Novel Approaches in Azide and Peptide Synthesis

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Novel Approaches in Azide and Peptide Synthesis
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El Khatib, Mirna
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Doctorate ( Ph.D.)
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University of Florida
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Chemistry
Committee Chair:
Katritzky, Alan R
Committee Members:
Mcelwee-White, Lisa A
Aponick, Aaron Steven
Ghiviriga, Ion
Narayanan, Ranganathan

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Subjects / Keywords:
acylation -- azide -- benzotriazole -- catalysis -- heterocycles -- ligation -- palladium -- peptide
Chemistry -- Dissertations, Academic -- UF
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Abstract:
The theme of this work is the development of novel methodologies for the efficient preparation of a variety of organic compounds.Chapter 1 presents a general overview of the work presented in subsequent chapters together with a brief discussion of the importance of benzotriazole methodology in organic synthesis. Chapter 2 describes the role of benzotriazole for the synthesis of a novel diazotransfer reagent and examines the utility of the reagent in the preparation of organic azide targets. This methodology is also employed in Chapter 3, where the tolerance of azide as a protecting group is examined in various acylation reactions. Chapter 4 presents the use of the microwave in organic synthesis. This chapter reports on the palladium-catalyzed reactions of N-acylbenzotriazoles with epoxides affording pseudohalohydrin ester surrogates as single regioisomers. Chapter 5 focuses on the development of a mild protocol towards the synthesis of O-acylisodipeptides from serine and threonine amino acid residues using benzotriazole methodology.This mild protocol is visited again in Chapter 6, where it was employed in peptide ligations via large transition states. Cysteine-free microwave-assisted chemical ligations are examined in this chapter using O-acylpeptides from serine residues. Chapter 7 summarizes the achievements together with the conclusions.
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by Mirna El Khatib.
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Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Katritzky, Alan R.
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1 NOVEL APPROACHES IN AZIDE AND PEPTIDE SYNTHESIS By MIRNA EL KHATIB 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 2012

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2 2012 Mirna El Khatib

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3 To my mother Dalal Al Omari, my father Basman El Khatib and to my siblings, Lina and Mohammad. I would never have achieved any of this without their love, support, and words of wisdom

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4 AC KNOWLEDGMENTS I ultimately thank my Lord for carrying me through this journey. I am heartily thankful to my supervisor, Prof essor Alan R. Katritzky, whose guidance and support from the initial to the final level enabled me to accomplish this work during th ese years I am grate ful to my committee members (Professor Lisa McElwee White, Dr. Aaron Aponick, Dr. Ion Ghiviriga, and Professor Ranga Narayanan ) for their contin uous assistance and support. I owe my deepest gratitude to Dr. C. Dennis Hall for the incredible help and support I received from him throughout the years and during the preparation of this thesis. I would like to thank former and current members of the Ka tritzky group, and professors of the Chemistry Department especially Dr. Ben Smith. I I would like to thank all members in Professor this work: Dr. Levan Khelashvili, Dr. Eka Todadze, Dr. Oleg Bol'shakov, Dr. Alex ander Oliferenko, Mohamed Elagawany, Eray Caliskan and undergraduate student Lilibeth Jauregui. I am in debted to my master advisor, Professor Makhluf J. Haddadin With out his help and encouragements, it would have been very difficult to me to achieve my g oals. Words are powerless to express my gratitude to my parents. They have always been there for me and encouraged me to find my own way. I would like to thank all the friends I have made along the se years in Gainesville for their full support and fun time spent outside of the lab: Davit Jishkariani, Dr. Ilker Avan, Dr. Bogdan Draghici, Khanh Ha, Dr. Maia Tsikolia, Dr. Bahaa El Dien El Gendy Dr. Siva S. Panda, Dr. Kiran Bajaj, Dr. Rajeev Sakhuja and Dr. Tarek S. Ibrahim. I would like to especially thank Da vit, who was always willing to help, support and give

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5 me his best suggestions during this time and for all the enjoyable time we have spent together.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF SCHEMES ................................ ................................ ................................ ...... 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 17 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .................. 19 2 BENZOTRIAZOL 1 YL SULFONYL AZIDE ................................ ............................ 24 2.1 Introduction ................................ ................................ ................................ ....... 24 2.2 Results and Discussion ................................ ................................ ..................... 26 2.2.1 Preparation and Characterization of Benzotriazol 1 yl sulfonyl Azide 2.7 ................................ ................................ ................................ ................. 26 2.2.2 Preparation of Azides from Primary Amines ................................ ............ 30 2.2.3 Azido Acids ................................ ................................ ... 31 2.2.4 Preparation of Diazo Compounds ................................ ........................... 32 2.3 Conclusions ................................ ................................ ................................ ...... 34 2.4 Experimental ................................ ................................ ................................ ..... 34 2.4.1 General Methods ................................ ................................ ..................... 34 2.4.2 Preparation of Benzotriazol 1 yl Sulfonyl Azide 2.7 ................................ 34 2.4.3 General Procedure for the Preparation of Azides 2.9a f .......................... 35 Azido Acids 2.11a e (2.11a+2.11a') ................................ ................................ ............................... 37 2.2.5 General Procedure for Preparation of Diazo Compounds 2.13a b .......... 39 3 BENZOTRIAZOL 1 YL SULFONYL AZIDE FOR PREPARATION OF AZIDOACYLBENZOTRIAZOLES ................................ ................................ ........... 40 3.1 Introduction ................................ ................................ ................................ ....... 40 3.2 Results and Discussion ................................ ................................ ..................... 43 Azido Acids ................................ ................................ .......................... 43 3.2.2 Preparation of N Azidoacyl)benzotriazoles ................................ ......... 43

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7 3.2.3 N Acylation ................................ ................................ .............................. 43 3.2.4 O S and C Acylation ................................ ................................ ........... 47 3.3 Conclusions ................................ ................................ ................................ ...... 49 3.4 Experimental ................................ ................................ ................................ ..... 49 3.4.1 General Methods ................................ ................................ ..................... 49 3.4.2 General Procedure for the Preparation of N Azidoacyl)benzotriazoles, 3.1a ................................ .............. 49 3.4.3 General Procedure for 3.3a k ................................ ................................ .. 52 3.4.4 General Procedure for 3.5a l ................................ ................................ ... 57 4 MICROWAVE ASSISTED REGIOSPECIFIC SYNTHESIS OF PSEUDOHALOHYDRIN ESTERS ................................ ................................ .......... 65 4.1 Introduction ................................ ................................ ................................ ....... 65 4.2 Results and Discussion ................................ ................................ ..................... 67 4.2.1 Optimization of reaction conditions for 4.4a. ................................ ............ 67 4.2.2 Synt (Benzotriazol 1 yl)ethyl esters 4.4 ................................ ... 69 4.3 Conclusions ................................ ................................ ................................ ...... 72 4.4 Experimental ................................ ................................ ................................ ..... 72 4.4.1 General Methods ................................ ................................ ..................... 72 4.4.2 General Procedure for the Preparation of N Acylbenzotriazoles 4.1 ....... 72 4.4 .3 General Procedure for the Preparation of (Benzotriazol 1 yl)ethyl Esters 4.4a I and 4.5 ................................ ................................ ..................... 76 5 SOLUTION PHASE SYNTHESIS OF CHIRAL O ACYL ISODIPEPTIDES ............ 82 5.1 Introductio n ................................ ................................ ................................ ....... 82 5.2 Results and Discussion ................................ ................................ ..................... 85 5.2.1 Synthesis of Serine based O Acylisodipeptides ................................ ...... 85 5.2.2 Synthesis of Threonine based O Acylisodipeptides ................................ 88 5.3 Conclusions ................................ ................................ ................................ ...... 90 5.4 Experimental ................................ ................................ ................................ ..... 90 5.4.1 General Methods ................................ ................................ ..................... 90 5.4.2 General Procedure for the Preparation of N (Pg Aminoacyl) Benzotriazoles 5.2 ................................ ................................ ......................... 90 5.4.3 General Procedure for the Preparation of O Acyl Isodipeptides 5.3 and 5.4 ................................ ................................ ................................ .......... 93 6 FREE CHEMICAL LIGATIONS FROM O ACYL SERINE SITES ................................ ................................ ................................ ..... 100 6.1 Introduction ................................ ................................ ................................ ..... 100 6.2 Results and Discussion ................................ ................................ ................... 104 6 O to N Acyl Shift via an eight membered TS ................................ ................................ ............................. 104 O to N Acyl Shift via an eleven membered TS ................................ ................................ ............................. 105

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8 O to N Acyl Shift ................................ ................................ ................................ ..... 107 6.3 Conclusions ................................ ................................ ................................ .... 108 6.4 Experimental ................................ ................................ ................................ ... 109 6.4.1 General Methods ................................ ................................ ................... 109 6.4.2 General Procedure for the Preparation of N (Z Aminoacyl)benzotriazoles 6.1' ................................ ................................ ..... 109 6.4.3 General procedure for the preparation of N (Boc aminoacyl)benzotriazoles 6.1' ................................ ................................ ..... 110 6. 4.4 General Procedure for the Preparation of Serine containing dipeptides 6.2a c ................................ ................................ ................................ .......... 111 6.4.5 General Procedure for the Preparation of O Acyl Isopeptides 6.3a c .... 112 6.4.6 General Procedure for the Preparation of O Acyl Isopeptides 6.4a c and 6.7a b ................................ ................................ ................................ ... 114 6.4.6.1 For deprotection of the Cbz protecting group .............................. 114 6.4.6.2 For deprotection of the Boc protecting group .............................. 114 6.4.7 General Procedure for the Preparation of O Acyl Isopeptides 6.6a b ... 116 ................................ ................................ 117 7 CONCLUSIONS AND SUMMARY OF ACHIEVEMENTS ................................ ..... 119 LIST OF REFERENCES ................................ ................................ ............................. 121 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 131

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9 LIST OF TABLES Table page 2 1 Optimization of reaction conditions for 2.7. ................................ ......................... 27 2 2 Synthesis of Azides from Primary Amines Utilizing 2.7. ................................ ..... 31 2 3 Synt Azido Acids 2.11a e,(2.11a + 2.11a') ................................ ......... 32 2 4 Synthesis of diazo compounds 2.13 Utilizing 2.7. ................................ ............... 33 3 1 Synthesis of N ( Azidoacyl)benzotriazoles 3.1 from Azido Acids 2.11. .......... 43 3 2 Synthesis of Amides 3.3 from N Azidoacyl)benzotriazoles 3.1. ..................... 45 3 3 O S and C Acylations Utilizing N ( Azidoacyl)benzotriazoles 3.1. ................ 48 4 1 Optimization of reaction conditions for the synthesis of 4.4a. ............................. 69 4 2 Synthesis of (Benzotriazol 1 yl)ethyl esters 4.4. ................................ ............. 70 5 1 The preparation of serine based O acyl isodipeptides 5.3a h. .......................... 87 5 2 The preparation of threonine based O acyl isodipeptides 5.4a h. ...................... 89

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10 LIST OF FIGURES Figure page 1 1 Number of publication w ith microwave irradiation. ................................ .............. 22 2 1 X ray of benzotriazol 1 sulfonyl azide 2.7. ................................ .......................... 28 2 2 Thermogravimetric analysis of benzotriazol 1 sulfonyl azide 2.7. ....................... 28 2 3 Differential scanning calorimetry of benzotriazol 1 sulfonyl azide 2.7: (a) cycle 1 and (b) cycle 2. ................................ ................................ ....................... 29 3 1 HPLC analysis for compounds 3.3a and 3.3g. ................................ .................... 47 5 1 Sheets. ................................ ................................ ................................ ............ 82 5 2 Pseudo prolines. ................................ ................................ ................................ 83 5 3 O Acyl isopeptide methodology. ................................ ................................ ......... 84 5 4 HPLC MS and ( )ESI MS analysis of 5.3b. ................................ ......................... 88 6 1 Peptide ligation. ................................ ................................ ................................ 101 6 2 Native Chemical Ligation. ................................ ................................ ................. 101 6 3 Auxiliary method. ................................ ................................ .............................. 102 6 4 O Acyl isopeptide methodology. ................................ ................................ ....... 103 6 2 Preor ganised conformer of O acyl peptides 6.4a. ................................ ............ 108

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11 LIST OF SCHEMES Scheme page 1 2 Synthesis of N acylbenzotriazole. ................................ ................................ ....... 21 2 1 Diazotransfer reagents. ................................ ................................ ...................... 26 2 2 Synthesis of benzotriazol 1 yl sulfonyl azide 2.7. ................................ ............... 26 2 3 Proposed mechanism for the formation of diazo compounds 2.13. .................... 33 4 1 Reported synthesis of a pseudohalohydrin ester from an N acylbenzotrizole and an epoxide. ................................ ................................ ................................ .. 66 4 2 Palladium catalyzed thermal reaction of N acylbenzotriazole 4.1a with epoxide 4.3a. ................................ ................................ ................................ ...... 67 4 3 Possible mechanism. ................................ ................................ .......................... 71 4 4 Acid halides and epoxides in halohydrin ester synthesis. 91 ................................ 71 4 5 Synthesis of pseudohalohydrin 4.5. ................................ ................................ .... 72 5 1 129 .............. 85 6 1 Preparation of O acyl isopeptides 6.4a c. ................................ ......................... 104 6 2 Chemical ligation of O acyl isopeptides 6.4a,c. ................................ ................ 105 6 3 Preparation of O acyl isopeptides 6.7a b and their ligation. ............................. 106

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12 LIST OF A BBREVIATIONS Alpha locant D Specific rotation A ngstrom(s) Ac A cetyl Ala Alanine Ar Aryl AZT 3 A zidothymidine Beta locant Bn Benzyl Boc t Butoxycarbonyl br Broad Bt Benzotriazol 1 yl C Carbon Degree Celcius Calcd Calculated Cbz Carboben zyloxy CDCl 3 Deuterated chloroform CTH Catalytic hydrogen transfer CuSO 4 .H 2 O Copper(II) sulfate pentahydrate Cys Cysteine Chemical shift in parts per million downfield from tetramethylsilane d D ays; Douplet (spectral) D Dextrorotatory (right) D BU 1,8 D iazabicyclo[5.4.0]undec 7 ene

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13 DCC N N' Dicyclohexylcarbodiimide DCM Dichloromethane DIPEA Diisopropylethylamine DMF Dimethylformamide DMSO Dimethylsulfoxide D 2 O Deuterium oxide EDC 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide (stands as an abbreviation for EDAC and EDCI as well) Et Ethyl et al. And others ESI Electrospray ionization Et 3 N Triethylamine EtOAc Ethyl acetate EtOH E thanol Equiv E quivalent(s) g Gram(s) Gly Glycine h Hour H Hydrogen HCl Hydrochloric acid HPLC High performance liquid chromatography HRMS High resolution mass spectrometry Hz Hertz IR Infrared J Coupling constant L Levorotatory (left)

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14 Leu Leucine Lit Literature m Multiplet M Molar Me Methyl MeCN Acetonitrile MeOH Methanol Met Methionine min Minute(s) mL milliliter MgSO 4 Magnesium sulfate mol Mole(s) mp Melting point MS Mass spectrometry/Mass spectra MW Microwave m/z Mass to charge ratio N Nitrogen Na 2 CO 3 Sodium carbonate NaOH Sodium hydroxide Na 2 SO 4 Sodium Su lfate NMR Nuclear magnetic resonance o Ortho locant O Oxygen OEt Ethoxy OH Hydroxyl group

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15 OMe methoxy p Para locant Pd Palladium Pd(OAc) 2 P alladium(II)acetate Pd(Ph 3 ) 4 Tetrakis(triphenylphosphine)palladium(0) Ph Phenyl Phe Phenylalanine PPh 3 Triphenylphosphine ppm Part per million Pro Proline Py Pyridine q Quartet R Rectus (right) ref. Reference rt Room temperature s Singlet S Si n ister (left) S Sulphur Ser Serine SOCl 2 Thionyl chloride SO 2 Cl 2 Sulfuryl chloride t Time; Triplet (spectral) t Tertiary TLC Thin layer chromatography TMS Trimethylsilane

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16 Trp Tryptophan Val Valine v/v volume per unit volume (volume to volume r atio) W Watt(s)

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17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NOVEL APPROACHES IN AZIDE AND PEPTIDE SYNTHESIS By Mirna El Khatib August 2012 Chair: Alan R. Katritzky Major: Chemistry The theme of this work is the development of novel methodologies for the efficient preparation of a variety of organic compounds. Chapter 1 presents a general overview o f the wor k pres ented in subsequent chapters together with a brief discussion of the importance of benzotriazole methodology in organic synthesis. Chapter 2 descri bes the role of benzotriazole for the synthesis of a novel diazotransfer reagent and examines th e util ity of the reagent in the preparation of organic azide targets. This methodology is also employed in C hapter 3, where the tolerance of azide as a protecting group is examined in various acylation reactions. Chapter 4 presents the use of the microwave in or ganic synthesis. This chapter reports on the palladium catalyzed reactions of N acylbenzotriazoles with epoxides affording pseudohalohydrin ester surrogates as single regioisomers. Chapter 5 focuses on the development of a mild protocol towards the synthes is of O acyliso dipeptides from serine and threonine amino acid residues using benzotriazole methodology. This mild protocol is visited again in C hapter 6, where it was employed in peptide ligations via large transition states Cysteine free microwave assis ted chemical ligation s are examined in this chapter using O acylpeptides from serine residues.

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18 Chapter 7 summarizes the achievements together with the conclusions.

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19 CHAPTER 1 GENERAL INTRODUCTION 1 H Benzotriazole 1 9 is an inexpensive, stable synthetic auxiliary that is soluble in many organic solvents, sparingly soluble in water and is highly soluble in basic solutions Since it is an acid of appreciable streng th ( pKa = 8.2 ), and a base (pKa = 1.6) benzotriazole can be readily removed from the reaction mixture by simply washing with a base or an acid 1 3 Benzotriazole displays the characteristics of a n ideal synthetic auxiliary and possesses both electron donor and electron acceptor properties 1 3 The application of benzotria zole in organic chemistry will be highlighted in many aspects in this thesis (Scheme 1 1). Scheme 1 1. Diverse application of benzotriazole highlighted in this thesis.

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20 The chemistry of azides s tarted with the preparation of the first organic azide, phenyl azide, the discovery of hydrazoic acid and Curtius rearrangement reported in 1890. 10 15 Since then several syntheses and applic ations of organic azides have been developed. These valuable intermediates have been used in the synthesis of various nitrogen containing heterocycles, in peptide chemistry, in azidonucleosides for the treatment of AIDS and for the preparation of bioconjug ates via Staudinger ligation. 16 18 In the course of investigations on the use of benzotriazole, C hapter 2 d emonstrates the use of benzotriazole in the sy nthesis of a novel diazotransfer reagent, benzotriazol 1 yl Chapter 3 examines the azido protecting group tolerance in various acylations using azidoacylbenzotriazoles. T he use of hetero cyclic acylazoles as acylating agents offer s many advantages 3 In particular, N acyl benzotriazole s are useful synthetic auxiliaries since they can be installed and removed readily. Benzotriazole is comparable in many ways to a halogen substituent because of its leaving group ability but regarded as a tame halogen substituent in vie w of its stability B enzotriazole can be acylated at N 1 or N 2 position In solution, it is substituted at the N 1 position. N A cylbenzotriazoles are: (i) solids (highly crystalline compounds), (ii) soluble in organic solvents and can be used in aqueous media (relatively stable to hydrolysis), (iii) non hygroscopic making them easy to handle and store, (iv) compatible with a wide range of functionality, (v) chirally stable for long periods and (vi) efficient neutral acylating agents where benzotriazole c an be easily recovered and recycled 3 5 19 More impo rtantly, they can be p repared directly from RCO 2 H in near quantitative yields

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21 (Scheme 1 2 ). Chapters 3 6 describe the synthesis and reactions of N acylbenzotriazoles demonstrating their application in microwave synthesis, azide and peptide chemistry. Scheme 1 2 Synthesis of N acylbenzotriazole. The use of microwave (MW) activation is of great interest as a non conventional energy source accelerating a wide range of organic reactions. 20 21 23 It is described as a green eco friendly approach since many reactions are run under solvent free conditions, with reduced reaction times enhanced conversions and sometimes selectivity. The number of publications using microwa ve irradiation in organic synthesis is growing exponentially (Figure 1 1 ). 20 21 23

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22 Figure 1 1 Number of publication with microwave irradiation. 24 25 M ost organic reactions have been performed using conventional heat transfer equipment such as oil baths, sand baths and heating jackets. However, thes e methods are rather slow and a temperature gradient can develop within the sample. In addition, local overheating may cause decomposition of the product, substrate or reagent. In contrast, in microwave dielectric heating, the microwave energy is introduce d into the chemical reactor remotely. The microwave radiation passes through the walls of the vessel and heats only the reactants and solvent, not the reaction vessel itself. 24 25 Chapters 4 and 6 de monstrate the application of microwave in the synthesis of pseudohalohydrin surrogates and in chemical ligations of peptides. Scientists have long sought to under stand how the structure of a protein molecule gives rise to its functional properties. Thus, the chemical synthesis of peptides and proteins is of great importance. A mild protocol towards the synthesis of chirally pure O acyldipeptides is described in C ha pter 5. This mild protocol is utilized in Chapter 6 for O acylpe ptides using serine

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23 Chapter 7 presents a summary of achievements together with conclusions.

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24 CHAPTER 2 BENZOTRIAZOL 1 YL SULFONYL AZIDE i 2.1 Introduction Organic azides 11 12 17 26 discovered by Peter Grie more than 140 years ago, play an important role at the interface between chemistry, biology, medi cine, and material science. These nitrogen rich molecules have been utilized : (i) as building blocks 12 27 28 exemplified by the synthesis of natural products 29 33 (ii) in photoaffinity labeling, 34 41 (iii) as drugs, such as anti HIV medication ( 3 azidothymidine or zidovudine, AZT), 42 and (iv) as masked amines as in the synthesis o f oseltamivir phosphate Tamiflu 12 43 Aliphati c azides 12 43 can be prepared by classical nucleophilic displacement (S N 2 type) with a highly nucleophilic azide anion 44 45 This is commonly performed with sodium azide (or other alkali azides, tetraalkylammonium azides, polymer bound azides or highly explosive silver azides) that displaces a good leaving group ( e.g. a halide, carboxylate sulfonat e, mesylate, nosylate or triflates) in a polar aprotic solvent. H owever such reactions at sp 3 carbon may cause inversion, epimerization, 46 or concurrent elimination and solvents such as DMF and DMSO can hinder iso lation of the azide product. O rganic azides can also be prepared by: (i ) reactions of aryldiazonium salts with inorganic azides; 47 (ii ) catalyzed c ross coupling of between sodium azide and aryl and vinyl boronic acids, 48 (iii ) catalyzed coupling reaction of aryl halides and vinyl halides with sodium azide 49 or (iv) diazo transfer of primary amines. i Reproduced with permission from The Journal of Organic Chemistry 2010 75 6532 6539 Copyright 201 0 American Chemical Society and E eros, Encyclopedia of Reagents for Organic Synthesis 2011 DOI:10.1002/047084289X.rn01342. Copyright 2011 John Wiley & Sons, Inc.

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25 P repa ration of azid es from amines by diazo transfer 50 avoids epimerization, inversion, and elimination. The conversion occurs efficiently in the presence of a catalytic amount of d ivalent metal ion that is believed to complex with the amine before attacking the electrophilic azide 50 53 An ideal diazotransfer reagent should be crystalline (for ease of purification, handling 54 and stability), nonexplosive, easily prepared, and of general applicability for diazotransfer. p Tosyl azide ( 2.1 ) the classical diazotransfer reagent, melts at 21 22 o C, 55 and requires relatively harsh conditions that limit its use. 56 Suggested replacements reported in the literature include : (i) mesyl azide ( 2.2 ) 57 an oil needing distillation at 56 o C (0.5 mm. Hg); (ii) polystyrene supported benzenesulfonyl azide ( 2.3 ) 58 a safe to handle but insoluble resin; (iii) oligomer bound benzenesulfonyl azide ( 2.4 ) 59 which is insoluble in most organic solvents, lacks long term stability, and needs to be utilized within 1 2 weeks; (iv) imidazole 1 sulfonyl azide ( 2.5 ) 50 54 a colorless oil used as crystalline hydrochloride salt; and (v) the transfer 3 2.6 ), 43 50 53 54 56 prepared from sodium azide and trifluoromethanesulfonic anhydride, which has a poor shelf life and must be used in situ as a solution because of its explosive nature (Scheme 2 1) 48 Thus, the synthesis of an improved diazotransfer reagent is of considerable interest. The preparation of benzotriazol 1 sulfonyl azide, 2 7 as an improved novel diazo donor is described (Scheme 2 2 ) 51 52

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26 Scheme 2 1. Diazotransfer reagents. 2.2 Results and Discussion 2.2.1 Pr eparation and Characterization of Benzotriazol 1 yl sulfonyl Azide 2 7 The react ion of chlorosulfonyl azide, prepared in situ from sodium azide and sulfuryl chloride, with benzotriazole (2 equiv) and pyridine (1 equiv) in MeCN ga ve benzotriazol 1 yl sulfo nyl azide 2 7 (70%, obtained after aqueous workup) as a white crystalline solid (mp 85.3 88.3 o C) requiring no further purification (Scheme 2 2 ). These conditions proved to be optimal. Initial investigation of the effect of various parameters toward reacti on optimization has shown that in the absence of pyridine, the reaction needed 2.5 days and gave 45% of 2 7 The use of other bases and solvent systems resulted in either partial or complete decomposition products (Table 2 1). Scheme 2 2 Synthesis of b enzotriazol 1 yl sulfonyl azide 2. 7

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27 Table 2 1. Optimization of reaction conditions for 2.7 Entry Reaction conditions Yield of 2. 7 (%) Decomposition a 1 Acetonitrile, BtH (2 equiv), 2.5 d 45 None 2 Acetone, BtH (2 equiv), 12h Decomposed 3 Acetonitrile, BtH (2 equiv), Et 3 N (2 equiv), 4h Decomposed 4 Acetonitrile, BtH (2 equiv), Et 3 N (1 equiv), 4h Decomposed 5 Acetonitrile, BtH (2 equiv), py (1 equiv), 12h 70 None a Determined by TL C and 1 H NMR. R eagent 2. 7 as a dry solid has a long shelf life at room temperature and can be ut ilized several months after its preparation. 1 H NMR on 2.7 after s torage in a closed clear glass vial at room temperature for a period of 6 weeks and even for s everal months, revealed no decomposition thus demonstrating its longevity. A moderate exothermic decomposition was noted when the hammer test was performed. Although no trouble was experienced when utilizing the reagent, appropriate safety measures must be taken at all times with high energy azides. 12 43 51 52 60 [An acidic workup should be avoided because of the possible formation of explosive hydrazoic acid resulting from trace amounts of residual sodium azide Procedure misuse has resulted in an explosion by a visiting research scholar ( in our laboratories ) ] Reagent 2. 7 is soluble in many organic solvents as well as in partially aqueous media (e.g., MeCN, CH 2 Cl 2 MeOH, EtOAc, MeCN/H 2 O (1:1)). The detaile d molecular structure of benzotriazol 1 yl sulfonyl azide 2. 7 was established by X ray diffraction analysis (Figure 2 1). Thermal properties of 2.7 were studied by TGA and DSC. Thermogravimetric analysis (TGA) shows that 64 wt% of 2. 7 is lost around 112 o C (Figure 2 2) Differential scanning calorimetry (DSC) shows that

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28 2. 7 is thermally stable below 95 o C melting and resolidifying (see Figure 2 3 which shows 2 cycles of heating to 95 o C and cooling to 100 o C). The heats of fusion (166.7 J/g for cycle 1 a nd 163.8 J/g for cycle 2) and heats of freezing (114.1 J/g for cycle 1 and 101.4 J/g for cycle 2) show that there is negligible material loss Figure 2 1. X ray of b enzotriazol 1 sulfonyl a zide 2. 7 Figure 2 2. T hermogravimetric analysis of benzotria zol 1 sulfonyl a zide 2. 7

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29 (a) (b) Figure 2 3. D ifferential scanning calorimetry of benzotriazol 1 sulfonyl a zide 2. 7 : (a) cycle 1 and (b) cycle 2.

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30 2.2.2 Preparation of Azides from Primary Amines Benzotriazol 1 yl sulfonyl azide 2. 7 was found to be an efficient diazo donor reagent with various aliphatic and aromatic amines. BtSO 2 N 3 2.7 converted amine compounds 2.8 a f into the corresponding azides 2.9 a f (in 47 85% yields), without requiring a base. In a typical reaction, benzotriazol 1 yl sulfonyl azi de 2. 7 reacted with an amine in methanol at room temperature in the presence of copper(II) sulfate (Table 2 2 ). It is noteworthy that the reaction also works in absence of catalyst ; for example, the reaction of 2. 7 with p methoxyphenylamine 2.8 a without ca talyst gave p methoxyphenyl azide 2.9 a (57%) after 24 h.

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31 Table 2 2. Synthesis of Azides from Primary Amines Utilizing 2. 7 E ntry S ubstrate 2 8 P roduct 2. 9 T ime (h) Y ield (%) 1 2. 2.8 a 2 .9 a 7 70 2 2.8 b 2 .9 b 12 75 3 2.8 c 2 .9 c 10 57 4 2.8 d 2 .9 d 8 47 5 2.8 e 2 .9 e 12 85 6 a 2.8 f 2 .9 f 7 51 a Et 3 N (1 equiv) was required. 2.2.3 Preparation of Azido Acids Reagent 2. 7 amino acids 2. 10 azido acids 2. 11 without any observable racemization. This mild transformation was achieved when 2. 7 was reacted with free amino acids 2.10 a e ( 2. 10 a + 2. 10 a') at 20 o C in a queous CH 3 CN in the presence of Et 3 N and catalytic amount of copper(II) sulfate to give corresponding azido acids 2.11 a e (2.11a + 2.11 a') in good yield (60 87%)(Table 2 3 ).

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32 Table 2 Azido Acids 2.11 a e (2.11a + 2.11 a') HPLC analysis [chirobiotic T column (250 mm 4.6 mm), detection at 254 nm, flow rate 0.5 mL/min, MeOH] on 2. 11 a (single peak, retention time 7.2 min) and (2.11a + 2.11 a') (two equal peaks, retention times 6.7 and 7.2 min) confirmed that product 2. 11 a is enantiomerical ly pure. The mechanistic details of this interconversion are not well established. A proposed mechanism for diazotransfer involves a tetra zole intermediate and incorporates a divalent metal ion that complexes with the amine. The amine in this complex is th en thought to attack the electrophilic azide. 50 2.2.4 Preparation of Diazo Compounds Diazo compounds a re versatile synthetic building blocks 61 with rich transition metal catalyzed chemistry. 62 64 Thus, carbene insertion into C H bon ds has increased in e ntry s ubstrate 2. 10 Product 2. 11 yield (%) 1 L Phe, 2 .10 a 2 .11 a 65 2 L Leu, 2 .10 b 2 .11 b 65 3 L Ala, 2 .10 c 2 .11 c 60 4 D L Phe, (2.10 a+2 .10 a') ( 2 .11 a+ 2 .11 a') 65 5 L Val, 2 .10 d 2 .11 d 87 6 ( L Cys) 2 2 .10 e 2 .11 e 77

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33 importance since its discovery by Meerwein and Werner. 65 We utilized benzotriazol 1 yl sulfonyl azide 2. 7 in the preparation of diazo compounds 2. 13 a,b containing activated methylene groups, and the results are summarized in Table 2 4 In general, yield s and reaction times compare favorably with those reported in the literature using the most recent diazotransfer reagent, imidazole 1 sulfonyl azide hydrochloride ( 2.5 .HCl) 54 Table 2 4. Synthesis of diazo c ompounds 2 .1 3 Utilizing 2.7 Product R R' time [lit] a (h) yield [lit] a (%) 2.13 a CN CO 2 Et 12[9] 65[61] 2.13 b SO 2 Ph CO 2 Et 14[48] 56[ ] b a Reaction with imidazole 1 sulf onyl azide ( 2.5 ) b No reaction was observed using imidazol e 1 sulfonyl azide ( 2.5 .HCl ) 16 I believe that the fo rmation of diazo compounds from activated CH 2 groups via diazotransfer occurs through nucleophilic attack of an intermediate enolate 2.14 onto b enzotriazol 1 ylsulfonyl azide 2. 7 followed by proton ation to give intermediate 2.15 In the presence of base, intermediate 2.16 is formed, which fragments to the desired diazo product 2.13 (Scheme 2 3 ). Scheme 2 3 Propo sed m echanism for the formation of diazo compounds 2.13

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34 2.3 Conclusions Benzotriazol 1 yl sulfonyl azide 2.7 is a new, thermally stable, and safe to handle crystalline diazotransfer reagent with a long shelf life and high solubility in organic and aqueo us solvents, which allows convenient and efficient synthesis of a wide range of azides and diazo compounds. 2.4 Experimental 2.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are unc orrected. The NMR spectra were recorded in CDCl 3 DMSO d 6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Silica was used as the stationary phase for column chromatography. Phosphomolybdic acid was used to detect compounds that were no t UV active. 2.4.2 Preparation o f B e nzotriazol 1 yl Sulfonyl A zide 2.7 Sulfuryl chloride (6.23 mL, 0.08 mol) was added portion wise to a suspension of NaN 3 (5 g, 0.08 mol) in MeCN (25 mL) at 0 o C, and the mixture was stirred overnight at room temperature. Benzotriazole (18.35 g, 0.15 mol) was dissolved in pyridine (6.46 mL, 0.08 mol) and acetonitrile (10 mL), and the solution was added to the suspension at 0 o C. The resulting suspension was stirred for 10 h at room temperature. The unreacted solid was filt ered, and the yellow orange filtrate evaporated and diluted with e thyl ace tate (~30 mL) The organic layer was washed with a saturated solution o f sodium carbonate to remove excess benzotriazole, dried over anhydrous MgSO 4 and filtered. The filtrate was c oncentrated under reduced pressure to give a light brown solid that was later used directly. Recrystallization using hexane/EtOAc 7:3 gave a white crystalline solid 2.7 (12.5 g, 70%); mp 85.3 88.3 o C; 1 H NMR (300 MHz, CDCl 3 ) : 8.17

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35 (d, J = 8.4 Hz, 1H), 7 .91 (d, J = 8.4 Hz,1H), 7.72 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H); 13 CNMR (75 MHz, CDCl 3 ) : 145.3, 131.6, 126.9, 121.3, 112.1. Anal. Calcd for C 6 H 4 N 6 O 2 S: C, 32.14; H, 1.80; N, 37.48. Found: C 32.18 ; H, 1.74; N, 37.27. NOTE: 1 H NMR on reagent 2.7 a fter storage in a closed clear glass vial at room temperature for a period o f 6 weeks, showing no decomposition A moderate exothermic d ecomposition was noted when the hammer test was performed on 2.7 [Caution: Although no trouble was experienced when uti lizing sodium azide, the in situ generated chlorosulfonyl azide, or any of the synthesized organic azides, appropriate safety measures must always be taken at all times because azides are often found to be high energy compounds. 10 12 51 52 60 ] 2.4.3 General P rocedur e for the P reparation of A zides 2.9 a f Benzotriazol 1 yl sulfonyl azide 2.7 (0.50 g, 2.23 mmol) was added to the amine (2.23 mmol) in MeOH (20 mL). CuSO 4 5H 2 O (2.5 the mixture was stirred at room temperature for the specified time (Table 2 1 ). The mixture was concentrated, diluted with water (20 mL), and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO 4 f iltered, and concentrated. Purification was performed via flash column chromatography to give the corresponding azide 2.9 1 Azido 4 methoxybenzene ( 2.9 a ). 4 Methoxyaniline (275 mg, 2.23 mmol) was treated according to the above procedure [flash chromatogra phy (hexane/EtOAc, 9:1)] to give 1 azido 4 methoxybenzene 2.9a as a pale yellow oil (233 mg, 70%); 1 H NMR (300 MHz, CDCl 3 ) : 6.98 6.87 (m, 4H), 3.80 (s, 3H); 13 CNMR (75 MHz, CDCl 3 ) : 156.9, 132.3, 120.0, 115.1, 55.6.

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36 1 Azido 4 bromobenzene ( 2.9 b ). 4 Bromoaniline (384 mg, 2.23 mmol) was treated according to the above procedure [flash chromatography (hexane)] to give 1 azido 4 bromobenzene 2.9 b as a pale yellow oil (332 mg, 75%) ; 1 H NMR (300 MHz, CDCl 3 ) : 7.47 7.44 (m, 2H), 6.92 6.88 (m, 2H); 13 C NMR (75 MHz, CDCl 3 ) : 139.2, 132.7, 120.6, 117.7. 1 Azido 3 nitrobenzene ( 2.9 c ). 3 Nitroaniline (308 mg, 2.23 mmol) was treate d according to the above procedure [flash chromatography (hexane/EtOA c, 9.8:0.2)] to give 1 azido 3 nitrobenzene 2.9c as a yellow solid (209 mg, 57%); mp 53.1 54.5 o C; 1 H NMR (300 MHz, CDCl 3 ) : 8.01 7.98 (m,1H), 7.89 (t, J = 2.1 Hz, 1H), 7.54 (t, J = 8.1 Hz, 1H), 7.35 (dd, J = 1.8, 7.5 Hz, 1H); 13 C NMR(75 MHz, CDCl 3 ) : 141.9, 130.6, 124.9, 119.7, 114.1. 3 Azidopyridine ( 2.9d ). Pyridin 3 amine (210 mg, 2.23 mmol) was treated according to the above procedure [flash chromatography (hexane)] to give 3 azi dopyridine 2.9d as a pale yellow oil (126 mg, 47%); 1 H NMR(300 MHz, CDCl 3 ) : 8.41 8.35 (m, 2H), 7.38 7.27 (m, 2H); 13 C NMR (75 MHz, CDCl 3 ) : 145.8,141.1, 137.0, 125.8, 124.0. (2 Azidoethyl)benzene ( 2.9 e ). 2 Phenylethanamine (270 mg, 2.23 mmol) was tr eated according to the above procedure [chromatography(hexane)] to give (2 azidoethyl)benzene 2.9 e as colorless oil (279 mg, 85%) ; 1 H NMR (300 MHz,CDCl 3 ) : 7.38 7.23(m, 5H), 3.52 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.2 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 ) : 137.9, 128.6, 128.5, 126.7, 52.4, 35.3. (1S,2S,3S,5R) 3 (Azidomethyl) 2,6,6 trimethylbicyclo[3.1.1] heptanes ( 2.9 f ). (+ ) 3 Pinanemethylamine hydrochloride (454 mg, 2.23 mmol) and triethyl amine (1 equiv)

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37 were treated according to the above procedure [flash chromatography (hexane)] to give (1 S ,2 S ,3 S ,5 R ) 3 (azidomethyl) 2,6,6 trimethylbicyclo [3.1.1]heptanes 2.9 f as a yellow oil (220 mg, 51%); [ ] D 20 + 6.9 ( c 1.5, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 3.32 (dd, J = 5.7, 11.7 Hz, 1H), 3.18 (dd, J = 7.5, 11.7 Hz, 1H), 2.35 2.26 (m, 1H), 2.22 2.12 (m, 1H), 2.04 1.89 (m, 2H), 1.81 1.68 (m, 2H),1.59 1.51 (m, 1H), 1.20 (s, 3H), 1.07 (dd, J = 1.5, 7.2 Hz, 3H),1.01 (s, 3H); 13 C NMR(75 MHz, CDCl 3 ) : 59.6, 47.7, 41.3, 40.5, 38.8, 36.5, 33.5, 32.2, 27.9, 22.9, 21.6; HRMS m/z for C 11 H 20 N [M N 2 + H] + calcd 166.1590, found 166.1593. 2.2.4 General P rocedure for t he P reparation of A zido Acids 2.11 a e ( 2.11 a +2.11a' ) Each amino acid (4.46 mmol, 2 equiv) was dissolved in MeCN/H2O (1:1, 20 mL) and triethyl amine (0.78 mL, 5.58 mmol). Benzotriazol 1 yl sulfonyl azide 2.7 (0.50 g, 2.23 mmol) was added to the solution followed by CuSO 4 5H 2 O(2.5 mg, 10 the mixture stirred at room temperature for 12 h. The mixture was acidified with 6N HCl, concentrated, diluted with ethyl acetate and washed with 6N HCl to remove benzotriazole. The organic layer was collected, dried over anhydrous MgSO 4 filtered, and con centrated to give the corresponding azido acid 2.11 (2S) 2 Azido 3 phenylpropanoic Acid ( 2.11a ). L Phenylalanine (736 mg, 4.46 mmol) was treated according to the above procedure to give (2 S ) 2 azido 3 phenylpropanoic acid 2.11a as a pale yellow oil (278 mg, 65%) ; 1 H NMR (300 MHz, CDCl 3 ) : 9.90 (s, 1H), 7.38 7.27 (m, 5H), 4.15 (dd, J = 4.8, 9.0 Hz, 1H), 3.22 (d, J = 5.1 Hz, 1H), 3.08 3.03 (m, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 175.7, 135.6, 129.2, 128.8, 127.4, 63.1, 37.5.

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38 (S) 2 Azido 4 methylpentanoic Acid ( 2.11 b ). L Leucine (586 mg, 4.46 mmol) was treated according to the above procedure to give ( S ) 2 azido 4 methylpentanoic acid 2.11b as a pale yellow oil (228 mg, 65%); 1 H NMR(300 MHz, CDCl 3 ) : 8.95 (s, 1H), 3.88 (dd, J = 5.7, 9.0 Hz, 1H), 1.89 1.77 (m, 1H), 1.76 1.64 (m, 2H), 1.00 0.96 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 177.0, 60.0, 39.8, 25.0, 22.7, 21.4. (S) 2 Azidopropanoic Acid ( 2.11c ). L Alanine (396 mg, 4.46 mmol) was treated according to the above procedure to give ( S ) 2 azidopropanoic acid 2.11c as a yellow oil (154 mg, 60 %) ; 1 H NMR (300 MHz, CDCl 3 ) : 4.56 (s, 1H), 4.03 (q, J = 7.1 Hz, 1H), 1.54 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) : 177.0, 57.0, 16.6. 2 Azido 3 phenylpropanoic Acid ( 2.11a+2.11a' ). D,L Phenylalanine (736 mg, 4.46 mmol) was treated according to the above procedure to give 2 azido 3 phenylpropanoic acid ( 2.11a+2.11a' ) as a pale yellow oil (278 mg, 65%); 1 H NMR (300 MHz, CDCl 3 ) : 7.38 7.25 (m, 5H), 4.17 (dd, J = 5.0, 8.9 Hz,1H), 3.25 (dd, J = 5.0, 14.0 Hz, 1H), 3.05 (dd, J = 8.9, 14.0 Hz, 1H); 13 C N MR (75 MHz, CDCl 3 ) : 175.6, 135.5 129.2, 128.8, 127.4, 63.0, 37.5. (S) 2 Azido 3 methylbutanoic Acid ( 2.11 d ). L Valine (523 mg, 4.46 mmol) was treated according to the above procedure to give ( S ) 2 azido 3 methylbutanoic acid 2 .11 d as a yellow oil (277 mg 87%); 1 H NMR(300 MHz, CDCl 3 ) : 11.8 (s, 1H), 3.76 (d, J = 5.7 Hz, 1H), 2.27 2.17 (m, 1H), 0.99 (d, J = 6.9 Hz, 3H), 1.0 4 (d, J = 6.9 Hz, 6H); 13 C NMR(75 MHz, CDCl 3 ) : 176.5, 67.8, 30.8, 19.3, 17.6. 2 Azido 3 2 azido 2 carboxyethyl)disulfanyl)pro panoic Acid ( 2.11 e ). L Cystine (268 mg, 1.12 mmol) was treated according to the above procedure to give 2 azido 3 ((( ) 2 azido 2 carboxyethyl)disulfanyl)propanoic acid 2.11 e as an orange

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39 brown oil (252 mg, 77%); [ ] D 20 37.7 ( c 1.3, CHCl 3 ); 1 H NMR (300 MH z, CDCl 3 ) : 7.59 (bs, 2H), 4.43 (dd, J = 5.0, 7.1 Hz, 2H), 3.32 (dd, J = 5.0, 14.0 Hz, 2H), 3.03 (dd, J = 7.2, 13.8 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 174.4, 60.8, 39.9; HRMS m/z for C 6 H 8 O 4 N 6 S 2 Na [M+Na] + calcd 314.9941, found 314.9940. 2.2.5 General P roc edure for P repar ation of D iazo C ompounds 2.13a b Pyridine (3.5 equiv) was added to the substrate 2. 12 (1 equiv) in MeCN (15 mL) and was stirred for 30 minutes before adding benzotriazol 1 yl sulfonyl azide 2.7 (0.50 g, 2.23 mmol). The mixture was stirred for the specified time (Table 2 3 ), concentrated under reduced pressure acidified with 4 N hydrochloric acid and extracted with ethyl acetate (50 mL) T he extracts were dried over anhyd MgSO 4 filtered and concentrated under reduced pressure Purification was performed via flash column chromatography to give the corresponding diazo compound 2.13 Ethyl Cyanodiazoacetate ( 2.13 a ) Ethyl cyanoacetate 2.12 a (0.2 mL, 2.23 mmol) was treated according to the above procedure, using pyridine (0.62 mL, 7.81 mmol) [fl ash chromatography (hexane:EtOAc, 9.8:0.2)], to give ethyl cyanodiazoacetate 2. 13a as a yellow oil (0.20 g, 65%); 1 H NMR (300 MHz, CDCl 3 ): 4.35 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ): 107.3, 63.5, 29.7, 14.3. Ethyl 2 dia zo 2 (phenylsulfonyl)acetate ( 2.13b ) Ethyl 2 (phenylsulfonyl)acetate 2.12 b (0.51 mg, 2.23 mmol) was treated according to the above procedu re, using pyridine (0.62 mL, 7.81 mmol) [flash chromatography (hexane:EtOAc, gradient)], to give ethyl 2 diazo 2 (phenylsulfonyl)acetate 2. 13 b as an orange solid (0.32 g, 56%); mp 52.0 54.0 o C; 1 H NMR (300 MHz, CDCl 3 ): 8.04 8.00 (m, 2H), 7.69 7.63 (m 1H), 7.58 7.53 (m, 2H), 4.21 (q, J = 6.9 Hz, 2 H), 1.24 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ): 159.5, 141.6, 134.0, 129.1, 127.8, 62.3, 14.1.

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40 CHAPTER 3 BENZOTRIAZOL 1 YL SULFONYL AZIDE FOR P REPARATION OF AZIDOACYLBENZOTRIAZO LES ii 3.1 Introduc tion Chemical synthesis is a powerful method for creating complex molecules with tailored biological and physical properties for drug discovery, engineering, nanotechnology, and the study of biological processes 66 72 and is based on the concourse of reagents and catalysts to att ain the clean formation of new bonds S uitable protecting groups are also often required to avoid the formation of undesired bonds and side r eactions. 73 74 Although, t he necessity to temporarily mask a functional group was first recognized by Emil Fischer, 75 it was not until 1932 that Bergmann and Zervas reported ycarbonyl (Z). 76 The use of protecting groups influe nce s the length, efficiency and complexity of the synthesis and are often responsible for its success or failure 68 In choosing a protecting group, the following characteristics should be kept in mind: (i) should be easily introduced into the functional group under mild conditions, in a selectiv e manner and in high yield (giving no additional stereocenters ) ; (ii) should be stable to a broad range of reaction conditions with a stabilizing effect on the molecule and s hould suppres s racemization or epimerization; and (iii) should be easy to remove at the end of the synthetic process or when the functional group requires manipulation Additionally other protecting groups present in the molecule and unprotected functiona lities should not be affe cted by the cleavage conditions 68 74 ii Reproduced with permission from The Journal of Organic Chemistry 2010 75 6532 6539 Copyright 2010 American Chemical Society

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41 O rthogonality, the concept of having two or more protecting groups that belong to independent classes and remove d by distinct mechanism s is of consideration in designing the retrosynthesis of a main target. 68 74 The differential protection of functional groups of comparable reactivity is a major challenge with conventional protecting group strategies, namely orthogonal protection and modula ted lability. In particular, the development of effective protective schemes for polyfunctional molecules is not trivial. 68 77 79 Chiral, nonracemic na tural products, including amino acids, have long been utilized as building blocks for organic synthesis. 80 Amino groups in peptide syntheses need protection 73 which is required to prevent polymerization of the amino acid once it is activated. 51 52 74 Azides have found application sensitive substrates such as oligosaccharides, aminoglycoside antibiotics, 12 27 28 glycosoaminoglycans, 8 1 and peptidonucleic acids (PNA) 82 and in solid phase pepti de synthesis. 83 In azido acyl groups the azide both masks the amine functionality and strongly activates the carboxyl moiety, 84 thus facilitating the formation of peptide bonds. 3 The small size of the azide unit in comparison to, e.g., Boc or Fmoc may be advantageous in the coupling of hindered compounds The azide group is stable under both ac idic and basic conditions and toward osmium 12 85 and ruthenium catalyzed dihydroxylation or alkylation. 12 86 Choice of activation for azido acid is important: (i) acyl halide s tend to be over activated 1 87 and require base for neutralizin g the hydrogen halide fo rmed; 88 (ii )

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42 acid anhydrides easily form imides with ammonia and primary amines; (iii) esters are frequently under activated and require basic cata lysts and/or high pressure. 1 87 T he use of acylazoles as acylating agents reported by offer several advantages 3 In particular, benzotriazole is an inexpensive, stable synthetic auxiliary that is soluble in many organic solvents (e.g. ethanol, benzene, toluene,chloroform, and DM F ). It is sparingly soluble in water but highly soluble in basic solutions. 3 I t is noteworthy that for a synthetic auxiliary group to be useful, it should demonstrate the following main characteristics: (i) be easy to remove at the end of the synthetic sequence (it is an added advantage if it can be recovered and used again); (ii) be able to be introduced readily at the beginning of the sequence ; and (iii) should be stable during various synthetic operations, and, if possible, exert an activating influence on other parts of the molecule. Benzotriazole displays all of these character istics to a high degree and possesses both electron donor and electron acceptor properties 1 3 N A cylbenzotriazoles are efficient neutral acylating agents and f orm amide bonds at ambient temperatures with unprotected amino acids in aqueous/organic solvents resisting side reactions in the preparation of N terminal protected peptides. 3 5 19 Thus N (protected aminoacyl)benzotriazoles have enabled fast preparations of biologically relevant peptides and peptide conj ugates in high yields and purity, under mild reaction conditions, with full retention of the original chirality. 89 Herein, the functional group tolerance of azido, as a protecting group, in N O S and C acylations is examined T he synthesis and the reliability of N ( azidoac yl)benzotriazoles as acylating agents is reported

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43 3.2 Results and Discussion 3 .2.1 Azido Acids Reagent 2.7 amino acids 2. 10 azido acids 2. 11 without any racemization at 20 o C in aqueous CH 3 CN in the presence of Et 3 N and copper(II) sulfate in good yield s (60 87%)(chapter 2, Table 2 3 ). 51 52 3 .2 .2 Preparation of N ( Az idoacyl) benzotriazoles N Azidoacyl)benzotriazoles 3.1a d (3.1a+3.1') were prepared in good yields (65 98%) by the treatment of the corresponding azido acids 2. 11 with 1.2 equiv of thionyl chloride and 2 equiv of benzotriazole in methylene chloride ( Table 3 1 ). Table 3 1 Synthesis of N ( Azidoacyl)benzotriazoles 3.1 from Azido Acids 2.11 azido acids, 2.11 product 3.1 yield (%) mp ( o C) N 3 L Phe, 2.11 a 3.1 a 98 66.1 68.3 N 3 L Leu, 2.11b 3.1 b 96 47.3 49.0 N 3 L Ala, 2.11 c 3.1 c 65 77.0 77.9 N 3 DL Phe, ( 2.11a+2.11 a') (3.1a+3.1 a') 98 Oil N 3 L Val, 2.11 d 3.1 d 72 Oil 3 .2 .3 N Acylation The reliability of N ( azidoacyl)benzotriazoles as acylating agents was tested on a variety of N nucleophiles 3.2 to provide ami des 3. 3 a k in 62 87% yields (Table 3 2 ). The azide demonstrated its functional group tolerance as a protecting group in N

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44 acylation of aromatic, aliphatic amines and free amino acids (including free cysteine), nucleobases, nucleosides, and sulfonamides. HP LC analysis [chiracel OD H column (250 mm 4.6 mm), detection at 254 nm, flow rate 0.5 mL/min, hexane/isopropyl alcohol (90:10)] on 3.3 a (single peak, retention time 50.8min) and 3.3 g (two equal peaks, retention times 47.6 and 51.4 min) confirmed that pro duct 3.3 a is enantiomerically pure (Figure 3 1 ) ; this result was further confirmed by a co injection Entries 9 and 10 (Table 3 2 ) were performed without added base with the objective of forming S acylated products according to a reported literature method by our group where S acylation was performed on aryl N acylbenzotriazoles. 90 As expected S acylation occurs first giving the thioester product, followed by S to N shift to provide the amide linkage. Interestingly, in this case ligation occurs spontaneously providing the N acylated dipeptides containing free thiol in the absence of base.

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45 Table 3 2 Synthesis of Amides 3. 3 from N ( Azidoacyl)benzotriazoles 3.1 entry N azidoacyl) benzotriazole, 3.1 N Nu, 3.2 optimized reaction conditions product, 3.3 yield (%) [m.p. ( o C)] 1 N 3 L PheBt, 3.1 a p Anisidine, 3.2 a MeCN, 12 h 3.3 a 79 [79 81] 2 N 3 L LeuBt, 3.1 b p Anisidine, 3.2 a MeCN, 12 h 3.3 b 84 [43 45] 3 N 3 L LeuBt, 3.1 b Adenine, 3.2 b DMSO, 8 h 3.3 c 68 [187 188] 4 N 3 L PheBt, 3.1 a p Toluene sulfonamide, 3.2 c Et 3 N (1.1 eq uiv), MeCN, 12 h 3.3 d 79 [120 122]

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46 Table 3 2 Synthesis of Amides 3. 3 from N ( Azidoacyl)benzotriazoles 3.1 (Continued) 5 N 3 L LeuBt, 3.1 b p Toluene sulfonamide, 3.2 c Et 3 N (1.1 equiv), MeCN, 8 h 3.3 e 77 [55 56] 6 N 3 DL PheBt, ( 3. 1 a+ 3.1 a ) Cytidine, 3.2 d D MF, 12 h 3.3 f 62 [glassy solid] 7 N 3 DL PheBt, ( 3 .1 a+ 3.1 a ) p Anisidine, 3.2 a MeCN, 12 h 3.3 g 79 [oil] 8 N 3 L PheBt, 3.1 a L Leu, 3.2 e H 2 O:MeCN (1:1), Et 3 N (2.5 equiv) N 3 L PheL Leu 3.3 h 87 [oil] 9 N 3 L PheBt, 3.1 a L Cys, 3.2 f H 2 O:MeCN (1:1) N 3 L PheL Cys 3.3 i 80 [112 115] 10 N 3 L LeuBt, 3.1 b L Cys, 3.2 f H 2 O:MeCN (1:1) N 3 L LeuL Cys 3.3 j 83 [oil] 11 N 3 L PheBt, 3.1a L Ala, 3.2 g H 2 O:MeCN (1:1), Et 3 N (2.5 equiv) N 3 L PheL Ala 3.3 k 80 [48 50]

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47 Figure 3 1. HPLC analysis for compounds 3.3a and 3.3g 3 .2 .4 O S and C Acylation Similar to N acylation the reactivity of N ( azidoacyl)benzotriazoles 3.1a c (3.1a + 3.1a ) was tested against a variety of O S and C nu cleophiles (Table 3 3 ). As expected, azide as a protecting group is well tolerated, and N ( azidoacyl)benzotriazoles 3.1 could be used in the acylation of phenols, alcohols (including sterols), thiols, and stabilized enolates (Table 3 3 ).

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48 Table 3 3 O S and C Acylations Utilizing N ( Azidoacyl) benzotriazoles 3.1 entry N azidoacyl) benzotriazole, 3.1 Nu 3.4 optimized reaction conditions product, 3.5 yield (%) [m.p. ( o C)] 1 N 3 L PheBt, 3.1 a Phenol, 3.4 a K 2 CO 3 ( 2 equiv) MeCN,12 h 3.5 a 84 [oil] 2 N 3 L LeuBt, 3.1 b Phenol, 3.4 a K 2 CO 3 (2 equiv) MeCN, 24 h 3.5 b 63 [oil] 3 N 3 DL PheBt, ( 3.1 a+ 3.1 a ) Cholesterol, 3.4 b THF, DMAP (cat.), 3 h 3.5 c 78 [56 58] 4 N 3 L LeuBt, 3.1 b Cholesterol, 3.4 b CHCl 3 Et 3 N (1 equiv), 72 h 3.5 d 61 [81 83] 5 N 3 L LeuB t, 3.1 b Sitosterol, 3.4 c CHCl 3 Et 3 N (1 equiv), 12 h 3.5 e 70 [57 60] 6 N 3 L LeuBt, 3.1 b Thiophenol, 3.4 d CH 2 Cl 2 py (1 equiv), 18 h 3.5 f 72 [oil] 7 N 3 L LeuBt, 3.1 b 2 Mercapto acetic acid, 3.4 e EtOAc, Et 3 N (2 equiv), 18 h 3.5 g 82 [o il] 8 N 3 L PheBt, 3.1 a Methyl 2 mercapto acetate, 3.4 f EtOAc, Et 3 N (1 equiv), 18 h 3.5 h 57 [oil] 9 N 3 L LeuBt, 3.1 b acid, 3.4 g CH 3 CN, Et 3 N (1 equiv), 14 h 3.5 i 72 [oil] 10 N 3 L LeuBt, 3.1 b Cyclohexane 1,3 dione, 3.4 h MeCN, Et 3 N (1 equiv), 14 h 3.5 j 95 [oil] 11 N 3 L LeuBt, 3.1 b Ethyl 2 cyanoacetate, 3.4 i CH 2 Cl 2 Et 3 N (1 equiv), 18 h 3.5 k 69 [oil] 12 N 3 L LeuBt, 3.1 b Ethanol, 3.4 j EtOH, Et 3 N (1 equiv), 12 h 3.5 l 95 [oil]

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49 3.3 Conclusions Benzotriazol 1 yl sulfonyl azide 2.7 allowed convenient and efficient synthesis of a wide range of azides, including N azidoacyl) benzotriazoles 3.1 which are efficient N S C and O acylating agents and enable facile preparation of azido peptides. In addition, the azido, as a protecting group, was shown to be well tolerated in these acylations. 3 .4 Experimental 3. 4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. The NMR spectra were recorded in CDCl 3 DMSO d 6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Silica was used as the stationary phase for column chromatography. Phosphomolybdic acid was used to detect compounds that were not UV active. [Caution: Although no trouble was experienced when utilizing sodium azide, the in situ generated chlorosulfonyl az ide, or any of the synthesized organic azides, appropr iate safety measures must be taken at all times because azides are often found to be high energy compounds. 10 12 51 52 60 ] 3.4.2 General Procedure for the P reparation of N A zidoacyl)benzotriazoles, 3.1a c, (3.1a+3.1 ) Thionyl chloride (0.01 mol, 1.2 equiv) was added to a solution of 1 H benzotriazole (2.38 g, 0.02 mol, 2 equiv) in methylene chloride (10 mL) to give a clear yellow solution and was s tirred for 15 min. at room azido acid 2.11 (0.01 mol, 1 equiv) was then added to give a suspension which was stirred for 2.5 h at room temperature. The precipitate was filtered, the filtrate evaporated, the residue dissolved in eth yl ace tate and the solution washed with a saturated solution of sodium carbonate.

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50 The organic portions were combined and dried over anhyd MgSO 4 filtered and dried to give the corresponding N azidoacyl)benzotriazoles 3.1 ( S ) 2 Azido 1 (1 H benzo[ d ][1,2,3]tria z ol 1 yl) 3 phenylpropan 1 one ( 3.1a ) (2 S ) 2 Azido 3 phenylpropanoic acid 2.11 a (1.91 g, 0.01 mol) was treated according to the above procedure to give ( S ) 2 azido 1 (1 H benzo[ d ][1,2,3]triazol 1 yl) 3 phenylpropan 1 one 3.1 a as a brown yellow solid (2.86 g, 98%); mp 66.1 68.3 o D 20 +32.5 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.20 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.42 7.18 (m, 5H), 5.48 (dd, J = 5.3, 8.9 Hz, 1H), 3.42 (dd, J = 5.1, 13.8 Hz, 1H), 3.30 3. 15 (m, 1H); 13 C NMR (75 MHz,CDCl 3 ) : 168.8, 146.0, 135.2, 130.9, 130.8, 129.2, 128.7, 127.4, 126.7, 120.4, 114.2, 62.4, 37.6; Anal. Calcd for C 15 H 12 N 6 O: C, 61.64; H, 4.14; N, 28.75. Found: C, 61.90; H, 4.04; N, 28.57. ( S ) 2 Azido 1 (1 H benzo[ d ][1,2,3]tria z ol 1 yl) 4 methylpentan 1 one ( 3.1b ) ( S ) 2 Azido 4 methylpentanoic acid 2.11 b (1.57 g, 0.01 mol) was treated according to the above procedure to give ( S ) 2 azido 1 (1 H benzo[ d ][1,2,3]triazol 1 yl) 4 methylpentan 1 one 3.1 b as a yellow solid (2.48 g, 96%) ; mp 47.3 49.0 o C; [ D 20 +42.4 ( c 0.6, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.30 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H) 5.28 (d, J = 9.3 Hz, 1H), 2.04 1.89 (m,3H), 1.08 (d, J = 5.4, Hz, 3H), 1.03 (d, J = 5.4 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ): 170.2, 146.1, 130.9, 126.7, 120.4, 114.3, 59.5, 39.9, 25.4, 22.9, 21.3; Anal. Calcd for C 12 H 14 N 6 O: C, 55.80; H, 5.46; N, 32.54. Found: C, 55.78; H, 5.76; N, 32.19. ( S ) 2 Azido 1 (1 H benzo[ d ][1, 2,3]triazol 1 yl)propan 1 one ( 3.1 c ) ( S ) 2 Azidopropanoic acid 2.11 c (1.15 g, 0.01 mol) was treated according to the above

PAGE 51

51 procedure to give ( S ) 2 azido 1 (1 H benzo[ d ][1,2,3]triazol 1 yl)propan 1 one 3.1 c as a brown solid (1.41 g, 65%); mp 77.0 77.9 o D 20 +32.4 ( c 1.0, CHCl 3 ); 1 H NMR (3 00 MHz, CDCl 3 ) : 8.28 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.73 7.67 (m, 1H), 7.57 7.51 (m, 1H), 5.35 (q, J = 7.0 Hz, 1H), 1.80 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz,CDCl 3 ) : 170.0, 146.1, 130.9, 126.7, 120.4, 114.3, 56.8, 17.0; HRMS m/z for C 9 H 9 ON 6 [M + H]+ calcd 217.0838, found 217.0826. 2 Azido 1 (1H benzo[d][1,2,3]triazol 1 yl) 3 phenylpropan 1 one (3.1a+3.1 a ) 2 Azido 3 phenylpropanoic acid (2.11a+2.11 a ) (1.91 g, 0.01 mol) was treated according to the above procedure to give 2 azido 1 (1 H benzo[ d ][1,2,3]triazol 1 yl) 3 phenylpropan 1 one (3.1a+3.1 a ) as a brown oil (2.86 g, 98%); 1 H NMR (300 MHz, CDCl 3 ) : 8.20 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.28 7.18 (m, 5H), 5.48 (d d, J = 5.1, 8.7 Hz, 1H), 3.42 (dd, J = 5.1,13.8 Hz, 1H), 3.20 (dd, J = 9, 13.5 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 168.9, 146.2, 135.2, 131.0, 131.0, 129.2, 128.8, 127.5, 126.8, 120.5, 114.3, 62.5, 37.7; Anal. Calcd for C 15 H 12 N 6 O: C, 61.64; H,4.14; N, 28.7 5. Found: C, 61.61; H, 4.13; N, 28.94. ( S ) 2 Azido 1 (1H benzo[d][1,2,3]tria zol 1 yl) 3 methylbutan 1 one ( 3.1 d ) ( S ) 2 Azido 3 methylbutanoic acid (2.11 d) (1.43 g, 0.01 mol) was treated according to the above procedure to give ( S ) 2 azido 1 (1H benzo[d][1 ,2,3]triazol 1 yl) 3 methylbutan 1 one 3.1 d as brown oil (1.76 g, 72%); [ D 20 + 0.6 ( c 6.5, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.85 (td, J = 0.9, 8.1 Hz, 1H), 7.69 7.66 (m, 1H), 7.28 7.22 (m, 1H), 7.13 7.07 (m, 1H), 4.76 (d, J = 6.9 Hz, 1H), 2.13 (octet, J = 6.6 Hz, 1H), 0.71 (dd, J = 6.9, 11.4 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 168.9, 145.7, 130.5, 126.3, 120.0, 114.0,

PAGE 52

52 66.4, 31.1, 19.3, 17.7; Anal. Calcd for C 11 H 12 N 6 O: C, 54.09; H, 4.95; N, 34.41. Found: C, 54.13; H, 4.94; N, 34.77. 3.4.3 General Procedure for 3.3 a k The appropriate N nucleophile 3.2 was reacted under the optimized conditions described in Table 3 2 N Azidoacyl)benzotriazoles 3.1 (1 equiv) was then added to the reaction mixture. This was stirred for the specified time (Table 3 2 ) at room temperature before isolation of products 3.3 a k in yields of 62 87%. ( S ) 2 Azido N (4 methoxyphenyl) 3 phenylpropanamide ( 3.3 a ) p Anisidine 3.2 a (260 mg, 2.11 mmol,1.5 equiv) was treated with 3.1 a (412 mg, 1.41 mmol) according to the above procedure. The solvent was then evaporated, the residue diluted with EtOAc and washed with 6N HCl. The organic portion was collected, dried over anhyd MgSO 4 filtered, and the filtrate concentrated under reduced pressure to give ( S ) 2 azido N (4 methoxyphenyl) 3 phenylpropanamide 3.3 a (328 mg, 1.11 mmol) as a beige solid: 79%; mp 79 .4 81.0 o D 20 14.6 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.00 (bs, 1H), 7.40 7.26 (m, 7H), 6.82 (d, J = 8.4 Hz, 2H), 4.27 (dd, J = 4.5, 7.8 Hz, 1H), 3.75 (s, 3H), 3.37 (dd, J = 4.1, 13.7 Hz, 1H), 3.08 (dd, J = 8.1, 14.1 Hz, 1H); 13 C NMR (75 M Hz, CDCl 3 ) : 166.6, 156.7, 135.9, 129.6, 129.3, 128.6, 127.1, 122.1, 114.0, 65.4, 55.3,38.5; Anal. Calcd for C 16 H 16 N 4 O 2 : C, 64.85; H, 5.44; N, 18.91. Found: C, 64.53; H, 5.59; N, 18.66. ( S ) 2 Azido N (4 metho xyphenyl) 4 methylpentanamide ( 3.3 b ) p Anisidi ne 3.2 a (251 mg, 2.04 mmol,1.5 equiv) was treated with 3.1 b (351 mg, 1.36 mmol) according to the above procedure. The solvent was then evaporated and the residue was diluted with EtOAc, washed with 6N HCl. The organic portion was then collected, dried over anhyd MgSO 4 filtered, and the filtrate concentrated under reduced pressure to give ( S ) 2

PAGE 53

53 azido N (4 methoxyphenyl) 4 methylpentanamide 3.3 b (300 mg, 1.14 mmol) as a white solid: 84%; mp 42.5 45.0 o D 20 +55.43 ( c 0.49, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.10 (s, 1H), 7.45 7.41 (m, 2H), 6.88 6.83 (m, 2H), 4.08 4.03 (m, 1H), 3.78 (s, 3H), 1.88 1.74 (m, 3H), 1.00 (d, J = 2.4 Hz, 3H), 0.98 (d, J = 2.4 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) : 167.8 156.7, 130.0, 121.8, 114.1, 63.1, 55.4, 41.3, 25.0, 23.0, 21.5; Anal. Calcd for C 13 H 18 N 4 O 2 : C, 59.53; H, 6.92; N, 21.36. Found: C, 59.86; H, 7.18; N, 20.99. ( S ) 2 Azido 4 methyl N (9 H purin 6 yl)pentanamide ( 3.3 c ) Adenine 3.2 b (268 mg, 1.99 mmol, 1 equi v) was treated with 3.1 b (514 mg, 1.99 mmol) according to the above procedure. The reaction mixture was diluted with ethyl acetate (30 mL) and washed with water (2 x 50 mL). The organic layer was dried over anhyd MgSO 4 filtered and the filtrate evaporated The residue was separated by column chromatography (acetonitrile) to give ( S ) 2 azido 4 methyl N (9 H purin 6 yl)pentanamide 3.3 c (370 mg,1.35 mmol) as a white solid: 68%; mp 187.0 188.0 o D 20 +47.5 ( c 0.3, CHCl 3 ); 1 H NMR (300 MHz, DMSO d 6 ) : 12.26 (bs, 1H), 11.60 (bs, 1H), 8.67 (s, 1H), 8.48 (s, 1H), 4.30 4.19 (m, 1H), 1.90 1.60 (m, 3H), 1.10 0.90 (m, 6H); 13 C NMR (75 MHz, DMSO d 6 ) : 170.7, 151.2, 146.4, 143.1, 59.7, 39.3, 24.9, 22.7, 21.5; Anal. Calcd for C 11 H 14 N 8 O: C, 48.17; H, 5.14; N, 40 .85. Found: C, 48.09; H, 4.96; N,40.61. ( S ) 2 Azido 3 phenyl N tosylpropanamide ( 3.3 d ) p Toluenesulfonamide 3.2 c (140 mg, 0.82 mmol, 1.2 equiv) was treated with 3.1 a (200 mg, 0.68 mmol) according to the above procedure. The solvent was evaporated, the res idue diluted with EtOAc and washed with 6N HCl. The organic portion was then collected, dried over anhyd MgSO 4 filtered and the filtrate concentrated under reduced pressure to give ( S ) 2 azido 3 phenyl N tosylpropanamide 3.3 d (185 mg, 0.54 mmol) as a beig e solid: 79%; mp 119.5

PAGE 54

54 121.7 o D 20 +4.9 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.76 (bs, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7.37 7.05 (m, 7H), 4.23 4.19 (m, 1H),3.21 (dd, J = 4.2, 14.1 Hz, 1H), 2.99 (dd, J = 7.5, 14.1 Hz, 1H), 2.47 (s, 3H); 13 C N MR (75 MHz, CDCl 3 ) : 166.7, 145.4, 134.8, 134.7, 129.6, 129.3, 128.7, 128.6, 127.4, 64.8, 38.0, 21.7; Anal. Calcd for C 16 H 16 N 4 O 3 S: C, 55.80; H, 4.68; N, 16.27. Found: C, 55.91; H, 4.74; N, 16.24. ( S ) 2 Azido 4 methyl N tosylpentanamide ( 3.3 e ) p Toluenes ulfonamide 3.2 c (119 mg, 0.70 mmol, 1.2 equiv) was treated with 3.1 b (151 mg, 0.58 mmol) according to the above procedure. The solvent was evaporated, the residue diluted with EtOAc and washed with 6N HCl. The organic portion was then collected, dried over anhyd MgSO 4 filtered and the filtrate concentrated under reduced pressure to give ( S ) 2 azido 4 methyl N tosylpentanamide 3.3 e (140 mg, 0.45 mmol) as white microcrystals: 77%; mp 55.3 56.0 o D 20 +69.1 ( c 0.3, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 ) : 9.29 (bs, 1H), 7.96 (dd, J = 1.8, 6.6 Hz, 2H), 7.39 7.33 (m, 2H), 3.96 3.38 (m, 1H), 2.44 (s, 3H), 1.86 1.60 (m, 3H), 1.00 0.87 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 168.0,145.5, 134.9, 129.6 128.5, 62.3 40.4, 24.7, 22.7, 21.7, 21.4; Anal. Calcd for C 13 H 18 N 4 O 3 S: C, 50.31; H, 5.85; N, 18.05. Found: C, 50.12; H, 6.02; N, 17.78. 2 Azido N (1 ((2 R ,3 R ,4 S ,5 R ) 3,4 dihydroxy 5 (hydroxymethyl)tetrahydrofuran 2 yl) 2 oxo 1,2 dihydropyrimidin 4 yl) 3 p henylpropanamide ( 3.3 f ): Cytidine 3.2 d (404 mg, 1.66 mmol, 1 equiv) was treated with (3.1a+3.1 a ) (486 mg, 1.66 mmol) according to the above procedure. DMF was evaporated and the residue chromotographed using MeOH:CH 2 Cl 2 (gradient) to give 2 azido N (1 ((2 R ,3 R ,4 S ,5 R ) 3,4 dihydroxy 5 (hydroxymethyl)tetrahydrofuran 2 yl) 2 oxo 1,2 dihydropyrimidin 4 y l) 3 phenylpropanamide 3.3 f (428 mg, 1.03 mmol) as a glassy white solid: 62%; mp (glassy

PAGE 55

55 D 20 +57.3 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 ) : 8.51 (d, J = 7.2 Hz, 1H), 7.36 7.31 (m, 5H), 7.17 (d, J = 7.5 Hz, 1H), 5.78 (d, J = 2.1 Hz, 1H), 5. 53 (d, J = 4.5 Hz, 1H), 5.21 (t, J = 5.0 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 4.32 (dd, J = 5.1, 9.3 Hz, 1H), 3.99 3.87 (m, 3H), 3.77 3.71 (m, 1H), 3.62 3.57 (m, 1H), 3.21 (dd, J = 5.0, 13.7 Hz, 1H), 2.96 (dd, J = 9.8, 13.7 Hz, 1H); 13 C NMR (75 MHz, DMS O d 6 ) : 170.6, 161.9, 154.4, 146.0, 136.4, 129.1, 128.5, 126.9, 95.3, 90.3, 84.3, 74.6, 68.6, 63.1, 62.8, 59.9, 36.6; Anal. Calcd for C 18 H 20 N 6 O 6 : C, 51.92; H, 4.84; N, 20.18. Found: C, 52.27; H, 4.35; N, 20.32. 2 Azido N (4 metho xyphenyl) 3 phenylpropanamide ( 3. 3 g ) p Anisidine 3.2 a (261 mg, 2.12 mmol, 1.5 equiv) was treated with (3.1a+3.1 a ) (411 mg, 1.41 mmol) according to the above procedure. The solvent was evaporated, the residue diluted with EtOAc and washed with 6 N HCl. The organic portion was then collec ted, dried over anhyd MgSO 4 filtered, and the filtrate concentrated under reduced pressure to give 2 azido N (4 methoxyphenyl) 3 phenylpropanamide 3.3 g (328 mg, 1.11 mmol) as a brown oil: 79%; 1 H NMR (300 MHz, CDCl 3 ) : 7.79 (b r s, 1H), 7.33 7.21 (m, 7H) 6.84 6.80 (m, 2H), 4.30 (dd, J = 4.4, 8.0 Hz, 1H), 3.75 (s, 3H), 3.38 (dd, J = 4.2, 14.1 Hz, 1H), 3.08 (dd, J = 8.3, 14.0 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 166.6, 156.8, 135.9,129.6, 129.4, 128.6, 127.2, 122.1, 114.1, 65.6, 55.4, 38.7; Anal. Calcd fo r C 16 H 16 N 4 O 2 : C, 64.85; H,5.44; N, 18.91. Found: C, 64.81; H, 5.51; N, 18.97. ( S ) 2 (( S ) 2 Azido 3 phenylpropan amido) 4 methylpentanoic acid ( 3.3 h ) L Leu 3.2 e (1.02 g, 7.74 mmol, 2 equiv) was treated with 3.1 a (1.13 g, 3.87 mmol) according to the above pr ocedure. The MeCN was evaporated, the residue diluted with EtOAc and washed with 6N HCl. The organic layer was then collected, dried over anhyd MgSO 4

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56 filtered and the filtrate concentrated under reduced pressure to give ( S ) 2 (( S ) 2 azido 3 phenylpropanam ido) 4 methylpentanoic acid 3.3 h (1.02 g, 3.37 mmol) as a bright D 20 +27.8 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 11.20 (bs, 1H), 7.28 7.25 (m, 5H), 6.72 (d, J = 8.1 Hz, 1H), 4.58 4.54 (m, 1H), 4.37 4.31 (m, 1H), 4.12 (d, J = 7.2 Hz, 1H), 3.31 (dd, J = 3.8, 14.3 Hz, 1H), 3.06 (dd, J = 7.5, 14.1 Hz, 1H), 1.68 1.58 (m, 1H), 1.54 1 .40 (m, 1H), 0.88 0.86 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 176.5, 169.0, 135.7, 129.5, 129.2, 128.6, 127.2, 65.0, 50.6, 40.9, 38.2, 24.6, 22.7, 21 .7; Anal. Calcd for C 15 H 20 N 4 O 3 : C, 59.20; H, 6.62; N, 18.41. Found: C, 59.19; H, 6.55; N, 18.60. ( R ) 2 (( S ) 2 Azido 3 phenylpropanam ido) 3 mercaptopropanoic acid ( 3.3 i ) L Cys 3.2 f (165 mg, 1.36 mmol, 2 equiv) was treated with 3.1 a (197 mg, 0.68 mmol) acco rding to the above procedure. The MeCN was evaporated, the residue diluted with EtOAc and the solution washed with 6N HCl. The organic layer was then collected, dried over anhyd MgSO 4 filtered and the filtrate concentrated under reduced pressure to give ( R ) 2 (( S ) 2 azido 3 phenylpropanamido) 3 mercaptopropanoic acid 3.3 i (160 mg, 0.54 mmol) as a beige solid: 80%; mp 159.0 161.0 o D 20 +2.5 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 8.51 (bs, 1H), 7.31 7.28 (m, 5H), 7.17 (d, J = 7.5 Hz, 1H), 4.85 ( t, J = 3.6 Hz, 1H), 4.43 4.39 (m, 1H), 3.29 (dd, J = 3.8, 14.1 Hz, 1H), 3.15 (dd, J = 6.6, 14.1 Hz, 1H), 3.00 2.92 (m, 1H), 2.84 2.70 (m, 1H); 13 C NMR (75 MHz,CDCl 3 ) : 172.9, 169.0, 135.2, 129.6, 128.7, 127.5, 64.7, 53.3, 38.1, 26.4; Anal. Calcd for C 12 H 14 N 4 O 3 S:C, 48.97; H, 4.79; N, 19.04. Found: C, 49.13; H, 4.47; N, 19.03. ( R ) 2 (( S ) 2 Azido 4 methylpentanam ido) 3 mercaptopropanoic acid ( 3.3 j ) L Cys 3.2 f (242 mg, 2.00 mmol, 2 equiv) was treated with 3.1 b (258 mg, 1.00 mmol) according

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57 to the above procedure. The MeCN was evaporated, the residue diluted with EtOAc and the solution washed with 6N HCl. The organic layer was then collected, dried over anhyd MgSO 4 filtered and the filtrate concentrated under reduced pressure to give ( R ) 2 (( S ) 2 azido 4 methylpentanamido) 3 mercaptopropanoic acid 3.3 j (216 mg, 0.83 D 20 15.2 ( c 0.3, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 ) : 10.22 (s, 2H), 8.68 (s, 1H), 4.54 4.40 (m, 1H), 3.87 3.78 (m, 1H), 3.00 2.80 (m, 2H), 1.80 1.50 (m 3H), 0.98 0.85 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 172.0, 170.5, 60.0, 52.9, 31.8, 31.0, 25.2, 23.2, 22.3; HRMS m/z for C 9 H 17 N 4 O 3 S [M + H]+ calcd 261.1016, found 261.1007. 2 (( S ) 2 Azido 3 phe nylpropanamido)propanoic acid ( 3.3 k ) L Ala 3.2 g (611 mg, 6. 86 mmol, 2 equiv) was treated with 3.1 a (1.00 g, 3.43 mmol) according to the above procedure. The MeCN was evaporated, the residue diluted with EtOAc and washed with 6N HCl. The organic layer was then collected, dried over anhyd MgSO 4 filtered and the fil trate concentrated under reduced pressure to give 2 (( S ) 2 azido 3 phenylpropanamido)propanoic acid 3.3 k (0.72 g, 2.74 mmol) as white microcrystals: 80%; mp 113.0 114.0 o D 20 10.7 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 ) : 7.58 (bs, 1H), 7.34 7.23 (m, 5H), 6.81 (d, J = 7.2 Hz, 1H), 4.61 4.50 (m,1H), 4.32 4.27 (m, 1H), 3.32 (dd, J = 3.9, 14.1 Hz, 1H), 3.05 (dd, J = 7.8, 14.1 Hz, 1H), 1.37 (s, 3H); 13 C NMR (75 MHz CDCl 3 ) : 176.3, 168.7, 135.6, 129.5, 128.6, 127.3, 65.0, 48.0, 38.4, 17.9; Anal.Calcd for C 12 H 14 N 4 O 3 : C, 54.96; H, 5.38; N, 21.36. Found: C, 55.25; H, 5.21; N, 21.13. 3.4.4 General Procedure for 3.5 a l The appropriate nucleophile 3.4 was reacted under t he optimized conditions described in Table 3 3 N Azidoacyl)benzotriazoles 3.1 (1 equiv) was added to the

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58 reaction mixture. This was allowed to stir for the specified time (Table 3 3 ) at room temperature before isolation of products 3.5 a l in yields of 57 95%. ( S ) Pheny l 2 azido 3 phenylpropanoate ( 3.5 a ) Phenol 3.4 a (70 mg, 0.75 mmol, 1.1 equiv) was treated with 3.1 a (198 mg, 0.68 mmol) according to the above procedure. The solvent was then evaporated, diluted with ether and washed with 1 M NaOH. The or ganic layer was then collected, dried over anhyd MgSO 4 filtered and the filtrate concentrated under reduced pressure to give ( S ) phenyl 2 azido 3 phenylpropanoate 3.5 a (153 mg, 0.57 mmol) as a brown oil: 84%; [ D 20 3.1 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.41 7.23 (m, 10H), 7.03 6.98 (m, 1H), 4.36 4.28 (m, 1H), 3.32 (dd, J = 6.0, 13.8 Hz, 1H), 3.19 (dd, J = 8.1, 13.8 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 168.4, 150.0, 135.4, 129.5, 129.3, 128.7, 127 .4, 126.3, 121.5, 121.1, 63.1, 37.7; Anal. Calcd for C 15 H 13 N 3 O 2 : C, 67.40; H, 4.90; N, 15.72. Found: C, 67.86; H, 5.09; N, 15.28. ( S ) Pheny l 2 azido 4 methylpentanoate ( 3.5b ) Phenol 3.4 a (104 mg, 1.10 mmol, 1.1 equiv) was treated with 3.1 b (258 mg, 1.00 m mol) according to the above procedure. The solvent was then evaporated, diluted with ether and washed with 1 M NaOH. The organic layer was then collected and chromatographed using EtOAc:Hexanes (1:5) to give (S) phenyl 2 azido 4 methylpentanoate 3.5 b (146 mg, D 20 +59.5 ( c 0.5, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.38 (d, J = 8.1 Hz, 2H), 7.25 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 8.7 Hz, 2H), 4.05 (t, J = 6.3 Hz, 1H), 1.99 1.80 (m, 3H), 1.05 1.00 (m, 6H); 13 C NMR (75 MHz CDCl 3 ) : 169.5, 150.2, 129.5, 126.3, 121.1, 60.3, 39.8, 25.1, 22.8, 21.6; Anal. Calcd for C 12 H 15 N 3 O 2 : C, 61.79; H, 6.48; N, 18.01. Found: C, 61.71; H, 6.75; N, 17.61.

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59 (8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 10,13 dimethyl 17 (( R ) 6 methylheptan 2 yl) 2,3,4,7,8,9,10,11 ,12,13,14,15,16,17 tetradecahydro 1 H cyclopenta[a]phenanthren 3 yl 2 azido 3 phenylpropanoate ( 3.5 c ) Cholesterol 3.4 b (178 mmol, 0.46 mmol, 1 equiv) was treated with (3.1a+3.1 a ) (135 mg, 0.46 mmol) according to the above procedure. The solvent was evapor ated, the oil obtained chromotographed using hexane to give (8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 10,13 dimethyl 17 (( R ) 6 methylheptan 2 yl) 2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro 1 H cyclopenta[a]phenanthren 3 yl 2 azido 3 phenylpropanoate 3.5 c (200mg, 0.3 6 mmol) as a white powder: 78%; mp 56.0 58.0 o C; D 20 10.5 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.15 7.03 (m, 5H), 5.19 (bs, 1H), 4.49 4.44 (m,1H), 3.82 (t, J = 6.3 Hz, 1H), 3.00 2.78 (m, 2H), 2.15 2.05 (m, 2H), 1.84 1.48 (m. 2H), 1.34 0.65 (m,42 H), 0.49 (s, 3H); 13 C NMR (75 MHz, C DCl 3 ) : 169.3, 139.1, 135.9, 129.2, 128.6, 127.2, 123.1, 75.8, 63.2, 56.6, 56.1, 49.9, 42.3, 40.3, 39.7, 39.5, 37.6, 36.9, 36.5, 36.1, 35.8, 31.8, 28.2, 28.0, 27.6, 24.3, 23.8, 22.8, 22.5, 21.0, 19.3, 18.7, 11.8; Anal. Calcd for C 36 H 53 N 3 O 2 : C, 77.24; H, 9 .54; N, 7.51. Found:C, 77.29; H, 9.37 ; N, 7.52. (8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 10,13 Dimethyl 17 (( R ) 6 methylheptan 2 yl) 2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro 1 H cyclopenta[a]phenanthren 3 yl 2 azido 4 methylpentanoate ( 3.5 d ) Cholesterol 3.4 b (3 87 mg, 1.00 mmol, 1 equiv) was treated with 3.1 b (259 mg,1.00 mmol) according to the above procedure. The solvent was evaporated, the oil obtained chromotographed using hexane:EtOAc (1:40) to give (8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 10,13 dimethyl 17 (( R ) 6 methylhept an 2 yl) 2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro 1 H cyclopenta[a]phenanthren 3 yl 2 azido 4 methylpentanoate 3.5 d (320 mg, 0.61 mmol) as a white powder: 61%; mp

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60 81.0 83.0 o D 20 29.2 ( c 0.6, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 5.50 5.35 ( m, 1H), 4.83 4.62 (m, 1H), 3.90 3.70 (m, 1H), 2.50 2.29 (m, 2H), 2.15 0.68 (m, 44H), 0.68 0.66 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 170.4, 139.2, 123.0, 75.6, 60.4, 56.7, 56.1, 50.0, 42.3, 39.9, 39.7, 39.5,38.0, 36.9, 36.6, 36.2, 35.8, 31.8, 28.2, 28.0, 27.7, 25.0, 24.3, 23.8, 22.8, 22.6, 21.6, 21.0, 19.3, 18.7, 11.9.; HRMS m/z for C 33 H 55 N 3 NaO 2 [M + Na]+ calcd 548.4186, found 548.4194. ( S ) (3 S ,8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 17 ((2 R ,5 R ) 5 Ethyl 6 methylheptan 2 yl) 10,13 dimethyl 2,3,4,7,8,9,10,11,12,13,14,1 5,16,17 tetradecahydro 1H cyclopenta[a]phenanthren 3 yl 2 azido 4 methylpentanoate ( 3.5 e ) Sitosterol 3.4 c (211 mg, 0.51 mmol, 1 equiv) was treated with 3.1 b (133 mg, 0.51 mmol) according to the above procedure. Chloroform was evaporated, residue diluted with EtOAc and flash chromotographed with hexanes:EtOAc (45:5). The organic fractions were evaporated to give ( S ) (3 S ,8 S ,9 S ,10 R ,13 R ,14 S ,17 R ) 17 ((2 R ,5 R ) 5 ethyl 6 methylheptan 2 yl) 10,13 dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro 1H cyclopen ta[a]phenanthren 3 yl 2 azido 4 methylpentanoate 3.5 e (200 mg, 0.36 mmol) as a white so lid: 70%; mp 57.1 60.0 o C; [ D 20 +43.7 ( c 1.0, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 5.40 (s, 1H), 2.36 (d, J = 6.9Hz, 2H), 1.99 0.68 (m, 54H); 13 C NMR (75 MHz, CDCl 3 ) : 170.5, 139.2, 123.1, 75.6, 61.7, 60.3, 56.7, 56.0, 50.0, 45.8, 42.6, 42.3, 39.9, 38.0, 36.9, 36.6, 33.7, 31.8, 28 .2, 27.7, 26.1, 25.0, 24.3, 22.8, 21.6, 21.0, 19.3, 18.8, 14.2, 11.9; Anal. Calcd for C 35 H 59 N 3 O 2 : C, 75.90; H, 10.74; N, 7.59. Found: C, 75.51; H, 10.60; N, 7.44. ( S ) S Phenyl 2 a zido 4 methylpentanethioate ( 3.5f ) Thiophenol 3.4 d (165 mg, 1.50 mmol, 1.5 e quiv) was treated with 3.1 b (258 mg, 1.00 mmol) according to the

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61 above procedure (pyridine (0.08 mL, 1 equiv) was added to a mixture of 3.4 d and 3.1 b at 0 o C and then left to stir at room temperature). The reaction mixture was filtered and filtrate evapora ted. The residue was diluted Et 2 O (30 mL) and washed with 5% solution of sodium hydroxide (2 x 50 mL) and water (2 x 30 mL). The organic layer was dried over anhyd MgSO 4 filtered and the filtrate evaporated. The residue was separated by column chromatogra phy using EtOAc:Hexanes (1:5) to give (S) S phenyl 2 azido 4 methylpentanethioate 3.5 f (179 mg, 0.72 mmol) as a colorless oil: 72%; [ D 20 +106.9 ( c 0.3, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.48 7.35 (m, 5H), 4.08 4.00 (m, 1H), 1.92 1.71 (m, 3H), 1.08 0.96 (m, 6H); 13 C NMR (75 MHz,CDCl 3 ) : 197.1, 134.5, 129.7, 129.3, 126.6, 67.5, 40.7, 25.0, 22.9, 21.5; Anal. Calcd for C 12 H 15 N 3 O S:C, 57.81; H, 6.06; N, 16.85. Found: C, 58.16; H, 6.18; N, 17.11. ( S ) 2 ((2 Azido 4 methylp entanoyl)thio)acetic acid ( 3.5g ). 2 Mercaptoacetic acid 3.4 e (183 mg, 1.99 mmol, 1 equiv) was treated with 3.1 b (513 mg, 1.99 mmol) according to the above procedure The solvent was evaporated, the residue diluted with EtOAc and washed with 6 N HCl. The organic portions were dried over anhyd MgSO 4 filtered, and the filtrate evaporated to give (S) 2 ((2 azido 4 methylpentanoyl)thio)acetic acid 3.5 g (376 mg, 1.63 mmol D 20 10.6 ( c 0.7, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 10.8 (bs, 1H), 4.04 (t, J = 7.2 Hz, 1H), 3.84 2.74 (m, 2H), 1.85 1.75 (m, 1H), 1.74 1.64 (m, 2H), 0.99 (d, J = 4.8 Hz, 3H), 0.97 (d, J = 4.8 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) : 190.8, 167.4, 60.5, 33.6, 24.1, 17.8, 15.9, 14.3; HRMS m/z for C 18 H 14 N 3 O 3 S [M+H]+ calcd 232.0750, found 232.0742. ( S ) Methyl 2 ((2 azido 3 phenylpropanoyl)thio)acetate ( 3.5 h ) Methyl 2 Mercaptoacetate 3.4 f (106 mg, 1.00 mmol, 1 equiv) was treated wit h 3.1 a (292 mg, 1.00

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62 mmol) according to the above procedure. The solvent was evaporated, the residue chromotographed using hexane: EtOAc (1:5) to give ( S ) methyl 2 ((2 azido 3 phenylpropanoyl)thio)acetate 3.5 h D 20 +19.2 ( c 0.8, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 7.36 7.18 (m, 5H), 4.25 4.20 (m, 1H), 3.75 3.70 (m, 5H), 3.25 3.21 (m, 1H), 3.05 2.87 (m, 1H); 13 C NMR (75 MHz, CDCl 3 ) : 196.7, 168.4, 135.4, 129.2, 128.6,127.2, 70.3, 52.8, 38.3, 31.1; Anal. C alcd. for C 12 H 13 N 3 O 3 S: C, 51.60; H, 4.69; N, 15.04. Found: C,51.99; H, 4.55; N,14.93. ( S ) 5 (2 Azido 1 hydroxy 4 methylpentylidene) 2,2 dimet hyl 1,3 dioxane 4,6 dione ( 3.5i ). 3.4 g (176 mg, 1.22 mmol, 1 equiv) was treated with 3.1 b (316 mg, 1 .22 mmol) according to the procedure above. The solvent was evaporated, the residue was separated by column chromatography [EtOAc] to give ( S ) 5 (2 azido 1 hydroxy 4 methylpentylidene) 2,2 dimethyl 1,3 dioxane 4,6 dione 3.5 i (250 mg, 0.88 mmol) as a yellow D 20 +113.0 ( c 0.5, CH 3 OH); 1 H NMR (300 MHz, Acetone d 6 ) : 5.08 5.02 (m, 1H), 1.77 (dd, J = 6.6, 13.2 Hz, 1H), 1.65 1.53 (m, 8H), 0.98 0.94 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 193.4, 170.4, 159.4, 105.6, 90.7, 58.6, 39.7, 26.9, 26.7, 25. 3, 23.0, 21.1; Anal. Calcd for C 12 H 17 N 3 O 5 : C, 50.88; H, 6.05; N, 14.83. Found: C, 51.01; H, 6.08; N,14.31. ( S ) 2 (2 Azido 4 methylpen tanoyl)cyclohexane 1,3 dione ( 3.5 j ) Cyclohexane 1,3 dione 3.4 h (449 mg,4.00 mmol, 2 equiv) was treated with 3.1 b (517 mg, 2.00 mmol) according to the procedure above. The solvent was then evaporated, the residue diluted with EtOAc (40 mL) and washed with 6 N HCl (3 x 50 mL ). The organic layer was dried with over anhyd MgSO 4 filtered and the filtrate evaporated. This residue was separated by column chromatography [EtOAc:Hexanes (1:1)] to give ( S ) 2 (2 azido 4

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63 methylpentanoyl)cyclohexane 1,3 dione 3.5 j (480 mg, 1.90 mmol) as a colorless oil; D 20 +70.3 ( c 0.3, CHCl 3 ) ; 1 H NMR (300 MHz, CDCl 3 ) : 17.34 (s, 1H), 5.07 (dd, J = 4.1, 9.8 Hz, 1H), 2.73 (t, J = 6.5 Hz, 2H), 2.51 (dd, J = 6.0, 7.2 Hz, 2H), 2.11 1.90 (m, 3H), 1.65 1.55 (m, 2H), 1.08 (d, J = 6.6 Hz, 3H), 1.00 (dd, J = 2.4, 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) : 203.7, 198.0, 194.9, 111.5, 62.7, 39.5, 38.4, 32.5 25.6, 23.3, 20.9, 18.9; Anal. Calcd for C 12 H 17 N 3 O 3 : C, 57.36; H, 6.82; N, 16.72. Found:C, 57.56; H, 7.10; N, 16.98. (4 S ) Ethyl 4 azido 2 cyano 6 methyl 3 oxoheptanoate ( 3. 10k ) Ethyl cyanoacetate 3.4 i (452 mg, 4.00 mmol, 2 equiv) was treated with 3.1 b (5 17 mg, 2.00 mmol) according to the procedure above. The solvent was evaporated, the residue diluted with EtOAc (40 mL) and washed with 6 N HCl (3 x 50 mL) The organic layer was dried with over anhyd MgSO 4 filtered and the filtrate evaporated. This resid ue was separated by column chromatography [EtOAc:Hexanes (1:1)] to give (4 S ) ethyl 4 azido 2 cyano 6 methyl 3 oxoheptanoate 3.5 k D 20 +125.9 ( c 0.4, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) : 13.84 (s, 1H), 4.42 4.3 5 (m, 2H), 4.30 4.26 (m, 1H), 1.89 1.67 (m, 3H),1.42 1.37 (m, 3H), 1.04 0.98 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 186.2, 169.7, 113.1, 81.3, 63.2,59.3, 39.7, 25.0, 22.6, 21.8, 14.0; Anal. Calcd for C 11 H 16 N 4 O 3 : C, 52.37; H, 6.39; N, 22.21. Found: C,5 2.67; H, 6.60; N, 22.14. ( S ) Ethy l 2 azido 4 methylpentanoate ( 3.5 l ) Ethanol 3.4 j (solvent, 10 mL) was treated with 3.1 b (39 mg, 0.85 mmol) according to the procedure above. The solvent was evaporated, the residue diluted with EtOAc (25 mL) and washed wit h saturated sodium carbonate solution to remove benzotriazole. The organic layer was dried with

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64 over anhyd MgSO 4 filtered and the filtrate evaporated to give ( S ) ethyl 2 azido 4 methylpentanoate 3.5 l (150 mg, 0.81 mmol) as a yellow oil; 95%; 1 H NMR (300 M Hz, CDCl 3 ) : 4.24 (q, J = 7.2 Hz, 2H), 3.81 3.75 (m, 1H), 1.81 1.58 (m, 3H), 1.33 1.24 (m, 3H), 0.98 0.93 (m, 6H); 13 C NMR (75 MHz, CDCl 3 ) : 171.0, 61.7, 60.3, 39.9, 25.0, 22.8, 21.5, 14.2.

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65 CHAPTER 4 MICROWAVE ASSISTED R EGIOSPECIFIC SYNTHES IS OF PSEUDOHALOHYDRIN ESTERS iii 4 .1 Introduction Halohydrin esters are important inte rmediates in the asymmetric synthesi s of a wide range of biologically active natural and synthetic products 91 96 including drugs, 97 aminoalcohols, 98 pyrrolidines 99 and functionalized cyclopropanes. 100 Halohydrin esters 101 103 are commonly prepared by (i) direct reaction of an epoxide with an acyl halide, 91 94 95 102 103 (ii) the ring opening of epoxides by halogen nucleophiles fol lowed by the O acylation of the resulting halohydrin derivatives 104 109 and (iii) from 1,2 diols. 96 110 In general, these strategies produce mixtures of regioisomers and side products. No previous general method has achieved the high regioselectivity which I now rep ort. Microwave heating is a powerful tool in pro moting a variety of reactions in organic synthesis and functional group transformations without solvents The use of a single mode cavity microwave synthesizer helps achieve reproducibility, safety, reduced pollution, and simplicity in processing and handling. 20 21 23 25 111 112 N Acylbenzotriazoles, are more stable than acid chlorides towards hydrolysis and have replaced them advantageously in many acylat ions, often reducing side reactions. 3 51 113 114 N Acylbenzotriazoles have thus enabled both Friedel Crafts and Vilsmeier Haack acylations. 3 115 Obase et al. 116 previously synthesized a pseudohalohydrin ester (Scheme 4 1) from an N acylbenzotriazole. However, the single example reported gave a mixture of three products, the parent benzotriazole, and two iii Reproduced with permission from SYNLETT 2012 23, 1384 1388 Copyright 2012 Georg Thieme Verlag Stuttgart New York

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66 isomeric pseudohalohydrin esters A and B. The structures of A (yield 13 %) and B (yield 19 %) were assigned only on substituted ben zotriazoles. I n addition to A and B, there is the third possible isomeric benzotriazole product C The only evidence advanced for the structures of A and B was derived from ultraviolet spectroscopy. This work 116 is limited to a single example and neither substrate scope nor optimization of reaction conditions were investigated. Scheme 4 1 Reported 116 synthesis of a pseudohalohydrin ester from an N ac ylbenzotrizole and an epoxid e. To the best of my knowledge, the palladium catalyzed synthesis of pseudohalohydrin esters from N ac ylbenzotri a zoles and epoxides has not been prev iously attempted. A n improved protocol for the regiospecific synthesis of (be nzotriazol 1 yl)ethyl pseudohalohydrin esters 4.4 with pall adium catalysis under microwave assisted, solvent free conditions is described Microwave heating is a powerful tool in pro moting a variety of reactions in organic synthesis and functional group transformations without solvents The use of a single

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67 mode cavity microwave synthesizer helps achieve reproducibility, safety, reduced pollution, and simplicity in processing and handling. 20 21 23 25 111 112 4.2 Results and Discussion 4.2.1 Optimization of reaction conditions for 4.4a I found that the palladium catalyzed thermal reaction of N acylbenzotriazole 1a 117 119 with epoxide 4. 3a gave single regioisomer (benzotriazol 1 yl)ethyl ester 4. 4a Although thermal isomerization of 4. 1a to 4. 1 a followed by oxidative addition of 4. 1a to palladium (0) was reported 33 recently to give 4. 1a I detected no 1 ,4 benzoxazine 4. 2a (Scheme 4 2). 120 Scheme 4 2 Palladium catalyzed thermal reaction of N acylbenzotriazole 4.1a with epoxide 4.3a R eaction of (1 H benzotriazol 1 yl)(4 ethylphenyl)methanone ( 4. 1a ) w ith styrene oxide 4. 3a in the presence of 10 mol% of Pd(PPh 3 ) 4 under microwave irradiation (130 o C, 50 W) for 30 minutes, gave 2 (1 H benzotriazol 1 yl) 1 phenylethyl 4 ethylbenzoate ( 4. 4a ) as a single regioisomer in 87% yield (Scheme 4 2). These conditions proved to

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68 be generally successful but in the absence of palladium catalyst no reaction occured during 30 min and prolonged reaction times resulted in decomposition. Other catalyst systems including CuSO 4 anhydrous, CuSO 4 .5H 2 O, Pd(OAc) 2 were less effective as were lower temperatures, power and catalyst loadings (100 o C, 20 W and 100 o C, 50 W, 130 o C, 50 W, 5 mol% Pd(PPh 3 ) 4 ) (Table 4 1).

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69 Table 4 1 Optimization of reaction conditions for the synthesis of 4.4a Entry Catal yst Reaction conditions 4.4a Yield (%) 1 None 130 o C, 50 W, 30 90 min 0 2 10 mol%Pd(PPh 3 ) 4 130 o C, 50 W, 30 min 87 3 10 mol% Pd(Ph 3 ) 4 rt, CHCl 3 12h 0 4 10 mol% Pd(Ph 3 ) 4 reflux, CHCl 3 12h 0 5 10 mol% Pd(Ph 3 ) 4 130 o C, 12h 0 6 5 mol% Pd(Ph 3 ) 4 130 o C, 50 W, 30 min 45 7 10 mol% CuSO 4 anhyd. 130 o C, 50 W, 30 min 0 8 10 mol% CuSO 4 5H 2 O 130 o C, 50 W, 30 min 20 9 10 mol% Pd(O a c) 2 130 o C, 50 W, 90 min 30 10 10 mol% PPh 3 130 o C, 50 W, 40 min 35 4.2. 2 Synthesis of (Benzotriazol 1 yl)ethyl esters 4.4 I examined the scope of the palladium catalyzed reaction of N acylbenzotriazoles 4.1a f with epoxides 4.3a c using the optimized conditions as detailed in Table 4 2.

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70 (Benzotriazol 1 yl)ethyl esters 4.4 were obtained as single isomers in 52 87% yields. The reaction proceeded faster with N aroylbenzotriazoles (reaction time, 30 min.) than with N alkylbenzotriazoles (reaction time, 60 min) and aromatic as well as alkyl substituted epoxides were tolerated (Table 4 2). Ta ble 4 2 Synthesis of (B enzotriazol 1 yl )ethyl esters 4.4 Entry Substrate 4. 1 Substrate 4.3 Product 4.4 yield(%) 1 p CH 3 CH 2 C 6 H 4 4.1a R =H, R =C 6 H 5 4.3a 4.4a 87 2 p CH 3 CH 2 C 6 H 4 4.1a R = C 6 H 5 R =C 6 H 5 4.3b 4.4b 62 3 C 6 H 5 CH 2 CH 2 4.1b R =H, R =CH 3 (CH 2 ) 3 4.3c 4.4c 72 4 1 Naphthyl 4.1c R =H, R =C 6 H 5 4.3a 4.4d 62 5 1 Naphthyl 4.1c R =H, R =CH 3 (CH 2 ) 3 4.3c 4.4e 73 6 p NO 2 C 6 H 4 4.1d R =H, R =C 6 H 5 4.3a 4.4f 70 7 C 6 H 5 4.1e R =H, R =C 6 H 5 4.3a 4.4g 75 8 C 6 H 5 4.1e R =H, R = CH 3 (CH 2 ) 3 4.3c 4.4h 76 9 1 Adamantane CH 2 4.1f R =H, R = CH 3 (CH 2 ) 3 4.3c 4.4i 52

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71 I propose initial oxidative addition of Pd(0) forming 4.6 121 The interaction of 4.6 with epoxide 4.3 forms 4. which is then attacked by benzotriazole giving 4 .4 as a single regioisomer (Scheme 4 3). Scheme 4 3 Possible mechanism The mechanism appears to be similar to the recent report 91 of coupling reaction of acyl halides and epoxides affording halohydrin esters as mixtures o f regioisomers (Scheme 4 4). 91 However, the palladium catalyzed path way proved to be regioselective providing a single regioisomer in each case Scheme 4 4. Acid halides and epoxides in halohydrin ester synthesis 91

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72 R eaction of Z L Phe Bt 4.1g with 4. 3c however, gave 4.5 the alcohol deri ved from ring opening of the epoxide by the benzotriazole anion (Scheme 4 5). In this case the epoxide oxygen is protonated rather than acylated. Scheme 4 5. Synthesis of pseudohalohydrin 4.5 4.3 Conclusions In conclusi on, an efficient Pd catalyzed one step, regioselective pathway towards pseudohalohydrin esters is reported. 4.4 Experimental 4.4.1 General Methods All reagents were available commercially. Melting points were determined on a capillary point apparatus equip ped with a digital thermometer and are uncorrected. The NMR spectra were recorded in CDCl 3 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Silica gel was utilized for column chromatography. Aliphatic and aromatic epoxides and carboxyli c acid groups were purchased from Aldrich and TCI America and used without further purification. 4.4.2 General P rocedure for the P reparation of N A cylbenzotriazoles 4. 1

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73 Thionyl chloride (0.6 mL, 8.00 mmol, 1.2 equiv) was added to a solution of 1 H benzotriazole (3.17 g, 26.67 mmol, 4 equiv) in methylene chloride to give a clear yellow solution that was stirred for 15 min at room temperature. The carboxylic acid (6.67 mmol, 1 equiv) was then added to give a suspension which was stirred for 2.5 h at room temperature. The suspension was filtered, the filtrate evaporated, the residue dissolved in EtOAc and the solution washed with a saturated solution of sodium carbonate. The organic portion was dried over anhydrous MgSO 4 filt ered, and dried to give the corresponding N acylbenzotriazoles 4.1 (1H Benzotriazol 1 yl)(4 ethylphenyl)methanone ( 4. 1a ) W hite microcrystals (91%); mp 112.0 113.0 o C; 1 H NMR (CDCl 3 ): 7.96 (d, J = 8.1 Hz, 1H), 7.88 7.64 (m, 3H), 7.26 (t, J = 7.1 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 7.01 6.98 (m, 2H), 2.36 (q, J = 7.3 Hz, 2H), 0.90 (t, J = 7.7 Hz, 3H); 13 C NMR (CDCl 3 ): 166.3, 150.7, 145.5, 132.2, 131.9, 130.1, 128.6, 127.9, 126.0, 119.9, 114.6, 28.9, 15.0; Anal. Calcd for C 15 H 13 N 3 O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.96; H, 5.56; N, 16.97. 1 (1H Benzotriazol 1 yl) 3 phenylpropan 1 one ( 4.1b ). W hite microcrystals (90%); mp 71.0 72.0 o C (lit. 23 mp 62.0 64 .0 o C); 1 H NMR (CDCl 3 ): 8.26 8.22 (m, 1H), 8.07

PAGE 74

74 8.04 (m, 1H), 7.59 (tt, J = 8.1, 1.5 Hz, 1H), 7.45 (tt, J = 7.2, 1.5 Hz, 1H), 7.28 7.24 (m, 4H), 7.21 7.15 (m, 1H), 3.72 (td, J = 7.8, 1.1 Hz, 2H), 3.19 (t, J = 7.7 Hz, 2H); 13 C NMR (CDCl 3 ): 171 .6, 146.1, 139.8, 131.0, 130.4, 128.6, 128.4, 126.5, 126.1, 120.1, 114.4, 37.1, 30.1; Anal. Calcd for C 15 H 13 N 3 O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.91; H, 5.59; N, 16.99. (1H Benzotriazol 1 yl)(naphthalen 2 yl)methanone ( 4. 1c) W hite microcrysta ls (76%); mp 140.0 142.0 o C (lit. 122 mp 136.0 137.0 o C) ; 1 H NMR (CDCl 3 ): 8.50 8.47 (m, 1H), 8.19 8.09 (m, 3H), 7.98 7.91 (m, 2H), 7.77 7.71 (m, 1H), 7.63 7.53 (m, 4H); 13 C NMR (CDCl 3 ): 167.8, 146.3, 133.7, 133.2, 132.2, 131.2, 130.7, 130.4, 129.5, 128.9, 128.1, 126.9, 126.7, 124.9, 124.5, 120.5, 114.9. (1H Be nzo[d][1,2,3]triazol 1 yl)(4 nitrophenyl)methanone (4.1d) W hite microcrystals (75%); mp 199.0 200.0 o C (lit. 123 mp 194.0 196.0 o C); 1 H NMR (CDCl 3 ): 8.42 8.34 (m, 5H), 8.18 (d, J = 8.1 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.59 (t, J =7.7 Hz, 1H); 13 C NMR (CDCl 3 ): 165.2, 150.6, 146.0, 137.1, 132.8, 131.1, 131.2, 127.2, 123.7, 120.7, 114.9.

PAGE 75

75 (1H Benzotriazol 1 yl)(phenyl)methanone ( 4. 1e) W hite microcrys tals (90%); mp 119.0 120.0 o C (lit. 124 mp 112.0 113.0 o C); 1 H NMR (CDCl 3 ): 8.36 (d, J = 8.4 Hz, 1H), 8.23 8.15 (m, 3H), 7.70 7.49 (m, 5H); 13 C NMR (CDCl 3 ): 166.8, 145.9, 133.8,132.5, 131.9, 131.6, 130.5, 128.6, 126.5, 120.3, 114.9. 2 ((3r,5r,7r) Adamantan 1 yl) 1 (1H benzo[d][1,2,3]triazol 1 yl)ethanone ( 4. 1f) W hit e microcrystals (74%); mp 92.0 93.0 o C (lit. 125 mp 84.0 85.0 o C); 1 H NMR (C DCl 3 ): 8.33 8.29 (m, 1H), 8.11 8.07 (m, 1H), 7.65 7.59 (m, 1H), 7.51 7.45 (m, 1H), 3.19 (s, 2H), 1.96 (br s, 3H), 1.73 1.74 (m, 12H); 13 C NMR (CDCl 3 ): 171.1, 146.5, 131.2, 130.4, 126.2, 120.3, 114.9, 48.4, 42.7, 36.8, 34.7, 28.8. (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate ( 4. 1g) W hite microcrystals (90%); mp 150.0 152.0 o C (lit. 5 mp 152.0 153.0 o C); 1 H NMR ( CDCl 3 ): 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t,

PAGE 76

76 J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.32 7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H), 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); 13 C NMR (CDCl 3 ): 170.8, 155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 128.1, 127.4, 126.5, 120.4, 114.3, 67.2, 55.6, 38.8. 4.4.3 General P rocedure for the P reparation of ( B enzotriazol 1 yl)ethyl E sters 4. 4 a I and 4.5 To a mixture of N acylbenzotriazoles 4.1 (0.20 mmol) and Pd(PPh 3 ) 4 (23.11 mg, 10 mol%) in a microwave tube was added epoxide 4.3 (1.5 equiv). The mixture was stirred at 130 o C and 50 W for 30 min. ( N aroylbenzotriazoles) or 60 min. ( N alkylbenzo triazoles). The residue was dissolved in MeOH and purified by silica gel column chromatography to obtain the corresponding hydrin esters 4.4 Table 4 1 summarizes optimization of the reaction condition s 2 (1H Benzotriazol 1 yl) 1 phenyleth yl 4 ethylbenzoate ( 4.4a ) P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 7:3) to obtain a yellow oil, (87%); 1 H NMR (CDCl 3 ): 7.83 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.27

PAGE 77

77 7.14 (m, 10H), 7.06 7.01 (m, 1H), 6.29 6.25 (m, 1H), 5.00 4.86 (m, 2H), 2.48 (q, J = 7.4 Hz, 2H), 1.03 (t, J = 7.5 Hz, 3H); 13 C NMR (CDCl 3 ): 165.3, 150.3, 136.7, 133.4, 129.9, 128.9, 128.0, 127.4, 126.3, 125.7, 123.9, 120.0, 109.3, 74.3, 52.8, 29.0, 15.2; HRMS m/z for C 23 H 22 N 3 O 2 [M+ H] + calcd. 372.1707, found 372.1703. 2 (1H Benzotriazol 1 yl) 1,2 diphenylethyl 4 ethylbenzoate ( 4.4b ) P urified by gradient silica gel column chromatography (hexanes to hexanes:CH 2 Cl 2 3:2, then hexanes:CH 2 Cl 2 1:1) to obtain beige microcrytals, (62% ), mp 109.0 110.0 o C; 1 H NMR (CDCl 3 ): 8.09 (dd, J = 8.3 ,1.4 Hz, 1H), 7.68 7.60 (m, 2H), 7.54 7.47 (m, 1H), 7.45 7.30 (m, 6H), 7.27 7.20 (m, 6H), 7.14 7.11 (m, 2H), 6.34 (dd, J = 9.2, 1.4 Hz, 1H), 2.82 2.73 (m, 1 H), 2.65 (q, J = 7.5 Hz, 2H), 1.35 1.28 (m, 2H), 1.22 (td, J = 7. 8, 2.1 Hz, 3H); 13 C NMR (CDCl 3 ): 165.0, 149.9, 136.8, 134.6, 133.3, 130.2, 129.6, 128.8, 128.7, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 127.7, 127.3, 127.2, 126.8, 125.9, 123.9, 120.1, 109.6, 76.8, 67.4, 28.8, 15.1; HRMS m/z for C 29 H 26 N 3 O 2 [M+H] + calcd .448.2020, found 448.2022.

PAGE 78

78 1 (1H Benzo[d][1,2,3]triazol 1 yl)hexan 2 yl 3 phenylpropanoate ( 4. 4c ). P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 4:1) to obtain yellow oil, (72%); 1 H NMR (CDCl 3 ): 8.05 8.01 (m, 1H) 7.49 7.42 (m, 2H), 7.36 7.31 (m, 1H), 7.29 7.07 (m, 5H), 5.28 5.21 (m, 1H), 4.74 (dd, J = 14.6, 4.8 Hz, 1H), 4.69 (dd, J = 14.5, 6.1 Hz, 1H), 2.97 2.40 (m, 4H), 1.59 1.52 (m, 2H), 1.30 1.19 (m, 4H), 0.83 (t, J = 7.0 Hz, 3H); 13 C NMR (CDCl 3 ) : 172.4, 145.9, 140.27, 133.7, 128.6, 128.4, 127.7, 126.5, 124.2, 120.2, 109.7, 72.5, 50.9, 35.9, 31.5, 30.9, 27.3, 22.6, 14.1. 2 (1H Benzo[d][1,2,3]triazol 1 yl) 1 phenylethyl 1 naphthoate ( 4.4d ). P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 9.3:0.7) to obtain white solid, (62%); mp 62.0 63.0 o C 1 H NMR (CDCl 3 ): 8.60 8.56 (m, 1H), 8.09 7.97 (m, 3H), 7.85 7.79 (m, 1H), 7.50 7.26 (m, 11H), 6.58 (dd, J = 7.5, 4.7 Hz, 1H), 5.19 (dd, J = 14.6, 7.5 Hz, 1H), 5.1 1 (dd, J = 14.6, 4.7 Hz, 1H); 13 C NMR (CDCl 3 ): 166.1, 146.0, 136.9, 134.0, 133.9, 133.6, 131.4, 130.6, 129.2, 128.7, 128.1, 127.7, 126.6, 126.5, 125.7, 124.6, 124.1, 120.3, 109.5, 74.7, 53.1; Anal. Calcd for C 25 H 19 N 3 O2: C, 76.32; H, 4.87; N, 10.68. Found : C, 75.97; H, 5.31; N, 10.45.

PAGE 79

79 1 (1H Benzo[d][1,2,3]triazol 1 yl)hexan 2 yl 1 naphthoate ( 4.4e ) P urified by gradient silica gel column chromatography (hexanes to EtOAc:hexanes, 9.3:0.7) to obtain a yellow oil (73%); 1 H NMR (CDCl 3 ): 8.64 8.59 (m, 1H), 8.02 7.89 (m, 3H), 7.79 7.75 (m, 1H), 7.51 7.17 (m, 6H), 5.61 5.53 (m, 1H), 4.88 (d, J = 5.3 Hz, 2H), 1.78 1.70 (m, 2H), 1.51 1.22 (m, 4H), 0.81 (t, J =7.2 Hz, 3H); 13 C NMR (CDCl 3 ): 166.8, 146.1, 133.9, 133.7, 131.5, 130.5, 128.7, 128.0, 127.6, 126.5, 126.4, 125.7, 124.6, 124.1, 120.2, 109.9, 72.8, 51.0, 31.7, 27.6, 22.6, 14.1; Anal. Calcd for C 69 H 73 N 9 O 8 : C, 71.67; H, 6.36; N, 10.90. Found: C, 71.53; H, 6.38; N, 10.93. 2 (1H Benzo[d][1,2,3]triazol 1 yl) 1 phenylethyl 4 nitrobenzoat e ( 4. 4f ) P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 9:1) to obtain a yellow oil (70%); 1 H NMR (CDCl 3 ): 8.31 8.11 (m, 7H), 7.52 7.34 (m, 5H), 6.44 (dd, J = 8.4, 3.6 Hz, 1H), 4.82 (dd, J = 12.0, 8.4 Hz, 1H), 4.71(dd, J =12.2, 3.8 Hz, 1H); 13 C NMR (CDCl 3 ): 164.5, 164.0, 151.0, 135.6, 135.2, 135.1, 131.1, 131.0, 129.5, 129.3, 126.9, 123.9, 75.1, 67.3.

PAGE 80

80 2 (1H Benzotriazol 1 yl) 1 phenylethyl benzoate ( 4.4g ). P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 9:1) to obtain a yellow oil (75%); 1 H NMR (CDCl 3 ): 8.02 7.93 (m, 3H), 7.55 7.27 (m, 11H), 6.46 (dd, J = 7. 5, 4.8 Hz, 1H), 5.15 (dd, J = 14.5, 7.3 Hz, 1H), 5.07 (dd, J = 14.5, 4.8, 1H); 13 C NMR (CDCl 3 ): 165.4, 145.9, 136.8, 133.6, 129.9, 129.5, 129.2, 129.1, 128.6, 127.6, 126.5, 124.1, 120.2, 109.4, 74.7, 52.9; HRMS m/z for C 21 H 18 N 3 O 2 [M+H] + calcd. 344.1394, found 344.1384. 1 (1H Benzo[d][1,2,3]triazol 1 yl)hexan 2 yl benzoate ( 4. 4h ) P urified by gradient silica gel column chromatography (hexanes to hexanes:EtOAc, 9:1) to obtain a yellow oil (76%); 1 H NMR (CDCl 3 ): 8.03 8.00 (m, 1H), 7.91 7.88 (m, 2H) 7.55 7.49 (m, 2H), 7.40 7.28 (m, 4H), 5.55 5.48 (m, 1H), 4.90 (d, J = 5.2 Hz, 2H), 1.76 1.67 (m, 2H), 1.47 1.26 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H); 13 C NMR (CDCl 3 ): 166.1, 146.1, 133.7, 133.5, 129.9, 129.7, 128.6, 127.6, 124.1, 120.2, 109.9, 7 2.9, 50.9, 31.5, 27.5, 22.6, 14.0; HRMS m/z for C 19 H 22 N 3 O 2 [M+H] + calcd. 324.1707, found 324.1719.

PAGE 81

81 1 (1H Benzo[d][1,2,3]triazol 1 yl)hexan 2 yl 2 ((3r,5r,7r) adamantan 1 yl)acetate ( 4.4i ). P urified by gradient silica gel column chromatography (hexane s to hexanes:EtOAc, 9.3:0.7) to obtain a yellow oil (52%); 1 H NMR (CDCl 3 ): 8.00 7.97 (m, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.47 7.41 (m, 1H), 7.33 7.28 (m, 1H), 5.27 5.19 (m, 1H), 4.72 4.69 (m, 2H), 2.05 1.88 (m, 3H), 1.84 1.80 (m, 2H), 1.63 1.55 (m, 8H), 1.49 1.46 (m, 2H), 1.37 1.18 (m, 8H), 0.82 (t, J = 7.0 H z, 3H); 13 C NMR (CDCl 3 ): 171.3, 146.0, 133.8, 127.7, 124.1, 120.2, 109.9, 71.9, 50.9, 49.0, 42.5, 42.4, 36.9, 36.8, 32.9, 31.7, 28.8, 28.7, 27.5, 22.6, 14.1; HRMS m/z for C 24 H 33 N 3 O 2 Na [M+Na] + calcd. 418.2465, found 418.2480. 1 (1H Benzotriazol 1 yl)h exan 2 ol ( 4.5 ) P urified by gradient silica gel column chromatography (hexanes to EtOAc:hexanes, 4:1) to obtain a yellow oil 126 (70%); 1 H NMR (CDCl 3 ): 7.96 (dd, J = 8.4, 0.9 Hz, 1H), 7.60 (dd, J = 8.7, 0.9 Hz, 1H), 7.50 7.45 (m, 1H), 7.36 7.30 (m, 1H), 4.71 4.64 (m, 2H), 4.56 4.49 (m, 1H), 4.25 (br s, 1H), 1.65 1.34 (m, 6H), 0.92 (t, J = 7.5 Hz, 3H); 13 C NMR (CDCl 3 ): 145.5, 133.8, 128.5, 127.4, 124.0, 1 19.7, 109.9, 71.0, 54.0, 34.3, 27.6, 22.6, 14.0; HRMS m/z for C 12 H 17 N 3 ONa [M+Na] + calcd. 242.1264, found 242.1266.

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82 CHAPTE R 5 SOLUTION PHASE SYNTHESIS OF C HIRAL O ACYL ISODIPEPTIDES iv 5.1 Introduction The synthesis of peptides and proteins is of great importance to the under standing of biological function Automated solid ph ase peptide synthesis has empowered rapid and convenient preparation of target peptides. However, the phenomena of aggregation in th e synthesis of long peptides, proteins and peptides quence problematic, resulting in low yields and purity. 127 137 The a ggregation is attributed to intermolecular hy in solution. H ydrogen bond networks in resin bound peptides can form extended structures sheets (Figure 5 1) 128 129 138 This phenomenon d epends on the nature of the peptide and side chain protecting groups, commonly occurring in peptides with sequences containing numerous Ala, Val, Ile, Asn and Gln residues. 139 Figure 5 1. Sheets. 128 129 138 Existing strategies to overcome such associations comprise iv Reproduced with permission from Med. Chem. Commun 2011 2 1087 1092. Copyright 2011 Th e Royal Society of Chemistry

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83 like solvent composition, elevated temperature, and use of chaotropic salts or solubilizing protecting groups but have been reported to have variable efficiencies. 136 Sheppard and Johnson et al. developed an N alkylation system, the 2 hydroxy 4 methoxybenzyl (Hmb) building block introduced as an amide protect ing group, thus preventing hydrogen bonding 140 Mutter introduced pseudo proline units which are essentially Ser/Thr derived oxazolidine and Cys derived thiazolodine derivatives. These onformation in the peptide backbone, originating in the preference for cis amide bond formation, thus, preventing peptide aggregation, self association. However, this metholodogy requires 2 6 step modification of Fmoc amino acids to synthesize the building blocks in solution. In addition, the removal of such building blocks proved to be difficult, requiring strong acid treatments (Figure 5 2) 139 141 142 Figure 5 2. Pseudo prolines. 128 130 131 Kiso et al. 130 demonstrated that the introduction of an O acyl in place of an N acy l residue within a peptide backbone significantly altered the secondary structure of native

PAGE 84

84 peptides O peptides (Kiso denotes O t native peptides under physiological condtions) 130 are more hydrophilic, and easier to purify by HPLC 130 The hydroxy amino acids have higher water solubility because of the newly formed and ionized amino group. 143 He found that a subsequent O N intramolecular acyl migration, triggered by change in pH, could rapidly generate a target natural peptide under physiological conditions (pH 7.4) (Figure 5 3 ). 129 144 O acyl 144 146 has been used to develop new water soluble taxoid prodrugs 147 148 HIV 1 protease inhibitors 149 the anti tumor agent, paclitaxel, 150 difficult sequence containing peptides including Ac Val Val Ser Val Val NH 2 128 139 147 148 Alzeheimer's disease related amyloid peptide (A ) 1 42, 90 91 94 131 143 151 154 and cyclic peptides 155 Figure 5 3 O Acyl isopeptide methodology However, epimerization during the esterification step in the solid phase synthesis of O acyl isopeptides remained a major problem. 1 29 131 156 Based on the hypothesis that epimerization during esterification should be suppressed in solution by a faster coupling rate compared to that on a solid support, Kiso 129 131 synthesized O acyl isodip eptides in three steps (Scheme 5 1 ): (i) p rotection of the carboxylic acid group in serine or threonine by benzyl esterification, (ii) O acylation and (iii) deprotection using

PAGE 85

85 Pd/C. Treatment of Cbz protected isodipeptides containing Cys and Met with H 2 over Pd/C failed, although catalytic hydroge n transfer (CTH) to Cys and Met containing protected isodipeptides gave 45% of the desired product. 129 Scheme 5 1. Literature reported s ynthetic scheme 129 The chapter report s an efficient single step preparation of chiral O acyl isodipeptides from serine and threonine. Advantageous N acylbenzotriaz ole methodology was used for such transformations. 19 51 89 N (Protected aminoacyl)benzotriazoles have enabled fast preparations of biologically relevant peptides and peptide conjugates in high yields and purity, under mild reaction conditions, with full retention of the original chirality. 89 5.2 Results and Discussion 5.2.1 Synthesis of Serine based O Acylisodipeptides O Acyl isoserinedipeptides 5.3 a h were prepared by O acylation of Boc protected serine 5. 1a with various N Pg (a aminoacyl)benzotriazoles 5. 2 in the

PAGE 86

86 presence of diisopropylethylamine in CH 3 CN at 23 o C fo r 12 h in yields of 74 90%. These proved to be the optimum condition s under which neither epimerization of 5. 3 nor hydrolysis of 5.2 occurred. The presence of water (MeCN/H 2 O, 9 : 1) in the reaction caused minimal hydrolysis of 5.2 (4%) but no epimerization was detected by HPLC MS analysi s on 5.3b and ( 5.3b + 5.3b ). The O acylated serine targets were characterized by NMR and elemental analysis (Table 5 1).

PAGE 87

87 Table 5 1 The preparation of serine based O acyl isodipeptides 5.3a h HPLC analysis [chirobiotic T column (250 mm 4.6 mm), detection at 254 nm, flow rate 2.5 mL min 1 MeOH] on 5.3b (single peak, retenti on time 1.3 min) and ( 5.3b + 5.3b ) (two peaks, retention times, 1.3 min and 1.5 min) as well as HPLC MS and ( ) ESI MS Entry O acyl isodipeptide s 5. 3a g Yield % mp/ o C 1 2 3 4 5 6 7 8 Boc L Ser(Cbz L Ala)OH 5. 3a Boc L Ser(Cbz L Phe)OH 5. 3b Boc L Ser(Cbz DL Phe)OH ( 5. 3b+ 5. 3b') Boc L Ser(Cbz L Trp)OH 5. 3c Boc L Ser(Cbz L Met)OH 5. 3d Boc L Ser(Cbz L Val)OH 5. 3e Boc L Ser(Cbz Gly)OH 5. 3f Boc L Ser(Cbz L Cys(Bzl)OH 5. 3g 84 81 81 87 74 90 87 85 oil oil 60 62 67 69 oil oil 57 58 oil 9 Boc L Ser(Boc Gly)OH 5. 3h 82 58 59

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88 on 5.3b (Figure 5 4 ) confirmed the absence of racemization in the targeted isodipeptides. Figure 5 4 HPLC MS and ( )ESI MS analysis o f 5.3b 5.2.2 Synthesis of Threonine based O Acylisodipeptides O Acylated threonine based isodipeptides 5.4a h were also prepared by O acylation of Boc protected threonine 5.1b with various N Pg ( aminoacyl)benzotriazoles 5.2 in the presence of diisopropy lethylamine in CH 3 CN at room temperature in yields of 86 91% (Table 5 2). The synthesized compounds were characterized by NMR and elemental analysis.

PAGE 89

89 Table 5 2. The preparation of threonine based O acyl isodipeptides 5.4a h HPLC analysis [chirobiotic T column (250 mm 4.6 mm),detection at 254 nm, flow rate 0.5 mL min 1 MeOH : H 2 O, 4 : 1] on 5.4b (single peak, retention time 7.23 min) and ( 5.4b + 5.4b ) (two peaks, retention times, 6.54 min and 7.20 min) confirmed the retention of chirality and lack of racemization in the desired isodipeptides. Entry O acyl isodipeptides 5. 4a f Yield ( % ) mp/ o C 1 2 3 4 5 6 7 Boc L Thr(Cbz L Ala) OH 5. 4a Boc L Thr(Cbz L Phe) OH 5. 4b Boc L Thr(Cbz D,L Phe) OH ( 5. 4b+ 5. 4b') Boc L Thr(Cbz L Trp) OH 5. 4c Boc L Thr(Cbz L Met) OH 5. 4d Boc L Thr(Cbz Gly) OH 5. 4e Boc L Thr(Cbz L Cys(Bzl)) OH 5. 4f 91 87 87 91 90 86 86 oil oil 60 62 89 90 oil 54 58 oil 8 Boc L Thr(Boc Gly ) OH 5. 4 g 85 64 65 9 Boc L Thr(Boc L Phe ) OH 5. 4 h 90 47 50

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90 5.3 Conclusions In conclusion, the mild protocol reported herein enables the efficient preparation of optically pure O acyl isopeptides from serine and threonine without protection of their carboxyl groups. 5.4 Experimental 5.4.1 General Methods Melting points were deter mined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. The NMR spectra were recorded in CDCl 3 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. 5.4.2 General P rocedure for the P reparation of N (Pg A minoacyl) B enzotriazoles 5. 2 N (Z Aminoacyl)benzotriazoles 5.2 Thionyl chloride (0.6 mL, 8.00 mmol, 1.2 equiv) was added to a solution of 1 H benzotriazole (3.17 g, 26.67 mmol, 4 equiv) in methylene chloride to give a clear yellow solution that was st irred for 15 min at room temperature. The amino acid (6.67 mmol, 1 equiv) was then added to give a suspension which was stirred for 2.5 h at room temperature. The suspension was filtered, the filtrate evaporated, the residue dissolved in EtOAc and the solu tion was washed with a saturated solution of sodium carbonate. The organic portion was dried over anhyd MgSO 4 filtered, and evaporated to give the corresponding N (Z aminoacyl)benzotriazole 5.2 N (Boc Aminoacyl)benzotriazoles 5.2 Boc protected amino acid (0.03 mol) was added to a solution of DCC (1 equiv) in methylene chloride under an atmosphere of nitrogen. After 30 min., BtH (1 equiv) was added and the mixture was stirred for 12 h. The suspension was filtered through a bed of silica and celite, the filtrate evaporated,

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91 and the residue dissolved in EtOAc, then filtered through a bed of silica and celite and washed with a solution of saturated sodium carbonate, t hen with water and brine. The organic portion was dried over anhyd MgSO 4 filtered on a bed of silica, and evaporated to give the corresponding N (Boc aminoacyl)benzotriazole 5.2 1 H NMR and mp of Boc Gly Bt 5.2h and Boc L Phe Bt 5.2i were found in agreement with that reported in the literature. 157 158 (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxopropan 2 yl)carbamate ( 5. 2a ) W hite solid (90%); mp 115 o C (lit. 159 mp 113 115 o C); 1 H NMR (CDCl 3 ): 8.16 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.40 7.03 (m, 6H), 5.80 5.60 (m, 2H), 5.10 4.99 (m, 1H), 1.59 (d, J = 6.3 Hz, 3H); 13 C NMR (CDCl 3 ): 172.2, 155.6, 145.9, 136.0,131.0, 130.6, 1 28.4, 128.1, 126.4, 120.2, 114.3, 67.1, 50.5, 19.0. (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate ( 5. 2b ). White solid (90%); mp 150 152 o C (lit. 160 mp 149.0 150.0 o C); 1 H NMR (CDCl 3 ): 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.32 7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H), 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); 13 C NMR (CDCl 3 ): 170.8,155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 128.1,127.4, 126.5, 120.4, 114.3, 67.2, 55.6, 38.8. Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate ( 5.2b + 5. 2b ) White solid (9 0%); mp 141 142 o C (lit. 22 mp 141 142 o C); 1 H NMR (CDCl 3 ): 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H) 7.32 7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H),

PAGE 92

92 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); 13 C NMR (CDCl 3 ): 170.8, 155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 1 28.1, 127.4, 126.5, 120.4, 114.3, 67.2, 55.6, 38.8. (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 3 (1H indol 3 yl) 1 oxopropan 2 yl)carbamate ( 5. 2c ) Yellow solid (80%); mp 101 o C (lit. 157 mp 98 100 o C); 1 H NMR (CDCl 3 ): 8.23 (br s, 1H), 8.14 8.06 (m, 2H), 7.57 (t, J = 7.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.31 7.24 (m, 4H), 7.20 (s, 1H), 7.10 7.04 (m, 1H), 6.95 6.90 (m, 2H), 6.87 6.85 (m, 1H), 6.14 6.10 (m, 1H), 5.70 (d, J = 7.5 Hz, 1H), 5.03 (s, 2H), 3.58 (dd, J = 15 .0 4.5 Hz, 1H), 3.40 (dd, J = 14.9, 7.7 Hz, 1H); 13 C NMR (CDCl 3 ): 171.1, 155.9, 145.8, 136.1, 131.0, 130.6, 128.4, 128.1,127.0, 126.4, 123.2, 122 .2, 120.2, 119.7, 118.3, 114.3, 111.2, 108.9, 67.2, 55.1, 28.7. (S) Benzy l (1 (1H benzo[d][1,2,3]triazol 1 yl) 4 (methylthio) 1 oxobutan 2 yl)carbamate ( 5. 2d ) Beige solid (85%); mp 108 109 o C (lit. 157 mp 108 109 o C); 1 H NMR (CDCl 3 ): 8.15 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.30 7.24 (m, 5H), 5.83 5.79 (m, 1H), 5.67 5.65 (m, 1H), 5.03 (s, 2H), 2.59 (t, J = 7.1 Hz, 2H), 2.35 (br s, 1H), 2.10 2.03 (m, 1H), 1.97 (s, 3H) ; 13 C NMR (CDCl 3 ): 171.2,155.7, 130.9, 146.0, 130.8, 128.4, 128.5, 128.3, 126.6, 120.4, 114.3, 67.4, 54.2, 32.4, 30.0, 15.4; Anal. Calcd for C 19 H 20 N 4 O 3 S: C, 59.36; H, 5.24; N, 14.57. Found: C, 59.69; H, 5.23; N, 14.54. (S) Benzyl (1 (1H benzo[d][1,2,3]tri azol 1 yl) 3 methyl 1 oxobutan 2 yl)carbamate ( 5. 2e ) White powder (87%); mp 107 o C (lit. 160 mp 73 74 o C); 1 HNMR (CDCl 3 ): 8.28 (d, J = 8.0 Hz, 1H), 8.15 ( d, J = 8.2 Hz, 1H), 7.68 (dd, J = 8.0, 7.4 Hz, 1H), 7.54 (dd, J =

PAGE 93

93 8.1, 7.3 Hz, 1H), 7.37 (br s, 5H), 5.78 5.74 (m, 1H), 5.57 (d, J = 9.2 Hz, 1H), 5.14 (s, 2H), 2.61 2.43 (m, 1H), 1.13 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H); 13 C NMR (CDCl 3 ): 17 1.5, 156.2, 146.0, 136.0, 131.0, 130.7, 128.5, 128.2, 126.5, 120.3, 114.3, 67.3, 59.4, 31.6, 19.7, 17.0. (S) Benzyl (2 (1H benzo[d][1,2,3]triazol 1 yl) 2 oxoethyl) carbamate ( 5.2f ) White solid (90%); mp 110 111 o C (lit. 161 mp 106 108 o C); 1 H NMR (CDCl 3 ): 8.24 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H),7.39 7.34 (m, 5H), 5.61 (br s, 1H), 5.19 (s, 2H), 5.09 (d, J = 5.7 Hz, 2H); 13 C NMR (CDCl 3 ): 168.3, 156.4, 145.9, 136.0, 130.8,128.5, 128.2, 128.1, 126.5, 120.3, 114.0, 67.4, 44.8. (R) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 3 (benzylthio) 1 oxopropan 2 yl)carbamate ( 5. 2g ) Y ellow oil (87%); 1 H NMR (CDCl 3 ): 8.31 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.60 (t, J = 8.1 Hz, 1H), 7.47 7.17 (m, 10H), 6.03 (br s, 2H), 5.22 (b r s, 2H), 3.79 (br s, 2H), 3.28 2.89 (m, 2H); 13 C NMR (CDCl 3 ): 169.8, 155.7, 145.9, 136.0, 135.9, 130.8,128.8, 128.4, 128.2, 128.1, 127.1, 126.6, 125.8, 120.3, 114.3, 67.4, 53.7, 36.1, 33.6; A nal. Calcd for C 24 H 22 N 4 O 3 S: C, 64.56; H, 4.51;N 12.55. Found: C, 64,26; H, 4.93; N 12.90. 5.4.3 General P rocedure for the P reparation of O A cyl I sodipeptides 5. 3 and 5.4 DIPEA (0.44 mL, 3 equiv) was added to a solution of Boc LSerOH or Boc L ThrOH (0.49 mm ol, 1 equiv) in MeCN (15 mL). The appropriate N (Pg a aminoacyl)benzotriazole 5. 2 (0.49 mmol,1 equiv) dissolved in MeCN (5 mL) was added to the clear solution and the mixture was stirred for 12 h at room temperature. Complete reaction was judged by the dis appearance of starting material. The sol ution was acidified with 1N HCl and evaporated; the residue was dissolved in EtOAc

PAGE 94

94 and washed with 1N HCl. The organic portion was dried over anhyd Na 2 SO 4 filtered and evaporated to give the corresponding O acyl iso dipeptide. All samples were then freeze dried and fully characterized. (R) 3 (((S) 2 (((Benzyloxy)carbonyl)amino)propanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5. 3a ) Prepared from 5. 2a C olorless oil (84%); 1 HNMR (CDCl 3 ): 10.2 (br s, 1H), 7.27 7.15 (m, 5H), 5.62 5.49 (m, 1H), 5.25 4.96 (m, 2H), 4.63 4.29 (m, 4H), 4.10 3.98 (m, 1H), 1. 36 (s, 9H); 13 C NMR (CDCl 3 ): 172.8, 172.4, 156.1, 155.6, 136.0, 128.4, 128.1, 80.6, 67.1, 64.9, 52.7, 49.7, 28.2, 18.1; Anal. Ca lcd for C 19 H 26 N 2 O 8 : C, 55.60; H, 6.39; N, 6.83. Found: C, 55.31; H, 6.41; N, 6.70. (R) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5. 3b ) P repared from 5. 2b Yellow transparent oil (81%); 1 H N MR (CDCl 3 ): 10.8 (bs, 1H), 7.31 7.18 (m, 10H), 7.11 7.05 (m, 2H), 5.53 5.44 (m, 1H), 5.10 4.97 (m, 2H), 4.64 4.55 (m, 2H), 4.41 4.32 (m, 2H), 3.04 2.98 (m, 1H), 1.42 (br s, 9H); 13 C NMR (CDCl 3 ): 172.6, 171.2, 156.0, 135.5, 129.3, 129.1, 128.6, 128.5, 128.4, 128.1, 128.0, 127.2, 80.6, 67.2, 65.2, 54.9, 52.5, 38.0, 28.2; Anal. Calcd for C 25 H 30 N 2 O 8 : C, 61.72; H, 6.22; N, 5.76. Found:C, 61.79; H, 6.34; N, 5.42. 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanoyl)oxy) 2 ((tert butoxycarbonyl)amino)prop anoic acid ( 5. 3b + 5. 3b ). P repared from ( 5. 2b + 5. 2b ). C olorless solid (81%); mp 60 62 o C; 1 H NMR (CDCl 3 ): 8.85 (br s, 1H), 7.33 7.10 (m, 10H), 7.05 6.95 (m, 2H), 5.59 5.41 (m, 1H), 5.03 (bs, 2H), 4.64 4.17 (m, 3H), 3.03 (bs, 2H), 1.41 (bs, 9H), 1.23 1.18 (m, 3H) ; 13 C NMR (CDCl 3 ): 175.2, 172.5, 171.1, 156.0, 155.5, 135.9, 135.5, 129.1,128.5, 128.4 128.0, 127.1, 80.5, 67.1, 65.2, 60.5,

PAGE 95

95 54.9, 53.6, 52.5, 38.8, 37.9, 37.7, 29.6, 28.2, Anal. Calcd for C 25 H 30 N 2 O 8 : C, 61.72; H, 6.22; N, 5.76. Found: C, 61.79; H, 6.35; N, 5.42. (S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 (1H indol 3 yl)propanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5. 3c ) Prepared from 5. 2c Y ellow gel (85%); 1 H NMR (CDCl 3 ): 8.37 8.31 (m, 1H), 7.92 (br s, 1H), 7.55 7.42 (m, 1H), 7.25 7.04 (m, 7H), 6.90 6.81 (m, 1H), 5.50 5.44 (m, 1H), 5.12 4.98 (m, 2H), 4.70 4.65 (m, 1H), 4.54 (br s, 1H), 4.38 4.28 (m, 1H), 3.31 3.21 (m, 2H), 1.44 (br s, 9H); 13 C NMR (CDCl 3 ): 175.9, 172.8, 171.6, 156.1, 155.7, 136.1, 128.5, 128.1, 127.3, 123.0, 122.0, 120.0,118.3, 111.4, 109.3, 80.7, 67.2, 65.0, 54.7, 52.7, 28.3; Anal. Calcd for C 27 H 31 N 3 O 8 : C, 61.71; H, 5.95; N, 8.00. Found: C, 61.96; H,6.00; N, 7.61. (R) 3 (((S) 2 (((Benz yloxy)carbonyl)amino) 4 (methylthio)butanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5. 3d ) Prepared from 5. 2d Y ellow oil (74%); 1 H NMR(CDCl 3 ): 9.40 (bs,1H), 7.27 7.12 (m, 5H), 5.72 5.62 (m, 1H), 5.08 4.97 (m, 2H), 4.57 4.42 (m, 3H), 2.49 2.41 (m, 2H), 2.22 1.84 (m, 5H), 1.36 (br s, 9H); 13 C NMR (CDCl 3 ): 175.7, 172.7, 171.4, 156.2, 155.5, 135.8, 128.4, 128.1, 80.6, 67.2, 65.1, 53.2, 52.7, 31.5, 29.7, 28.2,15.2; Anal. Calcd for C 21 H 30 N 2 O 8 S: C, 53.60; H, 6.43; N, 5.95.Found: C, 53. 94; H, 6.56; N, 5.52. (S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 methylbutanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5. 3e ) P repared from 5. 2e Y ellow oil (90%); 1 H NMR (CDCl 3 ): 9.54 (br s, 1 H), 7.34 7.22 (m, 5H), 5.80 5.51 (m, 1H), 5.12 5.00 (m, 2H),4.62 4.02 (m, 3H), 2.13 2.00 (m, 1H), 1.39 (br s, 9H), 0.96 0.83 (m, 6H); 13 C NMR (CDCl 3 ): 172.5, 171.4, 156.5, 155.5, 135.9,128.4, 128.1, 80.5, 67.2, 64.7, 59.1,

PAGE 96

96 52.7, 31.0, 28.2, 18.9, 17.4; Anal. Calcd for C 21 H 30 N 2 O 8 : C 57.52 ; H 6.90; N 6.39. Found: C:57.79, H 6.69, N 6.45. (S) 3 (2 (((Benzyloxy)carbonyl)amino)acetoxy) 2 ((tert butoxycarbonyl) amino)propanoic acid ( 5. 3f ) P repared from 5. 2f C olorless solid (87%); mp 57 58 o C; 1 H NMR (CDCl 3 ): 10.18 (br s, 1H),7.13 7.11 ( m, 5H), 5.62 5.47 (m, 1H), 4.90 (br s, 2H), 4.41 4.17(m, 2H), 3.73 (br s, 2H), 1.23 (br s, 9H); 13 C NMR (CDCl 3 ): 172.5, 169.7, 156.7, 155.5, 135.9, 128.4, 128.0, 80.5, 67.2, 65.0, 52.5, 42.5, 28.2; Anal. Calcd for C 18 H 24 N 2 O 8 : C, 54.54; H, 6.10; N, 7. 07. Found: C, 54.72; H, 6.04; N, 6.73. (S) 3 (((R) 2 (((Benzyloxy)carbonyl)amino) 3 (benzylthio)propanoyl)oxy) 2 ((tert butoxycarbonyl)amino)propanoic acid ( 5.3g ) P repared from 5.2g Y ellow gel (85%); 1 H NMR (CDCl 3 ): 10.9 (br s, 1H), 7.49 7.16 (m, 11H ), 5.81 5.71 (m, 1H), 5.09 4.98 (m, 2H), 4.61 4.32 (m, 3H), 3.66 (s, 2H), 2.79 2.73 (m, 2H), 1.36 (br s, 9H); 13 C NMR (CDCl 3 ): 172.3, 170.1, 156.0, 155.4,137.2, 135.7, 128.8, 128.4, 128.0, 127.1, 80.4, 67.2, 65.2, 53.3, 52.5, 36.1, 33.1, 28.1; An al. Calcd for C 26 H 32 N 2 O 8 S: C, 58.63; H, 6.06; N, 5.26. Found: C, 58.95; H, 6.08; N, 5.12. (S) 2 ((Tert butoxycarbonyl)amino) 3 (2 ((tert butoxycarbonyl) amino)acetoxy)propanoic acid ( 5.3h ) Prepared from 5.2h W hite microcrystals (82%); mp 58 59 o C; 1 H N MR (CDCl 3 ): 7.38 7.28 (s, 2H), 5.59 5.20 (m, 1H), 4.58 4.45 (m, 3H), 3.86 (d, J = 4.2 Hz, 2H), 1.38 (s,18H); 13 C NMR (CDCl 3 ): 172.3, 170.1,156.1, 155.6, 126.1, 114.9, 82.1, 80.5, 65.1, 52.7, 43.9, 42.3, 28.3; Anal. Calcd for C 15 H 26 N 2 O 8 : C, 49.72; H, 7.23; N, 7.73. Found:C, 50.41; H, 7.29; N, 8.16. (2S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino)propanoyl)oxy) 2 ((tert butoxycarbonyl)amino)butanoic acid ( 5. 4a ) Prepared from 5. 2a C olorless oil (91%); 1 H

PAGE 97

97 NMR (CDCl 3 ): 9.73 (bs, 1H), 7.13 7.10 (m, 6H), 5.56 5.5 3 (m, 1H), 5.28 (br s, 1H), 4.89 (br s, 2H), 4.31 3.82 (m,2H); 1.24 (s, 9H), 1.11 1.05 (m, 6H); 13 C NMR (CDCl3): 173.8, 173.2, 171.8, 156.1, 136.0, 128.4, 128.1, 80.3, 71.9, 67.0, 58.2, 57.0, 49.6, 28.2, 18.0, 16.6; Anal. Calcd for C 20 H 28 N 2 O 8 : C, 56. 60; H, 6.65; N, 6.60. Found: C, 56.21; H, 6.55; N, 6.40. (2S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanoyl)oxy) 2 ((tert butoxycarbonyl)amino)butanoic acid ( 5. 4b ). Prepared from 5. 2b C olorless oil (87%); 1 H NMR (CDCl 3 ): 9.86 (bs, 1H), 7.31 7.13 (m, 10 H), 7.13 (br s, 2H), 5.42 (br s, 1H), 5.10 4.99 (m, 2H), 4.79 4.40 (m, 2H), 3.03 (d, J = 6.0 Hz, 2H),1.46 (s, 9H), 1.28 1.18 (m, 3H); 13 C NMR (CDCl 3 ): 173.4,170.6, 156.1, 135.7, 129.2, 128.5, 128.0, 127.0, 80.3, 72.2, 67.1,56.8, 54.8, 38.1, 28.2, 16.6; Anal. Calcd for C 26 H 32 N 2 O 8 : C,62.39; H, 6.44; N, 5.60. Found: C, 61.99; H, 6.44; N, 5.53. (2S) 3 ((2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanoyl)oxy) 2 ((tert butoxycarbonyl)amino)butanoic acid ( 5. 4b + 5. 4b ) Prepared from ( 5. 2b + 5. 2b ). Y ellow solid (87%); mp 60 62 o C; 1 H NMR (CDCl 3 ): 9.56 (br s, 1H), 7.38 7.19 (m, 10H), 7.08 (br s, 2H), 5.36 5.30 (m, 2H), 5.01 (br s, 2H), 4.64 4.44 (m, 2H), 3.13 2.99 (m, 3H), 1.42 (br s, 9H), 1.19 (d, J = 22.8 Hz, 3H); 13 C NMR (CDCl 3 ): 175.7, 173.5, 170.8, 156.0, 136.0, 135.6, 129.3, 128.5, 128.4, 128.1, 127.0, 80.4, 72.3, 67.0, 56.8, 54.6, 38.1, 37.7, 28.2,16.7; Anal. Calcd for C 26 H 32 N 2 O 8 : C, 62.39; H, 6.44; N, 5.60. Found: C, 62.46; H, 6.50; N, 5.59. (2S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 3 (1H indol 3 yl)propanoyl)oxy) 2 ((tert butoxy carbonyl)amino)butanoic acid ( 5. 4c ) P repared from 5.2c Y ellow oil (90%); 1 H NMR (CDCl 3 ): 8.38 8.15 (m, 1H), 7.54 (d, J = 5.4 Hz, 1H), 7.42 6.90 (m, 12H),

PAGE 98

98 5.52 5.39 (m, 2H), 5.10 4.96 (m, 3H), 3.22 (s, 2H), 1.45 (s, 9H), 1.25 1.14 (m, 3H); 13 C NMR (CDCl 3 ): 172.7, 170.9, 156.1, 136.0,128.5, 128.1, 127.5, 122.9, 122.3, 119.7, 118.6, 111.2, 109.6, 80.5,72.0, 67.2, 56.8, 54.6, 28.3, 27.7, 16.5; Anal. Calcd for C 28 H 33 N 3 O 8 : C, 62.33; H, 6.16; N, 7.79. Found: C, 62.34; H, 6.30; N, 7.60. (2R) 3 (((S) 2 (((Benzyloxy)carbonyl)amino) 4 (methylthio)butanoyl)oxy) 2 ((tert butoxycarbonyl)amino)b utanoic acid ( 5.4d ) P repared from 5.2d Y ellow oil (90%); 1 H NMR (CDCl 3 ): 8.84 (br s, 1H), 8.58 (br s, 1H), 7.34 7.27 (m, 5H), 5.86 5.83 (m, 1H), 5.71 (t, J = 8.9 Hz, 1H), 5.52 (br s, 1H), 5.10 (br s 2H), 4.63 4.38 (m, 1H), 2.54 2.47 (m, 2H), 2.05 1.91 (m, 4H), 1.38 (s, 9H), 1.28 1.27 (m, 3H); 13 C NMR (CDCl 3 ): 17 3.6, 170.7, 156.3, 156.1,135.9, 128.4, 128.1, 80.4, 72.1, 67.2, 56.8, 53.2, 31.5, 29.8, 28.2, 16.7, 15.4; Anal. Calcd for C 22 H 32 N 2 O 8 S: C, 54.53; H, 6.66; N,5.78. Found: C, 54.87; H, 6.77; N, 5.76. (2S) 3 (2 (((Benzyloxy)carbonyl)amino)acetoxy) 2 ((tertbuto xycarbonyl)amino)butanoic acid ( 5. 4e ). P repared from 5. 2f C olorless solid (86%); mp 54 57 o C; 1 H NMR (CDCl 3 ): 7.33 7.31 (m, 5H), 7.01 (bs, 1H), 5.62 5.40 (m, 2H), 5.10 (s, 2H), 3.90 (d, J = 5.7 Hz, 2H), 1.45 (br s, 9H), 1.29 (d, J = 6.9 Hz,3H); 13 C NMR (CDCl 3 ): 173.3, 169.2, 156.5, 156.0, 136.0,128.5, 128.1, 80.5, 72.0, 67.2, 56.8, 42.6, 28.2, 16.8; HRMS m /z for C 19 H 26 N 2 O 8 Na [M + Na] + calcd 433.1581, found 433.1597 (2S) 3 (((R) 2 (((Benzyloxy)carbonyl)amino) 3 (benzylthio)propanoyl)oxy) 2 ((tert butoxycarbonyl)amino)butanoic acid ( 5. 4f ) P repared from 5. 2g Y ellow gel (86%); 1 H NMR (CDCl 3 ): 9.05 (br s, 1 H), 7.50 7.09 (m, 11H), 5.83 5.78 (m, 1H), 5.44 5.39 (m, 1H), 5.08 4.96 (m, 2H), 4.56 4.44 (m, 1H), 3.62 (s, 2H), 2.82 2.50 (m, 2H), 1.39

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99 (br s, 9H), 1.20 (d, J = 6.3 Hz, 3H); 13 C NMR (CDCl 3 ): 173.2, 169.5, 156.0, 137.4, 135.8, 128.8, 128.4,1 28.0, 127.1, 80.3, 72.4, 67.1, 56.7, 53.3, 36.2, 33.0, 28.2, 16.6; HRMS m/z for C 27 H 34 N 2 O 8 SNa [M + Na] + calcd. 569.1928, found 569.1939. (2S,3S) 2 ((Tert butoxycarbonyl)amino) 3 (2 ((tert butoxycarbonyl)amino)acetoxy)butanoic acid ( 5. 4g ). P repared from 5. 2 h W hite microcrystals (85%); mp 64 65 o C; 1 H NMR (CDCl 3 ): 7.89 (s, 2H), 5.58 5.38 (m, 3H), 4.47 4.45 (m, 1H), 3.84 (s, 2H),1.43 and 1.42 (overlapped s, 18H), 1.31 (d, J = 5.7 Hz, 3H); 13 C NMR(CDCl 3 ): 173.2, 169.7, 156.6, 156.1, 81.8, 80.4, 72.0, 56.9, 43.6, 28.3, 16.8; Anal. Calcd for C 16 H 28 N 2 O 8 : C, 51.05; H, 7.50; N, 7.44. Found: C, 50.72; H, 7.64; N, 7.41. (2S,3S) 2 ((Tert butoxycarbonyl)amino) 3 (((S) 2 ((tert butoxycarbonyl)amino) 3 phenylpropanoyl)oxy)butanoic acid ( 5. 4h ) Prepared from 5. 2i W hite microcrystals (90%); mp 47 50 o C; 1 H NMR (CDCl 3 ): 7.07 7.01 (m, 5H), 6.97 6.95 (m, 2H), 5.21 (s, 1H), 4.92 4.80 (m, 3H), 4.31 4.25 (m, 2H), 2.83 (s, 1H), 1.28 (s, 9H), 1.21 (s, 9H), 1.06 1.05 (m, 3H); 13 C NMR (CDCl 3 ): 171.0,156.1, 15 5.5, 129.2, 128.6, 127.1, 80.4, 72.1, 56.8, 54.4, 38.1, 28.3,16.6; Anal. Calcd for C 23 H 34 N 2 O 8 : C, 59.21; H, 7.35; N, 6.00. Found: C, 59.62; H, 7.92; N, 6.54.

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100 CHAPTER 6 FREE CHEMICAL LIGATI ONS FROM O ACYL SERINE SITES v 6.1 Introduction Proteins are biological macromolecules that are involved in most biochemical functions of the cell. 162 The total chemical synthesis of proteins has already contributed to knowledge of the relationship of protein structure to their function in important biological processes. 127 131 162 168 Once synthetic access to a protein has been established, chemical synthesis allows the researcher to: (i) effect, at will, any required change in the covalent structure of a protein molecule and (ii) label a protein without limitation as to the numbe r and kind of labels introduced. 162 Merr phase peptide synthesis (SPPS) is a common tool used in the synthesis of polypeptides. 134 However, the linear SPPS of a very large polypeptide can be expensive. The development of a technique to achieve a convergent synthesis using smaller polypeptide fragments then becomes critical in terms of both reducing the cost of production of peptide therapeutics and reali zing chemical synthesis of proteins. 169 173 The development of chemical ligation has facilitated the synthesis of large peptides by linking the C terminus of one unpro tected peptide with the N terminus of another (Figure 6 1) 162 167 v Reproduced with permission from Org. Biomol. Chem. 2012 10 4836 4838 Copyright 2012 The Royal Society of Chemistry

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101 Figure 6 1. Peptide ligation. 174 Native chemical ligation (NCL), first reported by Wieland 175 and later developed by Kent, 162 165 is a chemoselective a nd regioselective reaction of the thiolate of an N terminal Cys peptide with the carbon of a C terminal thioester in another peptide that results in a native ami de bond at the ligation site through a rapid NCL S to N acyl transfer via a cyclic transition state. 162 167 The bifunctional nature of the N terminal cysteine 1, 2 mercaptoamine moiety is responsible for the o bserved chemosele c tivity in NCL ( Figure 6 2 ) 169 176 Figure 6 2. Native Chemical Ligation. While of great importance, NCL has limitations that include the requirement of a N terminal cysteine residue at the ligation site to afford a peptide containing an internal

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102 cysteine. The low abundance of cysteine in human proteins (1.7% of the residues) also presents another limitation. 174 176 180 C onsiderable effort has been devoted to developing thiol auxiliary groups i n attempts to overcome the problem of low abundance of cysteine, (Figure 6 3) but, such ligations were found: (i) difficult to c omplete due to steric hindrance 174 178 186 and (ii) problematic since extraneous gro ups in the lig ated product can be troublesome to remove. 174 178 186 Another approach to overcome this limitatio n involves the conversion of a cysteine residue into a serine residue after NCL, 174 179 however, thi s requires post NCL modifications after NCL peptide synthesis. 179 Figure 6 3. Auxiliary method. To address these limitations, o ur group recently reported 159 187 188 lig ations of S acylated cysteine peptides to form native peptides through expanded transition states with 11 and 14 membered rings. The developed methodology required no auxiliary groups and enabled the selective S acylation of cysteine peptides by N acylbe nzotriazoles in good yields and under mild conditions followed by microwave assisted chemical ligations of S acyl isopeptides. However, the challenge of ligation through an 8 membered transition state and the low abundance of cysteine remained

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1 03 an obstacle. Our current approach is therefore focused on serine which possesses the 1,2 hydroxylamine bifunctionality 169 (mimic k ing the SH/NH 2 bifunctionality of cysteine) and th us offer s the possibility of chemoselective ligations by O to N acyl transfer without the need of cysteine residues. Initially, two problems existed for the acylation of the hydroxyl group of serine: (i) difficulty in achieving O acylation without epime rization (especially in solid phase synthesis) 127 130 168 and (ii) the facile hydrolysis of O acyl serine ester linkages 114 under the aqueous conditions of classical NCL. These problems were suc cessfully overcome by our recently reported methodology for the preparation of chirally pure O acyl isopeptides in a single step (74 91%) and under anhydrous conditions. 114 Kiso et al. 130 168 demonstrated that O acyl residues within a backbone s ignificantly O hydrophilic and easier to purify by HPLC 119 155 than their corresponding native peptides. N the corresponding native peptide via a 5 membered transition state (Figure 6 4 ). 128 Figure 6 4 O Acyl isopeptide methodology 128 T hat this classic O to N acyl shift via a 5 membered transition state can be extended to eight membered and eleven membered transition states is now described O to N acyl shift (at a Ser site) involving

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104 neither cysteine nor an auxiliary gro up at the ligation site can be utilized for the synthesis of longer peptides 6 .2 Results and Discussion 6 .2.1 O to N Acyl Shift via an eight membered TS Traceless chemical ligation by O to N acyl shift via an eight memb ered TS at Ser site was demonstrated in 6.4a,c Protected N (Pg aminoacyl )benzotriazoles 6. 1a c were coupled with L Ser OH using benzotriazole methodology 51 113 giving intermediate s 6. 2a c which on O acylation provided 6. 3a c Deprotection of the Cbz/Boc group of 6. 3 by hydrogenation with Pd/C or by HCl dioxane afforded O acy l iso peptides 6. 4a c (Scheme 6 1). Scheme 6 1. Preparation of O acyl iso peptide s 6.4a c

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105 Intermediates 6.4a,c underwent ligation under microwave irradiation in piperidine DMF 20 v/v%, 50 o C, 50 W, 1 h (Scheme 6 2). Anhydrous conditions were chosen t o avoid ester hydrolysis. Indeed, HPLC MS indicated the formation of the desired intra molecular ligated products 6.5a (57%, retention time 23.07 min.) and 6.5c (22%, retention time 39.50 min.) and the presence of starting materials 6.4a (43%, retention t ime, 19.61 min.) and 6.4c (78%, retention time, 38.65 min.). The retention times and fragmentation patterns of 6.4a and 6.4c were also studied by control experiments (HPLC MS of pure 6.4a or 6.4c ). HPLC MS, via ( )ESI MS/MS confirmed that compounds 6.4a an d 6.5a with MW 409 have different fragmentation patterns. This data indeed proved the formation of intramolecular ligated products 6.5a and 6.5c Moreover, product 6.5a was is olated and the structure confirmed by HRMS. Sch eme 6 2. Chemical ligation of O acyl iso peptide s 6. 4a,c 6 .2.2 O to N Acyl Shift via an eleven membered TS Traceless chemical ligation by O to N acyl shift from a Ser site via an 11 me mbered TS was achieved in iso pepti de s 6.7a b Amino unprotected O acyl iso peptides 6.4a b (Scheme 6 1) were coupled with Pg'' Gly Bt to give 6.6a b which

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106 after deprotection of the protecting group Pg'' provided O acyl iso peptides 6.7a b (Scheme 6 3). Schem e 6 3 Preparation of O acyl iso peptide s 6.7a b and their ligation Intermediates 6.7a b underwent ligation (Scheme 6 3) under anhydrous conditions (piperidine 20 v/v% in DMF, MW 50 o C, 50 W, 1 h (for 6.7a ) and 3h (for 6.7b ). HPLC MS showed formation of th e expected intramolecular ligated product 6.8a (99%, retention time 21.67 min.), hydrolysed form 6.9a (1%) and none of the intermolecular by product 6.10a As for ligation on 7b HPLC MS indicated the formation of the desired 6.8b (18%, retention time 17.8 6 min.), hydrolysed form 6.9b (8%) and intermoleculer by product 6.10b (31%). The retention times and fragmentation patterns of 6.7a and 6.7b were also studied by control experiments (HPLC MS of pure 6.7a and 6.7b ). HPLC MS, via ( )ESI MS/MS demonstrated t hat products 6.7a and 6.8a each of MW

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107 466, produced different fragmentation patterns. In addition, product 6.10a wa s isolated and structure confirmed by HRMS. The ligation of 6.7a was also examined under aqueous conditions, (pH 7.6, 1 M buffer strength, MW 50 o C, 50 W, 1 h). HPLC MS of the aqueous product, disclosed a small amount of the ligated product 6.8a and a major signal of MW 366 which corresponds to removal of the Boc group either from 6.7a or from the ligated product 6.8a 6 .2.3 Computational S tudies for O to N Acyl Shift The eight membered ring transition state in S acyl tripeptides is sterically hindered and poorly organized for binding 159 188 but the structurally similar O acyl tripeptide 6. 4a demonstrated a preferential internal O to N acyl shift. This counter intuitive reactivity of O acyl tripeptide s is however rationalized by the same computational protocol of virtual screening and quantum chemical calculations performed by Dr. Alex ander Oliferenko The effectiveness of conformational preorgani z ation was defined in terms of the b (N C) scoring functi on, i.e. the geometrical distance between the nucleophilic amine nitrogen and the electrophilic ester carbon atom. A f ull conformational search was performed using the MMX force field (as implemented in PCModel v.9.3 software), resulting in 572 conformatio ns which were subsequently ranked in descending order of the b (N C) scoring function. The best preorganised conformer shown in Figure 6 2 had a value for b (N the value of 3.591 found in the similar S acyl structur e (The GMMX routine of PCModel and MMX force field were used for scanning all rotatable bonds). 188

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108 Figures 6 2. Preorganised conformer of O acyl peptides 6.4a The quantum chemical reaction energy E react is defined as E react = E 2 E 1 where E 1 and E 2 are the energies of the starting and final, geometry optimised structures, respectively. Geometry optimization resulted in E react = 62 kcal/mol, which is abo ut 33 kcal/mol more favorable than the previously studied S acyl structure. 188 The cyclic transition state looks rather more organized than in the correspo nding S acyl structure ( previously reported in literature in Figure 2b in ref. 188 ) 6.3 Conclusions In conclusion, chemical ligation via O to N acyl transf er with 8 and 11 transition states occurs successfully without the use of either cysteine or an auxiliary group. The reactivity of O acyl peptides in traceless chemical ligation reactions is supported by theoretical and computational studies.

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109 6.4 Experim ental 6.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. The NMR spectra were recorded with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Starting m aterials were available commercially. HPLC MS analyses were performed on reverse phase gradient Phenomenex Synergi Hydro RP (C18): (2 x 150 mm; 4 um) + C18 guard column (2 x 4 mm) using 0.2% acetic acid in H 2 O/acetonitrile as mobile phases or 0.4 mM ammoni um formate in H 2 O/methanol; wavelength = 254 nm; flow rate 0.2 mL/min; and mass spectrometry was done with electro spray ionization (ESI). 6.4.2 General P rocedure for the P reparation of N (Z A minoacyl)benzotriazoles 6.1' Thionyl chloride (0.6 mL, 8.00 mmol, 1.2 equiv) was added to a solution of 1 H benzotriazole (3.17 g, 26.67 mmol, 4 equiv) in methylene chloride to give a clear yellow solution that was stirred for 15 min at room temperature. The amino acid 6.1 (6.67 mm ol, 1 equiv) was then added to give a suspension which was stirred for 2.5 h at room temperature. The suspension was filtered, the filtrate evaporated, the residue dissolved in EtOAc and the solution was washed with a saturated solution of sodium carbonate

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110 The organic portion was dried over anhyd MgSO 4 filtered, and dried to give the corresponding N (Z a aminoacyl)benzotriazole 6.1' (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate ( 6.1a' ). white solid (90%); mp 150 152 o C (lit. 160 mp 149.0 150.0 o C); 1 H NMR (CDCl 3 ): 8.23 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7 .32 7.23 (m, 7H), 7.14 (br s, 3H), 6.09 (d, J = 4.2 Hz, 1H), 5.57 (d, J = 6.6 Hz, 1H), 5.08 (s, 2H), 3.48 (d, J = 9.6 Hz, 1H), 3.24 (d, J = 7.8 Hz, 1H); 13 C NMR (CDCl 3 ): 170.8, 155.7, 146.0, 135.9, 134.9, 131.0, 130.8, 129.2, 128.7, 128.5, 128.1, 127.4 126.5, 120.4, 114.3, 67.2, 55.6, 38.8. (S) Benzyl (1 (1H benzo[d][1,2,3]triazol 1 yl) 1 oxopropan 2 yl)carbamate ( 6.1c' ). white solid (90%); mp 115 o C (lit. 159 mp 113 115 o C); 1 H NMR (CDCl 3 ): 8.16 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.40 7.03 (m, 6H), 5.80 5.60 (m, 2H), 5.10 4.99 (m, 1H), 1.59 (d, J = 6.3 Hz, 3H); 13 C NMR (CDCl 3 ): 172.2, 155.6, 145.9, 136.0, 131.0, 130.6, 128.4, 128.1, 126.4, 120.2, 114.3, 67.1, 50.5, 19.0. 6.4.3 General procedure for the preparation of N (Boc aminoacyl)benzotriazoles 6.1' Boc protected amino acid 6.1 (0.03 mol) was added to a solution of DCC (1 equiv) in methylene chloride under an atmosphere of nitrogen. After 30 min., BtH (1 equiv) was

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111 added and the mixture stirred for 12 h. The suspension was filtered on a bed of silica and celi te, the filtrate evaporated, the residue dissolved in EtOAc, filtered on a bed of silica and celite and washed with a solution of saturated sodium carbonate, then with water and brine. The organic portion was dried over anhyd MgSO 4 filtered on a bed of silica, and dried to give the corresponding N (Boc aminoacyl)benzotriazole. 1 H NMR and mp of Boc L Phe Bt and Boc Gly Bt matched that reported in the literature. 157 158 6.4.4 General Procedure for the Preparation of Serine containing dipeptides 6. 2a c N (Pg Aminoacyl)benzotriazoles 6.1' (1.0 m mol) in MeCN (5 mL) was added dropwise to a solution of L Ser (1.5 mmol) and Et 3 N (3.0 mmol) in MeCN/H 2 O (9:1, 15 mL) at room temperature and stirred for 4 h. MeCN was evaporated and the residue dissolved in EtOAc (50 mL) and washed with 3N HCl (5 x 50 mL) The organic portion was dried over anhyd. NaSO 4 filtered and concentrated to give 6.2a c (S) 2 ((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanamido) 3 hydroxypropanoic acid ( 6.2a ). W hite solid (85%); mp 156 157 o C; 1 H NMR (CD 3 OD) : 8.16 (d, J = 7 .8 Hz, 1H), 7.38 7.20 (m, 10 H), 5.05 4.80 (m, 2H), 4.52 4.42 (m, 2H), 3.95 3.80 (m, 2H), 3.23 3.16 (m, 1H), 2.90 2.81 (m, 1H); 13 C NMR (CD 3 OD) : 174.3, 173.2, 158.4, 138.7, 138.2, 130.5, 129.6, 129.0, 128.8, 127.8, 67.7, 63.0, 57.9, 56.2, 39. 3; Anal. Calcd for C 20 H 22 N 2 O 6 : C, 62.17; H, 5.74; N, 7.25; Found: C, 62.47; H, 5.82; N, 7.21.

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112 (S) 2 ((S) 2 ((Tert butoxycarbonyl)amino) 3 phenylpropanamido) 3 hydroxypropanoic acid ( 6.2b ). W hite solid (81%); 63.0 65.0 o C; 1 H NMR (CDCl 3 ) : 7.52 (br s, 1H ), 7.26 7.17 (m, 5H), 6.98 (br s, 1H), 4.62 4.57 (m, 2H), 4.05 3.87 (m, 2H), 3.16 2.88 (m, 2H), 1.34 (s, 9H); 13 C NMR (CDCl 3 ) : 172.7, 172.1, 156.1, 136.3, 129.4, 128.5, 126.9, 80.8, 62.6, 55.5, 54.7, 38.7, 28.2, 28.0; Anal. Calcd for C 17 H 24 N 2 O 6 : C, 57.94; H, 6.86; N, 7.95; Found: C, 57.83; H, 7.34; N, 7.47. (S) 2 ((S) 2 (((Benzyloxy)carbonyl)amino)propanamido) 3 hydroxypropanoic acid ( 6.2c ). W hite solid (73%); 195.0 197.0 o C; (lit. 19 mp 192.0 194.0 o C); 1 H NMR (DMSO d 6 ) : 7.99 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 7.29 (m, 5H), 5.02 (s, 2H), 4.29 4.23 (m, 1H), 4.17 4.10 (m, 1H), 3.72 (dd, J = 11, 5 Hz, 1H) 3.62 (dd, J = 11, 4 Hz, 1H), 1.21 (d, J = 7.1 Hz, 3H); 13 C NMR (DMSO d 6 ) : 172.5 171.9, 155.6, 137.0, 128.3, 127.8, 127.7, 65.4, 61.3, 54.6, 49.8, 18.3. 6.4. 5 General Procedure for the Preparation of O Acyl Isopeptides 6.3a c Compound 6.2 (1.0 mmol) was added to a solution of Pg' AA Bt (1.0 mmol) a nd DIPEA (3.0 mmol) in MeCN (20 mL) at room temperature and stirred for 12 h. MeCN was evaporated and the residue dissolved in EtOAc (50 mL) and washed with 2N HCl

PAGE 113

113 (3 x 50 mL). The organic portion was dried over anhyd. NaSO 4 filtered and concentrated to g ive 6.3 (S) 2 ((S) 2 (((Benzyloxy)carbonyl)amino) 3 phenylpropanamido) 3 (2 ((tert butoxycarbonyl)amino)acetoxy)propanoic acid ( 6.3a ). White solid. (86%); mp 86 90 o C; 1 H NMR (CD 3 OD) : 8.22 (d, J = 8.1Hz, 1H), 7.20 7.04 (m, 10 H), 4.88 4.85 (m, 2H ), 4.64 4.60 (m, 1H), 4.44 (dd, J = 11.4, 3.6 Hz, 1H), 4.35 4.23 (m, 2H), 3.65 (s, 2H), 3.05 (dd, J = 13.8, 4.5 Hz, 1H), 2.76 2.67 (m, 1H), 1.29 (s, 9H); 13 C NMR (CD 3 OD) : 174.3, 171.8, 158.6, 158.3, 138.6, 138.2, 130.5, 129.5, 129.0, 128.8, 127.8, 80.9, 67.7, 65.0, 57.8, 53.0, 43.0, 39.2, 28.9; Anal. Calcd for C 27 H 33 N 3 O 9: C, 59.66: H, 6.12; N, 7.73; Found C, 59.62; H, 6.13; N, 6.96. (S) 3 (((S) 2 (((Benzyloxy)carbonyl)amino)propanoyl)oxy) 2 ((S) 2 ((tert butoxycarbonyl)amino) 3 phenylpropanamido)pro panoic acid ( 6.3b ). White solid. (73%); mp 72.0 73.0 o C; 1 H NMR (CDCl 3 ) : 7.85 (br s, 2H), 7.36 7.16 (m, 10H), 5.65 (br s, 1H), 5.21 4.98 (m, 2H), 4.80 4.69 (m, 2H), 4.53 4.23 (m, 3H), 3.24 2.88 (m, 2H), 1.39 1.28 (m, 12H); 13 C NMR (CDCl 3 ) : 172.4, 172.1, 171.3, 156.4, 155.7, 136.7, 135.8, 129.3, 128.5, 128.2, 128.1, 126.7, 80.3, 67.3, 63.7, 55.8, 51.8, 49.9, 38.3, 28.2, 17.5; Anal. Calcd for C 28 H 35 N 3 O 9: C, 60.31: H, 6.33; N, 7.54; Found C, 60.05; H, 6.77; N, 7.39. (S) 2 ((S) 2 (((Benzyloxy) carbonyl)amino)propanamido) 3 (((S) 2 ((tert butoxycarbonyl)amino) 3 phenylpropanoyl)oxy)propanoic acid ( 6.3c ). White solid. (70%); mp 66.0 68.0 o C; 1 H NMR (CDCl 3 ) 7.03 7.09 (m, 10H), 5.17 4.83 (m, 3H), 4.53 4.36 (m, 4H), 3.07 2.78 (m, 2H), 1.3 8 (d, J = 2.5 Hz, 3H), 1.34 (s, 9H); 13 C NMR (CDCl 3 ) 173.3, 171.9, 171.6, 156.4, 155.9, 136.3, 136.0, 129.4, 128.9, 128.7, 128.4,

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114 128.3, 127.3, 80.8, 67.3, 64.0, 54.7, 51.8, 50.7, 38.0, 28.4, 18.8; Anal. Calcd for C 28 H 35 N 3 O 9: C, 60.31: H, 6.33; N, 7.54; Found C, 60.34; H, 6.74; N, 7.37. 6.4.6 General Procedure for the Preparation of O Acyl Isopeptides 6. 4a c and 6. 7a b 6.4.6.1 For deprotection of the Cbz protecting group Compound 6.3 (6.6) (1.0 mmol) was dissolved in anhydrous MeOH (30 mL) and stirred under an atmosphere of hydrogen in the presence of a catalytic amount of Pd/C for 4 h. Filtration through a bed of celite and evaporation afforded 6.4 (6.7) 6.4.6.2 For deprotection of t he Boc protecting group Compound 6.3 (6.6) (1.0 mmol) was dissolved in either HCl dioxane (4.0 M in dioxane) or freshly prepared HCl MeOH (prepared by bubbling HCl in MeOH) (30 mL) and stirred for 1 h. Solvent was evaporated, and ether was added to the r esidue and

PAGE 115

115 stirred for 2h. Filtration gave a white solid 6.4 (6.7) (when sticky solid resulted, decantation of ether several times was performed instead). (S) 2 ((S) 2 Amino 3 phenylpropanamido) 3 (2 ((tert butoxycarbonyl)amino)acetoxy)propanoic acid ( 6.4a ). White solid (80%);mp 170 o C; 1 H NMR (CD 3 OD) : 7.32 7.30 (m, 5H), 4.60 4.50 (m, 1H), 4.39 (s, 2H), 4.25 4.18 (m, 1H), 3.77 (s, 2H), 3.30 3.00 (m, 2H) 1.38 (s, 9H); 13 C NMR (CD 3 OD) : 174.2, 172.8, 169.5, 158.8, 135.5, 130.7, 130.3, 129.0, 81.5 66.5, 55.9, 55.6, 43.1, 38.2, 28.8; HRMS m/z for C 19 H 28 N 3 O 7 [M+H] + calcd. 410.1922, found 410.1909. (S) 2 ((S) 2 Amino 3 phenylpropanamido) 3 (((S) 2 (((benzyloxy)carbonyl)amino)propanoyl)oxy)propanoic acid ( 6.4b ). White microcrystals (79%);mp 103.0 10 4.0 o C; 1 H NMR (DMSO d 6 ) : 9.15 (d, J = 8.1 Hz, 1H), 8.37 (br s, 3H), 7.82 (d, J = 7.2 Hz, 1H), 7.39 7.23 (m, 10H), 4.99 (dd, J = 15.7, 12.6 Hz, 2H), 4.65 4.59 (m, 1H), 4.36 (dd, J = 11.3, 4.7 Hz, 1H), 4.27 (dd, J = 11.3, 5.9 Hz, 1H), 4.16 4.05 (m, 2H), 3.20 (dd, J = 14.3, 5.7 Hz, 1H), 3.03 (dd, J = 14.3, 7.5 Hz), 1.29 (d, J = 7.4 Hz, 3H); 13 C NMR (DMSO d 6 ) : 172.6, 170.0, 168.2, 155.9, 136.9, 134.8, 129.7, 128.5, 128.4, 127.8, 127.1, 66.4, 65.6, 53.2, 51.2, 49.3, 36.7, 16.9; Anal. Calcd for C 46 H 58 N 6 O 15: C, 54.93; H, 5.81; N, 8.35; Found C, 54.63; H, 6.27; N, 8.02. ( S ) 2 (( S ) 2 Aminopropanamido) 3 ((( S ) 2 ((tert butoxycarbonyl)amino) 3 phenylpropanoyl)oxy)propanoic acid ( 6.4c ). White solid (80%);mp 150.0 152.0 o C; 1 H NMR (CD 3 OD) : 7.26 7.19 (m, 5H), 4.53 4.33 (m, 4H), 3.18 (dd, J = 13.9, 4.7 Hz, 1H), 2.87 (dd, J = 13.9, 9.5 Hz, 1H), 1.53 (d, J = 6.7 Hz, 3H), 1.36 (s, 9H); 13 C NMR (CD 3 OD) : 174.2, 173.4, 170.8, 157.8, 138.5, 130.5, 130.3, 129.4, 127.7, 80.6, 66.5,

PAGE 116

116 56.5, 55.6, 50.4, 38.4, 28.7, 17.5; Anal. Calcd for C 20 H 29 N 3 O 7: C, 56.73; H, 6.90; N, 9.92; Found C, 56.61; H, 7.33; N, 9.18. (S) 2 ((S) 2 (2 Aminoacetamido) 3 phenylpropanamido) 3 (2 ((tert butoxycarbonyl)amino)acetoxy)propanoic acid ( 6.7a ). White solid (85%) yield; mp 168 173 o C; 1 H NMR (CD 3 OD) : 7.18 6.88 (m, 5H), 4.55 4.40 (m, 1H), 4.39 4.26 (m, 2H), 4.20 4.10 (m, 1H), 3.70 3.40 (m, 3H), 3.04 2.95 (m, 1H), 2.78 2.40 (m, 4H), 1.20 (s, 9H); 13 C NMR (CD 3 OD) : 174.9, 173.2, 172.2, 167.8, 158.7, 138.5, 130.5, 129.7, 128 .0, 80.9, 66.0, 56.6, 54.7, 44.8, 43.1, 38.8, 28.9; HRMS m/z for C 21 H 30 N 4 O 8 Na [M+Na] + calcd. 489.1956, found 489.1965. (5S,9S,12S) 12 Benzyl 9 carboxy 5 methyl 3,6,11,14 tetraoxo 1 phenyl 2,7 dioxa 4,10,13 triazapentadecan 15 aminium chloride ( 6.7b ). White solid (78%) yield; mp 93.0 94.0 o C; 1 H NMR (CD 3 OD) 7.35 7.18 (m, 10H), 5.14 5.02 (m, 3H), 4.61 (dd, J = 10.6, 4 Hz, 1H), 4.39 (dd, J = 10.6, 5 Hz, 1H), 4.27 4.22 (m, 1H), 3.75 3.67 (m, 2H), 3.59 3.55 (m, 1H), 3.24 (dd, J =13.7, 5 Hz, 1H), 2.9 2 (dd, J =13.7, 9.2Hz, 1H), 1.38 (d, J = 6.7 Hz, 3H); 13 C NMR (CD 3 OD) 174.5, 173.4, 172.0, 167.4, 158.8, 138.2, 138.0, 130.4, 129.6, 129.6, 129.2, 128.9, 128.0, 68.2, 68.0, 65.1, 56.2, 53.2, 41.6, 38.9, 17.6; HRMS m/z for C 25 H 30 N 4 O 8 [M+H] + calcd. 515.213 6, found 515.2137. 6.4.7 General Procedure for the Preparation of O Acyl Isopeptides 6. 6a b

PAGE 117

117 Pg'' Gly Bt (1.0 mmol) was added to a solution of 6. 4 (1.0 mmol) and DIPEA (3.0 mmol) in MeCN:H 2 O (9.5:0.5, 20 mL) at room temper ature and stirred for 12 h. MeCN was evaporated and the residue dissolved in EtOAc (50 mL) and washed with 2N HCl (3 x 50 mL). The organic portion was dried over anhyd. NaSO 4 filtered and concentrated to give 6. 6 (8S,11S) 8 Benzyl 11 ((2 ((tert butoxycar bonyl)amino)acetoxy)methyl) 3,6,9 trioxo 1 phenyl 2 oxa 4,7,10 triazadodecan 12 oic acid ( 6. 6a ). White solid (89%), converted to compound 6. 7 a after checking NMR 6. 6 a : mp 180 o C (decomposed). 1 H NMR (CD 3 OD) : 7.20 7.03 (m, 10H), 4.89 (s, 2H), 4.56 4.4 4 (m, 2H), 4.38 4.30 (m, 1H), 4.24 4.18 (m, 1H), 3.60 3.47 (m, 4H), 3.11 2.87 (m, 1H), 2.78 2.58 (m, 4H), 1.23 (s, 9H); 13 C NMR (CD 3 OD) 177.0, 173.6, 173.4, 172.1, 171.9, 171.8, 159.1, 138.3, 130.5, 129.6, 129.1, 129.0, 127.9, 127.2, 80.9, 68.0 65.0, 55.7, 53.0, 44.0, 43.0, 38.7, 28.6. (9S,12S) 9 Benzyl 12 ((((S) 2 (((benzyloxy)carbonyl)amino)propanoyl)oxy)methyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oic acid ( 6. 6b ). colorless oil (89%); converted to compound 6. 7 after chec king NMR of 6.6b ; 1 H NMR (CDCl 3 ) 9.70 (br s, 2H), 7.34 (d, J = 7.4 Hz, 1H), 7.25 7.07 (m, 10H), 6.03 (d, J = 7.4 Hz, 1H), 5.07 4.57 (m, 4H), 4.29 4.14 (m, 2H), 3.84 3.55 (m, 3H), 3.12 2.89 (m, 2H), 1.36 (d, J = 2.8 Hz, 3H), 1.32 (s, 9H); 13 C NM R (CDCl 3 ) : 155.5, 155.1, 135.1, 134.9, 128.2, 127.5, 127.4, 127.1, 127.0, 125.9, 124.4, 66.1, 62.6, 53.0, 50.9, 48.8, 42.8, 36.5, 29.3, 27.2, 16.4. 6.4.8 Chemical Ligation (9S,12S) 9 Benzyl 12 (hydroxymethyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oic acid ( 6.5 ). Compound 6.4 (20 mg, 0.05 mmol) was dissolved in

PAGE 118

118 piperidine 20 v/v% in DMF (1 mL) and stirred at 50 o C and 50 W for 1h. The mixture was then evaporated and purified by HPLC to give ligated product 6.5 (for example 6 .5a 57%); The sample was analyzed via reverse phase gradient C18 HPLC/UV/( )ESI MSn to give a retention time of 23.07 min (for 6.5a ). Structure 6 .5a was confirm ed by HRMS m/z for C 19 H 26 N 3 O 7 [M H] + calcd. 408.1776, found 408.1794. (12S,15S) 12 benzyl 15 ( hydroxymethyl) 2,2 dimethyl 4,7,10,13 tetraoxo 3 oxa 5,8,11,14 tetraazahexadecan 16 oic acid ( 6.8 ). Compound 6.7 (20 mg, 0.04 mmol) was dissolved in piperidine 20 v/v% in DMF (1 mL) and stirred at 50 o C and 50 W for 1h (3h for 6.8b ). The mixture was then e vaporated and purified by HPLC to give ligated product 8 (for example 6.8a 99.39%); The sample was analyzed via reverse phase gradient C18 HPLC/UV (254 nm/ESI MSn to give a retention time of 21.67 min (for 6.8a ). To confirm structure, HRMS for 6.8a m/z fo r C 21 H 29 N 4 O 8 [M H] + calcd. 465.2064, found 465.1992.

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119 CHAPTER 7 CONCLUSIONS AND SUMM ARY OF ACHIEVEMENTS Chapter 1 provides a general introduction to the main themes presented throughout this thesis. These include the chemistry of N subsituted benzotriazol e, azide s peptide s and the role of the microwave in organic synthesis. Chapter 2 pres ents the synthesis of a novel diazotransfer reagent, benzotriazol 1 yl sulfonyl azide 2.7 which was characterized by 1 H 13 C NMR, elemental analysis and X ray diffract ion. Thermogravimetric analysis, differential scanning calorimetry and the hammer test we re performed on the reagent. The reagent ( 2.7 ) proved to have a long shelf life and to be highly soluble in many organic and partially aqueous solvents. The synthesis of a wide range of azides and diazo compounds starting from aliphatic amino acids, and activated methylenes were examined to demonstrate the utility of the reagent. In Chapter 3, azide chemistry is again examined but in a protecting group context. Reagent 2.7 was used to prepare N azidoacyl) benzotriazoles 3.1 ; these proved to be efficient N S C and O acylating agents and enable d the facile preparation of azido peptides The utility of benzotriazole: (i) to activa azido acid carboxylic acid group and (ii) as a good leaving group is presented for the first time. In amino acids by an azide proved to be an excellent choice when performing acylation reactions. The pall adium catalyzed reaction of N acylbenzotriazoles with epoxide gave unexpected pseudohalohydrin ester surrogates 4.4 Chapter 4 describes t he reaction conditions and the scope of this effiecient, one step microwave assisted, solvent free

PAGE 120

120 reaction towards th e synthesis (benzotriazol 1 yl)ethyl esters 4.4 as single regioisomers. Peptide chemistry is introduced in C hapters 5 and 6. A mild protocol towards the synthesis of enantiomerically pure O acylisodipeptides from serine and threonine using benzotriazol e methodology is described in C hapter 5. This mild protocol was used to study eight and eleven membered transition states in cysteine free ligations using serine residues as described in C hapter 6

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121 LIST OF REFERENCES (1) Katritzky, A. R.; He, H. Y.; Suzuki, K. J Org Chem 2000 65 8210. (2) Katritzky, A. R.; Lan, X.; Yang, J. Z.; Denisko, O. V. Chem Rev 1998 98 409. (3) Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005 1656. (4) Katritzky, A. R. Chem Rev 2004 104 2125. (5) Katritzky, A. R.; Angrish, P.; Todadze, E. Synlett 2009 2392. (6) Katritzky, A. R.; Brzezinski, J.; Lam, J. N. Rev. Roum. Chim. 1991 36 573. (7) Katritzky, A. R.; Lan, X. Chem Soc Rev 1994 23 363. (8) Katritzky, A. R.; Rachwal, S. Chem Rev 2011 111 7063. (9) Katritzky, A. R.; Rachwal, S. Chem Rev 2010 110 1564. (10) Brse, S.; Banert, K. Organic Azides, Syntheses and Applications, 1 st ed.; John Wiley & Sons: West Sussex, 2009; pp 43 47 (pp 3 27 for safety meas ures). (11) Scriven, E. F. V.; Turnbull, K. Chem. Rev. 1988 88 297. (12) Brse S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem., Int. Ed. 2005 44 5188. (13) Huisgen, R.; Knorr, R.; Moebius, L.; Szeimies, G. Chem. Ber. 1965 98 4014. (14) Demk o, Z. P.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002 41 2110. (15) Demko, Z. P.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002 41 2113. (16) Lertpibulpanya, D.; Marsden, S. P.; Rodriguez Garcia, I.; Kilner, C. A. Angew. Chem., Int. Ed. 2006 45 500 0. (17) Hassner, A. Acc. Chem. Res. 1971 4 9. (18) Kirschning, A.; Monenschein, H.; Schmeck, C. Angew. Chem., Int. Ed. 1999 38 2594. (19) Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004 2645. (20) Varma, R. S. Green Chem 1999 1 43.

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122 (2 1) Hayes, B. L. In Microw ave Synthesis: Chemistry at the Speed of Light ; CEM Publishing: Matthews (NC, USA), 2002. (22) Katritzky, A. R.; Angrish, P. Steroids 2006 71 660. (23) Katritzky, A. R.; Cai, C.; Singh, S. K. J Org Chem 2006 71 3375. (24) Perreux, L.; Loupy, A. Tetrahedron 2001 57 9199. (25) Lidstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001 57 9225. (26) L'Abbe, G. Chem. Rev. 1969 69 345. (27) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org Lett 20 04 6 2853. (28) Kalisiak, J.; Sharpless, K. B.; Fokin, V. V. Org Lett 2008 10 3171. (29) Sugimori, T.; Okawa, T.; Eguchi, S.; Kakehi, A.; Yashima, E.; Okamoto, Y. Tetrahedron 1998 54 7997. (30) Airiau, E.; Spangenberg, T.; Girard, N.; Breit, B.; Mann, A. Org Lett 2010 12 528. (31) Snider, B. B.; Zhou, J. J Org Chem 2005 70 1087. (32) Campbell Verduyn, L. S.; Mirfeizi, L.; Dierckx, R. A.; Elsinga, P. H.; Feringa, B. L. Chem Commun 2009 2139. (33) Cassidy, M. P.; Oezdemir, A. D.; Pad wa, A. Org Lett 2005 7 1339. (34) Teixeira Clerc, F.; Michalet, S.; Menez, A.; Kessler, P. Bioconjugate Chem 2003 14 554. (35) Radominska, A.; Drake, R. R. Methods Enzymol. 1994 230 330. (36) Buchmueller, K. L.; Hill, B. T.; Platz, M. S.; Weeks K. M. J Am Chem Soc 2003 125 10850. (37) Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosenquist, B. E.; Pettit, G. R.; Verdier Pinard, P.; Hamel, E. Bio Med Chem 2000 8 2417. (38) Chambers, J. J.; Gouda, H.; Young, D. M.; Kuntz, I. D.; E ngland, P. M. J Am Chem Soc 2004 126 13886. (39) Voskresenska, V.; Wilson, R. M.; Panov, M.; Tarnovsky, A. N.; Krause, J. A.; Vyas, S.; Winter, A. H.; Hadad, C. M. J Am Chem Soc 2009 131 11535.

PAGE 123

123 (40) Sechi, M.; Carta, F.; Sannia, L.; Dallocchi o, R.; Dessi, A.; Al Safi, R. I.; Neamati, N. Antiviral Res 2009 81 267. (41) Okada, M.; Matsubara, A.; Ueda, M. Tetrahedron Lett 2008 49 3794. (42) Piantadosi, C.; Marasco, C. J., Jr.; Morris Natschke, S. L.; Meyer, K. L.; Gumus, F.; Surles, J. R. ; Ishaq, K. S.; Kucera, L. S.; Iyer, N. J Med Chem 1991 34 1408. (43) Yang, H.; Li, Y.; Jiang, M.; Wang, J.; Fu, H. Chem Eur J 2011 17 5652. (44) Baran, P. S.; Zografos, A. L.; O'Malley, D. P. J Am Chem Soc 2004 126 3726. (45) Tao, B.; S chlingloff, G.; Sharpless, K. B. Tetrahedron Lett 1998 39 2507. (46) Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. J Am Chem Soc 1990 112 4011. (47) Das, J.; Patil, S. N.; Awasthi, R.; Narasimhulu, C. P.; Trehan, S. Synthesis 2005 1 801. (48) Tao, C. Z.; Cui, X.; Li, J.; Liu, A. X.; Liu, L.; Guo, Q. X. Tetrahedron Lett 2007 48 3525. (49) Zhu, W.; Ma, D. Chem Commun 2004 888. (50) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J Am Chem Soc 2002 124 10773. ( 51) Katritzky, A. R.; El Khatib, M.; Bol'shakov, O.; Khelashvili, L.; Steel, P. J. J Org Chem 2010 75 6532. (52) Katritzk y, A. R.; El Khatib, M.; Khelashivili, L. In E eros, Encyclopedia of Reagents for Organic Synthesis 2011 DOI:10.1002/047084289X. rn01342 (53) Alper, P. B.; Hung, S. C.; Wong, C. H. Tetrahedron Lett 1996 37 6029. (54) Goddard Borger, E. D.; Stick, R. V. Org Lett 2007 9 3797. (55) Curphey, T. J. Org. Prep. Proced. Int. 1981 13 112. (56) Liu, Q.; Tor, Y. Org Lett 2003 5 2571. (57) Boyer, J. H.; Mack, C. H.; Goebel, N.; Morgan, L. R., Jr. J Org Chem 1958 23 1051.

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124 (58) Green, G. M.; Peet, N. P.; Metz, W. A. J Org Chem 2001 66 2509. (59) Harned, A. M.; Sherrill, W. M.; Flynn, D. L.; Hanson, P. R. Tetrahedron 2 005 61 12093. (60) Griffiths, J. J. Chem. Soc. C 1971 3191. (61) Maas, G. Angew. Chem., Int. Ed. 2009 48 8186. (62) Merlic, C. A.; Zechman, A. L. Synthesis 2003 1137. (63) Davies, H. M. L.; Antoulinakis, E. G. J. Organomet. Chem. 2001 617 618 4 7. (64) Davies, H. M. L.; Beckwith, R. E. J. Chem Rev 2003 103 2861. (65) Ye, T.; McKervey, M. A. Chem Rev 1994 94 1091. (66) Afagh, N. A.; Yudin, A. K. Angew. Chem., Int. Ed. 2010 49 262. (67) Miranda, L. P.; Meldal, M. Angew. Chem., Int. Ed 2001 40 3655. (68) Schelhaas, M.; Waldmann, H. Angew. Chem., Int. Ed. 1996 35 2056. (69) Shenvi, R. A.; O'Malley, D. P.; Baran, P. S. Acc Chem Res 2009 42 530. (70) Newhouse, T.; Baran, P. S.; Hoffmann, R. W. Chem Soc Rev 2009 38 3010. (71) Sartori, G.; Ballini, R.; Bigi, F.; Bosica, G.; Maggi, R.; Righi, P. Chem Rev 2004 104 199. (72) Gaich, T.; Baran, P. S. J Org Chem 2010 75 4657. (73) Ibrahim, T. S.; Tala, S. R.; El Feky, S. A.; Abdel Samii, Z. K.; Katritzky, A. R. Synlett 2011 2013. (74) Isidro Llobet, A.; Alvarez, M.; Albericio, F. Chem Rev 2009 109 2455. (75) Fischer, E.; Bergmann, M. Ber. Dtsch. Chem. Ges 1918 51 1760. (76) Bergmann, M.; Zervas, L. Ber. Dtsch. Chem. Ges 1932 65B 1192. (77) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis. 2nd Ed 1991. (78) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc. D 1971 636.

PAGE 125

125 (79) Barany, G.; Merrifield, R. B. J Am Chem Soc 1977 99 7363. (80) Reetz, M. T. Angew. Chem., In t. Ed. 1991 103 1559. (81) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H. Angew. Chem., Int. Ed. 2002 41 2128. (82) Debaene, F.; Winssinger, N. Org Lett 2003 5 4445. (83) Lundquist, J. T. I. V.; Pelletier, J. C. Org Lett 2001 3 781. (84) Rijkers, D. T. S.; van Vugt, H. H. R.; Jacobs, H. J. F.; Liskamp, R. M. J. Tetrahedron Lett 2002 43 3657. (85) Ainai, T.; Wang, Y. G.; Tokoro, Y.; Kobayashi, Y. J Org Chem 2004 69 655. (86) Plietker, B.; Niggemann, M. Org Lett 2003 5 3353. (87) March, J. Advanced Organic Chemistry, 4th ed.; John Wiley & Sons: New York, 1992; pp 416 425. (88) Carey, F. A.; Sundberg, R J. Advanced Organic Chemistry, Pt. B: Reactions and Synthesis. 2 nd Ed 1983. (89) Katritzky, A. R.; Khelashvili L.; Munawar, M. A. J Org Chem 2008 73 9171. (90) Katritzky, A. R.; Tala, S. R.; Abo Dya, N. E.; Gyanda, K.; El Gendy, B. E. D. M.; Abdel Samii, Z. K.; Steel, P. J. J Org Chem 2009 74 7165. (91) Nakano, K.; Kodama, S.; Permana, Y.; Nozaki, K. Chem Commun 2009 6970. (92) Berecibar, A.; Grandjean, C.; Siriwardena, A. Chem Rev 1999 99 779. (93) Tietze, L. F.; Ila, H.; Bell, H. P. Chem Rev 2004 104 3453. (94) Moghaddam, F. M.; Saeidian, H.; Mirjafary, Z.; Javan, M. J.; Farimani, M. M. ; Seirafi, M. Heteroat. Chem 2009 20 157. (95) Akiyama, Y.; Fukuhara, T.; Hara, S. Synlett 2003 1530. (96) Kolb, H. C.; Sharpless, K. B. Tetrahedron 1992 48 10515. (97) Watson, K. G.; Fung, Y. M.; Gredley, M.; Bird, G. J.; Jackson, W. R.; Gountzos H.; Matthews, B. R. J Chem Soc D 1990 1018. (98) Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998 37 1986.

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126 (99) Chung, J. Y. L.; Cvetovich, R.; Amato, J.; McWilliams, J. C.; Reamer, R.; DiMichele, L. J Org Chem 2005 70 3592. (100) Sin gh, A. K.; Rao, M. N.; Simpson, J. H.; Li, W. S.; Thornton, J. E.; Kuehner, D. E.; Kacsur, D. J. Org. Process Res. Dev. 2002 6 618. (101) Traeff, A.; Bogar, K.; Warner, M.; Baeckvall, J. E. Org Lett 2008 10 4807. (102) Gros, P.; Le Perchec, P.; Sen et, J. P. J Org Chem 1994 59 4925. (103) Azizi, N.; Mirmashhori, B.; Saidi, M. R. Catal. Commun. 2007 8 2198. (104) Bentley, P. A.; Mei, Y.; Du, J. Tetrahedron Lett 2008 49 1425. (105) Das, B.; Venkateswarlu, K.; Krishnaiah, M. Helv. Chim. Act a 2007 90 149. (106) Constantino, M. G.; Lacerda Junior, V.; Invernize, P. R.; Carlos da Silva Filho, L.; Jose da Silva, G. V. Synth Commun 2007 37 3529. (107) Shibata, I.; Baba, A.; Matsuda, H. Tetrahedron Lett 1986 27 3021. (108) Oriyama, T.; Ishiwata, A.; Hori, Y.; Yatabe, T.; Hasumi, N.; Koga, G. Synlett 1995 1004. (109) Stamatov, S. D.; Stawinski, J. Tetrahedron Lett 2006 47 2543. (110) Villorbina, G.; Tomas, A.; Escriba, M.; Oromi Farrus, M.; Eras, J.; Balcells, M.; Canela, R. Tetrah edron Lett 2009 50 2828. (111) Tanaka, K.; Toda, F. Chem Rev 2000 100 1025. (112) Pchelka, B. K.; Loupy, A.; Petit, A. Tetrahedron 2006 62 10968. (113) Katritzky, A. R.; Jishkariani, D.; Narindoshvili, T. Chem. Biol. Drug Des. 2009 73 618. ( 114) El Khatib, M. J., L.; Tala, S.; Khelashvili, L.; Katritzky, Alan R. Med. Chem. Commun. 2011 2 1087. (115) Katritzky, A. R.; Jiang, R.; Suzuki, K. J Org Chem 2005 70 4993. (116) Obase, H.; Tatsuno, H.; Goto, K.; Shigenobu, K.; Kasuya, Y.; Yam ada, Y.; Fujii, K.; Yada, S. Chem. Pharm. Bull. 1978 26 1443.

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127 (117) Katritzky, A. R.; Ji, F. B.; Fan, W. Q.; Gallos, J. K.; Greenhill, J. V.; King, R. W.; Steel, P. J. J Org Chem 1992 57 190. (118) Nakamura, I.; Nemoto, T.; Shiraiwa, N.; Terada, M Org Lett 2009 11 1055. (119) Katritzky, A. R.; Heck, K. A.; Li, J.; Wells, A.; Garot, C. Synth Commun 1996 26 2657. (120) Zhou, Y. G.; Yang, P. Y.; Han, X. W. J Org Chem 2005 70 1679. (121) Tsuji, J. Palladium Reagents and Catalysis: New Perspectives for the 21 st Century ; John Wiley & Son 2005. (122) Katritzky, A. R.; Suzuki, K.; Singh, S. K. Croat. Chem. Acta 2004 77 175. (123) Pardin, C.; Pelletier, J. N.; Lubell, W. D.; Keillor, J. W. J Org Chem 2008 73 5766. (124) Katritzky, A. R.; Pastor, A. J Org Chem 2000 65 3679. (125) Kreutzberger, A.; Schroeders, H. H. Arch. Pharm. 1976 309 330. (126) Li, Z.; Zhang, Y. J. Chem. Res. Synop 2001 522. (127) Bacsa, B.; Boesze, S.; Kappe, C. O. J Org Chem 2010 75 2103. (12 8) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Chem Commun 2004 124. (129) Sohma, Y.; Taniguchi, A.; Skwarczynski, M.; Yoshiya, T.; Fukao, F.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett 2006 47 3013. (130) Sohma, Y.; Yoshiya, T .; Taniguchi, A.; Kimura, T.; Hayashi, Y.; Kiso, Y. Biopolymers 2007 88 253. (131) Yoshiya, T.; Taniguchi, A.; Sohma, Y.; Fukao, F.; Nakamura, S.; Abe, N.; Ito, N.; Skwarczynski, M.; Kimura, T.; Hayashi, Y.; Kiso, Y. Org Biomol Chem 2007 5 1720. ( 132) Coin, I.; Doelling, R.; Krause, E.; Bienert, M.; Beyermann, M.; Sferdean, C. D.; Carpino, L. A. J Org Chem 2006 71 6171. (133) Carpino, L. A.; Krause, E.; Sferdean, C. D.; Schuemann, M.; Fabian, H.; Bienert, M.; Beyermann, M. Tetrahedron Lett 2 004 45 7519. (134) Merrifield, R. B. J Am Chem Soc 1963 85 2149.

PAGE 128

128 (135) Moore, J. A.; Dice, J. R.; Nicolaides, E. D.; Westland, R. D.; Wittle, E. L. J Am Chem Soc 1954 76 2884. (136) Woehr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter, M. J Am Chem Soc 1996 118 9218. (137) Thomas, G. L.; Payne, R. J. Chem Commun 2009 4260. (138) Tam, J. P.; Lu, Y. A. J Am Chem Soc 1995 117 12058. (139) Sohma, Y.; Hayashi, Y.; Kimura, M.; Chiyomori, Y.; Taniguchi, A.; Sa saki, M.; Kimura, T.; Kiso, Y. J. Pept. Sci. 2005 11 441. (140) Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. C. J Chem Soc D 1993 369. (141) Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wohr, T. Pept Res 1995 8 145. (142) Guichou, J F.; Patiny, L.; Mutter, M. Tetrahedron Lett 2002 43 4389. (143) Sohma, Y.; Taniguchi, A.; Yoshiya, T.; Chiyomori, Y.; Fukao, F.; Nakamura, S.; Skwarczynski, M.; Okada, T.; Ikeda, K.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. J. Pep t. Sci. 2006 12 823. (144) Skwarczynski, M.; Kiso, Y. Curr. Med. Chem. 2007 14 2813. (145) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos Santos, S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucede, L.; Tuchscherer, G. Angew. Chem., I nt. Ed. 2004 43 4172. (146) Dos Santos, S.; Chandravarkar, A.; Mandal, B.; Mimna, R.; Murat, K.; Saucede, L.; Tella, P.; Tuchscherer, G.; Mutter, M. J Am Chem Soc 2005 127 11888. (147) Sohma, Y.; Hayashi, Y.; Skwarczynski, M.; Hamada, Y.; Sasaki, M.; Kimura, T.; Kiso, Y. Biopolymers 2004 76 344. (148) Skwarczynski, M.; Sohma, Y.; Kimura, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Bio Med Chem Lett 2003 13 4441. (149) Hamada, Y.; Matsumoto, H.; Kimura, T.; Hayashi, Y.; Kiso, Y. Bioorg Med Che m Lett 2003 13 2727. (150) Hayashi, Y.; Skwarczynski, M.; Hamada, Y.; Sohma, Y.; Kimura, T.; Kiso, Y. J Med Chem 2003 46 3782. (151) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Tetrahedron Lett. 2004 45 5965.

PAGE 129

129 (152) Sohma, Y.; Chi yomori, Y.; Kimura, M.; Fukao, F.; Taniguchi, A.; Hayashi, Y.; Kimura, T.; Kiso, Y. Bioorg Med Chem 2005 13 6167. (153) Taniguchi, A.; Sohma, Y.; Kimura, M.; Okada, T.; Ikeda, K.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. J Am Ch em Soc 2006 128 696. (154) Taniguchi, A.; Skwarczynski, M.; Sohma, Y.; Okada, T.; Ikeda, K.; Prakash, H.; Mukai, H.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. Chembiochem 2008 9 3055. (155) Yoshiya, T.; Kawashima, H.; Hasegawa, Y .; Okamoto, K.; Kimura, T.; Sohma, Y.; Kiso, Y. J Pept Sci 2010 16 437. (156) Yoshiya, T.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett 2006 47 7905. (157) Masiukiewicz, E.; Rzeszotarska, B.; Wawrzycka Gorczyca, I.; Kolodziejczyk, E. Synth Commun 2007 37 1917. (158) Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K. Synthesis 2004 1806. (159) Katritzky, A. R.; Tala, S. R.; Abo Dya, N. E.; Ibrahim, T. S.; El Feky, S. A.; Gyanda, K.; Pandya, K. M. J Org Chem 2011 76 85. (1 60) Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K. ARKIVOC 2005 36. (161) Katritzky, A. R.; Vincek, A. S.; Suzuki, K. ARKIVOC 2005 116. (162) Kent, S. B. H. Chem Soc Rev 2009 38 338. (163) Merrifield, B. Science 1986 232 341. (164) Kaiser, E T. Acc Chem Res 1989 22 47. (165) Dawson, P. E.; Muir, T. W.; Clark Lewis, I.; Kent, S. B. H. Science 1994 266 776. (166) El Faham, A.; Albericio, F. Chem Rev 2011 111 6557. (167) Yeo, D. S. Y.; Srinivasan, R.; Chen, G. Y. J.; Yao, S. Q. Ch em Eur J 2004 10 4664. (168) Sakakibara, S.; Shin, K. H.; Hess, G. P. J Am Chem Soc 1962 84 4921. (169) Li, X.; Lam, H. Y.; Zhang, Y.; Chan, C. K. Org Lett 2010 12 1724.

PAGE 130

130 (170) Bruckdorfer, T.; Marder, O.; Albericio, F. Curr. Pharm. Biotec hnol 2004 5 29. (171) Bray, B. L. Nat. Rev. Drug Discovery 2003 2 587. (172) Albericio, F. Curr. Opin. Chem. Biol. 2004 8 211. (173) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys. and Biomol. Struct. 2005 34 91. (174) Hacke nberger, C. P. R.; Schwarzer, D. Angewandte Chemie, International Edition 2008 47 10030. (175) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H.; Schafer, W. Justus Liebigs Annalen der Chemie 1953 583 129. (176) Nilsson, B. L.; Kiessling, L L.; Raines, R. T. Organic Letters 2000 2 1939. (177) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Organic Letters 2001 3 9. (178) Restituyo, J. A.; Comstock, L. R.; Petersen, S. G.; Stringfellow, T.; Rajski, S. R. Organic Letters 2003 5 4357. (179) Okamoto, R.; Kajihara, Y. Angew. Chem., Int. Ed. 2008 47 5402. (180) Macmillan, D. Angew. Chem., Int. Ed. 2006 45 7668. (181) Hojo, H.; Ozawa, C.; Katayama, H.; Ueki, A.; Nakahara, Y.; Nakahara, Y. Angew. Chem., Int. Ed. 2010 49 5318. (182) Macmillan, D.; Anderson, D. W. Org Lett 2004 6 4659. (183) Kawakami, T.; Aimoto, S. Tetrahedron Lett 2003 44 6059. (184) Offer, J.; Boddy, C. N. C.; Dawson, P. E. J Am Chem Soc 2002 124 4642. (185) Botti, P.; Carrasco, M. R.; Kent, S. B. H Tetrahedron Lett 2001 42 1831. (186) Wu, B.; Chen, J.; Warren, J. D.; Chen, G.; Hua, Z.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2006 45 4116. (187) Katritzky, A. R.; Abo Dya, N. E.; Tala, S. R.; Abdel Samii, Z. K. Org Biomol Chem 2010 8 23 16. (188) Oliferenko, A. A.; Katritzky, A. R. Org Biomol Chem 2011 9 4756.

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131 BIOGRAPHICAL SKETCH Mirna was born in Beirut, Lebanon of Jordanian parents, but grew up in Cyprus. She graduated from the Grammar School in Nicosia, Cyprus in June 2003 w ith honors and also received the Ioan nis & Gregoriou Memorial Award as the top student in both c hemistry and in p ure m athematics as well as the London Examinations General Certificate ( a dvanc ed level in b iology, c hemistry and p ure m athematics). She receiv ed her Bachelor of Science degree in c hemistry with a minor in b iology in June 2006 and her Masters of Science in c hemistry in June 2008 from the American University of Beirut, in Lebanon. During her Masters, Mirna worked under the supervision of Professor Quinoxaline 1,4 Dioxides, and Quinoxalinocinnoline N joined the graduate program in the Department of Chemistry at the University of Florida and pursued her Ph.D. i n o rganic c hemistry under the guidance of Professor Alan R. Katritzky at the Center of Heterocyclic Compounds where she worked on the novel synthetic utility of benzotriazole as a synthetic auxiliary chiefly in azide and peptide chemistry. During her cours e of study, Mirna has participated in several internationally renowned conferences and delivered either a poster or an oral presentation. In addition, Mirna was recognized with the Certificate of Outstanding Achievement three times (2010 2012) for maintain ing a GPA of 4.0, the Proctor & Gamble Award for Research Excellence (2011), the GSC Travel Grant (2011) and the best FloHet 13 poster award (2012).