Hz, 1H), 8.33 7.71J = 6.0Hz, 1H), 4.50 (m, 1H), 4.14 (s, 2H), 4. 10 J = 7.1Hz, 3H); 13C NMR (DMSO -d6) 172.6, 171.9, 155.9, 145.4, 143.7, 140.7, 136.1, 131.1, 130.7, 127.6, 127.2, 127.1, 125.4, 125.3, 124.1, 120.8, 120.2, 120.1, 118.7, 118.2, 114.0, 111.3, 110.1, 65.7, 55.0, 48.7, 46.6, 27.8, 16.7. HRMS Calcd for [C35H30N6O4+H]+, 621.2221; found, 621.2232. (9 H -Fluoren -9 -yl)methyl( S ) -1 -((S )-1 -(1 H -benzo[d][1,2,3]triazol -1 yl) -1 oxopropan -2 ylamino) -4 methyl -1 -oxopentan -2 -ylcarbamate (Fmoc -L-Leu -L-Ala -Bt) 3.5f: White microcrystals (81 %); mp 153.0 154.2 C ; [ ]D 24 = 60.9 (c 1.5, DMF); 1H NMR (CDCl3) 8.82 (d, J = 5.5 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.80 (t, J = 8.0 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.64 (quin, J = 7.7 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.41 (t, J = 7.1 Hz, 2H), 7.31 (t, J = 7.1 Hz, 2H), 5.58 (t, J = 6.3 Hz, 1H), 4.32 (m, 4H), 1.75 1.62 (m, 1H), 1.56 (d, J = 6.9 Hz, 3H), 1.53 J = 6.9 Hz, 6H); 13C NMR (CDCl3) 172.9, 171.9, 156.0, 145.3, 143.9, 143.8, 140.7, 131.1, 130.6, 127.6, 127.0, 126.7, 125.4, 120.2, 120.1, 65.5, 52.3, 48.6, 48.6, 46.7, 24.1, 23.1, 21.4, 16.5. Anal. Calcd for C30H31N5O4: C, 68.55; H, 5.94; N, 13.32; found: C, 68.59; H, 5.94; N, 13.16

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62 3.4.5 H PLC Results of P eptide 3.6 -3.10 Figure 3 4 HPLC Profile of peptide 3.6 B ottom; the profile of crude peptide 3.6 (H -LLeu -LAla -LLeu NH2). Top; The profile of peptide 3.6 after purification. HRMS Calcd for [C15H30N4O3+H]+, 315.2391; found, 315.2401. Figure 3 5 HPLC Profile of peptide 3.7 Bott om; the profile of crude peptide 3.7 (H -LTrp -LAla -L-Met -L-Ala NH2). T wo diastereoisomers were obtained in ratio 2.3 : 1 (9.5 min : 10.2 min). Top; The profile of peptide 3.7 after purification. HRMS Calcd for [C22H32N6O4S+H]+, 477.2279; found, 477.2291.

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63 Figure 3 6 HPLC Profile of peptide 3.8 Bottom; the profile of crude peptide 3.8 (H -LTrp -LPhe -LMet -L -Leu -L-Ala NH2). Two diastereoisomers were obtained in ratio 4 : 1 (15.5 min : 16.1 min). Top; The profile of peptide 3.8 after purification. HRMS Ca lcd for [C34H47N7O5S+H]+, 666.3432; found, 666.3445. Figure 3 7 HPLC Profile of peptide 3.9 Bottom; the profile of crude peptide 3.9 (H -LLeu -LAla -L-Met -LPhe -L-Phe -L-Met NH2). Two diastereoisomers were obtained in ratio 2.5 : 1 (16.4 min : 16.9 min) Top; The profile of peptide 3.9 after purification. HRMS Calcd for [C37H55N7O6S2+H]+, 758.3728; found, 758.3707.

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64 Table 3 5 MS/MS Sequence of peptide 3.9 L A M F F M(NH 2 ) MW = 757.4 [M+H] + = 758.4 a ions NH 3 69.10 140.1 271.1 418.2 565.3 696.3 a i ons (loss of CO) 86.1 157.1 288.2 435.2 582.3 713.3 C term b ion N term 114.1 185.1 316.2 463.2 610.3 741.3 753.4 Residue H L A M F F M NH 2 Residue mass 1.0 113.1 71.0 131.0 147.1 147.1 131.0 16.0 y ions 758.4 645.3 574.3 443.2 296.2 149.1 Loss of NH 3 741.4 628.3 557.3 426.2 279.2 132.1 Highlighted numbers were determined during analysis Figure 3 8 HPLC Profile of peptide 3.10. Bottom; the profile of crude peptide 3.10 (H -LLeu -LAla -L-Met -L-Ala -L -Phe -L-Ala Gly NH2). Top; The profile of pe ptide 3.10 after purification. HRMS Calcd for [C31H50N8O7S+H]+, 679.3596; found, 679.3589.

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65 CHAPTER 4 COUMARIN LABELING OF PEPTIDES ON SOLID PH ASE1 4.1 Introduction Proteins modified with fluorescent dyes, enzymes, and other reporter groups are valuable to ols with widespread uses in immunology and biochemical research. Protein -capture microarrays have become a promising tool for protein analysis in drug discovery, diagnostics, and biological research. [04DDT24] Fluorescent derivatives of biologically active peptides are useful experimental tools for studying biological structure and function and for visualization of intracellular processes or molecular interactions. [95B3972, 97JPR444] Matrix metalloprotease (MMP) proteins are implicated in many diseases, in cluding arthritis, periodontal disease, tumor cell invasion, and metastasis. [93CROBM197] The detection of a protein bound by a specific capture agent is key to microarray based methods, for traditional immunoassays and biosensor applications. [02PS2655, 03CB53, 05CBC1043] Synthetic peptide based assays can differentiate enzyme types and monitor their activity. [89JBC4227, 91AB137, 93BJ601] Fluorogenic substrates can be monitored continuously and utilized at low concentrations, thus providing a particularly convenient enzyme assay method. [07AHC131] Fluorescent biosensors are composed of a binding molecule, such as an antibody or an enzyme, derivatized with a single fluorescent probe, which is sensitive to changes in the local environment. [02JMB429, 02JOC3120, 02PS2655] Fluorogenic groups can often be attached at the cleavage sites of proteases and esterases. However, this simple approach is not applicable if the enzyme requires binding interactions on both sides of the cleavage site. For such cases, quenche d fluorescent peptides are designed as short sequences of amino acids, containing 1Reproduced in part with permission from Organic & Biomolecular Chemistry 2008, 6, 4582. Copyright 2009 The Royal Society of Chemistry

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66 enzyme -cleavable specific sites, with fluorescent donor and acceptor probes linked at the N and C-termini. [93BC537, 93BJ601, 94JBC20952] In other biosensors [00BC71, 02JMR311] the binding molecule is labeled with two fluorophores, suitable for F ster (fluorescence) resonance energy transfer (FRET). The acceptor -quencher pair enables nonradiative energy transfer between an excited donor flu orophore to a proximal acceptor fluo rophore. [05ACIE2642] Usually, the donor fluorescence is quenched by the acceptor without subsequent fluorescence emission. Donor and acceptor groups, attached to a synthetic peptide undergo FRET, producing a unique fluorescence spectrum. Enhanced donor fl uorescence indicates proteolysis accompanied by the loss of FRET as a result of separation of the donor and acceptor groups (Scheme 4 1). Scheme 4 1. Mechanism of FRET Many donor and acceptor groups have been incorporated into quenched fluorogenic subst rates. [94JBC20952, 97FEBS379, 03AB141] Coumarins have extensive and diverse applications as fluorescent probes or labels; [97CR1515, 04CR3059] since they exhibit an extended spectral range, are photostable and have high emission quantum yields. 7 -Methoxycoumarin 4 ylacetyl (Mca, 325 = 14500 M1cm1 and f = 0.49) functional group was proposed as a fluorophore for thimet peptidase pitrilysin and MMP substrates (Figure 4 1) [92FEBS263,

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67 93BJ601, 94JBC20952] The combination of Mca as a fluorophore and a 2,4 -dinitrophenyl (Dnp) grou p as a quencher has several advantages over the more common fluorogenic substrate pair of Trp/Dnp; the Mca residue is more fluorescent and chemically more stable than Trp, and Mca is efficiently quenched by Dnp. Figure 4 1. Structure of Mca and Dnp moie ty Fluorescently -labeled peptides might be created by the reaction of the peptide in solution with an ac tivated form of the fluorophore but a potentially more effective approach is to assemble the peptide chain on solid phase and incorporate the fluorophor e into the peptide whilst attached to the solid support. [ 98BMCL597, 04TL6079] For the solid phase peptide labeling by Mca, the N termini of peptide resins are acylated with 7 -methoxycoumarin 4 ylacetic acid using standard synthetic cycles. [92FEBS263, 93B J601, 94JBC20952] However, inefficient acylation of the peptide resin led Malkar and Fields to incorporate Mca into NFmoc lysine molecules by a 4 -step method providing NFmoc -LLys(Mca) OH 4.4 (17 % overall) (Scheme 4 2). [01LPS263] Coumarin -labeled lysines are of considerable interest for the design and synthesis of fluorogenic triple -helical substrates for the analy sis of MMP family members. [01B5795, 03AB105, 05JSS1812] Thus, N-coumarin labeled -N-Fmoc lysines allow the successful labeling of peptide substrates by solid phase peptide synthesis for an e xtracellular MMP and represent a powerful tool for monitoring pr oteolysis. [01B5795, 03AB105, 05JSS1812]

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68 Scheme 4 2. Reported synthesis of N(fluoren 9 ylmethoxycarbonyl) N[(7 -methoxycoumarin4 yl)acetyl -Llysine 4.4 Our group has reported the extensive use of N acylbenzotriazoles for N acylation, [00JOC8210, 02A 39, 08JOC511] Cacylation, [00JOC3679, 03JOC4932, 03JOC5720] and O acylation [04CCA175, 07BC994] reactions. ( N-Fmoc aminoacyl)benzotriazoles and their Boc and Cbz analogs enabled the preparation of chiral di tri and tetrapeptides in average yields of 88% from natural amino acids in solution phase. [ 02A134, 04S2645, 09A47] The efficient fluorescent labeling of peptides on solid phase by acylation with benzotriazole activated derivatives of (i) coumarin 3 -ylcarboxylic acid, 7 -methoxycoumarin 4 ylacetic acid, (ii) coumarin 3 -ylcarbonyl (Cc) and Mca labeled lysines are r e ported below.

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69 4.2 Results and Discussion T he preparation of b enzotriazoleactivated fluorogenic substrates 4.5 4.6 4.7, 4.9 4.11 and their utilization as useful reagents for the efficie nt fluorescent labeling of pept ides by the solid phase method have been demonstrated 4.2.1 Preparation of N-Fmoc -N-[(7 methoxycoumarin -4 yl)acetyl] -L-lysine ( NFmoc -LLys(Mca) -OH) 4.4 and I ts Benzotriazole Derivative 4.6 7 Methoxycoumarin4 ylacetic ac id 4.2 was converted into crystalline, stable 4 (benzotriazole 1 -ylacetyl) 7 -methoxycoumarin 4.5 (78 %) by reaction with BtH and SOCl2 in DCM at 20 C (Scheme 4 3) Compound 4.5 was then coupled with NFmoc L -lysine in aqueous MeCN in the presence of Et3N for 20 min to afford NFmoc -L-Lys(Mca) OH 4.4 (overall 51 %). Compared with the recent literature procedure [01LPS263] for the preparation of N-Fmoc -LLys(Mca) OH 4.4 my two -step methodology using NFmoc -Llysine, offers simple preparative and workup procedures, short times to completion, the use of inexpensive reagents and high yields. Conventional benzotriazole activation of 4.4 gave NFmoc -LLys(Mca) -Bt 4.6 (70 % ). Scheme 4 3. Preparati on of N-Fmoc -L Lys(Mca) -Bt 4.6

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70 4.2.2 Preparation of N-Fmoc -N-(coumarin -3 ylcarbonyl) -L-lysine Benzotriazolide ( NFmoc -L-Lys(Cc)-Bt) 4.9 and N(Coumarin -3 -ylcarbonyl) -N-Fmoc -L-lysine Benzotriazolide ( N-Cc -L-Lys(Fmoc) -Bt) 4.11 3 (Benzotriazole1 ylcarbonyl)chromen2 -one 4.7 [08BC1471] was coupled with commercia lly available N-Fmoc -Llysine and N-Fmoc -Llysine in aqueous MeCN at 20 C in the presence of Et3N to provide lysine -scaffold based fluorescent building blocks 4.8 [08BC1471] and 4.10 (87 and 79 % respectively), that were converted into the corresponding N acylbenzotriazoles 4.9 and 4.11 (87 and 71 %) (Scheme 4 4). Scheme 4 4. Preparation of N-Fmoc -LLys(Cc) -Bt 4.9 and NCc -LLys(Fmoc) -Bt 4.11 4.2.3 Solid Phase Fluorescent Labeling with 4.6, 4.9, 4.11 to Synthesize Labeled Peptides 4.12-4.17 Solid ph a se peptide s ynthesis, microwave assisted as optimized previously in our group, [07CBDD465] enables efficient acylation of NH2groups on solid phase by benzotriazole activated fluorogenic substrates 4.5 4.6 4.7 4.9 4.11. Compounds 4.5 and 4.7 were used to

PAGE 71

71 couple the fluorophore directly to diverse peptides. Alternatively, 4.6 4.9, and 4.11 were used to couple the fluorophore already attached to a lysine moiety to the peptides. Coumarinlabeled peptides were synthesized as C terminal amides using Fmoc solid -phase methodology under microwave irradiation. T he utility of 4.6 for fluorescent labeling on solid phase was demonstrated for a model dipeptide H -L-Ala -L-Lys( N-Mca) NH2 4.12 (Scheme 4 5). Peptide 4.12 was synthesized using microwave assisted SPPS c onditions. [07CBDD465] After initial removal of the Fmoc protecting group, free Rink resin NH2 was coupled with 4.6 in DMF under microwave irradiation for 10 min at 70 C The second coupling was performed with the ( N-Fmoc aminoacyl)benzotriazole reagent derived from Fmoc -L-Ala. Finally the desired peptide was cleaved from the resin to produce pept ide amide H -L-Ala -L-Lys( NMca) NH2 4.12 (26 %) (Table 4 1). Conditions were optimized to maximize the rate while avoiding epimerization. Scheme 4 5. Synthesi s of coumarin -labeled dipeptide 4.12 In a similar manner, microwave assisted SPPS was effected (3 min coupling time for each step) with 4.9 to obtain the fluorescently labeled di -, tri tetra and hexapeptides: H -L-Ala -LLys( NCc) NH2 4.13, H -LPro -L-Ph e -LLys( NCc) NH2 4.14, H -LTrp -L-Lys( NCc) -L-Met -L-

PAGE 72

72 Phe NH2 4.15, H -LLys( NCc) -L -Pro Gly -LLeu -LMet -LTrp -NH2 4.16 in yields of 18 45 % (after HPLC purification) (Table 4 1). The successive coupling steps utilized the appropriate N acylbenzotriazoles derived from Fmoc -L-Met, Fmoc -LTrp, Fmoc -L-Phe, Fmoc -L-Leu, Fmoc -LPro and Fmoc Gly prepared by our published procedures. [05S397, 07CBDD465] The mild synthetic conditions allowed utilization of the unprotected indole NH of LTrp, and no complications wer e observed with L-Met or any other of the amino -N protected amino acids used. I achieved fluorescent labeling by coupling to N-coumarin attached lysine 4.11 through its free N-position to prepare labeled tripeptide H -LPhe -L -Leu -LLys( NCc) -NH2 4.17 (35 %) under microwave assisted SPPS (3 min coupling time for each step) and Fmoc strategy (Table 4 1). Table 4 1. Preparation o f fluorescent peptides 4.12 4.17 Labeled Peptide Structure (N to C terminus) Yield (%)a Purity (%)b tR (min)c HRMS [M+H]+ d 4.12 H -LAla -LLys( NMca) NH2 2 6 99 9.10 433.2103 4.13 H -LAla -LLys( NCc) NH2 45 99 9.82 389.1825 4.14 H L Pro L Phe L Lys( N Cc) NH 2 23 99 13.38 562.2680 4.15 H L Trp L Lys( N Cc) L Met L Phe NH 2 18 94 18.02 782.3328 4.16 H L Lys( N Cc) L Pro Gly L Leu L Met L Trp NH 2 20 99 17.00 902.4212 4.17 H L Phe L Leu L Lys( N Cc ) NH 2 35 99 14.28 578.2987 a Isolated yields after HPLC pu rification; b Purity after HPLC purification; c t R = retention time. For condition see the experimental section. dFor the calculated values, see the experimental section. 4.2.4 Solid Phase Fluorescent Labeling with 4.7 to Synthesize Labeled Dipeptide (Cc) -LLeu -L-Leu -NH2 4.18 and Labeling with 4.5 to Synthesize Labeled Dipeptide (Mca) -LLeu -L-Leu -NH2 4.19 I also demonstrated fluorescent labeling with benzotriazole activated coumarin 3 ylcarboxylic acid 4.7 by the preparation of Cc labeled dipeptide (Cc) -LL eu -LLeu NH2 4.18 (Scheme 4 6). After initial removal of the Fmoc protecting group from Rink amide resin, I

PAGE 73

73 utilized Fmoc -LLeu Bt for each of two successive coupling steps. After final coupling with 4.7 (10 min coupling time), the desired fluorescent labe led peptide 4.18 (29 %) (Table 4 2) was cleaved from the resin. Scheme 4 6. Preparation of peptide 4.18 Fluorescent labeling with benzotriazole activated 7 -methoxycoumarin 4 ylacetic acid 4.5 was used to prepare Mca l labeled dipeptide (Mca) -LLeu -LLeu NH2 4.19 (26 %) (Table 4 2), under similar conditions to those utilized for the preparation of 4.18. Table 4 2 Preparation of f luorescent p eptides 4.18 and 4.19 Labeled peptide Structure (N to C terminus) Yield (%)a Purity (%)b tR (min)c HRMS [M+H]+ d 4 .18 (Cc ) -LLeu -LLeu N H2 29 >99 20.67 438.2223 4.19 (Mca) -L-Leu -LLeu N H2 26 >99 17.47 460.2455 a Isolated yields after HPLC purification; b Purity after HPLC purification; c t R = retention time. For condition see the experimental section. dFor the calculat ed values, see the experimental section. 4.2.5 Fluorescence Measurement s of Peptides 4.12-4.19 Absorption ( Abs) and fluorescence ( Em) wavelength maxima were recorded for fluorescent peptides 4.12 4.19 (Table 4 3).

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74 Table 4 3 Absorption and fluorescen ce data of f luorescent la b eled p eptides 4.124.19 Entry 1 2 3 4 5 6 7 8 Peptide 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 Abs. [nm] a 323 294 295 289 290 299 295 322 Em [nm] a 383 409 407 407 405 407 413 383 a Determined in 95% methanol 0 0.2 0.4 0.6 0.8 1 1.2 220 270 320 370 420 Wavelength (nm) Absorption Intenisty 4.12 4.13 4.14 4.15 4.16 4.17 Figure 4 2. Absorption spectra of 4.12-4.17 0 0.2 0.4 0.6 0.8 1 200 250 300 350 400 450 Wavelength (nm) Apsorption Intensity 4.18 4.19 Figure 4 3. Absorption spectra of 4.18-4.19

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75 0 0.2 0.4 0.6 0.8 1 1.2 350 400 450 500 550 Wavelength (nm) Emission Intensity 4.12 4.17 4.19 Figure 4 4 Emission spectra of 4.12, 4.17, 4.19 4.3 Conclusion In conclusion a convenient and efficient preparation in solution phase of a variety of coumarin fluorescent probes is described, including Cc and Mca labeled lysines as fluorogenic substrates. Their benzotriazole derivatives are appropriate materials for peptide labeling thus enabling amino group acylation under microwave irradiation on solid phase without the use of coupling agents or additives and without side reactions or epimerization. 4.4 Experimental Section 4.4.1 Preparation of ( S )-2 -(((9 H F luoren -9 yl)methoxy)carbonylamino) 6 -(2 -(7 -methoxy -2 oxo -2 H -chromen -4 -yl)acetamido)pentanoic acid ( NFmoc -L-Lys(Mca) OH) 4.4 Compound 4.5 (1.1 mmol) was added in one portion to a solution of N-Fmoc -Llysine (1.8 mmol) in Me CN : H2O (24 mL : 5 mL) in the presence of Et3N (5 .4 mmol) (Scheme 4 3) The reaction mixture was stirred at 20 C for 15 min. A solution of 6M HCl aq (2 mL) was then added and MeCN was removed under reduced pressure. The residue was extracted with EtOAc (100 mL), and the organic extract was washed with 6 M HCl aq (50 mL x 2), brine (50 mL) and dried over MgSO4. Evaporation of the solvent gave 4.4 which was recrystallized from EtOAc/hexanes to give yellow microcrystals (65 %); mp 184.9 185.7 C ; 1H NMR (DMSO d6)

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76 8.31 8.24 (m, 1H) 7.93 (d, J = 7.4Hz, 2H), 7.76 (d, J = 7.4Hz, 2H), 7.74 7.64 (m, 2H), 7.45 (t, J = 7.2Hz, 2H), 7.36 (t, J = 7.2Hz, 2H), 7.04 6.97 (m, 2H), 6.28 (s, 1H), 4.36 4.20 (m, 3H), 3.98 3.90 (m, 1H), 3.88 (s, 3H), 3.70 (s, 2H), 3.15 3.02 (s, 2H), 1.80 1.46 (m, 2H), 1.46 1.20 (m, 4H); 13C NMR (DMSO d6) 174.0, 167.4, 162.4, 160.1, 156.2, 154.9, 151.2, 143.9, 143.8, 140.7, 127.7, 127.1, 126.5, 125.3, 120.1, 112.7, 112.6, 100.9, 65.6, 55.9, 53.8, 46.6, 30.4, 28.5, 23.1. Anal. Calcd for C33H32N2O8: C 67.80; H, 5.52; N, 4.79; found: C, 67.55; H, 5.60; N, 4.40. 4.4.2 General Procedure for the Preparation of 4.5, 4.6, 4.9, 4.11 SOCl2 (1.2 mmol) was added to a solution of BtH (4.0 mmol) in dry DCM (15 mL) at 20 C and the reaction mixture was stirred for 20 min (Scheme 4 3 and 4 4) C ompounds 4.2 4.4 4.8 4.10 (1.0 mmol) were each added separately to the above reaction mixture, and each mixture was stirred for 2 h at 20 C The white precipitate which formed in each case was filtered, the filtrate dilut ed with additional DCM (80 mL) and the solution washed with 6M HCl aq (50 mL x 3) (for 4.2 4.8 4.10), with 10 % Na2CO3 aq (50 mL x 3) (for 4.4 ), brine (50 mL), and dried over MgSO4. Removal of the solvent under reduced pressure gave 4.5 4.6 4.9 4.11 t hat were each recrystallized from DCM/hexanes. 4 -(2 -Benzotriazol -1 yl -2 -ox oethyl) -7 methoxy-chromen -2 -one (Mca -Bt) 4.5 : Y ellow microcrystals (78 %); mp 125.0 C ; 1H NMR (CDCl3) 8.24 (d, J = 8.2 Hz, 1H) 8.17 (d, J = 8.2 Hz, 1H), 7.74 2H), 3.88 (s, 3H); 13C NMR (CDCl3) 167.3, 163.0, 160.5, 155.6, 147.0, 146.4, 131.0, 130.9, 126.8, 125.5, 120.5, 114.7, 114.3, 112.8, 112.3, 101.2, 55.8, 38.4. HRMS Calcd for [C18H13N3O4+Na]+, 358.0798; found, 358.0784. (S )-(9 H -Fluoren -9 -yl)methyl1 -(1 H -benzo[d][1,2,3]triazol -1 -yl) -6 -(2 -(7 -methoxy -2 -oxo 2H -chromen -4 -yl)acetamido) -1 -oxohexan -2 -ylcarbamate ( NFmoc -L-Lys(Mca) -Bt) 4.6 :

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77 Yellow microcrystals (70 %); mp 144.0 C ; 1H NMR (DMSO d6) 8.32 7.89 (d, J = 7.4 Hz, 2H), 7.81 (t, J = 7.7 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 6.90 4.29 (m, 2H), 2.00 13C NMR (DMSO d6) 172.2, 167.5, 162.4, 160.2, 156.5, 155.0, 151.2, 145.4, 143.8, 140.8, 131.3, 130.5, 127.7, 127.1, 126.9, 126.5, 125.3, 120.3, 120.2, 114.0, 112.8, 112.6, 112.1, 100.9, 65.1, 55.9, 55.9, 54.3, 46.6, 38.6, 30.2, 28.4, 23.1. HRMS Calcd for [C39H35N5O7+Na]+, 708.2428; found, 708.2455. {(S )-1 -(Benzotriazole -1 -carbonyl) -5 -[(2 -oxo -2 H -chromene -3 -carbonyl) amino] pentyl} -carbamic acid 9 H -fluoren -9 -ylmethyl ester ( N -Fmoc -L-Lys(Cc) -Bt) 4.9 : White m icrocrystals (82 %); mp 113.0 115.0 C ; 1H NMR (DMSO d6) 8.80 (s, 1H) 8.70 (t, J = 5.5 Hz, 1H), 8.32 J = 9.6 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 6.7 Hz, 2H), 7.82 J = 7.4Hz, 1H), 7.52 7.35 2H), 2.0813C NMR (DMSO d6) 172.1, 161.1, 160.3, 156.4, 153.8, 147.3, 145.3, 143.7, 143.7, 140.7, 134.0, 131.2, 130.6, 130.2, 127.6, 127.1, 126.8, 125.3, 125.1, 120.2, 119.0, 118.5, 116.1, 114.0, 65.9, 54.3, 46.6, 30.3, 28.4, 23.1. Anal. Calcd for C37H31N5O6: C, 69.26; H, 4.87; N, 10.91; found: C, 69.01; H, 4.76; N, 11.03. (S )-6 -(((9 H -Fluoren -9 -yl)methoxy)carbonylamino) -2 -(2 -oxo -2 H -chromene-3 carboxamido)hexanoic acid ( N -(Cc)-L-Lys(Fmoc) -OH) 4.10 : Solid 4.7 (0.5 mmol) was added in one portion to a solution of N -F moc L -lysine (0.5 mmol) in Me CN : H2O (5 mL : 3 mL), in the presence of Et3N (0.5 mmol) (Scheme 4 4) The reaction mixture was stirred at 20 C for 30 min, 6M H Cl aq (2 mL) was added and the Me CN was removed under vacuum. The residue was

PAGE 78

78 dissolved in DCM (50 mL), and the organic extract was washed with 6M HCl aq (50 mL), brine (50 mL), and dried over MgSO4.Evaporation of the solvent gave 4.10 which was recrystall ized from DCM/hexanes to give white microcrystals (79 %); mp 87.9 C ; 1H NMR (DMOS d6) 13.00 (br s, 1H) 9.07 (d, J = 7.4 Hz, 1H), 8.89 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.85 (d. J = 7.1 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.65 (d, J = 7.1 Hz, 2H), 7.23J = 5.5Hz, 1H), 4.24 1.2613C NMR (DMSO d6) 172.9, 160.7, 160.6, 156.1, 154.0, 148.0, 143.9, 140.7, 134.3, 130.4, 127.6, 127.0, 125.2, 125.1, 120.1, 118.4, 118.2, 116.2, 65.2, 52.3, 46.7, 31.2, 29.0. Anal. Calcd for C31H28N2O7: C, 68.88; H, 5.22; N, 5.18; found: C, 68.59; H, 5.57; N, 4.97. (S )-(9 H -Fluoren -9 -yl)methyl -6 -(1 H -benzo[d][1,2,3]triazol -1 -yl) -6 -oxo -5 -(2 -oxo -2 H chromene -3 -carboxamido)h exylcarbamate ( N-(Cc)-L-Lys(Fmoc) -Bt) 4.11 : White microcrystals (71 %); mp 106.9 108.9 C ; 1H NMR (DMOS d6) 9.40 (d, J = 6.9 Hz, 1H) 8.92J = 7.7 Hz, 1H), 7.89 (m, 3H), 7.56 1H), 4.29 2.2513C NMR (DMSO d6) 170.9, 161.6, 160.5, 156.1, 154.0, 148.2, 145.4, 140.7, 134.5, 131.2, 130.7,130.5, 127.6, 127.0, 126.9, 125.3, 125.1, 120.3, 120.1, 118.4, 118.1, 116.3, 114.0, 65.2, 53.0, 46.7, 31.1, 31.0, 28.9, 22.4. HRMS Calcd for [C37H31N5O6+Na]+, 664.2167; found, 664.2125. 4.4.3 General Procedure of Solid Support Peptide Synthesis Labeled peptides were synthesized using Fmoc solidphase methodology as C-terminal amides utilizing Rink amide -HMBA resin (200 400 mesh, 0.35 meq/g). Standard removal of the Fmoc protecting group of rink amide resin (0.05 mmol) gave unprotected resin. Resin-NH2 was coupled with 5 equiv of NFmoc -protected (aminoacyl)benzotriazole reagent derived from

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79 Fmoc -protected amino acids, prepared following previously published procedures (Scheme 4 5 and 4 6) [09A47] When complete coupling was verified by a negative Kaizer (ninhydrin) test (10 min), t he solid resin was washed with DMF (5 mL x 3) and DCM (5 mL x 3) followed by another coupling. After the coupling step, the desired peptide was cleaved from the resin u sing cleavage cocktails: i) TFA : anisole : thioanisole : BAL (90 : 2 : 3 : 5) (for pept ide sequences i ncluding Trp or Met) or ii) TFA : water : TIPS (95 : 2.5 : 2.5) (for the other peptide sequences) at 20 C for 2 h The resin was filtered, the cocktail was concentrated under nitrogen and cold diethyl ether was added to achieve precipitated peptide ( 4.12 4.19), under conditions optimized to increase rate but avoid epimerization. 4.4.4 HPLC Profiles of Peptide 4.12-4.19 Figure 4 5 HPLC Profile of peptide 4.12. Bottom; the profile of crude peptide 4.12 (H -L-Ala -LLys( N-Mca) NH2). Top; The profile of peptide 4.12 after purification. HRMS Calcd for [C21H28N4O6+H]+, 433.2082; found, 433.2103.

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80 Figure 4 6 HPLC Profile of peptide 4.13. Bottom; the profile of crude peptide 4.13 (H -L-Ala -LLys( N -Cc) NH2). Top; the profile of peptide 4.13 a fter purification. HRMS Calcd for [C21H28N4O6+H]+, 389.1819; found, 389.1825. Figure 4 7 HPLC Profile of peptide 4.14. Bottom; the profile of crude peptide 4.14 (H -LPro -LPhe -LLys( NCc) NH2). Top; the profile of peptide 4.14 after purification. HRMS Calcd for [C30H35N5O6+H]+, 562.2660; found, 562.2680.

PAGE 81

81 Figure 4 8 HPLC Profile of peptide 4.15. Bottom; the profile of crude peptide 4.15 (H -LTrp -LLys( NCc) -L-Met -LPhe NH2). Top; The profile of peptide 4.15 after purification. HRMS Calcd for [C41H47N7O7S+H]+, 782.3300; found, 782.3328. Figure 4 9 HPLC Profile of peptide 4.16. Bottom; the profile of crude peptide 4.16 (H -LLys( NCc) -L-Pro -Gly -L-Leu -L-Met -LTrp NH2). Top; the profile of peptide 4.16 after purification. HRMS Calcd for [C45H59N9O9S+H]+, 902.4229; found, 902.4212.

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82 Table 4 4. MS/MS Sequence of peptide 4.16 K(der) P G L M W (NH 2 ) MW = 901.4 [M+H] + = 902.4 b ions H 2 O 681.3 867.4 a ions (loss of CO) 273.1 370.2 427.2 540.3 671.3 587.4 b ion N term 301.1 398.2 455.2 568.3 699.3 885.4 C term Residue H K(der) P G L M W NH 2 Residue mass 1.0 300.1 97.1 223.1 113.1 131.0 186.1 16.0 y ions 902.4 602.3 505.3 448.3 335.2 204.1 Loss of NH 3 885.4 585.3 488.3 431.3 318.2 187.1 Highlighted numbers were determined during analy sis Figure 4 10. HPLC Profile of peptide 4.17. Bottom; the profile of crude peptide 4.17 (H -L-Phe -LLeu -L-Lys( NCc) NH2). Top; the profile of peptide 4.17 after purification. HRMS Calcd for [C31H39N5O6+H]+, 578.2973; found, 578.2987. Figure 4 11. HPLC Profile of peptide 4.18. Bottom; the profile of crude peptide 4.18 ((Cc) -LLeu -LLeu NH2). Top; The profile of peptide 4.18 after purification. HRMS Calcd for [C22H29N3O5+H]+, 416.2180; found, 416.2223.

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83 Figure 4 12. HPLC Profile of peptide 4.19. Bo ttom; the profile of crude peptide 4.19 ((Mca) -LLeu -LLeu NH2). Top; The profile of peptide 4.19 after purification. HRMS Calcd for [C24H33N3O6+Na]+, 460.2442; found, 460.2455.

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84 CHAPTER 5 DESIGN AND SYNTHESIS OF PH SENSITIVE GFP CHROMOPHORE ANALOGUE S 5.1 Introduction Fluorescent peptide labeling is useful for monitoring biological activity : a fluorophore in a peptide or a protein enables ligands, inhibitors, and antigens to be detected at low concentration by introducing a fluorophore into a peptide or a p rotein. [06S217, 07AHC131] Natural aromatic amino acids (Phe, His, Trp, and Tyr) play key role s in the recognition of receptors ; they have frequently been replaced by unnatural highly fluorescent amin o acids in specific positions by bioactive peptides. [00B118, 0 1 AMB274, 04PS1489, 06TA2393] Despite many commercially available fluorophores, new examples are required (i) small enough to avoid misfolding of the protein or blockage of the binding site and (ii) that absorb above 320 nm to avoid interference from Trp residues [09OBC627] N ew fluorophores with wavelengths of absorption and emission that vary with media properties, e.g. the polarity and/ or pH, are neede d for a wide range of applications [06OBC4265] Enhanced sensitivity of a fluorophore in the pH r ange of 5 to 9 is important because most tumors develop a microenvironment characterized by low oxygen tension, high lactate concentration and/ or low extra cellular pH. [01CRLC295, 06C R6699] Green fluorescent protein (GFP) and similar proteins (CFP or YFP) are well established as fluorescent markers for monitoring biological activity because they have high light emission (quantum yields up to f = 0.8, Scheme 1) and work well in vitro and in living mammalian cells [97PNAS230, 98B509, 07B9865] However, the large size (up to 238 amino acids) of the GFP chromophore can cause misfolding or other structural changes in target proteins.

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85 Scheme 5 1. Intramolecular biosynthesis of imidazolinonyl chromophore in wildtype GFP Unlike t he chromophore of wild -type GFP, which is surrounded by its protein sequence (1 64 and 68238) and stabilized as the Z isomer, [97B9759, 07B9865] the GFP model chromophores of type 5. 1 show only low fluorescence at 20 C due to Z -E photoisomerization at the exo -methylene group (Scheme 5 2 a ). [03FEBS35, 06CP358]. Hydrogen bonding can control photochemical isome rization: Ar ai et al demonstrated hemi indigo derivatives 5. 2 [98CL1153] which exist as the Z isomers stabilized by six -membered ring intramolecular hydrogen bonding prevent or minimize photoisomerization (Scheme 5 2 b ). The Z isomer of the GFP chromophore analogue 5. 3 stabilized by boron l igation showed high fluore scent activity ( f = 0.89) compared to low fluorescence of the boron ligated E isomer (Scheme 5 2c) [08JACS4089] T he present work sought to construct new, pH -sensitive chromophores 5. 4 5. 6 (Figure 5 1) modeled on GFP and connected to lysine, glutamine and asparagine in which Z -E photoisomerization w ould be controlled by pH, thus allowing monitoring of pH in cells under natur al and pathological condition s Each chromophore contains five or six -membered heterocyclic ring s such as furyl, thienyl, or pyridyl groups, attach ed to the imidazolinone ring. Heteroatoms (O, S, or N) in aromatic rings on position 2 have a n import ant role in prevention of E -Z photoisomerization In acidic media, the basic heteroatom (the imidazolinone nitrogen if thienyl/furyl containing chromophore or the pyridyl nitrogen) gets protonated to form more stable six -membered intramolecular hydrogen bonding. To assess th e utility of such

PAGE 86

86 fluorophores, compounds 5. 9a -f and 5. 14a b were synthesized and their fluorescence record ed spectra over the pH range 1 7. Scheme 5 2. Literature examples Figure 5 1. T he proposed GFP based lysin (Lys), asparagine (Asn), and glutamine (Gln) analogues 5.4 5.6

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87 5.2 Results and Discussion 5.2.1 Synthesis of Imidazolinone Chromophore s 5.9a -f and Their Fluorescent Activit y A zalactones 5. 8a -c were each synthesized by reaction of hippuric acid 5. 7 with the appropriate aldehyde in the presence of sodium acetate and acetic anhydride (Scheme 5 3) [07SC1709, 05 AJC576] Compounds 5. 8a -c reacted under microwave conditions with p t oluidine to give 5. 9a and with N,N -dimethylethylenediamine to give 5. 9b -d in yields of 33 81% (Table 5 1). Scheme 5 3. Synthesis of GFP modified fluorophore 5.9a -d Simila l ly 5. 8e -f were obtained by reaction s of 5. 10 (prepared from 2 -naphthoyl chloride and glycine ) with the chosen aldehyde followed by treatment with N,N -dimethylethylenediamine under MW conditions to give 5. 9e and 5. 9f in yields of 51 and 30 % ( Scheme 5 4, Table 5 1). Scheme 5 4 Synthesis of GFP modified fluorophore 5.9e -f

PAGE 88

88 Table 5 1. Synthesis of GFP modified fluorophore 5.9a -f The exo -methylene group in GFP chromophores photoisomerises to the E isomer under U V irradiation, but reverts to the Z isomer on heating. [07S1103, 08JACS4089] Fluorophore 5. 9a was isolated as the Z -isomer a s revealed by 1H NMR (Figure 5 2 a) which showed on upfield resonance at 6.8 ppm for the olefinic proton compared to 5.3 -Z and 5.3 E [08JACS4089] After 1.5 5.5 h under UV light (365 nm) a solution of 5. 9a in DMSO d6, the formation of increasing amounts of the E isomer was observed between 7.9 ppm to 8.2 ppm (Figure 5 2 b,c). In the presence of concentrated HCl, the NMR spectrum showed o nly the Z -isomer even after 16 h under UV irradiation (Figure 5 2d) demonstrating stabilization of the Z isomer by intramole cular hydrogen bonding (Scheme 5 5 ). The slightly upfield shift in the presence of HCl is explained by formation of intramolecular h ydrogen bonding by the protonated Z -isomer (F i gure 5 2d) The absorption and emission spectra of the fluorophores 5. 9b d at 105 M in Britton Robinson Buffer solution [31JCS458] were recorded over the pH range 1 7 ; the 1H NMR dat a indicate that protonation restricts 5. 9a to the cis configuration (Figure 5 2). From pH 7 to pH 3 there is virtually no change in the absorption spectra of 5. 9b -f but a distinct decrease in the R1 R2 Yield (%)a mp (C) R3 Yield (%)a 5. 8a Ph 2 Thie nyl 69 180.0 182.0 5. 9a p (CH3)C6H4 56 5. 9b (CH2)2NMe2 33 5. 8b Ph 5 Methyl 2 furyl 59 150.5 152.0 5. 9c (CH2)2NMe2 81 5. 8c Ph 2 Pyrryl 35 155.5 15 6.5 5. 9d (CH2)2NMe2 55 5. 8d Napht h 2 yl 2 Thie nyl 50 212.0 214.0 5. 9e (CH2)2NMe2 51 5. 8e Naphth2 yl 5 Methyl 2 furyl 40 161.8 162.5 5. 9f (CH2)2NMe2 30 aIsolated yield

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89 intensity of the emission spectra by factors of between 2 and 4 (Fig ure 5 3 to 5 6 ). Th e origin of the fluorescence intensity decrease is clearly not associated with the furyl, thienyl or pyrryl units of 5. 9b -f or to protonation of N3 of the imidazolinone ring (pKa ca 1.4). Compounds 5. 9b and 5. 9e both containing the 2 thienyl group, gave v irtually identical absorption and emission spectra (shown only for 5. 9b Figure 5 3 ) that revealed a small (ca 20nm) bathochromic shift of the absorption spectra below pH 2.5 but no change in the emission spectra. The results are best explained by protonati on of N3 of the imidazolinone ring but only weak hydrogen bonding with sulfur that may twist the thiophene ring out of planarity with the imidazolinone system. Figure 5 2 Prevention of photoisomerization of imidazolinonyl compound 5.9a (a) Isolated product 5.9a (in DMSO d6), (b) UV irradiation (365 nm) after 1.5 h, (c) UV irradiation after 5.5 h, (d) addition of HCl, UV irradiation after 16 h (d) (c ) (b ) (a )

PAGE 90

90 Scheme 5 5 Prevention of photoisomerization of imidazolinonyl compound 5.9a by six membered rin g intramolecular hydrogen bond With compounds 5. 9c and 5. 9f containing the 5 -methyl 2 -furyl unit, the absorption spectra again showed bathochromic shifts (ca 40nm) below pH 2.5 but this was accompanied by significant increases of intensity in the emission spectra by factors of 7 ( for 5. 9c ) and 10 (for 5. 9f ). Clearly, protonation of N3 and hydrogen bonding with furyl oxygen enforces planarity on the cis configuration and the ensuing higher degree of conjugation enhances both the fluorescence wavelength and i ntensity (Scheme 5 6). Finally, compound 5. 9d containing the pyrrole system, shows similar behavior to that of 5. 9c and 5. 9f with emission intensity increasing 5 fold between pH 2.5 and 1 (Figure 5 5 ). This fluorescence increase is probably due to protonat ion of the carbonyl group in the imidazolinone ring since the N3 is already occupied with existing intramolecular hydrogen bonding with the NH of the pyrryl group (Scheme 5 7). Protonation of the imidazolinone ring at low pH was supported by the 15N NMR shift of N3 in 5.9c from 241.7 ppm in CDCl3 to 151.2 ppm in TFA d6. The bathochromc shift in the absorption and emission spectra of 5.9c (F i gure 5 4), may be due to protonation of the C=O

PAGE 91

91 oxygen (Scheme 5 6), but the NMR date do not provide unambiguous evi dence for the hypothesis (Figure 5 7 ). Scheme 5 6 Expected protonation of GFP analogues 5.9b 5.9c 5.9e 5.9f Scheme 5 7. Expected protonation of GFP analogues 5.9 d 0 0.5 1 200 250 300 350 400 450 500 550 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 0 50000 100000 150000 200000 250000 400 450 500 550 600 650 700 Wavelentgh (nm) Fluorescence Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 Figur e 5 3 Absorption ( left ) and emission spectra ( right ) of 5.9b (105 M in B ritton Robinson Buffer from pH 1 7 Raman peak of water has been fixed. )

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92 0 0.5 1 200 250 300 350 400 450 500 550 600 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 400 450 500 550 600 650 700 Wavelength (nm) Fluorescence Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 Figure 5 4 Absorption ( left ) and emis sion spectra ( right ) of 5.9c (105 M in Britton -Robinson Buffer from pH 1 7 Raman peak of water has been fixed. ) 0 0.5 1 200 250 300 350 400 450 500 550 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH3 pH4 pH6 pH7 0 100000 200000 300000 400000 500000 600000 430 480 530 580 630 680 Wavelength (nm) Fluorescence Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 Figure 5 5 Absorption (left) and emission spectra (right) of 5.9d (105 M in Britton Robinson Buffer from pH 1 7 Raman peak of water has been fixed. ) 0 0.5 1 200 250 300 350 400 450 500 550 600 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH4 pH5 pH6 pH7 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 400 450 500 550 600 650 Wavelength (nm) FluorescenceIntensity pH1 pH1.3 pH1.6 pH2 pH4 pH5 pH6 pH7 Figure 5 6 Absorption ( left ) and emission spectra ( right) of 5.9f (105 M in Britton -Robinson Buffer from pH 1 7 Raman peak of water has been fixed. )

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93 Figure 5 7 15N NMR study of 5.9c 5.2.2 Synthesis of Imidazolinone Chromophore 5.14a b and Their Fluorescent Activity Compound 5.14a and 5.14b were synthesized by a different route from com mercially available benz amide a nd c hloroacetyl chloride (Scheme 5 8 Table 5 2). [08JACS4089] Scheme 5 8 Synthesis of imidazolinone chlomophore 5.14a -b Table 5 2. Imidazolinone chromophore 5.14a-b Entry R Yield (%) a 1 5. 14a 6 Methyl 2 p yridyl 18 2 5. 14b 5 Methyl 2 fur yl 29 a Iso lated yield

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94 The absorption and emission spectra of 5. 14a and 5. 14b were recorded in 105 M in Brit ton -Robinson Buffer (Figure 5 8 and 59 ). Fluorescen ce of 5. 14a increas ed below pH 6 initially due to protonation of the pyridine nitrogen to 5. 14a followe d by tautomeri sm of the amide group to 5. 14a (Scheme 5 9 ) as indicated by the red shift in both absorption and emission spectra. This tautomerization at high pH can be explained by the free NH group of the imidazolinone ring and stronger intramolecular hyd rogen bonding between protonated pyridyl nitrogen and the imidazolinone N3. However, fluorescence of 5. 14b increased only below pH 2.5 thus exhibiting similar behavior to that of 5. 9c and 5. 9f Scheme 5 9 Possible mechanism of fluorophore 5.14a 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 200 250 300 350 400 450 500 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 400 450 500 550 600 650 Wavelength (nm) Fluorescent Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH4 pH5 pH6 pH7 Figu re 5 8 Absorption (left) and emission spectra (right) of 5.14a (105 M in Britton -Robinson Buffer from pH 1 7 Raman peak of water has been fixed. )

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95 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 200 250 300 350 400 450 500 550 600 Wavelength (nm) Absorption Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH3 pH5 pH6 0 50000 100000 150000 200000 250000 300000 350000 400000 390 440 490 540 590 640 690 Wavelength (nm) Fluorescence Intensity pH1 pH1.3 pH1.6 pH2 pH2.5 pH4 pH5 pH6 pH7 Figure 5 9 Absorption ( left ) and emission spectra ( right ) of 5.14b (105 M in Britton -Robinson Buffer from pH 1 7 Raman peak of water has been fixed. ) 5.2.3 Absorption and Emission Measurement of Chromophores 5.9b -f and 5.14a b T he absorption and emission spectra of 5.9b -f and 5.14a-b were also measur ed in Britton Robinson Buffer and quant um yield s (f) at pH 1 and excitation coefficients ( = M1nm1) were evaluated (Table 5 3). Quantum yields were calculated relatively to Coumarin 30 as a reference compound [ 85JPC294] according to the follwing equation (Eq.) where Aref, Sref, nref and Asample, Ssample, nsample represent the absorption at the exited wavelength, the integrated emission band area, and the solvent regractive index of the standard and the sample. [92JL269] f = ref (Ssample/ Sref) ( Aref/ Asample) (n2 sample/ n2 ref) (Eq.) The best potential fl uorescence marker, 5. 14a provides only 3 % of the fluorescence intensity of GFP and then only at pH 1, a condition that is highly unlikely to be of value in vivo Table 5 3. Quantum yields and excitation coe fficients of 5.9b -f and 5.14a b Entry Compound abs max (M1cm1) em max f pH 1 pH 7 pH 1 pH 1 pH 7 pH 1 1 5. 9b 409 394 24313 488 478 0.0009 2 5. 9c 444 411 22376 502 484 0.0036 3 5. 9d 462 423 45580 494 492 0.0018 4 5. 9e 410 26913 512 0.0008 5 5. 9f 4 51 416 28617 511 485 0.0060 6 5. 14a 4 05 380 20343 526 469 0.0304 7 5. 14b 449 420 20046 503 484 0.0072

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96 5.3 Conclusion GFP modified pH sensitive chromophores were synthesized and their fluorescence activit ies were measured in Britton -Robinson Buffer over the pH range 1 7. Chromophores contai ning five -membered heterocyclic rings ( 5. 9a-f and 5. 14b ) showed an increase in fluorescence below pH 2.5 and fluorescence increase below pH 6 for six -membered heterocyclic ring 5.14a but the best quantum yield (observed with 5. 14a ) was only 3 % of the fluo rescence value at pH 1 The results, however, demonstrate that high fluorescence intensity requires a cis configuration of the double bond exoc yclic to the imidazolinone ring that is enforced by hydrogen bonding between the protonated N3 of the imidazolino ne ring and nitrogen or oxygen atoms of the adjacent hetero ring. The p rotonation mechanism of 5.9d remains unconfirmed 5.4 Experimental 5.4.1 General Synthesis for the Preparation of Azalactone 5.8 A mixture of hippuric acid (5.0 mmol) (for 5.8a -c ) or 5.1 0 (5.0 mmol) (for 5.8d -e ) and sodium acetate (5.0 mmol) in acetic anhydride (3.0 mL) was stirred at room temperature for 30 min. Aldehyde (5.0 mmol) was added at room temperature and stirred for 30 min, and the reaction mixture was then heated at 80 C for 2 h. The reaction mixture was cool ed to room temperature and water (20 mL) was added. The p recipitate was filtered off, washed with water, dissolved in DCM and the DCM solutes were washed wish brine. The organic solvent was concentrated by reduce d pressu re and recrystallized from MeOH/DCM ( 5.6c was purified by silica gel column chromatography using DCM) to yield stereoisomeric mixture of azalactone 5.8 a -c (Scheme 5 3). 2 -P henyl -4 -(thiophen-2 -ylmethylene)oxazol -5(4 H )-one 5.8a : Yellow microcrystal (68 %); m p 180.0 C (Lit. [50JOC81] 174.5 175.5 C ); 1H NMR (CDCl3) 8.20 7.73 (d, J = 5.1Hz, 1H), 7.64 (d, J = 3.9 Hz, 1H), 7.62

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97 7.5213C NMR (CDCl3) 167.0, 162.5, 137.6, 135.4, 135.0, 133.2, 130.9, 128.9, 128.3, 127.9, 127.7, 125.6, 124.9. Anal. Calcd for C14H9NO2S: C, 65.87; H, 3.55; N, 5.49. Found: C, 65.70; H, 3.43; N, 5.45. 4 -((5 -M ethylfuran -2 yl)methylene) -2 phenyloxazol -5(4 H )-one 5.8b : Yellow microcrystal (59 %); mp 150.5 C (Lit. [06JICS98] 139 141 C) ); 1H NMR (CDCl3) 8.18 2H), 7.62J = 3.3 Hz, 1H), 2.45 (s, 3H); 13C NMR (CDCl3) 167.4, 162.1, 158.1, 149.3, 132.9, 128.9, 128.8, 128.1, 125. 7, 122.4, 118.5, 111.0, 14.2. Anal. Calcd for C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 70.90; H, 4.33; N, 5.57. 4 -((1 H -P yrrol -2 -yl)methylene) -2 -phenyloxazol -5(4 H )-one 5.8c : Yellow microcrystal (35 %); mp 155.5 C (Lit. [60DE1095833] 143 1 44 C) ; 1H NMR (CDCl3) 12.10 (br s, 0.5H), 10.81 (br s, 1H), 8.11 (dd, J = 1.2 & 8.2 Hz, 2H), 8.03 (dd, J = 1.2 & 8.0 Hz, 1H), 7.62 1H), 6.46 1 13C NMR (CDCl3) 169.9, 167.0, 134.5, 132.7, 132.1, 130.6, 128.9, 128.9, 128.5, 127.7, 127.3, 127.2, 126.8, 123.0, 121.4, 120.6, 112.7, 111.9. Anal. Calcd for C14H10N2O2: C, 70.58; H, 4.23; N, 11.76. Found: C, 70.25; H, 4.08; N, 11.59. 2 -(N aphthalen -2 -yl) -4 -(thiophen-2 ylmethylene)oxazol -5(4H) -one 5.8d : Yellow microcrystal (50 %); mp 212.0 214.0 C; 1H NMR (CDCl3) 8.64 (s, 1H), 8.28 J = 7.8 Hz, 2H), 7.91 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 5.1 Hz, 1H), 7.66 (d, J = 3.6 Hz, 1H), 7.64 7.58 (m, 2H), 7.52 (s, 1H), 7.21 13C NMR (CDCl3) 167.0, 162.6, 137.7, 135.6, 135.3, 134.9, 132.7, 131.1, 129.8, 129.3, 128.9, 128.6, 128.0, 128.0, 127.1, 124.7, 123.7, 122.8. Anal. Calcd for C18H11NO2S: C, 70.80; H, 3.63; N, 4.59. Found: C, 70.52; H, 3.82; N, 4.50.

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98 4 -((5 -Methylfuran -2 yl)methylene) -2 -(naphthalen-2 -yl)oxazol -5(4H) -one 5.8e : Yellow microcrystal (40 %); mp 161.8 1H NMR (CDCl3) 8.62 (s, 1H), 8.25 8.02 7.14 (s, 1H), 6.33 (d, J = 3.3 Hz, 1H), 2.47 (s, 3H); 13C NMR (CDCl3) 167.4, 162.3, 158.1, 149.4, 135.5, 132.8, 129.6, 129.3, 129.2, 129.0, 128.8, 128.8, 128.5, 128.3, 128.0, 127.1, 127.0, 124.0, 123.6, 123.2, 123.0, 122.4, 118.4, 111.3, 111.0, 14.2. An al. Calcd for C19H13NO3: C, 75.24; H, 4.32; N, 4.62. Found: C, 75.03; H, 4.39; N, 4.57. 5.4.2 General Synthesis for the Preparation of Imidazolinone 5.9 Unsym -N,N -dimethylmethylenediamine (3.0 mmol) was added to a solution of 5.8 (1.0 mmol) and sodium acet ate (2.0 mmol) in acetic acid (2 mL) at 25 C and the reaction mixture was then heated at 70 C under microwave irradiation (100 W). After 0.5 2.5 h, the reaction mixture was cool ed to room temperature, and 10% Na2CO3 aq was added to the reaction mixture. The precipitate was purified by silica gel column chlomatography (0% to 5% MeOH in DCM) to yield stereoisomeric mixtures of 5.9 a -f (Scheme 5 3). 1 -M ethyl -2 -phenyl -4 -(thiophen-2 -ylmethylene) -1 H -imidazol -5(4 H )-one 5.9a : Yellow microcrystals (56 %); mp 193.0 195.0 C ; 1H NMR (CDCl3) 7.67 (d, J = 4.8 Hz, 1H), 7.65J = 4.4 Hz, 1H), 7.10 13C NMR (CDCl3) 169.9, 159.1, 138.4, 138.2, 136.4, 134.7, 134.2, 132.1, 131.2, 130.1, 129.2, 128.8, 128.3, 127.6, 127.1, 122.5, 21.2,. Anal. Calcd for C21H16N2OS: C, 73.23; H, 4.68; N, 8.13. Found: C, 73.01; H, 4.56; N, 8.01. 1 -(2 -(D imethylamino)ethyl) -2 -phenyl -4 -(thiophen-2 -ylmethylene) -1 H -imidazol -5(4 H )-one 5.9b : Ye llow microcrystals (33 %); mp 103.0 105.0 C ; 1H NMR (CDCl3) 7.86 2H), 7.62 (d, J = 5.4 Hz, 1H), 7.57 (d, J = 3.9 Hz, 1H), 7.56 7.13J = 7.1 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H); 13C NMR (CDCl3) 171.0, 170.8, 162.2, 161.3, 151.1, 146.1, 138.0, 136.7, 136.1, 134.5, 133.9, 131.2,

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99 131.1, 129.9, 129.8, 128.8, 128.8, 128.5, 128.4, 127.5, 122.1, 119.2, 115.6, 113.3, 57.4, 57.3, 45.5, 45.4, 40.0, 39.9. 1 -(2 -(D imethylamino)ethyl) -4 -((5 -methylfuran -2 yl)methylene) -2 phenyl -1 H -imidazol 5(4 H )-one 5.9c : Y ellow microcrystals (81 %); mp 77.0 C ; 1H NMR (CDCl3) 7.82 (m, 2H), 7.56 J = 3.3 Hz, 1H), 7.11 (s, 1H), 6.21 (d, J = 3.3 Hz, 1H), 3.88 (t, J = 7.1 Hz, 2H), 2.46 13C NMR (CDCl3) 171.0, 161. 1, 157.1, 149.9, 134.9, 131.0, 130.0, 128.7, 128.4, 121.3, 116.0, 110.5, 57.4, 45.4, 39.9, 14.2. 4 -((1 H -P yrrol -2 -yl)methylene) -1 -(2 -(dimethylamino)ethyl) -2 -phenyl -1 H -imidazol -5(4 H )one 5.9d : Yellow sticky solid (55 %); 1H NMR (CDCl3) 13.20 (br s, 0.6H), 11.10 (br s, 0.4H),7.76 (m, 1H), 7.15 6.70 (m, 0.6H), 6.44 6.54 2.71 (t, J = 7.4 Hz, 2H), 2.34 (s, 6H); 13C NMR (CDCl3) 176.0,170.1, 131.1,130.7, 129.6, 129.5, 128.9, 128.4, 128.3, 128.2, 126.1, 126.0, 121.7, 119.2, 112.4, 111.3, 56.1, 44.3, 44.2, 38.9, 38.4, 21.1. HRMS Calcd for [C18H20N4O+H]+, 309.1710; found, 309.1717. 1 -(2 -(D imethylamino)ethyl) -2 -(naphthalen -2 -yl) -4 -(thiophen-2 -ylmethylene) -1 H -imidazol 5(4 H )-one 5.9e : Yellow microcrystals (51 %); mp 154.8 C ; 1H NMR (CDCl3) 8.30 (s, 1H), 7.93J = 4.1 Hz, 1H), 3.90 (t, J = 6.8 Hz, 2H) 2.42 (t, J = 6.8 Hz, 2H), 2.07 (s, 6H); 13C NMR (CDCl3) 170.9, 161.2, 138.2, 136.9, 134.4, 134.4, 133.9, 132.7, 128.6, 128.9, 128.6, 127.8, 127.5, 127.1, 126.9, 125.1, 122.0, 57.5, 45.5, 40.2. 1 -(2 -(D imethylamino)ethyl) -4 -((5 -methylfuran -2 yl)methylene )-2 -(naphthalen -2 -yl) -1 H imidazol -5(4 H )-one 5.9f : Yellow oil (30 %); 1H NMR (CDCl3) 8.28 (s, 0.72H), 8.24 (d, J =

PAGE 100

100 3.6 Hz, 0.28H), 8.21 (s, 0.28H), 7.92 J = 3.3 Hz, 0.72H), 7.19 (s, 0.28H), 7.06 (s, 0.72H), 6.20 ( d, J = 3.3 Hz, 0.28H), 6.15 (d, J = 3.3 Hz, 0.72H), 3.943.84 (m, 2H), 2.50 2.37 (m, 2H), 2.34 (s, 3H), 2.09 (s, 1.7H), 2.06 (s, 4.3H); 13C NMR (CDCl3) 171.1, 161.0, 157.1, 150.0, 134.3, 132.8, 128.8, 128.8, 128.7, 128.6, 128.5, 127.9, 127.7, 127.6, 127. 3, 126.9, 125.0, 124.8, 122.8, 121.5, 121.3, 115.9, 110.8, 110.5, 57.5, 45.5, 40.2, 14.2. HRMS Calcd for [C23H23N3O2+H]+, 374.1863; found, 374.1872. 5.4.3 Synthesis of 2 -(2 -Naphthamido)acetic acid 5.10 2 Napht h oylchloride (0.57g, 3.03mmol) and glycine (0. 25g, 3.03 mmol) were mixed and stirred in the presence of NaOH (0.12 g, 3.03 mmol) in aqueous Me CN ( Me CN : H2O = 2mL : 5mL) at room temperature. After 1 h, the organic solvent was evaporated under reduced pressure, washed with 6M HCl aq, and extracted with EtOAc. The organic layer was then w ashed with brine, and dried with MgSO4 to yield 5.10 as white microcrystals followed by recrystal l ization from EtOAc/h exanes (Scheme 5 4). (70%); mp 150.0 151.0 C; 1H NMR (DMSO d6) 12.64 (br s, 1H), 9.03 (t, J = 5.8 Hz, 1H), 8.49 (s, 1H), 8.057.50 (m. 4H), 7.657.55 (m, 2H), 3.99 (d, J = 5.7 Hz, 2H) ; 13C NMR (DMSO d6) 171.4, 166.8, 134.3, 132.2, 131.3, 129.0, 128.1, 127.8, 127.7, 127.7, 126.9, 124.1, 41.4. Anal. Calcd for C13H11NO3: C, 68.11; H, 4.84; N, 6.11. Fou nd: C, 67.78; H, 4.88; N, 6.49. 5.4.4 Synthesis of N -(2 -Chloroacetyl)benzamide 5. 11 Chloroacetyl chloride (2.9 mL, 0.04 mol) was added dropwise to a solution of benzamide (4.0 g, 0.03 mol) in toluene (80 mL) The reaction mixture was heated under reflux fo r 1.5 h, and then cooled to room temperature. The organic solvent was concentrated under reduced pressure. The re sidue was recrysta l lized in DCM/h exanes to yield N (2 -chloroacetyl)benzamide 5.11 as white microcrystals (Scheme 5 7 ). (70 %); mp 153.0 155.0 C ; 1H NMR ( CDCl3) 9.69 (br s, 1H), 7.93 (d, J = 7.8 Hz, 2H), 7.70 13C NMR

PAGE 101

101 (CDCl3) 168.8, 165.7, 133.6, 131.8, 129.0, 128.0, 45.4. Anal. Calcd for C9H8ClNO2: C, 54.70; H, 4.08; N, 7.09. Found: C, 54.71; H, 4.03; N, 7.01. 5.4.5 Synthesis of N -(2 Azidoacetyl)benzamide 5.12 A m ixture of 5.11 (1.0 g, 5.06 mmol) and sodium azide (0.7 g, 10.12 mmol) in DMSO (4.5 mL) was stirred at room temperature for 12 h, and ice -cold water was then added. The precipit ate was filtered off, and recrysta l lized from EtOAc/h exanes to yield 5.12 (Scheme 5 7 ). White microcrystals (66 %); mp 125.0 126.0 C ; 1H NMR (CDCl3) 9.62 (br s, 1H), 7.96 (d, J = 7.3 Hz, 2H), 7.687.60 (m, 1H), 7.54 (t, J = 7.1 Hz, 2H) 4.60 (s, 2H); 13C NMR (CDCl3) 171.6, 165.8, 133.8, 131.5, 129.1, 127.9, 54.3. Anal. Calcd for C9H8N4O2: C, 52.94; H, 3.95; N, 27.44. Fou nd: C, 53.08; H, 4.09; N, 27.46. 5.4.6 Synthesis of 2 -Phenyl -1 H -im idazol -5(4 H )-one 5.13 Triphenylphosphine (1.67 g, 6.37 mmol) was added to a solution of N (2 azidoacetyl) benzamide (1.0 g, 4.90 mmol) in toluene (50 mL). The mixture was stirred at room tem perature for 3 h, then evaporated under reduced pressure. The residue was washed with ether and recrystal l ized from DCM/h exanes to yield 5.13 (Scheme 5 7 ). Red microcrystals (66 %); mp 147.0 148.0 C ; 1H NMR (CDCl3) 10.59 (br s, 1H) 7.92 (d, J = 7.8 Hz, 2H), 7.40 3H), 4.42 (s, 2H); 13C NMR (CDCl3) 184.8, 161.3, 132.1, 129.0, 128.4, 126.7, 60.2. Anal. Calcd for C9H8N2O: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.16; H, 5.00; N, 17.22. 5.4.7 General Procedure for the Preparation of Imidazolinone 5.14 A solution of the appropriate aldehyde (0.85 mmol) and imidazolin4 one 5.13 (0.94 mmol) in piperidine (9 mL) was stirred at room temperature for 1 .0 1.5 h (Scheme 5 7) H2O was added and the mixture was extracted with D C M washed with brine, and dried with MgSO4. The solvent was removed under reduced pressure. The crude mixture was purified by neutral

PAGE 102

102 alumina chromatography (2% MeOH in DCM) to yield isomeric mixtures of imidazolinone c hromophore 5. 14 followed by recrystalization in MeOH. 4 -((6 -M ethylpyridin -2 yl)methylene) -2 phenyl -1 H -imidazol-5(4 H )-one 5.14a : White microcrystals (18 %); mp 189 C (decomposed); 1H NMR (CDCl3) 11.65 (br s, 0.67H) 10.87 (br s, 0.33H), 8.68 (d, J = 7.8 H z, 0.33H), 8.10 7.90 (m, 2H), 7.60 0 (m, 3.66H), 7.15 (d, J = 7.5 Hz, 0.67H), 7.11 (s, 0.67H), 6.99 (d, J = 7.7 Hz, 1.34H), 6.75 (s, 0.33H), 2.55 (s, 2H), 2.46 (s, 1H); 13C NMR (CDCl3) 180.1, 172.6, 158.6, 158.3, 153.6, 142.2, 137.5, 136.5, 134.5, 134.2, 132.9, 129.3, 129.1, 128.5, 128.3, 127.8, 127.6, 127.4, 125.1, 124.4, 123.4, 122.8, 113.2, 25.1, 24.5. Anal. Calcd for C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C, 72.75; H, 4.85; N, 15.66. 4 -((5 -M ethylfuran -2 yl)methylene) -2 phenyl -1 H -imidazol-5( 4 H )-one 5.14b : Yellow microcrystals (29 %); mp 230.0 C (decomposed); 1H NMR (DMSO d6) 12.05 (br s, 1H), 8.14 (dd, J = 7.9 & 1.4 Hz, 2H), 7.657.52 (m, 3H), 7.49 (d, J = 3.3 Hz, 1H), 6.80 (s, 1H), 6.44 (d, J = 1.8 Hz, 1H), 2.39 (s, 3H); 13C NMR (DMSO d6) 171.2, 159.0, 156.1 ,149.5, 136.7, 132.2, 128.9, 128.0, 127.2, 120.0, 112.3, 110.7, 13.7. Anal. Calcd for C15H12N2O2: C, 71.41; H, 4.79; N, 11.10. Found: C, 71.54; H, 4.77; N, 10.87.

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103 CHAPTER 6 SU MMARY OF AC H IEVEMENT S Synthetic organic chemistry plays an important ro le in the fields of material scien ce pharmaceut icals agricult ures and f ood chemical s Th e present work helps in the essential purs it of more efficient and environment ly -friendly intermadiates Chapter 1 provides an overview of the importance of BtH in organic syntheses. Previously, our group reported the synthesis of novel be nzotriazole intermediates, 1 (a lkyl/arylthio carbamoyl) benzotriazoles a nd b enzotriazole1 carboxamidine which were utilized in the synthesis of guanidine and thiourea analogues. [04JOC2976, 06A226, 06JOC6753] In Chapter 2 new C -C bond forming C aminoim idoylation and C thiocarbamoylation reactions with sulf ones and ketones were achieved under mild reaction conditions. [07JOC 6742] This approach provides easy access to interesting classes of compounds for further transformations. Furthermore, reactions of ketones with N -benzyl 1 H benzo[d][1,2,3] triazole1 carbothioamide surprisingly gave the isomeric 1,3 oxazolidine 2 thione followed by formation of isothiocyanates and cyclization due to deprotonation of benzyl proton s In Chapter 3 tri tetra p enta hexa and heptapeptides were prepared by solid phase segment condensation assisted by microwave irradiation. [08CBDD181] (N-Fmoc -prote cted dipeptidoyl)benzotriazoles were synthesized, in which the original chirality was maintained. (NFmoc -prote cted -dipeptidoyl) benzotriazoles are air and moisture insensitive acylation reagents which enabl e solid phase segment condensation wi thout the use of other coupling reagents or additives. Side reactions and epimerization were not obse rved in these sequences, some of which were previously reported as difficult during peptide synthesis by segment condensation In Chapter 4 a convenient and efficient preparation in solution phase of a variety of coumarin fluorescent probes is described with coumarin 3 ylcarboxyl (Cc) and 7 -

PAGE 104

104 methoxycoumarin 4 -ylacetyl (Mca) labeled lysines as fluorogenic substrates. [08OBC4582] Their benzotriazole derivativ es are appropriate materials for peptide labeling thus enabling amino group acylation under microwave irradiation on solid phase and witho ut the use of coupling agents and/or additives avoiding s ide reactions and epimerization. In C h a pter 5, GFP modified pH sensitive chromophores were synthesized and their fluorescent activities were measured in Britton Rob inson Buffer between pH 7 and pH 1 [Proj#1976] Chromophore s containing five -membered aromatic ring s showed increase of fluorescenc e below pH 2.5, but the chromophore containing 2 pyridyl group was more se nsitive at higher pH ( below pH 6 ) due to a strong intramolecular hydrogen bonding with imidazolinone nitrogen. This may be useful in the determination of the acidity of biological sy stem s Many novel reactions using benzotriazole methodology have been carried out by previous members of the Katritzky group, and my work has expanded their studies and presented further application s of BtH in organic synthesis

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105 LIST OF REFERENCES The ref erence citation system employed throughout this research report is from Comprehensive Heterocyclic Chemistry (vol.1); Pergamon Press: New York, 1996 (Eds. Katritzky, A. R.; Rees, C. W.; Scriven, E.). Each time a reference is cited, a number lett er code is designated to the corresponding reference with the first two or four if the referencec is before1910s number indicating the year followed by the letter code of the journal and the page number in the end. Additional notes to this reference system are as follows: 1 ) Each reference code is followed by conventional literature citation in the ACS style. 2 ) Journals which are published in more than one part including in the abbreviation cited the appropriate part. 3 ) Less commonly used books and journals are still a bbreviated as using initials of the journal name. 4 ) The list of the reference is arranged according to the designated code in the order of (i) year, (ii) journal/book in alphabetical order, (iii) part number or volume number if it is included in the code, a nd (iv) page number. 5 ) Project number is used to code the unpublished resul ts. [31JCS458] Britton, H. T. S.; Robinson, R. A. J. Chem. Soc 1931,458. [47JACS119] Shivers, J. C.; Dillon, M. L.; Hauser, C. R. J. Am. Chem. Soc 1947, 69, 119. [48JCS2240] Alber t, A.; Goldacre, R.; Phillips, J. J. Chem. Soc 1948, 2240. [50JOC81] Crowe, B. F.; Nord, F. F. J. Org. Chem 1950, 15, 81. [59JACS4882] Bachman, G. B.; Hokama, T. J. Am. Chem. Soc 1959, 81, 4882. [60DE1095833] Bohringer, H. Patent DE 1095833, 1960. [70J ACS5792] Kemp, D. S.; Rebek, J. J Am. Chem. Soc 1970, 92, 5792. [76CB3047] Hoppe, D.; Follmann, R. Chem. Ber 1976, 109, 3047.

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106 [77BCSJ1999] Sato, K.; Abe, H.; Kato, T.; Izumiya, N. Bull. Chem. Soc. Jap. 1977, 50, 999. [78JOC337] Larsen, C.; Steliou, K.; Harpp, D. N. J. Org. Chem 1978, 43, 337. [79S343] Kantlehner, W.; Mergen, W. W. Synthesis 1979, 5 343. [79ZOK2349] Martynov, A. V.; Mirskova, A. N.; Kalikhman, I. D.; Makerov, P. V.; Vitkovskii, V. Y.; Voronkov, M. G. Zh. Org. Khim 1979, 15, 2349. [8 3LAC290] Kantlehner, W.; Mergen, W. W.; Haug, E. Liebigs Ann. Chem 1983, 2 290. [84S572] Dourtoglou, V.; Bernard, G.; Lambropoulou, V.; Zioudrou, C. Synthesis 1984, 572. [85JPC294] Jones, G. II; Jackson, W. R.; Choi, C. Y.; Bergmark, W. R. J. Phys. Chem 1985, 89, 294. [89JBC4227] Stack, M. S.; Gray, R. D. J. Biol. Chem. 1989, 264, 4227. [91AB137] Birkett, A. J.; Soler, D. F.; Wolz, R. L.; Bond, J. S.; Wiseman, J.; Berman, J.; Harris, R. B. Anal. Biochem 1991, 196, 137. [92FEBS263] Knight, C. G.; Wille nbrock, F.; Murphy, G. FEBS Lett 1992, 296, 263. [92JL269] Pardo, A.; Reyman, D.; Payato, J. M. L.; Medina, F. J. Lumin. 1992, 51, 269. [93BC537] Geoghegan, K. F.; Emery, M. J.; Martin, W. H.; McColl, A. S.; Daumy, G. O. Bioconjugate Chem 1993, 4 537. [93BJ601] Anastasi, A.; Knight, C. G.; Barrett, A. J. Biochem. J. 1993, 290, 601. [93CROBM197] Birkedal Hansen, H.; Moore, W. G. I.; Bodden, M. K.; Windsor, L. J.; Birkedal Hansen, B.; DeCarlo, A.; Engler, J. A. Crit. Rev. Oral Biol. Med 1993, 4 197. [93 JACS4397] Carpino, L. A. J. Am. Chem. Soc 1993, 115, 4397. [93JACS7912] Hwang, B. K.; G u, Q. M.; Sih, C. J. J. Am. Chem. Soc 1993, 115, 7912. [93JPPS405] Mihara, H.; Xu, M.; Nishino, N.; Fujimono, T. J. Pept. Protein Res 1993, 41, 405. [94CSR363] Katri tzky, A. R.; Lan, X. Chem. Soc. Rev 1994, 363. [94IJPPR118] Obeyesekere, N. U.; Croix, J. N. L.; Budde, R. J. A.; Dyckes, D. F.; McMurray, J. S. Int. J. Pept. Protein Res 1994, 43, 118.

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107 [94JBC20952] Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem 1994, 269, 20952. [94JHC917] Katritzky, A. R.; Galuszka, B.; Rachwal, S.; Black, M. J. Het. Chem 1994, 31, 917. [95B3972] Turcatti, G.; Vogel, H.; Chollet, A. Biochem 1995, 34, 3972. [96JMS45] Hansen, P. E.; Duus, F.; Bolvig, S.; Jagodzinski, T. S J. M ol. Struct 1996, 378, 45. [97B9756] Wachter, R. M.; Ki ng, B. A.; Heim, R.; Kallio, K .; Tsien, R. Y.; Boxer, S. G.; Remington, S. J. Biochem 1997, 36, 9759. [97CR1515] Silva, A. P. D.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev ., 1997, 97, 1515. [97FEBS379] Gulnik, S. V.; Suvorov, L. I.; Majer, P.; Collins, J.; Kane, B. P.; Johnson, D. G.; Erickson, J. W. FEBS Lett 1997, 413, 379. [97JPR444] Cowley, D. J.; Schulze, A. J. Pept. Res 1997, 49, 444. [97PNAS2306] Brejc. K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien, R. Y.; Orm M.; Remington, S. J. Proc. Natl. Acad. Sci. USA 1997, 94, 2306. [97TL6771] Fustero, S.; Pina, B.; Simn -Fuentes, A. Tetrahedron Lett 1997, 38, 6771. [98B509] Tsien, R. Y. Annu. Rev. Biochem ., 1998, 67, 509. [98BMCL597] Weber, P. J. A.; Bader, J. E.; Folkers, G.; Beck Sickinger, G. Bioorg. Med. Chem. Lett 1998, 8 597. [98CL1153] Arai, T.; Hozumi, Y. Chem. Lett 1998, 1153. [98CR409] Katritzky, A. R.; Lan, X.; Yang, J. Z.; Denisco, O. V. Chem. Rev 1998, 98, 409. [99JACS1636] Hamuro, Y.; Scialdone, M. A.; DeGrado, W. F. J. Am. Chem. Soc 1999, 121, 1636. [99OL977] Fustero, S.; Pina, B.; Garcia de la Torre, M.; Navarro, A.; Ramrez de Arellano, C.; Simn A Org. Lett 1999, 1 977. [00B118] Murakami, H.; Hohsaka, T.; Ashizuka, Y.; Hashimoto, K.; Sisido, M. Biomacromol 2000, 1 118. [00B4423] Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochem 2000, 39, 4423. [00BC71] Geoghegan, K. F.; Rosner P. J.; Hoth, L. R. Bioconjugate Chem 2000, 11, 71.

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108 [00JACS5849] Wang, H.; Burda, C.; Persy, G.; Wirz, J. J. Am. Chem. Soc 2000, 122, 5849. [00JOC1583] Barun, O.; Ila, H.; Junjappa, H. J. Org. Chem. 2000, 65, 1583. [00JOC3679] Katritzky, A. R.; Pastor, A. J. Org. Chem 2000, 65, 3679. [00JOC8210] Katritzky, A. R.; He, H. Y.; Suzuki, K. J. Org. Chem 2000, 65, 8210. [00S2029] Katritzky, A. R.; Fang, Y.; Donkor, A.; Xu. L. Synthesis 2000, 14, 2029. [01AMB274] Sisido, M.; Hohsaka, T. Appl. Microbiol. Biotechnol 2000 57, 274. [01B5795] Lauer Fields, J. L.; Broder, T.; S ritharan, T.; Chung, L.; Nagase, H.; Fields, G. B. Biochem 2001, 40, 5795. [01BCSJ2133] Mashraqui, S. H.; Kumar, S.; Mudaliar, C. D. Bull. Chem. Soc. Jpn. 2001, 74, 2133. [01CRLC295] Metzler, D. E. Biochem: The Chemical Reactions of Living Cells 2nd Editio n, Harcourt/Academic Press 2001, 1 295. [01LPS263] Malkar, N. B.; Fields, G. B. Lett. Peptide Sci 2001, 7 263. [01RJBC306] Kolobanova, S. V.; Filippova, I. Y.; Lys ogorskaya, E. N.; Bacheva, A. V.; Oksenoit, E. S.; Stepanov, V. M. Rus. J. Bioorg. Chem 2001, 27, 306. [01P] Califano, J. C.; Devin, C.; Shao, J.; Blodgett, J. K.; Maki, R. A.; Funk, K. W.; Tolle, J. C. Peptide 2000, Proceedings of the European Peptide Symposium, 26th Montpellier, F rance 2001. [02A39] Katritzky, A. R.; Yang, H.; Zhang, S.; Wang, M. ARKIVOC 2002, xi 39. [02JMB429] Renard, M.; Belkadi, L.; Hugo, N;. England, P.; Altschuh, D.; Bedouelle, H J. Mol. Biol. 2002, 318, 429. [02JMR311] Wei A. P.; Herron, J. N. J. Mol. Re cognit. 2002, 15, 311. [02JOC3120] Enander, K.; Dolphin, G. T.; Andersson, L. K.; Liedberg, B.; Lundstrom, I.; Baltzer, L. J. Org. Chem 2002, 67, 3120. [02JOC4667] Fustero, S.; Pina, B.; Salavert, E.; Navarro, A.; Ramrez de Arellano, M. C.; Simn Fuentes A. J. Org. Chem 2002, 67, 4667. [02PS2655] Lorimier, R. M. D.; Smith, J. J.; Dwyer, M. A.; Looger, L. L.; Sali, K. M.; Paavola, C. D.; Rizk, S. S.; Sadigov, S.; Conrad, D. W.; Loew, L.; Hellinga, H. W. Protein Sci. 2002, 11, 2655.

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109 [03AB105] Lauer Fields J. L.; Kele, P.; Sui, G.; Nagase, H.; Leblanc, R. M.; Fields, G. B. Anal. Biochem 2003, 321, 105. [03AB141] Yan, Z. H.; Ren, K. J.; Wang, Y.; Chen, S.; Brock, T. A.; Rege, A. A. Anal. Biochem 2003, 312, 141. [03CB53] Takahashi, M.; Nokihara, K.; Mihar a, H. Chem. Biol. 2003, 1 0, 53. [03FBES35] He, X.; Bell. A. F.; Tonge, P. J. FEBS Lett 2003, 549, 35. [03JOC1443] Katritzky, A. R.; Abdel Fattah, A. A. A.; Wang, M. J. Org. Chem. 2003, 68, 1443. [03JOC4932] Katritzky, A. R.; Abdel Fattah, A. A. A.; Wang, M. J. Org. Chem. 2003, 68, 4932. [03JOC5720] Katritzky, A. R.; Suzuki, K.; Singh, S. K.; He, H. Y. J. Org. Chem. 2003, 68, 5720. [04CCA175] Katritzky, A. R.; Suzuki, K.; Singh, S. K. Croat. Chem. Acta 2004, 77, 175. [04CR3059] Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem. Rev ., 2004, 104, 3059. [04DDT24] Stoll, D.; Bachmann, J.; Templin, M. F.; Joos, T. O. DDT: Targets 2004, 3 24. [04JOC188] Shi, Y.; Zhang, J.; Grazier, N.; Stein, P. D.; Atwal, K. S.; Traeger, S. C.; Callahan, S. P.; Malley M. F.; Galella, M. A.; Gougoutas, J. Z J. Org. Chem 2004, 69, 188. [04JOC2976] Katritzky, A. R.; Ledouux, S.; Witek, R. M.; Nair, S. K. J. Org. Chem 2004, 69, 2976. [04PS1489] Filippis, V. D.; Boni, S. D.; Dea, E. D.; Dalzoppo, D.; Grandi, C.; Fontana A. Protein Sci 2004, 13, 1489. [04S1806] Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K. Synthesis 2004, 11, 1806. [04S2645] Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004, 2645. [04TL6079] Fernandez Carneado, J.; Giralt, E. Tetrahedron Le tt. 2004, 45, 6079. [05AJC576] Saravanan, V. S.; Kymar, S. P. V.; De, B.; Gupta, J. K. Asian J. Chem 2005, 17, 269. [05ACIE2642] Tinnefeld, P.; Sauer, M. Angew. Chem. Int. Ed. 2005, 44, 2642.

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110 [05B238] Makino, T.; Matsumoto, M.; Suzuki, Y.; Kit ajima, Y.; Yamamoto, K.; Kuramoto, M.; Minamitake, Y.; Kangawa, K.; Yabuta, M. Biopolymers 2005, 79, 238. [05CBC1043] Engfeldt, T.; Renberg, B.; Brumer, H.; Nygren, P. A.; Karlstrm, A. E. Chem. Bio. Chem 2005, 6, 1043. [05FSPPS215] Chan, W. C.; White, P. D. Fmoc S olid Phase Peptide Synthesis: A practice approach, Orxford University Press Inc. 2005, 9 215. [05HCA1664] Katritzky, A. R.; Khashab, N. M.; Bobrov, S. Helvetica Chim. Acta 2005, 88, 1664. [05JOC4993] Katritzky, A. R.; Jiang, R.; Suzuki, K. J. Org. Chem 2005, 70, 4993. [05JSS1812] Shi, Y.; Xiang, R.; Horvth, C.; Wilkins, J. A. J. Sep. Sci 2005, 28, 1812. [05S397] Katritzky, A. R.; Angrish, P.; Hur, D.; Suzuki, K. Synthesis 2005, 3 397. [05S1656] Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005, 1 1 1656. [06A226] Katritzky, A. R.; Khashab, N. M.; Gromova, A. V. ARKIVOC 2006, 3 226. [06 CBDD326] Katritzky, A.R.; Meher, G.; Angrish, P. Chem. Biol. Drug Des 2006, 68, 326. [06CP358] Nifos, R.; Tozzini, V. Chem. Phys 2006, 323, 358. [06CR6699] Rofs tad, E. K.; Mathiesen, B.; Kindem, K.; Galappathi, K. Cancer Res 2006, 66, 6699. [06JICS98] Salehi, P.; Dabiri, M.; Khosropout, A. R.; Roozbehniya, P. J. Iranian Chem. Soc ., 2006, 3 98. [06JOC6753] Katritzky, A. R.; Khashab, N. M.; Bobrov, S.; Yoshioka, M. J. Org. Chem 2006, 71, 6753. [06JPS116] Goulas, S.; Gatos, D.; Barlos, K. J. Peptide Sci 2006, 12, 116. [06N GIR] Khashab, N. M. Novel guanylating and imidoylating reagents University of Florida 2006. [06OBC4265] Wilson, J. N.; Kool, E. T. Org. B iomol. Chem ., 2006, 4 4265. [06S411] Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis 2006, 3 411. [06S217] Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312 217.

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111 [06 TA2393] Royo, S.; Jim nez, A. I.; Cativiela, C. Tet rahedron: Asymmetry 2007, 17, 2393. [06TL7905] Yoshiya, T.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett 2006, 47, 7905. [07AHC131] Suzuki, T.; Matsuzaki, T.; Hagiwara, H.; Aoki, T.; Takata, K. Acta Hiscochem. Cytochem 2007, 40, 131. [07 B9865] Malo, G. D.; Pouwels, L. J.; Wang, M.; Weichsel, A.; Mon t fort, W. R.; Rizzo, M. A.; Piston, D. W.; Wac hter, R. M. Biochem 2007, 46, 9865. [07BC994] Katritzky, A. R.; Angrish, P.; Narindoshvili, T. Bioconjugate Chem 2007, 18, 994. [07CBDD465] Katri tzky, A. R.; Khashab, N. M.; Yoshioka, M.; Haase, D. N.; Wilson, K. R.; Johnson, J. V.; Chung, A.; Haskell -Luevano, C. Chem. Biol. Drug Des. 2007, 70, 465. [07JOC6742] Katritzky, A. R.; Khashab, N. M.; Haase, D. N.; Yoshioka, M. Ghiviriga, I.; Steel, P. J. J. Org. Chem 2007, 72, 6742. [07S1103] Pr ger, B.; Bach, T. Synthesis 2007, 7 1103. [07S3141] Katritzky, A. R.; Le. K. N. B.; Mohapatra. P. P. Synthesis 2007, 20, 3141. [07SC1709] Jursic, B. S.; Sagiraju, S.; Ancalade, D. K.; Clark, T.; Stevens, E. D. S ynthetic Comm 2007, 37, 1709. [08CBDD181] Katritzky, A. R.; Yoshioka, M.; Narindoshvili, T.; Chung, A.; Khashab, N. M. Chem. Biol. Drug Des. 2008, 72, 181. [08JOC511] Katritzky, A. R.; Narindoshvili, T.; Draghici, B.; Angrish, P. J. Org. Chem 2008, 73, 5 11. [08JACS4089] Wu, L.; Burgess, K. J. Am. Chem. Soc 2008, 130, 4089. [08OBC4582] Katritzky, A. R.; Yoshioka, M.; Narindoshvili, T.; Chung, A.; Johnson, J. V. Org. Biomol. Chem 2008, 6 4582. [09A47] Katritzky, A. R.; Singh, A.; Haase, D. N.; Yoshioka, M. ARKIVOC 2009, viii, 47. [09OBC627] Katritzky, A. R.; Narindoshvili, T. Org. Biomol. Chem 2009, 7 627. [Proj#1979] Katritzky, A. R.; Yoshioka Tarver, M.; El Gendy, B. E. M.; Hall, C. D. In progress.

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112 BIOGRAPHICAL SKETCH Megumi Yoshioka Tarver, first daughter of Hitoshi and Kazuyo Yoshioka was born in Japan. Sh e received her Bachelor of Engineering from University of Fukui, Japan, in March 2004. During her senior year, she worked as an undergraduate researcher in a synthetic organic chemistry lab focu sed on supramoleculer chemistry, under the directi on of Dr. Yuji Tokunaga. Upon graduation, she continued her education at the Department of Chemistry, University of Florida from August 2005. Her doctor ate -level research focused on synthesis of heterocycl es and p eptides supervised by Dr. Alan R. Katritzky