Strategic Synthesis of Peptides, Labeled Peptides and Peptidomimetics

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Title:
Strategic Synthesis of Peptides, Labeled Peptides and Peptidomimetics
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1 online resource (193 p.)
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english
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Biswas, Suvendu
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University of Florida
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Gainesville, Fla.
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
APONICK,AARON
Committee Co-Chair:
CASTELLANO,RONALD K
Committee Members:
MILLER,STEPHEN ALBERT
SMITH,BEN W
MOUDGIL,BRIJ M

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Subjects / Keywords:
labeling -- peptide -- peptidomimetics
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
The theme of the current work is the design and development of strategicmethodologies for the synthesis of variety of peptide and peptide-like organic compounds. Chapter 1 represents a general overview of the work presented in followingchapters together with a brief discussion on the strategies of peptide synthesis and importance of peptide and peptidomimetics.Chapter 2 describes formation of native peptides via chemical ligations fromtryptophan containing isopeptide. These chemical ligations have been achieved the migration of an N-acyl tryptophan isopeptide unit using neither cysteine/serine/tyrosine residue nor an auxiliary group at the ligation site. Chapter 3 reports the efficient preparation of azodye labeled aminoxy acids and peptides. Aminoxy acids are the analogues of beta-amino acids. Azodyes are the widely used chromophoric unit to label peptides. Chapter 4 highlights on syntheses, absorption and fluorescence data of new fluorescent coumarin-labeled depsipeptides. A depsipeptide is a peptide in which one or more of the amide (-CONHR-) bonds are replaced by ester (COOR) bonds. These compounds exhibited high emission quantum yields in some particular solvents and emission absorption profile highly dependent on the chemical nature (electron donating or withdrawing) of coumarin derivative. Chapter 5 describes development of a mild protocol towards the synthesis of azapeptides from amino acid residues using benzotriazole methodology. The protocol was revisited in the following chapter and Chapter 6 focuses on de novo design and synthesis of oxy-azapeptides in which an amino acid is replaced by an aza-hydroxy acid. Calculations revealed that oxyazapeptides should occupy a beta-turn secondary structure and enjoy greater conformational freedom which could render them more adaptive to varying steric demand of biological interactions.Chapter 7 presents a summary of achievements together with conclusions.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Suvendu Biswas.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: APONICK,AARON.
Local:
Co-adviser: CASTELLANO,RONALD K.

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1 STRATEGIC SYNTHESIS OF PEPTIDES, LABELED PEPTIDES AND PEPTIDOMIMETICS By SUVENDU BISWAS 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 2014

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2 2014 Suvendu Biswas

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3 To my parents, wife Nandita and daughter Khushi

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4 ACKNOWLEDGMENTS First of all I would like to thank my supervisor the late Prof. Alan R. Katritzky, whose leadership, counsel and support throughout my graduate studies allowed this work to be accomplished His expert assistance encouraged me to continually improve both my research and my scientific writing. I am grateful to my committee members Dr. Ronald K. Castellano Dr. Stephen A. Miller and Dr. Brij M. Moudgil for their insightful suggestions and critiques, which have he lped me advance in my research. I am indebted to Dr. A a ron Aponick who served as the chair after death of Prof. Katritzky. I would also like to give my sincere thanks to Dr. Ben Smith Graduate C o ordinator and also my committee member, whose support and guidance facilitated my initial transition in to the graduate program in 2008. I would like to express my deepest gratitu de to Dr. C. Dennis Hall for his enormous help and support throughout the years and during the pr eparation of this thesis. I thank former and current members of the Katritzky group Dr. Srinivas a R. Tala, Dr. Nader Abo Dya, Dr. Ilker Avan Dr. Alexander Oliferenco, Dr. Iryna Lebe dyeva, Dr. Vadim Popov, Dr. Girinath G. Pillai, Mr. Akash K. Basak Mr. Am ir Khiabani, Mr. Roger Kayaleh and Mr. Christopher Seon who have contributed to this work. I would like to thank my previous advisor Dr. Pradeep Mathur whose instruction and encouragement during my Master inspired me to furt her my studies in the United States. I deeply express my appreciation to all the friends I have made along these years in Gainesville for their full support: Eray Caliskan, Khanh Ha, Peter Vertesaljai, Dr. Bahaa El Dien El Gendy, Dr. Anand Tiwari, Dr. Siv a S. Panda, Mr. Z. Wang Dr. Rachel

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5 A. Jones and Dr. Tarek S. Ibrahim. I am grateful for the ir love, support and friendships which have provided me with both a social outlet and support system. Last but not least, I would like to thank my parents and my family for shaping me into the individual I am today. Their continual love and support has motivated me to pursue a career in research and has given me the strength to persevere. My wife and daughter have stood as my inspiration to achieve my professional goals and without their support, completing my work would have been more difficult. My enormous debt of gratitude can hardly be repaid to them for their unconditional support.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .............. 4 LIST OF TABLES ................................ ................................ ................................ ....................... 10 LIST OF FIGURES ................................ ................................ ................................ ..................... 11 LIST OF SCHE ME ................................ ................................ ................................ ..................... 12 LIST OF ABBREVIATIONS ................................ ................................ ................................ ...... 14 ABSTRACT ................................ ................................ ................................ ................................ 18 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ ............................ 20 2 LONG RANGE CHEMICAL LIGATION FROM N N ACYL MIGRATIONS IN TRYPTOPHAN PEPTIDES VIA CYCLIC TRANSITION STATES OF 10 TO 18 MEMBERS ................................ ................................ ................................ ........................... 27 2.1 Introduction ................................ ................................ ................................ .................... 27 2.2 Results and Discussion ................................ ................................ ............................... 29 2.2.1 Preliminary Results on Acyl Migrations via 10 12 Membered Cyclic TS ................................ ................................ ................................ .................... 29 2.2.2 Feasibility of Acyl Migrations via 13 Membered Cyclic TS ............... 31 2.2.3 Feasibility of Acyl Migrations via 14 Membered Cyclic TS ............... 33 2.2.4 Feasibility of Acyl Migrations via 15 Membered Cyclic TS ............... 36 2.2.5 Feasibility of Acyl Migrations via 16 18 Membered Cyclic TS ......... 37 2.2.6 Isolation of Ligated Product ................................ ................................ .............. 39 2.2.7 Competitive Ligation Experiments ................................ ................................ .. 40 2.2.8 Computational Analysis ................................ ................................ .................... 41 2.2.9 Experimental Validation of Model ................................ ................................ .... 46 2.3 Conclusion ................................ ................................ ................................ ..................... 47 2.4 Experimental Section ................................ ................................ ................................ ... 48 2.4. 1 General Methods ................................ ................................ ............................... 48 2.4.2 General Procedure for Preparation of Boc Protected Isotetrapeptides 2. 13a o and Isopentapeptides 2. ................................ ................................ 48 2.4.3 General Procedure for Preparation of Unprotected Isotetrapeptides 2. 2. ................................ ................................ ... 49 2.4.4 General Procedure for Chemical Ligation of N Acylisopeptides 2. N Acylisopentapeptides 2. ................... 49 3 EFFICIENT PREPARATION OF AZODYE LABELED AMINOXY ACIDS AND PEPTIDES ................................ ................................ ................................ ............................ 65

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7 3.1 Introduction ................................ ................................ ................................ .................... 65 3.2 Results and Discussion ................................ ................................ ............................... 67 3.2.1 Preparation of Azodye Labeled Aminoxy Acids 3.4a i ................................ 67 3.2.2 Preparation of Fmoc Protected Aminoxy Hybrid Peptides 3. 6a e ............. 68 3.2.3 Preparation of Azodye Labeled Aminoxy Peptides 3. 7a c ......................... 68 3.3 Conclusion ................................ ................................ ................................ ..................... 69 3.4 Experimental Section ................................ ................................ ................................ ... 69 3.4.1 Synthesis of Azodye Carboxylic Acid Labeled Aminoxy Acids 3.4a i ...... 69 3.4.2 Synthesis of Fmoc Protected Aminoxy Peptides 3. 6a e ............................. 72 4 PHOTOPHYSICS OF NOVEL COUMARIN LABELED DEPSIPEPTIDES IN SOLUTION; SENSING INTERACTIONS WITH SDS MICELLE VIA TICT MODEL ................................ ................................ ................................ ................................ 75 4.1 Introduction ................................ ................................ ................................ .................... 75 4.2 Results and discussion ................................ ................................ ................................ 77 4.2.1 Preparation of Unprotected Depsidipeptides 4.5a b ................................ .... 77 4.2.2 Preparation of Coumarin Labeled Depsidipeptides 4.8a f .......................... 78 4.2.3 Preparation of Unprotected Depsitripeptides 4.11a c ................................ .. 79 4.2.4 Preparation of Coumarin Labele d Depsitripeptides 4.12a d ....................... 79 4.2.5 Photophysical Studies of Coumarin Labeled Depsipeptides 4.8 and 4.12 ................................ ................................ ................................ ............................. 80 4.2.5.1 Absorbance, fluorescence data for coumarin labeled depsipeptides ................................ ................................ ................................ ..... 80 4.2.5.2 Photophysical properties of 7 N N diethylaminocoumarin labeled depsipeptides 4.8c and 4.12d in SDS micellar microenvironment ................................ ................................ .............................. 84 4.3 Conclusion ................................ ................................ ................................ ..................... 87 4.4 Experimental Section ................................ ................................ ................................ ... 87 4.4. 1 General Preparation of Unprotected Depsidipeptides 5a b ........................ 88 4.4.2 General Preparation of Coumarin Labeled Depsidipeptides 4. 8a f ........... 89 4.4.3 General Preparation of Unprotected Depsidipeptides 4. 11a c ................... 92 4.4.4 General Preparation of N Coumarinoyl Labeled Depsitripeptides 4. 12a d ................................ ................................ ................................ ....................... 94 5 BENZOTRIAZOLE MEDIATED SYNTHESIS OF AZA PEPTIDES: EN ROUTE TO AN AZA LEUENKEPHALIN ANALOGUE ................................ ................................ 97 5.1 Introduction ................................ ................................ ................................ .................... 97 5.2 Results and Discussion ................................ ................................ ............................... 99 5.2.1 Preparation of Alkyl N (Pg) Hydrazines 5.10 c ................................ ........ 99 5.2.2 Construction of Protected Azadipeptides 5.18a i ................................ ....... 100 5.2.3 Preparation of N (N Pg Azadipeptidoyl)Benzotriazoles 5.20a e ............. 102 5.2.4 Coupling of 5.20a e with Various Nucleophiles to Form Longer Azapeptides ................................ ................................ ................................ ............. 104 5.2.5 Preparation of Free Azadipeptides 5.29a c and Coupling with N (N Pg Am inoacyl)Benzotriazoles ................................ ................................ .......... 106

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8 5.2.6 Alternative Facile Route to the Synthesis of N Pg Azatripeptides 5.33a,b and 5.35a,b ................................ ................................ ............................... 107 5.2.7 Solution Phase Synthesis of Hybrid Azapeptide 5.40; an Analogue of Leuenkephalin ................................ ................................ ................................ ......... 108 5.3 Conclusions ................................ ................................ ................................ ................. 109 5.4 Experimental Section ................................ ................................ ................................ 110 5.4.1 General Methods for the Preparation of 5.16a e ................................ ....... 113 5.4.2 General Methods for the Preparation of N Pg Azadipeptide 5.18a h ... 114 5.4.3 General Methods for OMe and O t Bu Group Deprotection .................... 117 5.4.4 General Method for the Prepa ration of Benzotriazolides of Azadipeptide 5.20a e ................................ ................................ ............................ 121 5.4.5 General Procedure for the Coupling of 5.20a e to Prepare 5.24a g, 5. 25a,b and 5.26a,b ................................ ................................ ............................... 123 5.4.6 General Method for the Preparation of 5. 33a,b ................................ .......... 130 5.4.7 General Method for the Preparation of 5.35a,b ................................ .......... 131 6 OXYAZAPEPTIDES: SYNTHESIS, STRUCTURE DETERMINATION AND CONFORMATIONAL ANALYSIS ................................ ................................ ................... 137 6.1 Introduction ................................ ................................ ................................ .................. 137 6.2 Result s and Discussion ................................ ................................ ............................. 139 6.2.1 Synthesis of Oxyaza Di Tri and Tetrapeptides ................................ ....... 139 6.2.2 Validation of the Synthetic Methodology ................................ ...................... 142 6.2.3 X Ray Structure Determination ................................ ................................ ...... 143 6.2.4 Conformational Analysis ................................ ................................ ................. 143 6.2.5 Computational Study of Turn Inducing ................................ ..................... 146 6.3 Conclusions ................................ ................................ ................................ ................. 149 6.4 Experime ntal Section ................................ ................................ ................................ 149 6.4.1 General Methods ................................ ................................ ............................. 149 6.4.2 Computational Details ................................ ................................ ..................... 150 6.4.3 General Methods for the Preparation of Oxyazadipeptide 6. 6a f ............ 150 6.4.4 General Methods for the Preparation of Oxyaza Tri and Tetrapeptide 6. 8a j, 6. 10a f ................................ ................................ ................................ ........ 152 7 SYNTHESIS OF TAURINE PEPTIDES, SULFONOPEPTIDES, AND N O CONJUGATES ................................ ................................ ................................ .................. 161 7.1 Introduction ................................ ................................ ................................ .................. 161 7.2 Results and Discussion ................................ ................................ ............................. 163 7.2.1 Synthesis of Taurine Containing Dipeptides ................................ ............... 163 7.2.2 Synthesis of Taurine Containing Tri and Tetrapeptides ........................... 165 7.2.3 Synthesis of Taurine Sulfonopeptides ................................ .......................... 166 7.2.4 Synthesis of N O Taurine Conjugates ................................ ....................... 167 7.3 Conclusion ................................ ................................ ................................ ................... 168 7.4 Experimental Section ................................ ................................ ................................ 168 7.4.1 General Methods for the Preparation of Taurine Containing Dipeptides 7.12a k, Tri and Tetrapeptides 7.14a k. ................................ .......................... 169

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9 7.4.2 General Methods for the Preparation of Sulphono Peptide 7.18a f and N O Acylated Taurine Conjugates 7.19a e ................................ .................... 176 8 CONCLUSIONS AND SUMMARY OF ACHIEVEMENTS ................................ .......... 180 LIST OF REFERENCES ................................ ................................ ................................ ......... 182 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ..... 193

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10 LIST OF TABLES Table page 2 1 Chemical ligation of N acyl isopeptide 2. 14a d via 13 mem TS ............................. 33 2 2 Chemical ligation of N acyl isopeptide 2.14e h via 14 mem TS ............................. 35 2 3 Chemical ligation of N acyl isopeptide 2.14i l via 15 mem TS ............................... 37 2 4 Chemical ligation of N acyl isopeptides 2.19a c via 16 18 mem TS ..................... 39 2 5 Computational data for pre organized (conformer) compounds 2.9a to 2.20c .... 42 2 6 Statistical model for relative abundance of the ligated peptide ............................... 44 2 7 Experimental and predicted relative abundance of ligated p eptide. ...................... 45 2 8 Chemical ligation of N acyl isopeptides 2. 14m o in DMF/piperidine ..................... 46 3 1 Preparation of azodye labeled aminoxy acids 3. 4a i ................................ ................ 67 3 2 Preparation of Fmoc protected aminoxy hybrid peptides 2.6a e ............................ 68 3 3 Preparation of azodye labeled aminoxy peptides 3.7a c ................................ ......... 68 4 1 Absorption and emission data in PBS buffer ................................ ............................. 81 4 2 Absorption and emission data in MeOH ................................ ................................ ..... 81 4 3 Absorption and emission data in DCM ................................ ................................ ....... 81 5 1 Preparation of protected azadipeptides 5. 18a i ................................ ..................... 102 5 2 Preparation of azadipeptide 5.19a h and azadipeptidoyl Bt 5.20a h .................. 103 5 3 Preparation of azapeptides 5. 24a g 5. 25a b 5. 26a b and 5. 28 ......................... 105 6 1 Construction of oxyaza dipeptides 6. 6a f ................................ ................................ 140 6 2 Preparation of oxyaza tripeptides 6.8a j ................................ ................................ ... 141 6 3 Preparation of oxyaza tetrapeptides 6.10a f ................................ ........................... 142 7 1 Preparation of taurine containing d ipeptides 7.12 ................................ .................. 164 7 2 Preparat ion of t auri ne containing tri and t etrapeptides 7.14 ................................ 165 7 3 Preparation of sulfono di and t ripeptides 7.18 ................................ ........................ 167 7 4 Preparation of N and O taurine b ioconjugates 7.19 ................................ .............. 167

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11 LIST OF FIGURES Figure page 2 1 Difference in 1 H spectra of isolated ligated peptide 2. 15f ( left) and starting compound 2. 14f (right) ................................ ................................ ................................ .. 40 2 2 Pre organized 3D representation with b (N C) for compound 2. 14f (left) and 2. 14h (right) ................................ ................................ ................................ ..................... 42 2 3 Correlation plot for relative abundance model of ligated peptide in Table 2 7 ..... 45 3 1 Azodye carboxylic acids. ................................ ................................ ............................... 65 3 2 Azodye labeled peptides. ................................ ................................ .............................. 65 4 1 Emission and absorption spectra of 4. 8b 4. 8e 4. 12a c ................................ ........ 82 4 2 Variation in e mission spectra of 4. 8c (left) and 4. 12d (right) in PBS buffer, MeOH and DCM ................................ ................................ ................................ ............. 84 4 3 Emission and absorption spectra (inset) in PBS buffer with different SDS concentrations for 4.8c (left) and for 4. 12d (right) ................................ .................... 85 4 4 Proof of interaction of coumarin A) binding of coumarin moiety to the Stern layer B) Blue shift in the emission spectra 4.8c and 4.12d in buffer and SDS s olution ................................ ................................ ................................ ............................. 86 5 1 Comparison of azadipeptide and native dipeptide ................................ .................... 97 6 1 General structures of peptide and peptide like molecules ................................ ..... 137 6 2 X ray crystal structure of 6.8h ................................ ................................ .................... 143 6 3 Optimized structures of azapeptide 6. 15 and oxyazapeptide 6. 16 ...................... 144 6 4 Torsional energy plots for dihedral angles (left) and (right) in azapeptide 6.15 (upper row) and oxyazapeptide 6.16 (lower row) ................................ ........... 146 6 5 Molecular structures of turn structures of oxyazapeptide 6.17 (left) and azapeptide 6.18 (right); the hydrogen bonds are indicated by the dotted lines 147 7 1 Some sulfonopeptides reported in the literature ................................ ..................... 162

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12 LIST OF SCHEME S Scheme page 1 1 Peptide synthesis by native chemical ligation ................................ ........................... 21 1 2 Peptide synthesis by chemical ligation developed by Katritzky group .................. 22 2 1 Synthesis of isodipeptide 2.4 and isotripeptides 2.9a c ................................ ........... 30 2 2 Chemical ligation of N acyl isopeptides 2. 9a c via 10 12 mem TS ........................ 31 2 3 Synthesis of isotetrapeptides 2. 14a d for ligation study via 13 mem TS .............. 32 2 4 Chemical ligation of N acyl isopeptides 2. 14a d via 13 mem TS ........................... 33 2 5 Synthesis of isotetrapeptides 2.14e h for ligation study via 14 mem TS .............. 34 2 6 Chemical ligation of N acyl isopeptides 2.14e h in via 14 mem TS ....................... 35 2 7 Synthesis of isotetrapeptides 2.14i l for ligation study via 15 mem TS ................. 36 2 8 Chemical ligation of N acyl isopeptides 2.14i l via 15 mem TS ............................. 37 2 9 Synthesis of isopentapeptides 2.19a c for ligation study ................................ ......... 38 2 10 Chemical ligation of N acyl isopeptides 2.19a c via 16 18 mem TS ..................... 39 2 11 Competitive Chemical ligation of 2.14f in DMF/piperidine ................................ ....... 41 2 12 Synthesis of 2. 14m o and Chemical ligation to validate the statistical model ...... 46 3 1 Synthesis of azodye labeled aminoxy acids 3. 4a i ................................ ................... 67 3 2 Synthesis of Fmoc protected aminoxy hybrid peptides 3.6a e ............................... 68 3 3 Synthesis of azodye labeled aminoxy peptides 3.7a c ................................ ............ 68 4 1 Preparation of unprotected depsidipeptides 4.5a b ................................ .................. 78 4 2 Preparation of N coumarinoyl labeled depsidipeptides 4.8a f ............................. 78 4 3 Preparation of unprotected depsitripeptides 4.11a c ................................ ............... 79 4 4 Preparation of N coumarinoyl labeled depsitripeptides 4.12a d. ......................... 80 4 5 TICT model ................................ ................................ ................................ ...................... 84 5 1 The constr uction of azapeptides by A) hydrazine pathway and B) peptide N terminus pathway ................................ ................................ ................................ ........... 98

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13 5 2 Preparation of N Alkyl N (Pg) hydrazines ................................ ................................ 99 5 3 Preparation of alkyl Pg carbazates 5. 14a 5. 14b and 5. 14c ............................ 100 5 4 Preparation of protected azadipeptides 5. 18a i ................................ ..................... 101 5 5 Preparation of N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e .......................... 103 5 6 Coupling reactions of N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e .............. 105 5 7 Preparation of free azapeptides 5. 29a c and coupling with N (N Pg aminoacyl)benzotriazoles to prepare 5.31a,b ................................ ......................... 107 5 8 Preparation of azatripeptide 5.33 and 5.35 from aminoacyl benzotriazolides ..... 108 5 9 Solution phase synthesis of azapeptide analog of Leuenkephalin 5.40 .............. 109 6 1 Synthesis of N benzylhydroxylamine ................................ ................................ ........ 139 6 2 Construction of oxyaza dipeptide ................................ ................................ .............. 140 6 3 Synthesis of oxyaza tri and tetra peptides ................................ .............................. 141 6 4 Synthesis of oxyaza analog of Leuenkephalin 6 14 ................................ ............... 143 7 1 Incorporation of taurine unit to prepare d ipeptides 7.12 ................................ ........ 164 7 2 Incorpora tion of the taurine u n it into tri and t etrapeptides 7.14 ........................... 165 7 3 Synthesis of taurine s ulfonopeptides 7.18 ................................ ............................... 166 7 4 Synthesis of t aurine N and O c onjugates 7. 19 ................................ ....................... 167

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14 LIST OF ABBREVIATIONS Alpha locant D Specific rotation Angstrom(s) Ac Acetyl Ala Alanine Beta locant Bn Benzyl Boc t Butoxycarbonyl br Broad Bt Benzotriazol 1 yl C Carbon Degree Cels ius Calcd Calculated Cbz Carbobenzyloxy CDCl 3 Deuterated chloroform Cys Cysteine Chemical shift in parts per million downfield from tetramethylsilane d Days; Doub let (spectral) D Dextrorotatory (right) DBU 1,8 Diazabicyclo[5.4.0]undec 7 ene DCC N,N' Dicyclohexylcarbodiimide DCM Dichloromethane DIPEA Diisopropylethylamine DMF Dimethylformamide

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15 DMSO Dimethylsulfoxide D 2 O Deuterium oxide EDC I 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide Et Ethyl et al. And others ESI Electrospray ionization Et 3 N Triethylamine EtOAc Ethyl acetate EtOH Ethanol Equiv. Equiv alent(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) Leu Leucine Lit Literature m Multiplet M Molar

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16 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 Sulfate NMR Nuclear magnetic resonance o Ortho locant O Oxygen OEt Ethoxy OH Hydroxyl group OMe M ethoxy p Para locant Pd Palladium Ph Phenyl

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17 Phe Phenylalanine ppm Part per million Pro Proline q Quartet R Rectus (right) ref. Reference rt Room temperature s Singlet S Sinister (left) S Sulphur Ser Serine SOCl 2 Thionyl chloride t Time;Triplet (spectral) t Tertiary TLC Thin layer chromatography TMS Trimethylsilane Trp Tryptophan Val Valine v/v volume per unit volume (volume to volume ratio) W Watt(s)

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18 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 STRATEGIC SYNTHESIS OF PEPTIDES, LABELED PEPTIDES AND PEPTIDOMIMETICS Suvendu Biswas M ay 2014 Chair: Aaron Apo nick Major: Chemistry Peptides are important class of organic molecules since they are essential for carrying out many biological functions. Recently, there have been increasing efforts to develop methodology for the synthesis of longer peptides and pepti de like molecules in solution The current study describes strategies for the peptide and peptide like molecule s synthesis, chemistry of N subsituted benzotriazoles and importance of peptide and peptidomimetics. Native chemical ligation (NCL), one of the well known strategies for the synthesis of peptides and proteins, is a chemoselective and regioselective reaction of peptide thioester and a terminal Cys peptide that gives a native amide. Based on the native chem ical ligation idea, we have developed the formation of native peptides via chemical ligations from tryptophan containing isopeptides. In this study, we report a statistical, predictive model using an extensive synthetic and computational approach to ration alize the chemical ligation. The feasibility of these traceless chemical ligations is supported by b (N C) bond distances in N acyl isopeptides. L abeled peptides and peptidomimetics have been found to be useful in chemistry, biology, and medicine for monit oring and detection perpose. Usually a chromophoric or fluorophoric unit is attached to a small peptides. Azodyes are a widely

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19 used chromophoric unit s to label peptides. In the current work we have synthesized novel a zodye labeled aminoxy acids and peptide s which can be used as probes for the same purpose As an extension to the idea of peptide labeling and potential application of labeled peptidomimetics, we described the synthesi s, absorption and fluorescence data of new fluorescent coumarin labeled depsi peptides. These novel compounds exhibit high emission quantum yields in certain solvents and the emission absorption profiles are highly dependent on the chemical nature (electron donating or withdrawing) of the coumarin unit. Azapeptides are can mimic natural peptides and can thus imitate or inhibit the same biological effect of a natural peptide. Here we report the development of a phosgene or isocyanate free mild protocol towards the synthesis of azapeptides from am ino acid residues using benzotriazole methodology. S table, crystalline and easy to handle azadipeptidoyl benzotriazoles were prepared and their synthetic utility was demonstrated by the synthesis of longer azapeptides. Th is novel pathway enabled the soluti on phase synthesis of an aza Leuenkephalin analogue. The protocol is revisited in the subsequent chapter which focuses on the de novo design and synthesis of oxy azapeptides in which an amino acid is replaced by an aza hydroxy acid. Calculations revealed t hat oxyazapeptides should occupy a turn secondary structure. We also designed and synthesized taurine containing water soluble peptidomimetics N T erminal and C terminal taurine acylations allowed the synthesis of a number of taurine containing peptides N O taurine conjugates and Sulfonopeptides which can be used for the preparation of bio active molecules.

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20 CHAPTER 1 GENERAL INTRODUCTION Peptide bonds are ubiquitous in cellular chemistry and they are cruc ial for carrying out most biological functions of living organisms. 1 In 1901 Fischer first reported the synthesis of a dipeptide gly gly 1 a Over the last century because of the remarkable advances in the peptide field the chemical synthesis of peptides and proteins is now possibl e. The most used and explored two methods for the preparation of peptides are conventional liquid phase and solid phase peptide synthesis. 1b Although the se two methods have enabled significant advancement in the technology of peptide synthesis, there remai n many limitations. 1 When longer peptides (more than 40 amino acids) are prepared using th e se techniques poor solubility and racemization issue make the peptide synthesis difficult. 1c In r ecent years modern methods such as na tive chemical ligation 11,12 and Staudinger traceless chemical ligati on 33 were developed to address the se problems. Native chemical ligation (NCL) is one of the best techniques for the synthesis of peptides and semi synthesis of proteins. It takes place in aqueous solution and is widely used. NCL was first reported by Wieland et al. and developed by Kent in 1992. 1 1 NCL is a chemoselective and regioselective reaction of peptide thioester and a terminal Cys peptide that gives a native amide bond at the ligation site through a rapid S to N acyl transfer via a five membered cyclic transition state ( Scheme 1 1). 12 NCL has been used in the synthesis of cancer protein NY ESO 1, c ytochrome b562, and dendrimers. 2 The proteins human matrix Gla protein (84 residues), 3a the anticoagulant microprotein S (116 residues), 3b defensin 1 (75 residues) have been synthesized successfully with native chemical

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21 ligation. 3 c NCL, one of the best techniques for the synthesis of peptides and proteins, whil e of great importance, is subject to limitations including (i) the requirement of a N terminal cysteine residue at the ligation site and (ii) the low abundance of cysteine in human proteins (1.7% of the residues). 19 21 In attempts to overcome the limitatio n of the low abundance of cysteine, considerable effort has been devoted to developing auxiliary thiol groups, 4 but t heir use in ligations was found to be (i) difficult to complete due to steric hindrance and (ii) problematic since extraneous groups presen t in the ligated product, can be difficult to remove. 22, 23 Scheme 1 1 Peptide synthesis by native chemical ligation T o address these limitations, our group has devoted considerable effort towards developing alternative techniques. 34 We have reported ligations of S acylated Cys containing peptides to form the corresponding native peptides through long range chemical ligations via 8 to 19 membered transition states. 35 38 This methodology utilizes the selective S acylation of Cys containing peptides by N acylbenzotriazoles followed by microwave assisted high yielding chemical ligations of the resulting S acyl

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22 isopeptides under mild conditions and with no auxiliary groups (Scheme 1 2) 34 35 However, the low abundance of Cys in the natural peptide sequence remains an obstacle. To address this challenge we reported recently the chemoselective O to N acyl migration of O acyl serines via 5 8 and 11 membered transition states. 39 and we successfully demonstrated the long range O to N acyl migrations of O acyl Tyr units via 10 to 18 membered transition states. 40 Scheme 1 2 Peptide synthesis by chemical ligation developed by Katritzky group Unlike the S to N acyl migration N to N acyl migration for the synthesis of native peptides has not been e xplored. In C hapter 2 we have demonstrated chemical ligati ons to form native peptides by N N acyl migrations in Trp containing peptides via 10 to 18 membered cyclic TS. In this study, we first report a statistical, predictive model using extensive synthetic and computational approach es to rationalize the chemical ligation. 41 The model was furt her supported by the synthetic and experimental ligation data. These ligations were achieved by the migration of an N peptidoyl unit of a

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23 Trp isopeptide unit to produce natural peptide and utilize neither a cysteine residue nor an auxiliary group at the ligation site. 41 1 H Benzotriazole 5 has been widely used as an inexpensive, stable synthetic auxiliary. Due to it s amphoteric nature (acidic pKa = 8.2 and basic pKa = 1.6) benzotriazole can be easily removed from the reaction mixture by washing with a base or an acid. Benzotriazole displays the characteristics of an ideal synthetic auxiliary and possesses b oth electron donor and electron acceptor properties. 5a We took the advantage of benzotriazole as a synthetic auxiliary on several occasions and t he application of benzotriazole in organic chemistry is shown in the light of activating the carboxylic group i n th e peptide sequence. N A cylbenzotriazoles can be regarded as a tame halogen substituent 5b but they are advantageous reageant s as the y posses the following characteristics : (i) solids (highly crystalli ne compounds), (ii) soluble in organic solvents and can be used in aqueous media ( very useful for peptide synthesis as free amino aci ds di s s olves in water ), (iii) non hygroscopic making them easy to handle and store, ( i v) can be prepared directly from RCO 2 H in near quantitative yields. More importantly, the y are chirally stable for long periods and are neutral acylating agents 5c Recently labeled peptides and peptidomimetics have been found to be useful in chemistry, biology, and medicine. 6 a In general, two types of peptide labeling are applied: i) a chromo phoric unit or ii) a fluorophoric unit is attached to the small peptides. 6b Both methods are advantageous in some respects. Fluorometric methods are potentially much more sens itive than colorimetric methods; h owever the colorimetric method is more useful w fluorescence or fluorescence created during sample preparation. 57 59 This can be

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24 avoided by the use of non fluorescent dyes. The two C hapters 3 and 4 of my research report highlight the labeling of peptidomimetics. Chapter 3 describes the efficient preparation of azodye labeled aminoxy acids and peptides. Aminoxy acids are the analogues of amino acids. 148a Azodyes are the most widely used chromophoric unit to label peptides. The la rge geometry and dipole change associated with azobenzene photoisomerization is the key factor which has been used to control protein activity with light. 61 aminoxy acids aminoxy a cid units are more rigid than the corresponding amino acid units, and aminoxy amide bonds RCONHOR' resist enzymatic degradation Aminoxy peptides also feature strong intramolecular hydrogen bonds between adjacent residues in peptidomimetic foldamers and may provide useful labels. 64 Chapter 4 reports on the synthesi s, absorption and fluorescence data of new fluorescent coumarin labeled depsipeptides. 44 A depsipeptide is a peptide in which one or more of the amide ( CONHR ) bonds are replaced by ester (COOR ) bonds. 87 These compounds exhibited high emission quantum yields in certain solvents and the emission absorption profile was highly dependent on the chemical nature (electron donating or withdrawing) of the coumarin derivative. These types of fluorescent compounds may be useful in monitoring biological and therapeutic activity. Peptides are widely used as drug delivery systems, in medicinal chemistry as prodrugs an d bioactive molecules. However when appie d to biological systems peptide s show rapid degrada tion which significantly limits their utilization in vivo 164 166 For decades considerable effort has been devoted to find peptide like molecules or

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25 peptidomimetics such as azapeptides, depsipeptides, pseudopeptides and peptoids that c ould address these pr oblems 7 a Peptidomimetics are small molecules that mimic natural peptides and can thus imitate or inhibit the same biological effect of a natural peptide. 7b The main advantages of using peptidomimet i cs as drugs are the i ncreased bioavailability, improved transport through cellular membranes and decreased rate of excretion and hydrolysis by peptidases. 8 Azapeptides are peptidomimetics in which the CH groups of one or more amino acid residues are replaced by a nitrogen atom. Azapeptides may exhibit better interactions with protein receptors and enhanced stability to enzymatic and chemical degradation. 9 Azapeptides are therefore potential leads for the generation of receptor ligands, enzyme inhibitors and clinically approved drugs. In C hapter 5 we have dev eloped a mild and nov el route towards azapeptides 43 This novel pathway enabled the solu tion phase synthesis of an aza Leu enkephalin analogue. It is possible to design small peptide like molecules that would show the same extent of biological effects like their natural peptide analogs. 10 In recent years many new peptidomimetics have been designed, synthesized and their potential application has been evaluated. Ho wever, res earchers are still in search of the new peptidomimet i cs with enhanced properties such as a high hydrolytic stability, higher bio availa bi li ty and also with the better selectivity. To design new peptidomimetics two very common modifications are i) amino acid side chain modification and i i) modification of the backbone of the peptide. 166 In Chapter 6 we describe the de novo design and synthesis of novel class of backbone heteroatom modifie d peptidomimetics, 42 which we have termed

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26 peptides Substituting the typical native N C oxy azapeptides in which an amino acid is replaced by an aza hydroxy acid. Oxyazapeptides can be considered as the depsipeptide analogues of azapeptides where the amino group of an aza amino acid to ester known limited conformational space of azapeptides was studied by computation al chemistry 138 Calculations revealed that oxy azapeptides should occupy a turn secondary structure and enjoy greater conformational freedom which cou ld render them more adaptive to varying steric demand of biological interactions. For decades, various peptidomimetics, including phosphonopeptides, ureidopeptides and sulfonopeptides, have been developed and reported. 184 Aminoalkanesulfonic acid peptidom imetics are hydrolysis resistant sulfono analogs of naturally occurring peptides. 185 187 Sulfonamide group increases the polarity and hydrogen bonding ability of the molecule since the SO 2 NH 2 group is more acidic (pKa 11 12) than the amide bond CO NH 2 In Chapter 7 a viable synthetic route towards sulfonopeptides, taurine peptides, and conjugates is reported. 148b Moisture sensitive taurine peptidomimetics with a series of amino acids, di and tripeptides are conveniently synthesized and isolated in high y ields. Taurine containing peptidomimetics and sulfonopeptides mimic natural peptides and therefore represent attractive scaffolds for drug delivery as well as prodrug and tool applications. Chapter 8 presents a summary of achievements together with conclu sions.

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27 CHAPTER 2 LONG RANGE CHEMICAL LIGATION FROM N N ACYL MIGRATIONS IN TRYPTOPHAN PEPTIDES VIA CYCLIC TRANSITION STATES OF 10 TO 18 MEMBERS 2.1 Introduction Native chemical ligation (NCL), first reported by Wieland et al and later developed by Kent, 1 1, 1 2 is a chemoselective and regioselective reaction of a peptide thioester and a terminal Cys peptide that gives a native amide bond at the ligation site through a rapid S to N acyl transfer via a cyclic transition state. Th is is one of the best techniques for the synthesis of peptides and semi synthesis of proteins It takes place in aqueous solution and is becoming widely used. 1 3 1 5 In biomedical research NCL has been used in the synthesis of cancer protein NY ESO 1, 1 6 cyto chrome b562, 1 7 and dendrimers, monodisperse macromolecules with highly branched three dimensional architectures. 1 8 While still of great use, NCL is limited by the need for an N terminal Cys residue at the ligation site to afford a peptide containing an internal Cys and the low abundance of Cys in globular proteins (1.7% of the residues). 19 2 1 To overcome the limitation of the low abundance of Cys, great effort has gone into developing thiol auxiliary groups. However, subsequent ligations were found to be hard to complete because of steric hindrance 2 2, 2 3 and problematic since extraneous groups in the ligated product may be challenging to remove. 21 2 5 There are number of approaches to circumvent the need for a N terminal Cys residue at the ligation site in classical NCL. These techniques include (i) use of an auxiliary group followed by removal of that group after ligation 8 (ii) NCL followed by Reproduced with permission from Chemistry A European Journal 201 4 DOI: 10.1002/chem.201400125 Copyright 2014 WILEY VCH Verlag GmbH & Co. KGaA, Weinheim

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28 the conversion of penicillamine to Val, 2 7 (iii) sugar assisted NCL, 1 (iv) Cys free 3 2 a nd (v ) traceless Staudinger ligation. 3 3 However, the development of new ligation methods is still an important area of research in order to access modified peptides and proteins. In an attempt to address these limitations, our group has devoted considerab le effort towards developing alternative techniques. We have reported ligations of S acylated Cys containing peptides to form the corresponding native peptides through long range chemical ligations via 8 to 19 membered transition states. 34 3 8 This methodo logy utilizes the selective S acylation of Cys containing peptides by N acylbenzotriazoles followed by microwave assisted high yielding chemical ligations of the resulting S acyl isopeptides under mild conditions and with no auxiliary groups. However, the low abundance of Cys in the natural peptide sequence still remains an obstacle. To address this challenge we reported recently the chemoselective O to N acyl migration of O acyl serines via 5 8 an d 11 membered transition states 3 9 and we successfully demonstrated the long range O to N acyl migrations of O acyl Tyr units via 10 to 18 membered transition states. 40 To the best of our knowledge N to N acyl migration for the synthesis of native peptides has not been explored. We di scovered recently the first examples of successful chemoselective N to N acyl migration involving Trp containing isopeptides via 10 11 and 12 membered cyclic transition states. 4 1 These chemical ligations were achieved by migration of the N peptidoyl u nit of a Trp isopeptide unit to produce natural peptides. They utilize neither Cys n or Ser residues, nor an auxiliary group at the ligation site. However this methodology still needs to be fully developed and explored by

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29 examining the following factors: i) the range of cyclic transition states; ii) the best conditions for ligation step; iii) effects of substituents in the amino acid residue and rationalization of the relative abundance of the ligated product. The purpose of this work is to identify structur al features controlling the ligation and to correlate variation in the reactivity of N acylpeptides. We have documented the synthetic and computational investigation of N acyl isopeptide ligations to form native peptides from non terminal Trp residues via 10 to 18 membered cyclic transition states. 2.2 Results and Discussion Intermediate isodipeptide 2. 4 was synthesized and served as the starting material for the investigation of N to N acyl migration via 10 18 membered cyclic transition states. In this study we aim to investigate a novel chemical ligation strategy for N to N acyl transfer by developing a general methodology and a computational model to predict the feasibility of N to N acyl migration in longer peptide synthesis. Compound 2. 4 was coupled with benzotriazolides of dipeptides 2. 12a n and tripeptides 2. 17a c of or amino acids to afford isotetrapeptides 2. 14a n and isopentapeptides 2. 19a c required for the ligation studies involving 1 3 18 membered cyclic transition states. To enhance the migration rates, possibly by lowering the steric hindrance at the ligation sites, we placed Gly, and amino acid units within the isopeptides in order to study the feasibility of N to N acyl migr ations in 13 18 membered cyclic transition states. A statistical model was generated using conformational analysis and molecular descriptors. 2.2.1 Preliminary Results on N N Acyl Migrations via 10 12 Membered Cyclic TS N A cylation of the indole nitrogen in Trp was challenging, but w as achieved when Boc protected Trp 2. 1 was treated with Cbz Ala Bt 2. 2 in MeCN in the presence of

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30 strong base such as DBU resulting in Boc protected N acylisodipeptide 2. 3 (80%). Subsequent Boc group deprotection was conducted using 4N HCl in dioxane solution to afford the hydrochloride salt of unprotected isodipeptide 2. 4 (91%). Scheme 2 1 Synthesis of isodipe ptide 2.4 and isotripeptides 2.9a c In our previous studies 4 1 we tried chemical ligation experiments on 2. 4 and observed that chemical ligation via 7 membered cyclic TS was not favored in either aqueous buffer or basic DMF/piperidine condition. However in longer isopeptides 2. 9a c which were prepared by the usual co upling and deprotection protocol (Scheme 2 1) under DMF/piperidine conditions, N to N acyl migration occurred via a 10 12 membered cyclic TS (Scheme 2 2) to give Z protected tripeptides 2. 10a c ( 44.4%, 71.4%, and 99.1% respectively) as the ligation produc ts. This N to N acyl chemical ligation occurs in Trp, one of the important natural amino acids present in peptides and proteins. This encouraged us to explore the area of chemical ligation by developing a general, high yielding and feasible pathway to the synthesis of T rp containing peptides via ligation techniques.

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31 Scheme 2 2 Chemical ligation of N acyl isopeptides 2. 9a c via 10 12 mem TS 2.2.2 Feasibility of N N Acyl Migrations via 13 Membered Cyclic TS The starting isotetrapeptides 2. 14a d for the N to N acyl transfer via 13 membered TS were prepared by a straight forward coupling reaction. N Acylbenzotriazoles are advantageous reagents to construct peptides, peptidomimetics and peptide conjugates. 42 4 4 Compound 2. 4 and four different Boc protected dipeptide benzotriazolides 2. 12a d were first coupled in MeCN/DIPEA to afford Boc protected isotetrapeptides 2. 13a d No chromatography was needed and compounds 2. 13 were purified by acidic and basic work ups. Boc deprotection of the Trp containing isotetrapeptides in 4N HCl/dioxane afforded the HCl salt of unprotected isotetrapeptides 2. 14a d (Scheme 2 3). The amino acids Gly, Ala, Phe and Pro were chosen for the isotetrapeptide sequence to enable a comparativ e study on the effect of the substituents at the chemical ligation site for the given 13 membered cyclic TS.

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32 Scheme 2 3 Synthesis of isotetrapeptides 2. 14a d for ligation study via 13 mem TS Initially, the chemical ligation experiments were carried out for 2. 14a under aqueous conditions (pH 7.4, 1M buffer strength, MW 50 C, 50 W, 3 h) to produce the expected ligation products. However the expected ligation product was observed in relatively l ow yield (2%). The ligation experiments were then switched to basic piperidine/DMF condition. To our delight this resulted in the expected ligation products, which, in some cases were produced in almost quantitative yield (Table 2 1). In the cases of Phe and Pro containing isotetrapeptides 2. 14c d the relative abundance of ligated peptides 2. 15c d was 5% and 17% respectively. This result was expected, as it was anticipated that the chemical ligation would be less feasible, due to steric hindrance by the CH 2 Ph group in the case of 2. 14c or, for 2. 14d as a result of the Pro residue inducing a turn in the peptide chain, resulting in too large a distance between the MS/MS, confirmed the formation of the ligated products 2. as these produce different MS fragmentation patterns from those of the starting isotetrapeptides 2. The relative abundance of the crude ligated mixtures were analyzed by HPLC (Table 2 1).

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33 Scheme 2 4 Chemical ligation of N acyl isopeptides 2 14a d via 13 mem TS Table 2 1 Chemical ligation of N acyl isopeptide 2. 14a d via 13 mem TS React Cyclic TS size Total crude yield (%) of products isolated Relative area (%) [a,b] Product characterization by HPLC MS Ligated peptide (LP) React (RT) LP (RT) BA (RT) LP [M+H] + found 2. 14a 13 87 39.11 (41.3) 60.89 (46.3) 0.00 2. 15a 614.3 2. 14b 13 88 27.20 (46.3) 72.80 (56.5) 0.00 2. 15b 628.3 2. 14c 13 84 94.99 (40.2) 5.01 (46.8) 0.00 2. 15c 704.3 2. 14d 13 84 83.05 (45.7) 16.95 (54.6) 0.00 2. 15d 654.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time 2.2.3 Feasibility of N N Acyl Migrations via 14 Membered Cyclic TS Coupling reactions between 2. 4 and four different Boc protected dipeptide benzotriazolides 2. 12e h were carried out to study N to N acyl transfer via a 14 membered TS. Boc deprotection of tryptophan tetrapeptides 2. 13e h in 4N HCl/dioxane gave the HCl salt of unprotected tetrapeptides 2. 14e h which were chosen as potential substrates for the ligation study via a 14 membered TS. The amino acids Gly, Ala,

PAGE 34

34 Ala, Phe and Pro and other amino acids in the isotetrapeptide sequence were chosen to enable a comparative study on the effect of chemical ligation between the given 14 membered cyclic TS and the 13 membered cyclic TS. Scheme 2 5 Synthesis of isotetrapeptides 2. 14e h for ligation study via 14 mem TS Chemical ligation via a 14 membered cyclic TS was investigated by subjecting isotetrapeptides 2. 14e h to microwave irradiation at 50 C, 50W for 3h using basic piperidine/DMF condition (Scheme 2 6). The reaction mixtures were cooled, the solvent was removed under reduced pressure, and the ligation mixtures (1.0 mg/mL in methanol) were analyzed by HPLC MS. The NH 2 site of unprotected N acyl isotetrapeptides 2. 14e h was attacked intramolecularly by the amide carbonyl carbon (C=O) linked to indole nitrogen of Trp via a 14 membered cyclic TS (Scheme 2 4) to give ligated peptide s 2. 15e h Formation of the expected ligation products in the cases of Gly and Ala 2. 15e,f was almost quantitative. However in case of 2. 14g (Phe at the N terminus), the ligation product was ob tained in only 30% yield, and in case of 2. 14h (Pro at the N terminus) only 2.3% of the ligated product 2. 15h was observed. This is consistent with our findings for 13 membered TS size for ligation products with similar MS/MS, confirmed that the ligated pro ducts

PAGE 35

35 2. each produced different MS fragmentation patterns from those of the starting isotetrapeptides 2. The relative abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 2. Scheme 2 6 Chemical ligation of N acyl i sopeptides 2. 14e h in via 14 mem TS Table 2 2 Chemical ligation of N acyl isopeptide 2. 14e h via 14 mem TS React Cyclic TS size Total crude yield (%) of products isolated Relative area (%) [a,b] Product characterization by HPLC MS Ligated peptide (LP) React (RT) LP (RT) BA (RT) LP [M+H] + found 2. 14e 14 83 11.06 (39.3) 88.94 (45.7) 0.00 2. 15e 628.3 2. 14f 14 88 1.75 (48.0) 98.25 (56.5) 0.00 2. 15f 642.3 2. 14g 14 87 69.50 (40.3) 30.50 (47.3) 0.00 2. 15g 718.3 2. 14h 14 84 97.37 (44.8) 2.63 (55.0) 0.00 2. 15h 668.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H]+ and [M+Na]+ ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time

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36 2.2.4 Feasibility of N N Acyl Migrations via 15 Membered Cyclic TS The starting isotetrapeptides for the N to N acyl transfer via a 15 membered TS were prepared following a similar protocol in to that of Scheme 2 5. Four different Boc protected dipeptide benzotriazolides 2. 12i l were first coupled with compound 2. 4 in Me CN/DIPEA to afford Boc protected isotetrapeptides 2. 13i l Boc deprotection in 4N HCl/dioxane gave the HCl salt of free isotetrapeptides 2. 14i l Scheme 2 7 Synthesis of isotetrapeptides 2. 14i l for ligation study via 15 mem TS Chemical ligation via a 15 membered cyclic TS was investigated under similar conditions to that of Scheme 2 4. The abundance of the expected ligation products in most cases was low. In the case of proline containing isotetrapeptide 2. 14l only 1% of the ligated product 2. 15l was observed. It is possible that t his result was due to a Pro MS/MS, confirmed that the ligated products 2. each produced different MS fragmentation pattern s from those of the starting isotetrapeptides 2. The relative abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 3.

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37 Scheme 2 8 Chemical ligation of N acyl isopeptides 2. 14i l via 15 mem TS Table 2 3 Chemical ligation of N acyl isopeptide 2. 14i l via 15 mem TS React Cyclic TS size Total crude yield (%) of products isolated Relative area (%) [a,b] Product characterization by HPLC MS Ligated peptide (LP) React (RT) LP (RT) BA (RT) LP [M+H]+ found 2. 14i 15 85 8.29 (37.7) 91.71 (43.8) 0.00 2. 15i 642.3 2. 14j 15 90 53.60 (45.4) 46.40 (54.1) 0.00 2. 15j 656.3 2. 14k 15 86 95.87 (40.7) 4.13 (47.4) 0.00 2. 15k 732.3 2. 14l 15 87 99.14 (45.4) 0.86 (55.9) 0.00 2. 15l 682.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time 2.2.5 Feasibility of N N Acyl Migrations via 16 18 Membered Cyclic TS N to N acyl transfer via 16 18 membered TS s would facilitate the synthesis of longer peptides. Coupling reactions between 2. 4 and three different sets of Boc protected tripeptide benzotriazolides 2. 17a c in MeCN in the presence of 3.0 equiv. of DIPEA at 20 o C gave Boc protected N acylisopentapeptides 2. 18a c Compounds 2. 18

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38 were purified by acidic and basic workups and no chroma tography was required. The HCl salts of unprotected isopentapeptides 2. 19a c were achieved upon Boc deprotection of N acylisopentapetides 2. 18a c in 4N HCl/dioxane. Scheme 2 9 Synthesis of isopentapeptides 2. 19a c for ligation study The chemical ligation experiments were performed first for 2. 19a under basic piperidine/DMF (MW 50C, 50W, 3 h) to produce the expected ligation products Ligation did not occur for 16 membered cyclic transition states (we recovered mainly starting material 2. 19a ). Compounds 2. 19b and 2. 19c were irradiated under microwave conditions in basic piperidine/DMF (MW 50C, 50 W, 3 h) and the reaction mixtures were analyzed by HPLC MS which show ed significant amounts of ligated products. The abundance of the expected ligation products in case of 2. 19b was 31% and for 2. 19c via MS/MS, confirmed that the ligated products 2. 20b,c each produced different MS fragmentation pattern s from those of the starting isotetrapeptides 2. 19b,c The abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 4. On combining all our experimental results, we observed that the intramolecular N to N acyl migration is highly favored for medium ring size cyclic transition states, while for larger ring size cyclic transition states there is a decrease in the expected ligation products.

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39 Scheme 2 10 Chemical ligation of N acyl isopeptides 2. 19a c via 16 18 mem TS Table 2 4 Chemical ligation of N acyl isopeptides 2. 19a c via 16 18 mem TS React Cyclic TS size Total crude yield (%) of products isolated Relative area (%)[a,b] Product characterization by HPLC MS Ligated peptide (L P) React (RT) LP (RT) BA (RT) LP [M+H]+ found 2. 19a 16 83 100 (49.4) 0 (NA) 0.00 2. 20a 2. 19b 17 86 69.40 (53.9) 30.60 (66.9) 0.00 2. 20b 739.3 2. 19c 18 87 79.30 (53.8) 20.70 (67.5) 0.00 2. 20c 753.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product 2.2.6 Isolation of Ligated Product The form ation of ligated product 2. 15f from compound 2. 14f was further confirmed by isolation via semi preparative HPLC and characterized by 1 H and 13 C NMR spectroscopy, elemental analysis and analytical HPLC. 1 H NMR spectra showed clear difference s in 11.00 7.00 ppm (Figure 2 1). The appearance of new peak in the case of 2. 15f at 10.85 ppm in the 1 H NMR (which was absent in 2. 14f ) indicated the formation of the desired ligation product. This peak at 10.85 ppm is a typical NH proton

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40 peak from the indole ring in a Trp moiety and clearly confirmed the intramolecular N N acyl migration of Z alanine to the N terminus forming native peptide 2. 15f (Scheme 2 6). Figure 2 1 Difference in 1 H spectra of isolated ligated peptide 2. 15f ( left ) and starting compound 2. 14f (right ) 2.2.7 Competitive Ligation Experiments To further support the intramolecular nature of the chemical ligation of unprotected isopeptides 2. 14a o and 2. 19a c we studied the chemical ligation of isotetrapeptide 2. 14f in t he presence of 20 equiv. of dipeptide 2. 22 (H Gly Gly OMe) under same reaction conditions to that of Scheme 2 isolated crude product confirmed the formation of 20% of the desired ligation product 2. 15f with a retention time at 48 .0 along with 80% of the starting material 2. 14f with a retention time at 56.5 min No bisacylated product 2. 16f was observed. It was also observed that there is no Cbz protected tripeptide 2. 23 (which is the N acylated product of dipeptide 2. 22 via competitive experiment supports the hypothesis that the N to N acylation is intramolecular rather than intermolecular.

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41 Scheme 2 11 Competitive Chemical ligation of 2. 14f in DMF/piperid ine 2.2.8 Com p utational A nalysis Internal chemical ligations can be considered mechanistically as intra molecular nucleophilic reactions between the amide carbonyl carbon (C=O) linked to the indole nitrogen of Trp and the unprotected N terminus (the distance between those two reactive terminus is represented as b (N C)). A cyclic transition state is attained for this transformation and therefore molecular structure is an important factor. The lowest energy conformer facilitates N to N acyl transfer due to formation of an amide bond in the ligated peptide. We applied techniques previously employed 37, 45 4 6 for similar ligation reactions including a full conformational search followed by scoring the conformers based on spatial distances b ( N C) between reactive termini. A full conformational search considering both rotatable bonds and the phenyl ring of twenty four related compounds 2. 9a to 2. 20c (including Leu/Val containing isopeptides which was used for prediction p urposes) and analyzed using the MMX force field, 4 7 in PCMODEL v.9.3 software. 4 8 The best pre organized conformer of each compound with b (N C) values, are shown in Table 2 5. Understanding the co linearity between the calculated 127 3D molecular descriptors using PADEL descriptors and relative abundance of ligated peptide, the highly contributing descriptors such as a) spatial distance b (N C) and b) B alaban index are shown in Table 2 5. Balaban Index estimates the isomeric discrimination ability of the pepti de. 4 9 Two of the pre organized 3D conformers with the highest and lowest b (N C) for given 14 membered TS compounds

PAGE 42

42 2. 14f and 2. 14h are shown in Figure 2 2 respectively. This confirms that b (N C) is highly dependent on the amino acid sequence chosen for the peptide chain. Figure 2 2 Pre organized 3D representation with b (N C) for compound 2. 14f (left ) and 2. 14h ( right ) Table 2 5 Computational data for pre organized (conformer) compounds 2. 9a to 2. 20c ID [a] TS [b] size Sequence Rel .[c] abund b(N C) [d] Bal. Ind. 2.9a 10 H Gly Trp(Z Ala) OBn 44.40 4.013 1.437 2.9b 11 H Ala Trp(Z Ala) OBn 71.40 3.171 1.444 2.9c 12 H Gaba Trp(Z Ala) OBn 99.10 3.010 1.447 2.14a 13 G H Gly Gly Trp(Z Ala) OBn 60.89 3.245 1.454 2.14b 13 A H Ala Gly Trp(Z Ala) OBn 72.80 3.328 1.456 2.14c 13 F H Phe Gly Trp(Z Ala) OBn 5.012 4.845 1.244 2.14d 13 P H Pro Gly Trp(Z Ala) OBn 16.95 3.995 1.253 13 V H Val Gly Trp(Z Ala) OBn NA 3.253 1.455 13 L H Leu Gly Trp(Z Ala) OBn NA 3.188 1.448 2.14e 14 G H Gly Ala Trp(Z Ala) OBn 88.94 2.996 1.444 2.14f 14 A H Ala Ala Trp(Z Ala) OBn 98.25 2.978 1.443 2.14g 14 F H Phe Ala Trp(Z Ala) OBn 30.50 3.731 1.221 2.14h 14 P H Pro Ala Trp(Z Ala) OBn 2.630 4.526 1.236 14 V H Val Ala Trp(Z Ala) OBn NA 3.233 1.437 14 L H Leu Ala Trp(Z Ala) OBn NA 3.555 1.426 2.14i 15 G H Gly Gaba Trp(Z Ala) OBn 91.71 3.126 1.430 2.14j 15 A H Ala Gaba Trp(Z Ala) OBn 46.40 3.655 1.428 2.14k 15 F H Phe Gaba Trp(Z Ala) OBn 4.130 4.045 1.198 2.14l 15 P H Pro Gaba Trp(Z Ala) OBn 0.861 4.324 1.218 15 V H Val Gaba Trp(Z Ala) OBn NA 3.887 1.417 15 L H Leu Gaba Trp(Z Ala) OBn NA 3.562 1.405 2.20a 16 H Ala Pro Ala Trp(Z Ala) OBn 0 4.911 1.281 2.20b 17 H Ala Pro Ala Trp(Z Ala) OBn 30.60 3.743 1.231 2.20c 18 H Ala Pro Gaba Trp(Z Ala) OBn 20.70 4.090 1.206 [a] ID represents the compounds,[b] represents the transition state size [c] represents the relative abundance of ligated product and [d] represents spatial distance b(N C).

PAGE 43

43 To justify the spatial distance b (N C) and stabilities of selected conformers, SVP level 50 5 2 of theory using Turbomole Software. 53,5 4 To determine whether these results are due to the congestion offered by the steric energy, we calculated those using molecular mechanics. Steric energy can also influence the relative abundance of ligated product, thus we determined steric energy to prioritize conformers in addition to the spatial distance b (N C). The steric energy of the pre organized conformers is deri ved as the difference between the global minimum energy and the energy of best pre organized conformer shown in Eq 2 1. E S = E (Global) E (conformer) ( 2 1 ) In general, the feasibility of ligation depends on the spatial distance between the reactive sites, steric hindrance and hydrogen bond distances. 3 7 We extended our studies using the data in Table 2 5 to design a predictive model using statistical techniques to correlate the Relative Abundance (ligated product %) with quantitative structural activi ty/property relationship (QSAR/QSPR). 5 5 The genetic algorithm linear regression method was performed using QSARINS software 5 6 which establishes a correlation between the dependent variable (property/response is relative abundance of ligated peptide) and in dependent variables (molecular descriptors or factors). The quality of the regression is reflected by the numerical values of statistical parameters including the coefficient of determination (R 2 ), the standard error (s), Fisher criterion (F), st (t) and cross validation coefficient of the determination (R 2 CV ). These statistical parameters for the model are shown in Table 2 6, SAR equation (ii) and regression plot in Figure 2 3. With reference to the generated model, spatial distance

PAGE 44

44 b (N C) has 85% of correlation with relative abunda nce and B alaban index with 71%. In this present study, the percentage of ligated peptide increases with a) shorter spatial distance b (N C) and b) higher B alaban index. Table 2 6 Statistical m odel for relative abundanc e of the ligated peptide ID Factors [a] Coeff [b] s [c] t [d] R2=0.93, F=106.1, s=9.92, n=2, N=18, R2cvloo=0.90, R2adj=0.88 I Intercept 0.1439 g1 Distance b(N C) 36.143 0.626 42.50 g2 Balaban Index 134.304 0.406 37.50 [a] represents the molecular descriptor based on BMLR stepwise model, [b] coefficients of respective factors test (t criterion). Relative Abundance = 0.1439 + ((g 1 x 36.1430.62) + (g 2 x 134.3040.4) ) ( 2 2 ) Using the Eq 2 2 model, the relative abundance of ligated product for twenty four compounds 2. 9a to 2. 20c (including Leu/Val containing isopeptides which were used for prediction purpose s ) was calculated. The cross validation (R 2 cv ) was very close to the r egression coefficient (R 2 ) which represents the quality of the model. To our delight that the experimental relative abundance of the ligated peptide for compounds 2. 9a, 2. 14b, 2. 14c, 2. 14e, 2. 14g, 2. 14h and 2. 20b was found to be very close to the predicted value. The experimental relative abundance of the ligated product for the remaining compounds was close to the predicted values and the results are shown in Table 2 7.

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45 Table 2 7 Experimental and predicted relative abundance of ligated p ept ide ID Cyclic TS size Ligated Peptide Sequence Exp. [a] Pred. [b] Error 2. 9a 10 Z Ala Gly Trp OBn 44.40 45.67 1.27 2. 9b 11 Z Ala Ala Trp OBn 71.40 79.50 8.1 2. 9c 12 Z Ala Gaba Trp OBn 99.10 85.98 13.12 2. 14a 13 G Z Ala Gly Gly Trp OBn 60.89 78.14 17.25 2. 14b 13 A Z Ala Ala Gly Trp OBn 72.80 75.41 2.61 2. 14c 13 F Z Ala Phe Gly Trp OBn 5.012 7.90 2.88 2. 14d 13 P Z Ala Pro Gly Trp OBn 16.95 24.03 7.08 13 V Z Ala Val Gly Trp OBn NA 77.29 NA 13 L Z Ala Leu Gly Trp OBn NA 77.57 NA 2. 14e 14 G Z Ala Gly Ala Trp OBn 88.94 85.79 3.15 2. 14f 14 A Z Ala Ala Ala Trp OBn 98.25 86.31 11.94 2. 14g 14 F Z Ala Phe Ala Trp OBn 30.50 29.28 1.22 2. 14h 14 P Z Ala Pro Ala Trp OBn 2.630 2.56 0.07 14 V Z Ala Val Ala Trp OBn NA 78.21 NA 14 L Z Ala Leu Ala Trp OBn NA 62.17 NA 2. 14i 15 G Z Ala Gly Gaba Trp OBn 91.71 79.21 12.5 2. 14j 15 A Z Ala Ala Gaba Trp OBn 46.40 59.83 13.43 2. 14k 15 F Z Ala Phe Gaba Trp OBn 4.130 14.84 10.71 2. 14l 15 P Z Ala Pro Gaba Trp OBn 0.861 7.44 6.579 15 V Z Ala Val Gaba Trp OBn NA 49.96 NA 15 L Z Ala Leu Gaba Trp OBn NA 60.10 NA 2. 20a 16 Z Ala Ala Pro Ala Trp OBn 0 5.31 5.31 2. 20b 17 Z Ala Ala Pro Ala Trp OBn 30.60 30.19 0.41 2. 20c 18 Z Ala Ala Pro Gaba Trp OBn 20.70 14.29 6.41 Relative abundance of ligated peptide [a] Experimental and [b] Predicted based on model Figure 2 3 Correlation plot for relative abundance model of ligated p eptide in Table 2 7

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46 2.2.9 Experimental Validation of M odel To test the statistical model, a further three compounds 2. 14m o (having Val at the N termini) were chosen for the isotetrapeptide sequence to enable a systematic investigation into the feasibility of chemical ligation for the 13 15 membered cyclic TSs. Scheme 2 12 Synthesis of 2. 14m o and c hemical ligation to validate the statistical model Table 2 8 Chemical ligation of N acyl isopeptides 2. 14m o in DMF/piperidine React Cyclic TS size Total crude yield (%) of products isolated Relative area (%) [a,b] Product characterization by HPLC MS Ligated peptide (LP) React (RT) LP (RT) BA (RT) LP [M+H] + found 2. 14m 13 83 2.50 (48.1) 97.5 (57.1) 0.00 2. 15m 656.3 2. 14n 14 88 1.80 (46.8) 98.2 (57.2) 0.00 2. 15n 670.3 2. 14o 15 87 91.65 (47.2) 8.35 (57.6) 0.00 2. 15o 684.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material), [b] LP = ligated peptide, BA = bisacylation product After the ligation experiment was carried out under similar conditions to those used previously, (Scheme 2 12) the crude reaction mixture was analysed by

PAGE 47

47 The expected ligated products 2. each produced different MS fragmentation patterns from those of the starting isotetrapeptides 2. and the relative abundances of the ligated peptides are shown in Table 2 8. The experimental and predicted abundances of the peptides were not a quantitative match, but a qualitative correlation was observed. These results indicate that the 13 and 14 membered cyclic TS are more feasible than the 15 membered cyclic TS. This was consistent with our findings in all other compounds for the 13 15 membered cyclic TS in Table 2 7. The chemical ligation via an intramol ecular N to N acyl transfer to form the native peptides was highly favorable. Intramolecular chemical ligation occurs smoothly in smaller (10 12 membered cyclic TS) isotripeptides, it is also highly favored in medium sized (13 15 membered cyclic TS) isote trapeptides but is highly dependent on the peptide sequence and spatial distance b (N C). When the terminal amino acids are Phe, and Pro; the large steric bulk of the substituents on those amino acids force the two reactive sites apart. When the cyclic TS g et larger (16 18 membered cyclic TS) the chemical ligation becomes even more difficult. 2.3 Conclusion We have demonstrated an efficient and convenient synthesis of novel N acyl isopeptides containing Trp residues. The subsequent intramolecular chemical ligation via an N to N acyl migration was favored through a long range 10 to 18 membered cyclic TS forming the native peptides. This novel methodology was achieved withou t using Cys/Ser/Tyr residues or an auxiliary group at the ligation site A statistical model was generated to predict and rationalize the feasibility of ligation. The m odel was further supported with synthe tic and experimental ligation data. Given that the re are an increasing number of studies involving the synthesis of longer peptides and isopeptides

PAGE 48

48 to evaluate their biological activities, we believe this new ligation approach represents a significant development in the field. 2.4 Experimental Section 2.4 .1 General M ethods reagent grade or HPLC grade. Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 and CD 3 OD using a 300 MHz and 500 MHz spectrometer (with TMS as an internal standard). All 13 C NMR spectra were recorded with complete proton decoupling. All microwave assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon sep tum and there action mixture was subjected to microwave irradiation ( Discover mode ; run time 60 s.; PowerMax analyses were performed on reverse phase gradient Phenomenex Synergi Hydro RP (2.1 150 mm; 5 m) + guard column (2 4 m m; 2.1 100 mm + guard column ) using 0.2% ac etic acid in H 2 O/methan ol as mobile phases; wavelength = 254 nm; and mass spectrometry was done with electrospray ionization ( ESI). 2.4.2 General Procedure for Preparation o f Boc Protected Isot etrapeptides 2. 13a o and Isopentapeptides 2. Boc protected benzotriazolides of dipeptides (Boc AA 1 AA 2 Bt) or tripeptides (Boc AA 1 AA 2 AA 3 Bt) (1.0 mmol, 1.0 equiv. ) in MeCN (10 mL) were added drop wise to a solution of 2. 4 (1.0 mmol, 1.0 equiv. 0.54 g) and DIPEA (3.0 mmol, 3.0 equiv. 0.52

PAGE 49

49 mL) at room temperature and stirred for 16 h until all the starting material was consumed. MeCN was evaporated and the residue dissolved in EtOAc (50 mL) and washed with 3N HCl (5 50 mL ), 5% sodium bicarbonate (2 50 mL) and brine (1 50 mL). The organic portion was dried over anhydrous Na 2 SO 4 filtered, concentrated and recrystallized from ether to give the corresponding Boc protected isotetrapeptides 2. 13a o 2.4.3 General Procedure for Preparation of Unprotected Isotetrapeptides 2. of Isopentapeptides 2. Boc protected isotetrapeptides 2. or i sopentapeptides 2. (0.5 mmol) were dissolved in 4 N HCl in 1 4 dioxane (15mL) at 20C and stirred f or 2 h. The reaction mixture were evaporated, and the residue was recrystallized from diethyl ether to give the corresponding hydrogen chloride salts of unprotected isotetrapeptides 2. or isopentapeptides 2. 2.4.4 General Procedure for Chemical Ligation of N Acyliso peptides 2. N Acylisopentapeptides 2. DMF / Piperidine N A cylisotetrapeptides 2. or N acylisopentapeptides 2. (0.20 mmol) mL/ 1.5 mL), and the mixture was irradiated under microwave (50 C, 50W, 3 h) in a microwave tube. After cooling to room temperature the reaction mixtures were acidified with 2 N HCl to pH = 1. Each mixture was extracted with ethyl acetate (3 10 mL), the combined organic extracts were dried over sodium sulfate, and the solvent was removed under reduced pressure. Each ligation mixture was weighed, and then absolution in methanol (1 mg mL 1 ) was Boc Gly Gly Trp(Z Ala) OBn ( 2. 13a 1 H NMR (300 MHz, CD 3 8.29 (d, J = 7.5 Hz, 1H), 7.58 7.35 (m, 2H), 7.24 6.88 (m, 12H),

PAGE 50

50 5.08 4.84 (m, 5H), 4.73 4.57 (m, 1H), 3.76 (d, J = 6.6 Hz, 2H), 3.60 (d, J = 7.2 Hz, 2H), 3.18 2.98 (m, 2H), 1.41 1.21 (m, 12H).; 13 C NMR (75 MHz, CDCl 3 171.0, 169.5, 156.6, 156.0, 136.3, 136.1, 135.1, 130.6, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 125.8, 124.3, 123.4, 118.9, 117.8, 117.1, 80.4, 67.7, 67.3, 52.3, 49.8, 44.3, 43.4, 28.5, 27.2, 19.7; Anal. Calcd for C 38 H 43 N 5 O 9 : C, 63.94; H, 6.07; N, 9.81. Found: C, 63.74; H, 6.46; N, 9.38 Boc Ala Gly Trp(Z Ala) OBn ( 2. 13b 1 H NMR (300 MHz, CDCl 3 J = 8.4 Hz, 1H), 7.50 (d, J = 11.1 Hz, 1H), 7.40 6.96 (m, 15H), 5.94 5.69 (m, 1H), 5.55 5.36 (m, 1H), 5.01 4.87 (m, 6H), 4.19 4.00 (m, 1H), 3.97 3.77 (m, 2H), 3.30 3.15 (m, 2H), 1.53 1.26 (m, 15H).; 13 C NMR (75 MHz, CDCl 3 ) 5.0, 130.6, 128.7, 128.7, 128.6, 128.4, 128.4, 128.2, 125.8, 124.3, 123.3, 118.9, 117.8, 117.1, 80.1, 67.6, 67.3, 52.3, 50.3, 49.8, 43.5, 28.5, 27.4, 19.5, 18.3; Anal. Calcd for C 39 H 45 N 5 O 9 : C, 64.36; H, 6.23; N, 9.62. Found: C, 64.52; H, 6.63; N, 9.38. Boc Phe Gly Trp(Z Ala) OBn ( 2. 13c 1 H NMR (300 MHz, DMSO d 6 J = 7.5 Hz, 2H), 8.08 7.94 (m, 1H), 7.92 7.80 (m, 1H), 7.71 7.55 (m, 2H), 7.39 7.24 (m, 14H), 7.22 7.13 (m, 4H), 5.09 4.95 (m, 5H), 4.84 4.59 (m, 1H), 4.2 9 4.03 (m, 1H), 3.94 3.66 (m, 2H), 3.79 (s, 2H), 3.17 (d, J = 6.9 Hz, 2H), 2.99 (d, J = 10.2 Hz, 1H), 2.72 (d, J = 10.8 Hz, 1H), 1.36 1.23 (m, 12H); 13 C NMR (75 MHz, CDCl 3 129.5, 129.4, 128.8, 128.7, 128.6, 128.4, 128.3, 128.1, 126.8, 125.9, 124.4, 120.4, 118.9, 117.1, 114.5, 80.4, 67.8, 67.2, 56.4, 52.6, 50.0, 39.2, 35.8, 28.5, 27.4, 19.8 ;

PAGE 51

51 Anal. Calcd for C 4 5 H 49 N 5 O 9 : C, 67.23; H, 6.14; N, 8.71. Found: C, 67.08; H, 6.36; N, 8.49. Boc Pro Gly Trp(Z Ala) OBn ( 2. 13d). 1 H NMR (300 MHz, CDCl 3 J = 8.4 Hz, 1H), 7.52 7.42 (m, 2H), 7.38 7.23 (m, 13H), 7.20 7.00 (m, 2H), 5.90 5. 65 (m, 1H), 5.18 4.92 (m, 6H), 4.23 3.76 (m, 3H), 3.48 3.13 (m, 5H), 1.91 1.63 (m, 3H), 1.42 1.26 (m, 12H); 13 C NMR (75 MHz, CDCl 3 171.4, 171.2, 169.8, 156.0, 155.6, 136.4, 136.1, 135.2, 130.6, 128.7, 128.5, 128.3, 128.2, 128.1, 125.8, 124.3, 123 .1, 118.9, 118.2, 117.0, 115.2, 80.6, 67.5, 67.3, 60.7, 52.4, 49.8, 47.5, 43.4, 29.8, 28.6, 27.2, 24.7, 19.2; Anal. Calcd for C 41 H 47 N 5 O 9 : C, 65.32; H, 6.28; N, 9.29. Found: C, 65.17; H, 6.62; N, 9.05. Boc Gly BAla Trp(Z Ala) OBn ( 2. 13e). 0.63 g, 86%: mp 88 1 H NMR (300 MHz, CD 3 J = 7.5 Hz, 1H), 7.61 7.35 (m, 2H), 7.24 6.97 (m, 12H), 5.08 4.84 (m, 5H), 4.73 4.57 (m, 1H), 3.54 (d, J = 8.7 Hz, 2H), 3.39 3.26 (m, 2H), 3.14 2.95 (m, 2H), 2.43 2.20 (m, 2H), 1.36 1.21 (m, 12H); 13 C NMR (75 MHz, CDCl 3 171.8, 171.4, 171.1, 169.7, 156.2, 155.8, 136.2, 136.1, 135.1, 130.7, 128.8, 128.7, 128.4, 128.3, 128.2, 126.0, 124.4, 120.5, 118.9, 117.1, 114.4, 80.3, 67.7, 67.4, 52.3, 49.7, 44.5, 35.9, 34.5, 28.5, 27.5, 19.4; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 39 H 45 N 5 O 7 Na750.3109; Found 750.3122. Boc Ala BAla Trp(Z Ala) OBn ( 2. 13f). 1 H NMR (300 MHz, CDCl 3 J = 5.7 Hz, 1H), 7.75 7.59 (m, 2H), 7.50 7.24 (m, 3H), 7.24 6.96 (m, 12H), 6.38 (d, J = 6.9 Hz, 1H), 5.70 (d, J = 7.2 Hz, 1H), 5.06 4.82 (m, 5H), 4.17 3.98 (m, 1H), 3.81 3.23 (m, 2H), 3.22 2.96 (m, 2H), 2.53 2.12 (m, 1H), 1.37 1.10 (m, 15H). 13 C NMR (75 MHz, CDCl 3

PAGE 52

52 155.8, 136.4, 136.1, 135.1, 130.5, 128 .7, 128.6, 128.2, 128.1, 125.7, 124.2, 122.8, 118.9, 118.5, 118.4, 117.0, 115.1, 79.9, 67.5, 67.2, 52.6, 50.7, 49.7, 36.3, 35.9, 28.4, 27.3, 19.0, 18.5; Anal. Calcd for C 40 H 47 N 5 O 9 : C, 64.76; H, 6.39; N, 9.44. Found: C, 64.36; H, 6.56; N, 9.46. Boc Phe Ala Trp(Z Ala) OBn ( 2. 13g). 1 H NMR (300 MHz, CD 3 J = 4.8 Hz, 1H), 7.63 7.40 (m, 2H), 7.31 6.88 (m, 17H), 5.14 4.83 (m, 5H), 4.77 4.63 (m, 1H), 4.28 4.06 (m, 1H), 3.37 3.25 (m, 2H), 3.23 3.06 (m, 2H), 2.96 (d, J = 7 .8 Hz, 1H), 2.70 (d, J = 7.8 Hz, 1H), 2.30 (t, J = 6.6 Hz, 2H), 1.31 (d, J = 6.3 Hz, 3H), 1.27 1.12 (m, 9H); 13 C NMR (75 MHz, CD 3 172.6, 171.9, 171.8, 157.1, 156.4, 137.5, 137.0, 136.4, 135.7, 130.6, 129.2, 128.5, 128.3, 127.8, 126.6, 125.3, 123.8, 123.3, 118.7, 118.1, 116.7, 79.5, 67.0, 66.7, 56.4, 52.9, 49.8, 38.3, 35.7, 35.1, 27.5, 27.0, 17.1; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 46 H 51 N 5 O 9 Na840.3579; Found 840.3555. Boc Pro Ala Trp(Z Ala) OBn ( 2. 13h). 1 H N MR (300 MHz, CDCl 3 J = 7.8 Hz, 1H), 7.83 7.72 (m, 1H), 7.52 7.09 (m, 15H), 5.98 (t, J = 10.8 Hz, 1H), 5.15 4.82 (m, 6H), 4.26 3.94 (m, 1H), 3.94 3.61 (m, 1H), 3.43 2.91 (m, 5H), 2.66 2.07 (m, 2H), 2.03 1.56 (m, 4H), 1.49 1.20 (m, 12H); 13 C NMR (7 5 MHz, CDCl 3 128.8, 128.7, 128.4, 128.2, 125.9, 125.7, 124.3, 122.7, 119.0, 117.1, 80.4, 67.6, 67.3, 60.7, 52.8, 49.7, 47.5, 36.8, 36.7, 30.0, 28.6, 27.2, 24.8, 19.5; Anal. Calcd for C 42 H 49 N 5 O 9 : C, 65.69; H, 6.43; N, 9.12. Found: C, 65.47; H, 6.71; N, 9.33. Boc Gly Gaba Trp(Z Ala) OBn ( 2. 13i). 1 H NMR (300 MHz, CD 3 J = 7.2 Hz, 1H), 7.54 (d, J = 9.9 Hz, 1H), 7.44 (d, J =

PAGE 53

53 6.9 Hz, 1H), 7.24 7.12 (m, 10H), 7.07 7.00 (m, 2H), 5.01 4.84 (m, 5H), 4.12 3.90 (m, 1H), 3.56 (s, 2H), 3.18 2.97 (m, 4H), 2.27 1.97 (m, 2H), 1.61 (t, J = 7.2 Hz, 2H), 1.36 1.23 (m, 12H); 13 C NMR (75 MHz, CDCl 3 1 36.2, 135.2, 134.2, 130.6, 128.7, 128.6, 128.4, 128.3, 128.1, 125.9, 124.3, 123.1, 120.3, 119.0, 117.0, 80.4, 68.5, 67.4, 49.8, 46.3, 45.3, 33.4, 33.3, 28.5, 26.0, 24.3, 19.8; Anal. Calcd for C 40 H 47 N 5 O 9 : C, 64.76; H, 6.39; N, 9.44. Found: C, 64.54; H, 6.42 ; N, 9.16. Boc Ala Gaba Trp(Z Ala) OBn ( 2. 13j). 1 H NMR (300 MHz, CDCl 3 J = 8.4 Hz, 1H), 7.54 7.39 (m, 2H), 7.34 7.10 (m, 14H), 6.73 (br s, 1H), 6.01 5.62 (m, 1H), 5.10 4.90 (m, 6H), 4.34 3.82 (m, 1H), 3.33 2.95 ( m, 4H), 2.42 1.89 (m, 2H), 1.78 1.65 (m, 2H), 1.47 1.24 (m, 15H). 13 C NMR (75 MHz, CDCl 3 128.80 128.7, 128.6, 128.4, 128.2, 125.9, 124.3, 122.7, 119.1, 119.0, 118.6, 117.0, 80.2, 67.5, 67.3, 52.6, 49.7, 45.8, 38.6, 33.1, 30.5, 28.5, 27.5, 19.5, 18.6; Anal. Calcd for C 41 H 49 N 5 O 9 : C,65.15; H, 6.53; N, 9.27. Found: C, 64.83; H, 6.74; N, 8.86. Boc Phe Gaba Trp(Z Ala) OBn ( 2. 13k). 0.71 g, 85%: mp 129.0 132.0 C; 1 H NMR (300 MHz, CD 3 J = 6.6 Hz, 1H), 7.55 7.40 (m, 2H), 7.26 6.88 (m, 17H), 5.14 4.83 (m, 5H), 4.28 4.04 (m, 1H), 3.99 3.74 (m, 1H), 3.23 3.27 (m, 5H), 2.71 (d, J = 12.9 Hz, 1H), 2.12 1.83(m, 2H), 1.52 (t, J = 7.2 Hz, 2H), 1.35 1.12 (m, 12H); 13 C NMR (125 MHz, C DCl 3 136.3, 136.2, 134.2, 130.7, 129.5, 128.7, 128.6, 128.4, 128.2, 127.0, 125.9, 124.4, 119.1, 118.6, 117.0, 114.6, 80.4, 67.6, 67.4, 56.3, 53.1, 50.0, 39.0, 38.4, 33.1, 28.5,

PAGE 54

54 28.4, 25.7, 19.8; Anal. Cal cd for C 47 H 53 N 5 O 9 : C, 67.85; H, 6.42; N, 8.42. Found: C, 67.71; H, 6.62; N, 8.37. Boc Pro Gaba Trp(Z Ala) OBn ( 2. 13l). 1 H NMR (300 MHz, CDCl 3 J = 8.4 Hz, 1H), 7.79 7.42 (m, 2H), 7.40 7.06 (m, 14H), 5.90 5.51 (m, 1H), 5.24 4.67 (m, 5H), 4.33 4.02 (m, 1H), 3.60 2.86 (m, 6H), 2.32 2.06 (m, 3H), 2.03 1.61 (m, 5H), 1.54 1.25 (m, 12H); 13 C NMR (75 MHz, CDCl 3 173.7, 173.4, 172.1, 171.1, 155.9, 155.5, 136.4, 136.1, 135.2, 130.6, 128.7, 128.5, 128.3, 128.2, 125.8, 124.3, 1 22.8, 119.0, 118.5, 117.0, 80.6, 67.4, 67.2, 60.6, 52.6, 49.8, 47.3, 38.5, 32.9, 29.5, 28.6, 27.5, 26.2, 24.7, 19.4; Anal. Calcd for C 43 H 51 N 5 O 9 : C, 66.05; H, 6.57; N, 8.96. Found: C, 65.68; H, 6.92; N, 8.78. H Gly Gly Trp(Z Ala) OBn ( 2. 14a ). 0.31 g, 94%: m 1 H NMR (300 MHz, CD 3 J = 7.5 Hz, 1H), 7.76 7.42 (m, 2H), 7.35 7.05 (m, 12H), 5.26 4.84 (m, 5H), 4.53 4.22 (m, 1H), 3.96 3.75 (m, 2H), 3.62 (d, J = 6.6 Hz, 2H), 3.39 2.24 (m, 2H), 1.33 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CD 3 168.7, 166.6, 157.1, 136.9, 136.2, 136.0, 129.8, 128.4, 128.3, 128.2, 127.8, 127.5, 125.5, 124.4, 124.0, 118.5, 117.8, 116.7, 66.9, 66.6, 52.7, 49.7, 42.0, 40.4, 26.7, 16.9; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 33 H 36 N 5 O 7 614.2609; Fo und 614.2614. H Ala Gly Trp(Z Ala) OBn hydrochloride salt ( 2. 14b). 0.30 g, 92%: mp 1 H NMR (300 MHz, DMSO d 6 8.66 (m, 2H), 8.47 8.19 (m, 4H), 8.05 (d, J = 6.6 Hz, 1H), 7.97 7.80 (m, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.44 7.12 (m, 12H), 5.2 2 4.90 (m, 5H), 4.81 4.5 (m, 1H), 4.00 3.68 (m, 3H), 3.51 3.29 (m, 2H), 3.29 3.07 (m, 2H), 1.43 1.28 (m, 6H). 13 169.0, 156.5, 137.5, 136.3, 136.0, 130.6, 129.0, 128.6, 128.5, 128.3, 128.1, 125.7,

PAGE 55

55 124.8, 124.3, 119 .5, 118.1, 116.8, 67.0, 66.3, 53.1, 50.1, 48.8, 42.3, 27.1, 18.1, 17.8; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 34 H 37 N 5 O 7 Na 650.2585; Found 650.2592. H Phe Gly Trp(Z Ala) OBn ( 2. 14c 1 H NMR (300 MHz, CD 3 J = 6.9 Hz, 1H), 7.78 7.44 (m, 2H), 7.36 7.05 (m, 17H), 5.26 4.82 (m, 5H), 4.72 4.60 (m, 1H), 4.12 3.96 (m, 1H), 3.87 (d, J = 9.9 Hz, 1H), 3.72 (d, J = 9.9 Hz, 1H), 3.18 3.05 (m, 3H), 2.98 2.88 (m, 1H), 1.36 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CD 3 171.0, 169.6, 169.0, 157.0, 136.9, 136.2, 134.3, 130.5, 129.3, 128.9, 128.3, 128.1, 127.8, 127.6, 127.5, 125.1, 123.8, 123.6, 118.6, 116.5, 67.0, 66.6, 54.6, 52.7, 49.7, 42.0, 37.2, 26.7, 17.4; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 40 H 42 N 5 O 7 704.3079; Fo und 704.3083. H Pro Gly Trp(Z Ala) OBn hydrochloride salt ( 2. 14d). 0.32 g, 92%: mp 1 H NMR (300 MHz, CD 3 J = 7.8 Hz, 1H), 7.96 7.55 (m, 1H), 7.72 7.40 (m, 3H), 7.38 7.07 (m, 10H), 5.24 4.89 (m, 6H), 4.41 4.08 (m, 1H), 3.90 (s, 2H ), 3.34 3.27 (m, 2H), 3.24 3.10 (m, 2H), 2.52 2.19 (m, 1H), 2.08 1.76 (m, 3H), 1.57 1.31 (m, 3H); 13 C NMR (75 MHz, CD 3 158.3, 138.2, 137.4, 136.9, 131.7, 129.6, 129.5, 129.4, 129.3, 129.1, 128.7, 126.4, 125.0, 124.8, 119.9, 119.0, 117.7, 68.3, 67.9, 61.1, 53.9, 50.9, 47.6, 43.5, 31.0, 27.9, 25.1, 18.5; Anal. Calcd for C 36 H 40 ClN 5 O 7 : C, 62.65; H, 5.84; N, 10.15. Found: C, 62.62; H, 6.22; N, 9.98. H Gly BAla Trp(Z Ala) OBn hydrochloride salt ( 2. 14e). 0.31 g, 93%: mp 1 H NMR (300 MHz, CD 3 J = 8.7 Hz, 1H), 7.59 7.41 (m, 2H), 7.32 6.79 (m, 12H), 5.16 4.84 (m, 5H), 4.55 4.18 (m, 1H), 3.74 3.42 (m, 2H), 3.39 3.24 (m, 3H), 3.14 2.98 (m, 1H), 2.52 2.21 (m, 2H), 1.31 (d, J = 6.6 H z, 3H); 13 C

PAGE 56

56 NMR (75 MHz, CD 3 128.4, 128.3, 128.0, 127.8, 127.6, 125.5, 125.2, 123.8, 123.2, 118.7, 116.7, 67.0, 66.6, 52.8, 49.7, 40.3, 35.8, 34.9, 26.9, 17.0; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 34 H 38 N 5 O 7 628.2776; Found 628.2777. H Ala BAla Trp(Z Ala) OBn hydrochloride salt ( 2. 14f). 0.33 g, 95%: mp 1 H NMR (300 MHz, DMSO d 6 J = 7.5 Hz, 1H), 8.70 8.53 (m, 1H), 8.40 8.27 (m, 3H), 8.08 7.85 (m, 5H), 7.69 7.54 (m 2H), 7.45 7.16 (m, 11H), 5.24 4.86 (m, 5H), 4.80 4.51 (m, 1H), 3.86 3.68 (m, 1H), 3.40 3.05 (m, 4H), 2.45 2.30 (m, 2H), 1.44 1.23 (m, 6H); 13 C NMR (75 MHz, CDCl 3 155.9, 138.7, 135.7, 135.4, 130.0, 128.4, 127.9, 127.8, 127.7, 127.4, 126.8, 126.4, 125.0, 124.0, 118.9, 117.6, 116.2, 66.4, 65.7, 52.5, 49.5, 48.2, 35.3, 34.7, 26.5, 17.5, 17.3; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 35 H 39 N 5 O 7 Na 664.2742; Found 664.2753. H Phe Ala Trp(Z Ala) OBn hydrochloride salt ( 2. 14g). 0.35 g, 92%: mp 1 H NMR (300 MHz, CD 3 J = 4.5 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.57 7.48 (m, 1H), 7.45 6.88 (m, 17H), 5.14 4.83 (m, 5H), 4.52 4.24 (m, 1H), 4.07 3.79 (m, 1H), 3.76 3.49 (m, 2H), 3.45 3.28 (m, 3H), 3.18 3.06 (m 1H), 2.56 2.10 (m, 2H), 1.36 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, CD 3 168.7, 156.8, 136.6, 135.9, 134.0, 130.2, 129.0, 128.6, 128.1, 128.0, 127.8, 127.5, 127.3, 127.2, 124.8, 123.5, 123.3, 118.3, 116.2, 66.7, 66.3, 54.3, 52.5, 49.4, 41.7, 37.0, 35.2, 26.4, 17.1;HRMS (ESI TOF) m/z: [M + H] + Calcd for C 41 H 44 N 5 O 7 718.3235; Found 718.3244.

PAGE 57

57 H Pro Ala Trp(Z Ala) OBn hydrochloride salt ( 2. 14h). 0.34 g, 94%: mp 1 H NMR (300 MHz, DMSO d 6 8.59 (m, 1H), 8.55 8.23 (m, 2 H), 8.18 7.78 (m, 3H), 7.59 (d, J = 6.6 Hz, 1H), 7.48 7.08 (m, 12H), 5.30 4.82 (m, 5H), 4.82 4.51 (m, 2H), 4.20 3.85 (m, 1H), 3.43 2.87 (m, 5H), 2.40 2.07 (m, 2H), 1.89 1.49 (m, 3H), 1.34 (d, J = 6.0 Hz, 3H); 13 1 67.9, 155.8, 136.8, 135.6, 135.4, 129.9, 128.3, 127.9, 127.8, 127.7, 127.6, 127.3, 125.2, 123.6, 118.8, 117.5, 116.1, 114.8, 66.3, 65.6, 58.5, 52.4, 49.4, 45.4, 35.4, 34.5, 30.6, 29.6, 23.5, 17.3; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 37 H 42 N 5 O 7 668.3040; Found 668.3055. H Gly Gaba Trp(Z Ala) OBn hydrochloride salt ( 2. 14i). 0.32 g, 94%: mp 1 H NMR (300 MHz, CD 3 J = 7.8 Hz, 1H), 7.65 7.43 (m, 2H), 7.29 7.03 (m, 12H), 5.12 4.84 (m, 5H), 4.51 4.22 (m, 1H), 3.56 (d, J = 5.4 Hz, 2H) 3.29 ( d, J =6.6 Hz, 2H), 3.18 2.97 (m, 2H), 2.31 1.97 (m, 2H), 1.62 (t, J = 6.2 Hz, 2H), 1.33 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 157.1, 136.9, 136.3, 135.7, 129.9, 128.4, 128.3, 128.2, 128.1,127.8, 127.6, 125.5, 12 5.2, 124.5, 124.0, 118.5, 116.6, 68.2, 66.6, 52.7, 49.7, 40.4, 38.6, 32.7, 26.9, 25.4, 17.1;HRMS (ESI TOF) m/z: [M + H] + Calcd for C 35 H 40 N 5 O 7 642.2922; Found 642.2916. H Ala Gaba Trp(Z Ala) OBn hydrochloride salt ( 2. 14j). 0.32 g, 92%: mp 1 H N MR (300 MHz, DMSO d 6 J = 11.1 Hz, 1H), 8.72 8.51 (m, 1H), 8.40 8.19 (m, 2H), 8.09 7.74 (m, 3H), 7.74 7.58 (m, 1H), 7.47 7.10 (m, 12H), 5.33 4.72 (m, 5H), 4.69 4.34 (m, 1H), 3.93 3.57 (m, 1H), 3.44 2.78 (m, 4H), 2.65 2.28 (m, 1H), 2.24 1.89 (m, 1 H), 1.86 1.46 (m, 2H), 1.46 1.25 (m, 6H); 13 C NMR (75 MHz,

PAGE 58

58 128.5, 128.4, 128.3, 128.1, 126.0, 125.6, 124.3, 119.6, 118.4, 116.8, 67.0, 66.3, 53.1, 50.1, 48.9, 38.8, 32.9, 2 8.9, 25.8, 18.0, 17.9; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 36 H 41 N 5 O 7 Na678.2898; Found 678.2897. H Phe Gaba Trp(Z Ala) OBn hydrochloride salt ( 2. 14k). 0.35 g, 90%: mp 1 H NMR (300 MHz, CD 3 J = 8.1 Hz, 1H), 7.65 7.40 (m, 2H), 7.26 6.88 (m, 17H), 5.14 4.83 (m, 5H), 4.54 4.25 (m, 1H), 4.10 3.74 (m, 1H), 3.46 3.27 (m, 1H),3.18 2.86 (m, 5H), 2.12 1.83 (m, 2H), 1.49 (t, J = 8.3 Hz, 2H), 1.33 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CD 3 .9, 168.7, 168.2, 157.1, 136.9, 136.2, 134.4, 129.3, 128.8, 128.4, 128.3, 128.2, 128.0, 127.8, 127.6, 125.5, 125.2, 124.4, 124.0, 123.2, 118.4, 116.7, 68.2, 66.6, 54.7, 52.7, 49.7, 38.6, 37.5, 32.6, 26.9, 25.1, 16.9; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 42 H 46 N 5 O 7 732.3392; Found 732.3385. H Pro Gaba Trp(Z Ala) OBn hydrochloride salt ( 2.14 l). 0.33 g, 92%: mp 1 H NMR (300 MHz, CD 3 J = 7.2 Hz, 1H), 7.97 7.81 (m, 1H), 7.65 7.44 (m, 3H), 7.33 7.15 (m, 10H), 5.20 4.90 (m, 6H), 4.22 4 .13 (m, 1H), 3.39 3.28 (m, 4H), 3.19 3.02 (m, 2H), 2.37 2.13 (m, 3H), 2.07 1.86 (m, 3H), 1.78 1.59 (m, 2H), 1.40 (d, J = 6.6 Hz, 3H), 13 171.5, 167.8, 155.9, 136.8, 135.7, 135.4, 130.0, 128.3, 127.9, 127.8, 127.7, 127.4 125.3, 123.6, 118.9, 117.7, 116.1, 114.9, 66.4, 65.6, 58.8, 52.5, 49.5, 45.5, 38.3, 32.3, 29.7, 26.4, 25.1, 23.6, 17.4; Anal. Calcd for C 32 H 43 N 3 O 7 : C,66.07; H, 7.45; N, 7.22. Found: C, 65.7; H,7.77; N, 7.58; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 38 H 44 N 5 O 7 682.3235; Found 682.3223.

PAGE 59

59 Boc Ala Pro Gly Trp(Z Ala) OBn ( 2. 18a). 1 H NMR (300 MHz, CDCl 3 8.21 (m, 1H), 7.58 7.36 (m, 2H), 7.33 7.13 (m, 15H), 5.99 5.63 (m, 1H), 5.30 4.69 (m, 7H), 4.63 3.98 (m, 2H), 3.71 2.90 (m, 5H), 2.30 1.50 (m, 2H), 1.47 0.95 (m, 18H); 13 C NMR (75 MHz, CDCl 3 172.1, 171.7, 169.0, 155.9, 155.5, 136.3, 136.1, 135.2, 130.6, 128.8, 128.7, 128.4, 128.3, 128.2, 128.0, 125.8, 124.3, 118.9, 118.3, 117.0, 114.5, 79.8, 67.5 67.3, 60.6, 52.6, 51.4, 49.9, 48.1, 47.5, 29.8, 28.6, 27.7, 25.4, 19.3, 18.4, 16.9; Anal. Calcd for C 45 H 54 N 6 O 10 : C, 64.42; H, 6.49; N, 10.02. Found: C, 64.42; H, 6.52; N, 9.68. Boc Ala Pro BAla Trp(Z Ala) OBn ( 2. 18b). 1 H NM R (300 MHz, CDCl 3 8.22 (m, 2H), 8.14 7.80 (m, 2H), 7.43 7.30 (m, 5H), 7.19 7.04 (m, 10H), 5.78 (t, J = 7.8 Hz, 1H), 5.31 5.01 (m, 3H), 5.95 4.73 (m, 6H), 4.67 4.32 (m, 2H), 3.58 3.23 (m, 5H), 2.29 1.83 (m, 3H), 1.63 0.88 (m, 15H); 13 C NMR (75 MHz, CDCl 3 135.1, 130.0, 128.9, 128.8, 128.7, 128.4, 128.2, 128.0, 125.7, 125.1, 124.4, 119.0, 118.7, 117.1, 79.5, 67.3, 66.1, 53.6, 49.5, 48.9, 47.9, 37.4, 29.6, 28.5, 28.2, 26.0, 25.5, 16.6, 15 .5; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 45 H 53 N 6 O 10 Na 861.3794; Found 861.3796. Boc Ala Pro Gaba Trp(Z Ala) OBn ( 2. 18c). 1 H NMR (300 MHz, CDCl 3 8.24 (m, 2H), 8.07 7.77 (m, 2H), 7.50 7.30 (m, 5H), 7.18 6.96 (m, 10H), 5.85 (t, J = 7.8 Hz, 1H), 5.29 5.02 (m, 3H), 5.00 4.70 (m, 6H), 4.68 4.20 (m, 2H), 3.91 2.89 (m, 5H), 2.13 1.85 (m, 5H), 1.55 1.10 (m, 15H); 13 C NMR (75 MHz, CDCl 3 135.2, 130.2, 128.9, 128.7, 128.4, 128.2, 125.9, 124.4, 123.0, 118.7, 118.6, 117.0, 79.5,

PAGE 60

60 67.8, 67.3, 60.3, 52.8, 49.6, 48.6, 47.7, 37.7, 32.2, 29.3, 28.6, 27.5, 25.2, 23.5, 20.1, 17.1; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 46 H 55 N 6 O 10 Na 875.3950; Found 875.3975. H Ala Pro Ala Trp(Z Ala) OBn hydrochloride salt ( 2. 19a). 0.37 g, 96%: mp 1 H NMR (300 MHz, CD 3 J = 7.5 Hz, 1H), 7.76 7.58 (m, 1H), 7.60 7.39 (m, 2H), 7.40 6.89 (m, 11H), 5.25 4.87 (m, 5H), 4.64 4.04 (m, 2H), 3.61 3.37 (m, 3H), 3.35 3.01 (m, 3H), 2.08 1.66 (m, 4H), 1.50 1.25 (m, 9H); 13 C NMR (75 MHz, CD 3 138.2, 137.5, 136.9, 131.8, 129.6, 129.5, 129.2, 128.9, 126.5, 125.1, 124.8, 120.0, 119.0, 117.8, 68.3, 67.9, 61.4, 54.0, 53.7, 51.2, 50.4, 30.9, 26.1, 24.4, 18.4, 18.0, 16.4; Anal. Calcd for C 40 H 47 ClN 6 O 8 : C, 61.97; H, 6.11; N, 10.84. Found: C, 61.61; H, 6 .21; N, 10.59. H Ala Pro Ala Trp(Z Ala) OBn hydrochloride salt ( 2. 19b). 0.36 g, 94%: mp 1 H NMR (300 MHz, DMSO d 6 8.30 (m, 2H), 8.29 8.22 (m, 1H), 8.08 7.98 (m, 1H), 7.97 7.86 (m, 3H), 7.61 (d, J = 7.2 Hz, 1H), 7.52 7.25 (m, 13H), 4 .83 4.52 (m, 5H), 4.70 4.00 (m, 1H), 3.70 3.35 (m, 5H), 3.29 2.90 (m, 3H), 2.37 2.18 (m, 1H), 1.99 1.50 (m, 5H), 1.38 1.25 (m, 6H); 13 171.4, 170.8, 170.7, 167.8, 155.8, 138.6, 135.6, 135.4, 129.9, 128.3, 127.7, 127.6, 127.3, 1 24.9, 123.8, 123.6, 118.8, 117.6, 116.1, 66.3, 65.6, 59.6, 52.3, 49.4, 47.0, 46.7, 34.9, 30.6, 29.1, 26.5, 24.5, 17.3, 15.5; Anal. Calcd for C 40 H 47 ClN 6 O 8 : C, 61.97; H, 6.11; N, 10.84. Found: C, 62.16; H, 6.47; N, 10.54. H Ala Pro Gaba Trp(Z Ala) OBn hydroc hloride salt ( 2. 19c). 0.36 g, 92%: mp 1 H NMR (300 MHz, CD 3 J = 7.5 Hz, 1H), 7.65 7.49 (m, 2H), 7.30 7.09 (m, 12H), 5.31 4.91 (m, 6H), 4.50 4.11 (m, 2H), 3.75 3.46 (m, 2H),

PAGE 61

61 3.26 2.92 (m, 5H), 2.37 2.04 (m, 3H), 1.94 1.56 (m, 4H), 1.46 (d, J = 6.6 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CD 3 158.4, 138.3, 137.6, 137.0, 131.8, 129.8, 129.6, 129.4, 129.2, 128.9, 128.1, 126.5, 125. 1, 124.5, 120.0, 117.9, 68.3, 67.9, 62.1, 54.1, 51.1, 47.4, 47.1, 39.8, 34.1, 31.0, 28.3, 27.3, 26.8, 26.3, 18.4, 16.3; Anal. Calcd for C 41 H 49 ClN 6 O 8 : C, 62.39; H, 6.26; N, 10.65. Found: C, 62.15; H, 6.30; N, 10.31. Boc Val Gly Trp(Z Ala) OBn ( 2. 13m ). 0.64 1 H NMR (300 MHz, CDCl 3 J = 8.1 Hz, 1H), 7.85 6.93 (m, 17H), 6.12 5.69 (m, 1H), 5.48 5.18 (m, 1H), 5.16 4.67 (m, 5H), 4.19 3.42 (m, 3H), 3.34 2.99 (m, 2H), 2.13 1.80 (m, 1H); 1.58 1.16 (m, 12H), 0.85 (d, J = 6.6 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 173.5, 171.5, 171.2, 169.3, 156.4, 155.8, 136.3, 136.1, 135.0, 130.5, 128.8, 128.7, 128.5, 128.4, 128.3, 125.8, 124.3, 123.5, 122.2, 118.9, 117.1, 80.4, 67.4, 67.2, 60.4, 52.3, 49.8, 43.7, 31.0, 28.6, 28.5, 19.5, 18.2; Anal. Calc d for C 41 H 49 N 5 O 9 : C, 65.15; H, 6.53; N, 9.27. Found: C, 64.85; H, 6.72; N, 9.20. Boc Va l Ala Trp(Z Ala) OBn ( 2. 13n). 1 H NMR (300 MHz, CDCl 3 J = 7.8 Hz, 1H), 7.57 6.98 (m, 17H), 5.96 (s, 1H), 5.57 5.18 (m, 1H), 5.17 4.80 (m, 6H), 3.98 3.42 (m, 2H), 3.34 3.09 (m, 2H), 2.56 2.09 (m, 2H),2.06 1.69 (m,1H), 1.49 1.26 (m, 12H), 0.90 0.73 (m, 6H); 13 C NMR (75 MHz, CDCl 3 56.3, 155.9, 136.3, 136.1, 135.1, 130.5, 128.8, 128.7, 128.6, 128.4, 128.2, 126.0, 124.3, 122.4, 118.9, 118.5, 117.1, 80.0, 67.7, 67.4, 60.5, 52.3, 49.7, 36.3, 36.2, 31.0, 28.5, 27.6, 19.5, 19.4, 18.2; Anal. Calcd for C 42 H 51 N 5 O 9 : C, 65.52; H, 6.68; N, 9.10. Found: C, 65.18; H, 6.97; N, 9.24.

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62 Boc Val Gaba Trp(Z Ala) OBn ( 2. 13o). 1 H NMR (300 MHz, CDCl 3 J = 6.6 Hz, 1H), 7.65 6.93 (m, 17H), 5.99 (s, 1H), 6.65 5.21 (m, 1H), 5.17 4.80 (m, 6H), 4.06 3.75 (m, 1H), 3.69 3.38 (m, 1H), 3.34 2.89 (m, 3H), 2.29 1.90 (m, 2H),1.88 1.58 (m,2H), 1.53 1.13 (m, 12H), 1.01 0.66 (m, 6H); 13 C NMR (75 MHz, CDCl 3 135.2, 130.6, 128.7, 128.6, 128.4, 128.2, 128.1, 125.9, 124.3, 122.8, 1 19.0, 118.6, 117.0, 80.1, 67.5, 67.3, 60.6, 52.5, 49.7, 38.4, 33.2, 31.1, 28.5, 27.4, 25.5, 19.6, 18.5; Anal. Calcd for C 43 H 53 N 5 O 9 : C, 65.88; H, 6.81; N, 8.93. Found: C, 65.54; H, 6.93; N, 8.77. H Val Gly Trp(Z Ala) OBn hydrochloride salt ( 2.14 m). 0.31 g, 90%: mp 1 H NMR (500 MHz, DMSO d 6 J = 8.0 Hz, 1H), 8.29 (s, 3H), 8.10 7.80 (m, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.48 7.12 (m, 14H), 5.12 4.90 (m, 4H), 4.81 4.60 (m, 1H), 3.92 3.77 (m, 2H), 3.64 (s, 2H), 3.32 3.05 (m, 2H), 1.34 (d, J = 8.5 Hz, 3H), 0.94 (s, 6H); 13 C NMR (75 MHz, CDCl 3 136.1, 135.9, 135.0, 130.7, 128.7, 128.4, 128.2, 128.1, 127.8, 125.6, 124.3, 123.4, 118.6, 117.0, 67.7, 67.3, 59.1, 53.0, 49.8, 43.4, 30.4, 26.4, 20.5, 18.6; HRMS (E SI TOF) m/z: [M + H] + Calcd for C 36 H 42 N 5 O 7 656.3079; Found 656.3098. H Val BAla Trp(Z Ala) OBn hydrochloride salt ( 2.14 n). 0.33 g, 93%: mp 115.0 1 H NMR (300 MHz, DMSO d 6 J = 8.1 Hz, 1H), 8.55 8.26 (m, 4H), 7.80 (s, 1H), 7.63 6.87 (m 14H), 6.26 5.77 (m, 1H), 5.30 4.64 (m, 6H), 4.32 3.73 (m, 2H), 3.57 3.07 (m, 3H), 2.69 2.36 (m, 1H), 2.34 1.97 (m, 2H), 1.42 (d, J = 6.3 Hz, 3H), 1.13 0.68 (m, 6H); 13 C NMR (75 MHz, CDCl 3 136.1, 136.1, 135.1, 130.3, 128.7, 128.4, 128.2, 128.0, 126.0, 125.8, 124.4, 118.9,

PAGE 63

63 118.4, 117.0, 68.0, 67.3, 59.9, 53.2, 49.9, 37.1, 36.3, 30.2, 26.4, 20.1, 18.9, 18.5; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 37 H 44 N 5 O 7 670.3235; Found 670.3264. H Val Gaba Trp(Z Ala) OBn hydrochloride salt ( 2. 14o). 0.33 g, 97%: mp 1 H NMR (300 MHz, DMSO d 6 8.26 (m, 3H), 7.96 7.70 (m, 1H),7.51 7.06 (m, 15H), 6.18 5.86 (m, 1H), 5.08 4.87 (m, 5H), 4.8 0 4.67 (m, 1H), 4.27 3.85 (m, 1H), 3.54 2.91 (m, 4H),), 2.57 1.97 (m, 3H),1.93 1.53 (m,2H), 1.41 (d, J = 6.6 Hz, 3H), 1.13 0.82 (m, 6H); 13 C NMR (75 MHz, CDCl 3 172.6, 171.2, 168.6, 155.9, 136.2, 136.0, 135.1, 130.4, 128.7, 128.5, 128.4, 128.2, 12 8.1, 128.0, 125.8, 124.3, 123.7, 118.9, 118.4, 117.1, 67.5, 67.3, 59.2, 53.0, 50.0, 38.8, 33.4, 30.4, 27.0, 25.7, 20.0, 18.8, 18.7; Anal. Calcd for C 38 H 46 ClN 5 O 7 : C, 63.37; H, 6.44; N, 9.72. Found: C, 63.07; H, 6.65; N, 9.67. Cbz Ala Ala Bala Trp OBn ( 2. 15f ). Compound 2. 14f (0.005 mmol, 1.0 equiv. 10 mg) in DMF/piperidine (5 mL) and irradiated in microwave for 3 h at 50 o C. The solvent was evaporated and dried overnight. The compound was isolated by HPLC chromatography to give ligated tripeptide Cbz Ala Ala Bala Trp OBn. 8.0mg, 92%: mp 1 H NMR (500 MHz, CD 3 J = 7.5 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.85 7.83 (m, 1H), 7.50 7.44 (m, 2H), 7.35 7.3 (m, 8H), 7.17 6.96 (m, 5H), 5.06 4. 98(m, 4H), 4.58 4.53 (m, 1H), 4.26 4.13 (m, 1H), 4.12 3.95(m, 1H), 3.43 2.89 (m, 5H), 2.27 (t, J = 1.5 Hz, 2H), 1.18 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 7.0 Hz, 3H); 13 C NMR (125 MHz, CD 3 170.9, 156.3, 137.4, 136.6, 136.3, 128.8, 12 8.4, 128.3, 128.2, 128.2, 128.1, 127.5, 124.2, 121.5, 118.9, 118.5, 111.9, 109.8, 66.3, 65.9, 53.8, 50.6, 48.6, 40.0, 35.7, 35.3,

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64 27.6, 18.8, 18.4 ; Anal. Calcd for C 35 H 39 N 5 O 7 : C, 65.51; H, 6.26; N, 10.65. Found: C, 65.32; H, 6.09; N, 10.55.

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65 CHAPTER 3 EFF ICIENT PREPARATION OF AZODYE LABELED AMINOXY ACIDS AND PEPTIDES 3.1 Introduction Photo isomerization and fluorescence resonance energy transfer (FRET) properties have led to the extensive use of azodye carboxylic acids such as 1a 1b and 1c (Figure 3 1) in biology and medicine. 57 59 Azodye labeled peptides are important for pharmaceutical and biological investigations (Figure 3 2). Peptide based azodye labeled molecules show substrate specificity for prostate membrane antigens 60 and in some cases are pote nt inhibitors of m calpain and chymotrypsin. 59 Azodye labeled peptides also serve as markers in biological applications. 61 Figure 3 1 Azodye carboxylic acids. Figure 3 2 Azodye labeled peptides. Reproduced with permission from ARKIVOC 20 11 212 220 Copyright 2011 ARKAT USA, Inc.

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66 Attaching azo dye carboxyli c acids to host molecules is key to the synthesis of azo photoresponsive systems. Amino acids/peptides or amines acting as links between azodye acyl groups and host mo lecules are common in many photo biological switches and bioprobes. Azo photoresponsive systems frequently incorporate azodye labeled ( ) amino acids/peptides, 62 amino alcohols or amines. 63 Aminoxy acids are analogs of amino acids in which the carbon atom is replaced by an oxygen atom. The incorporation of aminoxy acids into peptidomimetics has attracted interest, since aminoxy acid units are more rigid than the corresponding amino acid units, 64 and aminoxy amide bonds RCONHOR resist en zymatic degradation. 65 Aminoxy peptides have also attracted interest as novel foldamers 66 with unusual conformations and diverse bioactivity. 67 Aminoxy peptides also feature strong intramolecular hydrogen bonds between adjacent residues in peptidomimetic foldamers 68 and may provide useful labels. N Acylbenzotriazoles are easily prepared, non hygroscopic, chirally stable analogues of acid halides that are relatively insensitive to water, 69,70 and are therefore advantageous for N O C or S acylation; 6 9 75 especially where the corresponding acid chlorides are unstable or difficult to prepare. 76 77 We previously acylated amino acids and amines with N (4 arylazobenzoyl) 1 H benzotriazoles 78 and we recently synthesized azodye labeled peptides in good yields by a milder procedure than previously published methods. 79 We have not located any previous syntheses of azodye labeled aminoxy acids or peptides, however due to interesting properties of aminoxy peptides, we now report the synthesis of azodye labeled ami noxy acids and peptides by reaction of N (4

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67 arylazobenzoyl) 1 H benzotriazole with aminoxy acids and aminoxy peptides under mild conditions. 3.2 Results and Discussion 3.2.1 Preparation of Azodye Labeled Aminoxy A cids 3. 4a i N (4 Arylazobenzoyl) 1 H benzotriazoles 3. 2a c prepared by our previously reported method, 80 were treated with the appropriate aminoxy acids 3. 3a c in THF H 2 O (3 1, v/v) in the presence of triethylamine for 4 8 h at 20 o C (monitored by TLC) to afford azodye labeled aminoxy acid s 3. 4a i in yields of 65 80% (Scheme 3 1, Table 3 1). Novel products were characterized by 1 H NMR, 13 C NMR and elemental analysis. Scheme 3 1 Synthesis of azodye labeled aminoxy acids 3. 4a i Table 3 1 Preparation of azodye labeled aminoxy acids 3. 4a i Entry 3. 2 a minoxy acid 3. 3 3. 4 yield (%) a 1 3. 2a AO Glycine 3. 3a 4 Paba AO Glycine 3. 4a 74 2 3. 2a AO Alanine 3. 3b 4 Paba AO Alanine 3. 4b 75 3 3. 2a AO Phenylalanine 3. 3c 4 Paba AO Phenylalanine 3. 4c 70 4 3. 2b AO Glycine 3. 3a 4 Mpaba AO Glycine 3. 4d 65 5 3. 2b AO Alanine 3. 3b 4 Mpaba AO Alanine 3. 4e 65 6 3. 2b AO Phenylalanine 3. 3c 4 Mpaba AO Phenylalanine 3. 4f 75 7 3. 2c AO Glycine 3. 3a 4 Dpaba AO Glycine 3. 4g 80 8 3. 2c AO Alanine 3. 3b 4 Dpaba AO Alanine 3. 4h 70 9 3. 2c AO Phenylalanine 3. 3c 4 Dpaba AO Phenylalanine 3. 4i 80

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68 3.2.2 Preparation of Fmoc Protected Aminoxy Hybrid P eptides 3. 6a e Fmoc protected aminoxy hybrid peptides 3. 6a e were prepared by the reaction of benzotriazole derivatives of Fmoc Amino acids 3. 5a c with aminoxy acids 3. 3a,b in the presence of triethylamine in acetonitrile water at room temperature (Scheme 3 2, Table 3 2). Scheme 3 2 Synthesis of Fmoc protected ami noxy hybrid peptides 3 .6a e Table 3 2 Preparation of Fmoc protected aminoxy hybrid peptides 2. 6a e Entry 3. 5 3. 6 yield (%) 1 Fmoc Phe Bt 3. 5b Fmoc Phe AO Gly OH 3. 6a 92 2 Fmoc Phe Bt 3. 5b Fmoc Phe AO Ala OH 3. 6b 90 3 Fmoc Met Bt 3. 5c Fmoc Met AO Gly OH 3. 6c 92 4 Fmoc Leu Bt 3. 5a Fmoc Leu AO Gly OH 3. 6d 90 5 Fmoc Met Bt 3. 5c Fmoc Met AO Ala OH 3. 6e 91 3.2.3 Preparation of Azodye Labeled Aminoxy P eptides 3. 7a c Azodye labeled aminoxy peptides 3. 7a c we re prepared by the reaction of Fmoc aminoxy hybrid peptides 3. 6a c with azodye Bt 3. 2a in the presence of DBU in THF. (Scheme 3 3, Table 3 3) Scheme 3 3 Synthesis of azodye labeled aminoxy peptides 3.7a c Table 3 3 Preparation of azodye labeled aminoxy peptides 3. 7a c Entry 3. 6 7 yield (%) 1 3.6a 4 Paba Phe AO Gly OH 3. 7a 55 2 3.6b 4 Paba Phe AO Ala OH 3. 7b 58 3 3.6c 4 Paba Met AO Gly OH 3. 7c 65

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69 3.3 Conclusion In conclusion, we have synthesized novel azodye labeled aminoxy acids and aminoxy hybrid peptides in a convenient and efficient manner by reacting N (4 arylazobenzoyl) 1 H benzotriazoles with aminoxy acids and aminoxy hybrid peptides. All the azodye labeled aminoxy acids and aminoxy hybrid peptides were obtained under mild reaction conditions in moderate to good yields. These novel azodye labeled aminoxy acids and aminoxy hybrid peptides may be useful in the preparation of peptidomimetic foldamers and in bio logical applications. 3.4 Experimental Section Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. NMR spectra were recorded in Acetone d 6 CDCl 3 or DMSO d 6 with TMS for 1 H (300 MHz) and 1 3 C (75 MHz) as an internal reference. Elemental analyses were performed on a Carlo Erba 1106 instrument. CH 2 Cl 2 was dried and distilled over CaH 2 whereas THF was used after distillation over Na benzophenone. 3.4.1 Synthesis of Azodye Carboxylic Acid Labeled Aminoxy Acids 3.4a i N (4 arylazobenzoyl) 1 H benzotriazoles 3. 2a c (1.0 mmol) were added to a aminoxy acids 3. 3a c (1.2 mmol) in THF H 2 O (3:1) in the presence of Et 3 N (2.0 mmol). The reaction mixture w as stirred at 20 C for about 3 h until TLC showed the absence of 3. 2a c then the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (50 mL) and the solution was washed with 4 N HCl (3 50 mL), saturated NaCl solution (50 mL), and dried over anhydrous Na 2 SO 4 After evaporation of the solvent, th e residue was crystallized from methanol/hexanes to give 3. 4a i

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70 4 Phenylazobenzoyl AO Gly ( 3. 4a). Orange microcrystals (74%), mp 200 202 C. 1H NMR (DMSO d 6 ) 8.10 7.93 (m, 6H), 7.70 7.60 (m, 3H), 4.54 (s, 2H); 13C NMR (DMSO d 6 ) 171.3, 163.8 153.8, 152.2, 134.6, 132.5, 130.0, 128.8, 123.1, 122.9, 72.5. Anal. Calcd for C 15 H 15 N 3 O 5 : C, 56.78; H, 4.76; N, 13.24. Found: C, 56.72; H, 4.68; N, 12.94. 4 Phenylazobenz oyl AO Ala ( 3. 4b). Orange microcrystals (75%), mp 188 190 C. 1 H NMR (DMSO d 6 ) 12.05 (br s, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.97 7.91 (m, 6H), 7.60 (d, J = 1.8 Hz, 3H), 4.57 (q, J = 6.5 Hz, 1H), 1.43 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 ) 172.7, 166.7, 151.9, 132.1, 130.6, 129.6, 128.7, 122.8, 122.6, 122.5, 78.7, 16.5. Anal. Calcd for C 16 H 15 N 3 O 4 : C, 61.34; H, 4.83; N, 13.41. Found: C, 61.37; H, 4.91; N, 13.24. 4 Phenylazobenzoyl AO Phe ( 3. 4c). Orange microcrystals (70%), mp 178 180 C. 1 H NMR (Acetone d 6 ) 8.05 7.94 (m, 6H), 7.63 7.58 (m, 3H), 7.40 (d, J = 7.2 Hz, 2H), 7.33 7.23 (m, 3H), 4.93 (t, J = 5.1 Hz, 1H), 3.35 3.20 (m, 2H); 13 C NMR (Acetone d 6 ) 172.0, 155.2, 153.3, 137.5, 134.1, 132.8, 131.6, 130.5, 130.2, 129.3, 129.1, 127.4, 123.8, 123.5, 85. 7, 37.9. Anal. Calcd for C 22 H 19 N 3 O 4 : C, 67.86; H, 4.92; N, 10.79. Found: C, 67.62; H, 4.90; N, 10.79. 4 [(4 Methoxy)phenylazo]benzoyl AO Gly ( 3. 4d). Orange microcrystals (65%), mp 200 202 C. 1 H NMR ( DMSO d 6 ) 12.15 (br s, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.95 7.86 (m, 6H), 7.15 7.13 (m, 2H), 4.52 (s, 2H), 3.87 (s, 3H); 13 C NMR (DMSO d 6 ) 170.1, 162.5 146.2, 130.6, 128.6, 124.9, 122.3, 122.2, 114.7, 71.8, 55.8. Anal. Calcd for C 16 H 15 N 3 O 5 : C, 58.36; H, 4.59; N, 12.76. Found: C, 58.29; H, 4.42; N, 11.60.

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71 4 [ (4 Methoxy)phenylazo]benzoyl AO Ala ( 3. 4e). Orange microcrystals (65%), mp 182 184 C. 1 H NMR (DMSO d 6 ) 12.03 (br s, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.96 7.87 (m, 5H), 7.14 (d, J = 8.1 Hz, 2H), 6.88 (br s, 1H), 4.56 (d, J = 6.9 Hz, 1H), 3.87 (s, 3H), 1.42 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 ) 174.1, 163.4, 154.7, 147.0, 133.6, 129.3, 125.9, 123.1, 115.5, 79.8, 56.2, 17.1. Anal. Calcd for C 17 H 17 N 3 O 5 : C, 59.47; H, 4.99; N, 12.24. Found: C, 59.13; H, 4.86; N, 12.08. 4 [(4 Methoxy)phenylazo]benzoyl AO Phe ( 3. 4f). Orange microcrystals (75%), mp 192 194 C. 1 H NMR (Acetone d 6 ) 11.53 (br s, 1H), 8.03 7.92 (m, 7H), 7.42 7.25 (m, 5H), 7.14 (d, J = 9.0 Hz, 2H), 4.98 4.93 (m, 1H), 3.93 (s, 3H), 3.37 3.17 (m, 2H); 13 C NMR (Acetone d 6 ) 171.6, 167.6, 155.7, 147.7, 137.4, 133.2, 130.5, 129.5, 129.1, 127.5, 125.9, 123.2, 115.4, 86.6, 56.1, 38.0. Anal. Calcd for C 23 H 21 N 3 O 5 : C, 65.86; H, 5.05; N, 10.02. Found: C, 65.53; H, 5.08; N, 9.84. 4 [(4 Dimethylamino)phenylazo]benzoyl AO Gly ( 3. 4g). Brown microcrystals (80%), mp 189 191 C. 1 H NMR (DMSO d 6 ) 7.92 7.79 (m, 7H), 6.88 (d, J = 8.4 Hz, 2H), 4.52 (s, 2H), 3.08 (s, 6H); 13 C NMR ( DMSO d 6 ) 170.1, 163.7, 153.2, 142.5, 131.8, 128.5, 126.3, 121.3, 113.0, 71.8, 40.5. Anal. Calcd for C 17 H 19 ClN 4 O 4 : C, 53.90; H, 5.06; N, 14.79. Found: C, 53.78; H, 5.01; N, 14.53. 4 [(4 Dimethylamino)phenylazo]benzoyl AO Ala ( 3. 4h). Brown microcrystals (70%), mp 19 6 198 C. 1 H NMR (DMSO d 6 ) 8.01 7.95 (m, 2H), 7.90 7.80 (m, 4H), 6.9 (d, J = 9.0 Hz, 2H), 4.63 (q, J = 7.2 Hz, 1H), 3.13 (s, 6H), 1.47 (d, J = 6.9 Hz, 3H); 13 C NMR (DMSO d 6 ) 173.3, 164.7, 154.1, 153.5, 142.8, 132.1, 128.8, 126.2, 121.9, 112.6, 79.2, 40.5, 16.9. Anal. Calcd for C 18 H 21 ClN 4 O 4 : C, 55.03; H, 5.39; N, 14.26. Found: C, 54.78; H, 5.51; N, 13.99.

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72 4 [(4 Dimethylamino)phenylazo]benzoyl AO Phe ( 3. 4i). Brown microcrystals (80%), mp 222 224 C. 1 H NMR (DMSO d 6 ) 8.00 7.85 (m, 7H), 7.40 7.21 (m, 5H), 7.03 (d, J = 9.0 Hz, 2H), 4.85 (t, J = 6.6 Hz, 1H), 3.22 3.08 (m, 8H); 13 C NMR (DMSO d 6 ) 171.6, 153.8, 153.2, 136.7, 132.1, 129.7, 128.6, 128.2, 126.6, 126.2, 121.5, 113.2, 83.7, 45.5, 37.0. Anal. Calcd for C 24 H 25 ClN 4 O 4 : C, 61.47; H, 5.37; N, 11.95. Found: C, 60.69; H, 5.42; N, 11.64. 3.4.2 Synthesis of Fmoc Protected Aminoxy Peptides 3. 6a e Benzotriazole derivatives of Fmoc amino acids 3. 5a c (1 mmol) were added to a aminoxy acids 3. 3a b (1 mmol) in MeCN H 2 O (3:1) in the presence of Et 3 N (2.0 mmol). The reaction mixtures were stirred at 20 C for about 1 h until TLC showed the absence of 3. 5a c t hen the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (150 mL) and the solution was washed with 4 N HCl (3 50 mL), saturated NaCl solution (50 mL), and dried over anhydrous Na 2 SO 4 After evaporation of the solvent, the residue was crystallized from methanol/hexanes Fmoc Phe AO Gly OH ( 3. 6a). White microcrystals (92%), mp 106 108 C. 1 H NMR (Acetone d 6 ) 7.85 (d, J = 7.2 Hz, 2H), 7.68 7.61 (m, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.34 7.20 (m, 7H), 4.40 (br s, 2H), 4.28 4.16 (m, 3H), 3.22 2.90 (m, 2H); 13 C NMR (Acetone d 6 ) 154.6, 151.7, 147.6, 139.9, 138.9, 138.2, 137.6, 137.2, 135.8, 130.4, 83.7, 77.0, 57.5, 48.2, 40.3, 29.6. Anal. Calcd for C 26 H 24 N 2 O 6 : C, 67.82; H, 5.25; N, 6.08. Found: : C, 67.85; H, 5.49; N, 5.55 Fmoc Phe AO Ala OH ( 3. 6b). White microcrystals (90%), mp 102 104 C. 1 H NMR (CDCl 3 ) 10.04 (br s, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.60 7.00 (m, 12H), 5.77 (br

PAGE 73

73 s, 1H), 4.63 4.12 (m, 4H), 3.03 (d, J = 7.2 Hz, 2H), 1.44 (s, 3H); 13 C NMR (CDCl 3 ) 171.5, 169.1, 156.4, 143.3, 141.2, 135.4, 129.2, 128.7, 127.8, 127.3, 127.1, 124.9, 120.0, 72.8, 67.7, 60.4, 53.4, 46.8, 38.8. Anal. Calcd for C 27 H 26 N 2 O 6 : C, 68.34; H, 5.52; N, 5.90. Found: C, 67.85; H, 5.49; N, 5.55. Fmoc Met AO Gly OH ( 3. 6c). White micr ocrystals (92%), mp 70 72 C. 1 H NMR (CDCl 3 ) 10.32 (br s, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.60 7.50 (m, 2H), 7.42 7.25 (m, 6H), 4.51 (s, 2H), 4.50 4.38 (br s, 3H), 4.22 4.18 (m, 1H), 2.60 2.40 (m, 2H), 2.20 1.80 (m, 5H); 13 C NMR (Acetone d 6 ) 173.7, 170. 4, 157.0, 145.0, 142.0, 128.5, 127.9, 126.1, 120.8, 74.1, 67.3, 67.1, 53.7, 52.7, 47.9, 32.2, 32.1, 30.9, 15.1. Anal. Calcd for C 22 H 24 N 2 O 6 S: C, 59.45; H, 5.44; N, 6.30. Found: C, 60.44; H, 5.53; N, 5.89. Compound 3. 6c was used for the further reaction and the final compound 3. 7c was fully characterized by 1 H, 13 C NMR and HRMS. Fmoc Leu AO Gly OH ( 3. 6d). White microcrystals (90%), mp 110 112 C. 1 H NMR (CDCl 3 ) 10.54 (br s, 1H), 7.73 (d, J = 7.5 Hz, 2H), 7.60 7.45 (m, 2H), 7.4 1 7.25 (m, 5H), 5.69 (d, J = 9.6 Hz, 1H), 4.50 (s, 2H), 4.43 4.30 (m, 3H), 4.15 (t, J = 6.0 Hz, 1H), 1.57 (d, J = 3.9 Hz, 3H), 0.89 (d, J = 4.5 Hz, 6H); 13 C NMR (CDCl 3 ) 171.6, 170.1, 156.7, 143.3, 141.3, 127.8, 127.1, 124.9, 120.0, 72.8, 67.7, 50.4, 46.9, 41.5, 24.5, 22.6, 22.0. Anal. Calcd for C 23 H 26 N 2 O 6 : C, 64.78; H, 6.14; N, 6.57. Found: C, 64.63; H, 5.87; N, 6.61. Fmoc Met AO Ala OH ( 3. 6e). White microcrystals (91%), mp 88 90 C. 1 H NMR (CDCl 3 ) 10.98 (br s, 1H), 7.84 (d, J = 7.8 Hz, 2H), 7.75 7.60 (m, 2H), 7.45 7.25 (m, 6H), 4.52 (d, J = 6.9 Hz, 1H), 4.40 4.20 (m, 4H), 2.60 2.51 (m, 3H), 2.07 2.02 (m, 4H), 1.41 (d, J = 6.9 Hz, 3H); 13 C NMR (CDCl 3 ) 174.3, 169.6, 15 6.5, 143.4, 141.1, 127.7,

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74 127.0, 124.9, 119.9, 79.7, 67.5, 46.7, 31.7, 31.4, 29.7, 16.1, 15.1. Anal. Calcd for C 23 H 26 N 2 O 6 S: C, 60.25; H, 5.72; N, 6.11. Found: C, 60.05; H, 5.71; N, 5.99. 4 Phenylazobenzoyl Phe AO Gly ( 3. 7a). Orange microcrystals (55%), mp 178 180 C. 1 H NMR (Acetone d 6 ) 8.02 (d, J = 8.7 Hz, 2H), 7.99 7.91 (m, 4H), 7.58 7.56 (m, 3H), 7.35 7.17 (m, 7H), 6.85 (d, J = 8.7 Hz, 1H), 4.84 (br s, 1H), 4.40 (br s, 2H), 3.39 3.10 (m, 2H); 13 C NMR (Acetone d 6 ) 170.2, 155.0, 153.4, 138.1, 136.9, 132.7, 130.2, 129.4, 129.2, 127.5, 123.7, 123.3, 121.1, 120.5, 74.1, 53.7, 38.0. Anal. Calcd for C 24 H 22 N 4 O 5 : C, 64.57; H, 4.97; N, 12.55. Found: C, 64.81; H, 4.99; N, 11.81. 4 Phenylazobenzoyl Phe AO Ala ( 3. 7b). Orange microcry stals (58%), mp 192 194 C. 1 H NMR (DMSO d 6 ) 12.81 (br s, 1H), 8.79 (d, J = 8.7 Hz, 1H), 8.53 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 10.8 Hz, 1H), 8.04 7.91 (m, 5H), 7.61 (s, 3H), 7.42 7.16 (m, 5H), 4.79 (br s, 1H), 4.26 (d, J = 6.6 Hz, 1H) 3.20 3.00 (m, 2H), 1.35 (d, J = 6.6 Hz, 3H); 13 C NMR (DMSO d 6 ) 174.0, 171.2, 165.4, 153.2, 151.8, 138.4, 132.0, 130.6, 129.5, 129.1, 128.7, 128.0, 122.7, 122.5, 122.2, 54.7, 47.6, 37.1, 17.1. HRMS (ESI) Calcd for C 25 H 24 N 4 O 5 : [M + Na] + 483.1 639. Found: 483.1645. 4 Phenylazobenzoyl Met AO Gly ( 3. 7c). Orange microcrystals (65%), mp 185 187 C. 1 H NMR (DMSO d 6 ) 8.10 8.00 (m, 2H), 7.88 7.82 (m, 5H), 7.55 7.45 (m, 3H), 4.45 (d, J = 5.7 Hz, 1H), 4.31 (s, 2H), 2.51 2.45 (m, 4H), 2.00 1.94 (m, 3H); 13 C NMR (DMSO d 6 ) 173.9, 170.4, 167.0, 154.8, 153.2, 137.1, 132.9, 130.4, 130.1, 129.9, 123.9, 123.5, 123.4, 121.8, 118.0, 114.2, 110.4, 71.2, 52.8, 50.9, 50.6, 38.3. HRMS (ESI) Calcd for C 20 H 22 N 4 O 5 S: [M + Na] + 453.1203. Found: 453.1215.

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75 CHAPTER 4 PHOTOPHYSICS OF NOVEL COUMARIN LABELED DEPSIPEPTIDES IN SOLUTION; SENSING INTERACTIONS WITH SDS MICELLE VIA TICT MODEL 4.1 Introduction Proteins and peptides labeled with fluorescent groups are widely applied in biology, biotechnology and medicinal chemis try for the detection and monitoring of physiochemical activity 81 83 Fluorescent peptides have been used to selectively label human v1b va sopressin or oxytocin receptors 84 and used in the construction of highly sensitive fluorescent tags for the detection of vascular endothelial growth factor, a biomarker for angiogenesis 85 Protein engineering has inserted position specific, non natural amino acids into biosynthetic proteins 86 and peptide analogs containing non natural amino acids a re broadly applied in structure activity studies. Incorporation of non natural amino or hydroxy acids into peptides and depsides expands the scope of structural perturbation and can induce specific steric properties 87 Depsipeptides, containing both amino acid units linked by a mide bonds and hydroxy acid units linked by ester bonds, are analogs of peptides and differ significantly in hydrogen bonding capacity compared to natural peptides. Thus the incorporation of hydroxy acids into a peptide chain is a useful tool for the diversification of peptidomimetics and for gaining a better understanding of their structural properties 88,89 Depsipeptides exhibit useful biological (antimicrobial, antifungal, anti inflammatory) and therapeutic activity (anticancer and anti HIV) 90 ; dide mnin B and dolastatin 10 have anti carcinogenic activities 91 ,92 Cyclic depsipeptides such as mirabamides E H, callipeltins A and also quinoxapeptin show promising Reproduced with permission from Amino Acids 2013 45 159 170 Copyright Springer Verlag Wien 2013.

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76 inhibitory activities against HIV 93 95 Valinomycin 96 a natural ionophore, can act as a non metallic isoforming agent in potassium selective electrodes with the best K + /Na + selectivity of all K + ionophores to date 97 Coumarins a re that can be highly sensitive to their environment, 98 they poss ess good solubility in many solvents with extended spectral ranges, high emission quantum yields and photo stability 99,100 These compounds are used to investigate ultrafast solvation dynamics and various electron transfer processes. Fluorescent coumarin l abeled peptides provide a sensitive and specific assay of matrix metalloproteinases, cathepsin D and E activity in biological samples 101,102 They are hydrolyzed by leucine aminopeptidase and hence they are able to act as inhibitors of clostridial aminopep tidase 103 Sodium dodecyl sulfate (SDS) micelles are capable of mimicking the tertiary interactions of protein, lipid and aqueous exposed helical surfaces and they are used as membrane mimetics to study complex biological phenomena 104,105 They can solubilize proteins and stabilize the intramolecular interactions and hence they are important in biosensor studies 106,107 These special properties of SDS micelles are widely used to dissolve and denature proteins, 108, 109 to characterize membran e protein non native states 110 and to investigate solvation dynamics of coumarin dyes 111 Although several literature papers concern general methods and simple photophysical studies of coumarin labeled peptides and peptidomimetics 112 115 a report showing their potential application inside the biological system should be interesting and useful. In this respect depsipeptides show in contrast to their natural analogues (i) high affinity for specific receptors, (ii) good metabolic stability towards endogenous

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77 proteases, (iii) greater oral bio availability and (iv) longer duration of action. These features encouraged us to explore the fields of labeled peptidomimetics and their applicability in biological system s N o literature report was foun d on the synthesis and/or spectroscopical properties of labeled depsipeptides. Our a im in this work was to synthesize and explore the use of labeled depsipeptides thus showing their potential utility in biological system. We report herein t he efficient synthesis and studies of steady state absorption and fluorescence properties of coumarin labeled depsipeptides in polar protic and polar aprotic solvents as well as in the organized confined media of SDS micelles. 4.2 Results and discussion 4.2.1 Preparati on of Unprotected Depsidipeptides 4.5 a b Free or protected optically pure L dipeptides (dipeptides) are useful building blocks for longer peptide analogues. The functions and applications of dipeptides have re ceived little attention in the literature due to the lack of an efficient protocol for the synthesis of dipeptide s compared with proteins and amino acids 116 Here, an efficient route to synthesize unprotected depsipeptides is reported under mild reaction conditions. Boc protected amino acid 4. 1 was coupled with 1 H benzotriazole using DCC to obtain N aminoacyl)benzotriazole 4. 2 which was further reacted with L hydroxycarboxylic acids 4. 3a b in THF in the presence of 4 dimethylaminopyridine (DMAP) as a base to obtain Boc protected depsidipeptid es 4. 4a b Without isolation, 4. 4a b were Boc deprotected by 4N HCl solution in dry dioxane to yield unprotected depsidipeptides 4. 5a b as hydrochloride salts (Scheme 4 1). These free depsidipeptides were characterized by 1 H, 13 C NMR and reacted with

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78 coumarinoylbenzotriazoles 4. 7a c to prepare coumarin labeled fluorescent depsidipeptides 4. 8a f (Scheme 4 2) Scheme 4 1 Preparation of unprotected depsidipeptides 4. 5a b 4.2.2 Preparation of C oumarin Labeled Depsidipeptides 4.8a f Coumarinoylbenzotriazoles 4. 7a c were prepared from the corresponding coumarinoyl acids 4. 6a c using previously reported methodology 115 N Coumarinoyl labeled depsidipeptides 4. 8a f were obtained by treatment of coumarinoylbenzotriazoles 4. 7a c with various unprotected depsipeptides 4. 5a b in the presence of triethylamine and MeCN/H 2 O (3:1 v/v) at 20 o C (Scheme 4 2). The n ovel fluorescent compounds were characterized by 1 H, 13 C NMR and elemental analysis. Scheme 4 2 Preparation of N coumarinoyl labeled depsidipeptides 4. 8a f

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79 4.2.3 Preparation of Unprotected Depsitripeptides 4.11a c Boc protected depsidipeptides 4. 4a c ( 4c was prepared by literature method 112 ) were coupled with EDCI to obtain N Boc( aminoacyl)benzotriazoles 4. 9a b which were reacted with L phe and L met in THF in the presence of triethylamine to obtain the Boc protected depsitripeptides 4. 10a c Without is olation, 4. 10a c were Boc deprotected by 4N HCl solution in dry dioxane to yield the unprotected depsitripeptides 4. 11a c as hydrochloride salts (Scheme 4 3). The novel depsitripeptides 4. 11a c were characterized by 1 H, 13 C NMR and coupled with coumarinoylbenzotriazoles 4. 7b c to prepare coumarin labeled fluorescent depsitripeptides (Scheme 4 4). Scheme 4 3 Preparation of unprotected depsitripeptides 4. 11a c 4.2.4 Preparation of Coumarin Labeled Depsitripeptides 4.12a d N Coumarinoyl labeled depsitripeptides 12a d were prepared by treatment of coumarinoylbenzotriazoles 4. 7b c with unprotected depsitripeptides 4. 11a c in the presence of triethylamine and MeCN/H 2 O (4:1 v/v) at 20 o C (Scheme 4 4). The novel fluorescent compounds were characterized by 1 H, 13 C NMR and elemental analysis.

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80 Scheme 4 4 Preparation of N coumarinoyl labeled depsitripeptides 4. 12a d 4.2.5 Photophysical Studies of Coumarin Labeled Depsipeptides 4.8 and 4.12 Due to a change in the dipole moments between the ground and excited electronic states of the coumarin moiety, the absorption and fluorescence maxima of coumarin labeled conjugates are sensitive to solvent polarity and H bonding ability 117 120 The spectroscopic properties of coumarins can be tuned by substituents at the 6 or 7 positions which affect the energy of the excited states 121,122 This special property prompted researchers to use these fluorescent dyes as a probe to inve stigate many physiochemical processes. Photophysical properties of coumarin labeled depsipeptides 4. 8a f and 4. 12a d were investigated in both polar protic and polar aprotic solvents over a wide range of solvent polarity. To simulate the physiological pH and gain a better understanding of the solvent pol arity effects inside a membrane like environment, photophysical properties of two specific compounds 4. 8c and 4. 12d were studied within the micellar microenvironment of sodium dodecyl sulfate (SDS) in phosp hate buffered saline (PBS) solution at pH 7.4. 4.2.5.1 Absorbance, fluorescence data for coumarin labeled depsipeptides The absorption and emission spectra of 4. 8a f and 4. 12a d in polar protic (methanol and PBS buffer) and polar aprotic (dichloromethane ) solvents are shown in

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81 Figure 4 1 and wavelengths of absorption maxima ( abs ), fluorescence emission maxima ( em ), molar absorptivity ( ) and quantum yields ( are listed in Tables 4 1 3 Table 4 1 Absorption and emission data in PBS buffer Entry Comp. No. abs (nm) em (nm) (10 4 cm 1 M 1 ) quantum yield) 1 4. 8a 301 415 1. 6 0.0 1 2 4. 8b 343 407 2. 8 0.2 4 3 4. 8c 430 481 5.0 0.0 2 4 4. 8d 301 419 1.9 0.0 1 5 4. 8e 343 410 2. 4 0.23 6 4. 8f 430 480 4. 6 0.01 7 4. 12a 346 406 2.2 0.24 8 4. 12b 345 405 2.3 0. 20 9 4. 12c 346 406 2.0 0.22 10 4. 12d 428 480 6.6 0.02 Table 4 2 Absorption and emission data in MeOH Entry Comp. No. abs (nm) em (nm) (10 4 cm 1 M 1 ) quantum yield) 1 4.8a 291 411 1.6 0.0 1 2 4.8b 347 403 1. 9 0.5 2 3 4.8c 420 469 4.8 0.0 3 4 4.8d 291 410 1.5 0.0 1 5 4.8e 346 404 2.5 0.45 6 4.8f 420 467 4.2 0.0 5 7 4.12a 347 403 2.4 0.41 8 4.12b 348 404 3. 3 0.3 2 9 4.12c 347 404 3. 7 0.27 10 4.12d 420 468 4. 1 0.05 Table 4 3 Absorption and emission data in DCM Entry Comp. No. abs (nm) em (nm) (10 4 cm 1 M 1 ) quantum yield) 1 4.8a 291 409 1.4 0.01 2 4.8b 351 402 2. 3 0.36 3 4.8c 427 458 4. 9 0.8 5 4 4.8d 291 405 1.5 0.0 1 5 4.8e 350 402 3. 1 0.5 7 6 4.8f 425 458 6. 4 0.97 7 4.12a 351 404 2.9 0.49 8 4.12b 351 404 4. 3 0.26 9 4.12c 352 404 3. 7 0.36 10 4.12d 426 460 5.0 0.96

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82 Figure 4 1 Emission and absorption spectra of 4. 8b 4. 8e 4. 12a c A) in PBS buffer at pH 7.4 B ) in MeOH C ) in DCM and Emission and absorption spectra of 4. 8c, 4. 8f 4. 12d D ) in PBS buffer E) in MeOH F ) in DCM Electron donating substituents, methoxy ( OMe) and diethylamino ( NEt 2 ) at position 7 of the coumarin skeleton cause a bathochromic shift of the fluorescence emission maxima ( max em) and increase quantum yield s. As expected 7 methoxycoumarin 3 ylcarbonyl labeled depsipeptides 4.8b 4.8e 4.12a c showed A. D. E. F. B. C.

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83 high quantum yields in PBS buffer ( = 0.20 0.24), in MeOH ( = 0.27 0.52) and in CH 2 Cl 2 ( = 0.26 0.57) relative to unsubstituted coumarin labeled depsipeptides 4.8a and 4.8d ( = 0.004 0.006). The quantum yields of 7 methoxycoumarin labeled depsipeptides are interpreted in terms of emission from an intramolecular charge transfer (ICT) excited state 12 3 The methoxycoumarin labeled depsipeptides showed reductions in quantum yields with increased solvent polarity particularly in PBS buffer (Fig ure 4 1 ) Interestingly, the quantum yields of 7 N,N diethylaminocoumarin 3 ylcarbonyl containing 4. 8c 4. 8f and 4. 12d were significantly higher ( = 0.85 0.99) in C H 2 Cl 2 (polar aprotic solvent ) and sharply decreased in polar protic solvents ( = 0.01 0.02 in buffer and ( = 0.03 0.05 in MeOH) (Fig ure 4 1 B, D, F ). In polar aprotic solvent (CH 2 Cl 2 ) diethylaminocoumarin labeled depsipeptides fluoresce from a highly emissive intramolecular charge transfer (ICT) excited state, but in polar protic solvent (PBS buffer, MeOH) rotation of the diethylamino group of the ICT excited state leads to a twisted intramolecular charge transfer excited state (TICT) from which non radiative decay to the ground state occurs (Scheme 4 5) 124,125 The polar solvent stabilizes the charge in the twisted zwitterionic TICT and consequently the interconversion of the ICT TICT is facilitated by an increase of solvent polarity 117 Increasing solvent polarity stabilizes the TICT exited state relative to ground state which explains the bathochromic shift in emission maxima ( max em) (Fig ure 4 2 ). The solvatochromic shifts are direc tly proportional to the dipole moments of the excited and ground state.

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84 Scheme 4 5 TICT model Figure 4 2 Variation in e mission spectra of 4. 8c (left ) and 4. 12d (right ) in PBS buffer, MeOH and DCM 4.2.5.2 Photophysical properties of 7 N N diethylaminocoumarin labeled depsipeptides 4. 8c and 4. 12d in SDS micellar microenvironment Potential applications of the coumarin labeled depsipept ides were investigated by furthe r photophysical study inside a membrane like system. From the earlier experiments it was found that 7 N N diethylaminocoumarin labeled depsipeptides are highly sensitive to the solve nt polarity compared to other coumarin labeled depsipeptides. Two 7 N N diethylaminocoumarin labeled compounds 4 8 c and 4 12d were chosen to stud y any change in the photophysical behaviour inside a membrane like environment. Having observed TICT for depsipeptides, our study was extended to a SDS micelle microenvironment since the TICT state of fluorophores is sensitiv e to the polarity, H bonding ability and viscosity of the solvent 126,127 The steady state absorption and fluorescence spectra of 7 N,N diethylaminocoumarin labeled depsidipeptide 4 8c and depsitripeptide 4 12d were recorded in PBS buffer solution at

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85 physiological pH 7.4 with different concentration s of SDS. All data were taken with the SDS concentration (6.0 200 mM) kept well above the critical micellar concentration (CMC ) of SDS micelles in PBS buffer 129 while the concentration of 4 8c and 4 12d were very low (8.63 11.15 M); according to Poisson statistics this should allow not more than one labeled depsipeptide to interact with each SDS micelle 130 The absorption spectra of 4 8c and 4 12d were unchanged at different SDS concentrations, but increasin g SDS concentration resulted in a gradual increase in steady state emission intensity of both 4 8c and 4 12d (Fig ure 4 3 ). Figure 4 3 Emission a nd absorption spectra (inset) in PBS buffer with different SDS concentrations for 4.8c (left) and for 4. 12d (right) This result clearly indicates that there is interaction between the 7 N,N diethylaminocoumarin labeled depsipeptides and SDS micelle. To g et a better understanding of this phenomena the emission and absorption spectra of both the compounds were recorded in PBS buffer solution (SDS free condition) and in SDS solution made in PBS buffer keeping the same 50 nM concentration in both cases. It was found that the quantum yields of 4. 8c and 4. 12d were significantly higher (around 4 times) even in very low 50 mM concentration of SDS solution ( = 0.073 and 0.093 respectively) compared to SDS free PBS buffer solution ( = 0.018 and 0.020

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86 respectively). The concentration dependent enhancement of fluorescence by SDS can be explained by the di usion of the 7 N,N diethylaminocoumarin labeled depsipeptides to the micellar Stern layer. The hydrophobic depsipeptide chains of 4. 8c and 4. 12d make them less soluble in a polar solvent such as water, thereb y; preferring the hydrophobic inner core of the micelle. The microenvironment around the 7 N,N die th ylaminocoumarin moiety however, is polar and it remains bound to the Stern layer of the SDS micelle 131,132 (Fig ure 4 4A ). This hypothesis was further suppor ted for both compounds 4. 8c and 4. 12d by a blue shift ( = 15 nm) of the emission maxima in SDS solution compared to PBS buffer solution (Fig ure 4 4B ). Figure 4 4 Proof of interaction of coumarin A) b inding of coumarin moiety to the Stern layer B) Blue shift in the emission spectra 4. 8c and 4. 12d in buffer and SDS solution Since TICT requires twisting of the donor diethylamino group (Scheme 4 5), an organized SDS Stern layer will restrict this twisting motion and retard TICT. 153 Conversion of ICT to non emissive TICT state was restricted by simultaneous decreased polarity and increased confinement at the Stern layer of the SDS micelle resulting in increased emission intensity with increasing SDS concentration. This significant change of the fluor e scence property in the SDS micelle could make these A. B.

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87 compounds useful in monitor ing reaction dynamics of a peptide based drug inside a biological system. 4.3 Conclusion In this chapter we have documented the synthesis and photophy sical studies of nov el coumarin labeled depsipeptides. The present study is complements that on the dye labeled natural peptides. Examination of the function and application of labeled peptidomimetics or depsipeptides should now be facilitated. Variations of quantum yields in different solvents are reported and rationalized in terms of ICT TICT excited states. 7 Methoxycoumarin labeled depsipeptides ar e efficient as probes since they exhibit high quantum yield s Moreover 7 diethylaminocoumarin labeled depsipeptides, with their unique TICT state in polar protic solvents, are highly sensitive to solvent polarity, H bonding ability and organized nature of the solvent medium such as an SDS micelle. Thus 7 diethylaminocoumarin labeled depsipeptides may be good candidates for real ti me monitoring of physiological processes. Their drastic change in fluorescence properties due to binding with the Stern layer of SDS micelle suggests potential utility in investigating reaction dynamics in biological membrane interfaces as well as for the monitoring of drug delivery. 4.4 Experimental Section Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. NMR spectra were recorded in CDCl 3 and DMSO d 6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. Elemental analyses were performed on a Carlo Erba EA 1108 Elemental Analyzer. Mass spectrometry was done on Agilent 6210 TOF MS with electro spray ionization (ESI). CH 2 Cl 2 was dried and distilled over CaH 2 whereas THF wa s used after distillation

PAGE 88

88 over Na benzophenone. Unprotected amino acids L p he, L m et and N (protected) aminoacid Boc Gly OH 4. 1 were purchased from Sigma and were used without further purification. Boc Gly Bt 4. 2 h ydroxycarboxylic acids 4. 3 Boc protected depsidipeptide 4. 4c and coumarinoylbenzotriazoles 4. 7a c were prepared by previously reported method s 112 114 Unprotected depsidipeptides 4. 5a b and depsitripeptides 4. 11a c were characterized by 1 H, 13 C NMR and used for the coupling st ep without further purification Absorption spectra were recorded on a Lambda 25 (Perkin Elmer) and Fluorescence spectra were recorded on FluoroMax 3 JobinYuon Horiba Spectrofluoremeter at 23 o C. ected emission spectra of standard sample with solution of coumarin 30 in acetonitrile or stilbene in methanol. The concentration of the standard was adjusted to give the same absorbance, which is around 0.1 as the sample at the excitation wavelength. 4.4. 1 General Preparation of Unprotected Depsidipeptides 5a b 4 Dimethylaminopyridine (1.2 mmol) was added to a stirred solution of Boc Gly Bt 4. 2 (1.0 mmol) and hydroxycarboxylic acid 4. 3a b (1.2 mmol) in dry THF (5.0 mL) at 4 o C. The reaction mixture was stirred for 4 h at room temperature until the reaction was complete by TLC [EtOAc hexanes (1:2)]. The solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (10.0 mL), washed with saturated citric acid solution (3 x 5 mL) and brine (5 mL) and dried over MgSO 4 The solvent was evaporated under reduced pressure to yield the crude product as oil. Without isolation, Boc deprotection was conducted by 4N HCl solution in dry dioxane (5.0 mL) for 2 h. Boc protected depsidipeptides 4. 4 a (0.31 g, 1.0 mmol for 4. 4a ) or 4. 4b (0.27 g, 1.0 mmol for 4. 4b ) was dissolved in dry dioxane (2.0 mL) and cooled to 0 o C.

PAGE 89

89 Then dry 4N HCl in dioxane (3.0 mL) was added into the solution through a syringe for 5 minutes at 0 o C. The mixture was stirred for another 2 h at 0 o C. The solvent was evaporated and the precipitate was washed with dry diethyl ether to yield unprotected depsidipeptides 4. 5a b as hydrochloride salts. (S) 2 (1 carboxy 2 phenylethoxy) 2 oxoethanaminium chloride ( 4. 5a). White microcrystals (54%), mp 150 152 o C; D 23 = 29.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 3.08 (dd, J = 14.4, 8.1 Hz, 1H), 3.19 (dd, J = 14.2, 4.2 Hz, 1H), 3.75 3.88 (m, 2H), 5.23 5.27 (m, 1H), 7.19 7.35 (m, 5H), 8.54 (br s, 3H); 13 C NMR (DMSO d 6 ) 36.3, 73.9 126.9, 128.4, 129.4, 136.1, 167.3, 169.7. (S) 2 (1 carboxy 2 methylpropoxy) 2 oxoethanaminium chloride ( 4. 5b). White microcrystals (79%), mp 165 168 o C; D 23 = 35.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 0.91 0.98 (m, 6H), 2.14 2.24 (m, 1H), 3.78 4.00 (m, 2H), 4.83 (d, J = 3.9 Hz, 1H), 8.60 (br s, 3H); 13 C NMR (DMSO d 6 ) 17.0, 18.6, 29.5, 77.5, 167.5, 169.9. 4.4.2 General Preparation of Coumarin Labeled Depsidipeptides 4. 8a f Hydrochloride salts of Gly L ( O Phe) 4. 5a (0.04 g, 0.16 mmol, 1.2 equiv. ) or Gly L ( O Val) 4. 5b (0.03 g, 0.16 mmol) and TEA (0.04 g, 0.32 mmol, 2.0 equiv. ) were dissolved in minimum amount of cold water (2 mL). Acetonitrile (6 mL) was added to this solution and cooled to 10 o C. A sol ution of N acylcoumarinoyl Bt 4. 7a c (0.04 0.06 g, 1.0 equiv. ) was added and stirred for 1 h at 25 o C. The reaction mixture was monitored with TLC [EtOAc Hexanes (1:2)]. After completion of reaction, solvent was evaporated. 4N HCl solution (5 mL) was added drop wise just to acidify the reaction mixture. The precipitated was filtered and washed with 1N HCl ( 5 mL) and water (5 mL)

PAGE 90

90 to afford desired N c oumarinoyl labeled depsidipeptides 4. 8a f (Note: for 4. 8c and 4. 8f acidification was done carefully just to neutralize the solution). (S) 2 (2 (2 oxo 2H chromene 3 carboxamido)acetoxy) 3 phenylpropanoic acid ( 4. 8a). White microcrystals (86%), mp 185 187 o C; D 23 = 17.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 3.07 (dd, J = 14.6, 8.0 Hz, 1H), 3.14 (dd, J = 14.6, 4.7 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H), 5.14 (dd, J = 7.7, 4.5 Hz, 1H), 7.14 7.30 (m, 5H), 7.45 (t, J = 7.3 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.77 (t, J = 7.9 Hz, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H), 8.89 (s, 1H), 9.07 (t, J = 5.6 Hz, 1H), 13.22 (br s, 1H); 13 C NMR (DMSO d 6 ) 36.5, 41.3, 73.1, 116.2, 118.1, 118.4, 125.3, 126.7, 128.2, 129.4, 130.5, 134.4, 136.2, 148.3, 154.0, 160.3, 161.4, 169.0, 170.2; Anal. Calcd for C 21 H 17 NO 7 : C, 63.80; H, 4.33, N, 3.54. Found: C, 63.51; H, 4.50; N, 3.51. (S) 2 (2 (7 methoxy 2 oxo 2H chromene 3 carboxamido) acetoxy) 3 phenylpropanoic acid ( 4. 8b). White microcrystals (89%), mp 184 187 o C; D 23 = 20.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 3.00 3.1 8 (m, 2H), 3.91 (s, 3H), 4.16 (d, J = 5.7 Hz, 2H), 5.13 (dd, J = 7.7, 4.7 Hz, 1H), 7.02 7.14 (m, 2H), 7.15 7.29 (m, 5H), 7.93 (d, J = 8.7 Hz, 1H), 8.84 (s, 1H), 9.01 (t, J = 5.4 Hz, 1H), 13.25 (br s, 1H); 13 C NMR (DMSO d 6 ) 36.4, 41.2, 56.3, 73.1, 100.3, 112.1, 113.8, 114.0, 126.7, 128.2, 129.4, 131.8, 136.2, 148.5, 156.4, 160.7, 161.7, 164.7, 169.1, 170.2 Anal. Calcd for C 22 H 19 NO 8 : C, 62.12; H, 4.50, N, 3.29. Found: C, 61.97; H, 4.40; N, 3.24. (S) 2 (2 (7 (diethylamino) 2 o xo 2H chromene 3 carbox amido)acetoxy) 3 phenylpropanoic acid ( 4. 8c). Yellow microcrystals (86%), mp 191 193 o C; D 23 = 39.0 (c 1.0, CH 3 OH); 1 H NMR ( DMSO d 6 ) 1.14 (t, J = 6.9 Hz, 6H), 3.00 3.17 (m, 2H),3.48 (q, J = 6.9 Hz, 4H), 4.14 (d, J = 5.7 Hz, 2H ), 5.12 (dd, J = 7.4, 4.5 Hz, 1H), 6.63 (s, 1H),

PAGE 91

91 6.81 (d, J = 8.7 Hz, 1H), 7.17 7.26 (m, 5H), 7.69 (d, J = 9.0 Hz, 1H), 8.65 (s, 1H), 8.98 (t, J = 5.6 Hz, 1H), 13.22 (br s, 1H); 13 C NMR (DMSO d 6 ) 12.3, 36.4, 41.0, 44.4, 73.0, 95.8, 107.6, 108.5, 110.2, 126.7, 128.2, 129.4, 131.8, 136.2, 148.2, 152.6, 157.4, 161.6, 162.5, 169.3, 170.2; Anal. Calcd for C 25 H 26 N 2 O 7 : C, 64.37; H, 5.62, N, 6.01. Found: C, 64.21; H, 5.50; N, 6.08. (S) 3 methyl 2 (2 (2 oxo 2H chromene 3 carboxamido) acetoxy)butanoic acid ( 4. 8d). White microcrystals (69%), mp 137 140 o C; D 23 = 26.0 (c 1.0, CH 3 OH); 1 H NMR (CDCl 3 ) 1.03 (d, J = 6.6 Hz, 3H), 1.06 (d, J = 8.1 Hz, 3H), 2.26 2.40 (m, 1H), 4.28 4.46 (m, 2H), 5.04 (d, J = 3.9 Hz, 1H), 7.36 7.44 (m, 2H), 7.64 7.74 (m, 2H), 8.95 (s, 1H ), 9.35 (t, J = 6.0 Hz, 1H); 13 C NMR (CDCl 3 ) 17.2, 19.0, 30.2, 42.0, 77.4, 116.9, 117.8, 118.7, 125.6, 130.3, 134.6, 149.4, 154.8, 161.4, 162.5, 169.1, 173.1; Anal. Calcd for C 17 H 17 NO 7 : C, 58.79; H, 4.93, N, 4.03. Found: C, 58.53; H, 4.87; N, 4.07. (S) 2 (2 (7 methoxy 2 oxo 2H chromene 3 carboxamido) acetoxy) 3 methylbutanoic acid ( 4. 8e). White microcrystals (90%), mp 182 185 o C; D 23 = 24.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 0.91 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 2.11 2.22 (m, 1H),3. 90 (s, 3H), 4.22 (d, J = 5.7 Hz, 2H),4.76 (d, J = 4.2 Hz, 1H), 7.02 7.07 (m, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 8.6, 2.9 Hz, 1H), 8.86 (d, J = 2.7 Hz, 1H), 9.06 (t, J = 5.6 Hz, 1H), 13.11 (br s, 1H); 13 C NMR (DMSO d 6 ) 16.9, 18.6, 29.4, 41.2, 56 .3, 76.7, 100.3, 112.1, 113.7, 114.0, 131.7, 148.5, 156.3, 160.7, 161.8, 164.7, 169.3; 170.3, Anal. Calcd for C 18 H 19 NO 8 : C, 57.29; H, 5.08, N, 3.71. Found: C, 57.20; H, 5.08; N, 3.63. (S) 2 (2 (7 (diethylamino) 2 oxo 2H chromene 3 carbox amido)acetoxy ) 3 methylbutanoic acid ( 4. 8f). Yellow microcrystals (92%), mp 174 176 o C; D 23 = 19.0

PAGE 92

92 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 0.91 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 1.14 (t, J = 7.1 Hz, 6H), 2.12 2.19 (m, 1H), 3.48 (q, J = 7.0 Hz, 4H), 4.20 (d, J = 6.0 Hz, 2H), 4.75 (d, J = 4.2 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.79 6.83 (m, 1H), 7.69 (d, J = 9.0 Hz, 1H), 8.67 (s, 1H), 9.02 (t, J = 5.7 Hz, 1H), 13.07 (br s, 1H); 13 C NMR (DMSO d 6 ) 12.3,16.9, 18.5, 29.4, 41.1, 44.4, 76.6, 95.9, 107.5, 107.6, 110 .2, 131.7, 148.1, 152.6, 157.3, 162.6, 169.5, 170.3, Anal. Calcd for C 21 H 26 N 2 O 7 : C, 60.28; H, 6.26, N, 6.69. Found: C, 59.73; H, 6.12, N, 7.45 4.4.3 General Preparation of Unprotected Depsidipeptides 4. 11a c A solution of EDCI (1.10 g, 5.80 mmol, 1.0 equiv. ) was added to a stirr ing solution of 4. 4a and 4. 4c (1.0 equiv. ) and benzotriazole (0.69 g, 5.80 mmol) in CH 2 Cl 2 (50 mL) at 0 o C. The reaction mixture was stirred for 16 h at rt Then reaction was quenched with water. The reaction mixture was washed with 20% citric acid solution (3 x 10 mL), saturated Na 2 CO 3 (3 x 15 mL), water (2 x 10 mL) and brine (15 mL), dried over MgSO 4 and solvent was evaporated under reduced pressure to yield the desired product 4. 9a b L P he (0.50 g, 3.0 mmol, 1.5 equiv. ) or L met (0.22 g, 1.5 mmol) and TEA (300 mg, 3.0 mmol, 1.5 equiv. ) were dissolved in minimum amount of cold water (5 mL). Acetonitrile (10 mL) was added to the solution and cooled to 10 o C. A solution of 4. 9a b (1.0 equiv. 2.0 mmol) in acetonitrile (5 mL) was added and stirred for 2 h at 25 o C. The reaction mixture was monitored with TLC [EtOAc Hexanes (1:2)]. After the completion of the reaction, solvent was evaporated. The residue was dissolved in CH 2 Cl 2 (30 mL) and washed with saturated citric acid solution (4 x 10 mL), water (10 mL) and brine (10 mL), dried over MgSO 4 and evaporated to give 4. 10a c Crude 4. 10a c (1.0 mmol) was dissolved in dry dioxane (2 mL) and cooled to 0 o C. Then dry

PAGE 93

93 4N HCl in dioxane ( 3 mL) was added into the solution through a syringe for 5 min at 0 o C and stirred for another 2 h at 0 o C. The solvent was evaporated and the precipitate was washed with dry diethyl ether to give the desired product 4. 11a c as white microcrystals. (S) 2 ((S) 2 (2 aminoacetoxy) 4 methylpentanamido) 3 phenyl propanoic acid hydrochloride ( 4. 11a). White microcrystals (42%), mp 175 178 o C; D 23 = 43.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 0.82 (d, J = 5.1 Hz, 3H) 0.84 (d, J = 4.5 Hz, 3H) 1.40 1.64(m, 3H), 2.94 (dd, J = 13.8, 9.0, 1H), 3.07 (dd, J =13.7, 5.0 Hz, 1H), 3.70 3.90 (m, 2H), 4.40 4.56 (m, 1H), 5.04 (dd, J = 8.7, 3.5 Hz, 1H), 7.18 7.36 (m, 5H), 8.34 8.46 (br s, 3H), 8.52 (d, J = 7.2 Hz, 1H); 13 C NMR (DMSO d 6 ) 21.5, 23 .1, 23 .8, 24.5 36.5, 53.2, 73.0, 126.5, 128.2, 129.2, 137.5, 167.1, 168.9, 172.5. (S ) 2 ((S) 2 (2 aminoacetoxy) 4 methylpentanamido) 4 (methylthio)butanoic acid hydrochloride ( 4. 11b). White microcrystals (30%), mp 104 107 o C; D 23 = 32.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 0.84 (d, J = 5.7 Hz, 3H); 0.86 (d, J = 6.3 Hz, 3H); 1.44 1.74 (m, 3H), 1.80 2.00 (m, 5H), 2.34 2.45 (m, 2H), 3.70 3.93 (m, 2H), 4.23 4.32 (m, 1H), 5.01 (dd, J = 9.5, 4.1 Hz, 1H), 8.40 8.62 (m, 4H); 13 C NMR (DMSO d 6 ) 14.7, 21.6, 23.2, 23.8, 29.8, 30.7, 50.8, 73.1, 167.2, 169.1, 172.9. 2 (((S) 1 (((S) 1 carboxy 3 (m ethylthio)propyl)amino) 1 oxo 3 phenylpropan 2 yl)oxy) 2 oxoethanaminium chloride ( 4. 11c). White microcrystals (35%), mp 180 182 o C; D 23 = 21.0 (c 1.0, CH 3 OH); 1 H NMR (DMSO d 6 ) 1.80 2.02 (m, 2H), 2.04 (s, 3H), 2.32 2.48 (m, 2H), 2.99 (dd, J = 14.3, 8.9 Hz, 1H), 3.14 (dd, J = 14.4, 3.6 Hz, 1H), 3.72 (d, J = 17.3, 1H), 3.89 (d, J = 18.2, 1H), 4.31 4.39 (m, 1H), 5.29

PAGE 94

94 (dd, J = 8.1, 3.6 Hz, 1H), 7.20 7.36 (m, 5H), 8.65 (d, J = 7.8 Hz, 1H); 13 C NMR (DMSO d 6 ) 14.6, 29.7, 30.6, 37.1, 50.9, 75.0, 126. 7, 128.3, 129.4, 136.3, 167.1, 168.2, 172.9. 4.4.4 General Preparation of N Coumarinoyl Labeled Depsitripeptides 4. 12a d Hydrochloride salts of 4. 11a c (0.050 0.055 g, 0.11 mmol, 1.1 equiv. ) and TEA (0.04 g, 0.22 mmol, 2.0 equiv. ) were dissolved in the minimum volume of cold water (1 mL). Acetonitrile (4 mL) was added to the solution and cooled to 10 o C. A solution of N coumarinoyl Bt 4. 7a c (0.04 0.05 g, 1.0 equiv. ) was added and stirred for 1 h at 20 o C. The rea ction mixture was monitored by TLC [EtOA c Hexanes (1:2)]. After completion of reaction, solvent was evaporated. 4N HCl solution (5 mL) was added drop wise just to acidify the reaction mixture. The precipitated was fi ltered and washed with 1N HCl ( 5 mL) and water (5 mL) to afford desired N c oumar inoyl labeled depsidipeptides 4. 12a d (Note for 4. 12d acidification was done carefully to just neutralize the solution, at the neutralization point a thick precipitate was formed and it was filtered off). The crude compound was recrystallized from EtOAc hexanes. (S) 2 ((S) 2 (2 (7 methoxy 2 oxo 2H chromene 3 carboxamido)acetoxy) 4 methylpentanamido) 3 phenylpropanoic acid ( 4. 12a). White microcrystals ( 88%), mp 160 163 o C; D 23 = 22.0 (c 1.0, CH 3 OH); 1 H NMR (CDCl 3 ) 0.86 (d, J = 6.3 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H), 1.54 1.66 (m, 3H), 3.16 (dd, J = 13.8, 8.4 Hz, 1H), 3.32 (dd, J = 14.3, 5.3 Hz, 1H), 3.92 (s, 3H), 4.16 4.20 (m, 2H), 4.76 4.88 (m, 1H), 5.27 (t, J = 6.5 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 8.7, 2.4 Hz, 1H), 7.14 7.30 (m, 6H), 7.59 (d, J = 8.7 Hz, 1H), 8.75 (s, 1H), 9.31 (t, J = 5.9 Hz, 1H); 13 C NMR (CDCl 3 ) 21.9, 23.3, 24.7, 37.3, 40.6, 42.5, 53.7, 56.3, 73.8, 100.5, 112.4, 113.6, 114.5, 127.2, 128.7, 129.5,

PAGE 95

95 131.6, 136.4, 149.4, 157.0, 161.8, 163.7, 165.5, 168.5, 170.8, 173.4; HRMS, [M+Na] + : Calcd for [C 28 H 30 N 2 O 9 Na] + : 561.1844. Found: 561.1847. (S) 2 ((S) 2 (2 (7 methoxy 2 oxo 2H chromene 3 carbox amido)acetoxy) 4 methylpentanamido) 4 (methylthio) butanoic acid ( 4. 12b). White microcrystals (92%), mp 182 184 o C; D 23 = 17.0 (c 1.0, CH 3 OH); 1 H NMR (CDCl 3 ) 0.92 (d, J = 6 Hz, 3H), 0.94 (d, J = 6.3 Hz, 3H), 1.65 1.82 (m, 3H), 2.10 (s, 3H), 2.10 2.31(m, 2H), 2.55 2.61 (m, 2H), 3.92 (s, 3H), 4.17 4.35 (m, 2H), 4.60 4.69 (m, 1H), 5.35 (t, J = 6.5 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 8.7, 2.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 8.83 (s, 1H), 9.38 (t, J = 5.6 Hz, 1H); 13 C NMR (CDCl 3 ) 15.4, 21.6, 23.2, 24.6, 30.2, 30.8, 40.4, 42.4, 51.8, 56.1, 73.5, 100.3, 112.2, 113.3, 114.3, 131.5, 149.3, 156.9, 161.7, 163.8, 165.4, 168.3, 171.1, 173.7; HRMS, [M+Na] + : Calcd for [C 28 H 30 N 2 O 9 Na] + : 545.1564. Found:545.1562. (S) 2 ((S) 2 (2 (7 methoxy 2 oxo 2H chromene 3 carboxamido)acetoxy) 3 phenylpropanamido) 4 (methylthio)butanoic acid ( 4. 12c). White microcrystals (83%), mp 170 172 o C; D 23 = 43.0 (c 1.0, CH 3 OH); 1 H NMR (CDCl 3 ) 2.03 (s, 3H), 2.02 2.25 (m, 4H), 3.14 (dd, J = 14.7, 4.5 Hz, 1H), 3.24 (dd, J = 14.3, 5.2 Hz, 1H), 3.95 (s, 3H), 4.09 (dd, J = 17.4, 5.7 Hz, 1H), 4.32 (dd, J = 17.4, 5.7 Hz, 1H), 4.52 4.62 (m, 1H), 5.55 (t, J = 4.7 Hz, 1H), 6.80 7.14 (m, 7H), 7.39 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 8.69 (s, 1H), 9.39 (t, J = 5.4 Hz, 1H); 13 C NMR (CDCl 3 ) 15.4, 30.0, 30.6, 37.4, 42.7, 52.0, 56.4, 74.6, 100.6, 112.5, 113.5, 114.6, 127.0, 128.4, 130.0, 131.8, 135.5, 149.5, 157.1, 162.0, 164.3, 165.7, 167.7, 169.9, 173.7; Anal. Calcd for C 27 H 28 N 2 O 9 S: C, 58.26; H, 5.07, N, 5.03. Found: C, 57.97; H, 5.20; N, 4.60.

PAGE 96

96 (S) 2 ((S) 2 (2 (7 (diethylamino) 2 oxo 2H chromene 3 carboxamido)acetoxy) 4 methylpentanamido) 3 phenylpropanoic acid ( 4. 12d). Yellow microcrystals (90%), mp 162 165 o C; D 23 = 25.0 (c 1.0, CH 3 OH); 1 H NMR (CDCl 3 ) 0.83 (s, 3H), 0.91 (s, 3H), 1.23(t, J = 6.9, 6H), 1.57 (s, 3H), 3.17(dd, J =14.2, 8.4, 1H), 3.33 (dd, J =14.2 ,4.5,1H) 3.44 (d, J=6.9 4H), 4.11 (d, J = 4.8 Hz, 2H), 4 .72 4.85 (m, 1H), 5.25 (br s, 1H), 6.46 (s, 1H), 6.45 (d, J = 8.4 Hz, 1H), 7.10 7.28 (m, 6H), 7.35 7.45 (m, 2H), 8.56 (s, 1H), 9.37 (s, 1H); 13 C NMR (CDCl 3 ) 12.7, 21.9, 23.3, 24.7, 37.3, 40.5, 42.7, 45.4, 54.3, 73.6, 96.7, 108.5, 110.5, 127.1, 128.7, 12 9.5, 131.9, 136.6, 149.0, 153.3, 158.1, 162.8, 165.2, 168.7, 171.3, 172.8, HRMS, [M+Na] + : Calcd for: [C 31 H 37 N 2 O 8 Na] + : 602.2473. Found: 602.2467.

PAGE 97

97 CHAPTER 5 BENZOTRIAZOLE MEDIATED SYNTHESIS OF AZA PEPTIDES: EN ROUTE TO AN AZA LEUENKEPHALIN ANALOGUE 5.1 Introduction Azapeptides are peptidomimetics in which the CH groups of one or more amino acid residues are replaced by a nitrogen atom (Fig ure 5 1A, B). This decreases the electrophilicity of the (NR)CO carbonyl group and changes the geometry at the positions from tetrahedral to trigonal planar, hence eliminating chirality. Relative to the natural peptides, azapeptides occupy a limited conformational space with dihedral 90 30 and = 0 30 or 180 30) close to those in the 78, = 149) and other types of turns (Fig ure 5 1C). 1 34 137 The effect of the geometry of an aza amino acid is similar to that of a turn, in changing the chemical and the biological properties of the parent peptide. Azapeptides may exhibit better interactions with protein receptors and enhanced stability to enzymatic and chemical degradation. 138,139 Azapeptides are therefore leads for the generation of receptor ligands, enzyme inhibitors and clinically appro ved drugs. 140 Azapeptides possessing electrophilic moieties also act as inhibitors of cysteine proteases. 141 1 43 Figure 5 1 Comparison of azadipeptide and native dipeptide Reproduced with permission from The Journal of Organic Chemistry 2013 78 3541 3552 Copyright 2013 American Chemical Society

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98 Azapeptides can be synthesized both in solution an d on solid phase by combining hydrazine chemistry and conventional peptide synthesis. 1 44 The nitrogen atoms of hydrazine need to be differentiated to produce N alkyl N Pg hydrazines 5. 1 ; phosgene (or an equiv alent) introduces the carbonyl group into the s keleton by reacting with the hydrazine derivative 5. 1 giving 5. 2 which then reacts with dipeptide ester 5. 3 (Scheme 5 1A). Alternatively, the phosgene reacts with the peptide N terminus of dipeptide 5. 3 (Scheme 5 1B). 1 45 14 7 Route B can produce a hydantoin by product 5. 7 by intramolecular nucleophilic attack of the secondary amide nitrogen of the preceding amino acid residue on the activated isocyanate 5. 6 Lubell et al coupled N Boc azadi and tripeptide segments to the am ine terminus of a growing peptide chain to circumvent formation of the hydantoin byproduct. 1 46 Scheme 5 1 The construction of an azapeptides by A. hydrazine pathway and B. peptide N terminus pathway

PAGE 99

99 N Acylbenzotriazoles are advantageous for N O C and S acylation, especially when the corresponding acid chlorides are unstable or difficult to prepare and we have demonstrated the use of benzotriazolides for the synthesis of peptidomimetics such as am inoxypeptides and depsipeptides. 112,148, 1 49 In the present work stable, crystalline and easy to handle N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e were prepared and their synthetic utility demonstrated by the synthesis of N Pg azatripeptides 5. 24a h and N Pg azatetrapeptides 5. 25a b and hybrid azapeptides containing oxyamide 5. 26a or depsi bonds 5. 26b and 5. 28 In addition, we developed a new route towards N Pg azatripeptides 5. 33a ,b and 5. 35a b containing a natural amino acid at their N terminus. The novel pathway enabled the solu tion phase synthesis of aza Leu enkephalin analogue 5. 40 5.2 Results and Discussion 5.2.1 Preparation of N Alkyl N ( Pg ) Hydrazines 5.10 c Scheme 5 2 Preparation of N Alkyl N (Pg) hydrazines Reaction of Boc NHNH 2 5. 8a and Fmoc NHNH 2 5. 8b with the appropriate aldehyde or ketone, followed by reduction with NaCNBH 3 furnished N alkyl t butyl carbazates 5. 10a b and N alkyl fluorenyl 9 ylmethyl carbazates 5. 10c (Scheme 5 2 ). Selective protection of N methyl hydrazine with (Boc) 2 O 5. 11 afforded N Boc N methyl hydrazine 5. 12 which was coupled with benzyl chloroformate and fluorenyl 9 ylmethyl

PAGE 100

100 chloroformate to give N Boc N methyl N Cbz hydrazine 5. 13a and N Boc N methyl N Fmoc hydrazine 5. 13b Subsequent Boc deprotection of 5. 13a b afforded N methyl benzyl carbazates 5. 14a and N methyl fluorenyl 9 ylmethyl carbazates 5. 14b as their HCl salts in 70 73% overall yield (Scheme 5 3 ) Reaction of Cbz NHNH 2 5. 13c and methyl 2 bromoacetate in dry DMF in the presence of DIPEA gave benzyl 2 (2 methoxy 2 oxoethyl)hydrazine 1 carboxylate 5. 14c (Scheme 5 3 ). Scheme 5 3 Preparation of alkyl Pg carbazates 5. 14a 5. 14b and 5. 14c 5.2.2 Construction of Protected A zadipeptides 5.18a i The activation of N alkyl t butyl carbazates into N (Boc)aza amino acid building blocks using p nitrophenyl chloroformate, 1 50 bis(2,4 dinitrophenyl) carbonate and carbonyldiimidazole (CDI) 142 gave nitrophenylcarbazates and imidazolides which required long reaction times and high temperatures and gave aza peptides in poor yields. Phosgene and triphosgene activates N alkyl t butyl carbazates and N substituted fluorenylmethyl carbazates efficie ntly, but these reagents are extremely toxic. Although several literature papers describe synthetic protocols, a general, high yielding and efficient synthetic method has yet to be reported. After examination of the aza peptide synthetic literature, 138 we chose to synthesize azadipeptides via activation

PAGE 101

101 of the amino acid ester hydrochloride salts 5. 15a e by carbonyldiimidazole (CDI) in the presence of 2.5 equiv. of DIPEA ( Hnig's base) in dry DCM to afford the active carbamates 5. 16a e Stirring 5. 16a e wit h N alkyl N Pg hydrazines 5. 10a c or 5. 14a,b at 20 o C for 16 h in dry THF containing one equiv. of DIPEA provided protected azadipeptides 5. 18a h (Scheme 5 4 ). DIPEA establishes an equilibrium between the active carbamates 16 and the isocyanate intermediates 5. 17 which react quickly with N alkyl N Pg hydrazines 5. 10a c or 5. 14a b to afford 5. 18a h The method tolerated N alkyl N Fmoc hydrazines 5. 10c and 5. 14b without any sign of N Fmoc deprotection. A simple extractive wo rk up using 2 N HCl gave N Pg azadipeptide esters 5. 18a h (Table 5 1) with satisfactory 1 H NMR and 13 C NMR and were used in the next step without full characterization. In an attempt to show general applicability of the protocol, we chose to synthesize an azadipeptide containing a polar side chain. Compound 5. 14c was subjected to react with 5. 16b which gave the protected azadipeptide 5. 18i with the polar side chain (Cbz AzaAsp Val O t Bu). It proves the methodology is general and by choosing appropriate coupl ing partners and protection/deprotection methods when reacting with 5. 16a e other azapeptides with the polar side chains can be achieved. Scheme 5 4 Preparation of protected azadipeptides 5. 18a i

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102 Table 5 1 Preparation of protected azadipeptides 5. 18a i Entry Pg R 2 R R 1 5. 18 yield a (%) a Cbz CH 3 CH 2 Ph CH 3 5. 18a 90 b Cbz CH 3 CH(CH 3 ) 2 C(CH 3 ) 3 5. 18b 82 c Boc CH(CH 3 ) 2 CH 2 CH(CH 3 ) 2 CH 3 5. 18c 89 d Boc CH(CH 3 ) 2 CH 2 CH 2 SCH 3 CH 3 5. 18d 90 e Boc CH 2 Ph CH 2 CH(CH 3 ) 2 CH 3 5. 18e 95 f Boc H CH 2 Ph CH 3 5. 18f 80 g Fmoc CH 3 CH(CH 3 ) 2 C(CH 3 ) 3 5. 18g 85 h Fmoc CH(CH 3 ) 2 CH 2 CH(CH 3 ) 2 C(CH 3 ) 3 5. 18h 78 i Cbz CH 2 CO 2 CH 3 CH(CH 3 ) 2 C(CH 3 ) 3 5. 18i 92 a Isolated yield over two steps 5.2.3 Preparation of N ( N Pg Azadipeptidoyl )Benzotriazoles 5.20a e N Pg Azadipeptides 5. 19a h were prepared either by hydrolysis of N Pg azadipeptides methyl esters 5. 18a, 5. 18c f using lithium hydroxide in methanol/water mixture (10:1, v/v) or cleavage of the t butyl group of 5. 18b 5. 18g,h with trifluoroacetic acid in DCM (1:1 v/v) N (N Pg Azadipeptidoyl)benzotriazoles 20a e were synthesized in 81 92% yields by treatment of N Pg azadipeptides 5. 19a d, 5. 19h with 3.0 equiv. of 1 H benzotriazole, 1.0 equiv. of thionyl chloride and 2.0 equiv. DIPEA i n DCM at 30 o C (Scheme 5 5, Table 5 2). The presence of DIPEA neutralizes the HCl liberated from the reaction of benzotriazole and thionyl chloride thus preventing N Boc deprotection. N (N Pg Azadipeptidoyl)benzotriazoles 5. 20a e were characterized by 1 H N MR, 13 C NMR and elemental analysis.

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103 Scheme 5 5 Preparation of N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e Table 5 2 Preparation of N Pg azadipeptide OH 5.19a h and N (N Pg azadipeptidoyl)benzotriazoles 5.20a h a i solated yield # Azadipeptide OH, 5. 19a h yield a (%) A zadipept idoyl Bt 5. 20a e yield a (%) A Cbz AzaAla Phe OH 5. 19a 90 Cbz AzaAla Phe Bt 5. 20a 87 B Cbz AzaAla Val OH 5. 19b 83 Cbz AzaAla Val Bt 5. 20b 81 C Boc AzaVal Leu OH 5. 19c 79 Boc AzaVal Leu Bt 5. 20c 90 D Boc AzaVal Met OH 5. 19d 86 Boc AzaVal Met Bt 5. 20d 85 E Boc AzaPhe Leu OH 5. 19e 89 (Not attempted) -F Boc AzaGly Phe OH 5. 19f 76 (Not attempted) -G Fmoc AzaAla Val OH 5. 19g 69 (Not attempted) -H Fmoc AzaVal Leu OH 5. 19h 80 Fmoc AzaVal Leu Bt 5. 20e 92

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104 5.2.4 Coupling of 5.20a e with Various Nu cleophile s to Form Longer Azapeptides Dipeptides are useful building blocks for longer peptide analogues but the functions and applications of dipeptides have been previously neglected, probably because of the lack of an efficient protocol for the synthesis of dipeptides. 1 16 However we now find that N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e may be used to obtain longer azapeptide sequences by coupling with (i) amino acids 5. 21a e (ii) dipeptides 5. 22a b (iii) aminoxyacetic acid 5. 23a and (iv) depsidipeptide 5. 23b in aqueous acetonitrile triethyl amine (TEA) at 20 o C for 0.5 2 h. These coupling reactions afford respectively: (i) N Pg azatripeptides 5. 24a g (ii) N Pg azatetrapeptides 5. 25a b (iii) hybrid N Pg azatripeptides containing the oxyamide bond 5. 26a and (iv) hybrid azatetrapeptide with an ester bond 5. 26b in 77 90% yields (Scheme 5 6, Table 5 3). The coupling of hydroxy phenylpropionic acid 5. 27 with 5. 20b in dry THF containing 2.0 equiv. of 4 dimethylaminopyridine (DMAP) gave hybrid peptide 5. 28 The target compounds were all characterized by 1 H NMR, 13 C NMR and elemental analysis. To show that no racemization occurs in the synt hetic protocol used here we conducted reactions between the active benzotriazole dipeptide species 5. 20b and amino acid cysteine (both L and D L forms). The absence of racemization in the azapeptide ( 5.24c+ ) was d educed from the 1 H NMR, where the SH proton signal showed two separated triplets split in the D,L cysteine moiety at 1.63 ppm ( J = 9.0 Hz) and 1.49 ppm ( J = 9.0 Hz), while compound 5. 24c showed a clear triplet at 1.61 ppm ( J = 9.0 Hz). Retention of chirality in the products was further confirmed by chiral HPLC analysis using a (S,S) Welk O1column (MeCN/H 2 O 50:50, flow rate 0.25 mL/min, detection at 210 nm). The diastereomer 5. 24c showed a single retention time peak in

PAGE 105

105 chiral HPLC at 10.2 min, while its corresponding diastereomeric mixture ( 5.24c+ ) showed two peaks at 9.3 and 10.1 min. In previous studies on benzotriazole based peptide coupling we also demonstrated chirality of the reaction is retained on N acylation with N acylbenzotriazoles for peptides 149 depsipeptides 1 12 and aminoxy hybrid peptides. 80 Scheme 5 6 Coupling reactions of N (N Pg azadipeptidoyl)benzotriazoles 5. 20a e Table 5 3 Preparation of azapeptides 5. 24a g 5. 25a b 5. 26a b and 5. 28 Entry 20 nucleophiles products yield a % 1 5. 20a H Cys OH 5. 21a Cbz AzaAla Phe Cys OH 5. 24a 80 2 5. 20b H Ser OH 5. 21b Cbz AzaAla Val Ser OH 5. 24b 80 3 5. 20b H Cys OH 5. 21a Cbz AzaAla Val Cys OH 5. 24c 83 5. 20b (D,L) H Cys OH 5. 21a+ Cbz AzaAla Val (D,L)Cys OH 5. 24c+ 85 4 5. 20c H Trp OH 5. 21c Boc AzaVal Leu Trp OH 5. 24d 89 5 5. 20d H Ser OH 5. 21b Boc AzaVal Met Ser OH 5. 24e 83 6 5. 20c H Asp (OBn) OH 5. 21d Boc AzaVal Leu Asp (OBn) OH 5. 24f 90 7 5. 20e H Pro OH 5. 21e Fmoc AzaVal Leu Pro OH 5. 24g 86

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106 Table 5 3 Continued Entry 20 nucleophiles products yield a % 8 5. 20a H Gly Gly OH 5. 22a Cbz AzaAla Phe Gly Gly OH 5. 25a 77 9 5. 20c H Gly Phe OH 5. 22b Boc AzaVal Leu Gly Phe OH 5. 25b 87 10 5. 20a H AO Gly OH 5. 23a Cbz AzaAla Phe AO Gly OH 5. 26a 90 11 5. 20d H Gly O Phe 5. 23b Boc AzaVal Met Gly O Phe OH 5. 26b 85 12 5. 20b HO Phe OH 5. 27 Cbz AzaAla Val O Phe OH 5. 28 87 a isolated yield 5.2.5 Preparation of Free Azadipeptides 5.29a c and Coupling with N ( N Pg Aminoacyl )Benzotriazoles The addition of a new unit to a peptide (or peptidomimetics) chain is accomplished chemically by coupling. However, chain extension of a peptidomimetic by N acylation of an aza amino acid residue is hampered by the low reactivity of the sem icarbazide nitrogen. Such coupling difficulties have previously been addressed by prolonged coupling time 151 mixed coupling reagents and double coupling. 140 1 46 In the present study, we have investigated the ability of N acylbenzotriazoles to acylate hydr ochloride or p TsOH salts of free amino azadipeptides 5. 29a c Salts 5. 29a c were prepared in 90 100% yields according to Scheme 5 7 Azadipeptide 5. 29c was conveniently acylated with N (N Pg aminoacyl)benzotriazoles 5. 30a b in acetonitrile/water (3:1 v/v) in the presence of 2.0 equiv. of Et 3 N, to give azatripeptides 5. 31a b However, azapeptides 5. 29a b were recovered unreacted upon treatment with 30a b under the same reaction conditions, and hydrolysis of 5. 30a was the only reaction observed when the coupling between 5. 29a and 5. 30a was attempted under microwave irradiation (1 h, 50 W, 70 o C) (Scheme 5 7).

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107 Scheme 5 7 Preparation of free azapeptides 5. 29a c and coupling with N (N Pg aminoacyl)benzotriazoles to prepare 5. 31a,b 5.2.6 Alternative Facile Route to the Synthesis of N Pg Azatripeptides 5.33a,b and 5.35a,b The low reactivity of 5. 29a b is due to greater stabilization of the amide electron delocalized structure by the N alkyl groups thus inductively reduc ing the electron density at the terminal amino group and making it less nucleophilic. Steric hindrance arising from substitution on adjacent nitrogen could also play a part. To bypass the low coupling rates of the aza amino ac id residue, we developed a novel route to azatripeptides. Fi rst we converted N (N Pg aminoacyl)benzotriazoles 5. 30a c e to the corresponding hydrazides 5. 32a d Coupling active carbamates 16b d with hydrazides 5. 32c d then furnished azatripeptides 5. 33a b containing an azaglycine residue. Alternatively, hydrazides 5. 32a b were benzylated by reductive amination to give N benzyl N (N Pg glycyl)hydrazines 5. 34a b which upon coupling with 5. 16b,d gave azatripeptides 5. 35a b (Scheme 5 8).

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108 Scheme 5 8 Preparation of azatripeptide 5.33 and 5.35 from aminoacyl benzotriazol ides 5.2.7 Solution Phase Synthesis of Hybrid Azapeptide 5.40; an Analogue of Leuenkephalin Leuenkephalin is an endogenous opioid peptide neurotransmitter found naturally in the brains of many animals, including humans. The amino acid sequence of Leuenkephalin is Tyr Gly Gly Phe Leu. Azapeptide mimetics of Leuenkephalin were synthesized and their binding affi nity was examined in the context of monoclonal antibody 3 E7 known to strongly bind the [Leu 5 ] enkephalin sequence. 152 Our new route to the synthesis of azatripeptides (Scheme 5 8) inserted the aza amino acid in the middle of azatripeptides, thus, enabling the synthesis of the yet unknown Aza Leuenkephalin analogue 5. 40 by segment condensation of free amino azatripeptide fragment 5. 36 and the benzotriazolide 5. 39 (Scheme 5 9 ).

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109 Scheme 5 9 Solution phase synthesis of azapeptide analog of Leuenkephalin 5.40 5.3 Conclusions In conclusion, a mild and efficient method for the preparation of N Pg azatripeptides, N Pg azatetrapeptides, hybrid N Pg azatripeptides and hybrid N Pg azatetrapeptide has been developed by reacting N (N Pg azadipeptidoyl)benzotriazoles with aminoacids, dipeptides, aminoxyacetic acid, a depsidipeptide and hydroxy phenyl propionic acid. These azatripeptides and tetrapeptides could be valuable building blocks for the synthesis of longer or cyclic aza peptides. In addition, N ( aminoacyl)benzotriazoles were easily converted to N Pg amino acid hydrazides which were used either directly or after alkylation to construct N Pg azatripeptides, thus inserting an aza amino acid in the middle of the azatripeptide unit. An aza leuenkephalin analogue was synthesized by adopting this novel protocol. The new method avoids the low coupling rates of aza amino acids and provides an excellent

PAGE 110

110 alternative to the construction of azapeptides starting from N Boc and N Fmoc hydrazides. 5.4 Experimental Section Melting points were determined on a capillary melting point apparatus equipped with a digital thermometer and are uncorrected NMR spectra were recorded in a cetone d 6 CDCl 3 and DMSO d 6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. DCM was dried and distilled over CaH 2 whereas tetrahydrofuran (THF) was used after distillation over Na benzophenone. Carbonyldiimidazole (CDI), Boc hydrazide 5. 8a (Boc) 2 O 5. 11 L amino methyl/ tert butyl ester hydrochloride 5. 15a e amino acids 5. 21a e free dipeptides H Gly Gly OH 5. 22a H Gly Phe OH 5. 22b aminoxyacetic acid 5. 23a and hydroxy phenyl propionic acid 5. 27 were purchased from chemical supply companies and used without further pu rification. Free despsipeptide H Gly O Phe.HCl 5. 23b was prepared according to the lit. method. 1 12 Fmoc NH NH 2 ( 5. 8b). Compound 5. 8b was prepared according to the lit. method. 144 White microcrystals (8.756 g, 89%); mp 170.0 172.0 o C lit. 1 44 mp 172.0 173.0 o C; 1 H NMR (300 MHz, DMSO d 6 J = 7.8 Hz, 1H), 7.70 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 6.9 Hz, 1H), 7.33 (t, J = 6.9 Hz, 1H), 4.36 4.16 (m, 3H), 4.09 (br s, 2H); 13 C NMR (75 MHz, DMSO d 6 125. 2, 120.1, 65.6, 46.7. Boc NH N=CMe 2 ( 5. 9a). Compound 5. 9a was prepared according to the lit. method. 1 46 White microcrystals (1.156 g, 89%); mp 102.0 104.0 o C; lit. 153 mp 103.0 104.0 o C; 1 H NMR (300 MHz, CDCl 3 1.98 (m, 3H), 1.83 1.76

PAGE 111

111 (m, 3H), 1.52 1.45 (m, 9H); 13 C NMR (75 MHz, CDCl 3 15.9. Boc NH N=CHPh ( 5. 9b). Compound 5. 9b was prepared according to the lit. method. 1 46 White microcrystals (2.980 g, 89%); mp 199 202 o C; lit. 154 mp 203 o C; 1 H NMR (300 MHz, DMSO d 6 7.49 (m, 2H), 7.48 7.28 (m, 3H), 1.47 (s, 9H); 13 C NMR (75 MHz, DMSO d 6 79.4, 28.1. Fmoc N H N=CMe 2 ( 5. 9c). To a solution of benzaldehyde (10.0 mmol, 1.0 equiv. ) in diethyl ether (50 mL); (9H fluoren 9 yl)methyl hydrazinecarboxylate 5. 8b (10.0 mmol, 1.0 equiv. ) and two drops of glacial acetic acid were added. The reaction mixture was heated under reflux for 2 3 h then cooled to room temperature. The white solid precipitate was collected by filtration, washed with cold diethyl ether and dried under vacuum to yie ld 5. 9c. Compound 5. 9c was characterized by 1 H, 13 C NMR and taken to the next step without further purification. White microcrystals (2.796 g, 95%); mp 147.0 149.0 o C ; 1 H NMR (300 MHz, CDCl 3 J = 7.7 Hz, 2H), 7.63 (d, J = 7.1 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 4.52 (d, J = 7.2 Hz, 2H), 4.30 (t, J = 7.1 Hz, 1H), 2.07 (d, J = 2.1 Hz, 3H), 1.84 (d, J = 3.0 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 Comp ound 5. 9c was characterized by 1 H, 13 C NMR and taken to the next step without further purification. tert Butyl 2 isopropylhydrazinecarboxylate ( 5. 10a). Compound 5. 10a was prepared according to the lit. method. 1 46 White microcrystals (0.364 g, 70%); mp 45.0 47.0 o C; lit. 1 46 mp 47.0 49.0 o C; 1 H NMR (300 MHz, CDCl 3 2.97

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112 (m, 1H), 1.43 (s, 9H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 tert Butyl 2 benzylhydrazinecar boxylate ( 5. 10b). Compound 5. 10b was prepared according to the lit. method. 1 46 C olorless oil (0.310 g, 75%); lit. 1 46 reported as low melting solid ; 1 H NMR (300 MHz, CDCl 3 7.21 (m, 5H), 6.13 (br s, 1H), 4.21 (br s, 1H), 3.99 (s, 2H), 1.46 (s, 9H); 1 3 C NMR (75 MHz, CDCl 3 128.4, 127.4, 80.5, 55.8, 28.3. (9H Fluoren 9 yl)methyl 2 isopropylhydrazinecarboxylate ( 5. 10c). Compound 5. 10c was prepared according to the lit method. 1 44 White microcrystals (0.786 g, 68%); mp 161.0 163.0 o C ; lit. 11 mp 163.0 164.0 o C ; 1 H NMR (300 MHz, DMSO d 6 s, 1H), 7.84 (d, J = 7.4 Hz, 2H), 7.65 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.27 (t, J = 7.4 Hz, 2H), 4.42 4.21 (m, 3H), 4.18 (d, J = 6.5 Hz, 1H), 3.05 2.82 (m, 1H), 0.86 (d, J = 5. 6 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 125.2, 120.1, 65.4, 49.5, 46.7, 20.6. Benzyl 2 methylhydrazinecarboxylate hydrochloride ( 5. 14a). Compound 5. 14a was prepared according to the lit. method. 155 White microcrystals (3.0 0 g, 70%); mp 174.0 177.0 o C ; lit. 155 mp 175.0 177.0 o C 1 H NMR (300 MHz, DMSO d 6 1H), 7.55 7.05 (m, 5H), 5.13 (s, 2H), 2.67 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 155.4, 136.3, 129.1, 129.0, 128.7, 67.9, 36.4. (9H Fluoren 9 yl)methyl 2 methylhydrazinecarboxylate hydrochloride ( 5. 14b). Compound 5. 14b was prepared according to the lit. method. 1 5 6 White microcrystals (0.850 g, 73%); mp 160.0 161.0 o C ; lit. 1 5 6 mp 160.0 o C 1 H NMR (300 MHz, DMSO d 6 10.79 (br s, 1H), 7.90 (d, J = 7.4 Hz, 2 H), 7.72 (d, J = 7.4 Hz, 2H), 7.43 (t, J = 7.5 Hz,

PAGE 113

113 2H), 7.34 (t, J = 7.4 Hz, 2H), 4.52 (d, J = 6.5 Hz, 2H), 4.29 (t, J = 6.6 Hz, 1H), 2.70 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 67.7, 47.0, 36.3. 5.4.1 General Methods for the Preparation of 5.16a e To a suspension of L amino methyl/ tert butyl ester hydrochloride 5. 15a e (10.0 mmol, 1.0 equiv. ) in DCM (20 mL) at 20 o C, 2.5 equiv. of DIPEA and CDI (carbonyldiimidazole, 1.1 equiv. ) were added. The reaction mixture was stirred for 3 h at rt the organic layer was washed with water (2 20 mL), NaHCO 3 (3 20 mL) and brine solution (2 20 mL). The organic layer was dried over MgSO 4 and evaporated under vacuum to give oily mono substituted imidazole derivative 5. 16a e Im Phe OMe ( 5. 16a). Colorless oil 157 (2.596 g, 95%); D 20 20.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.17 (m, 3H), 7.15 7.03 (m, 2H), 7.00 (d, J = 1.7 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 4.90 4.80 (m, 1H), 3.75 (s, 3H), 3.30 3.11 (m, 2H); 13 C NMR (75 MHz, CDCl 3 129.0, 128.6, 127.6, 116.3, 55.0, 53.0, 37.6. Im Val O t Bu ( 5. 16b). Oil (2.406 g, 90%); D 20 28.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 1.5 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.69 (d, J = 8.1 Hz, 1H), 4.45 (dd, J = 8.1, 4.8 Hz, 1H), 2.30 2.18 (m, 1H), 1.48 (s, 9H), 0.99 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 148.7, 136.0, 130.5, 115.8, 83.0, 59.0, 31.4, 28.0, 18.8, 17.8. Compound 5. 16b was characterized by 1 H, 13 C NMR and taken to the next step without further purification. Im Met OMe ( 5. 16c). Colorless oil 158 (2.419 g, 94%); D 20 19 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.8 Hz, 1H), 7.44 (d, J = 1.2 Hz,

PAGE 114

114 1H), 7.04 (d, J = 1.5 Hz, 1H), 4.79 4.71 (m, 1H), 3.77 (s, 3H), 2.62 2.55 (m, 2H), 2.31 2.01 (m, 5H); 13 C NMR (75 MHz, CDCl 3 30.4, 30.1, 15.4. Im Leu OMe ( 5. 16d). White microcrystals (2.297 g, 96%); mp 80.0 82.0 o C; lit. 158 mp 80.0 81.0 o C D 20 47.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 8.15 (s, 1H), 8.00 (d, J = 6.0 Hz, 1H), 7.44 (d, J = 3.0 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 4.69 4.39 (m, 1H), 3.69 (s, 3H), 1.82 1.41 (m, 3H), 0.89 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.0 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 40.4, 24.8, 22.8, 21.4. Im Leu O t Bu ( 5. 16e). Colorless oil (2.589 g, 92%); D 20 23.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 1.1 Hz, 1H), 7.34 (s, 1H), 7.04 6.97 (m, 1H), 6.99 6.89 (m, 1H), 4.58 4.43 (m, 1H), 1.76 1.52 (m, 4H), 1.45 (s, 12H), 0.94 (d, J = 4.2 Hz, 4H), 0.91 ( d, J = 4.3 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 130.5, 115.8, 83.0, 59.0, 53.4, 31.4, 28.0, 18.8, 17.8. Compound 5. 16e was characterized by 1 H, 13 C NMR and taken to the next step without further purification. 5.4.2 General Methods for the Preparation of N Pg Azadipeptide 5.18a h The residue 5. 16a e (1.0 equiv. ) was dissolved in dry DCM (20 mL) and reacted with alkyl N Pg hydrazines 5. 10a c or 5. 14a,b (1.0 equiv. ; Pg = Boc Fmoc Cbz ) in the presence of DIPEA (1.0 equiv ) at 20 o C overnight. The reaction mixture was poured into a separatory funnel and washed with water (2 20 mL), 2 N HCl (3 20 mL), brine solution (2 20 mL), dried over MgSO 4 and evaporated under vacuum to give both NH 2 and CO 2 H side protected azad ipeptide 5. 18a h which were used in the next step without further purification.

PAGE 115

115 Cbz AzaAla Phe OMe ( 5. 18a). White microcrystals (1.734 g, 90%); mp 51.0 53.0 o C; lit. 159 mp not reported; D 20 25 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.31 (s, 5H), 7.27 7.11 (m, 3H), 7.02 (dd, J = 7.2, 2.4 Hz, 2H), 6.86 (s, 1H), 5.77 (d, J = 8.1 Hz, 1H), 5.13 (s, 2H), 4.72 4.64 (m, 1H), 3.62 (s, 3H), 3.13 2.94 (m, 5H); 13 C NMR (75 MHz, CDCl 3 128.4, 128.1, 126.9, 67.9, 54.2, 52.1, 38.1, 35.8; Anal. Calcd for C 20 H 23 N 3 O 5 : C, 62.33; H, 6.01; N, 10.90. Found: C, 62.42; H, 6.17; N, 10.84. Cbz AzaAla Val O t Bu ( 5. 18b). Low melting solid (1.556 g, 82%) ; D 20 22.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.18 (m, 5H), 7.07 (s, 1H), 5.81 (d, J = 8.8 Hz, 1H), 5.18 (s, 2H), 4.28 (dd, J = 8.8, 4.5 Hz, 1H), 3.10 (s, 3H), 2.20 1.96 (m, 1H), 1.43 (s, 9H), 0.87 (d, J = 6.0 Hz, 3H), 0. 78 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 7.4, 135.3, 128.5, 128.4, 128.2, 81.7, 67.9, 58.4, 35.9, 31.5, 28.0, 18.8, 17.3.Compound 5. 18b was characterized by 1 H, 13 C NMR and taken to the next step without further purification. Boc AzaVal Leu OMe ( 5. 18c). White microcrystals (1.537 g, 89%); mp 60.0 62.0 o C; D 20 27.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 (d, J = 9.1 Hz, 1H), 4.66 4.53 (m, 1H), 4.47 (q, J = 7.7 Hz, 1H), 3.68 (s, 3H), 1.76 1.48 (m, 3H), 1.44 (s, 9H), 1.09 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 5.7 Hz, 3H), 0.95 0.83 (m, 6H); 13 C NMR (75 MHz, CDCl 3 24.9, 23.2, 22.0, 19.9, 19.5; Anal. Calcd for C 16 H 31 N 3 O 5 : C, 55.63; H, 9.05, N, 12.16. Found: C, 55.99; H, 9.18; N, 12.09. Boc AzaVal Met OMe ( 5. 18d). Colorless oil (1.636 g, 90%); D 20 35.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 4.42 (m,

PAGE 116

116 2H), 3.74 (s, 3H), 2.54 (t, J = 7.5 Hz, 2H), 2.29 1.90 (m, 5H), 1.49 (s, 9H), 1.11 (d, J = 5.7 Hz, 3H), 1.08 ( d, J = 7.8 Hz, 3H). Compound 5. 18d was characterized by 1 H NMR and taken to the next step without further purification. Boc AzaPhe Leu OMe ( 5. 18e). Low melting solid (1.869 g, 95%); lit. 6 mp not reported D 20 25.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 ) 7.18 (m, 6H), 6.20 (s, 1H), 5.82 (d, J = 8.6 Hz, 1H), 4.60 4.51 (m, 1H), 3.75 3.65 (m, 5H), 1.70 1.51 (m, 3H), 1.44 (s, 9H), 0.96 (d, J = 6.0 Hz, 3H), 0.94 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 127.7, 82.2, 51.8, 41.8, 27.9, 24.7, 22.9, 21.8. Boc AzaGly Phe OMe ( 5. 18f). Colorless oil (1.350 g, 80%); D 20 16.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.17 (m, 3H), 7.13 7.05 (m, 2H), 6.71 (s, 1H), 5.91 (d, J = 8.1 Hz, 1H), 5.50 (d, J = 8.2 Hz, 1H), 4.80 4.71 (m, 1H), 3.65 (d, J = 7.2 Hz, 3H), 3.07 (d, J = 5.7 Hz, 1H), 3.01 (d, J = 6.0 Hz, 1H), 1.44 (s, 9H); 13 C NMR (75 MHz, CDCl 3 28.3. Compound 5. 18f was characteriz ed by 1 H, 13 C NMR and taken to the next step without further purification. Fmoc AzaAla Val O t Bu ( 5. 18g). Low melting solid (1.987 g, 85%); D 20 10.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.5 Hz, 2H), 7.58 (t, J = 6.1 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.13 (s, 1H), 5.85 (d, J = 7.8 Hz, 1H), 4.62 4.44 (m, 2H), 4.30 (dd, J = 8.1, 3.8 Hz, 1H), 4.23 (t, J = 6.9 Hz, 1H), 3.09 (s, 3H), 2.20 2.00 (m, 1H), 1.42 (s, 9H), 0.91 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H ); 13 C NMR (75 MHz, CDCl 3

PAGE 117

117 124.9, 120.0, 81.8, 67.9, 58.5, 46.9, 35.9, 31.5, 28.0, 18.9, 17.5. Compound 5. 18g was characterized by 1 H, 13 C NMR and taken to the next step without further purification. Fmoc AzaVal Leu O t Bu ( 5. 18h ). Low melting solid (1.988 g, 78%); D 20 4 .0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.5 Hz, 2H), 7.60 (t, J = 6.6 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 6.81 6.48 (m, 0.5H), 5.67 5.42 (m, 0.5H), 4.63 4.36 (m, 3H), 4.31 4.07 (m, 1H), 1.78 1.50 (m, 3H), 1.45 (s, 9H), 1.07 (d, J = 6.7 Hz, 6H), 1.00 0.84 (m, 6H); 13 C NMR (75 MHz, CDCl 3 157.0, 143.6, 141.6, 128.1, 127.4, 125.3, 120.3, 81.9, 52.7, 48.6, 47.3, 42.5, 28.2, 25.1, 23 .0, 22.4, 19.6; Anal. Calcd for C 29 H 39 N 3 O 5 : C, 68.34; H, 7.71, N, 8.24. Found: C, 68.04; H, 8.14; N, 8.47. Cbz AzaAsp(OMe) Val O t Bu ( 5. 18i ). Low melting solid (2.012 g, 92%); D 20 21.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.18 (m, 5H), 5.98 (d, J = 8.7 Hz, 1H), 5.13 (s, 2H), 4.22 (dd, J = 8.7, 4.5 Hz, 1H), 3.67 3.57 (m, 5H), 2.27 1.71 (m, 1H), 1.38 (s, 9H), 0.83 (d, J = 6.8 Hz, 3H), 0.77 (d, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 5.4, 128.8, 128.7, 128.4, 82.0, 68.3, 58.8, 52.4, 52.2, 31.9, 28.2, 19.0, 17.7; HRMS (ESI) calcd for C 21 H 31 N 3 O 7 Na [ M+Na] 460.2054 found 460.2075. 5.4.3 General Methods for OMe and O t Bu Group Deprotection Method 1: OMe deprotection : N Pg AzaAA 1 AA 2 OMe (4.0 mmol, 1.0 equiv. ) was dissolved in a solution of MeOH and water (10 mL, 9:1 v/v). LiOH (8.0 mmol, 2.0 equiv. ) was added and the mixture was stirred at room temperature for 2 h. After completion of the reaction (followed by TLC) MeOH was evaporated and water (5.0 mL) was added, the aqueous layer was washed with ether (2 20 mL) and then acidified

PAGE 118

118 with 2 N HCl. The aqueous layer was extracted with EtOAc (2 10 mL) solution, the organic layer was dried over anhyd. MgSO 4 and evaporated to give N Pg a zadipeptide. Method 2: O t Bu deprotection: N Pg AzaAA 1 AA 2 O t Bu (4.0 mmol) was dissolved in a solution of dry DCM (10 mL) containing trifluoro acetic acid (5.0 mL) and stirred for 2 h at room temperature. After completion of the reaction [followed by T LC] and TFA were evaporated, water (5 mL) was added and the aqueous layer was extracted with EtOAc ( 2 10 mL), organic layer was dried over anhy drous MgSO 4 and evaporated to give white solid. Compounds 5. 19b, d were characterized by 1 H and 13 C NMR were taken to the next step without further purification. Cbz AzaAla Phe OH ( 5. 19a). Compound 5. 19a was prepared from 5. 18a according to the method 1 for OMe deprotection. White microcrystals (1.337 g, 90%); mp 58.0 60.0 o C ; D 20 30.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 (br s, 1H), 9.50 9.39 (m, 1H), 7.47 7.29 (m, 5H), 7.27 7.15 (m, 5H), 6.67 6.50 (m, 1H), 5.13 (s, 2H), 4.36 4.28 (m, 1H), 3.00 (d, J = 6.5 Hz, 2H), 2.93 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 .5, 128.3, 128.1, 127.9, 126.4, 66.4, 54.5, 39.9, 36.9; Anal. Calcd for C 19 H 21 N 3 O 5 : C, 61.45; H, 5.70, N, 11.31. Found: C, 61.13; H, 5.90; N, 11.18. Cbz AzaAla Val OH ( 5. 19b). Compound 5. 19b was prepared from 5. 18b according to the method 2 for O t Bu deprot ection. White microcrystals (1.074 g, 83%); mp 65.0 67.0 o C ; D 20 15.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.41 (m, 1H), 7.34 (s, 5H), 5.99 5.90 (m, 1H), 5.19 (s, 2H), 4.30 4.20 (m, 1H), 3.09 (s, 3H), 2.18 2.07 (m, 1H), 0.89 (d, J = 7.5 Hz, 3H), 0.80 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3

PAGE 119

119 17.6. Compound 5. 19b was characterized by 1 H, 13 C NMR and taken to the next step without further purification Boc AzaVal Leu OH ( 5. 19c) Compound 5. 19c was prepared from 5. 18c according to the method for 5. 19a White microcrystals (1.047 g, 79%); mp 56.0 58.0 o C; D 20 25.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 0.3H), 6.05 5.90 (m, 1H), 4.50 4.25 (m, 1H), 4.24 3.97 (m, 1H), 1.80 1.46 (m, 3H), 1.41 (s, 9H), 0.97 (d, J = 6.4 Hz, 6H), 0.88 0.83 (m, 6H); 13 C NMR (75 MHz, DMSO d 6 ) Calcd for C 15 H 2 9 N 3 O 5 : C, 54.36; H, 8.82, N, 12.68. Found: C, 54.02; H, 9.15; N, 12.30. Boc AzaVal Met OH ( 5. 19d). Compound 5. 19d was prepared from 5. 18d according to the method for 5. 19a Low melting point solid (1.202 g, 86%); D 20 20.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MH z, DMSO d 6 6.12 (m, 1H), 4.52 3.98 (m, 2H), 2.44 (d, J = 7.4 Hz, 2H), 2.11 1.86 (m, 5H), 1.42 (s, 9H), 0.99 (d, J = 6.3 Hz, 6H). Compound 5. 19d was characterized by 1 H NMR and taken to the next step without further purification. Boc AzaPhe Leu OH ( 5. 19e). Compound 5. 19e was prepared from 5. 18e according to the method for 5. 19a White microcrystals (1.351 g, 89%); mp 60.0 62.0 o C; D 20 22.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz,CDCl 3 7.11 (m, 5H), 5.94 (d, J = 8.0 Hz, 1H), 4.60 4.38 (m, 1H), 1.78 1.49 (m, 3H), 1.40 (s, 9H), 0.96 (d, J = 6.6 Hz, 4H), 0.94 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, Acetone d 6 137.7, 128.9, 128.4, 127.4, 80.5, 52.0, 41.4, 27.6, 24.6, 22.8, 21.3. Compound 5. 19e was characterized by 1 H and 13 C NMR.

PAGE 120

120 Boc AzaGly Phe OH ( 5. 19f). Compound 5. 19f was prepared from 5. 18f according to the method for 5. 19a White microcrystals (0.983 g, 76%); mp 159.0 161.0 o C; lit. 13 mp 163.0 164.0 o C; D 20 69.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 7.06 (m, 5H), 6.26 (d, J = 7.6 Hz, 1H), 4.45 4.32 (m, 1H), 3.08 2.86 (m, 2H), 1.39 (s, 9H); 13 C NMR (75 MHz, DMSO d 6 ) .4, 28.1; Anal. Calcd for C 15 H 20 LiN 3 O 5 : C, 54.71; H, 6.12, N, 12.76. Found: C, 54.70; H, 6.41; N, 12.30. Fmoc AzaAla Val OH ( 5. 19g). Compound 5. 19g was prepared from 5. 18g according to the method for 19b White microcrystals (1.136 g, 69%); mp 178 .0 180.0 o C; D 20 7.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.5 Hz, 2H), 7.53 (d, J = 7.1 Hz, 2H), 7.35 (t, J = 7.0 Hz, 2H), 7.29 7.18 (m, 2H), 4.57 4.30 (m, 1H), 4.18 (t, J = 6.8 Hz, 2H), 3.03 (s, 3H), 2.23 2.00 (m, 1H), 0.89 (d, J = 6.3 Hz, 3H) 0.82 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 128.3, 127.7, 125.8, 120.8, 58.9, 47.2, 36.1, 31.3, 30.9, 19.7, 18.6. Compound 5. 19g was characterized by 1 H and 13 C NMR. Fmoc AzaVal Leu OH ( 5. 19h). Compound 5. 19h was prepared from 5. 18h according to the method for 5. 19b .White microcrystals (1.451 g, 80%); mp 157.0 159.0 o C; D 20 16.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 10.9 Hz, 1H), 7.90 (d, J = 7.2 Hz, 2H), 7.76 (d, J = 7.3 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.1 Hz, 2H), 6.40 6.20 (m, 1H), 6.29 (dd, J = 33.4, 8.1 Hz, H), 4.65 3.93 (m, 5H), 1.80 1.12 (m, 3H), 0.97 (dd, J = 13.2, 5.7 Hz, 6H), 0.82 (d, J = 6.7 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 156.5, 140.7, 127.7, 127.1, 125.3, 120.1, 66.1,

PAGE 121

121 64.9, 51.4, 47.6, 46.6, 24.2, 22.9, 21.5, 19.8, 19.4; Anal. Calcd for C 25 H 31 N 3 O 5 : C, 66.21; H, 6.89, N, 9.26. Found: C, 65.90; H, 6.98; N, 9.21. 5.4.4 General Method for the Preparation of Benzotriazolides of Azadipeptide 5 20a e Benzotriazole (6.0 mmol, 3.0 equiv. ) was dissolved in dry DCM ( 50 mL). SOCl 2 (2.2 mmol) was added by syringe and the mixture was stirred for 15 min at rt. under argon. The solution temp was lowered to 30 40 o C (dry ice + acetone) a nd 2.0 equiv. of TEA was added. After 5 mins of stirring, N Pg AzaAA 1 AA 2 OH (2.0 mmol) was added and the mixture stirred for 2 h keeping the temperature at 30 to 40 o C. After completion of the reaction, ice cold water (20 mL) was added and the organic l ayer was washed with water (20 mL 2); NaHCO 3 (20 mL 4) and then brine (20 mL 2). The organic layer was dried over MgSO 4 and evaporated to yield a white solid 5. 20a e. Cbz AzaAla Phe Bt ( 5. 20a). White microcrystals (0.822 g, 87%); mp 70.0 72.0 o C; D 20 15.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 8.4 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.30 7.15 (m, 5H), 7.09 6.92 (m, 5H), 6.14 (d, J = 7.8 Hz, 1H), 6.04 5.98 (m, 1H), 5.01 (s, 2H), 3. 30 (dd, J = 14.0, 5.2 Hz, 1H), 3.12 (dd, J = 14.0, 7.5 Hz, 1H), 2.98 (s, 3H); 13 C NMR (75 MHz, CDCl 3 127.9, 127.1, 126.3, 120.1, 114.1, 67.7, 55.1, 38.2, 35.7; Anal. Calcd for C 25 H 24 N 6 O 4 : C, 63.55; H, 5.12, N, 17.79. Found: C, 63.23; H, 5.14; N, 18.00. Cbz AzaAla Val Bt ( 5. 20b). White microcrystals (0.688 g, 81%); mp 57.0 59.0 o C; D 20 1 2.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 8.2 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.30 7.13 (m, 5H), 6.16 (d, J = 7.7 Hz, 1H), 5.69 (dd, J = 8.9, 5.4 Hz, 1H), 5.07 (s, 2H), 3.04 (s, 3H),

PAGE 122

122 2.40 2.26 (m, 1H), 0.91 (d, J = 6.9 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 120.0, 114.1, 67.7, 58.8, 35.8, 31.4, 19.5, 17.2; HRMS (ESI) calcd for C 21 H 24 N 6 O 4 Na [M+Na] + 447.1751 found 447.1771 Boc AzaVal Leu Bt ( 5. 20c). White microcrystals (0.779 g, 90%); mp 92.0 95.0 o C; D 20 34.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 8.2 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 6.52 6.25 (m, 1H), 5.94 5.78 (m, 2H), 4.70 4.56 (m, 1H), 1.92 1.65 (m, 3H), 1.49 (s, 9H), 1.18 1.0 (m, 9H), 0.94 (d, J = 4.3 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 131.4, 130.7, 126.5, 120.4, 114.6, 82.2, 53.1, 48.4, 41.9, 28.3, 25.5, 23.5, 21.7, 19.8, 19.5; HRMS (ESI) calcd for C 21 H 32 N 6 O 4 Na [M+Na] + 455.2377 found 455.2399 Boc AzaVal Met Bt ( 5. 20d). White microcrystals (0.766 g, 85%); mp 80.0 82.0 o C; D 20 22.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.7 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 7.50 (t, J = 8.1 Hz, 1H), 6.54 6.24 (m, 1H), 4.67 4.59 (m, 1H), 2.68 (q, J = 7.2, 6.1 Hz, 2H), 2.21 2.10 (m, 3H), 2.07 2.05 (m, 3H), 1.50 (s, 9H), 1.13 1.06 (m, 6H ); 13 C NMR (75 MHz, CDCl 3 145.9, 131.1, 130.6, 126.4, 120.2, 114.3, 83.1, 53.4, 48.2, 32.1, 30.1, 28.1, 19.6, 19.2, 15.4; Anal. Calcd for C 20 H 30 N 6 O 4 S: C, 53.32; H, 6.71, N, 18.65. Found: C, 53.04; H, 6.78; N, 18.33. Fmoc AzaVal Leu Bt ( 5. 20e). White microcrystals (1.021 g, 92%); mp 100.0 102.0 o C; D 20 1 9.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 4.3 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.85 7.70 (m, 3H), 7.63 (t, J = 7.7 Hz, 2H), 7.44 7.39 (m, 2H), 7.33 (t, J = 7.5 Hz, 2H),

PAGE 123

123 7.23 6.90 (m, 1H), 5.64 5.56 (m, 1H), 4.56 4.47 (m, 1H), 4.44 3.98 (m, 3H), 1.99 1.41 (m, 3H), 1.07 0.89 (m, 9H), 0.86 0.76 (m, 3H); 13 C NMR (75 MHz, DMSO d 6 157.2, 156.4, 145.3, 143.7, 143.5, 140.7, 131.0, 130.6, 127.7, 127.1, 126.6, 125.4, 125. 2, 120.1, 120.1, 113.9, 65.9, 52.5, 48.0, 46.7, 24.6, 22.9, 21.0, 19.8, 19.2; HRMS (ESI) calcd for C 31 H 34 N 6 O 4 Na [M+Na] + 577.2534 found 577.2526 5.4.5 General Procedure for the C oupling of 5. 20a e to P repare 5. 24a g, 5. 25a,b and 5. 26a,b Amino acids 5. 21a e or dipeptides 5. 22 5. 23 (1.2 mmol, 1.2 equiv. ) and TEA (1.2 mmol, 1.2 equiv. ) were dissolved in minimum amount of cold water (5.0 mL). Acetonitrile (10 mL) was added and the solutions cooled to 10 o C. A solution of N Pg azadipeptidoyl Bt (1.0 mmol, 1.0 equiv. ) in acetonitrile (5.0 mL) was added and stirred for 2 h at 20 o C. The reaction mixture was monitored by TLC [EtOAc hexanes (1:2)]. After completion of reaction, the solvent was evaporated. The residue was dissolved in DCM (30 mL) and washed with 2N HCl solution (4 x 10 mL), water (10 mL) and brine (10 mL). The solvent was dried over MgSO 4 and evaporated to give various N Pg aza tri and tetrapeptides. Cbz AzaAla Phe Cys OH ( 5. 24a). White microcrystals (0.379 g, 80%); mp 89.0 92.0 o C ; D 20 25.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 1H), 8.25 (br s, 1H), 7.48 7.33 (m, 5H), 7.28 7.08 (m, 6H), 6.62 (br s, 1H), 5.13 (s, 2H), 4.70 4.30 (m, 2H), 3.06 2.78 (m, 7H); 13 C NMR (75 MHz, DMSO d 6 157.1, 155.6, 137.7, 136.2, 129.4, 129.2, 128.5, 128.1, 127.9, 126.2, 66.4, 54.6, 54.4, 37.6, 35.5, 25.7; Anal. Calcd for C 22 H 26 N 4 O 6 S: C, 55.68; H, 5.52; N, 11.81. Found: C, 55.56; H, 5.64; N, 11.78.

PAGE 124

124 Cbz AzaAla Val Ser OH ( 5. 24b). White microcrystals (0.328 g, 80%); mp 50.0 52.0 o C; [ D 20 5.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.70 (m, 1H), 7.35 7.25 (m, 6H), 5.24 5.07 (m, 2H), 4.67 4.54 (m, 1H), 4.33 4.16 (m, 1H), 4.06 3.81 (m, 2H), 3.03 ( s 3H), 2.20 1.92 (m, 1H), 0.85 (d, J = 6.3 Hz, 3H), 0.77 (d, J = 5.4 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 135.3, 128.5, 128.0, 67.9, 62.5, 59.8, 54.7, 36.1, 31.2, 19.1, 17.9; HRMS (ESI) calcd for C 18 H 25 N 4 O 7 [M H] 409.1729 found 409.1726 Cbz AzaAla Val Cys OH ( 5. 24c). White microcry stals (0.354 g, 83%); mp 113 .0 115.0 o C; D 20 17.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.50 (m, 2H), 7.28 7.22 (m, 5H), 5.10 (s, 2H), 4.73 4.63 (m, 1H), 4.16 (t, J = 8.1 Hz, 1H), 3.00 (s, 3H), 2.96 2.78 (m, 1H), 2.07 1.81 (m, 1H), 1.61 (t, J = 9.0 Hz, 1H), 0.81 (d, J = 6.0 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 158.2, 135.2, 128.5, 128.4, 128.0, 67.9, 54.2, 53.4, 36.3, 30.6, 26.3, 19.1, 18.2; HRMS (ESI) calcd for C 18 H 25 N 4 O 6 S [ M H] 425.1500 found 42 5.1521 Cbz AzaAla Val (D,L)Cys OH ( 5. 24c+ White microcrystals (0.363 g, 85%); mp 90 .0 92.0 o C; 1 H NMR (300 MHz, CDCl 3 J = 7.1 Hz, 1H), 7.39 7.27 (m, 5H), 5.18 (s, 2H), 4.89 4.56 (m, 1H), 4.46 4.13 (m, 1H), 3.09 (s, 3H), 3.04 2.79 (m, 2H), 2.10 1.94 (m, 1H), 1.63 (t, J = 9.0 Hz, 0.5H), 1.49 (t, J = 8.9 Hz, 0.5H), 0.90 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 4.9 Hz, 3H); 13 C NMR was similar to the compound 5. 24c Anal. Calcd for C 18 H 26 N 4 O 6 S: C, 50.69; H, 6.15; N, 13.14. Found: C, 50.48; H, 6.21; N, 13.08. Boc AzaVal Leu Trp OH ( 5. 24d). White microcrystals (0.461 g, 89%); mp 110.0 113.0 o C ; D 20 4.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6

PAGE 125

125 1H), 8.58 (br s, 1H), 7.94 (d, J = 6.2 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.12 (s, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.89 (br s, 1H), 4.53 4.50 (m, 1H), 4.37 (qt, J = 6.6 Hz, 1H), 4 .28 4.13 (m, 1H), 3.18 (dd, J = 14.7, 5.9 Hz, 1H), 3.05 (dd, J = 14.7, 7.4 Hz, 1H), 1.66 1.55 (m, 1H), 1.50 1.38 (m, 10H), 1.00 (d, J = 6.6 Hz, 6H), 0.85 (d, J = 6.3 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 172.6, 156.2, 155.3, 136.0, 127.2, 123.6, 120. 9, 118.3, 118.2, 111.4, 109.7, 79.6, 52.9, 51.7, 47.1, 42.0, 27.9, 27.1, 23.7, 23.4, 22.0, 19.8, 19.3; Anal. Calcd for C 26 H 39 N 5 O 6 : C, 60.33; H, 7.59, N, 13.53. Found: C, 60.48; H, 8.07; N, 13.17. Boc AzaVal Met Ser OH ( 5. 24e). White microcrystals (0.362 g, 83%); mp 48.0 50.0 o C; D 20 21.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, Acetone d 6 8.10 (m, 1H), 7.83 7.73 (m, 1H), 7.56 (t, J = 8.2 Hz, 1H), 7.39 7.31 (m, 1H), 6.41 6.22 (m, 1H), 4.42 (dt, J = 14.8, 7.0 Hz, 3H), 3.84 (d, J = 1 1.7 Hz, 1H), 3.74 (d, J = 11.1 Hz, 1H), 2.43 (q, J = 7.4 Hz, 2H), 2.16 1.68 (m, 5H), 1.34 (s, 9H), 0.97 (d, J = 7.5 Hz, 6H ); 13 C NMR (75 MHz, CDCl 3 28.4, 19.7, 15.5; HRMS (ESI) calcd for C 17 H 31 N 4 O 7 S [ M H] 435.1919 found 435.1935 Boc AzaVal Leu Asp(OBn) OH ( 5. 24f). White microcrystals (0.483 g, 90%); mp 108.0 110.0; D 20 12.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 8.65 8.10 (m, 1H), 7.40 7.30 (m, 5H), 6.13 5.86 (m, 1H), 5.10 (s, 2H), 4.62 4.56 (m, 1H), 4.40 4.35 (m, 1H), 4.26 4.21 (m, 1H), 2.87 (dd, J = 17.1, 6.9 Hz, 1H), 2.73 (dd, J = 16.5, 7.2 Hz, 1H), 1.89 1.45 (m, 3H), 1.41 (s, 9H), 0.97 (d, J = 6.6 Hz, 6H), 0.86 (d, J = 7.1 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 128.4, 128.0, 127.9, 79.6, 65.8, 51.7, 48.6, 42.2, 35.9, 27.9, 23.2, 21.9, 19.7, 19.3; HRMS (ESI) calcd for C 26 H 39 N 4 O 8 [ M H] 535.2773 found 535.2782

PAGE 126

126 Fmoc AzaVal Leu Pro OH ( 5. 24g). White microcrystals (0.474 g, 86%); mp 210.0 212.0 o C; D 20 37.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz,DMSO d 6 Hz, 3H), 1.30 1.48 (m, 2H), (1.50 2.0, m, 4H), 2.0 2.2 (m, 1H), 3.7 (s, 1H), 4.1 4.4 (m, 4H), 4.44 (s, 2H), 6.10 6.40 (rotamars, 1H), 7.33 (t, 10 Hz, 7.42 (t, 7.5 Hz), 7.75 (d, 7.2 Hz, 2 H), 7.89 (d, 7.2 Hz, 2H), 9.30 (d, 13.2 Hz, 1H); 13 C NMR (75 MHz, DMSO d 6 172.6, 171.2, 156.8, 156.5, 143.7, 143.6, 143.5, 140.8, 129.0, 127.8, 127.4, 127.2, 1 25.4, 125.3, 121.5, 120.2, 120.1, 66.2, 52.3, 48.1, 47.7, 46.8, 40.8, 24.1, 23.3, 21.8, 19.8, 19.4, 18.4; Anal. Calcd for C 30 H 38 N 4 O 6 : C, 65.44; H, 6.96, N, 10.17. Found: C, 65.65; H, 7.36; N, 10.01. Cbz AzaAla Phe Gly Gly OH ( 5. 25a). White microcrystals (0.374 g, 77%); mp 72.0 74.0 o C ; D 20 = 22.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 s, 1H), 8.30 8.10 (m, 2H), 7.44 7.30 (m, 5H), 7.25 7.10 (m, 5H), 6.70 (br s, 1H), 5.12 (s, 2H), 4.45 4.35 (m, 1H), 3.89 3.59 (m, 4H), 3 .00 (d, J = 6.3 Hz, 1H), 2.94 (d, J = 6.3 Hz, 1H), 2.91 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 155.6, 137.9, 136.2, 129.3, 128.5, 128.1, 128.0, 127.9, 126.2, 66.5, 55.2, 41.9, 40.7, 37.6, 35.5; HRMS (ESI) calcd for C 46 H 53 N 10 O 14 [2 M H] 969.3748 found 969.3740 Boc AzaVal Leu Gly Phe OH ( 5. 25b). White microcrystals (0.466 g, 87%); mp 82.0 85.0; D 20 6.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 11.1 Hz, 0.7H), 8.37 (s, 0.3H), 8.24 7.94 (m, 2H), 7.35 7.15 (m, 5H), 6.30 (d, J = 8.1 Hz, 0.5H), 5.99 (d, J = 8.1 Hz, 0.5H), 4.45 4.35 (m, 2H), 4.25 4.16 (m, 1H), 3.77 (dd, J = 16.9, 6.2 Hz, 1H), 3.59 (dd, J = 17.0, 5.2 Hz, 1H), 3.04 (dd, J = 13.7, 4.8 Hz, 1H), 2.87 (dd, J = 13.7, 8.7 Hz, 1H), 1.73 1.43 (m, 3H), 1.42 (s, 9H), 0.97 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.3 Hz, 3H), 0.88 0.77 (m, 6H); 13 C NMR (75 MHz, DMSO d 6

PAGE 127

127 172.7, 168.6, 156.5, 155.3, 137.4, 129.1, 128.2, 126.4, 79.5, 53.5, 52.1, 47.1, 41.6, 36.9, 27.9, 23.9, 23.2, 21.8, 20.0, 19.3; Anal. Calcd for C 26 H 41 N 5 O 7 : C, 58.30; H, 7.72, N, 13.07. Found: C, 58.00; H, 7.98; N, 12.87. Cbz AzaAla Phe AOGly OH ( 5. 26a). White microcrystals (0.400 g, 90%); mp 57.0 59.0 o C; D 20 = 21.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 s, 1H), 9.40 (s, 1H), 7.92 (s, 1H), 7.49 7.38 (m, 5H), 7.25 7.15 (m, 5H), 6.62 (s, 1H), 5.13 (s, 2H), 4.40 4.20 (m, 3H), 3.42 2.75 (m, 5H); 13 C NMR (75 MHz, DMSO d 6 170.7, 169.4, 157.6, 156.1, 137.9, 136.9, 129.9, 129.1, 128.7, 128.4, 127.0, 72.3, 67.0, 53.5, 38.6, 36.1; HRMS (ESI) calcd for C 42 H 47 N 8 O 14 [2 M H] 887.3217 found 887.3214 Boc AzaVal Met Gly OPhe OH ( 5. 26b). White microcrystals (0.471 g, 85%); mp 4 4.0 46.0 o C; D 20 15.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.34 (m, 1H), 7.35 6.91 (m, 5H), 6.80 6.23 (m, 1H), 5.23 (dd, J = 8.6, 4.1 Hz, 1H), 4.75 4.30 (m, 2H), 4.21 3.63 (m, 2H), 3.04 (dd, J = 18.6, 4.1 Hz, 2H), 2.65 2.34 (m, 2H), 2.20 1.80 (m, 5H), 1.47 (s, 9H), 1.19 1.11 (m, 6H); 13 C N MR (75 MHz, Acetone d 6 171.5, 170.2, 169.4, 157.4, 136.7, 129.8, 128.8, 127.2, 73.6, 73.2, 53.5, 48.3, 42.9, 41.0, 37.3, 32.9, 28.0, 19.7, 19.4, 14.8; HRMS (ESI) calcd for C 25 H 37 N 4 O 8 S [ M H] 553.2338 found 553.2350 Cbz AzaAla Val OPhe OH ( 5. 28). 4 Dimethylaminopyridine (1.2 mmol) was added to a stirred solution of Cbz AzaAla Val Bt 5. 20b (1.0 mmol) and hydroxycarboxylic acid 5. 27 (1.0 mmol) in dry THF (5.0 mL) at 4 o C. The reaction mixture was stirred at room temperature for 4 h. After comp letion of the reaction the solvent was evaporated. The residue was dissolved in EtOAc washed with 2 N hydrochloric acid solution (3 x 5 mL) and brine (5 mL) and dried over MgSO 4 The

PAGE 128

128 solvent was evaporated to yield the hybrid peptides. Colorless oil (0.410 g, 87%); D 20 13.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.05 (m, 10H), 6.05 5.80 (m, 1H), 5.10 (t, J = 5.4 Hz, 1H), 5.04 (s, 2H), 4.34 4.25 (m, 1H), 3.03 2.79 (m, 5H), 2.01 (s, 1H), 0.71 (d, J = 6.9 Hz, 3H), 0.64 (d, J = 5.7 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 126.9, 126.6, 73.2, 70.9, 67.9, 39.9, 36.8, 30.9, 18.6, 17.0; HRMS (ESI) calcd for C 24 H 28 N 3 O 7 [ M H] 470.1933 found 470.1942 H AzaAla Phe OH ( 5. 29a ). Cbz AzaAla Phe OH 5. 19a (0.75 g, 2.0 mmol) was dissolved in THF (20 mL), treated with a suspension of 10 mol % of Pd on carbon (20 wt %) in THF, placed under hydrogen gas and stirred overnight at room temperature. The reaction mixture was filtered through Celite. The organic filtrate was evaporated to give H AzaAla Phe OH ( 5. 29a ). White microcrystals (0.427 g, 90%); mp 63 .0 65.0 o C; lit. 160 mp not reported; D 20 3.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 1 H NMR (300 MHz, DMSO d 6 7.08 (m, 5H), 7.07 6.87 (m, 1H), 4.44 4.07 (m, 1H), 3.03 2.73 (m, 5H); 13 C NMR (75 MHz, DMSO d 6 55.0, 38.0, 37.8; HRMS (ESI) calcd for C 11 H 15 N 3 O 3 Na [ M+Na] 260.1006 found. 260.1010. H AzaPhe Leu OH.HCl ( 5. 29b). Compound 5. 19e (1.0 mmol) was dissolved in a solution of dry 4N HCl/dioxane (10 mL) and stirred for 2 h at room temperature. After completion of the reaction [followed by TLC] ether (5 mL) was added and the white solid was filtered off to give H AzaPhe Leu OH.HCl ( 5. 29b ). White microcrystals ( 0.294 g, 93%); mp 170.0 172.0 o C ; D 20 31.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 ) J = 7.9 Hz, 1H), 7.47 7.16 (m, 5H), 4.92 (s, 2H), 4.17 4.07 (m, 1H), 1.82 1.40

PAGE 129

129 (m, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.1 Hz, 3H); 13 C NMR ( 75 MHz, DMSO d 6 ) 174.3, 157.0, 135.2, 128.5, 128.3, 127.9, 66.4, 52.1, 51.9,24.3, 23.1, 21.0; HRMS (ESI) ca lcd for C 14 H 20 N 3 O 3 [ M H] 278.1583 found 278.1564 H AzaGly Phe OH.TsOH ( 5. 29c). Compound 5. 19f (3.0 mmol, 1.0 equiv. ) was dissolved in a solution of dry DCM (10 mL) containing p TsOH (1.0 equiv. ) and stirred for 12 h at room temperature. After completion of the reaction [followed by TLC] ether (5 mL) was added and the white solid was filtered off to give H AzaGly Phe OH.TsOH ( 5. 29c ). White microcrystals (1.186 g, 100%); mp 159.0 160.0 o C ; D 20 36.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 8.0 Hz, 2H), 7.36 (d, J = 8.1 Hz, 1H), 7.32 7.18 (m, 5H), 7.14 (d, J = 8.3 Hz, 2H), 4.42 4.32 (m, 1H), 3.10 (dd, J = 13.9, 4.5 Hz, 1H), 2.90 (dd, J = 13.9, 9.4 Hz, 1H), 2.30 (s, 3H); 13 C (75 MHz, DMSO d 6 125.5, 54.3, 36.8, 20.9; Anal. Calcd for C 17 H 21 N 3 O 6 S: C, 51.64; H, 5.35, N, 10.63. Found: C, 51.85; H, 5.25; N, 10.52. Cbz Gly AzaGly Phe OH ( 5. 31a). The t osylate salt of free aza dipeptide 5. 29c (1.0 mmol, 1.0 equiv. ) and TEA (1.0 mmol, 1.0 equiv. ) were dissolved in minimum amount of cold water (5.0 mL). Acetonitrile (1 0 mL) was added and the mixture cooled to 10 o C. A solution of Cbz Gly Bt 5. 30a (1.0 mmol, 1.0 equiv. ) in acetonitrile (5.0 mL) was added and stirred for 2 h at 20 o C. The reaction mixture was monitored by TLC [EtOAc hexanes (1:2)] After completion of reaction the solvent was evaporated and the resid ue was dissolved in DCM (30 mL) and washed with 2 N HCl solution (4 10 mL), water (10 mL) and brine (10 mL). The solvent was dried over MgSO 4 and evaporated to give Cbz Gly AzaGly Phe OH ( 5. 31a ) White microcrystals (0.340 g, 82%); mp 205.0

PAGE 130

130 207.0 o C ; lit 161 mp not reported; D 20 12.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 12.70 (s, 1H), 9.59 (d, J = 2.1 Hz, 1H), 7.96 (s, 1H), 7.46 (t, J = 6.1 Hz, 1H), 7.32 7.11 (m, 10H), 6.38 (d, J = 8.0 Hz, 1H), 4.98 (s, 2H), 4.56 4.16 (m, 1H), 3.60 (d, J = 6.0 Hz, 2H), 3.29 (s, 3H), 2.97 (dd, J = 13.7, 5.2 Hz, 1H), 2.86 (dd, J = 13.7, 7.4 Hz, 1H). 13 C NMR (75 MHz, DMSO d 6 129.3, 128.4, 128.2, 127.8, 127.7, 126.5, 65.5, 53.9, 42.1, 37.3; Anal. Calcd for C 20 H 22 N 4 O 6 : C, 57.97; H, 5.35, N, 13.52. Found: C, 57.73; H, 5.17; N, 13.49. Cbz Met Ala AzaGly Phe OH ( 5. 31b). Compound 5. 31b was prepared according to the method for preparation of Cbz GlyAza Gly Phe OH ( 5. 31a ). White microcrystals (0.476 g, 85%); mp 185.0 187.0 o C ; D 20 = 40.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 11.0 Hz, 1H), 8.20 7.96 (m, 2H), 7.59 7.49 (m, 1H), 7.40 7.16 (m, 10H), 6.38 (dd, J = 12.5, 7.9 Hz, 1H), 5.03 (s, 2H), 4.42 4.20 (m, 2H), 4.18 4.05 (m, 1H), 3.10 2.90 (m, 2H), 2.56 2.47 (m, 2H), 2.02 (s, 3H), 1.96 1.73 (m, 2H), 1.23 (d, J = 6.5 Hz, 3H); HRMS (ESI) calcd for C 26 H 32 N 5 O 7 S [ M H] 558.2028 found 558.2029. 5.4.6 General Method for the Preparation of 5. 33a,b Hydrazine hydrate (1.0 mmol, 1.0 equiv. ) and DIPEA (1.0 mmol, 1.0 equiv. ) were dissolved in ether (5.0 mL). A solution of Cbz Phe Bt 5. 30c or Fmoc Cys(S trt) Bt 5. 30d (1.0 mmol, 1.0 equiv. ) in ether (5.0 mL) was added and stirred for 10 min at 20 o C. The white precipitate 5. 32a,b formed and was collected by filtration. Compounds 5. 32a,b were used for the next step without further purification. 162 The compounds 5.32 dissolved in dry DCM (20 mL) and reacted with 5. 16b d (1.0 equiv. ) in the presence of DIPEA (1.0 equiv. ) at 20 o C overnight. The reaction mixture was washed with water (2

PAGE 131

131 20 mL), NaHCO 3 (3 20 mL), brine (2 20 mL), dried over MgSO 4 and evaporated to give both NH 2 and CO 2 H side protected aza tripeptides 5. 33a b Cbz Phe AzaGly Leu OMe ( 5. 33a). Compound 5. 33a w as prepared according to the general method given for the preparation of 5. 33a,b White microcrystals (0.417 g, 86%); mp 179.0 181.0 o C ; D 20 20.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 9.35 (s, 0.5H), 7.97 (s, 0.5H), 7.34 7.02 (m, 11H), 6.40 (d, J = 7.9 Hz, 0.5H), 6.05 (d, J = 7.0 Hz, 0.5H), 4.99 (d, J = 12.6 Hz, 1H), 4.85 (d, J = 12.3 Hz, 1H), 4.63 4.27 (m, 2H), 3.59 (s, 3H), 3.13 (dd, J = 13.9, 5.4 Hz, 1H), 2.92 (dd, J = 14.0, 9.0 Hz, 1H), 1.77 1.42 (m, 3H), 0.85 (d, J = 6.2 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 156.6, 136.5, 136.2, 129.5, 128.7, 128.6, 128.2, 128.0, 127.0, 67.2, 55.1, 52.5, 51.8, 41.4, 38.1, 24.9, 23.0, 22.0; HRMS (ESI) calcd for C 25 H 32 N 4 O 6 Na [M+Na] + 507.2214 found 507.2232. Fmoc Cys(S trt) AzaGly Val O t Bu ( 5. 33b). Compound 5. 33b was prepared according to the method for preparation of 5. 33a White microcrystals (0.599 g, 75%); mp 79.0 81.0 o C ; D 20 23.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 7.4, 3.9 Hz, 2H), 7.59 7.46 (m, 2H), 7.45 7.30 (m, 8H), 7.30 7.08 (m, 12H), 6.74 (s, 1H), 6.23 6.02 (m, 1H), 4.45 4.24 (m, 3H), 4.14 4.10 (m, 2H), 2.80 2.55 (m, 2H), 1.50 1.38 (m, 9H), 1.29 1.23 (m, 1H), 0.93 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H ). 13 C NMR (75 MHz, CDCl 3 128.1, 127.7, 126.9, 125.0, 119.9, 82.0, 67.1, 65.8, 58.1, 47.0, 31.6, 31.3, 28.0, 19.0, 17.5; HRMS (ESI) calcd for C 47 H 50 N 4 O 6 S Na [M+Na] + 821.3343 found 821.3358. 5.4.7 G eneral Method for the P reparation of 5. 35a,b To a solution of benzaldehyde (1.0 equiv. ) in diethyl ether (10 mL), 5. 32c,d (1.0 equiv. ) (prepared using a lit. method) 162 and two drops of glacial acetic acid were

PAGE 132

132 added. Each reaction mixture was heated under reflux for 2 3 h then cooled to room temperature. The white solid precipitate was collected by filtration, washed with cold diethyl ether and dried under vacuum to yield the desired hydrazone. The hydrazone intermediate ( 1.0 equiv. ) was treated with sodium cyanoborohydride (NaBH 3 CN) (1.1 equiv. ) in absolute methanol (20 mL). 1N HCl in MeOH was added dropwise over 1 h to maintain the reaction pH in between 3.5 5.0. After stirring at room temperature overnight, the solvent w as removed and the residue was partitioned between ether (30 mL) and brine. The organic phase was washed with sat. NaHCO 3 (2 20 mL), brine (2 10 mL), dried over MgSO 4 and evaporated to give the 5. 34a b (yield 70 76%) C ompounds 5. 34a b were taken to t he next step without further purification. Each residue was dissolved in dry DCM (20 mL) and reacted with 5. 16b d (1.0 equiv. ) in the presence of TEA (1.0 equiv. ) at 20 o C overnight. The reaction mixture was poured into a separatory funnel and washed with water (2 20 mL), NaHCO 3 (3 20 mL), brine solution (2 20 mL), dried over MgSO 4 and evaporated under vacuum to give aza tripeptide 5. 35a b Boc Gly AzaPhe Leu OMe ( 5. 35a). Compound 5. 35a was prepared according to the general method for the preparation of 5. 35a b White microcrystals (0.405 g, 90%); mp 167.0 170.0 o C ; lit. 163 mp not reported; D 20 9.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, Acetone d 6 7.17 (m, 5H), 6.47 (d, J = 7.2 Hz, 2H), 4.54 4.29 (m, 1H), 3.69 (d, J = 5.5 Hz, 2H), 3.64 (s, 3H), 3.30 (s, 2H), 1.88 1.49 (m, 3H), 1.40 (s, 9H), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H); 174.8, 168.9, 157.4, 156.8, 136.7, 128.9, 128.8, 127.9, 81.1, 52.5, 52.4, 51.3, 44.0, 41.2, 28.5, 24.9, 23.1, 21.9; HRMS (ESI) calcd for C 22 H 34 N 4 O 6 Na [M+Na] + 473.2371 found 473.2395.

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133 Cbz Gly AzaPhe Val O t Bu ( 5. 35b). Compound 5. 35b was prepared according to the method for preparation of 5. 35a White microcrystals (0.410 g, 80%); mp 48.0 49.0 o C ; D 20 1 2.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.14 (m, 11H), 5.86 (d, J = 8.6 Hz, 1H), 5.61 (s, 0.5H), 5.15 4.94 (m, 2H), 4.96 4.77 (m, 0.5H), 4.49 (d, J = 14.6 Hz, 0.5H), 4.30 (d, J = 8.7 Hz, 1H), 4.28 (d, J = 8.7 Hz, 1H), 3.69 (d, J = 5.5 Hz, 2H), 2.19 1.98 ( m, 1H), 1.40 (s, 9H), 0.90 (d, J = 6.9 Hz, 3H), 0.83 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 129.9, 128.9, 128.8, 128.7, 128.5, 128.3, 127.9, 82.1, 67.6, 59.2, 51.5, 43.9, 31.7, 28.3, 19.2, 18.0; HRMS (ESI) calcd f or C 27 H 36 N 4 O 6 Na [M+Na] + 535.2527 found 535.2560. H Gly AzaPhe Leu OMe.TsOH ( 5. 36) Compound 5. 36 was prepared according to the method for preparation of 5. 29c White microcrystals (0.372 g, 95%); mp 155.0 157.0 o C ; D 20 26.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 7.43 (d, J = 8.1 Hz, 2H), 7.34 7.14 (m, 5H), 7.07 (d, J = 7.8 Hz, 2H), 4.25 4.04 (m, 1H), 3.74 3.52 (m, 7H), 2.24 (s, 3H), 1.70 1.37 (m, 3H), 0.82 (d, J = 6.3 Hz, 3H), 0.79 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 2, 166.7, 157.4, 146.2, 138.3, 138.2, 128.8, 128.7, 128.6, 126.1, 119.9, 79.0, 52.4, 51.7, 43.0, 28.8, 24.7, 23.4, 22.0, 21.5. C ompound 5. 36 was characterized by 1 H, 13 C NMR and used for the next step without further purification Cbz Tyr (OBn) Bt ( 5. 38). B enzotriazole (6.0 mmol, 3.0 equiv. ) was dissolved in dry DCM ( 50 mL); SOCl 2 (2.2 mmol) was added by syringe and the mixture was stirred for 15 min a t rt. under argon. The temp was lowered to 30 40 o C (dry ice + acetone) and 2.0 equiv. of TEA was added. After 5 mins of stirring, Cbz Tyr (OBn) OH 5. 37 (2.0 mmol, 1.0 equiv. ) was added and the mixture stirred for 2 h keeping the temperature at

PAGE 134

134 30 to 40 o C. After completion of the reaction ice cold water (20 mL) was added and the organic layer was washed with water (20 mL 2); NaHCO 3 (20 mL 4) and then brine (20 mL 2). The organic layer was dried over MgSO 4 and evaporated to yield a white solid. White microcrystals (0.841 g, 83%); mp 87.0 89.0 o C ; D 20 20.0 ( c 1.0, CH 3 OH); 1 H NMR (3 00 MHz, CDCl 3 J = 8.2 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.41 7.23 (m, 10H), 7.03 (d, J = 8.2 Hz, 3H), 6.82 (dd, J = 8.8, 3.1 Hz, 2H), 6.15 5.88 (m, 1H), 5.68 (d, J = 8.1 Hz, 1H), 5.07 (d, J = 3.5 Hz, 2H), 4.96 (d, J = 2.8 Hz, 2H), 3.40 (dd, J = 13.9, 5.2 Hz, 1H), 3.14 (dd, J = 14.1, 7.6 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 136.7, 135.9, 130.9, 130.6, 130.2, 128.4, 128.0, 127.8, 127.3, 127.1 126.4, 120.2, 114.9, 114.2, 69.8, 67.1, 55.7, 37.8. Compound 5. 38 was characterized by 1 H, 13 C NMR and taken to the next step without further purification. Cbz Tyr (OBn) Gly Bt ( 5. 39). Gly (1.2 mmol, 1.2 equiv. ) and TEA (1.2 mmol, 1.2 equiv. ) were dissolved in minimum amount of cold water (5.0 mL). Acetonitrile (10 mL) was added and the solution was cooled to 10 o C. A solution of Cbz Tyr (OBn Bt 5. 38 (1.0 mmol, 1.0 equiv. ) in acetonitrile (5.0 mL) was added and stirred for 2 h at 20 o C. The r eaction mixture was monitored by TLC [EtOAc hexanes (1:2)]. After completion of reaction, the solvent was evaporated. The residue was dissolved in DCM (30 mL) and washed with 2 N HCl solution (4 x 10 mL), water (10 mL) and brine (10 mL). The solvent was dr ied over MgSO 4 and evaporated to give Cbz Tyr (OBn) Gly OH, which was directly taken to the next step without further purification. B enzotriazole (3.0 mmol, 3.0 equiv. ) was dissolved in dry DCM ( 50 mL). SOCl 2 (1.1 mmol) was added by syringe and the mixture was stirred for 15 min at rt. under argon. The solution temp was lowered to

PAGE 135

135 30 40 o C (dry ice + acetone) and 2.0 equiv. of TEA was added. After 5 mins of stirring, Cbz Tyr (OBn) Gly OH was added and the mixture was stirred for 2 h keeping the temperature at 30 to 40 o C. After completion of the reaction ice cold water (20 mL) was added and the organic layer was washed with water (20 mL 2); NaHCO 3 (20 mL 4) and then brine (20 mL 2). The organic layer was dried over MgSO 4 and evaporated to yield Cbz T yr (OBn) Gly Bt ( 5. 39 ) White microcrystals (0.451 g, 80%); mp 132 133 o C ; D 20 19.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 9.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.1 Hz, 1H), 7.46 (t, J = 7.1 Hz, 1H), 7.36 7.29 (m, 5H), 7.28 7.22 (m, 6H), 7.19 7.00 (m, 3H), 6.91 6.75 (m, 2H), 5.74 (d, J = 7.1 Hz, 1H), 5.18 4.99 (m, 4H), 4.93 (s, 2H), 4.75 4.44 (m, 1H), 3.24 2.96 (m, 2H); 13 C NMR (75 MHz, CDCl 3 137.1, 136.1, 131.0, 130.6, 128.7, 128.3, 128.1, 127.6, 126.7, 126.2, 120.5, 115.2, 114.2, 70.1, 67.5, 56.6, 43.5, 37.9. C ompound 5. 39 was characterized by 1 H, 13 C NMR and taken to the next step without further purification. Leu enkephalin Aza Analog ( 5. 40). The t osylate salt of free aza tripeptide 5. 36 (0.5 mmol, 1.0 equiv. ) and DIPEA (1.0 mmol, 2.0 equiv. ) were dissolved in dry THF. A solution of Cbz Tyr (OBn) Gly Bt 5. 39 (0.5 mmol, 1.0 equiv. ) in THF (5.0 mL) was added and stirred for 12 h at 20 o C. The reaction mixture was monitored by TLC [EtOAc hexanes (1:2)]. After completion of the reaction, the solvent was evaporated. The residue was dissolved in DCM (30 mL) and washed with 2 N HCl solution (4 x 10 mL), water (10 mL) and brine (10 mL). The solvent w as dried over MgSO 4 and evaporated to give Leu enkephalin Aza Analog ( 5. 40 ). White microcrystals (0.278 g, 70%); mp 89.0 91.0 o C ; D 20 13.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6

PAGE 136

136 8.17 7.91 (m, 2H), 7.42 7.25 (m, 20H), 6.90 (dd, J = 5.8, 2.7 Hz, 2H), 6.46 (d, J = 7.9 Hz, 1H), 5.06 (s, 2H), 5.03 4.82 (m, 3H), 4.77 4.37 (m, 2H), 4.32 4.16 (m, 2H), 3.93 3.68 (m, 4H), 3.62 (s, 3H), 3.01 (dd, J = 13.9, 4.2 Hz, 2H), 2.94 2.65 (m, 2H), 1.77 1.41 (m, 3H), 0.91 0.84 (m, 6H); 13 C NMR (75 MHz DMSO d 6 170.3, 168.9, 157.4, 156.5, 138.4, 137.8, 137.5, 131.1, 130.9, 130.8, 129.0, 128.8, 128.7, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.6, 127.5, 127.3, 114.9, 70.5, 69.7, 67.0, 65.9, 57.0, 52.3, 42.7, 42.0, 37.1, 31.8, 2 4.6, 23.5, 21.9; HRMS (ESI) calcd for C 43 H 50 N 6 O 9 Na [M+Na] + 817.3531 found 817.3551.

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137 CHAPTER 6 OXYAZAPEPTIDES: SYNTHESIS, STRUCTURE DETERMINATION AND CONFORMATIONAL ANALYSIS 6.1 Introduction Peptides and proteins play vital roles in biologi cal and physiological processes and n atural peptides are widely used as drugs. However, they often need to be modified to circumvent certain problems related to drug delivery including; (i) affinity for specific receptors, (ii) metabolic stability towards endogeno us proteases, (iii) appropriate bio distribution and bio availability and (iv) duration of action. 1 64 165 Such problems have been addressed by the design of peptidomimetics which may be devoid of many of the undesirable properties of natural peptides. Once the structure of a natural active peptide is known, key amino acid residues necessary for receptor recognition can be identified by single amino acid modification of the peptide ligand using novel substituted amino acids and/or amide bond replacemen ts. Figure 6 1 General structures of peptide and peptide like molecules A ) Native peptides B ) Depsipeptides C ) Azapeptides D ) Hybrid azadepsipeptides E ) Oxyazapeptides Reproduced with permissi on from The Journal of Organic Chemistry 2013 78 8502 8509 Copyright 2013 American Chemical Society

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138 The modification of the peptide backbone (Figure 6 1A) with a heteroatom le ading to depsipeptides (Figure 6 1B) 166 and azapeptides (Figure 6 1C) 167 has p roved to be a useful strategy in the design peptidomimetics. Azapeptides are a family of peptidomimetics in which substitution of the easily rotatable C C(O) bond in natural peptides by a more rigid urea N C(O) bond caus es significant changes to both chemical and biological properties (Fig ure 6 1C). Azapeptides prefer a limited conformation al space with dihedral angle values close to those of a polyproline II helix and other types of turns. 138,170 A systematic study of their effects of sequential replacement of amino acid residues by their aza counterparts on backbon e conformation and acti vity is 138,144 The introduction of an hydroxy acid into a peptide sequence results in the formation of an ester bond, also called a depsipeptide bond (Fig ure 6 1B). 87 to or investigating the effect of backbone H bonds on the 3D structure formation and stability of proteins. 1 7 1 Recently, many depsipeptides, such as enniatins 1 7 2 and cycloocta depsipeptide PF1022A. 1 7 3 have been found to be biologically active. Thus, Dyker et al. synthesized a novel class of pseudopeptide s 6 1D) and applied the method to the synthesis of a bis aza analogue of the antiparasitic cyclooctadepsipeptide PF1022A. 1 7 4 In the present study, we describe the de novo de sign, synthesis, and characterization of oxyazapeptides in which an amino acid is repla ced by an aza hydroxy acid (Figure 6 1E). Oxyazapeptides can be considered as the depsipeptide analogues of azapeptides where the amino group of an aza amino acid is r eplaced by to known limited

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139 conformational space of azapeptides 1 75,137 has been studied computationally. Conformational analysis based on molecular mechanics calculations revealed that oxyazapeptides should adopt a turn secondary structure and enjoy greater conformational freedom render ing them more adaptive to varying steric demand s of biological interactions. Thus insertion of aza hydroxy acid units into biologically active peptides, activity relationships. The newly developed synthetic protocol was validated by the synthesis of an oxyaza analogue of leuenkephalin an endogen ous neurotransmitter. 6.2 Results and Discussion 6.2.1 Synthesis of Oxyaza Di Tri and T etrapeptides The reaction of hydroxylamine 6. 1a with benzaldehyde afforded corresponding oxime 6. 2 which was reduced with sodium cyanoborohydride (NaCNBH 3 ) to give N benzylhydroxylamine 6. 3 in 70% overal l yield (Scheme 6 1). Scheme 6 1 Synthesis of N benzylhydroxylamine Amino acid ester hydrochloride salts 6. 4a d were converted into active acyl imidazoles 6. 5a d by reaction with carbonyldiimidazole (CDI) in the presence of 2.5 equiv. 6. 5a d were taken to the next step without further purification. Stirring 6. 5a d with hydroxylamine 6. 1a N methylhydroxylamine 6. 1b or N benzylhydroxylamine 6. 3 at 20 o C for 16 h in dry THF containing one equiv. of DIPEA afforded free oxyaza dipeptides 6. 6a f in 85 92%

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140 yields (Scheme 6 2 Table 6 1 ). No column chromatography was needed to purify the products a nd a si mple extractive work up using 2 N HCl gave oxyaza dipeptides displaying satisfactory 1 H and 13 C NMR spectra. Scheme 6 2 Construction of oxyaza dipeptide Table 6 1 Construction of oxyaza dipeptides 6. 6a f Entry R 3 R 1 R 2 6. 6 ,Yield a % 1 H CH(CH 3 ) 2 C(CH 3 ) 3 HO Aza Gly Val O t Bu 6. 6a 88 2 CH 3 CH(CH 3 ) 2 C(CH 3 ) 3 HO Aza Ala Val O t Bu 6. 6b 90 3 CH 2 Ph CH(CH 3 ) 2 C(CH 3 ) 3 HO Aza Phe Val O t Bu 6. 6c 85 4 CH 3 CH 2 CH(CH 3 ) 2 CH 3 HO AzaAla Leu OMe 6. 6d 92 5 CH 2 Ph CH 2 CH(CH 3 ) 2 CH 3 HO AzaPhe Leu OMe 6. 6e 87 6 CH 3 CH 2 Ph CH 3 HO AzaAla Phe OMe 6. 6f 90 a Isolated yield N Acylbenzotriazoles are advantageous reagents to construct peptides, peptidomimetics and peptide conjugates. N Pg ( aminoacyl)benzotriazoles 6. 7a g were prepared following our reported procedure, 112,116 then coupled with free oxyaza dipeptides 6. 6a f in dry THF containing one equiv. of DIPEA and catalytic amount of DMAP (Scheme 6 3 Table 6 2 ) to give N Pg oxyaza tripeptide esters 6. 8a j in 85 93% yields. In an att empt to show that no racemization occurs during any stage of the reactions, we also conducted reactions between Cbz Ala Bt 6. 7b and 6. 7b+ (both L and DL forms) and oxyaza dipeptide 6. 6a The absence of racemization in the oxyazapeptide ( 6. 8b+ ) was deduced from the 1 H NMR and retention of chirality was further confirmed by chiral HPLC analysis using a ( S,S ) Welk O1column (MeCN/H 2 O 50:50, flow rate 0.15 mL/min, detection at 210 nm). The diastereomer 6. 8b showed a

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141 single retention t ime peak at 1 3.56 min, while the corresponding diastereomeric mixture ( 6. 8b+ ) showed two well defined separate peaks at 13.46 and 16. 52 min. In previous studies we have demonstrated that chirality of the reaction product is maintained on N acylation with N acylbenzot riazoles for peptides, depsipeptides and azapeptides. 112,116,176 Table 6 2 Preparation of oxyaza tripeptides 6. 8a j Entry RCOBt 6. 7a g 6. 6a f oxyaza tripeptide 8a j ,Yield a % 1 Cbz Gly Bt 6. 7a 6. 6b Cbz Gly O AzaAla Val O t Bu 6. 8a, 87 2 Cbz Ala Bt 6. 7b 6. 6a Cbz Ala O Aza Gly Val O t Bu 6. 8b, 87 3 Cbz DL Ala Bt 6.7b + 6. 6a Cbz DL Ala O AzaGly Val O t Bu 6.8b+ 90 4 Cbz Phe Bt 6. 7c 6. 6d Cbz Phe O AzaAla Leu OMe 6. 8c, 91 5 Cbz Phe Bt 6. 7c 6. 6e Cbz Phe O AzaPhe Leu OMe 6. 8d, 90 6 Cbz Phe Bt 6. 7c 6. 6f Cbz Phe O AzaAla Phe OMe 6. 8e, 95 7 Boc Gly Bt 6. 7d 6. 6c Boc Gly O AzaPhe Val O t Bu 6. 8f, 87 8 Boc Ala Bt 6. 7e 6. 6c Boc Ala O AzaPhe Val O t Bu 6. 8g, 85 9 Fmoc Leu Bt 6. 7f 6. 6b Fmoc Leu O AzaAla Val O t Bu 6. 8h, 89 10 Fmoc Leu Bt 6. 7f 6. 6c Fmoc Leu O AzaPhe Val O t Bu 6. 8i 93 11 Fmoc Phe Bt 6. 7g 6. 6c Fmoc Phe O AzaPhe Val O t Bu 6. 8j 92 a Isolated yield Scheme 6 3 Synthesis of oxyaza tri and tetra peptides

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142 Table 6 3 Preparation of oxyaza tetrapeptides 6. 10a f Entry 6. 9a e 6. 6b e oxyaza tetrapeptide 6. 10a f Yield a % 1 Cbz Ala Met Bt 6. 9a 6c Cbz Ala Met O AzaPhe Val O t Bu 6. 10a 90 2 Cbz Phe Met Bt 6. 9b 6b Cbz Phe Met O AzaAla Val O t Bu 6.10b 86 3 Cbz Ala Met Bt 6. 9a 6d Cbz Ala Met O AzaAla Leu OMe 6.10c 84 4 Boc Gly Gly Bt 6. 9c 6d Boc Gly Gly O AzaAla Leu OMe 6.10d 87 5 Boc Ala Ala Bt 6. 9d 6e Boc Ala Ala O AzaPhe Leu OMe 6.10e 84 6 Boc Ala Gaba Bt 6. 9e 6b Boc Ala Gaba O AzaAla Val O t Bu 6.10f 88 a Isolated yield Oxyazapeptide esters 6. 10a f were prepared in solution by treatment of N Pg ( dipeptidoyl)benzotriazoles 6. 9a e with free oxyaza dipeptides 6. 6b e in THF containing one equiv. of DIPEA and a catalytic amount of DMAP for 16 hr at 20 o C. All compounds were isolated without column chromatography (Scheme 6 3, Table 6 3). The target compounds were characterized by 1 H NMR, 13 C NMR, and elemental analysis. No detectable racemization of the N Pg oxyaza tetrapeptides esters was observed by chi ral HPLC analysis. 6.2.2 Validation of the Synthetic M ethodology We aimed to utilize our methodology and to show general applicability and scope of the biologically impor tant pentapeptide Leuenkephalin 15 2 Leuenkephalin is an endogenous opioid peptide neurotransmitter found naturally in the brains of many animals, including humans. Its amino acid sequence is Tyr Gly Gly Phe Leu. Protected tyrosine 6. 11 was first activated and coupled with Gly Gly to give tripeptide 6. 12 Then by using the standard benzotriazole methodology the acid group was activated and coupling of compound 6. 13 with free oxyaza dipeptide 6. 6e in dry THF in the presence of 1.0 equiv. of DIPEA and a catalytic amount of DMAP (10%) gave the target oxyaza analog of Leuenkephalin 14 (Scheme 6 4).

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143 Scheme 6 4 Synthesis of oxyaza analog of Leuenkephalin 6 14 6.2.3 X Ray Structure D etermination It was deemed important to confirm the structure of a representative example of this new family of peptidomimetics. Thus, an X ray crystal structure was obtained of compound 6. 8h (Fig ure 6 2), which crystallizes in the orthorhombic space group P2 1 2 1 2 1 Thi s unambiguously confirmed the structure and absolute configuration of 6. 8h Figure 6 2 X ray crystal structure of 6. 8h 6.2.4 Conformational A nalysis The c onformational behaviour of oxyazapeptides in comparison with azapeptides would be the most interesting to study. This can be done by rotations around the

PAGE 144

144 common dihedral angles Here, the angle denotes rotation about the N N (or O N ) bond, and the angle is rotation about the bond linking the N and the carbonyl carbon (Figure 6 3). Rotations around these angles are expected to proceed differently for aza and oxyazapeptides, because oxyazapeptides have a set of distinct features such as (i) the absence of hydrogen at the atom, (ii) shorter C O and O N bond lengths comparing to C N and N N bond lengths, and (iii) much less double bond character of the ester C O bond comparing to the amide C N bond. To make the calculations simpler, model aza 6. 15 and oxyaza dipeptide 6. 16 were drawn by removing the bulky protecting groups and substituting them with methyl groups. These structures were geometry optimized using the MMX force field (a s implemented in the PCModel v. 9.3 software). The optimized structures are displayed in Fig ur e 6 3. One can see that the main difference between 6. 15 and 6. 16 is the dihedral angle equal to 178 and 103.5 in 6. 15 and 6. 16 respectively. In azapeptide 6. 15 the dihedral angle is trans due to certain double bond character of the hydrazine N N bond. By contrast, oxyazapetide 6. 16 renders a gauche conformation, because rotation around the O N bond is much less hindered. Figure 6 3 Optimized structures of azapeptide 6. 15 and oxyazapeptide 6. 16

PAGE 145

145 Energy barriers to rotations can also provide interesting information. To calculate rotational barriers, the optimized structures 6. 15 and 6. 16 were subjected to the Dihedral Driver procedure, as implemented in PCModel. The torsional energy plots are shown in Fig ure 6 4. The most striking difference between the aza and oxyazapeptide is the barrier to rotation around the N(O) N bond. In azapeptide 6. 15 the barrier is as high as 35 kcal/mol, while in oxyazapeptide 6. 16 it is only 7 kcal/mol. The shape of the pote ntial energy profile also exhibits a sharp difference: it is a steep double maximum in 6. 15 and a shallow single maximum in 6. 16 The symmetric maxima on the torsional energy plot of 6. 15 occur almost exactly at 90 and 90 while the single maximum of 6. 16 corresponds to a pure cis conformation. In structure 6. 15 these maxima can be associated with the rehybridization in the hydrazine fragment and a significant repulsion by the methyl groups, while in 6. 16 the maximum is due to the shorter N O bond. As the influence of the varying heteroatom drops as the distance increases, rotation around N N proceeds in a very similar way in both structures. As seen in Fig ure 6 4, (right column) the barrie r heights (both close to 5 kcal/mol) and the shapes of the torsional energy profiles are very similar in structures 6. 15 and 6. 16 It was found that inclusion of implicit solvation does not significantly change the conformational behaviour, and the gas pha se and solvation calculations were in a good qualitative agreement. To test this, we capped the free amino groups in 6. 15 and 6. 16 with acetyl groups and carried out calculations with the GB/SA model Therefore the rest of the conformational analysis was do ne with gas phase force field calculations.

PAGE 146

146 Interestingly, the ray structure (Fig ure 6 2) of 6. 8h are in excellent agreement with these gas phase calculations. In the solid state, the angle is 91.3(2) o while o Figure 6 4 Torsional energy plots for dihedral angles (left) and (right) in azapeptide 6. 15 (upper row) and oxyazapeptide 6. 16 (lower row) 6.2.5 Computational S tudy of Turn I nducing It is known that azaamino acid residues are instrumental in inducing turns and other helical structures in peptides. 169,177 It is believed that this ability stems from a restricted rotation around the N partial double bonded character of the latter. Conformational analysis of a simple dipeptide with one azaamino unit (For Ala azaAla NH 2 ) revealed that a global minimum structure was one having a set of dihedral angles consistent with a turn structural m otif. 1 6 9 As the oxyaza unit enjoys at least o ne additional degree of freedom ( rot ation around the C O ester bond)

PAGE 147

147 one would expect oxyazapeptides to attain a turn structure with even greater ease. To explore the ability of the oxyaza unit to induce a t urn, we ran a full conformational search (MMX force field) of a model For Ala Ala NH 2 dipeptide in which one of the C atoms was replaced by nitrogen and the adjacent amide N was replaced by oxygen. For consistency, this model oxyaza dipeptide was made iso steric to For Ala azaAla NH 2 previously published by Lee et al. 169 The conformational search found that a turn conformer was the global minimum on the potential energy surface. Figure 6 5 Molecular structures of turn structures of oxyazapeptide 6. 17 (left) and azapeptide 6. 18 (right); the hydrogen bonds are indicated by the dotted lines This oxyaza dipeptide 6. 17 conformer is displayed in Figure 6 5, with the isosteric azadipeptide 6. 18 also shown for comparison. The dihedral angles 1 1 and 2 2 characterizing rotations in oxyaza dipeptide 6. 17 are found to be 67 109 109 and 2 which is very close to the type II turn structure. 1 37 ,1 75 As seen in Figure 6 5, the isosteric azadipeptide 6. 18 also makes a turn conformation, but, according to the set of the dihedral angles 77 76 179 and 3.7 the hydrazine moiety prefers a trans conformation, which can be explained by the steric preference of the adjacent urea group.

PAGE 148

148 Comparative analy sis of hydrogen bond contacts in structures 6. 17 and 6. 18 also to est As seen in Figure 6 5, an unobstructed C10 (ten membered intramolecular cycle) hydrogen bond is formed in th e oxyaza dipeptide 6. 17 which is characterized by the O1 H(N4) separation of 2.25 and the O1 H(N4) N4 angle of 167 In the X ray structure of oxyaza tripeptide 6. 8h (Fig ure 6 2), the respective contact length is slightly longer (2.66 ) and the O H N angle is somewhat smaller (155 which is probably due 6. 18 the separation and angle are 2.61 and 154.2 respectively which render the respective hydrogen bond a weak er one compared to that in 6. 17 Another structural feature of azapeptides, which oxyazapeptides are free of, is the O H(N2) contact. In structure 6. 18 this contact (2.10 and 135.7 ) making a C7 hydrogen bond, makes the structure a turn rather than a turn. On the other hand, O H(N2) should be a weaker hydrogen bond than O1 H(N4) (because of the strong angular dependence of hydrogen bond strength) and therefore can be considered as one exerting an assistance to the primary O1 H(N4) in keeping the tu rn conformation. In contrast to 6. 18 the turn conformation of oxyaza structure 6. 17 is supported by a single although stronger hydrogen bond O1 H(N4). As a result, the increased backbone flexibility and a stronger C10 hydrogen bond in oxyazapeptides can put them on a par with azapeptides or even make better turn inducers that can be used in the synthesis of artificial peptidomimetics.

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149 6.3 Conclusions We have designed, synthesized and structurally studied a new family of C bond is replaced by an O N bond. This class of compounds are conformationally more labile, due to a lower barrier to rotation around the O N and C O bonds, compared to those around the N C and C N bonds in native peptides As a result, oxyazapeptides enjoy a higher degree of conformational freedom that might make them potentially more adaptable to varying steric demand in receptor binding. The conformational analysis suggests that the oxyaza moiety can effectively induce turns in peptidomimetics and thus se rve as useful synthetic auxiliaries in the design of small peptide based drugs. More importantly, these newly discovered oxyazapeptides can be a useful tool for drug discovery and for the targeted design of biologics. 6 .4 Experimental Section 6.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. NMR spectra were recorded with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal referen ce. Reaction progress was monitored by thin layer chromatography (TLC) and visualized by UV light. Elemental analyses were performed on a Carlo Erba EA 1108 instrument. DCM was dried and distilled over CaH 2 whereas tetrahydrofuran (THF) was used after dis tillation over Na benzophenone. Carbonyldiimidazole (CDI), hydroxylamine 6. 1a N methylhydroxylamine 6. 1b sodium cyanoborohydride, benzaldehyde and L amino methyl/ tert butyl ester hydrochloride 6. 4a d were purchased from chemical supply

PAGE 150

150 companies and used without further purification. N Pg ( aminoacyl)benzotriazoles 7a g, N Pg ( dipeptidoyl)benzotriazoles 6. 9a d and Cbz Tyr (OBn) Gly Gly Bt 6. 13 were prepared according to literature methods. 6.4.2 Computational D etails All calculations were carried out with the PCModel software ver. 9.3, Serena Software. The MMX force field was used. The ground state structures were identified through full conformational searches using the GMMX routine of PCModel. The implicit solvent mo del used was GB/SA (Generalized Born/Surface Area) with the analytical method of Still. The solvent dielectric constant was taken 78.30 (water) and the internal dielectric constant was taken 1, which are default settings in PCModel. Full conformational sea rch was also done with the GB/SA solvent method. The torsional potentials were calculated with the Dihedral Driver procedure as implemented in PCModel; start and final angles were 180 and 180, respectively, with the step equal to 10 6.4.3 General Method s for the Preparation of O xyaza dipeptide 6. 6a f To a suspension of L amino methyl/ tert butyl ester hydrochloride 6. 4a d (1.0 mmol, 1.0 equiv. ) in DCM (20 mL) at 20 C were added 2.5 equiv. of DIPEA and CDI (carbonyldiimidazole, 1.1 equiv. ). The reaction mixture was stirred for 3 h at rt, and the organic layer was washed with water (2 20 mL), NaHCO 3 (3 20 mL) and brine solution (2 20 mL). The organic layer was dried over MgSO 4 and evaporated under vacuum to give oily mono substituted imidazole derivative s 6. 5a d The residue 6. 5a d (1.0 equiv. ) was dissolved in dry THF (20 mL) and reacted with N alkylhydroxylamine 6. 1a,b or 6. 3 (1.0 equiv. ) in the presence of DIPEA (1.0 equiv. ) at 20 o C overnight. Each

PAGE 151

151 reaction mixture s was poured into a separatory funnel and washed with water (2 20 mL), 2 N HCl (3 20 mL), brine solution (2 20 mL), dried over MgSO 4 and the solvent evaporated under vacuum to give oxyazadipeptide 6. 6a f HO AzaGly Val O t Bu ( 6. 6a). White microcrystals (0.408 g, 88%); mp 125.0 127.0 o C; D 20 10.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.30 (s,1H), 6.42 (d, J = 9.0 Hz, 1H), 4.28 (dd, J = 9.0, 4.5 Hz, 1H), 2.24 2.06 (m, 1H), 1.45 (s, 9H), 0.94 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 TOF) m/z: [M + Na] + Calcd for C 10 H 20 N 2 O 4 Na 255.1315; Found 255.1326. HO AzaAla Val O t Bu ( 6. 6b). White microcrystals (0.443 g, 90 %) ; mp 88.0 91.0 o C; [ D 20 4.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 8.7 Hz, 1H), 4.22 (dd, J = 8.9, 4.7 Hz, 1H), 3.13 (s, 3H), 2.18 2.11 (m, 1H), 1.46 (s, 9H), 0.95 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 161.3, 82.2, 58.7, 38.8, 31.4, 28.2, 19.2, 17.9; Anal. Calcd for C 11 H 22 N 2 O 4 : C, 53.64; H, 9.00; N, 11.37. Found: C, 53.52; H, 9.27; N, 11.04. HO AzaPhe Val O t Bu ( 6. 6c). White microcrystals (0.548 g, 85%) ; mp 90.0 92.0 o C; D 20 20.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.30 7.12 (m, 5H), 6.41 (d, J = 9.0 Hz, 1H), 4.64 4.45 (m, 2H), 4.19 (dd, J = 8.7, 4.8 Hz, 1H), 2.16 1.97 (m, 1H), 1.39 (s, 9H), 0.85 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H) ; 13 C NMR (75 MHz, CDCl 3 172.0, 160.7, 137.1, 128.9, 128.5, 127.5, 82.1, 77.7, 77.3, 76.9, 58.7, 54.8, 31.6, 28.2, 19.1, 17.9; Anal. Calcd for C 17 H 26 N 2 O 4 : C, 63.33; H, 8.13; N, 8.69. Found: C, 63.38; H, 8.40; N, 8.65.

PAGE 152

152 HO AzaAla Leu OMe ( 6. 6d). Low melting solid (0.402 g, 92%) ; D 20 26.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 8.1 Hz, 1H), 4.38 4.29 (m, 1H), 3.67 (s, 3H), 3.07 (s, 3H), 1.70 1.46 (m, 3H), 0.87 (d, J = 6.3 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 A nal. Calcd for C 9 H 18 N 2 O 4 : C, 49.53; H, 8.31; N, 12.84. Found: C, 49.34; H, 8.60; N, 12.46. HO AzaPhe Leu OMe ( 6. 6e). White microcrystals (0.256 g, 87%) ; mp 114.0 116.0 o C; [ D 20 12.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.13 (m, 5H), 6.30 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 15.0 Hz, 1H), 4.52 (d, J = 15.0 Hz, 1H), 4.42 4.34 (m, 1H), 3.65 (s, 3H), 1.70 1.48 (m, 3H), 0.88 (d, J = 6.3 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 21.9; Anal. Calcd for C 15 H 22 N 2 O 4 : C, 61.21; H, 7.53; N, 9.52. Found: C, 61.30; H, 7.92; N, 9.49. HO AzaAla Phe OMe ( 6. 6f). Low melting solid (0.454 g, 90%) ; [ D 20 20.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.18 (m, 3H), 7.16 7. 08 (m, 2H), 6.34 (d, J = 7.8 Hz, 1H), 4.76 4.60 (m, 1H), 3.68 (s, 3H), 3.10 3.02 (m, 5H); 13 C NMR (75 MHz, CDCl 3 38.4; Anal. Calcd for C 12 H 16 N 2 O 4 : C, 57.13; H, 6.39; N, 11.10. Found: C, 57.52; H, 6.69; N, 10.73. 6.4.4 General M et hods for the Preparation of Oxyaza Tri and Tetra peptide 6. 8a j, 6. 10a f N Pg ( A minoacyl) benzotriazoles 6. 7a g or N Pg ( dipeptidoyl)benzotriazoles 6. 9a d (1.0 mmol, 1.0 equiv. ) was dissolved in dry THF (20 mL) and reacted with oxyaza dipeptides 6. 6a f (1.0 equiv. ) in the presence of DIPEA (1.0 equiv. ) and a 10% cat alytic amount of DMAP at 20 o C overnight. Each reaction mixture was poured into a separatory funnel and washed with water (2 20 mL), 2 N

PAGE 153

153 HCl (3 20 mL), brine solution (2 20 mL), dried over MgSO 4 and evaporated under vacuum to give oxyaza tripeptide 6. 8a j or oxyaza tetrapeptide 6. 10a f which were characterized by 1 H, 13 C NMR and elemental analysis. Cbz Gly O AzaA la Val O t Bu ( 6. 8a). White microcrystals (0.381 g, 87%) ; mp 80.0 82.0 o C; [ D 20 13.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.17 (m, 5H), 6.01 (d, J = 8.4 Hz, 1H), 5.52 (t, J = 5.6 Hz, 1H), 5.07 (s, 2H), 4.22 (dd, J = 8.4, 4.8 Hz, 1H), 3.99 (d, J = 5.7 Hz, 2H), 3.15 (s, 3H), 2.15 2.04 (m, 1H), 1.38 (s, 9H), 0.87 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 158.8, 156.8, 136.1, 128.7, 128.5, 128.3, 82.1, 67.7, 58.9, 42.2, 38.6 31.6, 28.2, 19.1, 17.9; Anal. Calcd for C 21 H 31 N 3 O 7 : C, 57.65; H, 7.14; N, 9.60. Found: C, 57.75; H, 7.52; N, 9.40. Cbz Ala O AzaGly Val O t Bu ( 6. 8b). Low melting solid (0.381 g, 87%) ; D 20 17.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.20 (m, 5H), 6.28 (d, J = 8.7 Hz, 1H), 5.69 (d, J = 7.2 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 5.01 (d, J = 12.6 Hz, 1H), 4.45 4.35 (m, 1H), 4.26 (dd, J = 8.7, 4.8 Hz, 1H), 2.25 2.00 (m, 1H), 1.48 1.33 (m, 12H), 0.89 (d, J = 6.6 Hz, 3H ), 0.85 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 48.9, 31.6, 28.2, 19.0, 17.9, 15.4; Anal. Calcd for C 21 H 31 N 3 O 7 : C, 57.65; H, 7.14; N, 9.60. Found: C, 57.50; H, 7.40; N, 9.31. Cbz ( DL )Ala O AzaGly Val O t Bu ( 6. 8b Low melting solid (0.394 g, 90%) ; 1 H NMR (300 MHz, CDCl 3 7.15 (m, 5H), 6.24 (br s, 1H), 5.63 5.52 (m, 1H), 5.16 4.98 (m, 2H), 4.50 4.37 (m, 1H), 4.32 4.20 (m, 1H), 2.20 2.04 (m, 1H), 1.47 1.36 (m, 12H), 0.92 0.82 (m, 6H); 13 C NMR (75 MHz, CDCl 3

PAGE 154

154 158.6, 156.2, 136.1, 128. 7, 128.4, 128.3, 82.3, 67.6, 67.5, 58.5, 49.1, 48.9, 31.6, 28.2, 19.2, 19.1, 17.8, 15.5.; Anal. Calcd for C 21 H 31 N 3 O 7 : C, 57.65; H, 7.14; N, 9.60. Found: C, 57.55; H,7.42; N, 9.22. Cbz Phe O AzaAla Leu OMe ( 6. 8c). White microcystals (0.455 g, 91%) ; mp 123.0 125.0 o C; D 20 17.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.00 (m, 10H), 6.46 (d, J = 8.1 Hz, 1H), 5.31 5.24 (m, 1H), 5.08 (d, J = 12.0 Hz, 1H), 4.99 (d, J = 12.0 Hz, 1H), 4.50 4.32 (m, 2H), 3.68 (s, 3H), 3.10 3.03 (m, 2H), 2.85 (s, 3H), 1.70 1.55 (m, 3H), 0.87 (d, J = 5.4 Hz, 3H), 0.84 (d, J = 4.8 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 128.3, 127.9, 67.8, 54.9, 52.4, 52.1, 41.1, 38.0, 37.3, 24.8, 23.2, 21.9; Anal. Calcd fo r C 26 H 33 N 3 O 7 : C, 62.51; H, 6.66; N, 8.41. Found: C, 62.76; H, 6.80; N, 8.56. Cbz Phe O AzaPhe Leu OMe ( 6. 8d). Low melting solid (0.518 g, 90%) ; D 20 41.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 6.95 (m, 15H), 6.67 (d, J = 7.8 Hz, 1H), 5.62 (d, J = 4.5 Hz, 1H), 5.08 4.93 (m, 2H), 4.64 (d, J = 15.0 Hz, 1H), 4.50 (d, J = 15.0 Hz, 1H), 4.29 4.18 (m, 1H), 3.64 (s, 3H), 2.77 (d, J = 7.2 Hz, 2H), 1.70 1.52 (m, 3H), 0.87 (d, J = 5.4 Hz, 3H), 0.86 (d, J = 5.7 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 173.7, 170.4, 158.7, 156.7, 135.8, 135.4, 135.1, 129.3, 129.2, 129.2, 128.8, 128.6, 128.4, 128.3, 127.9, 127.8, 67.6, 54.9, 54.8, 52.4, 52.3, 40.9, 36.7, 24.8, 23.2, 21.9; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 32 H 37 N 3 O 7 Na 598.2524; Found 598.2550. Cbz Phe O Aza Ala Phe OMe ( 6. 8e). White microcystals (0.507 g, 95%) ; mp 132.0 134.0 o C; D 20 24.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 6.98 (m, 15H), 6.57 (d, J = 7.8 Hz, 1H), 5.57 (d, J = 5.4 Hz, 1H), 5.02 (d, J = 12.3 Hz, 1H), 4.93 (d, J = 12.3 Hz, 1H ), 4.63 (q, J = 7.2 Hz, 1H), 4.40 (q, J = 6.8 Hz, 1H), 3.60 (s, 3H),

PAGE 155

155 3.11 2.98 (m, 4H), 2.84 (s, 3H); 13 C NMR (75 MHz, CDCl 3 156.5, 136.7, 135.9, 134.9, 129.5, 129.4, 129.1, 128.8, 128.6, 128.6, 128.3, 127.8, 127.1, 67.7, 55.0, 54.9, 52.4, 38.1, 38.0, 37.3; Anal. Calcd for C 29 H 31 N 3 O 7 : C, .65.28; H, 5.86; N, 7.88. Found: C, 65.40; H, 6.11; N, 7.92. Boc Gl y O AzaPhe Val O t Bu ( 6. 8f). Low melting solid (0.417 g, 87%) ; D 20 11.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.20 (m, 5H), 6.03 (d, J = 8.4 Hz, 1H), 5.24 (t, J = 5.4 Hz, 1H), 4.81 (d, J = 15.3 Hz, 1H), 4.72 (d, J = 15.3 Hz, 1H), 4.27 (dd, J = 8.4, 3.3 Hz, 1H), 3.90 3.66 (m, 2H), 2.20 2.04 (m, 1H), 1.43 (s, 9H), 1.39 (s, 9H), 0.91 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 8.4 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 171.0, 168.4, 158.1, 155.8, 135.2, 128.9, 128.4, 127.8, 81.8, 80.4, 59.0, 54.5, 41.4, 31.2, 28.2, 28.0, 18.8, 17.8; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 24 H 37 N 3 O 7 Na 502.2543; Found 502.2524. Boc Ala O AzaPhe Val O t Bu ( 6. 8g). Low melting solid (0.420 g, 85%) ; D 20 20.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.20 (m, 5H), 5.76 (d J = 8.4 Hz, 1H), 5.16 (t, J = 5.7 Hz, 1H), 4.82 (d, J = 15.3 Hz, 1H), 4.69 (d, J = 15.3 Hz, 1H), 4.28 (dd, J = 8.4, 4.5 Hz, 1H), 3.30 (q, J = 6.0 Hz, 2H), 2.50 (dd, J = 6.8, 5.3 Hz, 2H), 2.15 2.03 (m, 1H), 1.41 (s, 9H), 1.37 (s, 9H), 0.88 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 128.0, 82.3, 79.6, 58.5, 54.3, 33.1, 31.6, 31.6, 28.4 28.0, 18.8, 17.7; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 25 H 39 N 3 O 7 Na 516.2687; Found 516.2680. Fmoc Leu O AzaAla Val O t Bu ( 6. 8h). White microcystals (0.518 g, 89%) ; mp 113.0 115.0 o C; D 20 +22.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 7.8 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H),

PAGE 156

156 6.29 (d, J = 8.7 Hz, 1H), 5.37 (d, J = 6.6 Hz, 1H), 4.40 4.22 (m, 4H), 4.16 (t, J = 7.2 Hz, 1H), 3.19 (s, 3H), 2.20 2.05 (m, 1H), 1.72 1.55 (m, 3H), 1.41 (s, 9H), 1.00 0.80 (m, 12H); 13 C NMR (75 MHz, CDCl 3 3.8, 141.5, 128.0, 127.3, 125.2, 120.2, 81.9, 67.6, 59.1, 52.0, 47.3, 40.5, 38.4, 31.6, 28.2, 25.1, 22.7, 22.2, 19.1, 18.0; Anal. Calcd for C 32 H 43 N 3 O 7 : C, 66.07; H, 7.45; N, 7.22. Found: C, 65.7; H,7.77; N,7.58. Fmoc Leu O AzaPhe Val O t Bu ( 6. 8i). White mic rocystals (0.612 g, 93%) ; mp 116.0 119.0 o C; D 20 13.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 7.5 Hz, 2H), 7.54 (d, J = 7.2 Hz, 2H), 7.44 7.20 (m, 9H), 6.41 (d, J = 8.4 Hz, 1H), 5.31 (d, J = 6.6 Hz, 1H), 4.95 (d, J = 15.3 Hz, 1H), 4.75 (d, J = 15.3 Hz, 1H), 4.40 4.30 (m, 3H), 4.22 4.08 (m, 2H), 2.21 2.10 (m, 1H), 1.47 (s, 9H), 1.42 1.17 (m, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.3, Hz, 6H); 13 C NMR (75 MHz, CDCl 3 8.6, 156.3, 143.6, 141.3, 135.3, 128.9, 128.3, 127.8, 127.1, 127.1, 125.0, 120.0, 81.6, 67.4, 59.2, 54.7, 51.7, 47.0, 39.8, 31.3, 28.0, 24.5, 22.4, 21.8, 18.9, 17.9; Anal. Calcd for C 38 H 47 N 3 O 7 : C, 69.38; H, 7.20; N, 6.39. Found: C, 69.18; H, 7.40; N, 6.60 Fmoc Phe O AzaPhe Val O t Bu ( 6. 8j). White microcystals (0.636 g, 92%) ; mp 107.0 109.0 o C; D 20 21.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 7.8 Hz, 2H), 7.55 7.44 (m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.35 7.22 (m, 10H), 7.11 (d, J = 7.8 Hz, 2H), 6.46 (d, J = 8.7 Hz, 1H), 5.33 (d, J = 5.7 Hz, 1H), 4.70 (s, 2H), 4.40 4.30 (m, 3H), 4.20 4.11 (m, 2H), 2.92 2.75 (m, 2H), 2.20 2.10 (m, 1H), 1.46 (s, 9H), 0.95 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 170.2, 158.4, 156.2, 143.6, 141.2, 135.3, 135.0, 129.1, 129.0, 128.3, 127.8, 127.6, 127.1,

PAGE 157

1 57 127.1, 125.0, 120.0, 81.6, 67.5, 59.2, 54.8, 54.3, 47.0, 36.7, 31.3, 28.0, 19.0, 18.0; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 41 H 45 N 3 O 7 Na 714.3150; Found 714.3186 Cbz Ala Met O AzaPhe Val O t Bu ( 6. 10a). White microcystals (0.593 g, 90%) ; mp 83.0 85.0 o C; D 20 31.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.14 (m, 11H), 6.46 (d, J = 8.7 Hz, 1H), 5.89 (d, J = 7.8 Hz, 1H), 5.16 4.99 (m, 2H), 4.93 (d, J = 15.3 Hz, 1H), 4.55 (d, J = 14.7 Hz, 1H), 4.40 4.05 (m, 3H), 2.37 2.10 (m, 3H), 2.09 1.71 (m, 5H), 1.43 (s, 9H), 1.28 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H).; 13 C NMR (75 MHz, CDCl 3 36.2, 135.4, 129.0, 128.6, 128.5, 128.3, 128.0, 127.9, 81.8, 67.1, 65.9, 59.3, 54.8, 51.4, 31.4, 29.8, 29.2, 28.1, 19.2, 19.1, 18.1, 15.2; Anal. Calcd for C 33 H 46 N 4 O 8 S: C, 60.16; H, 7.04; N, 8.50. Found: C, 60.10; H,7.14; N, 8.81. Cbz Phe Met O AzaAla Val O t Bu ( 6. 10b). White microcystals (0.567 g, 86%) ; mp 58.0 60.0 o C; D 20 23.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 6.3 Hz, 1H), 7.30 6.96 (m, 11H), 6.61 6.52 (m, 1H), 4.98 (d, J = 12.3 Hz, 1H), 4.89 (d, J = 12.3 Hz, 1H), 4.57 4.35 (m, 1H), 4.33 4.14 (m, 2H), 3.11 2.85 (m, 5H), 2.41 2.21 (m, 1H), 2.19 1.86 (m, 7H), 1.34 (s, 9H), 0.89 (d, J = 6.9 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 127.6, 126.7, 81.6, 66.7, 60.2, 58.8, 51.3, 37.8, 37.7, 31.3, 29.7, 29.2, 27.8, 18.8, 17.7, 15.1; Anal. Calcd for C 33 H 46 N 4 O 8 S: C, 60.16; H, 7. 40; N, 8.50. Found: C, 59.80; H, 7.14; N, 8.53. Cbz Ala Met O AzaAla Leu OMe ( 6. 10c). Low melting solid (0.466 g, 84%) ; D 20 36.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 5.4 Hz, 1H), 7.39 7.08 (m, 5H), 6.57 (d, J = 8.1 Hz, 1H), 5.93 (d, J = 7.5 Hz, 1H), 5.01 (s, 2H), 4.48

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158 4.14 (m, 3H), 3.59 (s, 3H), 3.08 (s, 3H), 2.58 2.31 (m, 2H), 2.22 1.87 (m, 5H), 1.68 1.42 (m, 3H), 1.27 (d, J = 7.2 Hz, 3H), 0.85 0.80 (m, 6H); 13 C NMR (75 MHz, CDCl 3 174.3, 173.9, 169.7, 158.9, 156.5, 136.2, 128.7, 128.4, 128.0, 67.2, 52.4, 52.1, 51.7, 50.2, 40.9, 38.1, 30.2, 29.5, 24.8, 23.1, 21.8, 17.9, 15.4; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 25 H 38 N 4 O 8 S Na 577.2303; Found 577.2331. Boc Gly Gly O AzaAla Leu OMe ( 6. 10d). White microcystals (0.376 g, 87%) ; mp 57.0 59.0 o C; D 20 6.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 J = 4.8 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.70 (t, J = 5.7 Hz, 1H), 4.54 4.30 (m, 1H), 4.06 (t, J = 5.3 Hz, 2H), 3.95 3.79 (m, 2H), 3.72 (s, 3H), 3.20 (s, 3H), 1.79 1.54 (m, 3H), 1.44 (s, 9H), 0.94 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 174.4, 171. 9, 167.7, 159.0, 156.5, 80.4, 52.5, 52.1, 44.0, 41.2, 40.9, 38.4, 28.5, 24.9, 23.1, 21.7; Anal. Calcd for C 18 H 32 N 4 O 8 : C, 49.99; H, 7.46; N, 12.95. Found: C, 50.41; H, 7.84; N, 12.58. Boc Ala Ala O AzaPhe Leu OMe ( 6. 10e). White microcystals (0.451 g, 84%) ; mp 61.0 63.0 o C; D 20 23.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 7.08 (m, 6H), 6.47 (d, J = 8.4 Hz, 1H), 5.44 (d, J = 7.8 Hz, 1H), 4.80 (d, J = 15.3 Hz, 1H), 4.63 4.42 (m, 2H), 4.11 3.95 (m, 1H), 3.62 (s, 3H), 3.52 3.20 (m, 2H), 2.45 2.28 (m, 2H), 1.60 1.47 (m, 3H), 1.32 (s, 9H), 1.19 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 5.4 Hz, 3H), 0.81 (d, J = 6.3 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 135.5, 129.0, 128.6, 128.0, 80.0, 54.3, 52.5, 52.0, 50.1, 41.2, 35.3, 33.0, 28.5, 24.9, 23.1, 21.9, 18.6; Anal. Calcd for C 26 H 40 N 4 O 8 : C, 58.19; H, 7.51; N, 10.44. Found: C, 58.18; H, 7.77; N, 10.60.

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159 Boc Ala Gaba O AzaAla Val OtBu ( 6. 10f). White microcystals (0.432 g, 86%) ; mp 124.0 126.0 o C; D 20 6 .0 ( c 1.0, methanol ); 1 H NMR (300 MHz, CDCl 3 s, 1H), 6.44 (br s, 1H), 5.64 (br s, 1H), 4.38 4.22 (m, 1H), 4.20 4.06 (m, 1H), 3.54 3.35 (m, 1H), 3.17 (s, 3H), 2.54 2.33 (m, 2H), 2.15 (d, J = 4.2 Hz, 2H), 2.01 1.71 (m, 2H), 1.45 (s, 9H), 1.41 (s, 9H), 1.29 (d, J = 6.9 Hz, 3H ), 0.92 (d, J = 7.5 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 58.9, 50.4, 38.1, 37.9, 31.8, 28.9, 28.5, 28.2, 24.9, 19.0, 18.5, 18.1; Anal. Calcd for C 23 H 42 N 4 O 8 : C, 54.96; H, 8.42; N, 11.15. Found: C, 54.96; H, 8.80; N, 11.08. Preparation of oxyaza analog of Leuenkephalin 6. 14 The oxyaza dipeptide 6. 6e (1.0 mmol, 1.0 equiv. ), DIPEA (1.0 mmol, 1.0 equiv. ) and cat alytic amount of DMAP ( 10%) were dissolved in dry THF. A solution of Cbz Tyr (OBn) Gly Gly Bt 13 (1.0 mmol, 1.0 equiv. ) in THF (5.0 mL) was added and the mixture stirred for 16 h at 20 C and monitored by TLC [EtOAc hexanes (1:2)]. After completion of the reaction, the solvent was e vaporated. The residue was dissolved in EtOAc (30 mL) and washed with 2 N HCl solution (4 10 mL), water (10 mL), and brine (10 mL). The solvent was dried over MgSO 4 and evaporated to give oxyaza analog of Leuenkephalin 6. 14 White microcystals (0.676 g, 85%) ; mp 88.0 90.0 o C; D 20 7.0 ( c 1.0, methanol); 1 H NMR (300 MHz, CDCl 3 8.32 (m, 2H), 7.56 7.17 (m, 19H), 6.91 (d, J = 8.3 Hz, 2H), 5.05 (s, 2H), 4.95 (s, 2H), 4.65 (d, J = 5.7 Hz, 1H), 4.24 (d, J = 12.3 Hz, 2H), 4.08 3.96 (m, 1H), 3.83 3.75 (m, 3H), 3.62 (s, 3H), 3.37 (s, 1H), 3.00 (dd, J = 15.3, 4.8 Hz, 1H), 2.72 (dd, J = 14.1, 3.3 Hz, 1H), 1.73 1.42 (m, 3H), 0.86 (d, J = 4.8 Hz, 3H), 0.82 (d, J = 5.4 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 71.6, 169.4, 167.7, 157.4, 156.5, 155.6, 136.8, 136.6, 135.5, 129.9, 128.3, 128.0, 127.9, 127.7, 127.4, 127.3, 127.2,

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160 127.1, 114.0, 68.8, 64.9, 56.1, 53.1, 51.5, 51.2, 41.4, 40.0, 36.2, 33.8, 23.8, 22.5, 20.7; Anal. Calcd for C 43 H 49 N 5 O 10 : C, 64.89; H, 6.21 ; N, 8.80. Found: C, 65.15; H, 6.35; N, 8.75.

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161 CHAPTER 7 SYNTHESIS OF TAURINE PEPTIDES, SULFONOPEPTIDES, AND N O CONJUGATES 7.1 Introduction Peptides are widely used as drug delivery systems, biopharmaceuticals, prodrugs and bioactive moieties. 178 However, when introduced into living systems, peptides show rapid enzymatic degradation which significantly limits their utilization in vivo 179 For decades, various peptidomimetics, including phosphonopeptides, 180 ureidopeptides 181 and sulfonopeptides, 18 2,183 have been developed and reported. Aminoalkanesulfonic acid peptidomimetics are hydrolysis resistant sulfono analogs of naturally occurring peptides. 184,185 Sulfonamide group increases the polarity and hydrogen bonding ability of the molecule since t he SO 2 NH 2 group is more acidic (pKa 11 12) than the amide bond CO NH 2 186 Accordingly, the conjugated base is weaker than that of the amide bond and therefore is less prone to protonation, the first step in acidic hydrolysis of amides and peptides. Synthe sis of or substituted aminoalkylsulfonates and sulfonamidopeptides has allowed preparation of a large number of oligomers which structurally mimic native peptides 187 and render extended conformation in contrast to cis or trans isomers of their native peptide analogs. 188 Sulfonopeptides can be prepared by the reaction of N protected aminoalkanesulfonyl chlorides with amino acid 189 o r peptide esters 185,190 or by condensation of N protected aminoalkanesulfinyl chlorides and amino or peptide esters followe d by oxidation. 191 Chlorination of sulfono acids usually requires harsh reaction conditions ( e.g. using of thionyl chloride, oxalyl chloride, phosgene, phosphorus Reproduced with permission from The Journal of Organic Chemistry 201 4 79, 2688 2693 Copyright 2014 American Chemical Society

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162 pentachloride and high temperatures) which may cause loss of chirality and affect N protecting groups. 192,193 Alternative routes to sulfonopeptides l ie in: i) NCS/HCl oxidative chlorination of xanthines and thioacetates into 7. 1 2 ; 182 ii) Mannich type reactions of N protected 2 aminoalkanesulfonamides, aldehydes, and aryldichlorophosphines, followed by aminolysis with amino esters to form 7. 3 ; 1 93 or iii) reaction of aminoalkanesulfone amides with C terminal aminoalkylphosphinic acids. 194 Liskamp synthesized or substituted aminoethane sulfonamide arginine glycine peptidomimetics 7. 4 and 7. 5 190 and also various peptidomimetics which contained mono and di sulfonyl moieties 7. 6 8 183 Gennari and colleagues reported the synthesis 184 and conformational study of chiral vinylogous aminosulfonic acids 195 ( vs amin o acids) and corresponding oligomers 7. 9 ( vs peptides) (Figure 7 1). 185 Figure 7 1 Some sulfonopeptides reported in the literature The synthesis and utility of taurine peptidomimetics and conjugates involving the sulfono group has not yet been explored in full. 196 199 The high hydrophilicity of taurine challenges its synthetic incorporation into peptides; therefore convenient methods to design aminosulfonic acid containing peptides need to be developed. The present

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163 work describes general and high yielding synthetic routes for incorporation of taurine unit into di tri and tetra peptides. The synthetic protocol was explored and utilized for a series of taurine acylations at its C terminus (SO 3 H group) which allowed synt hesis of sulfonopeptides and taurine N O conjugates. 7.2 Results and Discussion Our group has developed an expertise in the synthesis of peptides which contain non natural amino acid units such as: hydrazino aza aminoxy depsi and oxyazapeptides. 2 00 In this work, the replacement of amide bond by a sulfonamide unit led to facile synthesis of hybrid sulfonopeptidomimetics and conjugates composed of taurine and natural amino acids, peptides and bioactive moieties. 7.2.1 Synthesis of Taurine Containing Dipeptides N Acylbenzotriazoles have been advantageous reagents to construct peptides, peptidomimetics and peptide conjugates. 23 27 Several activated amino acids 7. 11 were prepared following our reported pro cedures. 24,27 Utilization of our well developed methodology showed general applicability and scope of this synthetic protocol to prepare water soluble taurine dipeptides 7. 12 (Scheme 7 1 ). MeCN was employed as solvent, and few drops of water were used to dissolve taurine 7. 10 using DIPEA as base. After completion of the reaction, the mixture was evaporated to dryness and diethyl ether was then added to the crude product and it was stirre d for 20 min, and dried again. During acylations of taurine N terminus we paid special attention to complete elimination of water from the reaction mixture. Due to high polarity of reaction products 7. 12 water would dissolve them lowering the reaction yie lds when present even at trace amounts. Acidification of reaction mixture was provided by drop wise addition of dry 4N HCl solution in dioxane for N Cbz and N Fmoc protected products.

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164 For N Boc protected products HCl was diluted in ether prior adding to the mixture and the solvent was then evaporated. C terminus taurine dipeptides 7. 12 were then isolated by flash column chromatography (1:1 EtOAc/Hexanes to remove any impurities other than re action products and then MeOH was passed to isolate products 7. 12 ). With this methodology we managed to synthesize taurine dipeptides in excellent yields ( 7. 12 Scheme 7 1, Table 7 1, 76 90%) and this purification concept has proved to be effective for all the reaction products. Scheme 7 1 Incorporation of taurine unit to prepare d ipeptides 7. 12 Table 7 1 Preparation of t a urine containing d ipeptides 7.12 # Pg R Target 7. 12 Yield% 7. 12a Cbz H Cbz Gly Tau OH 78 7. 12b Cbz Me Cbz L Ala Tau OH 87 7. Cbz Me Cbz DL Ala Tau OH 84 7. 12c Cbz CH 2 CH 2 SMe CbzL Met Tau OH 82 7. 12d Boc Me Boc L Ala Tau OH 88 7. 12e Boc CH(OBn)OMe Boc L Thr(OBn) Tau OH 86 7. 12f Boc H Boc Gly Tau OH 76 7. 12g Boc CH(Me)2 Boc L Val Tau OH 82 7. 12h Boc (3 indolyl)CH 2 Boc L Trp Tau OH 86 7. 12i Fmoc H Fmoc Gly Tau OH 90 7. 12j Fmoc CH 2 CH(Me) 2 Fmoc L Leu Tau OH 82 7. 12k Fmoc CH 2 Ph Fmoc L Phe Tau OH 88 It was observed that almost every coupling reaction were completed within 1 2 h and even they were done in presence of water/base the benzotriazole intermediates show significant stability in reaction condition and provided high yields for the reaction products. Again, stability of benzotriazole intermediates in water solutions has also been studie d in detail in a number of research papers from our group previously. 2 00

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165 7.2.2 Synthesis of Taurine Containing Tri and Tetrapeptides To prepare longer taurine containing tri and tetrapeptides, a series of peptidoyl benzotriazoles 7. 13 were synthesized u sing benzotriazole methodology. 23 Products 7. 14 were prepared in good to excellent yields by reacting Pg di tripeptidoyl benzotriazoles 7. 13 with taurine 7. 10 The synthetic route was applied to various Cbz, Boc, and Fmoc N protected di and tripeptides 7. 13 (Scheme 7 2, Table 7 2). Products 7. 14 were isolated in good to excellent yields (73 93%) which proved the general applicability and effectiveness of the developed methodology. Scheme 7 2 Incorporation of the Taurine Unit into Tri and Tetrapeptides 7. 14 Table 7 2 Preparation of taurine containing tri and t etrapeptides 7. 14 # Pg AA 1 AA 2 Products 7. 14 Yield% 7. 14a Cbz L Phe Gly Cbz L Phe Gly Tau OH 89 7. 14b Cbz L Ala Gly Cbz L Ala Gly Tau OH 91 7. 14c Cbz L Phe L Met Cbz L Phe L Met Tau OH 73 7. 14d Cbz L Val Gly Cbz L Val Gly Tau OH 93 7. 14e Cbz L Phe Gly Gly Cbz L Phe Gly Gly Tau OH 85 7. 14f Boc L Ala Gly Boc L Ala Gly Tau OH 75 7. 14g Boc L Pro L Ala Boc L Pro L Ala Tau OH 78 7. 14h Boc L Ala L Pro L Ala Boc L Ala L Pro L Ala Tau OH 80 7. 14i Fmoc L Val L Ala Fmoc L Val L Ala Tau OH 78 7. 14j Fmoc L Val Gly Fmoc L Val Gly Tau OH 90 7. 14k Fmoc L Leu Gly Gly Fmoc L Leu Gly Gly Tau OH 84 The efficiency of this coupling reactions seem to depend largely on a homogeneous system of reactants as most of the reactions proceeds homogeneously. It was observed that in some cases the starting material (especially Fmoc AA Bt compounds) did not dissol ve completely in the MeCN, however as the reaction proceeds (within 10 20 min) the reaction mixture becomes clear. The current protocol

PAGE 166

166 can also be applied to a longer peptide fragment with more hydrophobic sequence. We also observe that the some reaction proceed smoothly in MeCN with the addition of few drops of THF. So this methodology is very efficient even when the starting material is not completely soluble at the first place. 7.2.3 Synth esis of Taurine Sulfonopeptides A library of sulfono di and tr ipeptides has been synthesized via the N protection of taurine with Cbz to form 7. 15 followed by SO 2 activation with thionyl chloride to form 7. 16 (Scheme 7 3, Table 7 3). N Cbz protected taurine chloride 7. 16 was unstable oil that decomposed within 72 h. To stabilize the SO 2 active intermediate we employed benzotriazole and obtained N Cbz taurine sulfonyl benzotriazole 7. 17 in good yield (72%). Reaction of 7. 17 with a number of amino ester or dipeptide esters formed N protected taurine sulfonopeptides 7. 18 (Scheme 7 3 Table 7 3 ) in good to excellent yields (70 86%). Isolation of the final products was achieved by extraction with EtOAc from 3N HCl followed by either recrystallization (DCM/Hexanes) or flash chromatography (EtOAc/Hexanes) to give 7. 18 Synthe sis of SO 2 active taurine intermediate 17 via benzotriazole methodology allowed fast incorporation of the taurine unit as an N terminus amino acid in the peptide chains. Scheme 7 3 Synthesis of taurine s ulfonopeptides 7. 18

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167 Table 7 3 Preparation of sulfono di and t ripeptides 7. 18 # Amino ester Sulphono peptide 7. 18 Yield% 7. 18a H Gly OBn Cbz Tau Gly OBn 82 7. 18b H L Val O t Bu Cbz Tau L Val O t Bu 80 7. 18c H L Met OMe Cbz Tau L Met OMe 86 7. 18d H L Phe OBz Cbz Tau L Phe OBz 78 7. 18e H L Leu OMe Cbz Tau L Leu OMe 70 7. 18f H Gly Gly OMe Cbz Tau Gly Gly OMe 75 7.2.4 Synthe sis of N O Taurine Conjugates The synthetic route to 7. 19 employed stable N protected taurine benzotriazole 7. 17 to acylate N and O nucleophilic compounds. C oupling of N Cbz protected SO 2 Bt activated taurine 7. 17 with N nucleophiles allowed formation of sulfonoamides 7. 19a 7. 19b and 7. 19c The reactions proceeded at room temperature overnight forming products 19a c (Scheme 7 4, Table 7 4) in good yields (64 73%). For the O acylation of threonine Cbz N Tau Bt 7. 17 and Boc Thr were coupled (1:1, o/n, rt) using excess of DIPEA as base. The Boc group of the threonine moiety was displaced most probably due to the acidic silica column during the isolati on of the O acylated taurin e conjugate to give 7. 19d O Acylation of L menthol gave 7. 19e in 62% yield and the product was isolated via column chromatography. Scheme 7 4 Synthesis of t aurine N and O c onjugates 7. 19 Table 7 4 Preparation of N and O taurine b ioconjugates 7. 19 # Nu Product 7. 19 Yield% 1 NH 2 7. 19a 70 2 NHBn 7. 19b 73 3 N(CH 2 ) 2 O(CH 2 ) 2 7. 19c 64 4 O L Thr 7. 19d 78 5 O L Menthol 7. 19e 62

PAGE 168

168 Generally, isolation of 7. 19 was easily performed by extraction of the final products with EtOAc from 3N HCl solution followed by recrystallization from DCM/Hexanes or flash column chromatography. Thus, general SO 2 activating methodology proved to be effective for the synthesis of ta urine N O conjugates. 7.3 Conclusion In conclusion, a viable synthetic route towards sulfonopeptides, taurine peptides, and conjugates is reported. Moisture sensitive taurine peptidomimetics with a series of amino acids, di and tripeptides are conveni ently synthesized and isolated in high yields. Taurine containing peptidomimetics and sulfonopeptides mimic natural peptides and therefore represent attractive scaffolds for drug delivery as well as prodrug and tool applications. We believe that our genera l and straightforward synthetic approach represents a significant development in the peptide field which could further facilitate the synthesis and evaluation of sulfonopeptides and related bio conjugated systems. 7.4 Experimental Section reagent grade or HPLC grade. Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 CD 3 OD, and D 2 O using a 300 MHz and 500 MHz spectrometer (with TMS as an internal standard). All 13 C NMR spectra were recorded with complete proton decoupling. The couplin g constants were reported in Hz. Reaction progress w as monitored by thin layer chromatography (TLC) and visualized by UV light. Elemental analyses were performed on a Carlo Erba EA 1108 instrument. DCM was dried and distilled over CaH 2 whereas tetrahydrofuran (THF) was

PAGE 169

169 used after distillation over Na benzo phenone. N Pg ( aminoacyl)benzotriazoles 7. 11a k, N Pg ( di and tripeptidoyl)benzotriazoles 7. 13a k were prepared according to the literature methods. 23 7.4.1 General Methods for the Preparation of Taurine Containing Dipeptides 7.12a k Tri and Tetrapeptides 7.14a k N Pg ( peptidoyl)benzotriazoles 7. 11a k or 7. 13a k (1.0 equiv, 1.0 mmol) and taurine (1.1 equiv, 1.1 mmol, 0.14 g) were dissolved in MeCN (20 mL) and two drops of water. DIPEA (0.21 mL, 1.2 equiv) was added to the reaction mixture and stirred for 1 h at room temperature. After the reaction was complete [monitored by TLC] the solvent was evaporated and ether (20.0 mL) was added to the mixture and it was then acidified with 4N HCl in dioxane. The solvent was evaporated again and the r esidue was dried overnight under vacuum. The crude product was purified by column chromatography (1:1 EtOAc to remove any impurities and then 100% MeOH) to give corresponding taurine containing dipeptides 7. 12a k tri and tetrapeptides 7. 14a k Z Gly Tau OH ( 7. 12a). white solid, 1 H NMR (300 MHz, DMSO d 6 J = 5.7 Hz, 1H), 7.40 7.26 (m, 5H), 5.03 (s, 2H), 3.55 (d, J = 6.0 Hz, 2H), 3.35 3.27 (m, 2H), 2.56 (t, J = 7.0 Hz, 2H); 13 C NMR (75 MH z, DMSO d 6 HRMS (ESI TOF) m/z : [M H] Calcd for C 12 H 15 N 2 O 6 S 315.0656; Found 315.0667. Z L Ala Tau OH ( 7. 12b). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 5.4 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.42 7.24 (m, 5H), 5.01 (s, 2H), 4.03 3.86 (m, 1H), 3.37 3.24 (m, 2H), 2.59 (t, J = 7.0 Hz, 2H), 1.20 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6

PAGE 170

170 155.7, 137.0, 128. 3, 127.8, 127.7, 65.4, 50.4, 50.3, 35.5, 18.1; HRMS (ESI TOF) m/z : [M H] Calcd for C 13 H 17 N 2 O 6 S 329.0813; Found 329.0820. Z DL Ala Tau OH ( 7. white solid, 1 H NMR (300 MHz, DMSO d 6 J = 5.4 Hz, 1H), 7.43 7.25 (m, 6H), 5.03 (s, 2H), 4.08 3.88 (m, 1H), 3.44 3.29 (m, 1H), 2.61 (t, J = 6.9 Hz, 2H), 1.21 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 50.4, 35.6, 18.2; HRMS (ESI TOF) m/z : [M H] Calcd for C 13 H 17 N 2 O 6 S 329.0813; Found 329.0829. Z L Met Tau OH ( 7. 12c). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 5.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.45 7.23 (m, 5H), 5.01 (s, 2H), 4.11 3.92 (m, 1H), 3.40 3.23 (m, 2H), 2.59 (t, J = 7.0 Hz, 2H), 2.50 2.35 (m, 2H), 2.01 (s, 3H), 1.94 1.67 (m, 2H); 13 C NMR (75 MHz, DMSO d 6 5, 31.4, 29.8, 14.6; HRMS (ESI TOF) m/z : [M H] Calcd for C 15 H 21 N 2 O 6 S 2 389.0847; Found 389.0865. Boc L Ala Tau OH ( 7. 12d). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, D 2 3.82 (m, 1H), 3.60 3.41 (m, 2H), 2.99 (t, J = 7.8 Hz, 2H), 1.33 (s, 9H), 1.24 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, D 2 TOF) m/z : [M H] Calcd for C 10 H 19 N 2 O 6 S 295.0969; Found 295.09 78. Boc L Thr(OBn) Tau OH ( 7. 12e). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, D 2 7.11 (m, 5H), 4.44 (d, J = 12.0 Hz, 1H), 4.30 (d, J = 12.0 Hz, 1H), 4.07 3.80 (m, 2H), 3.53 3.34 (m, 2H), 2.91 (t, J

PAGE 171

171 = 6 .9 Hz, 2H), 1.32 (s, 9H), 1.09 (d, J = 5.4 Hz, 3H); 13 C NMR (75 MHz, D 2 157.4, 137.6, 128.6, 128.2, 128.1, 81.5, 74.3, 70.9, 59.3, 49.6, 35.2, 27.6, 16.0; HRMS (ESI TOF) m/z : [M H] Calcd for C 18 H 27 N 2 O 7 S 415.1544; Found 415.1558. Boc Gly Tau OH ( 7. 12f). white solid, 1 H NMR (300 MHz, D 2 3.42 (m, 2H), 3.08 2.88 (m, 2H), 1.33 (s, 9H); 13 C NMR (75 MHz, D 2 TOF) m/z : [M H ] Calcd for C 9 H 17 N 2 O 6 S 281.0813; Found 281.0823. Boc L Val Tau OH ( 7. 12g). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 7.8 Hz, 1H), 3.77 3.60 (m, 1H), 3.42 3.21 (m, 2H ), 2.59 (t, J = 7.2 Hz, 2H), 1.99 1.83 (m, 1H), 1.38 (s, 9H), 0.82 (d, J = 6.0 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 155.5, 78.0, 56.1, 50.5, 35.4, 30.2, 28.2, 19.2, 18.1; HRMS (ESI TOF) m/z : [M H] Calcd for C 12 H 23 N 2 O 6 S 323.1282; Found 323.1289. Boc L Trp Tau OH ( 7. 12h). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, D 2 J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.12 6.84 (m, 3H), 4.29 3.98 (m, 1H), 3.50 3.17 (m, 4H), 2.68 2.53 (m, 2H), 1.18 (s, 9H); 13 C NMR (75 MHz, D 2 121.6, 119.1, 111.7, 109.7, 79.8, 65.9, 50.2, 35.7, 28.8, 28.3; HRMS (ESI TOF) m/z : [M H] Calcd for C 18 H 24 N 3 O 6 S 410.1391; Found 410.1390. Fmoc Gly Tau OH ( 7. 12i). white solid, 0 1 H NMR (300 MHz, D 2 J = 6.6 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 7.25 7.05 (m, 4H), 4.81 4.69 (m, 3H), 4.14 (d, J = 8.1 Hz, 2H), 3.66 3.53 (m, 2H), 3.09 (t, J = 6.9 Hz, 2H); 13 C NMR (75 MHz, D 2

PAGE 172

172 66.6, 54.0, 49.5, 46.4, 34.9; HRMS (ESI TOF) m/z : [M H] Calcd for C 19 H 19 N 2 O 6 S 403.0969; Found 403.0973. Fmoc L Leu Tau OH ( 7. 12j). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.77 7.64 (m, 3H), 7.60 7.49 (m, 2H), 7.37 7.29 (m, 3H), 7.29 7.20 (m, 2H), 4.50 4.00 (m, 4H), 3.64 3.39 (m, 2H), 3.15 2.85 (m, 2H), 1.80 1.50 (m, 3H), 0.91 (d, J = 4.5 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 143.9, 141.3, 127.8, 127.2, 125.40, 120.0, 67.1, 58.1, 50.6, 47.3, 42.7, 35.8, 24.9, 23.2, 21.9; HRMS (ESI TOF) m/z : [M H] Calcd for C 23 H 27 N 2 O 6 S 459.1595; Found 459.1607. Fmoc L Phe Tau OH ( 7. 12k). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 5.7 Hz, 1H), 7.87 (d, J = 7.5 Hz, 2H), 7.73 7.58 (m, 3H), 7.40 (t, J = 7.4 Hz, 2H), 7.33 7.13 (m, 7H), 4.27 4.05 (m, 4H), 3.40 3.20 (m, 2H), 3.03 (dd, J = 13.8, 4.5 Hz, 1H), 2.79 (dd, J = 13. 6, 10.2 Hz, 1H), 2.57 (t, J = 6.3 Hz, 2H); 13 C NMR (75 MHz, DMSO d 6 155.8, 143.8, 143.7, 140.6, 138.3, 129.2, 128.0, 127.6, 127.1, 126.2, 125.4, 125.3, 120.0, 65.7, 56.5, 50.4, 46.6, 37.5, 35.6; HRMS (ESI TOF) m/z : [M H] Calcd for C 26 H 25 N 2 O 6 S 493.1439; Found 493.1450. Z L Phe Gly Tau OH ( 7. 14a). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 5.4 Hz, 1H), 7.87 (t, J = 5.4 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.37 7.17 (m, 10H), 4.97 (d, J = 12.9 Hz, 1H), 4.91 (d, J = 13.5 Hz, 1H), 4.37 4.21 (m, 1H), 3.68 (d, J = 5.4 Hz, 2H), 3.40 3.30 (m, 2H), 3.17 3.00 (m, 1H), 2.78 (dd, J = 13.6, 10.6 Hz, 1H), 2.63 (t, J = 7.2 Hz, 2H); 13 C NMR (75 MHz, DMSO d 6

PAGE 173

173 128.1, 127.7, 127.5, 126.2, 65.3, 56.3, 50.4, 42.3, 37.3, 35.5; HRMS (ESI TOF) m/z : [M H] Calcd for C 21 H 24 N 3 O 7 S 462.1340; Found 462.1353. Z L Ala Gly Tau OH ( 7. 14b). white solid, 0.35 g, 91%, mp 216. D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 7.58 6.95 (m, 6H), 4.92 (s, 2H), 4.14 3.81 (m, 1H), 3.68 3.43 (m, 2H), 3.43 3.02 (m, 4H), 1.12 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 55.9, 137.0, 128.4, 127.9, 65.6, 50.4, 50.3, 42.3, 35.5, 18.0; HRMS (ESI TOF) m/z : [M H] Calcd for C 15 H 20 N 3 O 7 S 386.1027; Found 386.1036. Z L Phe L Met Tau OH ( 7. 14c). white solid, D 20 +4.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CD 3 7.11 (m, 10H), 5.26 4.96 (m, 2H), 4.52 4.20 (m, 2H), 3.69 3.44 (m, 2H), 3.05 2.88 (m, 3H), 2.62 2.33 (m, 1H), 2.16 1.79 (m, 5H); 13 C NMR (75 MHz, D 2 130.5, 129.8, 129.6, 129.6, 129.1, 129.0, 128.9, 128.9, 127.9, 68.0, 58.9, 54.0, 51.3, 38.6, 36.8, 31.3, 31.1, 15.4; HRMS (ESI TOF) m/z : [M H] Calcd for C 24 H 30 N 3 O 7 S 2 536.1531; Found 536.1528. Z L Val Gly Tau OH ( 7. 14d). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR ( 300 MHz, DMSO d 6 J = 4.6 Hz, 1H), 7.87 7.78 (m, 1H), 7.51 7.23 (m, 6H), 5.03 (d, J = 5.4 Hz, 2H), 3.98 3.82 (m, 1H), 3.66 (d, J = 6.0 Hz, 2H), 3.42 3.27 (m, 2H), 2.62 (t, J = 6.9 Hz, 2H) 2.10 1.87 (m, 1H), 0.86 (d, J = 6.3 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 127.9, 127.8, 65.6, 60.4, 50.5, 42.2, 35.6, 30.1, 19.3, 18.2; HRMS (ESI TOF) m/z : [M H] Calcd for C 17 H 24 N 3 O 7 S 414.1340; Found 414.1348.

PAGE 174

174 Z L Phe Gly Gly Tau OH ( 7. 14e). white solid, 0.44 g, 85%, mp 232.0 234.0 D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 5.4 Hz, 1H), 8.18 (t, J = 5.6 Hz, 1H), 7.91 (t, J = 4.9 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.43 7.07 (m, 10H), 4.96 (d, J = 13.1 Hz, 1H), 4.91 (d, J = 13.1 Hz, 1H), 4.39 4.17 (m, 1H), 3.78 (d, J = 5.4 Hz, 2H), 3.65 (d, J = 5.7 Hz, 2H), 3.37 3.28 (m, 2H), 3.06 (dd, J = 13.7, 3.8 Hz, 1H), 2.86 2.68 (m, 1H), 2.58 (t, J = 7.2 Hz, 2H); 13 C NMR (75 MHz, DMSO d 6 171.9, 169.1, 168.2, 155.9, 138.2, 137.0, 129.2, 128.3, 128.0, 127.6, 127.4, 126.2, 65.2, 56.3, 50.3, 42.2, 42.1, 37.3, 35.4; HRMS (ESI TOF) m/z : [M H] Calcd for C 23 H 27 N 4 O 8 S 519.1555; Found 519.1548. Boc L Ala Gly Tau OH ( 7. 14f). white solid, 0.27 g D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, D 2 J = 7.2 Hz, 1H), 3.82 (s, 2H), 3.51 (t, J = 6.8 Hz, 2H), 3.00 (t, J = 6.9 Hz, 2H), 1.34 (s, 9H), 1.26 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, D 2 81.6, 49.5, 48.6, 42.5, 35.1, 27.6, 16.7; HRMS (ESI TOF) m/z : [M H] Calcd for C 12 H 22 N 3 O 7 S 352.1184; Found 352.1189. Boc L Pro L Ala Tau OH ( 7. 14g). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 7.4 Hz, 1H), 7.95 7.85 (m, 1H), 4.24 3.98 (m, 2H), 3.38 3.15 (m, 4H), 2.56 (t, J = 7.2 Hz, 2H), 2.19 1.63 (m, 4H), 1.30 (s, 9H), 1.19 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 30.8, 28.0, 23.2, 18.3; HRMS (ESI TOF) m/z : [M H] Calcd for C 15 H 26 N 3 O 7 S 392.1497; Found 392.1497. Boc L Ala L Pro L Ala Tau OH ( 7. 14h). white solid, 0.37 g, 80%, mp 130.0 D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 7.86 (m,

PAGE 175

175 1H), 7.75 (s, 1H), 6.97 (s, 1H), 4.43 3.91 (m, 3H), 3.56 (s, 2H), 3.33 (s, 2H), 3.12 2.95 (m, 1H), 2.59 (s, 2H), 2.10 1.80 (m, 4H), 1.37 (s, 9H), 1.25 (d, J = 4.8 Hz, 3H), 1.25 (d, J = 7.5 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 .0, 59.5, 53.0, 50.3, 48.4, 47.7, 35.5, 28.9, 28.2, 24.6, 18.0, 16.8; HRMS (ESI TOF) m/z : [M H] Calcd for C 18 H 31 N 4 O 8 S 463.1868; Found 463.1874. Fmoc L Val L Ala Tau OH ( 7. 14i). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 8.13 (d, J = 7.5 Hz, 1H), 7.96 7.56 (m, 5H), 7.55 7.14 (m, 5H), 4.35 4.08 (m, 3H), 4.07 3.94 (m, 1H), 3.93 3.67 (m, 1H), 3.45 3.02 (m, 2H), 2.67 2.39 (m, 2H), 2.10 1.75 (m, 1H), 1.34 0.98 (m, 3H), 0.95 0.65 (m, 6H); 13 C NMR (75 MHz, DMSO d 6 143.9, 143.8, 140.7, 127.6, 127.1, 125.4, 120.1, 65.7, 60.2, 50.5, 48.3, 46.7, 35.6, 30.4, 19.2, 18.2, 18.1; HRMS (ESI TOF) m/z : [M H] Calcd for C 25 H 30 N 3 O 7 S 516.1810; Found 516.1804. Fmoc L Val Gly Tau OH ( 7. 14j). wh ite solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 J = 4.5 Hz 1H), 7.90 7.60 (m, 5H), 7.51 (t, J = 9.0 Hz, 1H), 7.45 7.23 (m, 4H), 4.35 4.15 (m 3H), 3.95 3.82 (m, 1H), 3.76 3.64 (m, 2H), 3.43 3.23 ( m, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.10 1.92 (m, 1H), 0.86 (d, J = 5.4 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 143.9, 140.7, 127.7, 127.1, 125.4, 120.1, 65.7, 60.4, 50.5, 46.7, 42.1, 35.6, 30.3, 19.3, 18.3; HRMS (ESI TOF) m/z : [M H] Calcd for C 24 H 28 N 3 O 7 S 502.1653; Found 502.1649. Fmoc L Leu Gly Gly Tau OH ( 7. 14k). white solid, 0.48 g, 84%, mp D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6

PAGE 176

176 8.07 (m, 2H), 8.02 7.81 (m, 3H), 7.83 7.52 (m, 3H), 7.52 7.19 (m, 4H), 4.45 3.90 (m, 4H), 3.83 3.47 (m, 6H), 2.59 (t, J = 7.5 Hz, 2H), 1.75 1.22 (m, 3H), 0.85 (d, J = 5.7 Hz, 6H); 13 C NMR (75 MHz, DMSO d 6 127.1, 125.3, 120.1, 65.6, 53.2, 50.3, 46.7, 42.2, 42.1, 35.4, 24 .2, 23.1, 21.4; HRMS (ESI TOF) m/z : [M H] Calcd for C 27 H 33 N 4 O 8 S 573.2025; Found 573.2028. 7.4.2 General Methods for the Preparation of Sulphono Peptide 7. 18a f and N O Acylated Taurine Conjugates 7.19a e Cbz Tau Bt (0.36 g, 1 mmol, 1 equiv) and amino ester (1.1 mmol, 1.1 equiv) were dissolved in dry MeCN (20 mL). DIPEA (0.44 mL, 2.5 equiv) was added drop wise and the mixture was stirred overnight at room temperature. Evaporation of the solvent followed by column chromatography (EtOAc hexanes 1:2) gave N O acylated taurine conjugates 7. 18a f To prepare 19a e similar protocol was used with various N and O nucleophiles. Cbz Tau Gly OBn ( 7. 18a). white solid, 1 H NMR (300 MHz, CDCl 3 7.28 (m, 10H), 5.53 (t, J = 6.0 Hz, 1 H), 5.41 (t, J = 6.3 Hz, 1H), 5.18 (s, 2H), 5.11 (s, 2H), 3.97 (s, 2H), 3.73 3.65 (m, 2H) 3.25 (t, J = 6.0 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 128.3, 67.9, 67.2, 53.4, 44.4, 36.2; Anal. Calcd for C 19 H 22 N 2 O 6 S: C, 56.15; H, 5.46; N, 6.89. Found: C, 56.12; H, 5.71; N, 6.93. Cbz Tau L Val O t Bu ( 7. 18b). white solid, D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, CD 3 7.42 (m, 5H), 5.07 (s, 2H), 3.74 (d, J = 5.4 Hz, 1H), 3.58 (t, J = 6.0 Hz, 2H), 3.23 2.90 (m, 2H), 2.15 1.95 (m, 1H), 1.47 (s, 9H), 0.99 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CD 3

PAGE 177

177 32.4, 28.4, 19.8, 18.1 ; HRMS (ESI TOF) m/z : [M + Na] + Calcd for C 19 H 30 N 2 O 6 SNa 437.1717; Found 437.1715. Cbz Tau L Met OMe ( 7. 18c). D 20 +8.0 ( c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.12 (m, 5H), 6.20 (d, J = 9.0 Hz, 1H), 6.04 5.57 (m, 1H), 5.03 (d, J = 5.7 Hz, 2H), 4.43 4.16 (m, 1H), 3.85 3.53 (m, 5H), 3.24 (s, 2H), 2.55 (s, 2H), 2.19 1.81 (m, 5H); 13 C NMR (75 MHz, CDCl 3 156.9, 136.3, 128.7, 128.4, 128.2, 67.2, 55.0, 53.4, 53.2, 36.2, 32.1, 30.0, 15.4; HRMS (ESI TOF) m/z : [M H] Calcd for C 16 H 23 N 2 O 6 S 2 403.1003; Found 403.1000. Cbz Tau L Phe OBz ( 7. 18d). D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 6.97 (m, 16H), 5.87 5.67 (m, 1H), 5.21 4.94 (m, 4H), 4.53 4.22 (m, 1H), 3.45 3.25 (m, 2H), 3.18 3.04 (m, 1H), 3.03 2.76 (m, 3H); 13 C NMR (75 MHz, CDCl 3 128.9, 128.8, 128.7, 128.4, 128.2, 127.8, 127.5, 68.0, 67.2, 57.8, 53.4, 39.3, 36.0; HRMS (ESI TOF) m/z : [M + Na] + Calcd for C 26 H 28 N 2 O 6 S Na 519.1560; Found 519.1577. Cbz Tau L Leu OMe ( 7. 18e). D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 J = 9.2 Hz, 1H), 6.23 6.02 (m, 1H), 5.03 (s, 2H), 4.19 3.99 (m, 1H), 3.72 (q, J = 6.8 Hz, 2H), 3.64 (s, 3H), 3.33 3.16 (m, 2H), 1.84 1.47 (m, 3H), 0.92 0.67 (m, 6H); 13 C NMR (75 MHz, CDCl 3 157.1, 136.3, 128.7, 128.3, 128.1, 67.2, 54.9, 53.5, 52.9, 41.9, 36.2, 24.6, 22.9, 21.5; HRMS (ESI TOF) m/z : [M + Na] + Calcd for C 17 H 26 N 2 O 6 S Na 409.1404; Found 409.1417. Cbz Tau Gly Gly OMe ( 7. 18f). white solid, 0.29 g, 75%, mp 66.2 67.8 C; 1 H NMR (300 MHz, CDCl 3 7.08 (m, 5H), 6.71 6.47 (m, 1H), 6.25 6.01 (m, 1H), 5.04 (d, J = 3.9 Hz, 2H), 3.92 (t, J = 5.2 Hz, 2H), 3.85 3.68 (m, 2H), 3.60 (d, J = 4.0 Hz,

PAGE 178

178 3H), 3.47 3.14 (m, 2H); 13 C NMR (75 MHz, CDCl 3 128.3, 128.1, 67.3, 62.5, 53.3, 52.8, 44.3, 36.3; Anal. Calcd for C 15 H 21 N 3 O 7 S: C, 46.50; H, 5.46; N, 10.85. Found: C, 46.18; H, 5.31; N 11.14. Cbz Tau NH 2 ( 7. 19a). white solid, 1 H NMR (300 MHz, DMSO d 6 7.26 (m, 5H), 6.91 (s, 2H), 5.03 (s, 2H), 3.50 3.24 (m, 2H), 3.13 (t, J = 7.5 Hz, 2 H); 13 C NMR (75 MHz, DMSO d 6 127.8, 127.8, 65.5, 53.9, 35.8; Anal. Calcd for C 10 H 14 N 2 O 4 S: C, 46.50; H, 5.46; N, 10.85. Found: C, 46.47; H, 5.71; N, 10.75. Cbz Tau NH Bn ( 7. 19b). white solid, 1 H NMR (3 00 MHz, CDCl 3 7.12 (m, 10H), 5.48 (s, 1H), 5.22 (s, 1H), 5.07 (s, 2H), 4.25 (s, 2H), 3.65 3.43 (m, 2H), 3.08 (t, J = 8.7 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 136.8, 136.3, 129.1, 128.7, 128.4, 128.3, 128.3, 128.2, 67.2, 52.7, 47.3, 36.1; Anal. Calcd for C 17 H 20 N 2 O 4 S: C, 58.60; H, 5.79; N, 8.04. Found: C, 58.27; H, 5.91; N, 8.11. Cbz Tau NH morph ( 7. 19c). white solid, 1 H NMR (300 MHz, CDCl 3 7.28 (m, 5H), 5.55 (t, J = 6.3 Hz, 1H), 5.11 (s, 2H), 3.77 (t, J = 7.8 Hz, 4H), 3.65 (t, J = 6.2 Hz, 2H), 3.27 3.14 (m, 4H), 3.10 (t, J = 6.0 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 35.6; Anal. Calcd for C 14 H 20 N 2 O 5 S: C, 51.21; H, 6.14; N, 8.53. Found: C, 51.36; H, 6 .41; N, 8.14. Cbz Tau O L Thr ( 7. 19d). Cbz N Tau Bt 17 (0.36 g, 1 mmol, 1 equiv) and Boc Thr OH (0.24 g, 1.1 mmol, 1.1 equiv) were dissolved in dry MeCN (20 mL) and DIPEA (0.44 mL, 2.5 equiv) was added drop wise and the mixture was stirred overnight at room temperature. Evaporation of the solvent followed by column chromatography (EtOAc

PAGE 179

179 hexanes 1:2) gave O acylated taurin e conjugate 19d The Boc group of the threonine moiety was displaced due to the acidic silica column during the isolation of the product. white solid, 0.33 g, 78%, mp 156.0 D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, DMSO d 6 7.13 (m, 5 H), 7.06 (t, J = 5.1 Hz, 1H), 4.94 (s, 2H), 4.11 3.84 (m, 1H), 3.34 3.07 (m, 2H), 3.05 2.95 (m, 1H), 2.57 (t, J = 6.9 Hz, 2H), 1.20 (d, J = 6.3 Hz, 3H); 13 53.6, 51.3, 42.0, 37.9, 18.2; Anal. Calcd for C 14 H 20 N 2 O 7 S: C, 46.66; H, 5.59; N, 7.77. Found: C, 46.78; H, 5.83; N, 7.42. Cbz Tau O L Menthol ( 7. 19e). white solid, 0.25 g, 62%, mp 67.1 D 20 c 1.0, CH 3 OH); 1 H NMR (300 MHz, CDCl 3 7.19 (m, 5H), 5.58 (s, 1H), 5.10 (s, 2H), 4.71 4.43 (m, 1H), 4.23 3.93 (m, 1H), 3.81 3.50 (m, 2H), 3.36 3.12 (m, 2H), 2.34 2.13 (m, 1H), 2.10 1.93 (m, 2H), 1.8 1.58 (m, 2H), 1.55 1.30 (m, 2H), 1.33 1.14 (m, 2H), 0.97 0.87 (m, 6H), 0.81 (d, J = 4.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 156.3, 136.3, 128.6, 128.2, 128.1, 83.7, 67.0, 51.8, 47.5, 42.2, 35.9, 33.7, 31.7, 25.8, 23.1, 21.9, 20.9, 15.6; HRMS (ESI TOF) m/z : [M + Na] + Calcd for C 20 H 31 NO 5 S Na 420.1815; Found 420.1829.

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180 CHAPTER 8 CONCLUSIONS AND SUMMARY OF ACHIEVEMENTS Chapter 1 provides a g eneral introduction to the themes presented throughout this thesis. Th is represents a general overview of the work and include s strategies for the peptide synthesis, chemistry of N subsituted benzotriazoles and importance of peptide and peptidomimetics. Chapter 2 decribes the formation of native peptides via chemical ligations from trypto phan containing isopeptide s In this study, we report a statistical, predictive model using an extensive synthetic and com putational approach to rationalize the chemical ligation. We achieved N N acyl migrations which form longer native peptides without the use of Cys/Ser/Tyr residues or an auxiliary group at the ligation site. The feasibility of these traceless chemical liga tions is supported by b (N C) bond distance s in N acyl isopeptides. The intramolecular nature of the chemical ligations were justified using competiti on experiment s and theoretical calculations Chapter 3 reports the efficient preparation of azodye labeled aminoxy acids and peptides. Aminoxy acids are analogues of amino acids. Azodyes are a widely used chromophoric unit to label peptides. Chapter 4 highlights on syntheses, absorption and fluore scence data of new fluorescent coumarin labeled depsipeptides Variations of quantum yields in different solvents are reported and rationalized in terms of ICT TICT excited states. 7 Methoxycoumarin labeled depsipeptides are efficient for probes since they e xhibit high quantum yields Moreover 7 diethylaminocoumarin labeled depsipeptides, with their unique TICT state in polar protic solvents, are highly sensitive to solvent polarity, H bonding ability and the organized nature of the solvent medium such as a n SDS micelle.

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181 The present study complements that on the dye labeled natural peptides. Examination of the function and application of labeled peptidomimetics or depsipeptides should now be facilitated. Chapter 5 decribes the development of a mild protocol towards the synthesis of azapeptides from amino acid residues using benzotriazole methodology S table, crystalline and easy to handle azadipeptidoyl benzotriazoles were prepared and their synthetic utility was demonstrated by the synthesis of azatripeptides and azatetrapeptides. In addition, a new route to azatripeptides containing a natural amino ac id at their N terminus was developed .The novel pathway enabled the solution phase synthesis of an aza Leuenkephalin analogue In Chapter 6 we report the synthesis, X ray structure determination and conformational analysis of a novel class of heteroatom modi fied peptidomimetics, which we have termed C bond with an O N bond creates a completely new, previously unknown family of peptidomimetics, which are hydrolytically stable and display very interesting co nformational behaviour. C onformational analysis supported by X ray suggests that the oxyaza moiety can effectively induce turns, which can make the newly discovered oxyazapeptide scaffold a useful tool for drug discovery and for design of biologics. In Chapter 7 taurine containing water soluble peptidomimetics were designed and synthesized. N terminal taurine acylations allowed synthesis of a number of taurine containing peptides. N protection of taurine with Cbz and SO 2 activation with benzotriazole followed by coupling with various amino ester, dipeptides and nucleophiles provided N O taurine conjugates and sulfonopeptides.

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191 170) Bolla, M.; Collette, L.; Blank, L.; Warde, P.; Dubois, J. B.; Mirimanoff, R. O.; Storme, G.; Bernier, J.; Kuten, A.; Ste rnberg, C.; Mattelaer, J.; Torecilla, J. L.; Pfeffer, J. R.; Cutajar, C. L.; Zurlo A.; Pierart, M. Lancet 2002 360 103. 171) Deechongkit, S.; Dawson P. E.; Kelly, J. W. J. Am. Chem. Soc. 2004 126 16762. 172) Santini, A.; Meca, G.; Uhlig, S.; Ritieni, A. World Mycotoxin J. 2012 5 71. 173) Scherkenbeck, J.; Jeschke, P.; Harder, A. Curr. Top. Med. Chem. 2002 2 759. 174) Dyker, H.; Scherkenbeck, J.; Gondol, D.; Goehrt, A.; Harder, A. J. Org. Chem. 2001 66 3760. 175) Richardson, J. S. Adv. Protein Chem. 1981 34 167. 176) Katritzky, A. R.; Avan, I.; Tala, S. R. J. Org. Chem. 2009 74 8690. 177) Thormann M.; Hofman, H. J. J. Mol. Struct THEOCHEM 1999 469 63. 178) Danquah, M. K.; Agyei, D Biotechnology 2012 1, 5. 179) Ghale, G.; Kuhnert, N.; Nau, W. M. Nat. Prod. Commun. 2012 7 343. 180) Allen, J. G.; Atherton, F. R.; Hall, M. J.; Hassall, C. H.; Holmes, S. W.; Lambert, R. W.; Nisbet, L. J.; Ringrose, P. S. Nature 1978 272 56. 181) Rincn, A. M.; Prados, P.; de Mendoza, J. J. Am. Chem. Soc. 2001 123 3493. 182) Kakaei, S.; Xu, J. Tetrahedron 2013 69 9068. 183) Moree, W. J.; van der Marel, G. A.; Liskamp, R. J. J. Org. Chem. 1995 60 5157. 184) Gennari, C.; Salom, B.; Potenza, D.; Williams, A. Angew. Chemie Int. Ed. 1994 33 2067. 185) Gennari, C.; Salom, B.; Potenza, D.; Longari, C.; Fioravanzo, E.; Carugo, O.; Sardone, N. Chem. Eur. J. 1996 2 644. 186) Palakurthy, N. B.; Dev, D.; Rana, S.; Nadimpally, K. C.; Mandal, B. Eur. J. Org. Chem. 2013 2627. 187) Obreza, A.; Gobec, S. Curr. Med. Chem. 2004 11 3263. 188) Moree, W. J.; Schouten, A.; Kroon, J.; Liskamp, R. M. J. Int. J. Pept. Protein Res. 2009 45 501. 189) S. Tetrahedron 2006 62 10980.

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193 BIOGRAPHICAL SKETCH Suvendu Biswas was born in Kolkata, India. He received his Bachelor of Science degree in C hemistry in June 2006 from Narendrapur Ram a krishna Mission, Kolkata under Calcutta Univer s ity. During h is M S studies Suvendu worked under the supervision of Professor Pradeep Mathur In August 2008, Suvendu joi ned the graduate program in the Department of Chemistry at the Univ ersity of Florida and pursued his Ph.D. in organic chemistry under the guidance of Professor Alan R. Katritzky at the C enter of Heterocyclic Compounds. He worked on the design of novel synt hetic st r ateg ies for the synthesis of the peptide and peptide like organic molecules. During h is course of study, Suvendu has participated in various internationally renowned conferences and delivered poster presentation. He maintained a n overall GPA of 3. 7 9 during his graduate study in UF and received the Grinter Fellowship A ward from the D ept artment of Chemistry, University of Florida. He received his Ph.D. from the University of Florida in the spring of 2014.



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MicellarCatalysisUsingaPhotochromicSurfactant:Applicationto thePd-CatalyzedTsuji TrostReactioninWaterMurielBillamboz,FlorianeMangin,NicolasDrillaud,CaroleChevrin-Villette,EstelleBanaszak-Le onard, andChristopheLen *Universite deTechnologiedeCompie gne,TransformationInte gre edelaMatie reRenouvelable,EA4297UTC/ESCOM,Centrede recherchedeRoyallieu,BP20529,F-60205Compie gneCedex,France*SSupportingInformation ABSTRACT: The rstexampleofaPd-catalyzedTsuji Trostreaction, appliedinaphotochromicmicellarmediaunderconventionalheating andmicrowaveirradiation,isreported.Thesurfactantactivityand recyclingabilitywereinvestigatedandcomparedwiththoseofafew commerciallyavailablesurfactants.Thesyntheticphotochromic surfactantprovedtobee cient,recyclable,andversatileforPdcatalyzedcouplingreactions.INTRODUCTIONWiththenotionofgreenchemistry,organicchemistsare stronglyencouragedtodevelopsaferprotocols.Amongthe12 principlesthatgoverngreenchemistry,1organicreactions conductedinsafersolvents havereceivedconsiderable attention.2Inthiscontext,substantiale ortshavebeendevoted tothedevelopmentofe cientPd-catalyzedcross-coupling reactionsinaqueousmedia.3Waterasasolventhasmanyadvantagesoverusualorganic solvents:itistheleastexpensiveandsafestsolventthatis non ammable,inexplosive,andnontoxic.Waterhasunique propertiesinsolvatingorganicmolecules,leadingtopositive e ectsonreactivitiesandselectivities.4However,thepotential scopeofaqueousorganometalliccatalysisisdrasticallyreduced whenhighlyhydrophobicsubstratesareinvolvedinthe chemicaltransformations.Toovercomethisproblem,di erent strategieshavebeenstudied:theuseoforganiccosolvents5or ionicliquids6aswellasadditives,suchasphasetransferagents,7cyclodextrins,8polymers,9orsurfactants.10Recently,wehavereportedthesynthesisanduseofanovel photochromicazobenzene-basedsurfactantinacetylation reactionsinwater.11Thissurfactantwasdesignedto(i) photo-organizeanddisorganizeinaqueoussolution,(ii)allowa betterextractionoftheproductsformedduetoitsphotochromismproperty,(iii)facilitatethereactionstakingplacein anaqueousphase,and(iv)enabletherecyclingoftheaqueous phase.Onthebasisofthesefactors,theconceptof photochromicsurfactantforPd-catalyzedcross-coupling reactionsinwaterwasinvestigatedinoneofthemost importantreactionsforcarbon carbonbondformation,the Tsuji Trostreaction.Pd-catalyzedallylicsubstitutionreactions arewidelyemployedforconstructingC C,C N,C S,and C Obondswithhighchemo-,regio-,andstereoselectivities.12Surprisingly,thedevelopmentoftheTsuji Trostreactionin aqueousphotoresponsivemicellarmediawasnotdescribed. Herein,wereportthescopeandlimitationsofthismethodologyusingconventionalandmicrowaveheatingunderlow loadingconditions(1mol%Pd).RESULTSANDDISCUSSIONSeveralphotoresponsivesurfactantshavebeenreportedwith di erentphotoresponsivegroupsusedtoprovidestructural change.13ThenonionicsurfactantC4-Azo-PEG 1 wasselected forthisstudysinceionicsurfactantscaninhibitthereactiondue toelectrostaticrepulsionsatthemicelle waterinterface.14It possessesanazobenzenemoietyasthephotochromiccore,a C4alkylchainasahydrophobictail,andapolyethyleneglycol (PEG)asanonionichydrophilicheadgroup(Figure1). Thesynthesisof 1 was rstdescribedbyShangetal.in 2003,15buttheproposedthree-stepprotocolsu ersfroma verypooroverallyieldof3%.Forthepresentwork,amodi ed three-stepprotocolwasfollowed.16Azobenzene 3 was synthesizedbyoxidativecouplingof4-butylaniline 2 and phenolviathecorrespondingdiazoniumsalt.Then,aclassical Williamsonetheri cationofphenolderivative 3 withthe tosylate 4 obtainedbyselectivesulfonatationoftheglycol derivativePEG3furnishedthedesiredC4-Azo-PEG 1 in50% overallyield(Scheme1). Received: August24,2013 Published: December2,2013 Figure1. StructureofC4-Azo-PEG 1 Article pubs.acs.org/joc 2013AmericanChemicalSociety493dx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500

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Thesurfacetensionmeasurementsof 1 havebeenpreviously reported,beforeandafterUVirradiation.Ithasbeenclearly demonstratedthat 1 formsmicelles,andtheCMC(critical micellarconcentration)ofthe trans andthe cis formsare4.1 and8 M,respectively.15The cis / trans equilibriumof 1 wasalsopreviouslystudied witha200Wmercurylamp.15Tocon rmtheabilityofthe diazofunctiontoisomerizeunderUVexposure(Scheme2), 1 wasanalyzedbyUV/visabsorptionspectroscopy.Inourhands, usinga500Wmercurylamp,irradiationat365nmof 1 ( trans ) indeionizedwateratroomtemperatureresultedinasubstantial changeintheUV/visspectrumof 1 ,duetoitsswitchingfrom its trans to cis isomers.Theabsorptionbandataround320nm wasfoundtodecreasegraduallywithcontinuedirradiation.At thesametime,thebandsataround250and420nmslightly increase.Theabsorptionbandsat320,250,and420nmare ascribedto andn ofthe trans andthe cis formsofthe azomoiety,respectively,17whichisinaccordancewiththe trans photoisomer 1 ( trans )beingconvertedintoits cis form.The maximumisomerizationyieldwasobtainedafter14minof irradiationat365nm(Figure2). Whenirradiatedat254nm(Figure3),the cis isomerof 1 returnedgraduallytoits trans form,andthemaximum isomerizationyieldwasobtainedafter6minofirradiation. Finally,asalreadyshownfornumerousazoderivatives,itmust bepointedoutthatthe cis isomercouldnotbeisolatedinpure form.18Repeatingtheirradiationof 1 insolution,toswitchbetween isomersdidnotrevealanydegradationof 1 ,asthemaximum UVspectrumobtainedaftereachirradiationwasconsistentto thepreviousone(Figure4). Resultsobtainedareinaccordancewiththepreviously describedabilityof 1 tophotoisomerizewithoutdegradation regardlessofthepowerofthemercurylamp(500Wvs200 W),whichonlyalloweddecreasingthetimenecessaryto achieveequilibrium. ToevaluatethepotentialoftheC4-Azo-PEG 1 inmicellar catalysis,theprotocoldevelopedin2005byFelpinetal.forthe 10%Pd/C-mediatedallylicsubstitutioninpurewaterwastaken Scheme1.SyntheticPathwaystoC4-Azo-PEG1 Scheme2.IsomerizationEquilibriumbetween1( trans )and 1( cis ) Figure2. OverlaidUVspectra(1scanper15s,from0to18min) duringtheisomerizationof 1 at365nm(500Wlamp).Sample concentration:10 4mol/L. Figure3. OverlaidUVspectra(1scanper15s,from0to6min) duringtheisomerizationof 1 at254nm(500Wlamp).Sample concentration:10 4mol/L. Figure4. Reversibilityofphotoisomerizationin 1 solutionat2 10 4mol/Lforthe trans -tocis and cis -totrans processes.Thesolutionwas irradiatedalternatelywith365and254nmfor14and6min, respectively.Theabsorbanceatthe trans bandwasrecordedaftereach illumination. TheJournalofOrganicChemistry Articledx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500494

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asreference.19Thisheterogeneousprocessusesaninexpensive andenvironmentallyfriendlysourceofPdsinceitis immobilizedonasupportandcanbeeasilyremovedbysimple ltration.Nevertheless,theoptimizedreactionrequireda prolongedheatingover18h.Also,theauthorsdidnotevaluate therecyclabilityofthecatalyticsystem.Inthepresentstudy, environmentalaspectshavebeenrespectedbyminimizingthe reactiontimeandreusingthecatalyst. Cinnamylacetate 5 wasselectedasamodelsubstratewith the p -toluenesul nicacidsodiumsalt( p -TsNa)asnucleophile (Scheme3).Catalystloadingandtemperaturehavebeen optimizedpreviously(1mol%ofPdand70 C)andwillnot beextendeduponhere. Withoutanysurfactant,atotalconversionwasobservedafter 18handthedesiredsulfone 6 wasisolatedin86%yield.Asthe conceptofthisworkisbasedonthecapabilityofthesurfactant tophoto-organizeanddisorganizewhensubmittedto irradiation,theconcentrationof 1 needstobecomprised betweentheCMCofthe trans form(4.1 M)andtheCMCof the cis form(8 M).Inthisregard,the rsttrialwasconducted with6 Mof 1 ,butatthisconcentration,noenhancementof thereactionwasobserved.Onepotentexplanationwasthe charcoalcapacityinadsorbing 1 ,leadingtoadecreaseoffree C4-Azo-PEG 1 inwater.Indeed,UVanalysisexperiment showedthat 1 (CMC trans <6 M
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(80,71,and83%yields,respectively).Thegoodresultobtained withCTABwasnotsurprisingsinceithasbeenpreviously reportedthattheCTABadsorptiononthecatalystsurfacecan enhancethereactivity.20Forallexperiments,sampleswere submittedtothesamedirectethylacetateextractionexceptfor entry6.Itisnoteworthythatirradiationcoupledwithextraction increasedtheyieldto96%yield(Table1,entry6).This di erenceinyield(13%)couldbedirectlycorrelatedtothe photochromicpropertiesof 1 .Thephotoirradiationdirectly actsonthesurfactanttoenableabetterextractionof 6 fromthe media,duetoane cientbreakdownoftheemulsion,visually seenexperimentally(Table1,entries5and6). ToexplorefurtherthescopeofC4-Azo-PEG 1 andto developane cientgreensystem,recyclingexperimentswere performedforthereactionusing10%Pd/C(1mol%)andthe surfactantsat3CMCconcentration(Figure7). Aftercompletingeachcycle,organicproductswereextracted threetimeswithethylacetate,andcinnamylacetate 5 (1equiv), p -TsNa(1equiv),andtriphenylphosphine(4mol%)were addedagaintotheaqueousphase.Theirradiatione ectwas alsoevaluatedwhenthereactionwasconductedwithC4-AzoPEG 1 (30minat365nmjustbeforeextraction).Asexpected, the10%Pd/C-SDSsystemwasnotrecyclable.EvenifSDSis knowntobeaPdstabilizer,21electrostaticrepulsionswiththe anionicnucleophilearepredominantandnoconversionwas observedforthesecondrun.Onlytworunswithmoderate yields(71%and67%yield,respectively)couldbeperformed withTween20beforeadramaticdecrease.After verunswere performedwithCTAB,aslowdecreaseateachrunwas observeduntil0%yieldwasobtainedafterthe fthrun.When thedirectextractiontreatmentwasapplied,usingC4-Azo-PEG 1 ,fourcycleswereachievableingoodyieldsof,respectively,83, 99,92,and72%,afterwhichnofurthercatalytice ectwas observed.However,whenwepretreatedfor30minwith365 nmirradiation,anincreaseofyieldwasobservedforeachcycle andthecatalyticsystemcouldbereusedforfourconsecutive runswithoutanylossofactivity.Fromtheseresults,itshould behighlightedthattheuseofthephotochromicC4-Azo-PEG surfactant 1 enhancedthereactivityandrecyclabilityofthe catalyticsystem. Withitshighdielectricconstant,waterispotentiallyavery usefulsolventformicrowave-mediatedsynthesis.22Indeed, microwaveheatinghasbeenwidelyrecognizedasane cient tool,anditsbene tshavebeenwell-documented.23Sincemany reactionsareknowntoresultinhigheryieldand/orshorter reactiontimes,thisalternativetechnologywasdevelopedinour groupforSuzuki Miyauracross-couplingofvariousuridinesin purewater.24Nevertheless,tothebestofourknowledge,there areonlyafewreportsonusingmicrowaveheatingcoupledwith cross-couplingmicellarcatalysis,suchfocusonSuzukior Sonogashiracouplings.25Asinthermalactivation,di erent reactiontimesweretestedtodevelopoptimizedconditions undermicrowaveirradiation.Tohaveagoodenergysaving,we decidedtoruntheexperimentnolongerthan30min. AsdescribedinFigure8,thebestcompromisebetween e ciencyandenergysavingwastoreactfor15minat70 C. Itwasnotedthatincreasingthetimeto30minallowedonly amodest5%yieldgain.Table2showstheresultsobtained underthefollowingconditions: pTsNa(2equiv),10%Pd/C(1 mol%),PPh3(4mol%),surfactant(3CMC),70 C, microwaveirradiation,15min.Asinconventionalheating, particularattentionwaspaidtotherecyclingabilityofthe systemandtheroleofthephotochromicsurfactant 1 .Inthe rstinstance,weobservedthatthe rstrununderthese conditionsgavecomparableresultsbetweenthermalheating andmicrowaveactivationforallcommerciallyavailable Figure7. Comparisonofrecyclingabilitiesunderconventionalheating. Figure8. Evolutionofyieldvstimeofreactionundermicrowave irradiation. Table2.SurfactantActivitiesandRecyclingAbilitiesunder MicrowaveIrradiationyieldof 6 (%)bentrysurfactantrun1run2run3run4run5run6 1none2022160 2SDS260 3Tween20730 4CTAB80100260 5C4-Azo-PEG6497280 6C4-Azo-PEGa65942624120aIrradiationat365nmduring30minjustbeforeextraction.b5 (1 equiv), pTsNa(2equiv),10%Pd/C(1mol%),PPh3(4mol%), surfactant(3CMC),70 C,3h. TheJournalofOrganicChemistry Articledx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500496

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surfactants(Figure7;Table2,entries2 4).Whenno surfactantwasused,themediawasrecyclabletwicewithlow yield(Table2,entry1). Therefore,wecanassumethatthepalladiumsourcekeptits activitytothesameextentaswithmicrowaveirradiation.SDSis thelessactivesurfactantundertheseconditions(Table2,entry 2)andisnotrecyclable.Moresurprisingly,Tween20,which wasrecyclableinconventionalheating,performedagood rst run(73%yield)beforeshowingadramaticlossofactivity (Table2,entry3).Theselastcommerciallyavailablesurfactants SDSandTween20seemedtoleadtoamarkeddeactivationof palladiuminonlyonerun.CTAB,asinconventionalheating (Figure7),isaseriousconcurrenttoC4-Azo-PEG 1 intermsof activityandrecyclability.ThetwosurfactantsCTABandC4Azo-PEG 1 indicatedausefulpotential(Table2,entries4and 5).Thesecondrunwasquantitative,butyieldsdramatically decreasedforthethirdrun.Theirbehaviorwasthesamewhen noirradiationstepoftheC4-Azo-PEG 1 wasincludedinthe sampletreatment(Table2,entry5).C4-Azo-PEG 1 ,asin conventionalheating,provedtobethemostinteresting surfactanttested,whensubmittedtotheirradiation extraction treatment(i.e.,irradiationat365nmfor30minjustbefore extraction)(Table2,entry6). Accordingtotheresultspresentedherein,theincreaseofthe catalyticactivityobservedwhenthereactionmediumis subjectedtomicrowaveirradiation(i.e.,15minvs3hunder conventionalheating)isduepresumablytoelectricdischargeor hotspotscreatedwithintheheterogeneouscatalyst.This phenomenonhasbeenpreviouslyobservedandstudiedinC CandP Ccouplings.26Inbothconditions,recyclingofthe 10%Pd/C-C4-Azo-PEG 1 systemwasconducted(Figure9). The rstandthesecondcyclesgavesimilarresults. Nevertheless,theactivityofthecatalystdecreaseddrastically duringthethirdcycleforthemicrowaveheatedsystem.This tendencywascon rmedduringthefourthrun.Inthese conditions,themetalsu eredthefastestdeactivationunder microwaveirradiationthanconventionalheating.Thedecrease incatalyticactivitycouldresultfromtheaggregationof palladiumnanoparticlesorblockageoftheactivesites.21One potentexplanationwasthatmicrowaveheatingcreatessmall andlocallysuperheated,highlyactivesites,leadingtoan accelerationofthedeactiv ationwhenthetemperature increased.27Toexplorefurtherthescopeofthisnewprocess,thereaction ofthreedi erentallylicacetates 5 7 ,and 8 withsomedi erent nucleophileswasexamined(Table3). UnderouroptimizedmethodusingC4-Azo-PEG 1 in thermalactivation,alldesiredcompoundswereobtainedin moderatetoexcellentyields(36 97%).Linearcinnamyl acetate 5 havingan E con gurationreactedsmoothlywith p toluenesul nicacidsodiumsalttogivethedesiredsulfone 6 in 96%yield,withoutanylossofcon guration(Table3,entry1). Startingfromacetate 5 withmorpholineanddibenzylamineas N -nucleophilesledtothetargetcompounds 11 and 14 ingood yields(75%and62%,respectively)(Table3,entries4and7). Undertheseconditions,withnoadditionalbaseinthereaction medium,nosidereactionofsaponi cationwasobserved.When C-nucleophilesaretested,abaseisnecessaryinthereaction mediumbutledtosomecompetitivesaponi cationofthe startingacetate 5 (10 15%ofcinnamylalcoholisolated).In theseconditions,loweryieldscanbeachieved.However,dimer 17 wasobtainedwith2,4-pentanedioneinagoodyieldof80% (Table3,entry10).TheuseofahardierO-nucleophile,phenol, surprisinglyledtothetargetcompound 18 inexcellentyield (97%,Table3,entry11).Themuchhinderedallylicacetate 7 wasfoundtobeagoodsubstrateundertheoptimized conditions.Assuch,compounds 9 12 ,and 15 wereobtainedin goodtoexcellentyields(Table3,entries2,5,and8).Despite itshighsterichindrance,compound 15 wasobtainedin77% yield.Finally,thebranchedallylicacetate 8 ,asexpected,was foundtobelessreactive,andthesulfone 10 wasobtainedin 52%yield.Morpholineas N -nucleophilewasfoundtoreact betterwiththeacetate 8 ,leadingtothedesiredcompound 13 in60%yield.Themuchmorehindered,dibenzylamine,gave compound 16 butinamodest36%yield.Inthiscase,the unreactedacetate 8 wasrecoveredwithoutmodi cation.An importantpointthatdeservescommentistheregioselectivityof thereaction.SimilartomosthomogeneousPd-catalyzed reactions,onlysubstitutionattheleasthinderedallylicposition isobserved.Inthisrespect,thisreactionappliedtoallylic acetatesshowedagoodrangeofcompatibilitywithvarious nitrogenorsulfurnucleophiles.CONCLUSIONInaccordancewiththeobjectiveofgreenchemistry,theTsuji Trostreactioninaqueousphotoresponsivemicellarmedia provedtobee cientunderconventionalheatingand microwaveirradiation.AsC4-Azo-PEG 1 canorganizeand disorganizeinaqueoussolutionunderUVirradiation,itallows abetterextractionoftheproductsformedduetoits photochromismpropertyandanenhancementofreactivity andrecyclabilityofthecatalyticsystem.Forthisstudy, conventionalheatingseemedtobemorefavorablethan microwaveirradiation.Thisattractivetechniquealloweda decreaseinreactiontimeandasavingsinenergy,butthe downsidewasafasterdeactivationofcatalyst.Ouroptimized methodunderconventionalheatingwasextendedtomuch hinderedandbranchedallylicacetates,andthesystemshowed agoodrangeofcompatibilitywithvariousnucleophiles.As photochromicsurfactantsbasedonazobenzenemoietiesand,in particular,C4-Azo-PEG 1 arereallyinterestingandversatile compounds,theirpotentialincatalysisshouldbemore extensivelyexplored.EXPERIMENTALSECTIONMaterials. Allcommerciallyavailableproductsandsolventswere usedwithoutfurtherpuri cation.ReactionsweremonitoredbyTLC (Kieselgel60F254aluminumsheet)withdetectionbyUVlightor potassiumpermanganateacidicsolution.Columnchromatographywas Figure9. Comparisonofrecyclingabilitiesbetweenconventional heatingandmicrowaveirradiationinthepresenceofC4-Azo-PEG 1 afterirradiationat365nmduring30minjustbeforeextraction. TheJournalofOrganicChemistry Articledx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500497

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performedonsilicagel40 60 m.Flashcolumnchromatographywas performedonanautomaticapparatus,usingsilicagelcartridges.UV analyseswereperformedonaUV/visspectrophotometercoupledwith anoptic ber.A500Wmercuriclampwasusedfortheirradiationof theC4-Azo-PEGsolutions.1Hand13CNMRspectrawererecorded ona400MHz/54mmultralonghold.Chemicalshifts( )arequoted inpartspermillion(ppm)andarereferencedtoTMSasaninternal standard.Couplingconstants( J )arequotedinhertz.Microwave experimentswereconductedinacommercialmicrowavereactor especiallydesignedforsyntheticchemistry.Monowave300(Anton Paar,Austria)isamonomodecavitywithamicrowavepowerdelivery systemrangingfrom0to850W.Thetemperaturesofthereactions weremonitoredviaacontactlessinfraredpyrometer,whichwas calibratedincontrolexperimentswitha ber-opticcontact thermometer.Sealedvesselsandamagneticstirbarinsidethevessel wereused.Temperatureandpowerpro lesweremonitoredinboth casesthroughthesoftwareprovidedbythemanufacturer. SynthesisofC4-Azo-PEG1. 4-Butyl-4 -hydroxyazobenzene( 3 ). Toasolutionof4-butylaniline( 2 )(30g,0.20mol)in37%HCl(50 mL)andwater(25mL)wasaddedNaNO2(16.6g,0.24mol)inwater (25mL)dropwisein30min.Thereactionwasconductedinanice bath.Thecrudesolutionwasaddeddropwisetoasolutionofphenol (20.7g,0.22mol)insoda(100mL,6M).Finally,thesolutionwas quenchedwithHCl.Theproduct 3 wasobtainedafter ltrationand recrystallizationfrompentaneasaredsolid(40.1g,79%).Analyses areconsistentwiththeliterature.15mp=80 C.1HNMR(400MHz,CDCl3): 7.85(d, J =8.8Hz, 2H),7.79(d, J =8.3Hz,2H),7.30(d, J =8.3Hz,2H),6.92(d, J =8.8 Hz,2H),2.68(t, J =7.7Hz,2H),1.75(bs,1H),1.64(quin, J =7.6 Hz,2H),1.37(sex, J =7.5Hz,2H),0.94(t, J =7.3Hz,3H).13CNMR (100MHz,CDCl3): 158.0,150.8,147.1,145.9,129.0(2 ),124.7 (2 ),122.5(2 ),115.7(2 ),35.5,33.4,22.3,13.9. TriethyleneglycolMonotosylate( 4 ). Toasolutionoftriethylene glycol PEG3 (11g,73.3mmol),triethylamine(2.6mL,19.1mmol), andDMAP(45mg,0.37mmol)in95mLofDCMwasaddedtosyl chloride(3.5g,18.1mmol)at5 C,andthemixturewasstirredfor4 h.Theresultingmixturewaswashedwith1NHCL,H2O,andbrine. TheorganiclayerwasdriedoverMgSO4andconcentratedunder vacuum.Theresiduewaspuri edbysilicagelchromatography (EtOAc/cyclohexane7:3)toobtainthetitlecompound 4 asyellowoil (4.2g,75%).28 Table3.ScopeoftheTsuji TrostReaction TheJournalofOrganicChemistry Articledx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500498

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1HNMR(400MHz,CDCl3): 7.80(d, J =8.2Hz,2H),7.35(d, J =8.0Hz,2H),4.16(t, J =4.7Hz,2H),3.70(q, J =4.7Hz,4H),3.61 (s,4H),3.57(t, J =4.5Hz,2H),2.45(s,3H),2.32(s,1H).13CNMR (100MHz,CDCl3): 144.8,132.8,129.7(2 ),127.9(2 ),72.4, 70.7,70.2,69.1,68.6,61.6,21.6. TriethyleneGlycolMono(4-butylazobenzene)Ether(C4-Azo-PEG 1 ). Inaround-bottom askunderN2,triethyleneglycolmonotosylate ( 4 )(4.4g,14.5mmol),K2CO3(9.9g,72.3mmol),andLiCl(20mg,3 mol%)weredissolvedinMeCN(150mL).Asolutionof4-butyl-4 hydroxyazobenzene( 3 )(3.9g,15.43mmol)inMeCN(50mL)was addeddropwise.Themixturewasre uxedunderN2for18h.The solventwasthenevaporatedundervacuum.Theresiduewasdissolved inDCMandthenwashedwithbrine(3 100mL).Theorganiclayer wasdriedoverMgSO4andconcentratedundervacuum.Theproduct 1 waspuri edbysilicagelchromatography(EtOAC/cyclohexane7:3) toobtainthedesiredcompoundasayellowsolidafterrecrystallization frompentane(2.6g,85%).Analysesareconsistentwiththe literature.15mp=60 C.1HNMR(400MHz,CDCl3): 7.91(d, J =8.8Hz, 2H),7.82(d, J =8.2Hz,2H),7.31(d, J =8.2Hz,2H),7.04(d, J =8.8 Hz,2H),4.24(t, J =4.6Hz,2H),3.92(t, J =4.6Hz,2H),3.72 3.78 (m,6H),3.65(t, J =4.4Hz,2H),2.70(t, J =7.7Hz,2H),2.34(s, 1H),1.66(quin, J =7.6Hz,2H),1.39(sex, J =7.4Hz,2H),0.96(t, J =7.3Hz,3H).13CNMR(100MHz,CDCl3): 160.9,150.9,147.2, 145.8,129.0(2 ),124.5(2 ),122.5(2 ),114.7(2 ),72.4,70.8, 70.3,69.6,67.6,61.7,35.5,33.4,22.3,13.9. GeneralProcedurefortheTsuji TrostReaction. A30mL MWvesselwaschargedwith10%Pd/C(20mg,1mol%),PPh3(20 mg,4mol%),thedesiredallylicacetate(2mmol),andnucleophile(4 mmol).Whennecessary,potassiumcarbonate(4mmol)wasaddedin themedium.Thesolutionofthesurfactantinwater(10mL)wasthen added.Themixturewasheatedat70 Cfor3h(conventional heating)or15min(MWirradiation.The nalproductwasextracted withEtOAc(3 5mL)beforepuri cation.Whennecessary,the extractioncanbeprefacedbya30minirradiationundera365nm lamp.Forrecyclingtests,onlyallylicacetate(2mmol),PPh3(4mol %),andnucleophile(2mmol)wereintroduced. trans-Cinnamyl-p-tolylSulfone( 6 ).19Flashchromatography(0% EtOAc cyclohexaneto50%EtOAc cyclohexane)gavethedesired product 6 asawhitepowder(522mg,96%). mp=120 C.1HNMR(400MHz,CDCl3): 7.69(d, J =8.3Hz, 2H),7.20 7.27(m,7H),6.32(d, J =15.5Hz,1H),6.04(quin, J =7.5 Hz,1H),3.86(dd, J =0.8,7.5,2H),2.37(s,3H).13CNMR(100 MHz,CDCl3): 144.7,138.9,135.8,135.5,129.6(2 ),128.6(2 ), 128.5(2 ),128.4,126.5(2 ),115.3,60.5,21.5. 1,3-Diphenyl-2-propenyl-p-tolylSulfone( 9 ).19Flashchromatography(0%EtOAc cyclohexaneto100%EtOAc cyclohexane)gave thedesiredproduct 9 asacolorlesssolid(362mg,52%).Analysesare consistentwiththeliterature. mp=158 159 C.1HNMR(400MHz,CDCl3): 7.53(d, J =8.4 Hz,2H),7.29 7.36(m,10H),7,20(d, J =8.0Hz,2H),6.55 6.58 (m,2H),4.81(d, J =7.6Hz,1H),2.40(s,3H).13CNMR(100MHz, CDCl3): 144.5,137.9,135.9,134.4,132.5(2 ),129.7(2 ),129.3 (5 ),128.8,128.6(2 ),128.4(2 ),126.7,120.2,75.3,21.6. Cyclohex-2-enyl-p-tolylSulfone( 10 ).19Flashchromatography(0% EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethedesired product 10 asacolorlessoil(245mg,52%).Analysesareconsistent withtheliterature.1HNMR(400MHz,CDCl3): 7.74(d, J =8.3Hz,1H),7.33(d, J =7.9Hz,1H),6.02 6.09(m,1H),5.73 5.78(m,1H),3.69 3.72(m, 1H),2.44(s,3H),1.80 1.85(m,5H),1.46 1.49(m,1H).13CNMR (100MHz,CDCl3): 144.5,135.1,134.4(2 ),129.6(2 ),129.1 (2 ),61.8,24.3,22.7,21.6,19.5. 4-Cinnamylmorpholine( 11 ).19Flashchromatography(0% EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethedesired product 11 asacolorlessoil(304mg,75%).Analysesareconsistent withtheliterature.1HNMR(400MHz,CDCl3): 7.20 7.38(m,5H),6.52(d, J = 16,0Hz,1H),6.25(dt, J =6.8,13.6Hz,1H),3.73(t, J =4.4Hz,4H), 3.14(d, J =6,8,2H),2,50(m,4H).13CNMR(100MHz,CDCl3): 136.8,133.3,128.5,127.5(2 ),126.3,126.1(2 ),67.0(2 ),61.4, 53.6(2 ). 4-(1,3-Diphenylallyl)morpholine( 12 ).28Flashchromatography (0%EtOAc cyclohexaneto50%EtOAc cyclohexane)gavethe desiredproduct 12 asacolorlesssolid(502mg,90%).Analysesare consistentwiththeliterature. mp=65 C.1HNMR(400MHz,CDCl3): 7.21 7.44(m,10H), 6.60(d, J =15.8Hz,1H),6.32(dd,J=16,0,J=8,8Hz,1H),3,80(d, J = 8,8Hz,1H),3.74(t, J =4,8Hz,4H),2,56(m,2H),2.42(m,2H).13C NMR(100MHz,CDCl3): 168.4,167.8,140.3,137.0(2 ),132.0 (2 ),129.3(2 ),128.9,128.7,128.0(2 ),127.8,127.3(2 ),67.2, 61.4(2 ). 4-(Cyclohex-2-enyl)morpholine( 13 ).29Flashchromatography(0% EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethedesired product 13 asacolorlessoil(200mg,60%).Analysesareconsistent withtheliterature.1HNMR(400MHz,CDCl3): 5.89(d, J =10.4Hz,1H),5.60(d, J =10.4Hz,1H),3.66 3.69(m,4H),3.11 3.12(m,1H),2.50 2.52 (m,4H),1.94 1.95(m,2H),1.76 1.78(m,2H),1.50 1.53(m,2H).13CNMR(100MHz,CDCl3): 130.4(2 ),128.9(2 ),67.6,60.4, 49.3,25.3,23.1,21.5. N,N-Dibenzyl-3-phenylprop-2-enamine( 14 ).30Flashchromatography(0%EtOAc cyclohexaneto50%EtOAc cyclohexane)gavethe desiredproduct 14 asayellowishoil(388mg,62%).Analysesare consistentwiththeliterature.1HNMR(400MHz,CDCl3): 7.18 7.26(m,15H),6.52(d, J = 15.9Hz,1H),6.28(dt, J =6.6,13.4Hz,1H),3.61(s,4H),3.20(d, J = 6.2Hz,2H).13CNMR(100MHz,CDCl3): 139.6(2 ),137.2, 132.4,128.8,128.5(4 ),128.2(2 ),127.7(4 ),127.3,126.8(2 ), 126.2(2 ),57.9(2 ),55.7. Dibenzyl-(1,3-diphenylallyl)amine( 15 ).31Flashchromatography (0%EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethe desiredproduct 15 asawhitesolid(600mg,77%).Analysesare consistentwiththeliterature. mp=112 C.1HNMR(400MHz,CDCl3): 7.12 7.48(m, 20H),6.44(m,2H),4.36(d, J =6.9Hz,1H),3.65(d, J =13.8Hz, 2H),3.52(d, J =13.8Hz,2H).13CNMR(100MHz,CDCl3): 54.0 (2 ),65.1,126.5(2 ),126.8(2 ),127.1,127.6,127.7,128.2(2 ), 128.3(2 ),128.4(4 ),126.6(2 ),126.7(4 ),134.1,137.0,140.0 (2 ),141.9. N,N-Dibenzylcyclohex-2-enamine( 16 ).19Flashchromatography (0%EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethe desiredproduct 16 asacolorlessoil(199mg,36%).Analysesare consistentwiththeliterature.1HNMR(400MHz,CDCl3): 7.13 7.33(m,10H),5.65 5.76 (m,2H),3.68(d, J =14.1Hz,2H),3.46(d, J =14.1Hz,2H),3.45(m, 1H),1.80 1,92(m,3H),1.72(m,1H),1.53(m,2H).13CNMR(100 MHz,CDCl3): 141.1(2 ),131.0,130.2(4 ),128.6(4 ),128.3 (2 ),126.8,54.7(2 ),54.0,25.5(2 ),22.0. 3,3-Dicinnamylpentane-2,4-dione( 17 ).32Flashchromatography (0%EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethe desiredproduct 17 ascolorlesscrystals(265mg,80%).Analysesare consistentwiththeliterature. mp=60 61 C.1HNMR(400MHz,CDCl3):7.15 7.23(m, 10H),6.40(d, J =16.0Hz,2H),5.86(m,2H),2.78(dd, J =7.6Hz, J =1.2Hz,2H),2.10(s,6H).13CNMR(100MHz,CDCl3): 205.8 (2 ),136.8(2 ),134.3(2 ),128.6(4 ),127.6(2 ),126.2(4 ), 123.3(2 ),70.8,34.6(2 ),27.4(2 ). Cinnamylphenylether( 18 ).33Flashchromatography(0%EtOAc cyclohexaneto100%EtOAc cyclohexane)gavethedesiredproduct 18 asawhitepowder(408mg,97%).Analysesareconsistentwiththe literature. mp=64 65 C.1HNMR(400MHz,CDCl3):7.14 7.33(m,7H), 6.86 6.90(m,3H),6.64(d, J =15.8Hz,1H),6;33(m,1H),4.61(dd, J =5.6Hz, J =1.5Hz,2H).13CNMR(100MHz,CDCl3): 158.7, 136.5,133.0,129.5(2 ),128.6(2 ),127.9,126.6(2 ),124.6,120.9, 114.8(2 ),68.6. TheJournalofOrganicChemistry Articledx.doi.org/10.1021/jo401737t | J.Org.Chem. 2014,79,493 500499

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ASSOCIATEDCONTENT*SSupportingInformation1Hexperimentsspectraareprovided.Thismaterialisavailable freeofchargeviatheInternetathttp://pubs.acs.org.AUTHORINFORMATIONCorrespondingAuthor* E-mail:christophe.len@utc.fr.Fax:(+33)(0)344971591.NotesTheauthorsdeclarenocompeting nancialinterest.ACKNOWLEDGMENTSTheRe gionPicardieisgratefullyacknowledgedforits nancial support.F.M.thanksESCOM(EcoleSupe rieuredeChimie OrganiqueetMine rale)forherresearchfellowship.Wearealso gratefultosomefourthyearstudentsfortheirimplication duringpreliminaryworksoncatalysis.REFERENCES(1)Anastas,P.T.;Warner,J.C. GreenChemistry:TheoryandPractice ; OxfordUniversityPress:NewYork,1998;p30. (2)(a)Li,C.J.;Chan,T.H. OrganicReactionsinAqueousMedia ; Wiley:NewYork,1997.(b)Grieco,P.A. OrganicSynthesisinWater ; BlakieAcad.:London,1998. (3)(a)Lamblin,M.;Nassar-Hardy,L.;Hierso,J.-C.;Fouquet,E.; Felpin,F.-X. Adv.Synth.Catal. 2010 352 ,33.(b)Lipshutz,B.H.; Abela,A.R.;Boskovic,Z.V.;Nishikata,T.;Duplais,C.;Krasovskiy,A. 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