Citation
Development of Strategies for the Coupling and Cyclization of Peptides and Synthesis of Polyketide Fragments for Modified Peptides

Material Information

Title:
Development of Strategies for the Coupling and Cyclization of Peptides and Synthesis of Polyketide Fragments for Modified Peptides
Creator:
Ha, Khanh
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
LUESCH,HENDRIK
Committee Co-Chair:
CASTELLANO,RONALD K
Committee Members:
MCELWEE-WHITE,LISA ANN
MILLER,STEPHEN ALBERT
JAMES,MARGARET O

Subjects

Subjects / Keywords:
benzotriazole
cyclization
ligation
peptide
polyketide
staudinger

Notes

General Note:
From very early on, chemists have identified peptides and proteins as targets for the development of synthetic protocols. New and improved strategies lead to more efficient synthesis of complex peptide targets, opening the way to both new drug candidates and a deeper understanding of the intimate relation between sequence, conformation and properties. Despite recent progress and the arsenal of reagents available, peptide synthesis remains challenging: complex targets and regulatory authority constraints in terms of purity for drugs are continuously stimulating chemists to improve and rethink synthetic approaches. This thesis addresses the development of synthetic methods and approaches targeting medium sized cyclic peptides, and coupling of large peptide fragments. For coupling of large peptide fragments. Novel approach applying S to N long range acyl migration to synthesize peptide and peptide analogs and also provided mechanistic evidence for the ligation process is reported. A series of novel S acyl peptides containing beta or gama amino acid residues, which are useful intermediates in various synthetic and biological applications, were synthesized according to original S to N acyl migration protocols. Another challenge of NCL is the slow coupling rate at proline site. To address this problem, hydroxyproline ligation strategy utilizing hydroxyproline chemoselective bifunctionality was developed. In synthesis of cyclic peptides, ring size is a significant factor in the success of macrolactamisation in the synthesis of a cyclic peptide. 7 to 15 membered rings are less accessible and can frequently only be synthesized with difficulty, whether using solution phase or polymer-supported strategies. This dissertation describes the development of synthetic methods and approaches targeting mediumsized cyclic peptides including: intramolecular Staudinger ligation, cyclooligomerization, conformationally assisted marcocyclization. Among cyclic natural products, cyclodepsipeptide Apratoxins are intriguing marine natural products of mixed biogenetic origin. Apratoxins were isolated from cyanobacterial Lyngbya spp collected in Guam and Palau. In contrast to most known potent anticancer natural products, the cellular and molecular basis of apratoxins action is unknown at present. Part of this dissertation describes the large scale synthesis of polyketide fragment for further synthetic modification and biological studies of Apratoxins.

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UFRGP
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All applicable rights reserved by the source institution and holding location.
Embargo Date:
5/31/2018

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1 DEVELOPMENT OF METHODOLOGIES FOR COUPLING AND CYCLIZATION OF PEPTIDES AND SYNTHESIS OF POLYKETIDE FRAGMENTS FOR MODIFIED PEPTIDES By KHANH HA 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 2016

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2 2016 Khanh Quoc Ha

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3 To my family and my friends

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4 ACKNOWLEDGMENTS This dissertation is written i n appreciation of n umerous people who impacted me during this process. I am heartily thankful to Professor Alan R. Katritzky, and Professor Hendrik Luesch whose guidance and support from the initial to the final step enabled me to accomplish this work. I a m grateful to my committee members (Professor Lisa McElwee White, Professor Ronald K. Castellano Professor Stephen A. Miller and Professor Margaret O. James ) for their continuous assistance and support. I owe my deep gratitude to Dr. C. Dennis Hall for t he incredible help and support I received from him throughout the years and during the preparation of my publications I would like to thank former and current members of the Katritzky group, and professors of the Chemistry Department especially Dr. Ben Sm ith. I group who have contributed to this work: Dr. Siva S. Panda, Dr. Girinath G Pillai Dr. Mamta Chahar, Dr. Jean Christophe M. Monbaliu, Dr. Ekaterina Todadze, Dr. Finn K. Hansen and Dr. Oleg Bol and all of the undergraduate students: Sadra Hamedzadeh Grant Simpson Jay McDaniel Kristin Martin Charles Ocampo Byron Williams Amir Nasajpour Aaron Lillicotch Alexander Frey Eric Faby Jocelyn Macho Ryan Quiones and Jillian Eugatnom I also would like to thank members in Professor Luesch laboratory who have helped me : Dr. Qi Yin Chen and Dr. Ranjala Ratnayake I am indebted to my undergraduate honor thesis advisor Professor Galina G Levko vskaya and my mentors Dr. Kobelevskaya Valenti na and Dr. Elena V. Rudyakova at the Siberian Branch of the Russian Academy of Science. Without their help and encouragement it would have been very difficult to me to achieve my goals. Words are

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5 powerless to express my gratitude to my parents. They have always been there for me and encouraged me to find my own way. I woul d like to thank my friends Luyen Tran Lan Pham and their families for their continuous support for my education Thier tenacity and enthusiasm towards me being successful have been alway strong and it paid off for me M y passion for chemistry started many years ago when I realized that I love the pursuit to investigate and innovate various aspects of science. My efforts were pushed by Dr. Pham Thinh from Thai Nguyen University of Science, because he taught me to be eager about what I love and perform at the 110%. He taught me to expand my horizon by looking for research opportunities a n d motivated me to be courageou s He ispired me to go to Russia to obtain my undergraduate education. I am highly thankful for the foundation that he gave me. I would like to thank all the friends I have made through these years in Gainesville for their full support and fun time spent outside of the lab: Juan Serrano Dr. Mirna El Khatib Dr. Davit Jishkariani, Dr. I lker Avan, Dr. Bogdan Draghici, Philip Dmitriev Weijing Cai Danmeng Luo, Lorelie Imperial Thi Hoang Ha Nguyen Dr. Yen Tang and Dr. Kien Pham I would like to especially thank Dr. Ghebreghiorgis Thomas Dr. Amrita Mullick and Dr. Raghida Bou Zerdan who w ere always willing to help, support and give their best advice and support during my years at the University of Florida.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 11 LIST OF FIGURES ................................ ................................ ................................ ........ 13 LIST OF ABBREVIATIONS ................................ ................................ ........................... 19 ABSTRACT ................................ ................................ ................................ .................... 21 CHAPTER 1 OVERVIEW ................................ ................................ ................................ ............. 23 2 DEVELOPMENT OF NOVEL STRATEGIES FOR COUPLING OF PEPTIDE FRAGMENTS VIA S TO N LONG RANGE ACYL MIGRATION .............................. 30 Introducti on ................................ ................................ ................................ ............. 30 Results and Discussion ................................ ................................ ........................... 37 Microwave Assisted Chemical Ligation of S Acyl Peptides Containing Non Terminal Cysteine Residues ................................ ................................ .......... 37 Study of the feasibility of S N acyl migration via an 8 membered compared to a 5 membered cyclic transition state ................................ .. 37 Study of S to N acyl migration via an 11 membered cyclic transition state ................................ ................................ ................................ ........ 40 Study of S N acyl migration via a 14 membered cyclic transition state .... 42 Long Range Intramolecular S N Acyl Migration: a Study of the Formation of Native Peptide Analogs via 15 and 16 Membered Cyclic Transition States ................................ ................................ ................................ ............ 44 Demonstration of S N acyl migratio n in S acyl tetrapeptide via a 15 membered cyclic transition state. ................................ ............................ 44 Demonstration of S N acyl migration in S acyl tetrapeptides 23b,c each via distinct isomeric 16 membered cyclic transition states ............. 49 Study of pH Dependence on the Ligation Experiment. ................................ ..... 52 Conclusion ................................ ................................ ................................ .............. 54 Experimental ................................ ................................ ................................ ........... 5 4 General Methods ................................ ................................ .............................. 54 Experimental Details for Compounds 2.1.2, 2.1.3, 2.1.4, 2.1.8, 2.1.9, 2.1.15, 2.1.1 6, 2.1.21 and 2.1.22 ................................ ................................ ............... 55 General Procedure for Boc Deprotection of Peptides 2.1.4, 2.1.11, 2.1.18 and 2.1.24 to Give the Corresponding Unprotected Peptides 2.1.5, 2.1.12, 2.1.19 and 2.1.25 ................................ ................................ ........................... 60 Chemical Ligation of Cys ( S (Fmoc L Ala)) Gly O Me Hydrochloride (2.1.5) to Form Native Tripeptide (2.1.6) ................................ ................................ ... 62

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7 General Procedure for the Synthesis of Peptides 2.1.10, 2.1.17 and 2.1.23 .... 62 General Procedure for the Preparation of Dimer Peptides 2.1.9 and 2.1.16 ..... 64 General Procedure for the Preparation of Boc Protected Dipeptides 2.1.15 ..... 65 General Procedure for the Preparation of S Acyl Peptides 2.1.11, 2.1.18 and 2.1.24 ................................ ................................ ................................ ...... 66 General Procedure for Chemical Ligation of S Acyl Peptides 2.1.12, 2.1.19 and 2.1.25 ................................ ................................ ................................ ...... 67 Preparations of Compounds 2.2.2a, 2.2.3a, 2.2.4a, 2.2.5a, 2.2.6a, 2.2.7a 2.2.8a and 2.2.9a ................................ ................................ ........................... 68 Preparations of Compounds 2.2.2b, 2.2.3b, 2.2.4a, 2.2.5b, 2.2.6b, 2.2.7b, 2.2.8b and 2.2.9b ................................ ................................ ........................... 73 Preparations of Compounds 2.2.2b, 2.2.3b, 2.2.4a, 2.2.5b, 2.2.6b, 2.2.7b, 2.2.8b and 2.2.9b ................................ ................................ ........................... 76 General Procedure for Long Range Acyl Migration of S (Pg aminoacyl)tetrapeptide 2.2.9a,b,c to Form Native P eptides 2.2.10, and 2.2.12a,b ................................ ................................ ................................ ........ 79 3 SCOPE AND MECHANISTIC ASPECTS OF S TO N LONG RANG E ACYL MIGRATION ................................ ................................ ................................ ............ 82 Introduction ................................ ................................ ................................ ............. 82 Results and Discussion ................................ ................................ ........................... 84 The F easibility of S Acyl Monoisotripeptide to Undergo S N Acyl Migration v ia a 9 Membered Cyclic Transition State ................................ ..................... 84 Demonstration of S N acyl Migration in S acyl Monoisotetrapeptide via a 13 Membe red Cyclic Transition State ................................ ............................ 87 Conclusion ................................ ................................ ................................ .............. 88 Experimental ................................ ................................ ................................ ........... 89 Genera l Methods ................................ ................................ .............................. 89 Procedures for P reparatio ns of C ompounds 3.4, 3.6 and 3.7 ........................... 89 Procedures for Preparations of C ompounds 3.11 and 3.12 .............................. 91 General Procedure for C hemical Ligation of S Acyl P eptides 3.7 and 3.12 ...... 92 4 LIGATION AT HYDROXYPROLINE SITE VIA SALICYLALDEHYDE CAPTURE AND IMINIUM INDUCED REARRANGEMENT ................................ ...................... 93 Introduction ................................ ................................ ................................ ............. 93 Results and Discussion ................................ ................................ ........................... 97 Primary Study ................................ ................................ ................................ ... 97 Mechanistic Study of Formation of the Aminal Intermediate ............................. 99 Salicylaldehyde Iminium Induced Ligation of Dipeptide via Hydroxyproline Capture Rearrangement ................................ ................................ .............. 100 Conclusion ................................ ................................ ................................ ............ 102 Experimental ................................ ................................ ................................ ......... 103 General Methods ................................ ................................ ............................ 103 General Preparations for Salicylaldehyde Ester ................................ ............. 103 Synthesis of Aminal, Mecha nistic Study ................................ ......................... 107 General P rocedure Hydroxyproline Ligation 4.5a j ................................ ......... 107

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8 5 A CONVENIENT SYNTHESIS OF MEDIUM_SIDED CYCLIC PEPTIDES BY STAUDINGER MEDIATED RING CLOSURE ................................ ....................... 113 Introduction ................................ ................................ ................................ ........... 113 Results and Discussion ................................ ................................ ......................... 116 Synthesis of 7 Membered and 8 Membered Cyclic Dipeptide Using Staudinger Assisted Ring Closure ................................ ............................... 116 Synthesis of a 10 Membered Cyclic Tripeptide Using Staudinger Assisted Ring Clos ure ................................ ................................ ................................ 119 Conformational Studies of 7 Membered Cyclic Dipeptides ............................. 120 Conclusion ................................ ................................ ................................ ............ 121 Experimental ................................ ................................ ................................ ......... 122 General Methods ................................ ................................ ............................ 122 General Procedure I for the Preparation of Compounds 5.3a f ....................... 123 General Procedure II for the Preparation of Compounds 5.4a f ...................... 125 General Procedure III for the Preparation of Compounds 5.5a f ..................... 127 General Procedure for the Cyclization of Azido Dipeptidoyl Thioesters (5.5e f) to Form Compounds 5.6a f ................................ ................................ ....... 130 General Procedure IVA for the Prepa ration of Compounds 5.6a c. .......... 130 General Procedure IVB for the Preparation of Compounds 5.6d,e. .......... 131 En Route for the Preparation of Cyclic Tripeptide 5.1 2 ................................ 132 Crystal Data for C ompound s 5.6a and 5.6c ................................ .................... 134 Computational Details ................................ ................................ ..................... 136 Computational details for the cyclization of aza ylides and cartesian coordinates of stationary points ................................ ............................. 136 Conformational analysis of compounds 5.6a, 5.6c, 5.6d and 5.6e ........... 148 6 SYNTHESIS OF CYCLIC PEPTIDES BY CYCLO OLIGOMERIZATION OF DIPEPTIDOYL BENZOTRIAZOLIDES ................................ ................................ 149 Introduction ................................ ................................ ................................ ........... 149 Results and Discussion ................................ ................................ ......................... 151 Synthesis of Benzotriazolide Cyclization Substrate ................................ ........ 151 Cyclization of Dipeptidoyl Benzotriazolides ................................ ..................... 152 Dependence of Cyclo Oligomerization on Substrate Concentration ............... 156 The Tur n Introducer Effect on Cyclooligomerization of Dipeptidoyl Benzotriazols ................................ ................................ ............................... 158 Conclusion ................................ ................................ ................................ ............ 159 Experimental ................................ ................................ ................................ ......... 160 General Methods ................................ ................................ ............................ 160 General Procedure for the Preparation of Benzotriazolides 6.2a c ................. 161 General Procedure for the Preparation of Dipeptides 6.4a g .......................... 162 General Procedure for the Preparation of Dipeptidoyl Bentrotriazolides 6.5a g ................................ ................................ ................................ ................... 164 General Procedure for the Cyclizations of Dipeptides 6.6a g ......................... 166 Cyclizations of dipeptide Cbz Ala Pro Bt (6.5a) ................................ .... 168 Cyclizations of dipeptide Cbz D,L Homo Ala D Pro Bt (6.5b) ............... 169 Cyclizations of dipeptide Cbz D,L Homo Ala D Pro Bt (6.5c) ............... 170

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9 Cyclizations of dipeptide Cbz Ala 3 Aze Bt (6.5e) ................................ 171 Cyclizations of dipeptide Cbz Ala L 2 Aze Bt (6. 5f) .............................. 172 7 CONFORMATIONALLY ASSISTED LACTAMIZATIONS FOR THE SYNTHESIS OF BIS 2,5 DIKETOPIPERAZINES ................................ ................................ ...... 173 Introduction ................................ ................................ ................................ ........... 173 Results and Discussion ................................ ................................ ......................... 176 Synthesis of Symmetrical Bis DPKs ................................ ............................... 176 Further Devel opment of the Method for Synthesis of Symmetrical Bis 2,5 DPKs ................................ ................................ ................................ ........... 179 Synthesis of Unsymmetrical Bis 2,5 DKPs ................................ ..................... 181 Conclusion ................................ ................................ ................................ ............ 184 Experimental Section ................................ ................................ ............................ 185 General Methods ................................ ................................ ............................ 185 General Procedure I for th e Preparation of Bis Benzotriazolides 7.2a b and 7.13a b ................................ ................................ ................................ ........ 185 General Procedure II for the Preparation of Di sulfide Dipeptides 7.3a e ....... 187 General Procedure III for the Preparation of Di Sulfide Dipeptidoyl Bentrotriazolides 7.4a e ................................ ................................ ............... 189 General Procedure IV for the Cyclization of Di Sulfide Dipeptidoyl Bentrotriazolides 7.4 a c to Form Symmetrical Bis DKPs 7.5a e .................. 192 General Procedure V for the O S and N Acylations of Bis Benzotriazolides 7.6a b and 7.13a b for Preparation of Compounds 7. 7a b and 7.15a e ...... 194 General Procedure VI for the Preparation of Bis Benzotriazolides 7.8a b and 7.16a e ................................ ................................ ................................ ........ 198 General Procedure VII for the Coupling of Bi s Benzotriazolides 8a b and 16a e with Proline to Prepare Compounds 7.9a c and 7.17a e ................... 201 General Procedure VIII for the Preparation of Cyclization Precursors 7.10a c and 7.18a e ................................ ................................ ................................ 205 General Procedure IX for the Cyclization of Precursor 7.10a c to Form Symmetrical Bis DKPs 7.11a c and 7.18a e to Form Unsymmetrical Bis DKPs 7.19a e ................................ ................................ .............................. 209 8 SYNTHESIS OF POLYKETIDE FRAGMENT OF APRATOXINS .......................... 214 Introduction ................................ ................................ ................................ ........... 21 4 Results and Discussion ................................ ................................ ......................... 218 Multigram Synthesis of the Key Aldehyde ................................ ....................... 218 Anti Crotylation of the C35 44 Aldehyde Fragment ................................ ......... 224 Conclusion ................................ ................................ ................................ ............ 227 Experimental ................................ ................................ ................................ ......... 228 General Methods ................................ ................................ ............................ 228 Pre paration of Compounds 8.6, 8.14 17 ................................ ......................... 229 Synthesis of Key Aldehyde 8.5 ................................ ................................ ....... 232 General procedure for RCM ................................ ................................ ..... 232 Procedure for conjugated methyl addition ................................ ................ 233 Procedure for ring opening of lactone 8.10 ................................ ............... 234

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10 Ge neral procedure for PMB protection ................................ ..................... 234 Procedure for reduction of Weinreb amide ................................ ............... 236 Synthesis of Leighton Anti Crotylating Reagent ................................ .............. 236 Synthesis of Compound 8.19 ................................ ................................ .......... 238 APPENDIX A : NMR SPECTRA ................................ ................................ ................... 240 LIS T OF REFERENCES ................................ ................................ .............................. 266 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 275

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11 LIST OF TABLES Table page 2 1 Chemical lig ation of isopeptides 2.1.25 ................................ ............................... 44 2 2 Characterization data of ligation product mixtures ................................ .............. 47 2 3 Characterization data of the isolated ligation products 2.2.10 and 2.2.12a,b ...... 47 2 4 Dependence of pH on the product ratio 2.2.10:2.2.11 in the ligation reaction of 2.2.9a ................................ ................................ ................................ .............. 53 3 1 Attempted c hemical ligation of S acyl isopeptides 3.7 and 3.12 ......................... 86 4 1 Scope of the r eaction between Hyp peptides and salicylaldehyde e sters ........... 99 5 1 En route to 7 and 8 membered cyclic dipeptides. ................................ ............ 118 5 2 Activation parameters ................................ ................................ ....................... 136 5 3 Absolute energi es (Hartree) ................................ ................................ .............. 137 5 4 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly Gly SPh ................................ ................................ ....... 138 5 5 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly Ala SPh 5.5.a ................................ ........................... 139 5 6 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly Ala SPh 5.5.a ................................ ........................... 141 5 7 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Ala Aib SPh 5.5c ................................ ............................. 142 5 8 Cartesian coordinates for the TS in the cyclizat ion of the aza ylide thioester derived from azido Ala Aib SPh 5.5c ................................ ............................. 144 5 9 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Ala Ala SPh 5. 5d ................................ ......................... 145 5 10 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly GABA SPh 5.5e ................................ ........................... 147 5 11 Similarity analysis between ball and stick model of both Xray crystal structure (yellow) and theoretical conformer (green) ................................ ........................ 148 6 1 Benzotriazole auxilary route to homo diketopiperazines ................................ ... 152

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12 6 2 Product distributions of Pd promoted tamdem deprotection/cyclization reactions of various concentrations of Cbz Ala L Pro Bt 6.5a ....................... 157 6 3 Product distributions of Pd promoted tamdem deprotection/cyclization reactions of Cbz protected dipeptidoyl benzotriazolides 6.5b d, 6.5g,h ............ 159 7 1 En route to symmetrical bis DKPs ................................ ................................ .... 178 7 2 En route to symmetrical bis DKPs 7.11a c ................................ ........................ 180 7 3 En route to unsymmetrical bis DKPs 7.19a e ................................ ................... 183 8 1 Conditions for ring closing metathesis of diene 8. 8 ................................ ........... 220 8 2 Ring closing metathesis of compound 8. 5 ................................ ......................... 221 8 3 Copper (I) mediated 1,4 unsaturated lactone 8.9 ..................... 222

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13 LIST OF FIGURES Figure page 1 1 Top selling peptide drugs ................................ ................................ .................... 24 1 2 Chemoselective ligation and modification strategies for peptides ....................... 25 1 3 The four possible ways to construct a peptide macrocycle ................................ 26 1 4 Lactamization method for cyclo peptide ring contractions ................................ ... 27 1 5 Structure of natural apratoxins A and E and synthetic apratoxin S4 ................... 28 2 1 Solid phase peptide synthesis ................................ ................................ ............. 31 2 2 Mechanistic pathway of Native Chemical Ligation ................................ .............. 32 2 3 Early observation on aminolysis of thioesters by Wieland ................................ ... 33 2 4 Total chemical synthesis of native HIV 1 protease via NCL ................................ 34 2 5 Synthesis of homogeneous EPO glycoform via NCL ................................ .......... 35 2 6 NCL with conversion of cysteine residues into other amino acids ....................... 36 2 7 Sugar assisted ligation (left) and traceless Staudinger ligation (right) ................. 36 2 8 Chemical ligation of S acyl dipeptide 2.1.5 ................................ ......................... 38 2 9 Synthesis and attempted chemical ligation of S acyl tripeptide 2.1.12 ................ 39 2 10 Synthesis of isopentapeptides 2.1.19 ................................ ................................ .. 41 2 11 Chemical ligation of S acyl mono isopentapeptides 2.1.19. ................................ 41 2 13 Chemical ligation of S acyl mono isohexapeptides 2.1.25. ................................ 43 2 14 Preparation of S acyl tetrapeptide 2.2.9a ................................ ............................ 45 2 15 Proposed 15 membered cyclic transition states of A) the second generation Sugar assisted Ligation (SAL) and (B) the long range intramolecular acyl migration. ................................ ................................ ................................ ............ 46 2 16 Chemical ligation of S acyl tetrapeptide 2.2.9a .... The ligation compound 2.2.10 is drawn as a monomer for clarity ................................ ................................ ........... 47 2 17 13 C NMR carbonyl signals in (A) ligated pentapeptide 2.2.10 and in (B) starting S acylated tetrapeptide 2.2.9a ................................ ............................... 48

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14 2 18 Preparation of S acyl tetra peptide 2.2.9a ................................ ............................ 50 2 19 Acyl migration of S acyl tetrapeptides 2.2.12b ... The ligation compound 2.2.13a is drawn as a monomer for clarity ................................ ................................ ........... 51 2 20 Acyl migration of S acyl tetrapeptide 23c The ligation compound 26b is drawn as a monomer for clarity ................................ ................................ ............................. 51 2 21 Effect of pH on S N acyl migration in S acyl tetrapeptide 2.2.9a. ..................... 53 3 1 Long range S N acyl migration to form native peptide analogs. ....................... 82 3 2 Cyclization reaction to form lactone ................................ ................................ .... 83 3 3 Reactivity profile for lactone formation ................................ ................................ 84 3 4 Chemical ligation of S acyl monoisotripeptide 3.7. ................................ .............. 85 3 5 A rotaxane based molecular machine for the synthesis of small peptides .......... 87 3 6 Chemical ligation of S acyl monoisotetrapeptide 3.12. ................................ ....... 88 4 1 The unique challenges of proline ligation arise from an n electron density. ................................ ................................ ................................ .. 93 4 2 The n N C antibond orbital ... 94 4 3 (A)Schemes of the previous advanced proline ligation strategy .......... Our reaction represented in (B) exhibiting the similarities in strategy ................................ ...... 95 4 4 Scheme for hydroxyproline site ligation After installation of O salicylaldehyde, the proline is captured forming an iminium, which then rearranges ultimately allowing for the native peptide bond to be formed ................................ ............... 96 4 5 Synthesis of salicylaldehyde ester. ................................ ................................ ..... 98 4 6 Model Hyp Ligation reaction. ................................ ................................ ............... 98 4 7 Formation of the aminal intermediate. ................................ ............................... 100 4 8 Hyp Ligation for synthesis of native peptides ................................ .................... 101 4 9 Hyp ligatio n to synthesis a hexapeptide. ................................ ........................... 102 5 1 Challenges in chemical synthesis of small cyclopeptides. ................................ 113 5 2 A pincer auxiliary to for ce difficult lactamization. ................................ ............... 114 5 3 Wedge shaped carbosilane dendrimeric carbodiimides to cyclize homodiketopiperazines through a site isolation mechanism ............................. 115

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15 5 4 Ring construction to form cyclic oligopeptides by Staudinger ring closure. ....... 116 5 5 Synthesis of starting azido dipeptide thioesters (Table 5 1). ............................. 117 5 6 Solution phase synthesis of a 10 membered cyclic tripeptide. .......................... 120 5 7 X ray structures, traditional representations and computed stru ctures of A) 1,5 diazocane 2,6 dione (5.6a) and B) 3,3 dimethyl 1,4 diazepane 2,5 dione (5.6c). ................................ ................................ ................................ ................ 121 5 8 Predicted conformers for compounds A) 1,5 diazocane 2,6 dione (5.6d) and B) 1,4 diazocan e 2,5 dione (5.6e). ................................ ................................ ... 121 5 9 TS in the cyclization of the aza ylide thioester corresponding to azido Gly Gly SPh ................................ ................................ ................................ ............. 137 5 10 TS in t he cyclization of the aza ylide thioester corresponding to azido Gly Ala SPh 5.5.a ................................ ................................ ................................ .... 140 5 11 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Gly SPh ................................ ................................ ................................ ............. 140 5 1 2 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Aib SPh 5.5c ................................ ................................ ................................ ..... 143 5 13 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Phe SPh 5.5b ................................ ................................ ................................ .... 143 5 14 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Ala SPh 5.5d ................................ ................................ ................................ .. 145 5 15 TS in the cycliz ation of the aza ylide thioester corresponding to azido Gly GABA SPh 5.5e ................................ ................................ ................................ 146 6 1 Cyclooligomerization method to give fast access to different peptide macro cycles. ................................ ................................ ................................ ............... 1 50 6 2 Cyclooligomerization method to give fast access to different peptide macro cycles ................................ ................................ ................................ ................ 150 6 3 Synthesis of N protected dipeptidoyl benzotriazolides. ................................ ..... 151 6 4 Intramolecular cyclization of N protected dipeptidoyl benzotriazolides: a) using Pro as turn introducer; b) using Hyp as turn introducer. .......................... 152 6 5 A) cis/trans conformers of proline containing amide bonds;B) C4 endo conformations of L Hyp Bt with H bond donor stabilization at C4. .................... 153 6 6 Intermolecular cyclodimeri zation of Cbz Ala 3 Aze Bt 6.5e. .......................... 154

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16 6 7 Structures of compounds cyclooligomerization products of 6.5a ..................... 155 6 8 Intermolecular cyclo oligomerization of N protected dipeptidoyl benzotriazolides ................................ ................................ ................................ 156 7 1 Representative of bioactive natural products containing DKP ........................... 174 7 2 Synthesis of bis DKP via head to tail condensation between the N and C termini ................................ ................................ ................................ ............... 175 7 3 Radical dimerization in synthesis of bis DKP ................................ .................... 175 7 4 Synthesis of bis DKP using carbanion chemistry ................................ .............. 175 7 5 Ring construction strategy to form DKPs ................................ .......................... 176 7 6 Cyclization of bis Cbz dipeptidoyl benzotriazoles 7.4a e ................................ .. 177 7 7 Synthesis of bis benzotriazoles 7.10a c ................................ ............................ 179 7 8 Synthesis o 2,5 DKPs 7.11a c ........................... 179 7 9 Preparation of 7.15a e ................................ ................................ ...................... 182 8 1 Structures of natural apratoxins ................................ ................................ ........ 214 8 2 Activities of synthetic apratoxins ................................ ................................ ....... 215 8 3 Retrosynthetic analysis of apratoxin A. ................................ ............................. 216 8 4 Detailed retro synthetic analysis of synthetic apratoxin S4 ............................... 217 8 5 Synthesis of aldehyde 8.5 ................................ ................................ ................. 218 8 6 Synthesis of alcohol 8.6 ................................ ................................ .................... 219 8 7 ................................ ................................ .............................. 220 8 8 1,4 Addition to lactone 8. 8 in the presence of Cu (I) salt ................................ ... 221 8 9 Stereoselectivity in 1,4 addition of lactone 8.9 ................................ ................. 222 8 10 PMB protection of alcohol 8. 11 ................................ ................................ ......... 223 8 11 Contemporary crotylating reagents for anti addition of aldehyde ...................... 224 8 12 Asymmetric crotylation of the key aldehyde 8.5 ................................ ................ 225 8 13 Transition state model in diastereoselection in reactions of EZ CrotylMix ........ 225

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17 8 14 Synthesis of Leighton crotylating reagent ................................ ......................... 226 8 15 Synthesis of polyketide fragment of apratoxins ................................ ................. 227 A 1 1 H NMR spectrum of compound 8.14 in CDCl 3 (400 MHz) ............................... 240 A 2 13 C NMR spectrum of compound 8.14 in CDCl 3 (100 MHz) .............................. 241 A 3 1 H NMR spectrum of compound 8.6 in CDCl 3 (400 MHz) ................................ 242 A 4 1 H NMR spectrum of compound 8.8 in CDCl 3 (400 MHz) ................................ 243 A 5 1 H NMR spectrum of compound 8.9 in CDCl 3 (400 MHz) ................................ 244 A 6 13 C NMR spectrum of compound 8.9 in CDCl 3 (100 MHz) ................................ 245 A 7 1 H NMR spectrum of compound 8.10 in CDCl 3 (400 MHz) ............................... 246 A 8 13 C NMR spectrum of compound 8.10 in CDCl3 (100 MHz) ............................. 247 A 9 1 H NMR spectrum of compound 8.11 in CDCl3 (400 MHz) ............................... 248 A 10 13 C NMR spectrum of compound 8.11 in CDCl 3 (100 MHz) .............................. 249 A 11 1 H NMR spectrum of compound 8.12 in CDCl 3 (400 MHz) ................................ 250 A 12 13 C NMR spectrum of compound 8.12 in CDCl 3 (100 MHz) .............................. 251 A 13 1 H NMR spectrum of compound 8.5 in CDCl 3 (400 MHz) ................................ .. 252 A 14 13 C NMR spectr um of compound 8.5 in CDCl 3 (100 MHz) ................................ 253 A 15 1 H NMR spectrum of PMB Br in CDCl 3 (400 MHz) ................................ ............ 254 A 16 13 C NMR spectrum of PMB Br in CDCl 3 (100 MHz) ................................ .......... 255 A 17 1 H NMR spectrum of PMB trichloroacetamide in CDCl 3 (400 MHz) .................. 256 A 18 13 C NMR spectrum of PMB trichl oroacetamide in CDCl 3 (100 MHz) ................. 257 A 19 1 H NMR spectrum of compound 8.21 in CDCl 3 (400 MHz) ................................ 258 A 20 13 C NMR spectrum of compou nd 8.21 in CDCl 3 (100 MHz) .............................. 259 A 21 1 H NMR spectrum of compound 8.22 in CDCl 3 (400 MHz) ................................ 260 A 22 13 C NMR spectrum of compound 8. 22 in CDCl 3 (100 MHz) .............................. 261 A 23 1 H NMR spectrum of tricloro( E crotyl)silane in CDCl 3 (400 MHz) ...................... 262

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18 A 24 1 H NMR spectrum of compou nd 8.18 in CDCl 3 (400 MHz) ................................ 263 A 25 1 H NMR spectrum of compound 8.18 in CDCl 3 (100 MHz) ................................ 264 A 26 1 H NMR spectrum of compound 8.1 9 in CDCl 3 (400 MHz) ................................ 265

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19 LIST OF ABBREVIATIONS Alpha locant Angstrom(s) Ac Acetyl Ala Alanine Ar Aryl AZT Azidothymidine Beta locant Bn Benzyl Boc t Butoxycarbonyl br Broad Bt Benzotriazol 1 yl C Carbon Degree Celcius Calcd Calculated Cbz Carbobenzyloxy CDCl 3 Deuterated chloroform CTH Catalytic hydrogen transfer CuSO 4 .H 2 O Copper(II) sulfate pentahydrate Cys Cysteine Chemical shift in parts per million downfield from tetramethylsilane d Days ; Doub let (spectral) DBU 1,8 Diazabicyclo[5.4.0]undec 7 ene DCC N N' Dicyclohexylcarbodiimide DCM Dichloromethane

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20 DIPEA Diisopropylethylamine DMF Dimethylformamide D 2 O Deuterium oxide EDC 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide (stands as an abbreviation for EDAC and EDCI as well)

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21 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 Chemistry DEVELOPMENT OF METHODOL OGIES FOR COUPLING AND CYCLIZATION OF PEPTIDES AND SYNTHESIS OF POLYKETIDE FRAGMENTS FOR MODIFIED PEPTIDES By Khanh Quoc Ha May 2016 Chair: Hendrik Luesch Major: Chemistry From very early on, chemists have identified peptides and proteins as targets for the development of synthetic protocols. New and improved strategies lead to more efficient synthesis of complex peptide targets, opening the way to both new drug candidates and a deeper understanding of the intimate relation between sequence, conformation and properties. Despite recent progress and the arsenal of reagents available, peptid e synthesis remains challenging; complex targets and regulatory authority requirements in terms of purity for drugs are continuously stimulating chemists to improve and re think synthetic approaches. This dissertation addresses the development of synthetic methods and approaches targeting medium sized cyclic peptides, and coupling of large peptide fragments. A n ovel approach applying S to N long range acyl migration to synth esize peptide and peptide analog ues and also provided mechanistic evidence for the ligation process are reported A series of novel S amino acid residues were sy nthesized according to S to N acyl migration protocols. An other challenge of NCL is the slow coupling rate at

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22 proline site. To address this problem a hydroxyproline ligation strategy utilizing chemoselective bifunctionality was developed. R ing size is a significant factor in the success of macro lactamiz ation in the synthesis of a cyclic peptide. Ring sizes of 7 15 membered rings are less accessible and can frequently only be synthesized with difficulty, whether using solution phase or polymer supported strategies. This dissertation describes the development of synthetic methods and approaches targeting medium sized cyclic peptides including: intramolecular Staudinger ligation, cyclooligomerization, conformational ly assisted cyclization. Among cyclic natural products, cyclodepsipeptide a pratoxins a re intriguing marine natural products of mixed biogenetic origin. a pratoxins w ere isolated from cyanobacteria Lyngbya spp. ( now known as Moorea bouillonii ) collected in Guam and Palau Apratoxins were found to deplete cancer cells of several cancer associa ted receptor tyrosine kinases by preventing their N glycosylation, leading to their rapid proteasomal degradation. It has been shown that apratoxin prevents co translational translocation of proteins destined for the secretory pathway. Part of this dissert ation describes the large scale synthesis of polyketide fragment for further synthetic modification and biological stud ies of a pratoxins

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23 CHAPTER 1 OVERVIEW Peptide s have found applications that range from catalysis 1 2 th r ough nano material 3 4 to drug discovery. 5 6 Peptides can catalyze enantioselectiv e addition of HCN to benzaldehyde 7 or asymmetric Strecker amino acid synthesis. 2 The peptide catalyst was designed to be a small molecule preferred alternative to oxynitrilase, an enzyme that was a known catalyst for the same process 1 Peptide derivatives have been utilized as scaffolds for the self assembly of nanotubes with controllable internal diameter, pore chemistry, and exterior functionality. 4 8 By modification of the pepti de structure peptide nanotubes have been designed for a wide range of applications from ion channels and antibacterial s to photoactive materials and sensors. 3 4 9 However, peptides are mainly used in pharmaceutical research (Figure 1 1) 10 11 12 13 b ecause of their size and relative c omplexity. Peptide s can show greater specificity and potency for biological targets compared to smaller acyclic compounds 14 15 16 Peptide scaffolds can modulate more challenging biological targets such as protein protein interactions or biomolecules that lack well define d small molecule binding si tes. 16 Although m any peptide s have b een used as therapeutic age nts including: octreotide, calcitonin, cyclosporine A, nisin, polymixin and colistin 15 16 10 pep tides are underrepresented in clinical use mainly due to difficulties associated with their synthesis New and improved strategies lead to more efficient synthese s of complex peptide targets, opening the way to both new drug candidates and a deeper underst anding of the relationship between sequence, conformation and properties. Despite recent progress and the arsenal of reagents available, peptide synthesis remains challenging C omplex targets and regulatory authority constraints in terms of drug purity are

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24 continuously stimulating chemists to improve and rethink synthetic approaches. 17 Reducing the number of steps is usually a synonym of better yields and ease of purification, which explains the success of convergent peptide synthesis. My thesis addresses the development of synthetic methods and approaches targeting small and medium sized cyclic peptides peptidomimetics, 18 19 20 and coupling of large peptide fragments. Figure 1 1. T op selling peptide drugs

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25 Figure 1 2 Chemoselective l igation and modification strategies for peptides For coupling of large peptide fragments, native chemical ligation (NCL), has become a widely used chemoselective technique to synthesize large peptides based on a capture/rearrangement concept (Figure 1 2). 21 The full potential of native chemical ligation was shown in 1994 by Kent and co workers for the reaction of unprotected thioesters with N terminal Cys peptides NCL is nowadays the most widely used chemoselective ligation technique. 22 The impact NCL made on chemistry and biochemistry is illustrated by more than 2000 citations this original article has received so far The classical NCL method is limited to peptides possessing an N terminal cysteine residue. 17 23 To overcome this requirement, a novel approach was developed

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26 applying S to N long range acyl migration to synthesize peptide and peptide analogs and also provided mechanistic evidence for the ligation process. A series of novel S amino acid residues were synthesized according to original S to N acyl migration protocols. Another challenge of NCL is the slow coupling rate at a proline site. 24 To address this problem, we developed a hydroxyproline ligation strategy, based on native chemical bifunctionality. In the synthesis of cyclic peptides, a peptide can be cyclized in four different ways: head to tail (C terminus to N terminus), head to side chain, side chain to tail or side chain to side chain d epending on its functional groups involved (Figure 1 3). 5 25 Figure 1 3. The f our possible ways to construct a peptide macrocycle Cyclic peptides and peptidomimetics have received the m ost attention in drug discovery, 10 which is explained by the existence of powerful synthetic and biological methods to rapidly put together the required amino acid building blocks. Although, chemical synthesis of cyclic peptides benefits from the availability of relatively

PAGE 27

27 inexpensive orthogonall y protected amino acids, many cyclic peptides are no toriously difficult to prepare. Figure 1 4. Lactamization method for cyclo peptide ring contractions R ing size is a significant factor in t he success of macrolactamiz atio n in the synthesis of a cyclic peptide. Cyclo peptides of 7 15 members are less accessible and can frequently be difficult to synthesize 26 The main hurdle to the cyclization of peptides to afford 7 to 15 membered rings is the preferential transoid alignment of amide bonds in their acyclic precursors which leads to a preferred extended structure, placing the termini far apart. 25 27 28 This dissertation describes the development of synthetic methods and approaches targeting medium sized cyclic peptides including: intramolecular Staudinger ligation, 20 cyclooligomerization, 18 conformationally assisted ma c r o cyclization ( Figure 1 4 ) 29 These novel methodologies in combi nation with

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28 convergent synthetic schemes can provide the key elements for the total chemical synthesis of n atural or fully unnatural linear and medium sized cyclic peptides with yet to be discovered properties. Among cyclic natur al products, the cyclodepsipeptide a pratoxins are intriguing marine natural products of mixed biogenetic origin. Apratoxins ( Figure 1 5) comprise a family of cyclic depsipeptides isolated from the Lyngbya species of cyanobacteria. 30 The fi rst member of the family to be discovered, apratoxin A, was isolated in 2001 by Moore, Paul and co workers. 31 Figure 1 5 Structure of n atural apratoxins A and E a nd synthetic apratoxin S4 It was isolated from the marine cyanobacterium Lyngbya majuscula from Finger's Reef, Apra Harbor, Guam. Structure determination revealed that apratoxin A was composed of discrete polyketide and po lypeptide domains, joined via amid e and ester linkage s Apratoxin A was found to deplete cancer cells of several cancer associated receptor tyrosine kinases by preventing their N glycosylation, leading to their rapid proteasomal degradation Through rational design, tota l synthesis of apra toxin S4 (Figure 1 5 ), hybrid of apratoxins A and E, has been previously achieved with improved antitumor activity and tolerability in vivo 32 The new apratoxin compound was found to be stable during purifications and stability tests. I t is important to develop synthetic

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29 approaches to the novel apratoxin derivatives to further study their biological applications. Part of this dissertation describes the multigram synthesis of the major C35 C44 subunit for the preparations of a pratoxins for further biology studies

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30 CHAPTER 2 DEVELOPMENT OF NOVEL STRATEGIES FOR COUPLING OF PEPTIDE FRAGMENTS VIA S TO N LONG RANGE ACYL MIGRATION Introduction Due to the significance and diver sity of proteins in living organisms, there has been an increasing amount of interest to stu dy their biochemical activity. Scientists desired a technique that would allow them to alter pr otein structure by changing the amino acid sequence. Such a techniq ue would provide a means to observe protein function after structural manipulation. During the past few decades, studies of proteins generated by Escherichia coli have been made through techniques of DNA based molecular biology. 33 Specifically, scientists systematically changed the amino a cid sequence of protein s and analyzed how function was affected. Comparisons in function were made to the ori ginal protein, and correlated with the specific changes that were made along the polypeptide sequence. Although this molecular biology approach provided useful insight to the chemical structure s and functions of proteins, 33 it came with major limitations. This methodology was able to utilize only the twenty genetically encoded amino acids, and modifications to the protein aft er translation could be difficult to control. Chemists sought to find a new approach to overcome the limitations of the molecular biology approach. If total chemical synthesis of a protein could be achieved, the manipulation of any amino acid residue alon g the polypeptide of a protein could be Reproduced in part with permission from Ha K.; Cha har M; Monbaliu M. J C.; Todadze E .; Finn H. K.; Oliferenko A. A; Ocampo C. E.; Leino D .; Lillicotch A .; Stevens C. V. ; Katritzky A. R. The Journal of Organic Chemistry 2012 6 2637 2648 Copyright 2012 American Chemical Society and Hansen, F K.; Ha, K ; Todadze, E ; Lillicotch, A ; Frey, A ; Katritzky, A R Organic & Biomolecular Chemistry 2011, 9 7162 7167 Copyright 2011 The Royal Society of Chemistry

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31 carried out with relative ease and high control. In 1963, Merrifeld discovered a technique called solid phase peptide synthesis (SPPS), which greatly facilitated the synthesis of polypeptides and overcame the major shortcomings of previous synthetic based methodologies (Figure 2 1) 34 35 Figure 2 1 Solid phase peptide synthesis

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32 Specifically, SPPS initially involves th e C terminus of an amino acid covalently linked to an insoluble polymeric resin. Deprotection of an N protecting group on the first amino aci d residue is then followed by purification, filtration and washing, and the addition of another N protected amino acid. This process is repeated until the desired amino acid sequence is achieved. In the final step, all protecting group s are removed, and the covalent bond to the insoluble resin is cleaved to yield the desired pe ptide product Decades of optimization have made stepwise SPPS a reliable tool for the preparation of peptides up to around 25 amino acids long; 35 34 but yields generally decrease as the length of the polypeptide increases. In 1992, the concept of chemoselecti ve condensation of unprotected peptides was intr oduced by Kent and Schnolzer. 21 23 Specifically, their novel concept used unprotected peptide fragments, each containing a uni que, mutually reactive functional group. These two functional groups were designed to react strictly with each other to produce a single polypeptide product. To simplify the total synthesis of proteins through this principle, the condition s to form a pep tide bond between the two peptides fragments were removed from consideration. If instead an analog structure at the ligation site was acceptable, a wide variety of known chemistries could easily be applied to covalently link the two peptides (Figur e 2 2 ). This technique was termed native chemical ligation. Figur e 2 2. Mechanistic pathway of Native Chemical Ligation

PAGE 33

33 Primary studies that led to the capture/rearrangement method were re ported in 1953 by Wieland who investiga ted the chemical properties of amino acid thioesters. 36 In the study, they reported that thi ophenol thioesters can undergo intermolecular aminolysis in the presence of amines to give amides. In contrast to these thiophenol thioesters, a glycine thioester of cysteamine could not be synthesized and isolated as such under neutral pH conditions. This observation was attributed to the additional amino group of cyst amine located in proximity to the thiol moiety. As a result, rearrangement occurred under mild acidic conditio ns a nd was accelerated at higher pH T he previously discussed intramolecular rearrangement was combined with an intermolec ular thiol thioester exchange: s pecifically, a Val thiophenol thioester was synthesized and treated with cysteine ( Figur e 2 3 ). The hi ghly reactive aryl thioester rapidly exchanged with the thiol moiety of Cys, which represents the capture step. The resulting Val ( S Cys ) thioester subsequently rearran ged to form a native Val Cys dipeptide linked to a native peptide bond ( Figur e 2 3 ) Figur e 2 3 Early observation on a minolysis of thioesters by Wieland Chemical ligation proved to be a breakthrough for the synthesis of many protein molecules and even enzymes. Nowadays, this method has been used to achieve the total synthesis of hundreds of proteins. For example, the complete synthesis of a fully

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34 functional HIV 1 protease synthetic analog was able to be achieved through the principles of chemical ligation (Figure 2 4 ) 21 22 Figure 2 4 Total chemical synthesis of native HIV 1 protease via NCL Recently, the total synthesis of Erythropoietin, which is a polyglycopeptide containing 166 amino acids i n the peptide sequence, has bee n achieved using NCL (Figur e 2 5 ). 37 38 Erythropoietin is a signaling gly coprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domai ns. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild type primary sequence, which were obtained by SPPS. The glycopeptidyl constructs were joined by chemical ligation. However, scie ntists viewed the requirement of having an N terminal cysteine residue in one of the peptide fragment for coupling as a major limitation of NCL since cysteine is one of the least common natural amino acid in proteins. Cysteine is a relatively rare amino a cid (1.3% average content) 39 and is not always available in a

PAGE 35

35 terminal position. Moreover, some amino acid cysteine bonds such as Pro Cys, Asp Cys, Glu Cys and Lys Cys can be difficult to access by chemical ligation. 39 40 Figure 2 5 Synthesi s of homogeneous EPO glycoform via NCL

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36 Figure 2 6 NCL with conversion of cysteine residues into other amino acids Figure 2 7 Sugar assisted ligation (left) and traceless Staudinger ligation (right) To overcome the requirement of a specifically placed cysteine residue one approach is the use of thiol ligation auxiliaries, but unfortunately, removable cysteine mimics can sterically hinder ligation and difficulties can arise at the stage of auxi liary removal (Figure 2 6). 41

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37 Other methodologies such as sugar assisted ligation 42 and traceless Staudinger ligation 43 were develo ped to provide alternative methods to achieve the total chemical synthesis of native proteins (Figure 2 7) A useful approach for the synthesis of cysteine containing peptides is isopeptide ligation methodology. Yoshiya et al. sy nthesized S acyl peptides containing N terminal cysteine residues; subsequent S O intramolecular acyl migration then furnished native peptide bonds. 44 This disertation describes the chemical ligation approach using S acylated cysteine peptides to form native peptides via 11 13 14 15 and 16 membered cyclic transition states via long range acyl migration. Th e method allows the synthesis of native peptides from S acyl isopeptides with a C terminal cysteine without utilizing auxiliaries. Our method utilizes non terminal cysteine residues that could significantly expand the applicab ility of this i sopeptide approach. We describe the first acyl migration in S acyl isopeptide to form native peptides from non terminal cysteine residues. Results and Discussion Microwave Assisted Chemical Ligation o f S Acyl Peptides Containing Non Termina l Cysteine Residues Study of the feasibility of S N acyl migration via an 8 membered compared to a 5 membered cyclic transition state The protected dipeptide dimer 2 1.2 was prepared in 79% yield by mixed anhydride coupling of bis Boc cystine 2.1.1 and H Gly OCH 3 according to a literature procedure. To investigate the feasibility of acyl migration through an 8 membered transition state, the Boc protected dipeptide dimer 2.1.2 was first reacted with tributylphosphine to afford the dipeptide monomer 2.1. 3 in 55% yield. Subsequent S

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38 acylation of 2.1. 3 with Fmoc Gly Bt furnished the mono isotetrapeptide 2.1. 4 in 88% yield. Next, 2.1. 4 was Boc deprotected selectively with methanol saturated with hydrochloric acid gas. The crude HCl salt 2.1. 5 was treated with tr iethylamine to give the corresponding free base which as expected underwent classical S to N acyl migration via a 5 membered transition state to furnish the native tripeptide 2.1. 6 in 52% yield ( Figure 2 8 ). Figure 2 8 C hemical ligation of S acyl dipeptide 2.1. 5 Attempts to prepare the Fmoc protected isotetrapeptide 2.1.7 from 2.1.6 failed (Figure 2 8). We therefore deprotected the Boc protected dipeptide dimer 2.1.2 utilizing saturated hydrochloric acid in methanol to af ford the dipeptide dimer 2.1.8 as dihydrochloride (85% yield) followed by mixed anhydride coupling of 2.1.8 with 2 equiv. of Boc glycine to furnish the novel tripeptide dimer 2.1.9 in 67% yield. Treatment of the disulfide 2.1.9 with tributylphosphine gave the tripeptide monomer 2.1.10 (Scheme 2 9 ).

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39 S Acylation of 2.1.10 with Cbz Ala Bt in the presence of triethylamine provided the key intermediates 2.1.11 (72% yield) which on deprotection by MeOH HCl gave the desired isotetrapeptide 2.1.12 in 82% yield (Sch eme 2 9 ). Figure 2 9 Synthesis and attempted chemical ligation of S acyl tripeptide 2.1.12 We then investigated the chemical ligation of 2.1.12 via an 8 membered cyclic transition state (TS). The isotetrapeptide 2.1.12 wa s dissolved in a mixture of 0.4 M NaH 2 PO 4 /Na 2 HPO 4 buffer (pH = 7.8) and acetonitrile (7:1) and subjected to microwave irradiation for 3 h at 70 C and 50 W (Scheme 2 3). Interestingly, the HPLC MS (ESI) analysis of the reaction mixture revealed that the ma jor component was unreacted starting materia l 2.1.12. The (+)ESI MSn spectrum of the major product (m/z 455 [M+H] + ion) w as nearly identical to those of 2.1.12. Moreover, the HPLC MS (ESI) analysis of a mixture of 2.1.12 and the product from the ligation e xperiment detected only one MW 454 compound, whose characteristics matched those of the mono

PAGE 40

40 isotetrapeptide 2.1.12. Absence of the S to N acyl migration of 2.1.12 suggests that the S N acyl migration via an 8 membered cyclic transition state for 2.1.12 is disfavored. Study of S to N acyl migration via an 11 membered cyclic transition state To study possible chemical ligation via an 11 membered cyclic transition state, we first prepared the N Boc protected dipeptides 2.1.15 in 69% yield by peptide couplin g reactions of Boc Gly Bt with p henylalanine (Phe) in partially aqueous solution in the presence of triethylamine (Figure 2.10). Mixed anhydride couplings of the dipeptide dimer 2.1.8 with 2 equiv. of the dipeptide 2.1.15 furnished the tetrapeptide dimers 2. 1.16 in 72% yield (Scheme 2 4). Next, the cleavage of the disulfide 2.1.16 afforded the monomer tetrapeptide 2.1.17 (62%), which w as subsequently S acylated with Cbz Ala Bt in the presence of triethylamine. The Boc protected mono isopentapeptide 2.1.18 w as purified by recrystallization and then isolated in 92% yield. Acid catalyzed deprotection of Boc group proceeded smoothly to form the desired mono isopentapeptides 2.1.19 (86%, Figure 2.10). We investigated S N acyl migration in compound 2.1.19 via an 11 m embered cyclic transition state The chemical ligation experiment on 2.1.19 was carried out under microwave irradiation at 50 C and 50 W for 1 h. After workup, HPLC MS (ESI) analysis of the crude l igation mixture showed that the ligation experiment yield ed the expected ligation product 2.1. 20a and an intermolecular transacylation product 2.1. 20b in ratio of 33:67 (86% combined crude yield ) ( Figure 2 11 ). The product mixture was subsequently purified by semi preparative HPLC, which allowed the isolation o f the desired ligation product 2.1.20 a as well as isolation of the

PAGE 41

41 intermolecular transacylation product 2.1. 20b in yields of 23% and 52%, respectively. Both products were characterized by analytical HPLC and HRMS. Figure 2 10 Syn thesis of isopentapeptides 2.1.19 Figure 2 11 Chemical ligation of S acyl mono isopentapeptides 2.1.19

PAGE 42

42 The formation of the side product 2.1. 20b could be explained as follows. In the fir st step the intramolecu lar S N acyl migration provides the desired product 2.1. 20 a which is later S acylated by another equivalent of the starting material 2.1. 19 to form the transacylation product 2.1. 20b by in termolecular reaction Study of S N acyl migration via a 14 membere d cyclic transition state The synthesis of starting material 2.1. 25 for a possible S N ligation via a 14 membered transition state was accomplished in a five step procedure starting from the tripeptide dimer 2.1. 9. After initial deprotection of 2.1. 9 to fo rm 2.1. 21, subsequent mixed anhydride coupling of 2.1. 21 with Boc Gly Phe OH afforded the pentapeptide dimer 2.1. 22 in 74% yield. Cleavage of the disulfide bond in 2.1. 22 furnished the monomer 2.1. 23 which was S acylated with Cbz Ala Bt to provide the Boc protected isohexapeptide 2.1. 24 (86%). Final deprotection of the Boc group in 2.1. 24 afforded the desired starting material 2.1. 25 ( Figure 2.12 ). Chemical ligation from isopeptide 2.1.25 through a 14 membered cyclic transition state was investigated by dis solving 2.1.25 in 0.4 M phosphate buffer (pH = 7.8) and acetonitrile (7:1) and subjecting the mixture to microwave irradiation (50 C, 50 W, 1h) ( Figure 2 13 ). HPLC MS (ESI) analysis of the crude ligation product mixture confirmed the major component (57%, Table 2 1) to be the expected ligation product 2.1.26. Again, the intermolecular transacylation product 2.1.27 was formed as side product (Table 2 1). Our results indicate that transition states studied in this present paper are favored in decreasing orde r 5>>14>11>>8. These are the first examples of successful isopeptide ligations starting from non terminal S acyl peptides and the results strongly

PAGE 43

43 suggest the chemical long range ligation via 11 and 14 membered cyclic transition states is a promising appr oach for the synthesis of native peptides. Figure 2 12 Synthesis of isohexapeptide 2.1. 25 Figure 2 13 Chemical ligation of S acyl mono isohexapeptides 2.1.25

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44 Table 2 1. Chemical ligati on of isopeptides 2.1.25 Product Isolated yield [%] Purity [%] a HRMS [M+Na] + found 2.1. 26 41 >95 % 1337.4629 b,c a Purity based on analytical HPLC b Isolated as disulfide dimer c HRMS calc. [M+Na] + : 1337.4578 Long Range Intramolecular S N Acyl Migra tion: a Study of the Formation of Native Peptide Analogs via 15 and 16 Membered Cyclic Transition States Demonstration of S N acyl migr ation in S acyl tetrapeptide via a 15 membered cyclic transition state Unnatural amino acids have been used to prepare peptide analogs with en hanced enzymatic stability, 45 and b ioavailability. 46 Amino acid residues, which are often en countered in natural products, 47 have been used for obtaini ng peptidomimetics and for affecting secondary and tertiary stru ctures of synthetic peptides. 48 49 To investigate the fur ther applicability of our methodology to access peptide analogs 50 we studied long range S N acyl m igration s of S a cylated c ysteine peptides containing residues via a 1 5 membered cyclic transition states leading to the formation of the corresponding native tetra and pentapeptide analogs. For the S N ligation via a 15 membered cyclic transition state, the starting material 2.2.9a was obtained in a si x step procedure starting from the L cystine dimethyl ester dihydrochloride ( 2.2.1 ). The protected dipeptide dimer 2.2.2 a was prepared in 67% yield from the mixed anhydride coupling of 2.2.1 with Boc Gly OH following a literature procedure ( Figure 2.14 ). 51 D eprotection of 2.2.2 a using HCl( g ) in methanol gave 2.2.3 a in 84% yield. The coupling of 2.2.3 a with Boc protected dipeptide Boc Ala L Leu OH ( 2.2.5a ) provided the tetrapeptide dimer 2.2.6 a in 65% yield, which was subsequently treated with tributylphosphine to afford the tetrapeptide monomer 2.2.7 a in

PAGE 45

45 75% yield. S Acylation of N Boc protected cysteine tetrapeptide 2.2.8 a with Cbz L Ala Bt in acetonitrile water (10:1) in the presence of triethylamine gave S acyl tetrapeptide 2.2.8a in 86% yield. Finally, compound 2.2.8a was Boc deprotected using HCl( g ) in methanol to give the HCl salt 2.2.9a in 82% yield ( Figure 2 14 ). Figure 2 14 Prepar ation of S acyl tetrapeptide 2.2.9a The intramolecular S N acyl migration experiment 2.2.9a .2.10 would proceed through a 15 membered ring transition state. P recedents for successful S N acyl shift in SAL proceeding (Figure 2 15 ) through transition states with 15 membered rings encouraged us to pursue this approach. 42

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46 The chemical ligation experiment was carried out at 2 mM c oncentration under microwave irradiation at 50 C and 50 W for 1 h at pH = 7.6. HPLC MS (ESI) A nalysis of the crude reaction mixture revealed the presence of two major products in a 35:65 ratio (Table 2 2 ). The HPLC MS (ESI) analysis identified the major reaction products as the desired ligation product 2 .2.10 and the minor product as the intermolecular transacylation product 2 .2.11 (Figure 2.16) Figure 2 15 Proposed 15 membered cyclic transition states of A) the second gene ration Sugar assisted Ligation (SAL) and (B) the long rang e intramolecular acyl migration Compound 2.2.10 was isolated by semi preparative HPLC and fully characterized by 1 H and 13 C NMR spectroscopy, HRESI MS and analytical HPLC (Table 2 3). 1 H NMR s pectra indicated for the formation of the desired ligation product 2.2.10 (appearance of five amide proton signals), while 13 C NMR provided further evidence. Indeed, S aminoacyltetrapeptide 2.2.9a has four typical amide 13 C signals with chemical shifts ran ging from 171.5 to 175.4 ppm and a very typical 13 C thioester 13 C NMR resonances for five amide bonds at 169.1, 170.7, 170.8, 172.4 and 172.5 ppm (Figure 2 17 ). These results confirme d the S N migration of Cbz alanine to the N terminus.

PAGE 47

47 Table 2 2. Characterization data of ligation product mixtures Entry Starting material Crude a yield (%) Product ratio b (%) [M+H] + c Ligated peptide Transacylation product Ligated peptide d Transacylati on product 1 2.2.9a 92 35, 2.2.10 65, 2.2.11 1183 809 2 2.2.9b 94 64, 2.2.12 a 36, 2 .2.13 a 1257 835 3 2.2.9c 82 57, 2.2.12 b 43, 2.2.13 b 1257 835 a The combined crude yield of ligated product was calculated based on the isolated amount of the product mix ture ligated peptide : transacylation product according to the following equation: combined crude yield = ([ ligated peptide ] + 2 x [ transacylation product ]) /[ starting material ]. The S deacylated peptide side products were removed during the work up. b Determ ined 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. c HPLC MS (ESI). d Analyzed as disulfide dimer. Table 2 3. Characterization data of the isol ated ligation products 2 .2.10 and 2 .2.12 a,b Ligation product After semi preparative HPLC HRMS [M+Na] + b Retention time c Purity a Isolated yield (%) Calculated Found 2.2.10 >98% 31 1183.4774 1183.4733 19.42 2.2.12 a >99% 42 1279.4774 1279.4718 20.89 2.2 .12 b >95% 37 1279.4774 1279.4775 18.64 a Purity based on analytical HPLC. b Analyzed as disulfide dimer. c 254 nm, MeOH/H 2 O (65:35), 0.15 mL/min. Figure 2 16 Chemical ligation of S acyl tetrapeptide 2.2.9a. The ligation compound 2.2.10 is drawn as a monomer for clarity

PAGE 48

48 Figure 2 17 13 C NMR carbonyl signals in (A) ligated pentapeptide 2 .2.10 and in (B) starting S acylated tetrapeptide 2.2.9 a To understand the dependence of the substrate concentration on the formation of the ligated product vs. the transacylation product, we compared the initial product ratio 2.2.10:2.2.11 in the ligation reaction of 2.2.9a at pH = 7.1 as a function of concentration of reactant under identical reaction conditions. Interestingly, HPLC MS (E SI) analysis revealed that the concentration of the substrate doe s not significantly influence the product ratio. At 2 mM concentration of the substrate 2.2.9a the ligated product vs. the transacylation product ratio was 45:55 while at 0.5 mM and 6 mM conc entrations of 2.2.9a the product ratios were 43:57 and 55:45 respectively.

PAGE 49

49 Demonstration of S N acyl migration in S acyl tetrapeptides 23b,c each via distinct isomeric 16 membered cyclic transition states To study the chemical ligation via a 16 membered cy clic transition state, we first prepared the tetrapeptide dimer 2.2.6 b in 52% yield by coupling unprotected cystine dipeptide dimer 2.2.3 b with Boc Ala L Phe OH ( 2.2.5 b). Then, tetrapeptide dimer 2.2.6 b was treated with tributylphosphine to release the t etrapeptide monomer 2.2.7 b in 68% yield. S Acylation of N Boc protected cysteine tetrapeptide 2.2.7 b with Cbz L Ala Bt in a acetonitrile water mixture (10:1) in the presence of triethylamine afforded S acyl tetrapeptide 2 2 .8 b in 88% yield. Boc deprotectio n of 2 2 .8 b using HCl( g ) in methanol gave the hydrochloride 2 .2.9 b in 81% yield ( Figure 2 18 ). Similarly, 2.2.3 b was coupled with Boc GABA L Phe OH ( 2.2.5 c) to afford Boc protected tetrapeptide dimer 2.2.6 c in 57% yield. Cleavage of the disulfide bond in 2 .2.6 c furnished the monomer 2.2.7 c (66%), which was S acylated with Cbz L Ala Bt to provide the Boc protected S acyl tetrapeptide 2 2 .8 c (92%). Final Boc deprotection afforded the desired amino unprotected S acylated tetrapeptide 2 .2.9 c in 87% yield ( Figur e 2 1 8 ). We then investigated S N acyl migration for compounds 2.2.9b and 2.2.9c which proceeds in each case via isomeric 16 membered cyclic transition states. Chemical ligation on 2.2.9b was carried out under microwave irradiation at 50 C and 50 W for 1 h in NaH 2 PO 4 /Na 2 HPO 4 buffer (1 M, pH 7.6) and acetonitrile (8:1 mixture). HPLC MS (ESI) analysis of the crude ligation mixture revealed the major component (64%) to be the expected ligation product 2.2.12a (molecular ion of the disulfide dimer [M+H] + m/z: 1257, vs 630 for the [M+H] + molecular ion of the starting S (Pg aminoacyl)tetrapeptide 2.2.9b). The HPLC MS (ESI) confirmed that the second major

PAGE 50

50 product is 2.2.13a formed by intermolecular trans acylation (Table 2 2 ; Figure 2 19 ). Separation of 2.2.12a by semi preparative HPLC allowed isolation of 42% of pure p entapeptide 2.2.12a, which was further characterized by analytical HPLC and HRMS analysis (Table 2 3). Figure 2 18 Prepar ation of S acyl tetrapeptide 2.2.9a

PAGE 51

51 Figure 2 19 Acyl migration o f S acyl tetrapeptides 2.2.12b. The ligation compound 2.2.13a is drawn as a monomer for clarity Figure 2 20 Acyl migration of S acyl tetrapeptide 23c. The ligation compound 26b is drawn as a monomer for clarity Similarl y, the acyl migration experiment of S acylpeptide 2.2.9 c was carried out under the conditions described above. HPLC MS (ESI) analysis showed the presence of two main products in a 57:43 ratio (Table 2 2 Figure 2 20 ). As expected, the major product of this experiment was the desired ligation product 2.2.12 b. The subsequent separation of 2.2.12 b by semi preparative HPLC provided the purified ligation product 2.2.12 b in 37% yield. The product was characterized by HPLC ana lytical and HRMS (Table 2 3 ).

PAGE 52

52 Study of p H Dependence on t he Ligation Experiment Chemical ligation is pH sensitive: ligation usually proceeds rapidly around pH 7 but is rendered less efficient at pH < 5.5. 52 In addition, in some cases the yields for trace less Staudinger ligation in water increased at higher pH, but decreased drastically 53 The ratio of the ligation product 2.2.9a versus the intermolecular transacylated compound 2.2.10 was studied under different pH conditions (Table 2 4, Figure 2 21 ). Chemic al ligation experiments on 2.2.9a were carried out under microwave irradiation at 50 C and 50 W for 1 h in 1M phosphate buffer with pH values ranging from 6.2 8.2. After workup, the crude ligation mixtures were subjected to HPLC MS (ESI) analysis. The res ults are summarized in Table 2 4 and Figure 2 9. HPLC MS (ESI) analysis of the crude ligation mixture at pH = 6.2 showed the presence of the major intermolecular transacylation product 2.2.11 and the desired ligation product 2.2.10 in a 80:20 ratio. When t he ligation experiment was conducted at pH = 8.2, HPLC MS (ESI) analysis identified the same two products: the transacylated product 2.2.11 and the desired ligated product 2.2.10 in a similar 85:15 ratio. In contrast, when ligation was carried out at pH 7. 0 and at pH 7.6, HPLC MS (ESI) showed a significant increase in ligation product 2.2.10 with calculated 2.2.10:2.2.11 ratios of 45:55 and 36:64, respectively. In case of a lower pH, the starting material 2.2.9a exists partially in the unreactive protonated form and the acyl migration reaction is relatively slow, which favors the intermolecular S acylation of 2.2.10 by the excess of starting material 2.2.9a to form the undesired product 2.2.11. At pH > 8 the thiol group of the ligation product 2.2.10 is part ially deprotonated, which again favors the formation of the transacylation product 2.2.11 in a subsequent intermolecular reaction of 2.2.10 with unreacted 2.2.9a. At pH=7.3, a 2.2.10:2.2.11 ratio of 43:57 was obtained. The S N acyl migration in S

PAGE 53

53 acyl tetr apeptide 2.2.9a via a 15 membered cyclic transition state to give 2.2.10 is therefore favored at a pH range from 7.0 7.6. Table 2 4 Dependence of pH on the product ratio 2.2.10:2.2.11 in the ligation reaction of 2.2.9a Entry pH Product ratio a Ligated p eptide b (2 .2.10 ) Transacylation product (2 .2.11 ) 1 6.2 20 80 2 7.0 45 55 3 7.3 43 57 4 7.6 36 64 5 8.2 15 85 a Determined by HPLC MS semiquantitation. The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of e ach compound was integrated. b Analyzed as disulfide dimer. Figure 2 21 Effect of pH on S N acyl migration in S acyl tetrapeptide 2.2.9a 2.2.10 (%)

PAGE 54

54 Conclusion In summary, this work demonstrates an efficient and convenient synthetic pathway for the preparation o f several S acyl isopeptides containing internal cysteine residues. The chemical ligation studies of these S acyl peptides via 5 8 11 and 14 membered cyclic transition states show that the 8 membered transition state is clearly disfavored, whereas the 11 and 14 membered transition states are relatively favored for long range ligation approach. These results indicate that the transition states studied in this present paper are decreasingly favored in the order 5>>14>11>>8. Furthermore, a series of nove l S acyl peptides containing and/or amino acid residues which are useful intermediates in various synthetic and biological applications, were synthesized according to original protocols. The ligation step was investigated under MW heating and was fou nd to be sensitive to concentration of the ligating fragments. The observed variation in ligation yield allows for reactivity scaling. The native peptides obtained after chemical ligation of tetrapeptides via 15 and 16 membered cyclic transition states we re isolated in modest to good yields. The experimental results show that the extent of chemical ligation via 14 TS, 15 TS and 16 TS are similar. Experimental General Methods Melting points were determined on a capillary point apparatus equipped with a digi tal thermometer and are uncorrected. NMR spectra were recorded in CDCl 3 DMSO d 6 or CD 3 OD d 4 on Gemini or Varian NMR operating at 300 MHz for 1 H and 75 MHz for 13 C with TMS as an internal standard. Elemental analyses were performed on a Carlo Erba 1106 ins trument. All microwave assisted reactions were carried out with a single

PAGE 55

55 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 ba r. The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 sec.; PowerMax cooling mode). L Cystine, Boc Gly OH and L Leucine were purchased from Sigma Aldrich. Cbz L Ala OH and Fmoc Gly OH were purchased from Chem Impex International. L Phenylalanine and glycine methyl ester hydrochloride were purchased from TCI US. All commercially available starting materials were used without further purification. Fmoc Gly Bt ( Bt = benzotriazo l 1 yl), Cbz L Ala Bt Boc Gly Bt and N N di Boc cystine 4 were prepared according to known procedures. The phosphate buffer (NaH 2 PO 4 /Na 2 HPO 4 ) (0.4 M, pH 7.8) was degassed by bubbling argon through the buffer. HPLC MS analyses were performed on reverse pha se gradient Phenomenex Synergi Hydro RP (2.1 x 150 mm; 5 m) + guard column (2 x 4 mm) or Thermoscientific Hypurity C8 (5 m; 2.1 x 100 mm + guard column) using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; and mass spectrometry wa s done with electro spray ionization (ESI). Product ratios were obtained from HPLC MS semiquantitation. 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. Semi preparative and anal ytical HPLC were carried out on Phenomenex Luna 10 m C18(2) columns. Methanol:water (70:30) was used as eluent for the isolation of compounds. Experimental D etails for C ompounds 2.1.2, 2.1.3, 2.1.4, 2.1.8, 2.1.9, 2.1.15, 2.1.16, 2.1.21 and 2.1.22 (6 R ,11 R ) methyl 11 ((tert butoxycarbonyl)amino) 6 ((2 methoxy 2 oxoethyl) carbamoyl) 2,2 dimethyl 4,12 dioxo 3 oxa 8,9 dithia 5,13 diazapentadecan 15 oate

PAGE 56

56 (2.1.2). A solution of N N di Boc cystine (1.32 g, 3 mmol) in dry THF (10 mL) under argon was cooled to 15 o C in an ice bath with stirring. N Methylmorpholine (0.67g, 6.6 mmol), followed by isobutylchloroformate (0.91 g, 6.6 mmol) were added. After 5 min, a solution of glycine methyl ester hydrochloride (0.75 g, 6 mmol) and N methylmorpholine (0.67 g, 6.6 mmol ) in DMF (15 mL) was added. The ice bath was removed after 5 min and the solution was allowed to stir for 24 h at room temperature. The solution was concentrated under vacuum and the residue was dissolved in a mixture of ethyl acetate (20 mL) and water (5 mL). After extraction, the aqueous phase was discarded and the organic phase washed successively with saturated Na 2 CO 3 (2 x 10 mL), water (10 mL) and 2N HCl (10 mL). The solution was dried over dry MgSO 4 filtered and concentrated under vacuum. The peptide was recrystallized from ethyl acetate:hexanes to give desired product. White microcrystals, 79% yield, mp 140 143 C; Anal. Calcd for C 22 H 38 N 4 O 10 S 2 : C 45.35; H 6.57; N 9.62. Found: C 45.68; H 6.62; N 9.18; 1 H NMR (300 MHz, CDCl 3 ) 1.41 (s, 18H), 2.89 (dd J = 14.3, 10.2 Hz, 2H), 3.04 (dd, J = 14.7, 3.9 Hz, 2H), 3.70 (s, 6H), 3.87 (dd, J = 17.6, 5.3 Hz, 2H), 4.11 (dd, J = 18.0, 6.3 Hz, 2H), 4.90 (br s, 2H), 5.56 (d, J = 9.6 Hz, 2H), 8.13 (s, 2H); 13 C NMR (75 MHz, CDCl 3 ) 28.5, 41.1, 46.9, 52.4, 54.7, 80.5 156.1, 169.9, 171.0. ( R ) Methyl 2 (2 ((tert butoxycarbonyl)amino) 3 mercaptopropanamido) acetate (2.1.3). (6 R ,11 R ) methyl 11 ((tert butoxycarbonyl)amino) 6 ((2 methoxy 2 oxoethyl)carbamoyl) 2,2 dimethyl 4,12 dioxo 3 oxa 8,9 dithia 5,13 diazapentadecan 15 oate (400 mg, 0.69 mmol) was treated with PBu 3 (277 mg, 0.34 mL, 1.37 mmol) in 12 mL of MeOH:water (9:1) for 2h at room temperature. The reaction mixture was concentrated under vacuum and the residue was dissolved in diethylether (15 mL). The

PAGE 57

57 solution was dried over magnesium sulfate, and then concentrated under vacuum. The peptide was purified by column chromatography on silica gel (Eluent hexane:AcOEt, 2:1) and recrystallized from Et 2 O:hexane to give 3. White microcrystals, 55% yield, mp 55 57 C; Anal. Calcd for C 11 H 20 N 2 O 5 S: C 45.19; H 6.90; N 9.58. Found: C 45.48; H 7.16; N 9.45; 1 H NMR (300 MHz, CDCl 3 ) 1.44 (s, 9H), 1.70 (dd, J = 10.4, 7.6 Hz, 1H), 2.70 2.80 (m, 1H), 3.09 3.18 (m, 1H), 3.76 (s, 3H), 3.98 4.15 (m, 2H), 4.45 (br s, 1H), 5.58 (d, J = 8.1 Hz, 1H), 7.0 (br s, 1H); 13 C NMR (75 MHz, CDCl 3 ) 27.3, 28.5, 41.4, 52.6, 55.7, 80.8, 155.6, 170.2, 1 70.8. ( R ) Methyl 2 (2 ((tert butoxycarbonyl)amino) 3 mercapto propanamido)acetate (2.1.4). Dipeptide Boc L Cys Gly OMe (0.29 g, 1 mmol) together with an equimolar amount of Fmoc Gly Bt (0.40 g, 1 mmol) was suspended in acetonitrile (20 mL) at 25 o C. Then, 1 2 mL of 0.1 N KHCO 3 in water was added dropwise. The solution was stirred at the same temperature and monitored by TLC for starting material consumption. After completion of the reaction, the solution was acidified with 2 N HCl (15 mL), acetonitrile was remo ved under reduced pressure. The residue formed was dissolved in ethyl acetate (40 mL), extracted with 2 N HCl (3 15 mL), sat. NaCl solution (20 mL) and dried over MgSO 4 Ethyl acetate was removed under reduced pressure and the residue was dissolved in dic hloromethane (15 mL), hexanes were added until the solution is turbid and the solution was left to crystallize in the freezer. The solid obtained was filtered, dried to give the corresponding target. White microcrystals, 88% yield, mp 108 112 o C; Anal. Cal cd for C 28 H 33 N 3 O 8 S: C 58.83; H 5.82; N 7.35. Found: C 58.93; H 5.87; N 7.38; 1 H NMR (300 MHz, CDCl 3 ) 1.44 (s, 9H), 3.25 (dd, J = 14.1, 7.6 Hz, 1H), 3.40 (dd, J = 14.0, 5.0 Hz, 1H), 3.71 (s, 3H), 4.01 (d, J = 5.4 Hz, 2H), 4.14 (d, J = 5.5 Hz,

PAGE 58

58 2H), 4.23 (t J = 6.9 Hz, 2H), 4.42 (s, 1H), 4.44 (s, 1H), 5.42 (d, J = 7.9 Hz, 1H), 5.63 (br s, 1H), 6.94 (br s, 1H), 7.30 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.59 (d, J = 7.1 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 ) 28.5, 30.9, 41.4, 4 7.3, 50.9, 52.6, 54.2, 67.5, 80.9, 120.2, 125.2, 127.3, 128.0, 141.5, 143.9, 155.8, 156.5, 170.1, 170.5, 198.5. ( 7 R ,12 R ) 3,6,13,16 Tetraoxo 2,17 dioxa 9,10 dithia 5,14 diazaoctadecane 7,12 diaminium chloride (2.1.8). HCl gas was passed through a solution o f peptide 2.1.2 (4 mmol) in methanol (15 mL) for 30 minutes. The methanol solution was concentrated under vacuum and diethyl ether was added. The turbid solution was left to crystallize in the freezer overnight. The solid formed was filtered and washed wit h dry ethyl acetate (10 mL) and then with diethyl ether (10 mL) dried to give the corresponding deprotected peptide 7. White solid, 85% yield, mp 112 116 C; Anal. Calcd for C 12 H 24 N 4 O 6 S 2 2HCl: C 31.65; H 5.75; N 12.30. Found: C 31.25; H 5.63; N 10.94; 1 H N MR (300 MHz, DMSO d 6 ) 3.18 3.37 (m, 4H), 3.64 (s, 6H), 3.93 (d, J = 5.4 Hz, 4H), 4.21 4.22 (m, 2H), 8.63 (br s, 6H), 9.38 (d, J = 5.4 Hz, 2H); 13 C NMR (75 MHz, CDCl 3 ) 38.5, 40.8, 51.1, 52.0, 167.5, 169.7. (Gly L Cys Gly OCH 3 ) 2 hydrochloride ( 2.1. 21). HCl gas was passed thro ugh a solution of peptide 2.1.9 (4 mmol) in methanol (15 mL) for 30 minutes. The methanol solution was concentrated under vacuum and diethyl ether was added. The turbid solution was left to crystallize in the freezer overnight. The solid formed was filtere d and washed with dry ethyl acetate (10 mL) and then with diethyl ether (10 mL) dried to give the corresponding deprotected peptide 21. White microcrystals, 92% yield, mp 209 212 C; Anal. Calcd for C 16 H 30 Cl 2 N 6 O 8 S 2 .H 2 O: C 32.71; H 5.49; N 14.30. Found: C 3 2.50; H

PAGE 59

59 5.27; N 13.13; 1 H NMR (300 MHz, CD 3 J = 13.7, 6.5 Hz, 1H), 3.23 3.28 (m, 1H), 3.69 (s, 3H), 3.87 (s, 2H), 3.96 (s, 2H); 13 C NMR (75 MHz, CD 3 42.2, 53.0, 54.1, 168.1, 171.8, 172.6. (Boc Gly L Phe Gly L Cys Gly OCH 3 ) 2 (2.1.2 2). A solution of 2 (2 ((tert butoxycarbonyl)amino)acetamido) 3 phenylpropanoic acid 2.1.21 (0.97 g, 3 mmol) in dry THF (10 mL) under argon was cooled to 15 C in a ice bath with stirring. N Methylmorpholine (0.33g, 3.2 mmol), followed by isobutylchlorofo rmate (0.45 g, 3.2 mmol) were added. After 3 min, a solution of 2.1.21 (0.85 g, 1.5 mmol) and N methylmorpholine (0.7 g, 1.6 mmol) in DMF (10 mL) was added. The ice bath was removed after 5 minutes and the solution was allowed to stir for 12 h at room temp erature. The solution was concentrated under vacuum and the residue was dissolved in ethyl acetate (30 mL) and water (5 mL). After extraction, the aqueous phase was discarded and the organic phase washed successively with saturated Na 2 CO 3 (2 x 15 mL), wate r (10 mL), 2N HCl (15 mL) and water (10 mL). The solution was dried over dry MgSO 4 filtered and then concentrated under vacuum. The peptide was recrystallized from ethyl acetate/hexane to give desired (Boc Gly L Phe Gly L Cys Gly OCH 3 ) 2. White microcrysta ls, 74% yield, mp 180 185 C; Anal. Calcd for C 48 H 68 N 10 O 16 S 2 : C 52.16; H 6.20; N 12.67. Found: C 52.44; H 6.60; N 11.83; 1 H NMR (300 MHz, DMSO d 6 2.89 (m, 4H ), 2.99 3.15 (m, 4H), 3.44 3.54 (m, 4H), 3.61 (br s, 6H), 3.67 3.74 (m, 2H), 3.84 3.91 (m, 6H), 4.52 (br s, 2H), 4.62 (br s, 2H), 6.88 (t, J = 5.5 Hz, 2H), 7.22 (br s, 10H), 8.03 (d, J = 7.8 Hz, 2H), 8.30 (d, J = 8.1 Hz, 2H), 8.38 (br s, 2H), 8.52 (br s, 2H). 13 C NMR (75 MHz, DMSO d 6 28.2, 37.6,

PAGE 60

60 40.8, 41.9, 43.1, 51.8, 53.9 78.1, 126.2, 128.0, 129.2, 137.7, 155.7, 168.9, 169.3, 169.9, 170.4, 171.4. General Procedure for Boc Deprotection of P eptides 2.1.4, 2.1.11 2.1.18 and 2.1.24 to Give the Corresponding Unprotected P eptides 2.1.5, 2.1.12, 2.1.19 and 2.1.25 HCl gas was passed through a solution of peptide 2.1.4, 2.1.1 1 2.1.18 or 2.1.25 in methanol (15 mL) for 30 minutes. The methanol solution was concentrated under vacuum and diethyl ether (20 mL) was added. The turbid solution was left to crystallize in the freezer ove rnight. The solid formed was filtered and washed with dry ethyl acetate (10 mL) and diethyl ether (10 mL) dried to give the corresponding deprotected peptide 2.1.5, 2.1.12 2.1.19 and 2.1.25 (R) 1 (9H Fluoren 9 yl) 3,6,10,13 tetraoxo 2,14 dioxa 7 thia 4,11 diazapentadecan 9 aminium chloride (2.1.5). White solid, 85% yield, mp 203 206 C; Anal. Calcd for C 23 H 25 N 3 O 6 S.HCl 2H 2 O: C 50.78; H 5.56; N 7.72. Found: C 50.81; H 5.07; N 7.47; 1 H NMR (300 MHz, DMSO d 6 3.42 (m, 2H), 3.61 (s, 3H), 3.90 (d, J = 5.4 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 4.08 (br s, 1H), 4.24 (t, J = 6.8 Hz, 1H), 4.34 (d, J = 6.8 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.88 (d, J = 7.4 Hz, 2H), 8.13 (t, J = 5.9 Hz, 1H), 8.54 (br s, 3H), 9.12 (t, J = 5.4 Hz, 1H); 13C NMR (75 MHz, DMSO d 6 125.3, 127.2, 127.8, 140.8, 143.8, 156.6, 167.3, 169.6, 197.9. (5S,9R) methyl 9 (2 aminoacetamido) 5 met hyl 3,6,10 trioxo 1 phenyl 2 oxa 7 thia 4,11 diazatridecan 13 oate hydrochloride (2.1.12). White microcrystals, 85% yield, mp 130 135 C; Anal. Calcd for C 19 H 27 ClN 4 O 8 S 1.5H 2 O: C 44.06; H 5.84; N 10.82. Found: C 43.95; H 6.05; N 10.78; 1 H NMR (300 MHz, DMSO d 6 ) J = 7.0 Hz,

PAGE 61

61 3H), 2.73 2.77 (m, 1H), 3.00 3.06 (m, 1H), 3.62 (br s, 5H), 3.85 (d, J = 5.2 Hz, 2H), 4.20 (t, J = 7.1 Hz, 1H), 4.54 4.56 (m, 1H), 5.02 5.11 (m, 2H), 7.37 (br s, 5H), 8.08 8.10 (m, 1H), 8.15 (br s, 3H), 8.70 8.72 (m, 1H), 8.82 (d, J = 8.5 Hz, 1H); 13 C NMR (75 MHz, DMSO d 6 166.1, 169.7, 169.9, 201.7. H Gly L Phe L Cys(S L Cbz Ala) Gly OCH 3 hydrochloride (2.1.19). White microcrystals, 86% yield, mp 172 175 C; Anal. Calcd for C 28 H 36 ClN 5 O 8 S 2H 2 O: C 49.88; H 5.38; N 10.39. Found: C 50.06; H 5.70; N 10.76; 1 H NMR (300 MHz, DMSO d 6 J = 7.0 Hz, 3H), 2.72 2.80 (m, 1H), 3.07 3.11 (m, 2H), 3.23 (dd, J = 13.0, 5.7 Hz, 1H), 3.62 (br s, 5H), 3.86 (d, J = 5 .1 Hz, 2H), 4.20 (t, J = 7.2 Hz, 1H), 4.40 4.44 (m, 1H), 4.61 4.64 (m, 1H), 5.05 (dd, J = 17.1, 12.4 Hz, 2H), 7.14 7.44 (m, 10 H), 8.11 (d, J = 7.3 Hz, 1H), 8.18 (br s, 3H), 8.49 (br s, 1H), 8.71 (d, J = 7.9 Hz, 1H), 8.79 (d, J = 8.0 Hz, 1H); 13 C NMR (75 MHz, DMSO d 6 65.8, 126.4, 127.8, 127.9, 128.1, 128.4, 129.3, 136.7, 137.5, 155.8, 165.7, 169.9, 170.7, 201.9. H Gly L Ph L Gly Cys(S L Cbz Ala) Gly OCH 3 hydrochloride ( 2.1. 25). White microcrystals, 85% yield, mp 131 134 C ; Anal. Calcd for C 30 H 39 ClN 6 O 9 S: C 51.83; H 5.65; N 12.09. Found: C 51.78; H 5.76; N 12.26; 1 H NMR (300 MHz, CD 3 J = 7.1 Hz, 3H), 2.98 3.06 (m, 1H), 3.24 3.28 (m, 2H), 3.43 3.50 (m, 1H), 3.66 3.91 (m, 5H), 3.98 4.06 (m, 4H), 4.35 (t, J = 6.9 Hz, 1H), 4.60 4.67 (m, 2H), 5.14 (s, 2H), 7.29 7.39 (m, 10H); 13 C NMR (75 MHz, CD 3 53.9, 57.0, 58.4, 68.0, 128.0, 128.9, 129.2, 12.6, 129.7, 130.4, 138.2, 138.4, 158.5, 167.9, 171.6, 171.7, 172.5, 173.8, 203.7

PAGE 62

62 Chemical L igation of Cys ( S (Fmoc L Ala)) Gly O Me H ydrochloride ( 2.1. 5 ) to Form Native T ripeptide (2.1.6) ( R ) 1 (9 H Fluoren 9 yl) 3,6,10,13 tetraoxo 2,14 dioxa 7 thia 4,11 diaza pentadecan 9 aminium chloride 2.1.5 (0.58 g, 1 mmol) was dissolved in a m ixture of water (8 mL) and acetonitrile (24 mL). Triethylamine (0.168 mL, 1.2 mmol) was added and the mixture was stirred at room temperature for 1 h under argon. The reaction was acidified to pH 1 using 2N HCl and extracted with ethyl acetate (2 10 mL). The ethyl acetate layer was washed with 2N HCl (3 15 mL), sat. NaCl solution (20 mL) and dried over MgSO 4 The ethyl acetate solution was concentrated under vacuum and hexane was added. The turbid solution was left to crystallize in the freezer overnigh t. The solid formed was filtered and dried to give the corresponding native tripeptide 2.1 .6. White microcrystals, 72% yield, mp 88 92 C; Anal. Calcd for C 23 H 25 N 3 O 6 S: C 58.59; H 5.34; N 8.91. Found: C 58.72; H 5.17; N 8.62; 1 H NMR (300 MHz, CDCl 3 ) 2.89 2.97 (m, 1H), 3.02 3.07 (m, 1H), 3.64 (s, 3H), 3.70 (t, J = 7.3 Hz, 1H), 3.93 9.99 (m, 2H), 4.05 4.09 (m, 2H), 4.21 4.24 (m, 1H), 4.39 (d, J = 6.9 Hz, 2H), 5.54 5.59 (m, 1H) 6.17 (br s, 1H), 7.13 7.19 (m, 1H), 7.28 (t, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H), 8.47 (br s, 1H); 13 C NMR (75 MHz, DMSO d 6 ) 40.8, 43.4, 46.7, 51.8, 62.2, 65.8, 74.9, 78.6, 120.1, 125.3, 127.1, 127.7, 140.7, 143.9, 156.6, 169.4, 170.0, 170.5. General Procedure for the Synthesis of P eptides 2.1.10, 2.1.17 and 2.1.23 A mixture of tributylphosphine (0.607 g, 3 mmol) and dimer peptide 2.1.9 2.1. 1 6 or 2.1. 22 (1.5 mmol) in MeOH:water (9:1, 20 mL) was stirred at rt for 2 h under argon. The solvent was evaporated and the resid ue was dissolved in diethylether (15 mL). The solution was dried over magnesium sulfate and concentrated under reduced pressure.

PAGE 63

63 The crude peptides were purified according to the following procedures. Peptide 2.1.10 was recrystallized from diethyl ether:he xanes. Compounds 2.1.17 and 2.1. 23 were recrystallized from MeOH:diethyl ether. The precipitates were washed with cold hexanes (5 mL) and CH 2 Cl 2 (5 mL) and dried under reduced pressure. (R) Methyl 9 (mercaptomethyl) 2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oate ( 2.1.10 ). White microcrystals, 67% yield, mp 77 79 C; Anal. Calcd for C 13 H 23 N 3 O 6 S: C 44.69; H 6.63; N 12.03. Found: C 44.29; H 6.77; N 11.67; 1 H NMR (300 MHz, CDCl 3 J = 11.3, 6.5 Hz, 1H), 2.64 2.72 (m, 1H), 3.3 (br s, 1H), 3.74 (s, 3H), 3.82 (t, J = 5.2 Hz, 2H), 3.87 3.95 (m, 1H), 4.10 4.18 (m, 1H), 4.75 4.79 (m, 1H), 5.28 (br s, 1H), 7.19 (br s, 1H), 7.30 (br s, 1H); 13 C NMR (75 MHz, CDCl 3 B oc Gly L Phe L Cys Gly OCH 3 (2.1.17 ). White microcrystals, 62% yield, mp 158 162 C; Anal. Calcd for C 22 H 32 N 4 O 7 S: C 53.21; H 6.50; N 11.28. Found: C 52.83; H 6.43; N 10.91; 1 H NMR (300 MHz DMSO d 6 J = 8.2 Hz, 1H), 2.72 2.84 (m, 3H), 3.01 3.05 (m, 1H), 3.45 3.60 (m, 2H), 3.63 (br s, 3H), 3.87 (br s, 2H), 4.42 4.44 (m, 1H), 4.58 (br s, 1H), 6.90 (br s, 1H), 7.22 (br s, 5H), 7.97 (d, J = 7.6 Hz, 1H), 8.30 (d, J = 7.6 Hz, 1H), 8.37 (br s, 1H); 13 C NMR (75 MHz, DMSO 28.2, 37.4, 40.7, 43.1, 51.8, 53.8, 54.9, 78.1, 126.3, 128.0, 129.3, 137.6, 155.7, 169.3, 170.1, 171.1. Boc Gly L Phe Gly L Cys Gly OCH 3 ( 2.1. 23). White microcrystals, 67% yield, mp 188 193 C; Anal. Calcd for C 24 H 35 N 5 O 8 S 1 : C 52.07; H 6.37; N 12.65. Found: C 52.00; H 6.62; N 11.79; 1 H NMR (300 MHz, DMSO d 6 2.83 (m, 3H), 3.00 3.05 (m, 1H), 3.62 (br s, 5H), 3.72 4.00 (m, 4H), 4.46 4.50 (m, 2H),

PAGE 64

64 6.88 (b r s, H), 7.23 (br s, 5H), 8.07 (d, J = 5.6 Hz, 2H), 8.41 (br s, 1H), 8.52 (br s, 1H); 13 C NMR (75 MHz, CD 3 128.0, 129.7, 130.4, 138 .4, 158.7, 171.8, 172.8, 174.5. General Procedure for the Pr eparation of Dimer P eptides 2.1.9 and 2.1.16 Isobutyl chloroformate (0.6 g, 4.4 mmol) was added to a solution of N Boc Gly OH or the respective Boc protected dipeptide 2.1.15 (4 mmol) and N methylmorpholine (0.45 g, 4.4 mmol) in dry THF (30 mL) at 10 C. After 5 min a mixture of {2 amino 3 [2 amino 2 (methoxycarbonylmethyl carbamoyl) ethyldisulfanyl] propionylamino} acetic acid methyl ester dihydrochloride 2.1.8 (0.91 g, 2 mmol) and N methylmorpholine (0.45 g, 4.4 mmol) in dry DMF (10 mL) was added at 10 C. The reaction mixture was stirred at rt for 12 h under argon. The THF was removed under reduced pressure, water (30 mL) was added and the resulting solution was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were subsequently wash ed with 2N HCl (2 x 50 mL), water (30 mL), 5% Na 2 CO 3 (2 x 50 mL), brine (30 mL) and dried over MgSO 4 Evaporation of the solvent gave the desired products, which were purified by recrystallization from CH 2 Cl 2 :hexanes. (9 R ,9' R ) Dimethyl 9,9' (disulfanediylb is(methylene))bis(2,2 dimethyl 4,7,10 trioxo 3 oxa 5,8,11 triazatridecan 13 oate) ( 2.1.9 ). White microcrystals, 62% yield, mp 88 92 C; Anal. Calcd for C 26 H 44 N 6 O 12 S 2 H 2 O: C 43.69; H 6.49; N 11.76. Found: C 43.81; H 6.58; N 11.49; 1 H NMR (300 MHz, CDCl 3 ) 1.44 (s, 18H), 2.89 3.08 (m, 4H), 3.76 (s, 6H), 3.82 3.96 (m, 6H), 4.01 (d, J = 4.8 Hz, 2H), 4.09 4.17 (m, 2H), 5.43 (br s, 2H), 5.64 (br s, 2H), 7.26 (s, 2H), 8.41 (br s, 2H); 13 C NMR (75 MHz, CDCl 3 ) 28.5, 41.6, 44.4, 45.2, 52.6, 53.2, 80.4, 156.3, 170 .2, 170.5, 170.6.

PAGE 65

65 (Boc Gly L Phe L Cys Gly OCH 3 ) 2 ( 2.1.16 ). White microcrystals, 72% yield, mp 155 159 C; Anal. Calcd for C 44 H 62 N 8 O 14 S 2 : C 53.32; H 6.71; N 11.31. Found: C 52.93; H 6.49; N 10.85; 1 H NMR (300 MHz, DMSO d 6 2.95 (m, 4H), 3.03 3.16 (m, 4H), 3.45 3.56 (m, 4H), 3.62 (br s, 6H), 3.86 (br s, 4H), 4.58 4.63 (m, 4H), 6.90 (br s, 2H), 7.22 (br s, 10H), 7.95 7.97 (m, 2H), 8.38 (br s, 2H), 8.48 (d, J = 7.3 Hz, 2H); 13 C NMR (75 MHz, DMSO d 6 9, 53.8, 78.1, 126.2, 128.0, 129.3, 137.5, 155.7, 169.3, 169.9, 170.2, 171.1. General Procedure for the Preparation of Boc Protected D ipeptides 2.1.15 Boc Gly Bt (10 mmol) was added at 25 C to a solution of the respective amino acid 2.1.14 (10 mmol) in Me CN:H 2 O (30 mL : 10 mL) in the presence of Et 3 N (10 mmol). The reaction mixture was stirred at 25 C for 2 h. Aq. 4N HCl solution (5 mL) was added and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), and th e organic extract was washed with 4N HCl (3 30 mL), brine (30 mL) and dried over MgSO 4 Evaporation of the solvent gave the desired product, which was purified by recrystallization from ethyl acetate:hexanes to yield the desired Boc protected dipeptides as solid compounds. ( S ) 2 (2 (( tert Butoxycarbonyl)amino)acetamido) 3 phenylpropanoic acid 2.1.15. White microcrystals, 69% yield, mp 147 148 C; Anal. Calcd for C 13 H 24 N 2 O 5 : C 59.62; H 6.88; N 8.69. Found: C 59.58; H 7.02; N 8.64; 1 H NMR (300 MHz, DMSO d 6 ) 1.36 (br s, 9H), 2.84 2.95 (m, 1H), 3.00 3.06 (m, 1H), 3.35 (br s, 3H), 3.48 3.60 (m, 2H), 4.41 4.46 (m, 1H), 6.93 (t, J = 5.9 Hz, 1H), 7.19 7.26 (m, 5H), 8.02 (d, J = 8.0 Hz, 1H); 13 C NMR (75 MHz, DMSO d 6 ) 28.2, 36.8, 43.0, 53.4, 78.0, 78.0, 126.5, 128.2, 129.1, 137.4, 155.7, 169.2, 172.8.

PAGE 66

66 General Procedure for the P reparation of S Acyl P eptides 2.1.11 2.1.18 and 2.1. 24 Cbz L Ala Bt (0.325 g, 1 mmol) was added to a mixture of 2.1.10, 2.1.17 or 2.1.23 (1 mmol) and triethylamine (0.1 g, 1 mmol) in ac etonitrile (20 mL). The mixture was stirred for 3 h at rt and the solvent was removed under reduced pressure. The crude compounds were purified according to the following procedure. The residue was dissolved in ethyl acetate (20 mL), extracted with 2N HCl (2 x 20 mL), water (15 mL), and brine (10 mL). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Compound 2.1.11 was recrystallized from CH 2 Cl 2 :hexanes. The S acyl peptides 2.1.18 and 2.1.24 were recrystallized fr om ethyl acetate:hexanes. The solids obtained were filtered, washed with ether (5 mL) and dried. S ) S (( R ) 2 (2 ((tert butoxycarbonyl)amino)acetamido) 4 ((methoxycarbonyl)amino) 3 oxobutyl) 2 (((benzyloxy) carbonyl)amino)propanethioate (10). White microcry stals, 82% yield, mp 62 67 C; Anal. Calcd for C 24 H 34 N 4 O 9 S: C 51.98; H 6.18; N 10.10. Found: C 51.70; H 5.87; N 9.75; 1 H NMR (300 MHz, CDCl 3 ) 1.44 (s, 12H), 3.37 3.41 (m, 2H), 3.72 (br s, 4H), 3.81 3.88 (m, 1H), 3.95 4.08 (m, 2H), 4.38 (t, J = 7.2 Hz, 1 H), 4.66 (br s, 1H), 5.06 5.20 (m, 2H), 5.43 (d, J = 6.6 Hz, 1H), 5.55 (br s, 1H), 6.89 (d, J = 7.4 Hz, 1H), 7.18 (br s, 1H), 7.36 (br s, 5H); 13 C NMR (75 MHz, CDCl 3 ) 18.2, 28.5, 29.7, 41.4, 44.5, 52.5, 53.4, 57.2, 67.6, 80.6, 128.3, 128.5, 128.8, 136. 1, 156.3, 156.6, 170.0, 170.1, 170.8, 202.5. Boc Gly L Phe L Cys( S L Cbz Ala) Gly OCH 3 (2.1.18) White microcrystals, 92% yield, mp 164 167 C; Anal. Calcd for C 33 H 43 N 5 O 10 S: C 56.48; H 6.18; N 9.98. Found: C 56.17; H 6.20; N 9.97; 1 H NMR (300 MHz, DMSO d 6 ) J = 6.9 Hz, 3H), 1.35 (s, 9H), 2.77 (dd, J = 13.7, 9.3 Hz, 1H), 3.00 3.06 (m, 2H), 3.21 (dd, J = 13.2, 5.4 Hz, 1H), 3.63 (br s, 5H), 3.87 (br s, 2H), 4.19 (t, J = 7.3 Hz, 1H), 4.38 4.47 (m, 1H), 4.54 (br s,

PAGE 67

67 1H), 5.01 5.10 (m, 2H), 6.90 (t, J = 5.4 Hz, 1H), 7.17 7.28 (m, 5H), 7.35 (br s, 5H), 7.92 (d, J = 7.7 Hz, 1H), 8.09 (d, J = 7.3 Hz, 1H), 8.38 (t, J = 5.1 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H); 13 C NMR (75 MHz, DMSO d 6 56.2, 58.4, 68.0, 80.9, 1 28.0, 128.9, 129.2, 129.6, 129.7, 130.5, 138.1, 138.3, 158.4, 171.5, 172.2, 172.6, 173.4, 203.6. Boc Gly L Phe Gly L Cys( S L Cbz Ala) Gly OCH 3 (2.1.24). White microcrystals, 86% yield, mp 126 128 C; Anal. Calcd for C 35 H 46 N 6 O 11 S: C 55.40; H 6.11; N 11.07. Found: C 55.02; H 6.05; N 11.00; 1 H NMR (300 MHz, CD 3 J = 7.3 Hz, 3H), 1.43 (s, 9H), 2.96 (dd, J = 13.9, 8.7 Hz, 1H), 3.17 3.24 (m, 2H), 3.43 (dd, J = 13.9, 5.1 Hz, 1H), 3.60 3.69 (m, 1H), 3.70 (br s, 5H), 3.97 (br s, 3H), 4.29 (q, J = 7 .3 Hz, 1H), 4.56 4.66 (m, 2H), 5.11 (s, 2H), 7.19 7.36 (m, 10H); 13 C NMR (75 MHz, CD 3 ppm 18.0, 28.9, 31.0, 38.4, 42.2, 43.8, 44.7, 52.8, 54.0, 56.5, 58.4, 68.0, 81.0, 127.9, 128.9, 129.2, 129.7, 130.4, 138.2, 138.5, 158.5, 17 1.6, 172.5, 172.8, 174.2, 203.4 General Procedure for Chemical L igation of S Acyl P eptides 2.1.12, 2.1.19 and 2.1. 25 The respective S acyl peptide hydrochloride 2.1.12, 2.1.19 or 2.1.25 ( 0.05 mmol) was suspended in degassed phosphate buffer (NaH 2 PO 4 /Na 2 HPO 4 ) (0.4 M, pH 7.8, 7 mL) and acetonitrile (~1 mL) was added dropwise until the starting material was dissolved. The mixture was subjected to microwave irradiation (for compounds 2 .1.19 and 2.1.25 : 50 C, 50 W, 1 h; for compound 2.1. 12 : 70 C, 50 W, 3 h) under argon. The reaction w as allowed to cool to room temperature, acetonitrile was removed under reduced pressure and the residue was acidified with 2N HCl to pH = 1. The mixture was extracted with ethyl acetate (3 x 20 mL), the combined organic extracts were dried over MgSO 4 and t he solvent was removed under reduced pressure. The ligation mixture was

PAGE 68

68 weighed and then a solution in methanol (1 mg/mL) was analyzed by HPLC MS. Compounds 2.1.20a, 2.1.20b and 2.1.26 were subsequently isolated by semi preparative HPLC and characterized b y an alytical HPLC and HRMS analysis Preparations of Compounds 2.2.2a, 2.2.3a, 2.2.4a, 2.2.5a, 2.2.6a, 2.2.7a, 2.2.8a and 2.2.9a Boc Ala Bt (2.2.4b). A solution of N Boc Ala OH (3.00 g, 15.9 mmol) in CH 2 Cl 2 (30 mL) was added to a solution of dicyclohexy lcarbodiimide (3.27 g, 15.9 mmol) and 1H benzotriazole (1.89 g, 15.9 mmol) in CH 2 Cl 2 (10 mL). The reaction mixture was stirred at room temperature for 1.5 h. The precipitate was filtered and the solution was passed through 6.00 g of celite. The solution w as evaportated under reduced pressure and the crude mixture obtained was dissolved in EtOAc (50 mL). The organic layer was washed with saturated Na 2 CO 3 (3 x 30 mL), brine (20 mL) and dried over MgSO 4 Concentration under reduced pressure produced the final product. Recrystallization from CH 2 Cl 2 :hexanes gave Boc Ala Bt White microcrystals, 3.46g, 63% yield; mp 112 115 C; 1 H NMR (CDCl 3 300 MHz): 1.43 (s, 9H), 3.66 3.70 (m, 4H), 5.11 (br s, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 8.14 ( d, J = 8.2 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H); 13 C NMR (CDCl 3 75 MHz): 28.5, 35.8, 36.6, 79.8, 114.4, 120.4, 126.4, 130.7, 131.1, 146.3, 155.9, 171.5; Anal. Calcd for C 14 H 18 N 4 O 3 : C 57.92, H 6.25, N 19.30. Found: C 58.00, H 6.33, N 19.31. Boc Ala L Leu OH (2.2.5a ). A solution of L Leucine (0.81 g, 6.20 mmol) and triethylamine (0.63 g, 6.20 mmol) in acetonitrile (10 mL) and water (5 mL) was added to the suspension of Boc Ala Bt (1.50 g, 5.17 mmol) in acetonitrile (30 mL). The solution was stirred for 15 h and then the solvent was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate. The organic phase was washed with 2.0 N

PAGE 69

69 HCl (3 x 10 mL) and brine (10 mL). The solution was dried over MgSO 4 filtered and then concentrated un der vacuum. The solid was recrystallized from ethyl acetate:hexanes to yield Boc Ala L Leu OH White microcrystals, 1.13 g, 72% yield; mp 124 127 C; 1 H NMR (DMSO d 6 300 MHz): 0.84 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H), 1.37 (s, 9H), 1.45 1.53 (m, 2H), 1.54 1.68 (m, 1H), 2.21 2.32 (m 2H), 3.10 (dd, J = 13.3, 7.2 Hz, 2H), 4.15 4.23 (m, 1H), 6.68 (t, J = 5.2 Hz, 1H), 8.11 (d, J = 7.9, Hz 1H), 12.50 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 21.3, 22.9, 24.3, 28.2, 35.5, 36.7, 39.9, 50.1, 77.6, 155.4, 170.4, 174.2; Anal. Calcd for C 14 H 26 N 2 O 5 : C 55.61, H 8.67, N 9.26. Found: C 55.98, H 8.91, N 9.22. (Boc Gly L Cys OCH 3 ) 2 (2.2.2a ). A solution of Boc Gly OH (1.13 g, 6.4 mmol) in dry THF (10 mL) under argon was cooled to 15 C in an ice bath with stirring. N methylmorpholine (0.65 g, 6.4 mmol), followed by isobutylchloroformate ( 0.84 g, 6.4 mmol) were added. After 5 minutes, a solution of L Cystine dimethyl ester dihydrochloride (1.00 g, 2.9 mmol) and N methylmorpholine ( 0.65 g, 6.4 mmol) in DMF (5 mL) were added. The ice bath was removed after 5 minutes and the solution was allo wed to stir for 24 h at room temperature. The solution was concentrated under vacuum; the residue was taken up in ethyl acetate (20 mL) and 2N HCl (5 mL). The organic phase was washed successively with saturated Na 2 CO 3 (3 x 10 mL), and 2N HCl (3 x 10 mL). The solution was dried over dry MgSO 4 filtered and then concentrated under vacuum. The peptide was recrystallized from diethyl ether:hexanes to give (Boc Gly L Cys OCH 3 ) 2 White microcrystals, 1.13 g, 66% yield; mp 51 54 C; 1 H NMR (DMSO d 6 300 MHz): 1.38 (br s, 18H), 2.96 (dd, J = 13.9, 8.3 Hz, 2H), 3.12 (dd, J = 13.4, 4.8 Hz, 2H), 3.58 (d, J = 5.8 Hz, 4H), 3.65 (s, 6H), 4.55 4.65 (m, 2H), 6.94 (t, J

PAGE 70

70 = 5.8 Hz, 2H), 8.35 (d, J = 7.9 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 28.2, 39.1, 42.9, 51.2, 52.2, 78.1, 155.7, 169.6, 170.8; Anal. Calcd for C 22 H 38 N 4 O 10 S 2 : C 45.35, H 6.57, N 9.62. Found: C 45.64, H 6.76, N 9.45. (Gly L Cys OCH 3 ) 2 hydrochloride (2.2.3a). HCl gas was passed through a solution of (Boc Gly L Cys OCH 3 ) 2 (0.58 g, 1.0 mmol) in methanol (15 m L) for 30 minutes. The methanol solution was concentrated under vacuum and ether was added. The turbid solution was left to crystallize in the freezer overnight. The solid formed was filtered and washed with dry diethyl ether (10 mL) dried to give the corr esponding (Gly L Cys OCH 3 ) 2 hydrochloride White microcrystals, 0.38 g, 83% yield; mp 185 189 C; 1 H NMR (DMSO d 6 300 MHz): 2.98 (dd, J = 13.9, 8.5 Hz, 2H), 3.15 (dd, J = 13.7, 5.0 Hz, 2H), 3.60 (s, 4H), 3.66 (s, 6H), 4.57 4.68 (m, 2H), 8.36 (s, 6H), 9. 31 (d, J = 7.4 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 39.9, 51.5, 52.4, 166.3, 170.4; Anal. Calcd for C 12 H 24 Cl 2 N 4 O 6 S 2 .H 2 O: C 30.45, H 5.54, N 11.84. Found: C 30.33, H 5.53, N 11.98. (Boc Ala L Leu Gly L Cys OCH 3 ) 2 (2.2.6a). A solution of Boc Ala L Leu O H (0.91 g, 3 mmol) in dry THF (10 mL) under argon was cooled to 15 o C in an ice bath with stirring. N methylmorpholine (0.33g, 3.2 mmol), followed by isobutylchloroformate (0.45 g, 3.2 mmol) were added. After 4 min, a solution of (Gly L Cys OCH 3 ) 2 hydroch loride (0.68 g, 1.5 mmol) and N methyl morpholine (0.7 g, 1.6 mmol) in DMF (5 mL) was added. The ice bath was removed after 5 min and the solution was allowed to stir for 12 h at room temperature. The solution was concentrated under vacuum; the residue was taken up in ethyl acetate (30 mL) and water (5 mL). The organic phase was washed successively with saturated Na 2 CO 3 (2 x 15 mL), water (10 mL), 2N HCl (15 mL) and water (10 mL). The solution was dried over MgSO 4 filtered and then

PAGE 71

71 concentrated under vacuu m. The peptide was recrystallized from ethyl acetate:hexanes to give (Boc Ala L Leu Gly L Cys OCH 3 ) 2 White microcrystals, 0.93 g, 65% yield; mp 105 111 C; 1 H NMR (DMSO d 6 300 MHz): 0.83 (d, J = 6.4 Hz, 6H), 0.88 (d, J = 6.6 Hz, 6H), 1.36 (s, 18H), 1.44 (t, J = 7.1, 4H), 1.53 1.63 (m, 2H), 2.25 2.31 (m, 4H), 2.95 (dd, J = 13.8, 8.5 Hz, 2H), 3.07 3.16 (m, 6H), 3.65 (s, 6H), 3.70 3.75 (m, 4H), 4.19 4.26 (m, 2H), 4.58 (dd, J = 13.3, 8.2 Hz, 2H), 6.70 (t, J = 5.5 Hz, 2H), 8.07 (d, J = 7.3 Hz, 2H), 8.22 (t, J = 5.6 Hz, 2H), 8.30 (d, J = 7.9 Hz, 2H); 13 C NMR (Acetone d 6 75 MHz): 19.4, 22.2, 23.4, 25.4, 28.7, 36.9, 37.8, 40.6, 41.3, 43.5, 52.8, 53.4, 78.8, 156.7, 170.2, 171.5, 173.2, 174.1; Anal. Calcd for C 40 H 70 N 8 O 14 S 2 : C 50.51, H 7.42, N 11.78. Found: C 50.35, H 7.56, N 11.39. Boc Ala L Leu Gly L Cys OCH 3 (2.2.7a). (Boc Ala L Leu Gly L Cys OCH 3 ) 2 (1.36 g, 1.43 mmol) was treated with P(Bu) 3 (0.58 g, 0.71 mL, 2.86 mmol) in 20 mL of 9:1 MeOH:water for 2 h at rt under argon gas. The reaction mixture was concentrated under vacuum and the residue was taken up in ethyl acetate (15 mL). The solution was dried over MgSO 4 and then concentrated under vacuum. The peptide was recrystallized from ethyl acetate:hexane to give Boc Ala L Leu Gly L Cys OCH 3 White microcrystals, 1.02 g, 75% yield; mp 140 142 C; 1 H NMR (DMSO d 6 300 MHz): 0.83 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H), 1.25 (br s, 1H), 1.37 (s, 9H), 1.44 (t, J = 7.3 Hz, 2H), 1.53 1.65 (m, 1H), 2.23 2.34 (m, 2H ), 2.74 2.90 (m, 2H), 3.11 (dd, J = 12.9, 7.0 Hz, 2H), 3.65 (s, 3H), 3.73 (d, J = 4.6 Hz, 2H), 4.16 4.24 (m, 1H), 4.46 4.53 (m, 1H), 6.72 (t, J = 6.1 Hz, 1H), 8.08 8.13 (m, 2H), 8.32 (t, J = 6.1 Hz, 1H); 13 C NMR (Acetone d 6 75 MHz): 22.2, 23.4, 25.4, 26 .7, 28.7, 36.9, 37.8, 41.1, 43.5, 52.6, 53.6,

PAGE 72

72 55.5, 78.8, 156.7, 169.9, 171.3, 173.2, 173.8; Anal. Calcd for C 20 H 36 N 4 O 7 S: C 50.40, H 7.61, N 11.76. Found: C 50.45, H 7.85, N 11.38. Boc Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 (2.2.8 a). (Boc Ala L Leu Gly L Cys OCH 3 (500 mg, 1.25 mmol) were suspended in acetonitrile (15 mL) and a solution of (10S,16R) methyl 10 isobutyl 16 (mercaptomethyl) 2,2 dimethyl 4,8,11,14 tetraoxo 3 oxa 5,9,12,15 tetraazaheptadecan 17 oate (596 mg, 1.25 mmol) in acetonitrile:water (1 0 mL 4:1) containing an equivalent amount of triethylamine (127 mg, 1.26 mmol) was added. The mixture was stirred at 25 o C for 3 h until completion. Acetonitrile was removed under reduced pressure and the residue was taken in ethyl acetate (30 mL), extract ed with 2N HCl (2 x 20 mL), water (15 mL) and brine (10 mL). Ethyl acetate was concentrated under reduced pressure and hexane was added; the turbid solution was left to crystallize overnight at 20 o C. The solid obtained was filtered, washed with diethyl e ther (3 mL), CH 2 Cl 2 (3 mL) and dried to give the corresponding Boc Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 White microcrystals, 739 mg, 87% yield; mp 100 105 C; 1 H NMR (DMSO d 6 300 MHz): 0.83 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H), 1.25 (d, J = 7.3 Hz, 3H), 1.36 (s, 9H), 1.44 (t, J = 7.2 Hz, 2H), 1.52 1.64 (m, 1H), 2.28 (t, J = 6.5 Hz, 2H), 3.06 3.15 (m, 3H), 3.24 (dd, J = 14.1, 6.4 Hz, 1H), 3.62 (s, 3H), 3.70 (t, J = 6.1 Hz, 2H), 4.14 4.26 (m, 2H), 4.36 4.43 (m, 1H), 5.06 (s, 2H), 6.70 (t, J = 5.0 Hz, 1H), 7.30 7.42 (m, 5H), 8.07 (t, J = 6.8 Hz, 1H), 8.18 (t, J = 5.6 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H); 13 C NMR (Acetone d 6 75 MHz): 18.0, 22.2, 23.4, 25.4, 28.7, 37.8, 41.2, 43.2, 52.7, 53.3, 57.9, 67.1, 78.8, 128.7, 129.3, 138.0, 156.7, 156.9, 169.9, 171.2, 173.0, 173.4, 202.0; Anal. Calcd for C 31 H 47 N 5 O 10 S: C 54.61, H 6.95, N 10.27. Found: C 54.58, H 6.80, N 10.08.

PAGE 73

73 Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 hydrochloride ( 2.2.9a ). HCl gas was passed through a solution of Boc Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 (600 mg, 0.88 mmol) dissolved in methanol for 1.5 h at rt. The solvent was then evaporated under reduced pressure and the so lid was recrystallized from methanol:ether to yield (5S,9R,15S) 15 isobutyl 9 (methoxycarbonyl) 5 methyl 3,6,11,14,17 pentaoxo 1 phenyl 2 oxa 7 thia 4,10,13,16 tetraazanonadecan 19 aminium chloride. White microcrystals, 0.45 g, 82% yield; mp 174 185 C; 1 H NMR (DMSO d 6 300 MHz): 0.86 (dd, J = 12.9, 6.4 Hz, 6H), 1.25 (d, J = 7.2 Hz, 3H), 1.42 1.50 (m, 2H), 1.54 1.64 (m, 1H), 2.92 3.00 (m, 2H), 3.09 (dd, J = 13.4, 7.4 Hz, 1H), 3.23 (dd, J = 14.6, 6.7 Hz, 1H), 3.62 (br s, 3H), 3.65 (br s, 2H), 3.70 3.76 (m, 2H), 4.14 4.22 (m, 1H), 4.29 (dd, J = 15.0, 7.5 Hz, 1H), 4.32 (q, J = 6.8 Hz, 1H), 5.06 (s, 2H), 7.30 7.42 (m, 5H), 7.90 (br s, 3H), 8.10 (d, J = 7.6 Hz, 1H), 8.26 (t, J = 5.4 Hz, 1H), 8.33 8.37 (m, 1H), 8.40 (d, J = 7.3 Hz, 1H); 13 C NMR (CD 3 OD, 75 MHz): 18.0, 22.2, 23.5, 26.0, 30.7, 32.8, 37.2, 41.4, 41.9, 43.3, 53.3, 54.0, 58.4, 67.9, 128.8, 129.2, 129.6, 138.2, 158.4, 171.5, 172.0, 172.9, 175.4, 203.2; Anal. Calcd for C 26 H 40 ClN 5 O 8 S.3H 2 O: C 46.46, H 6.90, N 10.42. Found: C 46.53, H 6.72, N 10.54. Preparations of Compounds 2.2. 2b, 2.2.3b, 2.2.4a, 2.2.5b, 2.2.6b, 2.2.7b, 2.2.8b and 2.2.9b Boc Ala L Phe OH (2.2.5b). The compound was prepared according to the method for preparation of Boc Ala L Leu OH (2.2.5a) White microcrystals, 1.02 g, 65% yield; mp 154 157 C; 1 H NMR (DMSO d 6 300 MHz): 1.37 (br s, 9H), 2.19 2.25 (m, 2H), 2.84 (dd, J = 13.7, 9.5, Hz, 1H), 3.00 3.11 (m, 3H), 4.37 4.44 (m, 1H), 6.63 (br s, 1H), 7.17 7.30 (m, 5H), 8.23 (d, J = 8.0 Hz, 1H); 13 C NMR (DMSO d 6 75 MHz):

PAGE 74

74 28.3, 35.5, 36.6, 36.7, 53.4, 77.6, 126.4, 128.1, 129.1, 137.7, 155.4, 170.3, 173.0; Anal. Calcd for C 17 H 24 N 2 O 5 : C 60.70, H 7.19, N 8.33. Found: C 60.29, H 7.22, N 8.19. (Boc Ala L Cys OCH 3 ) 2 (2.2.2 b). The compound was prepared according to the method for preparation of (Boc Gly L Cys OCH 3 ) 2 ( 2.2.2a ) White microcrystals, 1.27 g, 72% yield; mp 117 119 C; 1 H NMR (DMSO d 6 300 MHz): 1.37 (s, 18H), 2.29 (t, J = 7.3 Hz, 4H), 2.92 (dd, J = 13.8, 8.8 Hz 2H), 3.06 3.13 (m, 6H), 3.64 (s, 6H), 4.53 (dd, J = 13.2, 8.1 Hz, 2H), 6.72 (br s, 2H), 8.46 (d, J = 7.4 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 28.2, 35.4, 36.5 51.1, 52.2, 77.6, 155.4, 170.6, 170.9; Anal. Calcd for C 24 H 42 N 4 O 10 S 2 : C 47.20, H 6.93, N 9.17. Found: C 47.33, H 7.12, N 9.01. ( Ala L Cys OCH 3 ) 2 hydrochloride ( 2.2.2b ). The compound was prepared according to the method for preparation of (Gly L Cys OCH 3 ) 2 hydrochloride ( 2.2.2a ). White microcrystals, 0.20 g, 82% yield; mp 87 92 C; 1 H NMR (DMSO d 6 300 MHz): 2.58 (t, J = 7.4 Hz, 4H), 2.92 3.00 (m, 6H), 3.12 (dd, J = 14.1, 4.9 Hz, 2H), 3.66 (br s, 6H), 4.53 4.60 (m, 2H), 8.04 (br s, 6H), 8.81 (d, J = 7. 7 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 32.6, 32.7, 51.9, 52.9, 170.3, 171.4; Anal. Calcd for C 14 H 28 Cl 2 N 4 O 6 S 2 .0.75H 2 O: C 33.84, H 5.98, N 11.27. Found: C 34.06, H 6.01, N 10.88. (Boc Ala L Phe Ala L Cys OCH 3 ) 2 ( 2.2.6b ). The compound was prepared accord ing to the method for preparation of (Boc Ala L Leu Gly L Cys OCH 3 ) 2 ( 2.2.6a ) White microcrystals, 0.45 g, 52% yield; mp 187 197 C; 1 H NMR (DMSO d 6 300 MH): 1.35 (s, 18H), 2.13 2.23 (m, 4H), 2.28 (t, J = 7.5 Hz, 4H), 2.73 (dd, J = 13.6, 9.6 Hz, 2H), 2.91 3.03 (m, 8H), 3.10 (dd, J = 13.7, 5.3 Hz, 2H), 3.16 3.27 (m, 4H), 3.63 (s, 6H), 4.38 4.45 (m, 2H), 4.52 4.59 (m, 2H), 6.61 (t, J = 5.3 Hz, 2H), 7.12 7.27 (m, 10H), 8.09 (bs, 2H), 8.15 (d, J = 8.5 Hz, 2H), 8.57 (d, J = 7.6 Hz, 2H); 13 C NMR (DMSO d 6 75

PAGE 75

75 MHz): 28.2, 34.8, 35.2, 35.5, 36.6, 37.8, 51.2, 52.2, 54.0, 77.6, 126.2, 127.9, 129.1, 138.0, 155.3, 170.1, 170.6, 170.9, 171.1; Anal. Calcd for C 48 H 70 N 8 O 14 S 2 : C 55.05, H 6.74, N 10.70. Found: C 55.00, H 7.15, N 10.72. Boc Ala L Phe Ala L Cys OCH 3 ( 2.2.7b ) The compound was prepared according to the method for preparation of Boc Ala L Leu Gly L Cys OCH 3 (2.2.7a) White microcrystals, 0.21 g, 68% yield; mp 137 142 C; 1 H NMR (DMSO d 6 300 MHz): 0.84 0.94 (m, 1H), 1.36(br s, 9H), 2.10 2.25 (m, 2H), 2.31 ( t, J = 7.2 Hz, 2H), 2.68 2.86 (m, 3H), 2.93 3.04 (m, 3H), 3.15 3.30 (m, 2H), 3.64 (s, 3H), 4.38 4.49 (m, 2H), 6.62 (bs, 1H), 7.17 7.27 (m, 5H), 8.04 (bs, 1H), 8.12 (d, J = 8.6 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H); 13 C NMR (CD 3 OD, 75 MHz): 26.7, 28.9, 36.3, 37.1, 37.2, 38.0, 39.1, 53.2, 56.3, 56.4, 80.3, 127.9, 129.6, 130.4, 138.7, 158.3, 172.3, 173.7, 173.9; Anal. Calcd for C 24 H 36 N 4 O 7 S: C 54.95, H 6.92, N 10.68. Found: C 55.02, H 7.01, N 11.00. Boc Ala L Phe Ala L Cys( S L Cbz Ala) OCH 3 ( 2.2.8b ). The comp ound was prepared according to the method for preparation of Boc Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 ( 2.2.8a ) White microcrystals, 0.25 g, 81% yield; mp 158 162 C; 1 H NMR (DMSO d 6 300 MHz): 1.24 (d, J = 7.2 Hz, 3H), 1.35 (br s, 9H), 2.12 2.30 (m, 4H), 2.71 (dd, J = 13.6, 9.2 Hz, 1H), 2.91 3.10 (m, 4H), 3.16 3.26 (m, 3H), 3.61 (s, 3H), 4.14 4.22 (m, 1H), 4.34 4.48 (m, 2H), 5.05 (s, 2H), 6.61 (br s, 1H), 7.16 7.30 (m, 5H), 7.30 7.42 (m, 5H), 7.98 8.02 (m, 1H), 8.07 8.12 (m, 2H), 8.47 (d, J = 7.6 Hz, 1H); 13 C NMR (CD 3 OD, 75 MHz): 18.0, 28.9, 30.7, 36.2, 37.1, 37.3, 38.0, 39.1, 53.3, 53.4, 56.4, 58.4, 67.9, 80.3, 127.9, 128.9, 129.2, 129.6, 130.4, 138.2, 138.7, 158.4, 172.2, 173.8, 203.3; Anal. Calcd for C 35 H 47 N 5 O 10 S: C 57.60, H 6.49, N 9.60. Found: C 57.42, H 6.70, N 9.44.

PAGE 76

76 Ala L Phe Ala L Cys( S L Cbz Ala) OCH 3 hydrochloride ( 2.2.9b ). The compound was prepared according to the method for preparation of Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 hydrochloride (2.2.9a) White microcrystals, 0.16 g, 81% yield; mp 158 162 C; 1 H NMR ( DMSO d 6 300 MHz): 1.24 (d, J = 7.2 Hz, 3H), 2.24 2.30 (m, 2H), 2.40 (dd, J = 15.4,7.7 Hz, 2H), 2.74 (dd, J = 13.5, 9.6 Hz, 1H), 2.82 2.88 (m, 2H), 2.97 (dd, J = 13.7, 4.7 Hz, 1H), 3.08 (dd, J = 13.6 8.0 Hz, 1H), 3.18 3.29 (m, 4H), 3.62 (br s, 3H), 4.1 3 4.23 (m, 1H), 4.34 4.48 (m, 2H), 5.06 (s, 2H), 7.16 7.28 (m, 5H), 7.28 7.37 (m, 5H), 7.91 (br s, 3H), 8.10 (d, J = 7.3 Hz, 1H), 8.17 (t, J = 5.4 Hz, 1H), 8.43 (d, J = 8.5 Hz, 1H), 8.53 (d, J = 7.7 Hz, 1H); 13 C NMR (CD 3 OD, 75 MHz): 17.9, 30.6, 32.8, 36. 1, 36.9, 37.1, 38.9, 53.2, 56.5, 58.4, 67.8, 127.9, 128.8, 129.1, 129.5, 130.3, 138.1, 138.5, 158.4, 172.1, 173.8, 203.5; Anal. Calcd for C 30 H 40 ClN 5 O 8 S.H 2 O: C 52.66, H 6.19, N 10.24. Found: C 52.64, H 6.62, N 10.13. Preparations of Compounds 2.2.2b, 2.2.3b 2.2.4a, 2.2.5b, 2.2.6b, 2.2.7b, 2.2.8b and 2.2.9b Boc GABA Bt ( 2.2.4b ). A solution of N Boc GABA OH (3.23 g, 15.9 mmol) in CH 2 Cl 2 (30 mL) was added to a solution of dicyclohexylcarbodiimide (3.27 g, 15.9 mmol) and 1H benzotriazole (1.89 g, 15.9 mmol) in CH 2 Cl 2 (10 mL). The reaction mixture was stirred at room temperature for 1.5 hours. The precipitate was filtered and the solution was passed through 6.00 g of celite. The solvent was evaportated under reduced pressure and the crude mixture obtained was d issolved in EtOAc (50 mL). The organic layer was washed with saturated Na 2 CO 3 (3 x 30 mL), brine (20 mL) and dried over MgSO 4 Recrystallization from CH 2 Cl 2 :hexanes gave Boc GABA Bt. White microcrystals, 4.68 g, 97% yield; mp 108 110 C; 1 H NMR (CDCl 3 300 MHz): 1.41 (br s, 9H), 2.12 (p, J = 7.0 Hz, 2H), 3.29 3.36 (m, 2H), 3.49 (t, J = 7.2 Hz, 2H), 4.72 (br s,

PAGE 77

77 1H), 7.52 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.67 (ddd, J = 8.2, 7.2, 1.0 Hz, 1H), 8.13 (ddd, J = 8.2, 0.8, 0.8 Hz, 1H), 8.29 (ddd, J = 8.6, 1.1, 0. 8 Hz, 1H); 13 C NMR (CDCl 3 75 MHz): 25.0, 28.5, 32.9, 39.9, 79.5, 114.6, 120.3, 126.3, 130.6, 131.3, 146.3, 156.2, 172.2; Anal. Calcd for C 15 H 20 N 4 O 3 : C 59.20, H 6.62, N 18.41. Found: C 59.34, H 6.71, N 18.39. Boc GABA L Phe OH (2.2.5c) The compound was prepared according to the method for preparation of Boc Ala L Leu OH ( 2.2.5a ) White microcrystals, 1.82 g, 68% yield; mp 78 81 C; 1 H NMR (DMSO d 6 300 MHz): 1.37 (br s, 9H), 1.49 (p, J = 7.2 Hz, 2H), 1.98 2.05 (m, 2H), 2.79 2.87 (m, 3H), 3.04 (dd, J = 13.9, 5.0 Hz, 1H), 4.36 4.44 (m, 1H), 6.76 (t, J = 5.5 Hz, 1H), 7.16 7.30 (m, 5H), 8.15 (d, J = 8.2 Hz, 1H); 13 C NMR (DMSO d 6 75 MHz): 26.0, 28.4, 32.7, 36.9, 53.5, 77.7, 126.5, 128.3, 129.2, 137.8, 155.7, 172.1, 173.3; Anal. Calcd for C 18 H 26 N 2 O 5 : C 61.70, H 7.48, N 7.99. Found: C 61.32, H 7.55, N 7.59. (Boc GABA L Phe G ly L Cys OCH 3 ) 2 (2.2.6c) The compound was prepared according to the method for preparation of (Boc Ala L Leu Gly L Cys OCH 3 ) 2 ( 2.2.6c ) White microcrystals, 0.33 g, 52% yield; mp 11 0 115 C; 1 H NMR (DMSO d 6 300 MHz): 1.37 (br s, 18H), 1.41 1.52 (m, 4H), 2.02 (t, J = 7.2 Hz, 4H), 2.70 2.82 (m, 6H), 2.93 3.06 (m, 4H), 3.15 (dd, J = 13.7, 4.3 Hz, 2H), 3.65 (br s, 6H), 3.72 (dd, J = 16.5, 5.0 Hz, 2H), 3.82 (dd, J = 17.0, 5.8 Hz, 2H), 4.43 4.54 (m, 2H), 4.55 4.64 (m, 2H), 6.74 (br s, 2H), 7.14 7.32 (m, 10H), 8.12 (d, J = 8.2 Hz, 2H), 8.33 (br s, 2H), 8.40 (d, J = 7.7 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 25.7, 28.3, 32.6, 37.4, 41.6, 51.3, 52.3, 54.1, 77.4, 126.2, 128.0, 129.1, 138.0, 155.5, 169.0, 170.7, 171.7, 172.0; Anal. Calcd for C 48 H 70 N 8 O 14 S 2 : C 55.05, H 6.74, N 10.70. Found: C 55.12, H 6.46, N 10.56.

PAGE 78

78 Boc GABA L Phe G ly L Cys OCH 3 (2.2.7c ) The compound was prepared according to the method for preparation of Boc Ala L Leu Gly L Cys OCH 3 (2.2.7a ) White microcrystals, 0.27 g, 66% yield; mp 84 90 C; 1 H NMR (DMSO d 6 300 MHz): 0.88 (dd, J = 13.5, 7.1 Hz, 1H), 1.37 (br s, 9H), 1.42 1.51 (m, 2H), 2.02 (t, J = 7.4 Hz, 2H), 2.71 2.88 (m, 5H), 3.02 (dd, J = 13.5, 4.2 Hz, 1H), 3.65 (br s, 3H), 3.69 3.83 (m, 2H), 4.46 4.53 (m, 2H), 6.75 (br s, 1H), 7.15 7.26 (m, 5H), 8.15 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.38 (d, J = 5.4 Hz, 1H); 13 C NMR (DMSO d 6 75 MHz): 25.4, 25.7, 28.3, 32.6, 37.3, 41.8, 51.2, 54.2, 54.5, 77.4, 126.2, 128.0, 129.1, 138.0, 155.6, 16.9, 170.5, 171.9, 172.2; Anal. Calcd for C 24 H 36 N 4 O 7 S: C 54.95, H 6.92, N 10.68. Found: C 54.84, H 6.59, N 10.67. Boc GABA L Phe Gly L Cys( S L Cbz Ala) OCH 3 (2.2.8c). The compound was prepared according to the method for prepa ration of Boc Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 ( 22a ). White microcrystals, 0.27 g, 87% yield; mp 91 96 C; 1 H NMR (DMSO d 6 300 MHz): 1.26 (d, J = 7.2 Hz, 3H), 1.37 (br s, 9H), 1.42 1.50 (m, 2H), 2.01 (t, J = 7.4 Hz, 2H), 2.70 2.82 (m, 3H), 3.02 (dd, J = 14.0, 3.8 Hz, 1H), 3.10 (dd, J = 13.7, 7.7 Hz, 1H), 3.25 (dd, J = 12.9, 5.0 Hz, 1H), 3.63 (br s, 3H), 3.65 3.82 (m, 2H), 4.15 4.25 (m, 1H), 4.38 4.54 (m, 2H), 5.06 (br s, 2H), 6.74 (br s, 1H), 7.14 7.40 (m, 10H), 8.08 8.11 (m, 2H), 8.29 (t, J = 5.4 Hz, 1H), 8.40 (d, J = 7.5 Hz, 1H); 13 C NMR (Acetone d 6 75 MHz): 18.1, 26.9, 28.8, 33.4, 37.9, 40.3, 52.8, 56.3, 57.9, 67.1, 78.7, 127.3, 128.7, 129.2, 129.3, 130.1, 138.0, 138.8, 156.9, 157.2, 169.9, 171.3, 172.6, 174.0, 202.1; Anal. Calcd for C 35 H 47 N 5 O 10 S: C 57.60, H 6.49, N 9.60. Found: C 57.49, H 6.66, N 9.46.

PAGE 79

79 GAB A L Phe G ly L Cys( S L Cbz Ala) OCH 3 hydrochloride (2.2.9c). The compound was prepared according to the method for preparation of Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 hydrochloride ( 23a ). White microcrystals, 0.17 g, 87% yield; mp 125 131 C; 1 H NMR (CD 3 OD, 300 MHz): 1.36 (d, J = 7.2 Hz, 3H), 1.79 1.88 (m, 2H), 2.24 2.41 (m, 2H), 2.76 2.85 (m, 2H), 2.91 (dd, J = 13.7, 9.4 Hz, 1H), 3.16 3.27 (m, 2H), 3.43 (dd, J = 13.8, 5.3 Hz, 1H), 3.67 3.74 (m, 5H), 4.29 (q, J = 7.3 Hz, 1H), 4.56 4.72 (m, 2H), 5.06 5.1 6 (m, 2H), 7.18 7.36 (m, 10H); 13 C NMR (CD 3 OD, 75 MHz): 18.0, 24.3, 30.7, 33.5, 38.6, 40.3, 43.3, 53.4, 56.7, 58.4, 68.0, 128.0, 128.8, 129.2, 129.6, 130.4, 138.2, 138.6, 158.5, 171.4, 171.9, 17.3, 174.8, 203.3; Anal. Calcd for C 30 H 40 ClN 5 O 8 S.3H 2 O: C 50.0 3, H 6.44, N 9.72. Found: C 50.06, H 6.24, N 9.55. General Procedure for Long Range Acyl Migration of S (Pg aminoacyl)tetrapeptide 2.2.9a,b,c to Form Native P eptides 2.2.10, and 2.2.12 a,b The N terminusunprotected S (Pg aminoacyl)peptide 2.2.9a,b,c (0. 02 mmol) was suspended in degassed phosphate buffer (NaH 2 PO 4 /Na 2 HPO 4 ) (1 M, pH = 6.2 8.2 for 2.2.9a and 7.6 for 2.2.9b and c 9.6 mL) and acetonitrile (0.4 mL) was added dropwise until the starting material was dissolved. The mixture was subjected to microw ave irradiation 50 C, 50 W, 1 h. The reaction was allowed to cool to rt and acetonitrile was removed under reduced pressure and the residue was acidified with 2N HCl to pH = 1. The mixture was extracted with ethyl acetate (3 x 10 mL), the combined organic extracts were dried over MgSO 4 and the solvent was removed under reduced pressure. Native peptides 2.2.10, and 2.2.12a,b were subsequently isolated as disulfide dimer by semi preparative HPLC on Phenomenex Luna C18(2) columns. Cbz L Ala Ala L Leu Gly L Cys OCH 3 (2 .2.10 ) The compound was prepared from Ala L Leu Gly L Cys( S L Cbz Ala) OCH 3 hydrochloride according to general

PAGE 80

80 p procedure for long range acyl migration. Cbz L Ala Ala L Leu Gly L Cys OCH 3 was subsequently isolated as disulfide dimer by semi preparative HPLC. Colorless oil, 3.1 mg, 31% yield; 1 H NMR (DMSO d 6 300 MHz): 0.83 0.88 (m, 12H), 1.17 (d, J = 7.2 Hz, 6H), 1.45 (t, J = 7.2 Hz, 4H), 1.56 1.64 (m, 2H), 2.26 3.36 (m, 4H), 2.97 (dd, J = 13.8, 8.4 Hz, 2H), 3.13 (dd, J = 13.9, 5.1 Hz, 2H) 3.21 3.28 (m, 4H), 3.65 (br s, 6H), 3.69 3.80 (m, 4H), 3.95 4.01 (m, 2H), 4.25 (q, J = 7.4 Hz, 2H), 4.57 4.61 (m, 2H), 4.97 5.05 (m, 4H), 7.28 7.32 (m, 2H), 7.32 7.38 (m, 10H), 7.87 (t, J = 5.8 Hz, 2H), 8.09 (d, J = 7.4 Hz, 2H), 8.21 (t, J = 5.8 Hz, 2H), 8.32 (d, J = 7.7 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 18.2, 21.5, 23.0, 24.2, 35.0, 35.3, 40.5, 41.5, 50.0, 51.2, 51.3, 52.2, 65.4, 127.7, 127.8, 128.3, 137.0, 155.6, 169.1, 170.7, 170.8, 172.4, 172.5; ESI MS m/z : 1183 (M+Na). HRMSI (ESI) calcd for C 52 H 76 N 10 O 16 S 2 Na [M+Na] + 1183.4774 found 1183.4733. Cbz L Ala Ala L Phe Ala L Cys OCH 3 (2.2.12a) The compound was prepared from Ala L Phe Ala L Cys( S L Cbz Ala) OCH 3 hydrochloride according to general p procedure for long range acyl migration. Cbz L Ala Ala L Phe Ala L Cys OCH 3 was subsequently isolat ed as disulfide dimer by semi preparative HPLC. Colorless oil, 2.2 mg, 42% yield; ESI MS m/z : 1256 (M+H). HRMS (ESI) calcd for C 60 H 76 N 10 O 16 S 2 Na [M+Na] + 1279.4774 found 1279.4718. Cbz L Ala GABA L Phe Gly L Cys OCH 3 ( 2.2.12b ) The compound was prepared fro m GABA L Phe G ly L Cys( S L Cbz Ala) OCH 3 hydrochloride according to general p procedure for long range acyl migration. Cbz L Ala GABA L Phe Gly L Cys OCH 3 was subsequently isolated as disulfide dimer by semi preparative HPLC.

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81 Colorless oil, 1.9 mg, 42% yiel d; ESI MS m/z : 1256 (M+H). HRMS (ESI) calcd for C 60 H 76 N 10 O 16 S 2 Na [M+Na] + 1279.4774 found 1279.4775.

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82 CHAPTER 3 SCOPE AND MECHANISTIC ASPECTS OF S TO N LONG RANGE ACYL MIGRATION Introduction S A cyl isopeptide methods 54 55 have the major advantages of utilizing conventional amino acids and providing an isomer of the native peptide tha t can be isomerized under a variety of controlled conditions. In contrast to conventional NCL, in which two unprotected peptides are condensed by thioester mediated amide bond formation S isopeptide based strategies (Figure 3 1) require conventional chemi cal synthesis of an amide or (thio)ester linked peptide which subsequently rearranges to a native peptide bond. 56 Figure 3 1 Long range S N acyl migration to form native peptide analogs. The re sults for the long range acyl migration via 5 8 11 14 15 and 16 Reproduced in part with permission from Bol'shakov, O ; Kovacs, J ; Chahar, M ; Ha, K ; Khel ashvili, L ; Katritzky, A R. Journal of Peptide S cience 2012 18 704 70 9 C opyright 2012 European Peptide Society and John Wiley & Sons, Ltd.

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83 previous studies on the long range ligation through cyclic intermediates. 57 58 HPLC MS used to provide preliminary analys is of the formation rate of each desired product. The result indicate s that the rate of long range S to N acyl migration is strongly dependent on the size of the cyclic transition state involved in precursor peptide Studies on the rates of lactone format ion have shown a similar dependence on ring size. 59 The formation of five an d six membered lactones is favorable but i n contrast, seven and eight membered lactones are difficult to form However, cyclization becomes more favorable as the size of the lactone ring increases (Figure 3 2, 3 3 ). 59 Figure 3 2 Cyclization reaction to form lactone O ur long range ligation experiments show a similar trend Th is chapter discusses the long range acyl migration via different transition state sizes by replacing one or aminoacyl units. The purpose of this study is to identify sequence and geometry requirements that enable long range acyl migration by a study of amino acid units via 9 and 13 membered cyclic TS to further understand scope and mechanistic aspect of the S to N long range acyl migation. 60

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84 Figure 3 3 Reactivity profile for lactone formation Results and Discussion The Feasibility o f S Acyl Monoisotripeptide to Undergo S N Acyl Migration v ia a 9 Membered Cycl ic Transition State Monoisotripeptide 3.7 was prepared for the study of S N acyl migration via a 9 membered cyclic transition state as illustrated in Scheme 3 2. Boc protected alanine 3.1 was converted into the corresponding benzotriazolide 3.2 by the standard method. 61 62 L Cysteine was reacted with the Boc alanine benzotriazolide 3.2 in aqueous acetonitrile (MeCN/H 2 O, 7/3) containing 1 equiv of Et 3 N for 1 h at 20 o C to give the Boc alanyl cysteine dipeptide 3.4 (68%). Subsequent S acylation of 3.4 with Z L Ala Bt 3.5 at room temperature in the presence of KHCO 3 furnished the Boc protected S acyl monoisotripeptide 6 (70%), and Boc group deprotection of 3.6 with dioxane saturated with hydrochloric acid gas gave S acyl monoisotripeptide 3.7 as its hydrochloride salt (Figure 3 4) -4 -3 -2 -1 0 1 2 3 2 5 8 11 14 17 20 23 log k intra Ring Size

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85 Chemical ligation via the 9 membered cyclic transition state was attempted by microwave irradiation of a solution of 3.7 at a 2 mM concentration in 0.4 M NaH 2 PO 4 /Na 2 HPO 4 buffer (pH = 8.2) and acetonitrile (24:1) for 4 h at 50 C. However, HPLC MS (ESI) analysis of the reaction mixt ure ( Figure 3 4 Table 3 1) revealed that the major product 3.9 arose from intermolecular indicating favored over intramolecular attack throu gh a 9 membered ring. Ligated product 8 was also present (Table 3 1) but was formed in only 4% yield. Figure 3 4 Chemical ligation of S acyl monoisotripeptide 3.7

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86 Table 3 1 Attempted c hemical ligation of S acyl isopept ides 3.7 and 3.12 Product a and relative amounts of each product (%) b c d product characterization by HPLC MS [M+H] + found transacylation product (TA) R LM LD TA structur e ligated peptide monomer (LM) ligated peptide dimer (LD) structur e [M+H] + found 3.7 (5) 3.8 (4) 3.10 (0) 3.9 (91) 3.8 397 792 3.9 602 3.12 (1) 3.13 (5) 3.15 (77) 3.14 (17) 3.13 468 934 3.14 673 a the combined crude yield was calculated according to the following equation: combined crude yield = ([ligated peptide] + 2 x [transacyla tion product])/[starting material] b Determined by HPLC MS semiquantitative. 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 c R=recovered reactant, LM=ligation monomer, LD=ligatio n dimer, TA=transacylation d amounts are corrected for LD = 2 mmol, LM = 1 mmol Similar observation was also reported in the study of molecular machine s for peptide synthesis, in which the amino acids are preorganized in a supramolecular architecture for t he synthesis of small peptides (Figure 3 5). 63 64 Leigh and co workers reported a conceptually new approach to peptide synthesis, in which the amino acids are preorganized in a supramolecular architecture for the synt hesis of small peptides. 64 For r eactions in this molecular machine, close analogies can be drawn to natural ribosomal and nonribosomal peptide biosynthesis. Ribosomal peptide s are synthesized by trans lation of the mRNA, whereas nonribosomal machinery fo r peptide synthesis uses multi enzyme complexes as an assembly line to catalyze the peptide condensa tion in a stepwise manner. This molecular machine design can be considered a major breakthrough in the area of supramolecular chemistry that w ill open up new methods for peptide synthesis using artificial nanomachines. The design was based on the S to N long range acyl transfer via 11 14 and 17 membered cyclic TS a nd in order for the machine operate su ccessfully, a Gly Gly dipeptide spacer between the

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87 cysteine residue and the amine of the peptide elong ation site was required to avoid the slow ligation reaction via small c yclic TS. 59 Figure 3 5 A rot axane based molecular machine for the synthesis of small peptides Demonstration of S N acyl M igration in S acyl M onoisotetrapeptide via a 13 M embered Cyclic Transition S tate Coupling of N terminal amino unprotected S acyl monoisotripeptide 3.7 with Boc Ala Bt 3.2 gave 3.11. Deprotection of the Boc group in 3.11 produced S acyl monoisotetrapeptide 3.12 ( Figure 3 6 ), which was subjected to microwave irradiation at 50 o C for 4 h in NaH 2 PO 4 /Na 2 HPO 4 buffer at pH 8.2. HPLC MS (ESI) analysis of the crude liga tion mixture revealed the successful ligation of 3.15 via a 13 membered cyclic transition state. Ligation product 3.15 was the major component (82%) (molecular ion of the disulfide dimer [M+H] + m/z: 934, vs 468 for the [M+H] + molecular ion of the starting S (Z Ala)tripeptide 3.12). The HPLC MS (ESI) also confirmed that a substantial amount of 3.14 was formed by intermolecular trans acylation (Table 3 1, Figure 3 6 ). Thus the feasibility of long range acyl migration via a 13 membered cyclic transition state is

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88 confirmed favorable in complete agreement with a previous study. 55 The findings of this investigation hence offer prospects for a convergent assembly of peptides and proteins with amino acid architecture. Figure 3 6 Chemical ligation of S acyl monoisotetrapeptide 3.12 Conclusion In summary, s table, amino unprotected S acyl monoisotri and S acyl monoisotetra cy steine and/or amino acid residues undergo chemical lig ations in which the cysteine S acyl groups migrate to the N terminal amino acid s via 9 and 13 membered cyclic transition states to form the corresponding native tri and tetrapeptide analogs. The work describ es a range of novel long range S to N acyl mig rations and offers evidence that relates to the challenging problem of coupling large peptides and peptide analog fragments.

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89 Experimental General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. N MR spectra were recorded in CDCl 3 DMSO d 6 or CD 3 OD d 4 operating at 300 MHz for 1 H and 75 MHz for 13 C with TMS as an internal standard. All microwave assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer. 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 septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 sec.; PowerMa x cooling mode). The starting Boc Ala Bt 3.2 and Z L Ala Bt 3. 5, and Boc Gly Bt were prepared in 90 95% yields from corresponding N protected amino acids following previously published one step procedures P rocedures for Preparations of C ompounds 3.4, 3 .6 and 3.7 Synthesis of peptide 3. 4. N protected Bt derivative 3. 2 (2 mmol) was dissolved in acetonitrile (20 mL) was added to a solution of L cysteine (2 mmol) and Et 3 N (2 mmol) in water (10 mL) at rt and stirred for 3 hours. Mixture was then evaporated, acidified with 1N citric acid solution to pH = 2 and extracted with ethyl acetate (2x30 mL). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Compounds 3.4 was recrystallized from CH 2 Cl 2 :hexanes. Boc Ala L Cys OH ( 3. 4). White microcrystals, 68% yield, mp 94 96 oC; 1 H NMR (300 MHz, CDCl 3 4.85 (m, 1H), 3.45 3.43 (m, 2H), 3.10 3.03 (m, 2H), 2.58 2.50 (m, 2H), 1.42 (s, 9H); 13 C NMR (75 MHz, CDCl 3 )

PAGE 90

90 3.1, 156.8, 80.4, 54.0, 37.2, 36.6, 28.4, 26.6. Anal. Calcd. For C 11 H 20 N 2 O 5 S: C 45.19; H 6.90; N 9.58. Found: C 45.50; H 7.11; N 9.40. Preparation of S acyl peptides 3. 6 (0.33 g, 1 mmol) was added to a mixture of 3 .4 (1 mmol) and KHCO 3 (0.1 g, 1 mmol) in acetonitrile (20 mL). The mixture was stirred for 3 h at rt and solvent was then removed under reduced pressure. The residue was dissolved in ethyl acetate (20 mL), extracted with 2N HCl (2 x 20 mL), water (15 mL), and brine (10 mL). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Compound 3. 6 was recrystallized from CH 2 Cl 2 :hexanes. Boc Ala L Cys( S Z L Ala) OH ( 3. 6). White microcrystals, 70% yield, mp 65 69 o C; 1 H NMR (300 MHz, CD 3 OD) 7.26 7.15 (m, 5H), 4.99 ( s, 2H), 4.51 4.48 (m, 1H), 4.19 4.15 (m, 1H), 3.41 3.34 (m, 1H), 3.18 3.15 (m, 2H), 3.10 3.00 (m, 1H), 2.27 (t, J = 6.9 Hz, 2H), 1.29 (s, 9H), 1.22 (d, J = 7.2 Hz, 3H ) ; 13 C NMR (75 MHz, CD 3 OD) 203.4, 174.0, 173.2, 158.4, 138.2, 139.6, 129.2, 128.9, 80.3, 68.0, 58.5, 53.3, 50.0, 38.0, 37.1, 32.9, 30.9, 28.9; Anal. Calcd. For C 22 H 31 N 3 O 8 S: C, 53.11; H, 6.28; N, 8.45. Found: C 53.44; H 6.46; N 8.40. Procedure for Boc deprotection of peptides 3.6 to give the corresponding unprotected peptides 3.7. HCl gas was passed through a solution of peptide 3. 6 in dioxane (15 mL) for 30 minutes. The dioxane solution was concentrated under vacuum and diethyl ether (20 mL) was added. The turbid solution was left to crystallize in the freezer overnight. The solid formed was f iltered and washed with dry ethyl acetate (10 mL) and diethyl ether (10 mL) to give corresponding deprotected peptide 3. 7

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91 H Ala L Cys( S Z L Ala) OH hydrochloride (3.7). White microcrystals, 100% yield, mp 120 123 o C; 1 H NMR (300 MHz, CD 3 OD) 7.23 7.14 (m, 5H), 4.98 (s, 2H), 4.60 4.55 (m, 1H), 4.15 4.12 (m, 1H), 3.42 3.35 (m, 1H), 3.17 3.14 (m, 1H), 3.05 2.98 (m, 3H), 2.48 (t, J = 6.6 Hz, 2H), 1.20 (d, J = 7.5 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) 203.7, 173.0, 172.3, 158.6, 138.2, 129.6, 129.2, 128.8, 68.0 58.5, 53.1, 37.1, 32.7, 30.9, 17.9; Anal. Calcd. For C 17 H 24 ClN 3 O 6 S: C 47.06; H 5.58; N, 9.68. Found: C 46.69; H 5.48; N 9.69. P rocedures for Preparations of C ompounds 3.11 and 3.12 S ynthesis of S Acyl isotetrapeptides 3.11 1 mmol of 3. 2 was added to sol ution of 3. 7 (1 mmol) and DIPEA (2 mmol) in acetonitrile (20 mL). Mixture was stirred for 4 h at rt. Solvent was then evaporated and residue diluted with ethyl acetate (50 mL) followed by washing with 1N HCl. Organic layer was dried over anhydrous sodium s ulfate and evaporated. Residue was separated by column chromatography using ethyl acetate to give S acyl isotetrapeptide 3.11 Boc Ala Ala L Cys( S Z L Ala) OH ( 3.11 ). White microcrystals, 62% yield, mp 108 112 o C; 1 H NMR (300 MHz, CD 3 OD) 7.11 7.05 (m 5H), 4.89 4.82 (m, 2H), 4.42 4.38 (m, 1H), 4.08 4.05 (m, 1H), 3.50 3.37 (m, 3H), 3.31 3.18 (m, 3H), 2.99 2.91 (m, 1H), 2.23 2.09 (m, 4H), 1.18 (s, 9H), 1.12 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) 203.4, 174.0, 173.8, 173.1, 158.3, 138.1, 129.6, 1 29.1, 129.0, 128.9, 80.2, 67.9, 58.4, 53.3, 38.1, 37.4, 37.0, 36.4, 30.9, 28.9, 18.0. H Ala Ala L Cys( S Z L Ala) OH hydrochloride ( 3.12 ). The compound was prepared according to the method for preparation of H Ala L Cys( S Z L Ala) OH hydrochloride (3 .7) White microcrystals, 97% yield, mp 95 98 o C; 1 H NMR (300 MHz, CD 3 OD) 7.22 7.11 (m, 5H), 5.00 4.84 (m, 2H), 4.53 4.46 (m, 1H), 4.20 4.08 (m, 1H),

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92 3.40 3.20 (m, 4H), 3.16 2.92 (m, 6H), 2.51 2.42 (m, 2H), 2.35 2.23 (m, 2H), 1.19 (t, J = 7.4 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) 203.5, 173.8, 173.1, 172.3, 158.4, 138.1, 129.6, 129.1, 129.0, 128.8, 67.9, 58.4, 53.2, 48.0, 37.4, 37.0, 36.3, 33.1, 30.9; HRMS (ESI) calcd. for [C 20 H 2 9 N 4 O 7 S] + m/z 469.1751, found 459.1774. General Procedure for Chemical L igation of S Acyl P eptides 3.7 and 3.12 The respective S acyl peptide hydrochloride 3.7 or 3.12 ( 0.05 mmol) was suspended in degassed phosphate buffer (NaH 2 PO 4 /Na 2 HPO 4 ) (0.4 M, pH 7.8, 7 mL) and acetonitrile (~1 mL) was added dropwise until the starting material was dissolved. The mixture was subjected to microwave irradiation (50 C, 50 W, 1 h) under argon. The reaction was allowed to cool to room temperature, acetonitrile was removed under reduced pressure and the residue was acidified with 2N HCl to pH = 1. The mi xture was extracted with ethyl acetate (3 x 20 mL), the combined organic extracts were dried over MgSO 4 and the solvent was removed under reduced pressure. The ligation mixture was weighed and then a solution in methanol (1 mg/mL) was analyzed by HPLC MS. Ligation products were subsequently purified by semi preparative HPLC and characterized by an alytical HPLC and HRMS analysis. Products were characterized as dimer ic sulfides

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93 CHAPTER 4 LIGATION AT HYDROXYPROLINE SITE VIA SALICYLALDEHYDE CAPTURE AND IMINIUM INDUCED REARRANGEMENT Introduction NCL involves the reaction between an unprotected peptidyl C terminal thioester with another peptide bearing an N terminal cysteine. Following rate determining trans gh a five membered tetrahedral intermediate leads to product. 65 Currently, there is much interest in the development of chemoselective l igations at a hydroxyproline site to access l arger peptide s for pharmacological targets. Proline site ligation in general is among the most challenging, as evident by its possessing the longest reaction times as well as the paucity of current synthetic protocols. The unique cyclic struc ture of a prol yl residue orientates the N carbonyl on oxygen in close proximity to the thioester carbonyl C atom, 66 67 and consequent n increases electron density and reduces electrophilicity of thioester carb onyl (Figure 4 1 and 4 2) 68 69 Figure 4 1. The unique challenges of proline ligation arise from an n electron density.

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94 Figure 4 2 The n C antibond orbital Incidentally, this same effect has been postulated to confer the unique structural rigidity that is imp licated in the polyproline triple helices 69,70 of collagen and the PrAMPs. A way to realize derivatized proline ligations was recently advanced by Danishefsky et al. using thio and selenoprolines 71,72 As mentioned previously, NCL is predicated on the bifunctional 1,2 mercaptoamine group of cysteine, which is present in the thioproline. This worked showed that ligation at a derivatized proline site was achievable. However, this advancement in proline site ligation has limited applications : thio proline is not a naturally occurring amino acid ; it requires a thiol installation; and later, a dethylization involving metals c atalysis, which has drawbacks. We were attracted by the imine induced proximity acyl transfer approach. Such a strategy was originally introduced by Kemp 10 and fully developed by Tam to ligate a C terminal glycolaldehyde peptide with another peptide contai ning a Cys, Thr, or Ser residue at the N terminus to furnish a coupled product with a pseudoproline structure (thiazolidine or oxazolidine) at the ligation site. 73 Taken together, we proposed an imine induced Figure 4 3 ).

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95 Figure 4 3 (A) Schemes of the previous advanced proline ligation strategy. Our re action represented in ( B ) exhi biting the similarities in strategy In this study (F igure 4 2A ), salicaldehyde was installed at the phenolic position of a mono or dipeptide using conventional EDCI/DMAP coup ling techniques, which furnished an electrophilic aldehyde group that is receptiv e amine capture of 4 hydroxyproline. An iminium species was type mechanism. Owing to the second functional group of 4 hydroxyproline, the compound rearranges chemoselective ly because the iminium formed induce s the nucle ophilic attack of the 4 hydroxy group forming a stable bridged bicyclic intermediate. This

PAGE 96

96 removed charges from nitrogen and restores its nucleophilicity. The N terminal carboxyl group is brought into proximity by the phenolic attachment of sali cylaldehyde, which undergoes an O N trans esterification with the now amine of 4 hydroxyproline connected to SAE as an aminal. The aldehyde c an then reform at the 4 hydroxy position, and this can then be acidolyzed, yielding the nat ive peptide bond at the hydroxy position ( Figure 4 4). Figure 4 4. Scheme for hydroxyproline site ligation. After inst allation of O salicylaldehyde, the proline is captured forming an iminium, which then rearranges ultimately allowing for the native peptide bond to be formed

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97 Results and Discussion Primary S tudy In order to dev elop a practical chemoselective Hyp peptide ligation based on the imine induced strategy two questions must be addressed: (i ) whether the imine capture step or the acyl transfer step can b e accelerated (it is not known which step is t he rate determining step) and (ii ) whether the formed pseudo bridged proline moiety can be readily transformed into natural peptide linkages. The second question is more challenging and critical because the rem oval conditions should ideally be compatible with ot her functionalities in peptides. Among many candidates, we tentatively identified the salicylaldehyde ester as a potential functional donor at C terminal. We hypothesized that, when the benzaldehyde est er is used, either the hydroxy attack to the imine or O to N acyl transfer would proceed faster. However, as the penalty, the acyl transf er has to progress through a dis favored long range acyl transfer via a 6 membered TS On the other hand, the formed N,O b enzylidene acetal group is expected to be readily removed under weak acidic conditions. The hypothesis was first tested with the commercially available salicylaldehyde. As a model study, we prepared Fmoc Ala salicylaldehyde ester 4.3a by coupling Fmoc pro tected alanine 4.1a with a salicylaldehyde 4.2 at the C terminus using coupling reagent EDCI ( Figure 4 5 ). Installing salicylaldehyde (SAE) phenolically on the commercially procured Fmoc protected amino acid was ca r ried out in anhydrous DCM (dried refluxed under calcium hydride) under N 2 gas pressure The reaction mixture was left overnight to ensure reac tion completion, with yields >77%.

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98 Figure 4 5 Synthesis of salicylaldehyde ester. Compound 4.3a was purified by recrysta llization from DCM/hexanes and fully characterized by 1 H and 13 C NMR spectroscopy. 1 H NMR spectra indicated the formation of the desired ligation product salicylaldehyde ester with appearance of newly formed adehyde proton signals. Having Fm oc Ala SAE in h and we next studied the ligation of 4.3a with Hyp OMe (4.4a). The ligation step was achieved by introducing the hydroxyproline to a solution of the O salicylaldehyde ester and pyridine/acetic acid ( Figure 4 6 ). After 5 hours, the solvent was removed, and i n the second step, the aminal is treated with a mixture of TFA/H 2 O/i Pr 3 SiH yielding the native peptide bond at the hydroxyproline ligation site. The products were purified by recrystallization and preparative HPLC. Figur e 4 6 Model Hyp Ligation reaction Proton spectra of the ligation product 4.5a revealed the major component to be the expected ligation product 3a with the absence of the aldehyde peak around 10 ppm. Purification of 4.5a by semipreparative HPLC allowed is olation of 91% of pure proline containing dipeptide 4.5a (Table 4.1 Entry 1 ).

PAGE 99

99 Table 4 1. Scope of the r eaction between Hyp peptides and salicylaldehyde e sters Entr y Peptide at C side Ligation Product (Yield [%]) HRMS [M+Na] + found 1 Ala Fmoc Ala Hyp OMe (4.5a) 91 461.1677 2 Val Fmoc Val Hyp OMe (4.5b) 85 481.2623 3 L e u Fmoc Leu Hyp Ome (4.5c) 88 503.2146 4 Val Fmoc Val Phe Hyp OMe (4.5d) 86 636.2685 5 Phe Fmoc Ala Phe Hyp OMe (4.5e) 91 608.2372 6 Leu Fmoc Leu Phe Hyp OMe (4.5f) 85 650.2837 7 Ala Fm oc Leu Ala Hyp OMe (4.5g) 92 574.2524 8 Phe Fmoc Gly Phe Hyp OMe (4.5h) 78 594.2211 9 Phe Fmoc Val Phe Hyp Leu OMe (4.5i) 81 739.4247 10 Phe Fmoc Val Phe Hyp B Ala Phe Leu OMe (4.5j) 88 967.4611 Mechani stic Study of Formation of the Aminal Intermediat e The key steps in formation of the native peptide bond in the Hyp ligation are the formation of the bridged bicyclic aminal and the phenolic ester has been known to directly condense with amines to afford amides. To rule out this direct condensation poss ibility and evaluate the chemoselectivity, two experiments were carried out to confirm the formation of the aminal intermediate. A ccording to our hypothesized mechanism, a stable bridged bicyclic aminal should be formed ( Figure 4 7 ). There general scheme f or these studies is shown below.

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100 Figure 4 7 Formation of the aminal intermediate. We attempted to isolate this intermediate via a reaction of reactants in dry DCM for 3 hours before adding a catalytic amount of p toluenes ulfonic acid, and then heated under reflux for at 45 C additional 60 h. But formation of the bicyclic aminal intermediate was not observed Next a study was attempted which produced the aminal. A solution of salicylaldehyde Fmoc Ala ester (4.3a), Hyp OMe (4.4a), and a catalytic amount of p toluenesulfonic acid combined in dry toluene (70 80 mL) was heated. The formatio n of the aminal was observed by HPLC MS analysis HPLC MS (ESI) analysis of the crude reaction mixture showed the presence of the MW expecte d with the aminal compound to provide evidence the presence of the intermediate The aminal 4.6 was latter treated with a mixture of TFA/H 2 O/i Pr 3 SiH leading to formation of the native peptide 4.5a. The results of these studies suggested that the native am ide bond proceeds though an aminal intermediate following the capture/rearrangement process. S alicylaldehyde Iminium I nduced Ligation of D ipeptide via H ydro xyproline Capture R earrangement We next explored the scope of the coupling reaction between N termin al Hyp containg peptides and the C terminal salicylaldehyde ester of hindered branched amino branched amino acids when used at the C terminus generally reduce the rate of peptide coupling step dramatically; thus, mos t known ligation methods require a prolonged time for completion at these amino acid sites or are limited to less hindered amino acids at the C terminal site. 39 However, these

PAGE 101

101 amino acids possessing a salicylaldehyde ester at the C terminus react with Hyp derived peptides resulted in >70% conversion after 30 min and completion within 5 to 8 h (Table 4.1 Figure 4 8 ). Treatment with (TFA/H2O/i Pr3SiH) gave rise to peptides with natural peptide bonds at the ligation sit e (Table 4.1). The products were isolated in diastereomerically pure form, and the epimerized diastereomer were not detected by LCMS and NMR. Figure 4 8. Hyp Ligati o n for synthesis of native peptides To demonstrate the feasibility of this ligation strategy in peptide segment ligation, a two step sequential ligation was carried out between the O salicylaldehyde ester dipeptide 4.3d and N terminal Hyp derived peptide 4.4c possessing ( Table 4 1, entry 10 ). With only one p urification, the desired hexapeptide 4.5j with a natural peptide bond (Phe Hyp) at the ligation site was obtained in 88% yield. It is noteworthy that peptide 4.5j contain s Hyp which possesses a hydroxy group at the fourth position. Hyp can be modified to a ccess functionalized proline residues that have diverse applications (Figure 4 9 ) 74

PAGE 102

102 Figure 4 9 Hyp ligation to synthesis a hexapeptide Conclusion S alicylaldehyde capture iminium induced rearrangement, chemoselective ligation at the hydroxyproline site was demonstrated by forming a series oligopeptides. In t his study, salicaldehyde was installed at the phenolic position of a mono or dipeptide using conventional coupling techniques, which furnishes an electrophilic aldehyde group that amine capture of 4 hydroxyproline. An iminium species is thereby formed Owing to the second functional group of 4 hydroxyproline, the compound rearranges chemoselective ly because iminium intermediate induces the nucleophilic attack of the 4 hydroxy group forming a stable bridged bicyclic intermediate. This removed the charge on the nitrogen and restores its nucleophilicity. The N terminal carboxyl group is brought into proximity by the phenolic attachment of salicylaldehyde, which undergoes an O to N trans esterification with the now amine of 4 hydroxyproline connected to SAE as an aminal. The aldehyde can then reform at the 4 hydroxyl position, and this can then be acidolyzed, yielding the native peptide bond at the N terminal position.

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103 This study represents the first repo rted ligation at the hydroxyproline site by employing the above described chemoselective capture/rearrangement methodology. Furthermore, the relatively high yields suggest this methodology is worthy of further study. Our novel methodologies in combination with convergent s ynthesis schemes should provide the key elements for the total chemical synthesis of natural or fully unnatural linear and cyclic peptides containing proline derivatives with yet to be discovered properties. Experimental General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 acetone d 6 or CD 3 OD using a 300 or 500 MHz spectrometer (with TMS as an internal standard). The following abbreviat ions are used to describe spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet, dd = doublet of doublets, ddd = doublet of doublets of doublets, and dt = doublet of triplets. HPLC MS analyses were perfo rmed on a reverse phase gradient using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; mass spectrometry was done with electrospray ionization (ESI), matrix assisted laser desorption/ionization time of flight (MALDI TOF) or a tmosphe ric pressure chemical ionization ( APCI ). Ether refers to diethyl ether. General Preparations for Salicylaldehyde Ester A stirred suspension of protected amino acid 1 (1 equiv.) and 4 dimethylaminopyridine (DMAP) (15% mole) in dry Dichloromethane (DCM) (100 ml/0.4 g of the protected peptide substrate was treated with 1 Ethyl 3 (3

PAGE 104

104 dimethylaminopropyl)carbodiimide (EDCI) (1.3 equiv.). After cooling at 0 o C for 10 minutes, salicylaldehyde (SAE) (1.1 equiv.) was added to the reaction. After 24 h stirring at roo m temperature the reaction mixture was diluted with DCM (150 ml). The organic layer was then washed successively with brine (3 x 250ml) and HCl (2N) (3 x 250ml). The organic layer was dried over magnesium sulfate, filtered, and evaporated under reduced pr essure resulting in the SAE coupled, protected amino acid 3a Fmoc L Ala SAE (4.3a ) 1.03 g, 2.47 mmol, 77% yield, sticky orange oil. 1 H NMR (300 MHz, Chloroform d 7.69 7.53 (m, 3H), 7.48 7.35 (m, 3H), 7.34 7.24 (m, 2H), 7.20 (d, J = 7.9 Hz, 1H), 5.48 (d, J = 7.7 Hz, 1H), 4.72 (p, J = 7.4 Hz, 1H), 4.50 4.33 (m, 2H), 4.23 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H). 13 C NMR (75 MHz, Chloroform d 155.9, 143.7, 135.4, 131.8, 127.7, 127.1, 126.8, 125.1, 123.3, 120.0, 67.2, 50.0, 47.1, 18.5, 18.1. Fmoc L Val SAE (4.3b), 0.35 g, 0.57 mmol; 77% yield White microcrystals,. mp 74.4 78.9 C. 1 H NMR (300 MHz, Chloroform d 9.99 (br s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.1 Hz, 2H), 7.36 (p, J = 7.5 Hz, 3H), 7.43 7.29 (m, 3H), 7.31 7.22 (m, 2H), 7.22 7.12 (m, 1H), 5.62 (d, J = 8.3 Hz, 1H), 4.69 4.56 (m, 1H), 4.44 (d, J = 7.1 Hz, 2H), 4.23 (t, J = 4.7 Hz, 1H), 2.51 2.33 (m, 1H), 1.11 (d, J = 6.1 Hz, 3H), 1.06 (d, J = 6.5 Hz, 2H). 13 C NMR (75 MHz, Chloroform d 155.0, 144.0, 141.4, 127.8, 127.2, 127.2, 125.3, 125.3, 120.1, 73.6, 70.3, 67.3, 58.0, 57.8, 55.8, 55.5, 52.4, 47.2, 43.2, 37.7, 31.3, 19.7, 19.3, 17.7, 14.7. Fmoc L Leu SAE (4.3c), 0.28 g, 0.62 mmol; Sticky oil. 81% yield. 1 H NMR (300 MHz, Chloroform d J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 2H),

PAGE 105

105 7.61 (d, J = 7.5 Hz, 3H), 7.40 (q, J = 7.2 Hz, 3H), 7.29 (t, J = 7.5 Hz, 2H), 7.20 (d, J = 8.2 Hz, 1H), 5.41 (d, J = 8.9 Hz, 1H), 4.66 (m, 1H), 4.46 (d, J = 7.0 Hz 2H), 4.25 (t, J = 7.0 Hz, 1H), 2.22 2.06 (m, 1H), 1.71 1.51 (m, 1H), 1.42 1.21 (m, 1H), 1.11 (d, J = 6.8 Hz, 3H), 1.01 (t, J = 8.9 Hz, 3H). 13 C NMR (75 MHz, Chloroform d 171.7, 156.4, 151.5, 143.8, 141.5, 135.5, 131.4, 127.9, 127.2 126.9, 125.2, 123.5, 120.1, 67.3, 53.0, 47.3, 41.1, 25.1, 23.2, 21.8. Fmoc Val P he SAE (4.5d) 0. 48 g, 0.81 mmol; 82 % yield White microcrystals, mp = 155.5 160.0 C. 1 H NMR (300 MHz, Chloroform d 7.72 (m, 2H), 7.65 7.49 (m, 4H) 7.39 (dd, J = 8.0, 4.7 Hz, 2H), 7.34 7.10 (m, 6H), 7.09 6.90 (m, 3 H), 5.66 5.32 (m, 1H), 4.82 (dq, J = 12.4, 6.4 Hz, 1 H), 4.37 (ddd, J = 39.7, 16.7 7.3 Hz, 2H), 4.26 3.97 (m, 1 H), 3.42 3.13 (m, 1H), 3.12 2.85 (m, 1H), 2.13 1.92 (m, 1H), 1. 17 0.55 (m, 6 H). 13 C NMR (75 MHz, Chloroform d 171.51, 156.77, 143.92, 141.49, 136.12, 135.82, 135.57, 131.95, 129.51, 129.03, 128.69, 128.00, 127.32, 126.95 125.23, 123.35, 120.23, 67.59, 60.53, 53.44, 47.31, 37.44, 31.25, 19.30, 18. 18. Fmoc Ala Phe SAE (4.5e) 0. 52 g, 0.92 mmol; 91 % yield White microcrystals, mp = 95.7 100.2 C. 1 H NMR (300 MHz, Chloroform d 9.52 (m, 1H), 7.89 7.62 (m, 3H), 7.60 7.49 (m, 4H), 7.44 7.33 (m, 3H), 7.33 7.16 (m, 4H), 7.09 6.96 (m, 3 H), 5.85 5.47 (m, 1H), 4.55 4.25 (m, 3H), 4.20 3.98 (m, 1H), 3.41 2.99 (m, 2H), 1.40 1.26 (m, 3H). 13 C NMR (126 MHz, cd 3 145.32, 142.69, 138.22, 137.89, 130.89, 130.71, 130.50, 129.54, 128.91, 128.30, 127.92, 126.35, 121.04, 70.84, 59.63, 52.86, 32.88, 23.83, 14.57.

PAGE 106

106 Fmoc Leu Phe SAE (4.5f) 2.18 g, 3.60 mmol, 81% yield; Sticky Oil. 1 H NMR (300 MHz, Chloroform d 7.70 (m, 2H), 7.62 7.48 (m, 5H), 7.37 (dd, J = 7.8, 5.2 Hz, 2H), 7.33 7.16 (m, 4H), 7.08 6.93 (m, 4H), 5.32 5.13 (m, 1H), 5.18 5.02 (m, 1H), 4.45 4.38 (m, 1H), 4.27 4.16 (m, 3H), 4.24 4.14 (m, 1H), 3.40 3.29 (m, 1H), 3.29 3.18 (m, 1H), 2.04 (s, 1H), 1.76 1.30 (m, 2H), 1.19 1.07 (m, 1H), 1.02 0.89 (m, 3H), 0 .89 0.64 (m, 3H). 13 C NMR (75 MHz, Chloroform d 174.2, 169.9, 143.9, 143.8, 137.2, 135.4, 133.9, 130.4, 129.9, 128.9, 128.0, 127.5, 126.8, 125.3, 123.3, 120.1, 117.8, 70.2, 60.6, 53.8, 47.3, 41.5, 37.7, 29.9, 24.7. Fmoc Leu Ala SAE (4.5g) 0.45 g, 0. 85 mmol; 78 % yield White microcrystals, mp = 119.8 121.7 C. 1 H NMR (300 MHz, Chloroform d J = 7.4, 4.1 Hz, 2H), 7.61 7.47 (m, 3H), 7.47 7.34 (m, 2H), 7.34 7.21 (m, 5H), 5.37 5.08 (m, 1H), 4.59 4.31 (m, 3H), 4.30 4.04 (m, 4H), 1.81 1.45 (m, 9H), 1.16 0. 58 (m, 6H). 13 C NMR (75 MHz, Chloroform d 141.45, 135.62, 132.03, 127.92, 127.25, 126.96, 125.21, 123.50, 120.16, 67.32, 53.57, 48.64, 47.28, 41.35, 24.80, 23.09, 22.19, 17.68. Fmoc Gly Phe SAE (4.5h) 0. 27 g, 0. 49 mmol; 85 % yield White microcrystals, mp = 90.7 95.2 C. 1 H NMR (300 MHz, Chloroform d J = 10.8 Hz, 1 H), 7.76 (d, J = 7.6 Hz, 2H), 7.67 7.51 ( m 4H), 7.46 7.14 (m, 8 H), 7.15 6.84 (m, 3H), 4.55 4.30 (m, 2H), 4.22 (t, J = 7.0 Hz, 2 H), 4.03 3.57 (m, 3H), 3.31 (dd, J = 13.0, 6.7 Hz, 1 H). 13 C NMR (75 MHz, Chloroform d 141.43, 137.18, 135.51, 133.92, 130.21, 129.48, 128.98, 128.71, 127.94, 127.28, 125.24, 123.35, 120.17, 117.78, 115.81, 69.68, 67 .51, 54.00, 47.18, 34.19.

PAGE 107

107 Synthesis of Aminal, Mechanistic Study A solution of salicylaldehyde Fmoc Ala ester 415.14 mg (1 m Mol), 145.07 mg Hyp OMe (1 m Mol ) and a catalytic amount of p toluenesulfonic acid (14 l, 0.1 m Mol, 10 mol%) where combined in d ry toluene ( 60 mL) was heated under reflux at a temperature of 100 125 C for 3 hours. The solution mixture was evaporated to 5 mL, replaced with 100 mL of DCM, and washed successively with NaHCO 3 water, and brine solution, then dried over MgSO 4 and evapo rated to yield final crude product The crude product was then directly subjected to HPLC MS analysis. Mass Spectrometry: ThermoFinnigan (San Jose, CA) LCQ Classic with electrospray ionization (ESI ). ESI: sheath gas(N2) = 65; aux gas(N2) = 5; heated capil lary temperature = 250 o C (+)ESI: spray voltage = 3.3 kV; heated capillary voltage = 12.5 V; tube lens offset = 0 V HPLC: Agilent (Palo Alto, CA) 1100 series system consisting of G1322A degasser and G1312A binary pump Column : Hypurity C8 (5um; 2.1 x 100 mm + guard col) Mobile Phases #1: A = Water + 0.2% acetic acid + 2.0 mM NH 4 OAc B = methanol + 0.2% acetic acid + 2.0 mM NH 4 OAc Compound 4.6, 2.5 uL of a 1/10 th dilution via C8 HPLC/254 nm UV/(+)ESI MSn. [M+H] + MW 542 isomers were detected: There were t wo m/z 543 ion peaks MW 542, RT 34.44 min. This isomer produced m/z 543 (top) which was dissociated t o m/z 265, 220, and 178 G eneral P rocedure Hydroxyproline Ligation 4.5a j The hydroxyproline segment 4.4 (1.1 equiv.) and the O salicylaldehyde ester segm ent 4.3 (1.0 equiv.) were dissolved in pyridine/acetic acid (1:1 mole/mole) at a concentration of 0.05 M. The reaction was stirred at room temperature. The reaction was monitored using TCL After the completion of the reaction (within 5 hou rs), the

PAGE 108

108 solvent was removed by reduced pressure the residue obtained was treated with TFA/H2O/i Pr3SiH (94/5/1, v/v/v) for 5 to 10 min to give the coupled product with a natural peptide bond at the ligation site. The solvent could be blown off by a stream of air for puri fication. The products were purified by HPLC or recrystallization from DCM/hexanes. Fmoc Ala Hyp OMe ( 4.5a ) 1.05 g, 2.25 mmol; 91% yield, sticky oil. 1 H NMR (500 MHz, DMSO d 6 J = 7.5 Hz, 2H), 7.72 (t, J = 6.2 Hz, 2H), 7.62 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 4.40 4.17 (m, 4H), 4.35 (d, J = 8.0 Hz, 1H), 4.33 4.27 (m, 2H), 4.26 4.17 (m, 2H), 2.56 2.47 (m, 1H), 4.00 (m, 1H), 3.67 (br s, 3H), 3.33 (br s, 2H), 2.10 (dd, J = 12.7, 8.8 Hz, 1H), 1.89 (ddd, J = 13.0, 8.4, 4.7 Hz, 1H), 1.24 (m, 3H). 13 C NMR (126 MHz, CDCl 3 143.9, 141.4, 127.9, 127.3, 125.3, 120.1, 70.5, 67.4, 58.0, 55.4, 52.6, 49.7, 48.5, 47.3, 37.6, 29.9, 18.6, 18.0. Fmoc Val Hyp OMe ( 4.5b ) 0.33 g, 0.67 mmol. White microcrystals mp 107.2 115.6 C 85% yield. 1 H NMR (500 MH z, Chloroform d 7.68 (m, 2H), 7.63 7.50 (m, 2H), 7.44 7.35 (m, 2H), 7.35 7.27 (m, 2H), 5.54 (d, J = 8.9 Hz, 0H), 5.45 5.25 (m, 1H), 4.75 4.59 (m, 1H), 4.58 4.48 (m, 1H), 4.46 4.28 (m, 2H), 4.26 4.12 (m, 1H), 3.75 (br s, 3H), 3.33 (p, J = 6.9 Hz, 1H), 2.75 (d, J = 13.9 Hz, 1H), 2.47 2.13 (m, 1H), 2.11 1.98 (m, 1H), 1.70 (br s, 1H), 1.37 1.15 (m, 1H), 1.12 1.01 (m, 2H), 1.01 0.86 (m, 3H). 13 C NMR (75 MHz, CDCl 3 143.9, 141.5, 135.5, 131. 0, 128.2, 127.9, 127.3, 126.9, 125.2, 123.4, 67.4, 59.6, 47.4, 31.1, 19.6, 17.8.

PAGE 109

109 Fmoc Leu HYPOMe ( 4.5c ) 0.36 g, 0.71 mmol. Sticky oil; 88% yield. 1 H NMR (300 MHz, Methanol d 4 7.69 (m, 2H), 7.68 7.50 (m, 2H), 7.43 7.14 (m, 4H), 4.59 4.42 (m, 1H), 4.41 4.24 (m, 2H), 4.23 4.04 (m, 2H), 3.89 3.76 (m, 1H), 3.75 3.63 (m, 2H), 2.44 2.19 (m, 1H), 2.19 1.91 (m, 1H), 1.84 1.67 (m, 1H), 1.63 1.44 (m, 2H), 1.00 0.91 (m, 3H), 0.90 0.63 (m, 3H). 13 C NMR (75 MHz, Methanol d 4 ) 74.2, 173.9, 173.6, 158.7, 145.6, 145.5, 145.3, 145.2, 142.7, 128.9, 128.3, 128.2, 126.4, 121.2, 121.0, 75.4, 71.2, 68.3, 68.1, 59.5, 59.4, 56.4, 54.2, 53.9, 53.0, 52.6, 49.1, 41.6, 41.4, 38.4, 35.6, 26.0, 25.8, 23.7, 23.6, 22.4, 21.9. Fmoc Val Phe Hyp OMe (4.5d) 0. 120 m g, 0. 19 mmol; 88 % yield White microcrystals, mp = 181.1 181.5 C. 1 H NMR (300 MHz, Chloroform d 7.73 (m, 2H), 7.65 7.52 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2 H), 7.28 7.08 (m, 5H), 5.54 5.29 (m, 1H), 5.0 0 4.80 (m, 1H), 4.69 4.26 (m, 4 H), 4.26 3.91 (m, 2H), 3.86 3.60 (m, 3H), 3.33 2.79 (m, 3H), 2.33 1.81 (m, 3 H), 1.16 0.47 (m, 6H). 13 C NMR (75 MHz, Chloroform d 141.47, 136.25, 129.82, 129.53, 128.82, 128.70, 127.91, 127.28, 125.30, 120.16, 70.30, 67.32, 58.00, 55.52, 52.56, 47.34, 38.46, 31.78, 22.85, 19.34, 17.60, 14.32. Fmoc Ala Phe Hyp OMe (4.5e) 0. 62 m g, 0. 1 mmol; 85 % yield Sticky oil. 1 H NMR (300 MHz, Chloroform d J = 7.3 Hz, 2H), 7. 64 7.51 (m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.36 7.29 (m, 2H), 7.29 7.07 (m, 4H), 5.45 (t, J = 8.4 Hz, 1H), 5.06 4.62 (m, 1H), 4.47 4.26 (m, 2H), 4.26 4.13 (m, 2H), 3.82 3.53 (m, 3H), 3.35 2.82 (m, 4H), 1.54 1.23 (m, 3H). 13 C NMR (75 MHz, Methanol d 4 156.85, 144.01, 141.37, 136.57, 129.18, 128.22, 127.59, 126.99, 126.61, 125.04,

PAGE 110

110 119.73, 69.66, 66.85, 58.35, 55.00, 51.55, 37.10, 31.56, 28.97, 22.51, 17.00, 13.27. MS (ESI) ( m/z ): [M+Na ] + calcd for C 4 3 H 5 35 N 3 O 7 Na 608.24, found 608.2. Fmoc Leu Phe Hyp(OMe) ( 4.5f ) 0. 55 m g, 0. 09 mmol; 85 % yield Sticky oil 1 H NMR (300 MHz, Methanol d 4 J = 7.4 Hz, 2H), 7.70 7.59 (m, 2H), 7.40 7.35 (m, 4H), 7.20 (m, 5H), 4.51 4.40 (d, J = 6.4 Hz, 3H), 4.17 4.29 (m, 1H), 4.18 (dd J = 6.9, 6.9 Hz, 1H), 4.13 4.03 (m, 1H), 3.67 (br s, 3H), 3.33 3.29 (m, 1H), 3.08 (m, 1H), 2.96 2.75 (m, 1H), 2.25 2.06 (m, 1H), 2.03 1.88 (m, 1H), 1.74 1.52 (m, 1H), 1.51 1.39 (m, 1H), 1.37 1.17 (m, 2H), 1.00 0.88 (m, 3H), 0.88 0.75 (m, 3H). 13 C NMR (75 MHz, Methanol d 4 128.9, 128.3, 128.0, 126.4, 121.1, 71.0, 68.0, 59.7, 56.3, 55.05, 53.9, 52.9, 49.2, 42.2, 38.9, 38.5, 25.9, 23.6, 22.0. Fmoc Leu Ala Hyp OMe (4.5g) 0. 28 m g, 0. 05 mmol; 77 % yield White microcrystals, mp = 106.3 126.5 C. 1 H NMR (300 MHz, Chloroform d 7.68 (m, 2H), 7.57 (dd, J = 7.4, 4.5 Hz, 2H), 7.43 7.33 (m, 2H), 7.32 7.23 (m, 2H), 5.77 5.44 (m, 1H), 4.83 4.57 (m, 1H), 4.55 4.09 (m, 5H), 3.94 3.48 (m, 4H), 2.39 2.18 (m, 1H), 2. 07 1.84 (m, 1H), 1.73 1.41 (m, 4H), 1.30 (t, J = 12.8, 6.9 Hz, 3H), 1.05 0.71 (m, 6H). 13 C NMR (75 MHz, Methanol d 4 145.28, 142.73, 128.92, 128.29, 126.33, 121.05, 71.09, 68.02, 59.46, 56.19, 54.78, 52.94, 48.12, 42 .16, 38.41, 25.98, 23.67, 21.97, 17.62, 17.19. HRMS (ESI) ( m/z ): [M+Na] + calcd for C 30 H 37 N 3 O 7 Na, 574.2534, found 574.2524. Fmoc Gly Phe Hyp OMe (4.5h) 0. 75 m 0.92 mmol; 78 % yield White microcrystals, mp = 105.7 108.5 C. 1 H NMR (300 MHz, Methanol d 4 ) J = 7.8 Hz, 2H), 7.68 (d, J = 6.6 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.34 (dd, J = 7.4, 1.6 Hz,

PAGE 111

111 2 H), 7.29 7.15 (m, 5H), 4.71 (dd, J = 7.9, 5.3 Hz, 1 H), 4.55 4.32 (m, 3H), 4.25 (d, J = 7.0 Hz, 1H), 3.87 3.63 (m, 5 H), 3.26 2.84 (m, 3H), 2. 35 1.72 (m, 2 H). 13 C NMR (75 MHz, Methanol d 4 129.58, 128.91, 128.30, 127.99, 126.35, 121.04, 70.69, 68.34, 59.67, 56.11, 55.00, 53.71, 52.88, 44.85, 43.66, 38.47. HRMS (ESI) ( m/z ): [M+Na ] + calcd for C 32 H 33 N 3 O 7 Na, 594.2211, found 594.2233. Fmoc Val Phe Hyp Leu OMe (4.5i) 4.5e) 0. 52 g, 71.5 mol; 81 % yield Sticky oil 1 H NMR (300 MHz, Methanol d 4 7.73 (m, 2H), 7.65 (t, J = 6.8 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.34 7.24 (m, 4H ), 7.16 7.07 (m, 3H), 4.53 4.25 (m, 3H), 4.25 4.16 (m, 1H), 3.87 3.75 (m, 2H), 3.75 3.51 (m, 3H), 3.25 3.11 (m, 1H), 3.05 2.76 (m, 1H), 2.36 2.10 (m, 1H), 2.00 1.84 (m, 7H), 1.79 1.66 (m, 1H), 1.60 (dd, J = 14.1, 7.1 Hz, 1H), 1.19 0.5 4 (m, 12H). 13 C NMR (75 MHz, DMSO d 6 172.47, 170.33, 170.26, 156.03, 154.23, 143.92, 143.76, 140.70, 129.43, 129.32, 127.62, 127.04, 125.35, 120.06, 69.91, 67.74, 65.69, 57.91, 51.78, 50.28, 46.74, 30.15, 27.67, 27.56, 24.14, 22.78, 21.34, 21.19, 19.34, 18.85, 18.16. HRMS (ESI) ( m /z ): [M+H ] + calcd for C 41 H 5 1 N 4 O 8 727.3701, found 727.3877 Fmoc Val Phe Hyp B Ala Phe Leu OMe (4.5j) 0. 12 m g, 12.4 mol; 88 % yield Sticky oil 1 H NMR (300 MHz, Methanol d 4 J = 7.5 Hz, 2H), 7.65 (t, J = 6.9 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7. 33 7.23 (m, 6H), 7.23 6.97 (m, 6H), 4.71 4.47 (m, 2H), 4.45 4.25 (m, 3H), 4.25 4.07 (m, 3 H), 3.95 3.74 (m, 3H), 3.74 3.57 (m, 4H), 3.15 (dd, J = 13.9, 5.2 Hz, 1H), 3.02 2.77 (m, 2H), 2.55 2.12 (m, 3H), 2.08 1.79 (m, 2H), 1.76 1.53 (m, 4 H), 1.21 1.12 ( m, 2 H), 0.99 0.88 (m, 12H). 13 C NMR (75 MHz, Methanol d 4

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112 145.57, 145.35, 142.82, 139.41, 138.68, 130.60, 130.48, 129.70, 129.42, 129.06, 128.46, 128.06, 127.69, 126.47, 73.27, 68.27, 62.20, 61.00, 57.02, 55.97, 55.09, 52.99, 52.47, 41.75, 39.38, 37.00, 32.25, 29.48, 28.87, 26.14, 23.63, 22.11, 20.07, 19.67, 18.32. HRMS (MALDI) ( m/z ): [M+Na] + calcd for C 53 H 64 N 6 O 10 Na, 967.4576, found 967.4611

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113 CHAPTER 5 A CONVENIENT SYNTHESIS OF MEDIUM_SIDED CYCLIC PEPTIDES BY STAUDINGER MEDIATED RING CLOSURE Introduction Small cyclic peptides have attracted attention for decades 75 76 10 Due to their metabolic stability defined conformations and importance as a source of new drug candidates. 16 77 Natural cyclic dipeptides possess diverse biological activities including antibiotic / antifungal, 78,79 plan t growth retardation 79 and cytot oxicity 80 Small cyclic peptides show potent cytotoxic effects by a variety of mechanisms 81 82 83 and dis play neuroprotective properties. 84 Ring size is a significant factor in the success of macrolactamisation in the synthesis of a cyclic peptide. 5 Six m embered dipeptides (2,5 diketopiperazines) are readily accessible 77 85 86 and often observed as undesired side products in the synthesis of peptide constructs. 5 In sharp contrast, synthesis of their 7 and 8 membered analogs is much more challenging. 87 88 Figure 5 1. Challenges in chemi cal synthesis of small c yclopeptides Reproduced in part with permission from Ha, K.; Monbaliu, J. C M.; Williams, B C.; Pillai, G G.; Ocampo, C E.; Zeller, M ; Stevens, C V.; Katritzky, A R. Organic & Biomolecular Chemistry 2012 10, 8055 8058 Copyright 2012 The Royal Society of Chemistry

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114 Ring closure to give small cyclic peptide by direct head to tail lactamiz ation is difficult and no cyclisation could be accomplished using the most powerful coupling reagents such as EDC HOBt BOP, TBTU and PfPyU at high dilution (Figure 5 1). 88 89 90 To overcome problems in synthesis of small and medium cyclo peptides, an auxiliary based method f or the synthesis of bis lactams was developed (Figure 5 2) 89 The novel auxiliary was designed to be inserted in to the backbone of a linear peptide facilitating the mutually reactive terminal groups to approach one another for a cyclization reaction. A subsequent ring contraction mechanism leads to formation of the desired cyclic products with the remainings of t he auxiliary still attached. Functionalized seven and eight membered cyclic dipeptides have been synthesized Figure 5 2 A pincer auxil iary to force difficult lactamiz ation However, r emoval of the auxiliary from the fi nal cyclic products can be difficult; moreover, possible side reactions that can occur Other strategies are bas ed on cyclizations on solid phase, 91 chemoenzymatic 92 and dendrimeric nanoreactors (Figure 5 3). 93 Bu t these methods are still limited to specific sequences

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115 Figure 5 3 Wedge shaped carbosilane dendrimeric carbodiimides to cyclize homodiketopiperazines through a site isolation mechanism The cyclization of linear pepti des containing more than seven amino acids usually proceed s well As a rule of thumb, di and tripeptides possessing 7 15 membered rings are less accessible and can frequently only be synthesized with difficulty. The main hurdle to the cyclization of pepti des to afford 7 to 15 membered rings is the preferential transoid alignment of amide bonds in their acyclic precursors which leads to a preferred extended structure placing the termini which need to react far apart 94 95 Among other strategies, t raceless Staudinger ligation has been the focus of numerous attempts to overcome problems i n the coupling of large peptide fragments 53 Intramolecular traceless Staudinger ligation has been applied to access cyclic polypeptides and medium sized membered) lactams, but this methodology required protection of the phosphi ne containing auxiliary by a borane complex to avoid undesired premature Staudinger reaction. 96 The cleavage of the borane protecti ng group may require harsh conditions and, the phosphinothioester peptide can undergo oxidation during the borane deprotection process to produce undesired by prod ucts. This chapter focuses on the development of innovative and efficien t cyclization procedu res to prepare 7 and 8 membered cyclic di and 10 membered cyclic

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116 tripeptides containing or amino acid residues via a solution phase Staudinger ligation like reaction (Fig ure 5 4 ). Figure 5 4. Ring construction to form cyclic oligopeptides by Staudinger ring closure. Results and Discussion Synthesis o f 7 Membered and 8 Membered Cyclic Dipeptide Using Staudinger Assisted Ring Closure In the first phase of the project, the synthesis of 7 and 8 membered cyclic dipeptides was investigated from azidopeptidoyl thioesters. The general procedure for the synth esis of the starting azidopeptidoyl thio e sters 5.5a f is illustrated in Figure 5 5 First, chloroalkylcarbonyl chlorides 5.1a d were reacted with amino acids 5.2a e to form dipeptide derivatives 5.3a f which with an excess of sodium azide in DMF at 80 o C or MeOH at reflux, afforded the corresponding azidoprotected dipeptides 5.4a f. Mixed anhydride coupling of 5.4a f with 1.2 equiv. of thiophenol furnished the azidopeptidoyl thio e sters 5.5a f (45% to 72%). Preparative conditions for the Staudinger mediated ring closure were first optimized on compound 5.5a: treatment with 1.5 equiv. of PBu 3 in dry THF under microwave heating at 50 C and 50 W for 5 min. The desired cyclic peptide 5.6a was precipitated from the crude mixture on addition of hexanes and obtain ed in high purity

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117 Figure 5 5 Synthe sis of starting azido dipeptide thioesters ( Table 5 1). Next synthesis of ( S ) 3 benzyl [1,4]diazepane 2,5 dione (5.6b), a challenging target difficult to obtain by direct macrolactam ization was studied. 6d,6f R eaction of 5.5b with tributylphosphine using the optimized conditions described above gave product 5.6b, isolated in 63% yield after purification. This protocol was then applied to the synthesis of 3,3 dimethyl 1,4 diazepane 2,5 dione (5.6c) by cyclization of precursor 5.5c. Novel cyclic dipeptide 5.6c was obtained in 72% yield after purification. These three examples show the advantages of our microwave assisted Staudinger mediated cyclization for synthesis of difficult diketopip erazines. The higher yield in the reaction of 5.5a in comparison to 5.5b suggests that the cyclization is sensitive to steric congestion at the C terminal fragment. 10 The unexpected high yield obtained for compound 5.5c is attributed to a Thorpe Ingold eff ect ( C dimethyl substitution). The few literature examples of 8 membered cyclic dipeptide are endowed with promising biological activity 12 To broaden the scope of our methodology, 1,4 diazocane 2,5 dione 5.6d and 1,5 diazocane 2,6 dione 5.6e were conside red as potential targets. The macrolactamizations of 5.5d f were each initiated by treatment with 1.5 equiv. of PBu 3 in dry THF at room temperature. The unfunctionalized 8 membered bislactams 6d and 6e were isolated after purification in 55% and 57% yield, respectively.

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118 Table 5 1. En route to 7 and 8 membered cyclic dipeptides From 5.5 X n m R 1 R 2 Structure of 5.6 Yield (%) a Cl 1 1 H H 73 b Br 2 0 Ph H 63 c Cl 2 0 Me Me 72 d Cl 2 1 H H 55; 68 a e Cl 1 2 H H 57 f Cl 3 0 H H 51 a The yield of 5.6d in italics was calculated from 1 H NMR spectrum (500 MHz) of the crude mixture. The impact of the ring size and the sequence on the activation barrier were studied. Transition states for the cyclization of model aza ylide thioester intermediates (PH 3 as model phosphine) were calculated at the B3LYP/6 31+G** level of theory and

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119 the nature of the stationary points was determined by analytical calculation of the Hessian. A model sequence N 3 Gly Gly SPh was selected as a reference. Its cyclization through a six membered TS required an activation barrier of 16.3 kcal mol 1 while the cyclization of the sequence N 3 Gly Ala SPh through a 7 membered TS required 25.1 kcal mol 1 Interestingly, the cyclization of the reversed sequence (N 3 Ala Gly SPh, leading to 5.6a) proceeded with a lower activation barrier ( G = 14.7 kcal mol 1 ). The conformational flexibility given to the aza ylide nucleophile by the Ala residue at the N terminus of the azido dipeptide thioester seemed to have great impact on the activation barrier. Incorporation of C thioester substituent l ed a slight increase of the activation barrier: 15.1 and 20.2 kcal mol 1 for the cylization of N 3 Ala Phe SPh (leading to 5.6b) and for the N 3 Ala Aib Sph (leading to 5.6c), respectively. The cylization of the eight membered cyclic peptide 5.6d require d 22.0 kcal mol 1 Synthesis o f a 10 Membered Cyclic Tripeptide Using Staudinger Assisted Ring Closure Th e procedure was applied to the cyclization of azidotripe ptidoyl thioster 5.11 for synthesis of the cyclic 10 membered tripeptide 5. 12 Cyclotripeptides constitute a very important class of compounds with high potential in drug discovery but their synthesis using traditional activated carboxylic acid s led to low yield and a number of side products 10 Azido tripeptide thioester 5. 11 was prepared according to the strategy used for compounds 5. 5a f The conditions for the cyclization of 5. 11 were similar to those used for the cyclization of 5. 5 d f and g ave cyclo(Gly Phe Ala ) 5. 12 in 48% yield (Figure 5 6 )

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120 Figure 5 6 Solution phase synthesis of a 10 membered cyclic tripeptide. Conformational Studies o f 7 Membered Cyclic Dipeptides Monocrystals were grown from acetone/methanol mixture s for compounds 5.6a and 5.6c, the structures of which were unambiguously assigned by X ray diffraction. The conformational behavior of medium sized cyclic peptides has been rarely studied in contrast to the decades of research dedicated to biological acti vity of cyclic peptides. 6f 6k,14 We now find that, in cyclic dipeptides 5.6a and 5.6c, the seven member ed ring is formed by two puckered planes (envelope like), in agreement with reported data for similar compounds (F igure 5 7 ). 97 Empirical force field calculations were performed to predict the conformational preferences of 1,5 diazocane 2,6 dione 5.6a and 3,3 dimethyl 1,4 diazepane 2,5 dione 5.6c. A high overlay similarity (93.7 and 98.5%, respectively) betwee n the X ray structure and the predicted conformers was found and encouraged us to study the conformational preferences of the eight membered cyclodipeptides 5.6d and 5.6e, for which no X ray data were available. These cyclic dipeptides could adopt two poss ible conformers: a C 2 symmetric twisted boat or a centro symmetric chair. Analysis of the empirical force field calculations showed an exclusive chair like conformation for both compounds 5.6d and 5.6e (Figure 5 8 ).

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121 Figure 5 7 X ray structures, traditi onal representations and computed structures of A) 1,5 diazocane 2,6 dione (5.6a) and B) 3,3 dimethyl 1,4 diazepane 2,5 dione (5.6c). Figure 5 8 Predicted conformers for compounds A) 1,5 diazocane 2,6 dione (5.6d) and B) 1,4 diazocane 2,5 dione (5.6e). Conclusion In summary we have developed a new straightforward and powerful strategy towards small cycl ic di and tripeptides that relies on a Staudinger assisted ring closure This was demonstrated by the successful ring closure of a series of azido pep tide thioesters yielding a small library of 7 8 and 10 membered cyclopeptides which could not be prepared efficiently using the previously reported methods A model of reactivity

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122 based on ring size and sequence was developed using computational chemist y. The c onformational behav ior of cyclodipeptides was studied by X ray on the homodiketopiperazine series. The conformers obtained by e mpirical force field calculations showed high levels of similarity with the X ray results E mpirical force field calculat ions were also used to predict the conformational behavior of diazocanes. H omodiketopiperazine s have showed envelope like conformational preferences while diazocanes prefer chair like comformations. Experimental General Methods All commercial materials ( Aldrich, Fluka) were used without further purification. All solvents were reagent grade or HPLC grade (Fisher Solvents were dried using standard protocols kept under a dry atmosphere of nitrogen. Melting points were determined on a capillary point apparatu s equipped with a digital thermometer and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 Acetone d 6 or CD 3 OD d 4 using a 300 or 500 MHz s pectrometer (with TMS as an internal standard) at ambient temperature unless otherwise sta ted. Chemical shifts are reported in parts per million relative to residual solvent CDCl 3 ( 1 H, 7.26 ppm; 13 C, 77.23 ppm). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet, dd = doublet of doublets, quint = pentet. All 13 C NMR spectra were recorded with complete proton decoupling. The data have been reported in order to provide the maximum amount of information regarding coupling constants, which has necessarily led to integrals reported following a group of peaks in some instances. High resolution and high performance liquid chromatography mass spectral analyses were performed by the University of Florida chemistry department

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123 facility staff. Reactions were carried out in oven dr ied glassware under an argon or nitrogen atmosphere unl ess otherwise noted. 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 m L glass pressure microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 sec.; PowerMax cooling mode). Analytical TLC was pe rformed on E. Merck silica gel 60 F254 plates and visualized by UV and potassium permanganate staining. Flash column chromatography was performed on E. Merck silica gel 60 (40 63 mm). Yields refer to chromatographically and spectroscopically pure compound General P rocedure I for the Preparation of C ompounds 5. 3a f To a suspension of amino acid ( 5. 2a e) in dry THF (20 mL/1 mmol) at 0 C was added halogen acyl chloride ( 5. 1a d) (l.5 eq.) and the resulting mixture was r efluxed for 2.5 h. The reaction mixtur e was filtered hot and the solve nt was evaporated under reduced pressure. Then diethyl ether (30 mL/1 mmol 2a e) was added to the residue and left in t he fridge to recrystallize. The solid obtained was washed with hexanes (5 mL/1 mmol), cold diethyl ether (5 mL/1 mmol) and dried to give desired products ( 5. 3a f). 3 (2 Chloroacetamido)propanoic acid ( 5. 3a) The compound was synthesized following the general procedure I from chloroacetyl chloride (4.77 mL, 60 mmol) and alanine (3.56 g, 40 mmol) in 75% yield (5.06 g, 28 mmol). White microcrystals, mp o C. 1 H NMR (DMSO d 6 300 MHz): 2.40 (t, J 4.04 (s, 2H), 8.26 (br s, 1H), 12.27 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 33.5,

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124 35.2, 42.5, 165.9, 172.8; Anal. Calcd for C 5 H 8 ClNO 3 : C, 36.27; H, 4.87; N, 8.46. Found: C, 36.33; H, 4.95; N, 8.33. (S) 2 (3 Bromoro propanamido) 3 phenylpropanoic acid ( 5. 3b) The compound was synthesized following the general procedure I from 3 bromopropy l chloride (3.63 mL, 36 mmol) and L phenylalanine (4.00 g, 24 mmol) in 88% yield (6.30 g, 21 mmol). White microcrystals, mp 94 98 C. 1 H NMR (DMSO d 6 300 MHz): 2.57 ( dt, J = 6.5, 1.9 Hz, 2H), 2.86 (dd, J = 14.0, 9.2 Hz, 1H), 3.05 (dd, J = 13.8, 5.1 Hz, 1H), 3.69 (t, J = 6.3 Hz, 2H), 4.41 4.50 (m, 1H), 7.16 7.30 (m, 5H), 8.37 (d, J = 8.0 Hz, 1H), 12.75 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 36.9, 38.0, 40. 7, 53.5, 126.5, 128.2, 129.2, 137.5, 168.9, 172.9; Anal. Calcd for C 12 H 14 BrNO 3 : C, 48.02; H, 4.70; N, 4.67. Found: C, 47.94; H, 4.50; N, 4.91. 2 (3 Chloropropanamido) 2 methylpropanoic acid (5.3c). The compound was synthesized following the general procedu re I from 3 chloropropyl chloride (5.6 g, 44 mmol) and 2 methylalanine (3.00 g, 29 mmol) in 73% yield (4.10 g, 21 mmol). White microcrystals, mp 167 169 C. 1 H NMR (DMSO d 6 300 MHz): 1.33 (br s, 6H), 2.56 (t, J = 6.3 Hz, 2H), 3.74 (t, J = 6.5 Hz, 2H), 8.18 (br s, 1H), 12.16 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 25.0, 38.0, 40.9, 54.9, 168.3, 175.4. HRMS ( m/z ): [M H] calcd for C 7 H 11 ClNO 3 192.0433, found 192.0442. 3 (3 Chloropropa namido)propanoic acid (5.3d). The compound was synthesized following the general procedure I from 3 chloropropyl chloride (7.48 g, 59 mmol) and 3 aminopropanoic acid (3.50 g, 39 mmol) in 56% yield (3.95 g, 22 mmol). White microcrystals, mp 116 120 C. 1 H N MR (DMSO d 6 300 MHz): 2.37 (t, J = 6.9 Hz, 2H),

PAGE 125

125 2.54 (t, J = 6.3 Hz, 2H), 3.25 (q, J = 6.4 Hz, 2H), 3.76 (t, J = 6.5 Hz, 2H), 8.09 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 33.9, 34.9, 38.2, 41.1, 169.0, 172.9. 4 (2 Chloroacetamido)butanoic acid (5.3e). The compound was synthe sized following the general procedure I from chlororacetyl chloride (2.31 mL, 29 mmol) and aminobutyric acid (2.00 g, 19 mmol) in 72% yield (2.51 g, 14 mmol). White microcrystals, mp 75 79 C. 1 H NMR (DMSO d 6 300 MHz): 1.64 (quint, J = 7.2 Hz, 2H), 2. 22 (t, J = 7.4 Hz, 2H), 3.06 3.13 (m, 2H), 4.03 (s, 2H), 8.23 (br s, 1H), 12.08 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 24.3, 30.9, 38.3, 42.7, 165.9, 174.1; Anal. Calcd for C 6 H 10 ClNO 3 : C, 40.13; H, 5.61; N, 7.80. Found: C, 39.78; H, 5.73; N, 7.45. 2 (4 Chlorobutanamido)acetic acid (5.3f). The compound was synthesized following the general procedure I from 4 chlorobutanoyl chloride (4.79 mL, 50 mmol) and glycine (2.50 g, 33 mmol) in 38% yield (2.27 g, 13 mmol). Sticky solid. 1 H NMR (DMSO d 6 300 MHz) : 1.93 (q, J = 6.9 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.72 (d, J = 6.0 Hz, 2H), 8.24 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 28.3, 32.1, 40.6, 45.0, 171.4, 171.6. General Procedure II for the Preparation of Compounds 5.4a f D ipeptide derivatives 5. 3a f was dissolved in a minimum amount of methanol ( 5. 4a) or DMF (4b f) and then sodium azide (5 eq.) was added to the solution. The suspension was heated at 80 C for 72 h (methanol) or 24 h (DMF). The solvent was removed under redu ced pressure and brine was added to the residue until extra sodium azide was dissolved. The resulting mixture was acidified slowly with HCl to pH 5 and extracted with ethyl acetate (3 x 10 mL/1 mmol 5. 3a f). The organic layers were combined, then dried ove r anhydrous MgSO 4 and filtered. The solvent was evaporated

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126 under reduced pressure to give the desired products 5. 4a f. Compounds 5. 4b and 5. 4c were recrystallized from a CH 2 Cl 2 :hexanes mixture 3 (2 Azidoacetamido)propanoic acid (5.4a). The compound was syn thesized following the general procedure II from 3 (2 chloroacetamido)propanoic acid (5. 3a ) (2.00 g, 12.1 mmol) in 63% yield (1.31 g, 7.6 mmol). Colorless oil. 1 H NMR (DMSO d 6 300 MHz): 2.62 (t, J = 6.0 Hz, 2H), 3.53 3.60 (m, 2H), 3.99 (s, 2H), 6.87 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 33.7, 34.9, 52.8, 167.4, 176.5. HRMS ( m/z ): [M H] calcd for C 5 H 7 N 4 O 3, 171.0524, found 171.0531. (S) 2 (3 Azidopropanamido) 3 phenylpropanoic acid (5.4b). The compound was synthesized following the general procedure II from (S) 2 (3 bromoropropanamido) 3 phenylpropanoic acid (5. 3b ) (3.51 g, 11.7 mmol) in 54% yield (1.66 g, 6.3 mmol). White microcrystals, mp 128 129 C. 1 H NMR (DMSO d 6 300 MHz): 2 .37 (t, J = 6.2 Hz, 2H), 2.86 (dd, J = 13.8, 9.3 Hz, 1H), 3.06 (dd, J = 13.7, 5.0 Hz, 1H), 3.42 (t, J = 6.3 Hz, 2H), 4.41 4.49 (m, 1H), 7.17 7.31 (m, 5H), 8.35 (d, J = 8.1 Hz, 1H), 12.72 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.3, 36.8, 46.8, 53.5, 126. 4, 128.1, 129.1, 137.5, 169.5, 172.9. (3 Azidopropanamido) 2 methylpropanoic acid (5.4c). The compound was synthesized following the general procedure II from (2 (3 chloropropanamido) 2 methylpropanoic acid (5. 3c ) (3.00 g, 15.5 mmol) in 82% yield (2.54 g, 12.7 mmol). Yellow microcrystals, mp 145 152 C. 1 H NMR (DMSO d 6 300 MHz): 1.33 (s, 6H), 2.36 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 6.5 Hz, 2H), 8.17 (br s, 1H), 12.30 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 24.9, 34.4, 46.8, 54.8, 168.9, 175.4. HRMS ( m/z ): [M H] calcd for C 7 H 11 N 4 O 3 199.08371, found 199.0843.

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127 3 (3 Azidopropanami do)propanoic acid (5.4d). The compound was synthesized following the general procedure II from 3 (3 chloropropanamido)propanoic acid (5. 3d ) (2.00 g, 11.1 mmol) in 63% yield (1.31 g, 7.0 mmol). Yellow oil. 1 H NMR (CDCl 3 300 MHz): 2.41 (t, J = 6.3 Hz, 2H ), 2.60 (t, J = 5.8 Hz, 2H), 3.54 (q, J = 6.0 Hz, 2H), 3.60 (t, J = 6.5 Hz, 4H), 6.38 (br s, 1H); 13 C NMR (CDCl 3 75 MHz): 33.9, 35.2, 36.0, 47.5, 170.9, 176.3. HRMS ( m/z ): [M H] calcd for C 6 H 9 N 4 O 3, 185.0680, found 185.0686. 4 (2 Azidoacetamido)bu tanoic acid (5.4e). The compound was synthesized following the general procedure II from 4 (2 chloroacetamido)butanoic acid (5. 3e ) (1.50 g, 8.35 mmol) in 68% yield (1.06 g, 5.68 mmol). Yellow sticky solid. 1 H NMR (CDCl 3 300 MHz): 1.89 (quint, J = 6. 8 Hz, 2H), 2.51 (t, J = 6.9 Hz, 2H), 3.36 (q, J = 6.6 Hz, 2H), 4.00 (s. 2H), 6.59 (br s 1H); 13 C NMR (CDCl 3 75 MHz): 24.2, 32.7, 38.5, 52.8, 167.3, 169.1 2 (4 Azidobutanamido)acetic acid (5.4f). The compound was synthesized following the general proced ure II from 2 (4 chlorobutanamido)acetic acid (5. 3f ) (2.00 g, 11.2 mmol) in 63% yield (1.31 g, 7.0 mmol). Yellow oil. 1 H NMR (CDCl 3 300 MHz): 1.75 (q, J = 7.1 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 3.33 (t, J = 6.9 Hz, 2H), 3.73 (d, J = 6.0 Hz, 2H), 8.22 ( br s, 1H), 12.52 (br s, 1H); 13 C NMR (CDCl 3 75 MHz): 24.5, 31.9, 40.6, 50.1, 171.4, 171.7. General Procedure III for the Preparation of Compounds 5.5a f A solution of azidoprotected dipeptides 5. 4a e and N methylmorpholine (1 eq.) in dry THF (10 mL/1 mm ol) was cooled to 0 C under argon atmosphere. To the resulting solution, isobutyl chloroformate was added (1 eq.). After 5 min, thiophenol (1.1 eq.) was added and the mixtured was stirred for 24 h at room temperature. The solvent was

PAGE 128

128 evapo rtated under red uced pressured and the residue was taken up with ethyl acetate (30 mL/ 1 eq. ). The organic layer was washed with Na 2 CO 3 (3 x 15 mL/ 1 eq.), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude product was recrystallized f rom a CH 2 Cl 2 /hexanes mixture to give 5. 5a f. S Phenyl 3 (2 azidoacetamido)propanethioate (5.5a). The compound was synthesized following the general procedure III from 3 (2 azidoacetamido)propanoic acid (5. 4a ) (0.93 g, 5.38 mmol) in 57% yield (0.81 g, 3.0 7 mmol). White microcrystal, mp 63 64 C. 1 H NMR (CDCl 3 300 MHz): 2.93 (t, J = 6.2 Hz, 2H), 3.55 3.65 (m, 2H), 3.96 (s, 2H), 6.79 (br s, 1H), 7.40 7.46 (m, 5H); 13 C NMR (CDCl 3 75 MHz): 35.3, 42.8, 52.7, 127.1, 129.5, 129.9, 134.7, 166.9, 197.3; Anal. Calcd for C 11 H 12 N 4 O 2 S: C, 49.99; H, 4.58; N, 21.20. Found: C, 50. 11; H, 4.57; N, 21.24. (S) S Phenyl 2 (3 azidopropanamido) 3 phenylpropanethioate (5.5b) The compound was synthesized following the general procedure III from (S) 2 (3 azidopropanamido) 3 phenylpropanoic acid (5. 4b ) (1.41 g, 5.4 mmol) in 66% yield (1.26 g, 3.6 mmol). White microcrystals, mp 86 87 C. 1 H NMR (DMSO d 6 300 MHz): 2.54 2.73 (m, 2H), 2.92 (dd, J = 13.8, 10.2 Hz, 1H), 3.15 (dd, J = 14.0, 5.0 Hz, 1H), 3.71 3.78 (m, 2H), 4.67 4.76 (m, 1H), 7.18 7.31 (m, 5H), 7.34 7.39 (m, 2H), 7.45 7.48 (m, 3H), 8.90 (d, J = 8.1 Hz, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.4, 36.6, 46.7 60.6, 126.6, 127.2, 128.2, 129.1, 129.3, 129.5, 134.5, 136.9, 170.2, 198.3. HRMS ( m/z ): [M+Na] + calcd for C 18 H 16 N 4 O 2 SNa, 370.1043, found 370.0656. S Phenyl 2 (3 azidopropanamido) 2 methylpropanethioate (5.5c) The compound was synthesized following the g eneral procedure III from 2 (3 azidopropanamido) 2 methylpropanoic acid (5. 4c ) (1.75 g, 5.99 mmol) in 64% yield (1.12 g, 3.83 mmol).

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129 Yellow microcrystals, mp 55 57 C. 1 H NMR (CDCl 3 300 MHz): 1.58 (br s, 6H), 2.43 (t, J = 6.5 Hz, 2H), 3.61 (t, J = 6.3 Hz, 2H), 6.41 (br s, 1H), 7.34 7.39 (m, 5H); 13 C NMR (CDCl 3 75 MHz): 25.6, 36.3, 47.3, 63.1, 127.6, 129.3, 129.5, 135.2, 169.7, 201.3. HRMS ( m/z ): [M+Na] + calcd for C 13 H 16 N 4 O 2 SNa, 315.0886, f ound 315.0894. S Phenyl 3 (3 azidopropanamido)propanethioate (5.5d). The compound was synthesized following the general procedure III from 3 (3 azidopropanamido)propanoic acid (5. 4d ) (0.20 g, 1.07 mmol) in 65% yield (0.19 g, 0.70 mmol). Pale yellow oil. 1 H NMR (CDCl 3 300 MHz): 2.34 (t, J = 6.5 Hz, 2H), 2.89 (t, J = 5.9 Hz, 2H), 3.55 (q, J = 5.9 Hz, 4H), 6.21 (br s, 1H), 7.39 7.43 (m, 5H); 13 C NMR (CDCl 3 75 MHz): 35.4, 36.0, 42.9, 47.5, 127.2, 129.5, 129.9, 134.7, 170.1, 197.9. HRMS ( m/z ): [M+Na] + calcd for C 12 H 14 N 4 O 3 Na, 301 .0730, found 301.0739 S Phenyl 4 (2 azidoacetamido)butanethioate (5.5e). The compound was synthesized following the general procedure III from 4 (2 azidoacetamido)butanoic acid (5. 4e ) (0.20 g, 1.07 mmol) in 55% yield (0.16 g, 0.59 mmol). Pale yellow oil. 1 H NMR (CDCl 3 300 MHz): 1.89 (quint, J = 7.1 Hz, 2H), 2.67 (t, J = 7.2 Hz, 2H), 3.27 3.33 (m, 2H), 3.89 (s, 2H), 6.58 (br s, 1H), 7.37 (s, 5H); 13 C NMR (CDCl 3 75 MHz): 25.7, 34.0, 47.5, 51.1, 127.3, 129.5, 129.9, 134.7, 170.2, 197.9. HRMS ( m/z ): [M+Na] + calcd for C 12 H 14 N 4 O 3 Na, 301.0730, found 301.0736. S Phenyl 2 (4 azidobutanamido)ethanethioate (5.5f). The compound was synthesized following the general procedure III from 2 (4 azidobutanamido)acetic acid (5. 4f ) (1.50 g, 8.06 mmol) in 65% yield (1.46 g, 5.25 mmol). Pale yell ow oil. 1 H NMR (CDCl 3 300 MHz): 1.92 (q, J = 6.8 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 6.5 Hz, 2H), 4.24 4.27 (m, 2H), 6.43 (br s, 1H), 7.35 7.43 (m, 5H); 13 C NMR (CDCl 3 75

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130 MHz): 24.8, 33.0, 49.1, 50.8, 127.3, 129.5, 130.0, 134.9, 172.4, 195.7. HRMS ( m/z ): [M+Na] + calcd fo r C 12 H 14 N 4 O 3 Na, 301.0730, found 301.0738. General Procedure for the Cyclization of Azido Dipeptidoyl Thioesters (5.5e f) to Form Compounds 5.6a f General Procedure IVA for the Preparation of C ompounds 5.6a c. To a solution of azido dipeptidoyl thioesters 5 5a c in dry THF (10 mL/1 mmol), PBu 3 (1.5 eq.) was added under argon atmosphere. The solution was subjected to microwave irradiation (50 C, 50 W, 5 min). The reaction was allowed to cool to room temperature, and then diluted with CH 2 Cl 2 (10 mL/1 mmol). H exanes was added until the solution turned turbid and was then left to crystallize in the freezer. The solid obtained was filtered off, washed with diethyl ether (2 mL/1 mmol) and dried under high vaccuum yielding pure products 5. 6a c 1,4 Diazepane 2,5 di one (5.6a). The compound was synthesized following the general procedure IVA from ( S ) phenyl 3 (2 azidoacetamido)propanethioate (5. 5a ) (200.00 mg, 0.76 mmol) in 73% yield (70.43 mg, 0.55 mmol). White microcrystals, mp 247 248 C. 1 H NMR (DMSO d 6 300 MHz) : 2.52 2.59 (m, 2H), 3.26 3.30 (m, 2H), 3.70 (d, J = 2.7 Hz, 2H), 7.73 (br s, 1H), 7.81 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 35.5, 37.3, 45.5, 170.6, 172.4; HRMS ( m/z ): [M+Na] + calcd for C 5 H 8 N 2 O 2 Na, 151.0479, found 151.0482. (S) 3 Benzyl 1,4 diazepan e 2,5 dione (5.6b). The compound was synthesized following the general procedure IVA from ( S ) phenyl 2 (3 azidopropanamido) 3 phenylpropanethioate (5.5b) (200.00 mg, 0.56 mmol) in 66% yield (80.75 mg, 0.37 mmol). White microcrystals, mp 222 223 C. 1 H N MR (DMSO d 6 300 MHz): 2.31 2.39 (m, 1H), 2.58 2.66 (m, 1H), 2.77 (dd, J = 8.6, 4.7 Hz, 1H), 3.03 3.12 (m, 2H), 3.61

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131 3.67 (m, 1H), 4.52 4.58 (m, 1H), 7.19 (t, J = 4.2 Hz, 1H), 7.28 (t, J = 4.4 Hz, 2H), 7.35 (d, J = 4.5 Hz, 2H), 7.45 (br s, 1H), 7.84 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.9, 35.8, 36.1, 52.9, 126.2, 128.1, 129.4, 138.3, 171.5, 172.0; Anal. Calcd for C 12 H 14 N 2 O 2 : C 66.04, H 6.47, N 12.83. Found: C 65.74, H 6.61, N 12.60. HRMS ( m/z ): [M+Na] + calcd for C 12 H 14 N 2 O 2 Na, 241.0947, found 241.0955. 3,3 Dimethyl 1,4 diazepane 2,5 dione (5.6c). The compound was synthesized following the general procedure IVA from ( S ) phenyl 2 (3 azidopropanamido) 3 phenylpropanethioate (5 .5c ) (0.19 g, 0.68 mmol) in 75% yield (79.61 mg, 0.51 mmol). White microcrystals, mp 253 256 C. 1 H NMR (DMSO d 6 300 MHz): 1.34 (s, 6H), 2.44 2.51 (m, 2H), 3.15 3.21 (m, 2H), 7.34 (s, 1H), 7.86 (br s, 1H); 13 C NMR (DMSO d 6, 75 MHz): 29.3, 37.0, 37.4, 57.4, 173.7, 173.9; HRMS ( m/z ): [M+Na] + calcd for C 7 H 12 N 2 O 2 Na, 179.0791, found 179.0792. Genera l Procedure IVB for the Prepa ration of C ompounds 5.6d,e To a solution of thioesters 5. 5d f in dry THF (100 mL/1 mmol), PBu 3 (1.5 eq.) was added dropwise under argon atmosphere. The mixture was stirred for 24 h at room temperature. The solvent was evaporated under reduced pressu re and the residue was taken up with ethyl acetate and washed with an aqueous solution of Na 2 CO 3 The organic layer was dried with anhydrous MgSO 4 filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatogr aphy using ethyl acetate:hexanes (gradient) as eluent to yield pure 5. 6d,e. 1,5 Diazocane 2,6 dione (5.6d). The compound was synthesized following the general procedure IVB from ( S ) phenyl 4 (2 azidoacetamido)butanethioate (5. 5e ) (200.00 mg, 0.72 mmol) in 55% yield (56.83 mg, 0.40 mmol). White microcrystals, mp

PAGE 132

132 295 300 C. 1 H NMR (DMSO d 6 300 MHz): 2.57 (t, J = 7 Hz, 4H), 3.38 (q, J = 4.3 Hz, 4H), 7.54 (t, J = 4.2 Hz, 2H); 13 C NMR (DMSO d 6 75 MHz): 36.2, 37.4, 172.4; HRMS ( m/z ): [M+Na] + calcd for C 6 H 10 N 2 O 2 Na 165.0634, found 165.0641 1,4 Diazocane 2,5 dione (5.6e) The compound was synthesized fol lowing the general procedure IVB from ( S ) phenyl 4 (2 azidoacetamido)butanethioate (5. 5e ) (200.00 mg, 0.72 mmol) in 57% yield (58.23 mg, 0.41 mmol) or S phenyl 2 (4 azidobutanamido)ethanethioate (5. 5f) (0.20 g, 0.72 mmol) in 51% yield (52.57 mg, 0.37 mmol ). Colorless oil. 1 H NMR (DMSO d 6 300 MHz): 1.97 2.04 (m, 2H), 2.58 (t, J = 4.8 Hz, 2H), 3.70 (t, J = 4.4 Hz, 2H), 4.11 (br s, 2H), 8.43 (br s, 2H); 13 C NMR (DMSO d 6 75 MHz): 17.1, 32.5, 42.9, 44.8, 167.1, 176.4; MS ( m/z ): [M+H] + calcd for C 6 H 11 N 2 O 2 143.16, found 143.0. En Route for the Prep aration of Cyclic Tripeptide 5. 1 2 ( S ) 2 (3 Amino propanamido) 3 phenylpropanoic acid hydrochloride ( 5. 8) HCl gas was passed through a solution of Boc Ala L Phe ( 5. 7) (336.17 mg, 1.00 mmol) in dioxane (25 mL) for 1 h. The dioxane solution was concentrated under vacuum and ether was added. The turbid solution was left to crystallize in the freezer overnight. The solid formed was filtered and washed with dry diethyl ether (5 mL), dried under high vacuum to give the corresponding Ala L Phe hydrochloride 5. 8 in 88% yield (239.44 mg, 0.88 mmol). White sticky solid. 1 H NMR (DMSO d 6 300 MHz): 2.41 2.60 (m, 2H), 2.81 3.00 (m, 3H), 3.06 (dd, J = 8.6, 5.3 Hz, 1H), 4.38 4.46 (m, 1H), 7.12 7.30 (m, 5H), 8.09 (br s, 3H), 8.57 (d, J = 8.1 Hz, 1H); 13 C NMR (DMSO d 6 7 5 MHz): 32.5, 35.8, 37.3, 54.3, 127. 1, 128.8, 129.7, 138.2, 170.0, 173.5

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133 ( S ) 2 (3 (2 Chloroacetamido)propanamido) 3 phenylpropanoic acid (5.9). The compound was prepared according to the general method I for the preparation of compounds 5. 3a f from chlo roacetyl chloride (0.12 mL, 1.5 mmol) and ( S ) 2 (3 aminopropanamido) 3 phenylpropanoic acid hydrochloride (5. 8 ) (272.09 mg, 1.00 mmol) in 81% yield (5.06 g, 0.81 mmol). White microcrystals, mp 188 189 C. 1 H NMR (DMSO d 6 300 MHz): 2.18 2.36 (m, 2H), 2 .85 (dd, J = 13.7, 9.5 Hz, 1H), 3.05 (dd, J = 14.0, 5.0 Hz, 1H), 3.17 3.24 (m, 2H), 4.01 (s, 2H), 4.39 4.47 (m, 1H), 7.16 7.30 (m, 5H), 8.17 (t, J = 5.6 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 12.72 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.6, 35.6, 36.8, 42. 6, 53.4, 126.4, 128.2, 129.1, 137.6, 165.8, 170.3, 173.1. HRMS ( m/z ): [M H] calcd for C 14 H 16 ClN 2 O 4, 311.0804, found 311.0813. ( S ) 2 (3 (2 Azidoacetamido)propanamido) 3 phenylpropanoic acid (5.10). The compound was prepared according to the general met hod II for the preparation of compounds 5. 4a f from ( S ) 2 (3 (2 chloroacetamido)propanamido) 3 phenylpropanoic acid (5.9 ) (624.18 mg, 2.00 mmol) in 61% yield (389.34 mg, 1.22 mmol). White microcrystals, mp 160 161 C. 1 H NMR (DMSO d 6 300 MHz): 2.18 2.34 (m, 2H), 2.84 (dd, J = 13.8, 9.6 Hz, 1H), 3.04 (dd, J = 13.7, 5.0 Hz, 1H), 3.16 3.23 (m, 2H), 3.75 (s, 2H), 4.37 4.46 (m, 1H), 7.17 7.30 (m, 5H), 8.07 (t, J = 5.6 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 12.7 (br s, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.8, 35.2, 36.7, 50.7, 53.4, 126.4, 128.1, 129.0, 137.7, 167.2, 170.2, 173.1. HRMS ( m/z ): [M H] calcd for C 14 H 16 N 5 O 4, 318.1208, found 318.1223. (S) S Phenyl 2 (3 (2 azidoacetamido)propanamido) 3 phenylpropanethioate (3.11). The compound was prepared ac cording to the general method III for the preparation of compounds 5. 5a f from ( S ) 2 (3 (2 azidoacetamido)propanamido) 3

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134 phenylpropanoic acid (5. 10 ) (500 mg, 1.57 mmol) in 51% yield (328.91 mg, 0.80 mmol). White microcrystals, mp 94 97 C. 1 H NMR (DMSO d 6 300 MHz): 2.26 2.46 (m, 2H), 2.93 (dd, J = 13.7, 10.4 Hz, 1H), 3.15 (dd, J = 13.8, 4.8 Hz, 1H), 3.23 3.31 (m, 2H), 3.76 (s, 2H), 4.66 4.74 (m, 1H), 7.19 7.32 (m, 5H), 7.35 7.39 (m, 2H), 7.45 7.48 (m, 3H), 8.16 (t, J = 5.6 Hz, 1H), 8.84 (d, J = 8.0 Hz, 1H); 13 C NMR (DMSO d 6 75 MHz): 34.8, 35.1, 36.6, 50.7, 60.5, 126.6, 127.3, 128.3, 129.1, 129.3, 129.4, 134.5, 137.0, 167.3, 170.9, 198.5; Anal. Calcd for C 20 H 21 N 5 O 3 S: C, 58.38; H, 5.14; N, 17.02. Found: C, 58.50; H, 5.26; N, 16.94. ( S ) 3 Benzyl 1, 4,7 triazecane 2,5,8 trione (5.12) The compound was prepared according to the general method IVB for the preparation of compounds 5. 6e d from ( S ) phenyl 2 (3 (2 azidoacetamido)propanamido) 3 phenylpropanethioate (5. 11 ) (205.57 mg, 0.50 mmol) in 48% yield (66.03 mg, 0.24 mmol). Colorless oil. 1 H NMR (DMSO d 6 300 MHz): 2.92 (dd, J = 8.3, 6.2 Hz, 1H), 3.13 (dd, J = 8.4, 3.0 Hz, 1H), 3.41 (q, J = 7.4 Hz, 2H), 4.15 (t, J = 3.9 Hz, 2H), 4.66 4.71 (m, 1H), 5.37 (dd, J = 3.6, 1.2 Hz, 2H), 7.34 7.37 (m, 2H), 7.42 7.48 (m, 2H), 7.49 7.52 (m, 1H), 8.13 (t, J = 3.0 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 8.82 (d, J = 4.5 Hz, 1H); ESI MS m/z : 298 (M+Na) + Crystal D ata for C ompound s 5.6a and 5.6c Compounds 5.6a and 5.6c were recrystallized from DMSO/Acetone. C ompound 5.6a : C olorless (block), dimensions 0.55 0.45 0.41 mm, crystal system monoclinic, space group P 2(1)/ c Z = 4, a = 9.243(3), b = 7.153(2), c = 8.902(3) , = 91.489(4), V = 588.3(3) 3 = 1.447 g cm 3 T = 100(2) K, max = 31.42, Crystal data were obtained by Professor Matthias Zeller at Department of Chemistry of Youngstown State University, Youngstown, USA

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135 radiation Mo scans with CCD ar ea detector covering a whole sphere in reciprocal space, 6608 reflections measured, 1871 unique ( R int = 0.0245), 1728 observed ( I > 2 ( I )), intensities were corrected for Lorentz and polarization effects, an empirical absorption correction was applied us ing SADABS22 based on the Laue symmetry of the reciprocal space, = 0.113 mm 1 T min = 0.6701, T max = 0.7462, structure solved by direct methods and refined against F 2 with a Full matrix least squares algorithm using the SHELXL 97 software package, 164 par ameters refined, hydrogen atoms were treated using appropriate riding models, goodness of fit = 1.053 for observed reflections, final residual values R 1 ( F ) = 0.0346, w R ( F 2 ) = 0.0943 for observed reflections. CCDC 881662 C ompound 5 6c : colorless (block), dimensions 0.55 0.36 0.32 mm, crystal system triclinic, space group P Z = 2, a = 5.6500(16), b = 7.884(2), c = 9.777(3) , = 90.330(4), V = 381.49(19) 3 = 1.360 g cm 3 T = 100(2) K, max = 30.80, radiation Mo scans with CCD area detector covering a whole sphere in reciprocal space, 7746 reflections measured, 2304 unique ( R int = 0.0160), 2184 observed ( I > 2 ( I )), intensities were corrected for Lorentz and polarization effects, an empirical absorption correction was applied using SADABS22 based on the Laue symmetry of the reciprocal space, = 0.101 mm 1 T min = 0.6852, T max = 0.7462, structure solved by direct methods and refined against F 2 with a Full matrix least squares algorithm using the SHELXL 97 software package, 164 parameters refined, hydrogen atoms were treated using appropriate riding models, goodness of fit = 1.039 for observed reflections, final residual values R 1 ( F ) = 0.0353, w R ( F 2 ) = 0.0954 for observed reflections. CCDC 881663

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136 Computational D etails Computational details for the c yclization of aza ylides and cartesian coordinates of stationary points All calculations were performed with t he density functional theory (DFT) using the B3LYP functional with the GAUSSIAN 03 series of programs, 98 using the Pople 6 31+G** polarized basis set. The analytical second derivatives are used to determine the nature of the stationary points and a full thermochemical analysis was performed (Table S1). Computations were performed on model compounds (PH3 as model phosphine) Table 5 2 Activation parameters S equences D H D (kcal) T D (kcal) D (kcal) D (kcal) T D (kcal) N 3 Gly Gly SPh 0,01886 0,02605 0,00719 11,8 16,3 4,5 N 3 Gly Ala SPh (5.5a) 0,03157 0,03995 0,00838 19,8 25,1 5,3 N 3 Ala Gly SPh 0,01679 0,02335 0,00657 10,5 14,7 4,1 N 3 Ala Aib SPh (5.5c) 0,02605 0,03219 0,00613 16,3 20,2 3,8 N 3 Ala Phe SPh (5.5b) 0,01721 0,02412 0,00691 10,8 15,1 4,3 N 3 Ala B Ala SPh (5.5d) 0,02966 0,03505 0,00539 18,6 22,0 3,4 Computational studi es were performed Dr. Girinath G. Pillai at the Department of Chemistry of University of Tartu, Tartu, Estonia and Dr. Jean Christophe M. Monbaliu at the Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent U niversity, Ghent, Belgium

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137 Table 5 3 Absolute energies (Hartree) S equences H G S H G S N 3 Gly Gly SPh 1.388,12800 6 1.388,19593 8 142,97500 0 1.388,1091 44 1.388,16989 1 127,8550 00 N 3 Gly Ala SPh (5.5a) 1.427,42458 5 1 .427,49624 3 150,81600 0 1.427,3930 15 1.427,45629 2 133,1780 00 N 3 Ala Gly SPh 1.427,40983 0 1.427,47966 5 146,97900 0 1.427,3930 41 1.427,45631 1 133,1620 00 N 3 Ala Aib SPh (5.5c) 1.505,98693 6 1.506,06181 0 157,58500 0 1.505,9608 84 1.506,02962 5 144,67 70 00 N 3 Ala Phe SPh (5.5b) 1.697,67003 4 1.697,75563 8 180,17000 0 1.697,6528 26 1.697,73151 9 165,6240 00 N 3 Ala B Ala SPh (5.5d) 1.466,70168 3 1.466,77312 2 150,35500 0 1.466,6720 20 1.466,73807 1 139,0160 00 Cartesian coordinates for the TS associate d with the cyclization of the aza ylide thioester derived from azido Gly Gly SPh (6 membered ring) Figure 5 9. TS in the cyclization of the aza ylide thioester corresponding to azido Gly Gly SPh

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138 Table 5 4 Cartesian coordinates for the TS in the cycliz ation of the aza ylide thioester derived from azido Gly Gly SPh 29 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.527477 O 1.066202 0.000000 2.139070 N 1.202581 0.061268 2.163652 C 2.542196 0.029058 1.582322 C 2.569062 0.641935 0.178003 O 2.281842 1.811733 0.054747 N 1.285478 0.477282 0.534087 P 1.473435 0.639510 2.135258 S 4.167801 0.140936 0.719511 C 4.731748 1.192798 1.766415 C 4.906447 2.500472 1.286690 C 5.397523 3.494442 2.136490 C 5.740040 3.197093 3.459199 C 5.580674 1.893055 3.936057 C 5.069081 0.898094 3.098735 H 2.019959 0.445884 2.856592 H 2.236873 1.747814 2.521369 H 0.224424 0.850060 2.760956 H 0.836125 0.633581 0.311847 H 0.217093 1.035842 0.300511 H 1.124636 0.104390 3.172194 H 2.931944 0.994276 1.545638 H 4.647010 2.739033 0.262745 H 5.520914 4.504999 1.757029 H 6.131524 3.973257 4.110458 H 5.850404 1.647542 4.959666 H 4.950758 0.115913 3.471807 H 3.207155 0.634138 2.207450 -----E(B3LYP/6 31+G**)= 1388 .34371254 Hartree Cartesian coordinates for the TS associated with the cyclization of the aza ylide thioester derived from azido Gly bAla SPh (7 membered ring)

PAGE 139

139 Table 5 5 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly Ala SPh 5.5.a 32 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.530226 O 1.073669 0.000000 2.134046 N 1.189255 0.045861 2.192812 C 2.572031 0.055428 1.695138 C 2.817281 0.982718 0.502342 C 2.560718 0.408733 0.8983 32 O 2.320315 1.147084 1.849214 N 1.128252 0.739148 0.579023 P 0.890480 1.654851 1.895371 H 1.441907 2.942605 1.856760 H 1.303434 1.118603 3.132852 H 0.491810 1.887257 2.072930 H 0.972960 0.415154 0.280340 H 0.012081 1.044592 0. 348957 H 1.066056 0.038141 3.197590 H 3.178976 0.405409 2.535458 H 2.907543 0.958890 1.454240 H 2.218250 1.895917 0.595627 H 3.867583 1.294115 0.494844 S 3.790389 1.139411 1.240030 C 5.006985 0.409518 2.328318 C 6.358921 0.455405 1.9 49117 C 7.346521 0.056804 2.794086 C 6.993969 0.635867 4.016769 C 5.648267 0.693248 4.391905 C 4.657075 0.165465 3.560653 H 6.630417 0.895412 0.994136 H 8.389271 0.009193 2.492228 H 7.761252 1.038933 4.671704 H 5.366257 1.145488 5. 338864 H 3.615072 0.220423 3.851736 -----E(B3LYP/6 31+G**)= 1427.65765139 Hartree

PAGE 140

140 Figure 5 10. TS in the cyclization of the aza ylide thioester corresponding to azido Gly Ala SPh 5.5.a Cartesian coordinates for the TS associated with the cyclization of the aza ylide thioester derived from azido Ala Gly SPh (7 membered ring) Figure 5 11. TS in the cyclization of the aza ylide thioester corresponding to azid o Ala Gly SPh

PAGE 141

141 Table 5 6 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Gly Ala SPh 5.5.a 32 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.564761 C 1.413779 0.000000 2.142895 O 1.863689 0.967 982 2.755230 N 2.160511 1.133276 1.949270 C 1.859096 2.157487 0.973171 C 2.125979 1.799794 0.509460 O 1.690711 2.504711 1.401313 N 1.340014 0.084398 0.580251 P 2.210657 1.207522 0.988409 H 2.879968 1.105240 2.217000 H 1.325829 2.295118 1.1 89178 H 3.175721 1.757857 0.117785 H 0.553087 0.866406 0.379159 H 0.532439 0.887749 0.364815 H 0.483925 0.896362 1.957097 H 0.553163 0.866432 1.945893 H 3.115283 1.059348 2.280859 H 0.809765 2.458574 1.041940 H 2.463174 3.042628 1.188263 S 4.020381 1.145078 0.615168 C 4.573700 1.780882 2.187316 C 3.801113 1.686143 3.356275 C 4.311574 2.156179 4.569623 C 5.593992 2.707103 4.636065 C 6.365323 2.800274 3.473261 C 5.856760 2.349542 2.253641 H 2.795753 1.284996 3.313374 H 3.700 041 2.090281 5.465422 H 5.987161 3.065339 5.583160 H 7.361118 3.233372 3.511061 H 6.450082 2.438090 1.348479 -----E(B3LYP/6 31+G**)= 1427.65788122 Hartree Cartesian coordinates for the TS associated with the cyclization of the aza ylide thio ester derived from azido Ala Aib SPh 5.5C (7 membered ring)

PAGE 142

142 Table 5 7 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Ala Aib SPh 5. 5c 38 C 2.912189 1.215281 0.735446 C 3.816311 0.109360 0.12830 8 C 3.130234 0.820018 0.874706 O 3.620876 0.980103 1.992130 N 1.998662 1.515318 0.525286 C 1.106153 1.408036 0.627540 C 0.316519 0.044082 0.683301 O 0.188009 0.338454 1.727948 N 1.611067 1.241002 0.061016 P 0.871040 2.628092 0.29733 7 H 0.308735 2.939149 0.409079 H 0.546082 2.881041 1.640511 H 1.728604 3.707395 0.026082 H 2.761954 1.059200 1.811294 H 3.430627 2.178017 0.638167 H 4.634708 0.555976 0.439033 H 4.269309 0.495967 0.921785 H 1.624250 2.013928 1.324225 C 1.825824 1.651272 1.972532 C 0.057615 2.532209 0.476087 H 0.555831 3.503578 0.555988 H 0.453778 2.474847 0.487905 H 0.692693 2.455866 1.266334 H 1.087270 1.666274 2.776340 H 2.561464 0.890580 2.225188 H 2.332305 2.620204 1.930739 S 0.718045 0.250057 1.006032 C 2.419996 0.073774 0.480798 C 3.251127 0.797820 1.204930 C 4.602058 0.924675 0.873357 C 5.135331 0.196905 0.194764 C 4.309554 0.664373 0.923171 C 2.961662 0.811485 0.584615 H 2.835646 1.371162 2.028525 H 5.23418 0 1.597903 1.445962 H 6.184631 0.299642 0.456463 H 4.715414 1.231795 1.756373 H 2.325926 1.472951 1.160419 -----E(B3LYP/6 31+G**)= 1506.28401397 Hartree

PAGE 143

143 Figure 5 12 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Aib SPh 5. 5c Cartesian coordinates for the TS associated with the cyclization of the aza ylide thioester derived from azido Ala Phe SPh 5.5b (7 membered ring) Figure 5 13 TS in the cyclization of the aza ylide thioester corresponding to azid o Ala Phe SPh 5.5b

PAGE 144

144 Table 5 8 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Ala Aib SPh 5.5c 45 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.561633 C 1.412714 0.000000 2.144422 O 1.832789 0.945108 2.810196 N 2.188211 1.106332 1.909522 C 1.937260 2.115789 0.895515 C 2.254608 1.639840 0.554286 O 1.934887 2.296872 1.525635 N 1.352604 0.013691 0.554536 P 2.053898 1.301321 1.205284 H 2.662797 1.095734 2.454247 H 1.054994 2.265 581 1.484418 H 3.011043 2.048044 0.486263 H 0.494992 0.899948 0.383388 H 0.588081 0.851261 0.366131 H 0.481453 0.897168 1.954994 H 0.555313 0.865555 1.942683 H 3.145721 1.001560 2.228338 H 0.870377 2.358721 0.898193 C 2.720411 3.410831 1 .239059 C 2.168544 4.682268 0.626595 C 1.065094 5.321928 1.212673 C 0.543732 6.498792 0.669793 C 1.127502 7.060920 0.469422 C 2.231561 6.437040 1.056761 C 2.746826 5.257758 0.512649 H 3.769311 3.273792 0.954483 H 0.613921 4.899969 2.108850 H 2. 693463 3.502278 2.330923 H 0.309823 6.979600 1.140077 H 0.727503 7.978041 0.892839 H 2.691945 6.865595 1.942699 H 3.594658 4.771542 0.985787 S 4.095308 0.781283 0.538072 C 4.927651 1.626781 1.871862 C 6.201778 2.167198 1.626565 C 6.915551 2. 786669 2.654596 C 6.359773 2.891179 3.933749 C 5.087891 2.365892 4.178008 C 4.374808 1.728272 3.158861 H 6.628894 2.097080 0.630534 H 7.902509 3.193738 2.452391 H 6.912811 3.378317 4.731795 H 4.645490 2.448225 5.167103 H 3.383062 1.339172 3.354232 -----E(B3LYP/6 31+G**)= 1698.03267142 Hartree

PAGE 145

145 Cartesian coordinates for the TS associated with the cyclization of the aza ylide thioester derived from azido Ala Ala SPh 5.5d (8 membered ring) Figure 5 14 TS in the cyclization of the aza ylide thioester corresponding to azido Ala Ala SPh 5.5d Table 5 9 Cartesian coordinates for the TS in the cyclization of the aza ylide thioester derived from azido Ala Ala SPh 5.5d 35 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.563200 C 1.442048 0.000000 2.036596 O 2.076300 1.054306 2.151589 N 2.037927 1.207544 2.233533 C 1.510215 2.560952 2.028760 C 0.813286 2.903164 0.694805 C 1.392962 2.466731 0.658365 O 0.895558 2.868103 1.697814 N 1.213493 0.592215 0.567214 P 2.369584 0.303232 1.237612 H 3.555312 0.601924 0.536465 H 2.821369 0.156840 2.485008 H 1.844920 1.584604 1.523558 H 0.863621 0.560874 0.377107 H 0.123107 1. 032707 0.348324 H 0.552913 0.856753 1.956846 H 0.475192 0.908877 1.939512 H 3.025755 1.133832 2.442791 H 0.809489 2.814437 2.836354 H 2.372211 3.220163 2.145820 H 0.232959 2.582952 0.693943

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146 Table 5 9. Continued 35 H 0.783542 3.997587 0.633 861 S 3.412646 2.432671 0.552286 C 3.891409 3.548992 1.860616 C 3.444588 3.393339 3.182671 C 3.882174 4.273114 4.176468 C 4.779978 5.299936 3.871504 C 5.232029 5.453510 2.557158 C 4.783467 4.590790 1.554852 H 5.122837 4.719325 0.531324 H 5 .926909 6.250796 2.307641 H 5.122982 5.975504 4.650009 H 3.520393 4.150829 5.193814 H 2.737224 2.610243 3.426264 -----E(B3LYP/6 31+G**)= 1466.96681296 Hartree Cartesian coordinates for the TS associated with the cyclization of the aza ylid e thioester derived from azido Gly GABA SPh 5.5e (8 membered ring) Figure 5 15 TS in the cyclization of the aza ylide thioester corresponding to azido Gly GABA SPh 5.5e

PAGE 147

147 Table 5 10 Cartesian coordinates for the TS in the cyclization of the aza ylide t hioester derived from azido Gly GABA SPh 5.5e 35 C 0.000000 0.000000 0.000000 C 0.000000 0.000000 1.531642 O 1.064314 0.000000 2.156103 N 1.204978 0.029873 2.169371 C 2.538754 0.234314 1.592434 C 2.839606 1.714636 1.219754 C 2.935677 2.052480 0.289188 C 1.986236 1.246492 1.189077 O 2.277555 0.100397 1.537040 N 0.478109 1.273649 0.560891 P 0.495757 2.577787 0.562659 H 0.661299 3.266677 0.664057 H 0.126225 3.424386 1.525594 H 1.820431 2.240899 0.906960 H 1.027865 0.206563 0.3 13001 H 0.651911 0.770100 0.423115 H 1.114753 0.107181 3.175615 H 2.662007 0.414858 0.722795 H 3.248082 0.113811 2.347675 H 2.064382 2.333043 1.686650 H 3.785081 2.021091 1.682855 H 2.780578 3.129609 0.415997 H 3.935692 1.828446 0.672 648 S 1.802000 2.663226 2.904751 C 0.640066 1.764127 3.908608 C 0.901046 0.436674 4.297698 C 0.021365 0.260676 5.081402 C 1.205591 0.351744 5.505695 C 1.465154 1.675178 5.137114 C 0.554381 2.373990 4.339220 H 0.751145 3.410023 4.074025 H 2.374274 2.167468 5.473090 H 1.912785 0.192633 6.125144 H 0.193068 1.286117 5.371053 H 1.819160 0.038767 3.972995 -----E(B3LYP/6 31+G**)= 1466.96642431 Hartree

PAGE 148

14 8 Conformational analysis of compounds 5. 6a, 5. 6c, 5. 6d and 5. 6e A series of conformers were generated using Marvin Suite 99 (ChemAxon Kft, Hungary) and each conformer was optimized using HyperChe m. 100 For both 5. 6a and 5. 6c, the most stable conformer was envelop like. A similarity analysis between the predicted conformers for 5 .6a and 5.6c and the Xray results show in each case high levels of overlay similarity (Table 5 11). Table 5 11 Similarity analysis between ball and stick model of both Xray crystal structure (yellow) and theoretical conformer (green) Entry 3D Superimpose Similarity (%) 5.6a 93.7 5.6c 98.5

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149 CHAPTER 6 SYNTHESIS OF CYCLIC PEPTIDES BY CYCLO OLIGOMERIZATION OF DIPEPTIDOYL BENZOTRIAZOLIDES Introduction Among the various methods for the synthesi s of cyclic peptides, a common approach is the head to tai l ring closing of the linear precusor. 5 Macrocyclizations are best performed on an insoluble polymer using peptide coupling reagents. 101 A protecting group strategy of at least three dimensions of orthogonality is required to c onstruct the linear peptide, deprotect the N and C termini, cyclize in a head to t ail fashion, and finally cleave the product from the solid support. 5 102 Ho wever, the reaction set ups are often rather sophisticated, insertion of the solid support into the peptide backbone requires more extended synthetic procedure and low yields and partial epimerizations are observed in many cases. In addition, t he direct he ad to tail macro lac tamization for small and medium ring sized cyclic peptides failed even when using the most powerful coupling reagents at high dilution 26 103 20 Among contemporary strategies for co nstruction of peptide ma c r o cycles, c yclooligomerization has proved to be a powerful method to get fast access to different peptide ma c r o cycles. 104 105 106 Cyclooligomerization is a valuable entry to large macrocycles and has been used to provide libraries of metal binding macrocyclic ligands t hat are likely to reveal novel ion binding and transport interactions and can serve as scaffolds for supramolecular and combinatorial chemistry 107 108 109 110 111 In most cases, cyclic dimer s trimer s and tetramers are the major products and t he ratio of dimer trimer tetramer and larger Reproduced in part with permission from Ha, K.; Lebedyeva, I.; Hamedzadeh, S.; Li, Z.; Quinones, R.; Pillai, G. G.; Williams, B.; Nasajpour, A.; Martin, K.; Asiri, A. M. ; Katritzky A. R. Chemistry A European Journal 2014 20 4874 4879 Copyright 2014 WILEY VCH Verlag GmbH & Co. KGaA, Weinheim

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150 macrocycles obtained in c yclooligomerization process is kinetically controlled and carbons (Fig 1) 3 4 113 105 107 Peptides containing turn inducing constraint s like oxazolines, oxazoles, thiazolines, thiazoles and imidazoles, are ideal precusors for cyclooligomerization re a ction 109 114 for formation of peptide macrocycle s because the imidate linkage fused into t he ir fi ve membered ring s has a rigid transoid conformation leading to an extended structure with the mutually reactive terminal groups far apart ( Fig ure 6 1 ). 105 109 Figure 6 1 C yclooligomerization method to give fast access to different peptide ma c r o cycles Figure 6 2 C yclooligomerization method to give fast access to different peptide ma c r o cycles

PAGE 151

151 This chapter describes the development of innovative and efficient cycli zation procedures for construction of peptide macrocycles 115 116 117 A versatile strategy for the synthesis of different small and medium ring sized cyclo pe p tide from di peptidoyl benzotriazolides by a palladium promoted tandem deprotection/cyclization method is reported. O pen chain N Cbz dipeptidoyl benzotriazole sequences containing a turn inducing constraint ( Figure 6 2 ) were convert ed into either the corresponding homo diketopiperrazines in a intramolecular cyclization pathway or a library of macrocyclic scaffolds via cyclooligomerization. Results and Discussion Synthesis of B enzotr iazolide Cyclization Substrate N dipeptidoyl benzotriazolide precursors for the cyclization study. As outlined in Scheme 1, Cbz N dipeptidoyl benzotriazolides 6.5a g were synthesized in a 3 step procedure starting f amino acids 6.1a c. Cbz N amino acid 6.1a c was first converted into the benzotriazolides 6.2a c; reaction with turn introducers 6.3a c gave dipeptides 6.4a g, which were converted into Cbz dipeptidoyl benzotriazoli des 6.5a g ( Figure 6 3 ). 118 Figure 6 3 Synthesis of N protected dipeptidoyl benzotriazolides.

PAGE 152

152 Cyclization of Dipeptidoyl Benzotriazolides P reparative conditions for intramolecular cyclization were first investigated using compound 6.5a Cbz Ala D Pro Bt. To avoid intermolecular oligomerization, a dilute solution of 6.5a was added dropwise to a suspension of Pd/C in dry methanol. On stirring at a concentration of 0.1 mM under hydrogen in the presence of Pd/C (10 wt%) at 20 o C, Cbz Ala D Pro Bt 6.5a was cyclized into homo diketopiperazine 6.6a ( Figure 6 4 Table 6 1). HPLC MS analysis confirmed the major product is the 7 membered cyclic dipep tide cyclo ( Ala D Pro ) 6.6a Similarly, cyclo ( Homo Ala L Pro ) 6.6b was synthesized in 68% yield by deprotection/cyclization of Cbz Homo Ala L Pro Bt 6.5b ( Figure 6 4A Table 6 1). Figure 6 4 Intramolecular cycli zation of N protected dipeptidoyl benzotriazolides: a) using Pro as turn introducer; b) using Hyp as turn introducer. Table 6 1 Benzotriazole auxilary route to homo diketopiperazines Entry n R 1 R 2 Product a HMRS [M+H]+ Yield (%) calcd. found 1 1 H H cyclo [ Ala D Pro ], 6.6a 169.0972 169.0985 76 2 1 Me H cyclo [ H Ala L Pro ], 6.6b 183.1128 183.114 68 3 1 H OH nd NA NA NA [a] The purity was determined by HPLC MS of the crude product.

PAGE 153

153 Next, we studied cis L 4 hydroxyproline as a turn inducing unit in cy clization of Cbz Ala L Hyp(Bn) Bt 6.5d; however, ring closure to give 7 membered ring cyclic dipeptide cyclo ( Ala L H yp ) 6d by head to tail lactamiz ation of 6.5d ( Figure 6 4B ) is difficult and no cycliz ation was accomplished. The main reason for the c ycliz ation failure may the predominant trans arrangement of the amide bond in the linear hydroxyproline precursor, preventing a correct spatial positioning of the terminal amine and the activated carboxylic group. 119 120 Due to a strong noncovalent interaction between endopuckered of C4 hydroxyproline, C4 OH substituted proline preferres endo ring pucke r of with trans amide bonds which prevent the cyclization ring contruction event to form the 7 membered ring cyclic dipeptide cyclo ( Ala L Hyp ) 6.6d (Figure 6 5 ). In addition, an endo ring pucker comformation of cis L Hyp is more favored by an attractive H bonding between the hydroxyl substituent at C4 and the carbonyl group of the be nzotriazolide moiety (Figure 6 5 B). 119 121 Figure 6 5 A) cis/tra ns conformers of proline containing amide bonds ;B) C4 endo conformations of L Hyp Bt with H bond donor stabilization at C4. Unexpectedly, treatment of Cbz Ala 3 Aze Bt 6.5e under similar reaction condition s to formation of cyclo dimerization product cyclo ( Ala 3 Aze) 2 6.7g as a

PAGE 154

154 result of LCMS and MS MS analysis ( Figure 6 6 ). P robably this is due to the unfavored eight membered ring as a result of intramo lecular reaction pathway since formation of the 16 membered cyclic tetrapeptide dimer would avoid the ring strain of the eight membered cyclo dipeptide. 89 122 Figure 6 6 Intermolecular cyclodimeri zation of Cbz Ala 3 Aze Bt 6.5e Next we studied the cyclo oligomerization of N Cbz protected dipeptidoyl benzotriazolides 6.5a c,h to access different peptide macro cycles. Since the cyclooligomerization cascade is kinetically controlled, 105 109 we po stulate that at higher concent ration of the substrate, the preferred ring size of the macrocycles is determined by the relative configurations of the cyclization precursors 6.5a turn introducer. Increased substrate concentration 113 with higher Pd/C load should allow for the spontaneous formation of larger macrocycles. During formation of intra mollecular 7 membered rin gs, the ground state E geometry of the peptide bond prevents the peptides from attaining the ring like conformation conducive to cyclization. However, larger ring sized product s of cyclo oligomerization do not pose this problem because they accommodate E p eptide bonds more easily To test the above hypothesis, we prepared a 15 mM solution of Cbz Ala D Pro Bt 6.5a and examined its reaction with Pd/C in the present of hydrogen gas. The reaction was monitored by TLC and analyzed

PAGE 155

155 and analyzed by HPLC and MS. After stirring at rt for 36 h, the reaction products were isolated and characterized. HPLC revealed that the one pot cyclooligomerization of the benzotriazolide Cbz Ala D Pro Bt 6.5a produced a novel series of constrained macrocycles cyclo Ala D Pro ] n (n=2 6) containing up to 12 amino acids with 42 atoms in the cycle. Molecular weights were confirmed by the precise match of peak shapes to calculated isotopic distribution patterns and by their HR ESI mass spectra. Thus, incremental differences in mass units of cyclo Ala D Pro ] and unique isotopic patterns characterized each macrocycle (Figure 6 7 ). Figure 6 7 Structures of compounds cyclooligomerization products of 6. 5a action, including the cyclic dimer 6.7a (72%), cyclic trimer 6.8a (15%), and cyclic tetramer 6.9a (10%) with formula cyclo Ala D Pro ] n in which n=2 4 (Table 6 2, Scheme 6 4). No direct intra molecular cyclization product of 6.5a cyclo Ala D Pro] was observed. All of the products were MS and HMRS analysis. Cyclic dimer 6.7a was purified by gradient

PAGE 156

156 chromatography (MeOH/ether) and obtained in 68% isolated yield and characterized characterized fully by 1 H and 13 C NMR spectroscopy and HRMS (ESI). Figure 6 8 Intermolecular cycl o oligomeri zation of N protected dipeptidoyl benzotriazolides Dependence of Cyclo Oligomerization on Substrate Concentration substrate concentration on the Pd promoted macrocyclization reaction of Cbz Ala D Pro Bt 6.5a and the product distribution. It was expected that substrate concentration w intermolecular and intramolecular reac tions For these studies, we used higher load of Pd/C in order to shorten the reaction time and facilitate the monitoring. As shown in Table 6 2, with higher substrate concentration, the yields of cyclic dimer 6.7a increased. The formation of cy c lic oligomeric peptides 6.7a, 6.8a and 6.9a in the product distribution suggests again that the relative ring size of the peptidoyl macrocycles is determined by kinetic control effected by the relative concentration of Cbz Ala D Pro Bt 6.5a. Another notable trend revealed in Table 6 2 is tha t the reaction gave a higher percentage of dimeric cyclo Ala L Pro) 2 to total cyclo oligomeric products with the increased concentration of 6.5a. This trend was expected because increased substrate concentrations should favor intermole cular reactions significantly.

PAGE 157

157 The main factor contributing to the formation of cyclo tetrapeptide 6.7a may be the relative energies of transition state during the ring construction event of cyclic dimer 6.7a. Figure 6 7 implies that Cbz deprotection of Cb z 1 N benzotriazolyl dipeptide 6.5a forms N Ala D Pro Bt which could coordinate to the Pd could form a pre organized tetrapeptide complex. The formation of the Pd complex in turn could lower the activation energy in the formation of the intermolecular dimerization/cyclization product 6.7a. 18 123 Table 6 2 Product distributions of Pd promoted tamdem deprotection/cyclization reactions of various concentrations of Cbz Ala L Pro Bt 6.5a Concentration of 6.5a (mM) Intramolecular cyclization product 6.6a b (%) Cyclic dimer 6.7a (%) Cyclic trimer 6.8a (%) Cyclic trimer 6.9a (%) 0.1 a 99.9 0.1 ND c ND 0.2 a 91 d 3 ND ND 13 ND 72 15 10 15 ND 82 12 5 50 ND 55 5 ND e a Reaction were performed under drop wised condition over 24 h with 16 36 h additional stirring b Yields derived from HPLC analysis of crude products; c Not detected; d 6% is hydrolyzed side product; e many linear oligo mers was detected To prove the generality of our approach, we studied the cyclo oligomerization of 6.5b amino acids and turn inducing constraint units (Table 3). Our one pot tandem oligomerization/cyclization procedure was employed to perform cyclization of N Cbz protected dipeptidoyl benzotriazolide 6.5b ( Figure 6 7 Table 6 3). The reaction was carried out under optimized conditions using 0.15 mM of Cbz Homo D,L Ala L Pro Bt. After reaction at room temperature for 204 h in the pres e ce of Pd/C and H 2 gas, the anticipated dimeric cyclic tetrapeptide 6.7b was detected in 78% crude yield, along with a small amount of the cyclic trimer 6.8b and

PAGE 158

158 tetramer 6.9b, in 8% and 2% yields, respectively (Table 3, entry 1). Cyclo tetrapeptide 6.7b wa s later obtained in 69% yield after purification. The cyclo oligomerization of 6.5c Cbz D,L Phe L Pro Bt offered the cyclo dimerization product as the major product (88%), and the trimeric cyclic product was also observed in a small quantity (<3%) (Table 6 3, entry 2). However, the reaction of peptide Cbz Ala L Hyp(Bzl) Bt gave only methyl ester and hydrolyzed side products with little dimerization or trimerization (Table 6 3, entry 3). The Turn Introducer E ffect on Cyclooligomerization of Dipeptidoyl B enzotriazol s To determine the turn introducer effect required for a dipeptidoyl benzotriazole to prefer cyclooligomerization vs direct head to tail cyclization, we then prepared benzotriazolides 6.5f,g containing a 4 membered ring L 2 Aze and 6 membered ri ng L Homo Pro. Subsequently, 0.12 0.15 mM peptide solutions were treated with catalytic amount s of Pd/C under hydrogen gas, and the reactions were monitored by HPLC and HR ESI MS Interestingly, HPLC MS analysis showed that Cbz Ala L Aze Bt 6f formed only trimeric cyclic product as the major product (90%) (Table 6 3, entry 4) and no dimerization, tetramerization or higher oligomerization products were detected. The change of ring size from 5 membered ring in proline to 4 membered ring in azetidine results in N C for the Aze peptide increased by 2 9 from those of the Pro containing peptide, 124 which could result in more favorable formation of lager ring sized trimeric cyclic product vs the dimer product in cyclooligomerization rea ction of Cbz Ala L Aze Bt 6.5f. No cyclooligomerization products were observered, when 0.12 0.15 mM peptide solutions of 6.5g were treated with similar conditions.

PAGE 159

159 Table 6 3. Product distributions of Pd promoted tamdem deprotection/cyclization reactions of Cbz protected dipeptidoyl benzotriazolides 6.5b d, 6.5g,h Entr y Cyclization substrates Cyclic dimer 6.7 (%) a Cyclic trimer 6.8 (%) Cyclic tetramer 6.9 (%) 1 78 8 2 2 a 88 trace 2 3 ND b ND ND 4 ND 90 c N D 5 ND b ND ND a Ratios derived from HPLC analysis of crude products; b Only trace amount was detected, mainly methyl ester and hydrolyzed side product were detected; c Only cyclic dimer was found Conclusion W e have de monstrated that open chain N Cbz dipeptidoyl benzotriazole sequences containing a turn inducing constraint were selectively converted into either the corresponding homo diketopiper azines in a n intramolecular cyclization pathway or macrocyclic scaffolds via cyclooligomerization. The relative ring size of the macrocyc les is under kinetic control determined by the concentration of the substrate and the relative configurations of turn amino acid containing N Cbz dipeptidoyl benzotriazole s formed 7 membered ring under

PAGE 160

160 treatment with Pd/C in the present of hydrogen gas. As concentration of the substrate increases, kinetic preference shifts to the formation of larger 14 21 and 28 membered ring systems to avoid the ring strain of small peptide cycles. Dipeptidoyl benz o triazolides co ntaining proline as turn introducer favor formation of cylic dimer. In contrast, the use of azetidine building block allows the formation of even larger trimeric peptide macrocycle s The approach described here provide s a convenient access to generate comb inatorial petide macrocycle libraries with conceivable uses as new antibiotics, new metal sequestrants, or as scaffolds for developing macromolecular devices and protein mimetics. Experimental General Methods Melting points were determined on a capillary p oint apparatus equipped with a digital thermometer. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 acetone d 6 or CD 3 OD using a 300 or 500 MHz spectrometer (with TMS as an internal standard). The following abbreviations are used to describe sp in multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet, dd = doublet of doublets, ddd = doublet of doublets of doublets, and dt = doublet of triplets. HPLC MS analyses were performed on a reverse phase grad ient using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; mass spectrometry was done with electrospray ionization (ESI), matrix assisted laser desorption/ionisation time of flight (MALDI TOF) or a tmospheric pressure chemical ioniza tion ( APCI ). Ether refers to diethyl ether.

PAGE 161

161 General Procedure for the Preparation of Benzotriazolides 6.2a c A stirred solution of 1H benzotriazole (BtH) (4 equiv.) in dry tetrahydrofuran (THF) (10 mL/1 g) was treated at 20 C with thionyl chloride (SOCl 2 ) (1 equiv.). After 20 minutes, a solution of (Cbz protected amino acid OH) 1 equiv.) in dry THF (10 mL/1 g) was added drop wise and the resulting solution was then stirred for 2 h at 20 C. Upon completion, the mixture was filtered, and THF was removed und er reduced pressure. The residue was dissolved by dichloromethane (CH 2 Cl 2 50 mL/1 g of 1a b) and washed successively with HCl (4N, 2 1 mL/1 mL CH 2 Cl 2 ), aq. Na 2 CO 3 (10%, 2 1 mL/1 mL CH 2 Cl 2 ) and brine (1 mL/1 mL). The organic layer was dried over magnesi um sulfate (MgSO4), filtered and evaporated to give the crude product. The solid was recrystallized from CH2Cl2/hexanes to yield benzotriazolides 6.2a c. (Cbz Ala Bt) 6.2a. White solid, 4.61 g, 5.10 mmol, 82% yield; mp 111.0 112.0 C. 1 H NMR (CDCl 3 3.70 (m, 2H), 3.75 (t, J = 6.0 Hz, 2H), 5.07 (br s, 2H), 5.36 (br s, 1H), 7.24 7.32 (m, 5H), 7.50 (dt, J = 7.2, 1.0 Hz, 1H), 7.64 (dt, J = 7.2, 1. 0 Hz, 1H), 8.10 (dd, J = 8.2, 0.9 Hz, 1H), 8.23 (dd, J = 8.2, 0.9 Hz, 1H). 13 C NMR (CDCl 3 136.5, 146.3, 156.4, 171.5. Anal. Calcd for C 17 H 16 N 4 O 3 : C 62.95, H 4.97, N 17.27. Found : C 63.01, H 4.94, N 17.61. (Cbz D,L Phe Bt) 6.2b. White solid, 1.10 g, 2.75 mmol, 82% yield; mp 166.8 167.5 C. 1 H NMR (CDCl 3 J = 16.7, 5.6 Hz, 1H), 4.02 4.18 (m, 1H), 5.02 5.51 (m, 2H), 5.59 (dd, J = 13.5, 6.6 Hz, 1H), 7.21 7.45 (m, 10H), 7.46 7.55 (m, 1H), 7.59 7.67 (m, 1H), 8.08 8.14 (m, 1H), 8.18 8.25 (m, 1H). 13 C NMR (CDCl 3 75 155.6, 169.6.

PAGE 162

162 (Cbz H Ala Bt) 6.2c. White Solid, 1.25 g, 3.70 mmol, 88% yield; mp 99.8 100.9 C. 1 H NMR (CDCl 3 J = 6.4 Hz, 3H), 3.58 (dd, J = 16.4, 5.9 Hz, 1H), 3.70 (dd, J = 12.8, 6.0 Hz, 1H), 4.40 4.55 (m, 1H), 4.97 (br s, 2H), 5.05 (br s, 2H), 5.20 5.42 (m, 1H), 7.20 7.42 (m, 5H), 7.47 7.54 (m, 1H), 7.62 7.68 (m, 1H), 8.09 8.13 (m, 1H), 8.24 8.28 (m, 1H). 13 C NMR (CDCl 3 120.4, 126.4, 128.3, 128.5, 128.6, 128.8, 130.7, 136.5, 146.3, 155.7, 170.4. Anal. Calcd for C 18 H 18 N 4 O 3 : C 63.89, H 5.36, N 16.56. F ound: C 64.11, H 5.49, N 16.70. General Procedure for the Preparation of Dipeptides 6.4a g Benzotriazolides 6.2a c (1 equiv.) were each suspended in acetonitrile/water (3:1) (25 mL/1 g) and a solution of amino acid (1 equiv.) in water (5 mL/1 g of amino ac id) containing triethylamine (1.0 1.1 equiv.) was added slowly. The mixtures were stirred at 20 C for 16 24 h until TLC revealed consumption of the starting materials. The solvent was removed under reduced pressure and the residue was dissolved in ethyl a cetate. The solution was washed with 4N HCl (3 1.5 mL/1 mL of ethyl acetate) and brine (1 mL/1 mL of ethyl acetate). Recrystallization from ethyl acetate/hexanes yielded dipeptides 6.4a g Cbz Ala L Pro OH (6.4a). 1 H NMR (CDCl 3 1 H NMR (300 MHz, Methanol d 4 J = 11.1, 3.6 Hz, 5H), 5.30 4.92 (m, 2H), 4.44 4.36 (m, 1H), 3.63 3.45 (m, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.56 (t, J = 6.7 Hz, 2H), 2.17 (qd, J = 9.7, 8.1, 3.6 Hz, 1H), 2.03 1.80 (m, 3H). 13 C NMR (CDCl 3 2.8, 172.7, 171.9, 171.4, 60.8, 60.7, 58.6, 47.6, 45.1, 44.7, 42.8, 29.4, 29.2, 24.7, 24.4, 23.6, 21.0. Cbz Homo Ala D Pro OH (6 .4b). 1 H NMR (300 MHz, Methanol d 4 7.19 (m, 5H), 5.04 (d, J = 8.0 Hz, 2H), 4.49 4.22 (m, 1H), 4.05 (dt, J = 13.6, 5.8 Hz, 1H), 3.80 3.65 (m, 2H), 2.66 (d, J = 17.1 Hz, 1H), 2.43 (p, J = 7.8 Hz, 1H), 2.21 (dt, J

PAGE 163

163 = 15.8, 7.4 Hz, 1H), 2.05 1.91 (m, 3H), 1.25 1.12 (m, 3H). 13 C NMR (75 MHz, .9, 45.5, 42.2, 41.9, 32.2, 32.2, 30.4, 30.4, 26.6, 25.8, 25.7, 23.6, 20.9. Cbz Phe L Pro OH ( 6.4 c). 1 H NMR (500 MHz, Methanol d 4 7.17 (m, 10H), 5.22 5.08 (m, 1H), 5.08 4.97 (m, 2H), 4.34 (ddd, J = 26.8, 8.7, 3.4 Hz, 1H), 3.60 3.40 (m, 2H ), 2.99 2.52 (m, 2H), 2.19 2.03 (m, 1H), 1.97 1.69 (m, 3H). 13 C 125.8, 64.9, 57.9, 50.8, 46.1, 28.4, 26.2, 23.9. Cbz Ala L Hyp(O Bn) OH (6.4d). 1 H NMR (299 MHz, Met hanol d 4 7.23 (m, 10H), 5.06 (d, J = 3.1 Hz, 2H), 4.61 4.49 (m, 2H), 4.45 (t, J = 8.0 Hz, 1H), 4.26 (d, J = 9.4 Hz, 1H), 3.77 3.62 (m, 1H), 3.42 3.34 (m, 3H), 2.52 (t, J = 6.7 Hz, 2H), 2.52 2.32 (m, 1H), 2.07 (ddd, J = 13.0, 8.0, 4.9 Hz, 1H). 13 C NMR (75 MHz, Methanol d 4 72.2, 67.5, 59.1, 53.8, 37.9, 36.2, 35.5. Cbz Ala 3 Aze OH (6.4e). 1 H NMR (300 MHz, Methanol d 4 7.26 (m, 5H), 5.07 (s, 2H), 4.35 4.24 (m 1H), 4.19 4.02 (m, 1H), 3.36 (td, J = 6.8, 4.3 Hz, 2H), 3.07 (dt, J = 3.1, 1.5 Hz, 1H), 2.50 (t, J = 6.8 Hz, 2H), 2.34 (t, J = 6.7 Hz, 2H). 13 C NMR (75 MHz, Methanol d 4 53.95, 51.82, 37.83, 32.9 5, 32.38. Cbz Ala L 2 Aze OH (6. 4 f). 1 H NMR (300 MHz, Methanol d 4 7.23 (m, 5H), 5.07 (s, 2H), 4.69 (dd, J = 9.6, 5.4 Hz, 1H), 4.24 4.02 (m, 1H), 3.93 (q, J = 6.9, 6.3 Hz, 1H), 3.38 (t, J = 5.0 Hz, 2H), 2.76 2.53 (m, 1H), 2.45 2.26 (m, 2H) 2.28

PAGE 164

164 2.12 (m, 1H). 13 C NMR (75 MHz, Methanol d 4 129.0, 127.2, 67.5, 62.8, 60.8, 37.8, 32.4, 21.1. Cbz Ala D,L Homo Pro OH (6. 4 g). 1 H NMR (300 MHz, Methanol d 4 J = 4.5 Hz, 5H), 5.07 (s, 2H), 4.12 3.7 6 (m, 1H), 3.46 3.34 (m, 2H), 2.76 2.57 (m, 1H), 2.52 (t, J = 6.7 Hz, 1H), 2.25 (d, J = 13.4 Hz, 1H), 1.53 1.22 (m, 4H), 0.94 (dd, J = 9.7, 6.5 Hz, 3H). 13 C NMR (75 MHz, CD 3 129.1, 128.9, 67.5, 53.4, 44.8, 34.5 27.9, 26.4, 22.0, 19.5. General Procedure for the Preparation of Dipeptidoyl B entrotriazolides 6.5a g A stirred solution of BtH (4 equiv.) in dry tetrahydrofuran (THF) (15 mL/1 g) was treated at 20 C with SOCl 2 (1 equiv.). After 20 minutes, the solutio n of BtH and SOCl 2 was cooled down on ice and salt (NaCl) for 5mins and a solution of 6.4a g (1 equiv.) in dry THF (20 mL/1 g) was added drop wise and the resulting solutions were then stirred for 4 h at 20 C. THF was removed under reduced pressure and t he residue was dissolved ethyl acetate (85 mL/1 g) and washed successively with Na 2 CO 3 10 wt. % in water (3 1 mL/1 mL of ethyl acetate), HCl (4N, 1 1 mL/1 mL of CH 2 Cl 2 ), and brine (1 30 mL). The organic layer was dried over magnesium sulfate, filtered and evaporated. The crude product was then recrystallized from CH 2 Cl 2 /hexanes to yield dipeptidoyl bentrotriazolides 6.5a g Cbz Ala L Pro Bt ( 6 .5a). 1 H NMR (300 MHz, Chloroform d J = 8.2 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.42 7.22 (m, 5H), 5.93 (dd, J = 9.0, 3.9 Hz, 1H), 5.56 (t, J = 6.3 Hz, 1H), 5.09 (s, 2H), 3 .83 3.58 (m, 2H), 3.58 3.43 (m, 2H), 2.71 2.58 (m, 2H), 2.58 2.47 (m, 1H), 2.26 2.06 (m, 3H). 13 C NMR (75 MHz, CDCl 3

PAGE 165

165 130.2, 128.2, 127.8, 127.7, 126.2, 126.0, 119.9, 119.8, 114.2, 114.1, 66.3, 5 9.2, 53.5, 47.3, 36.5, 34.2, 29.6, 24.8, 23.0. Cbz Homo Ala D Pro Bt (6.5 b). 1 H NMR (300 MHz, DMSO d 6 J = 8.3 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.42 7.18 (m, 6H), 5.72 (dt, J = 8.6, 4.1 Hz, 1H), 5.01 (d, J = 4.2 Hz, 2H), 3.98 3.82 (m, 1H), 3.79 3.62 (m, 2H), 2.65 (dt, J = 15.3, 5.5 Hz, 1H), 2.42 (dt, 2H), 2.26 2.11 (m, 1H), 2.11 1.97 (m, 2H), 1.11 (dd, J = 6.6, 2.1 Hz, 3H). 13 C NMR (75 MHz, DMSO) 0, 130.6, 128.3, 127.7, 126.7, 120.2, 114.0, 65.1, 58.8, 58.7, 47.1, 29.1, 29.0, 24.8, 24.7. Cbz Phe L Pro Bt (6 .5c). 1 H NMR ( 300 MHz, Chloroform d 8.19 (m, 1H), 8.17 7.98 (m, 1H), 7.71 7.59 (m, 1H), 7.58 7.46 (m, 1H), 7.46 7.14 (m, 1 0H), 5.90 5.74 (m, 1H), 5.23 5.10 (m, 1H), 5.10 4.98 (m, 3H), 3.78 3.32 (m, 3H), 2.95 2.80 (m, 1H), 2.81 2.32 (m, 2H), 2.23 1.96 (m, 2H), 1.96 1.65 (m, 2H), 1.18 0.78 (m, 1H). 13 C NMR (75 MHz, CDCl 3 1 36.7, 131.4, 131.3, 130.7, 130.6, 129.1, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.0, 127.8, 127.7, 127.6, 127.5, 126.6, 126.5, 126.3, 126.2, 125.8, 120.3, 120.1, 115.0, 114.6, 114.5, 109.9, 73.0, 66.8, 59.6, 59.5, 52.4, 47.8, 45.1, 29.8, 29.6, 27.2, 2 5.1, 25.0, 23.3. Cbz Ala L Hyp(O Bn) Bt (6.5d). 1 H NMR (299 MHz, Chloroform d J = 8.2 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.1 Hz, 1H), 7.46 7.24 (m, 10H), 5.66 5.42 (m, 1H), 5.08 (s, 2H), 4.71 4.41 (m, 2H), 3.90 3.61 (m, 1H), 3.61 3.24 (m, 4H), 2.68 2.16 (m, 3H), 2.12 2.01 (m, 1H). 13 C NMR (75 MHz, CDCl 3

PAGE 166

166 128.83, 128.78, 128.64, 128.26, 128.16, 127.83, 126.63, 120.45, 114.71, 71.55, 66.69, 57.78, 5 2.51, 36.71, 35.03, 34.54, 29.89 Cbz Ala 3 Aze Bt (6.5e). 1 H NMR (299 MHz, Chloroform d 8.22 (m, 1H), 8.17 8.10 (m, 1H), 7.76 7.65 (m, 1H), 7.60 7.50 (m, 1H), 7.39 7.27 (m, 5H), 5.58 5.45 (m, 1H), 5. 09 (br s, 2H), 4.79 4.64 (m, 1H) 4.59 4.36 (m, 2H), 3.78 (q, J = 5.7 Hz, 1H), 3.69 (t, J = 5.7 Hz, 1H), 3.49 (q, J = 5.9 Hz, 2H), 2.37 (t, J = 5.4 Hz, 2H). 13 C NMR (75 MHz, CDCl 3 127.8, 126.5, 126.1, 120.2, 114.0, 66.5, 51.5, 49.9, 36.3, 33.2, 31.1 Cbz Ala L 2 Aze Bt (6.5f). 1 H NMR (299 MHz, Chloroform d 8.18 (m, 1H), 8.16 8.05 (m, 1H), 7.73 7.59 (m, 1H), 7.57 7.42 (m, 1H), 7.38 7.22 (m, 5H), 6.09 (dd, J = 9.8, 5.5 Hz, 1H), 5.72 5.53 (m, 1H), 5.15 5.00 (m, 2H), 4.31 4.07 (m, 1H), 3.80 3.60 (m, 1H), 3.57 3.40 (m, 2H), 3.09 2.81 (m, 1H), 2.55 2.28 (m, 3H). 13 C NMR (75 MHz, Chloroform d 128.3, 127.8, 126.7, 126.4, 120.0, 114.1, 66.3, 59.6, 48.2, 36.4, 31.0, 21.0. Cbz Ala D,L Homo Pro Bt (6.5g). 1 H NMR (500 MHz, Chloroform d J = 8.4, 0.9 Hz, 1H), 7.78 7.66 (m, 1H), 7.50 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.43 7.29 (m, 6H), 6.52 (dd, J = 6.8, 2.4 Hz, 1H), 536 5.30 (m, 1H), 5.11 ( br s, 2H), 4.48 4.25 (m, 1H), 4.08 (dtd, J = 31.2, 8.2, 6.2 Hz, 3H), 3.49 (q, J = 6.1 Hz, 1H), 3.16 (dddd, J = 13.0, 8.2, 4.5, 2.4 Hz, 1H), 2.64 2.46 (m, 2H), 2.45 2.31 (m, 1H), 2.28 2.11 (m, 2H), 1.01 0.74 (m, 1H). G eneral Procedure for the Cycl izations of Dipeptides 6. 6a g The dipeptidoyl benzotriazolides 6 g (100 mg) were dissolved in methanol with the desired concentration (0.1 50 mM) To the solutions, 150 mg of 10 wt % Pd/C was added slowly. The mixture was stirred for 24 72h at room tempera ture, under 40 psi

PAGE 167

167 pressure of hydrogen gas. After the completion of reaction, the mixture was filtered through a celite pad and washed thoroughly with methanol to separate Pd/C. The solvent was evaporated under reduced pressure to yield the crude mixture of products. For 6.5a,b,c the mixture was separated to its components by gradient column chromatography. For 6 .5d,e,f,g the crude product mixture was analyzed by HPLC MS to determine the percentage of each component. The samples were analyzed via reverse p hase gradient C 18 HPLC/UV(254 nm)/(+)ESI MSn. The details of mass spectrometry and HPLC. Mass Spectrometry: ThermoFinnigan (San Jose, CA) LCQ with electrospray ionization (ESI ESI: sheath gas(N2) = 65; aux gas(N2) = 3; heated capillary temperature = 250 C (+)ESI: spray voltage = 3.3 kV; heated capillary voltage = 12.5 V; tube lens offset = 0 V. HPLC: Agilent (Palo Alto, CA) 1100 series binary pump. Column: thermoScientific Hypurity C8 (2.1 x 100 mm; 5 um) + Phenomenex (Torrace, CA) C18 guard column (2mm x 4 mm) .Mobile Phases: A = 0.2% Acetic acid in H2O (HPLC grade, B&J HPLC, Honeywell Burdick & Jackson) B = 0.2% acetic aicd in acetonitrile (LC MS grade, Honeywell Burdick & Jackson). Acetic Acid (glacial, ACS Certified PLUS, Thermo Scientific). Gradient: @ 0.25 mL/min: A:B(min) = 100:0(0) => 5:95(45 60). Injector: Rheodyne 7125 manual injector, 25 mL injection loop; 25 mL Hamilton 1702 gastight syringe. UV: Agilent 1100 G1314A UV/Vis detector; wavelength = 254 nm For Cbz Ala L Hyp(O Bn) Bt (6.5d) and Cbz Ala D,L Homo Pro Bt (6.5g) no significant cyclization was observed at low concentrations of 0.1 mM or higher concentrations of 50 mM.

PAGE 168

168 Cyclizations of dipeptide Cbz Ala Pro Bt (6.5a) [ Ala D Pro ] n. Cyclization of Cbz Ala D Pro 6.5a. 0.1, 0.2, 13, 15 and 50 mM of 6.5a was treated with conditions stated in general procedure. The sample was analyzed via reverse phase gradient C18 HPLC/UV/(+)ESI TOF HRMS or via reverse phase gradient C8 HPLC/254 nm UV/(+)ESI MSn. All of the products 6.6a, 6.7a 6.8a and 6 .9 a of interest were detected with 13 and 15 mM concentrations. Intramolecular cyclization product and 6.6a were detected with 0.1 and 0.2 mM. Products 6. 7 a 6.8a and 6 .9 a were detected with 50 mM of 6.5a. Intramolecu lar cyclization product MW 168 eluted at RT 14.98 min. Intramolecular cyclization product MW 168 (RT 14.98 min) produced an abundant m/z 169 [M+H] + ion. Dimeric products 6. 7 a MW 336 eluted at RT 43.65 min. 6. 7 a MW 336 produced an m/z 337 [M+H]+ ion and m/z 359 [M+Na]+ ions (top) along with several fragment ions. Trimeric 6 8 a MW 504, eluted at RT 49.60 min. MW 504, RT 49.60 min. 6. 8 a the MW 504 produced m/z 505 [M+H]+ and m/z 527 [M+Na]+ ions. Tetrameric 6. 9 a MW 67 2 eluted at RT 55.59 min. The MW 672 pro duced m/z 673 [M+H]+ and m/z 695 [M+Na]+ ions. Products distributions were calculated based on mass area. Cyclo ( Ala D Pro) 3 .(6 8 a) was purified by gradient chromatography DCM:Ether 1:9 to 1:1. Colorless oil, 139 m g, 0. 41 mmol 35 % yield. 1 H NMR (500 MHz, Methanol d 4 5.42 (m, 3H), 4.43 (dddd, J = 15.1, 12.0, 5.3, 3.4 Hz, 3H), 4.28 4.20 (m, 3H), 4.16 (t J = 6.8 Hz, 3H), 3.86 (dq, J = 15.4, 5.3 Hz, 3H), 3.47 (t, J = 3.8 Hz, 3H), 3.44 (dd, J = 4.3, 3.3 Hz, 3H), 3.24 3.12 (m, 6H), 2.72 (dq, J = 13.6, 7.0 Hz, 3H), 2.50 (p, J = 6.9 Hz, 6H). 13 C NMR (75 MHz, Methanol d 4 23 .6. Anal Calcd. for HRMS ( MALDI TOF MS ) C 24 H 37 N 6 O 6 : 505.2769. Found: 505.2771.

PAGE 169

169 Cyclo ( Ala D Pro) 5 Cyclic pentamer was detected using MALDI TOF MS. HRMS MALDI TOF MS calcd. for C 40 H 61 N 10 O 10 [M + H] + : 841.4572. Found: 841.4521. Cyclo ( Ala D Pro) 6 Cyclic hexamer was detected using MALDI TOF MS. HRMS MALDI TOF MS calcd. for C 48 H 73 N 12 O 12 [M + H] + : 1009.5470. Found: 109.5428 Cyclizations of dipeptide Cbz D,L Homo Ala D Pro Bt (6.5b) Cyclo oligomerization of Cbz D,L Homo Ala D Pro 6.5b 0.1 mM or 15 mM of 6 .5a was treated with conditions stated in general procedure. The sample was analyzed via reverse phase gradient C18 HPLC/UV/(+)ESI TOF HRMS. All of the products 6.6b, 6.7b 6.8a and 6 .9 b of interest were detected. HPLC: Agilent (Palo Alto, CA) 1100 se ries binary pump Mobile Phases: MP A: 0.2% HOAc in H 2 O MP B: 0.2% HOAc in Methanol. Gradient: @ 0.17 mL/min: A:B(min) = 95:5(0) => 5:95(65 80). Product distributions were determined based on weight area. At 0.1 mM concentration only intramolecular cycli zation product Cyclo (D,L Homo Ala D Pro) MW 182 detected at RT 13.6 min. HRMS (ESI) calcd for C 9 H 15 N 2 O 2 [M + H] + 183.1128 found 183.114 At higher concentration of 15 mM higher oligomers were detected. Cyclo (D,L Homo Ala D Pro) 2 6.7 b MW 364: With a 20 ppm window about the m/z 365.2183 and m/z 387.2003 ions expected of MW 364, there were a number of ion peaks due to combination of diastereomers detected. RT 15.9 min. HRMS ESI MS calcd. for C 18 H 29 N 4 O 4 [M + H] + 365.2183. Found: 365.2204. RT 21.8 min. HRMS ESI MS calcd. for C 18 H 29 N 4 O 4 [M + H] + 365.2183. Found: 365.2198. RT 21.8 min. HRMS ESI MS calcd. for C 18 H 29 N 4 O 4 [M + H ] + 365.2193 found 365.2183 Cyclo ( Ala D Pro) 3 .(6 8 a) was purified by gradient chromatography DCM:Ether 1:9 to 1:1.

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170 C yclo ( D,L H omo Ala D Pro ) 2 (6.7b) was purified by gradient chromatography DCM:Ether 1:9 to 1:1. Colorless oil, 146 mg, 0.40 mmol, 68% yield. 1 H NMR (CDCl 3 1.23 (d, J = 6.4 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H), 1.76 1.88 (m, 4H), 2.04 2.15 (m, 2H), 2.37 2.45 (m, 2H), 2.48 2.61 (m, 2H), 2.89 (dd, J = 17.3, 2.9 Hz, 1H), 3.12 (t, J = 13.4 Hz, 1H), 3.43 3.53 (m, 3H), 3.54 3.60 (m, 1H), 3.69 3.76 (m, 1H), 4.03 4.09 ( m, 1H), 4.71 (dd, J = 7.9, 4.3 Hz, 1H), 4.77 (t, J = 6.9 Hz, 1H). 13 C NMR (CDCl 3 75 MHz): 21.0, 23.6, 24.4, 24.7, 29.2, 29.4, 42.8, 44.7, 45.1, 47.7, 58.6, 60.7, 171.4, 172.0, 172.7, 172.9. HRMS (ESI) calcd for C 18 H 29 N 4 O 4 [M + H] + 365.2193, found 365.2183 Cyclo ( D,L H omo Ala D Pro) 3 6.8 b MW 546, RT 26.5 min: m/z 547.3220 [M+H]+ ion. HRMS ESI MS calcd. for C 27 H 43 N 6 O 6 [M + H] + : 547.3239. Found: 547.322. Cyclo ( D,L H omo Ala D Pro) 4 6.9 b MW 728, RT 28.9 min: There were several m/z 751 ion peaks (botto m) but only the RT 28.8 ion peak correlated with the m/z 729 ion peak. HRMS ESI MS calcd. for C 36 H 56 N 8 O 8 [M + H] + : 729.4294. Found: 729.429 Cyclizations of dipeptide Cbz D,L Homo Ala D Pro Bt (6.5c) Cyclo oligomerization of Cbz D,L Phe D Pro 6 .5c. 15 mM of 6 .5a was treated with conditions stated in general procedure. The samples were analyzed via reverse phase gradient C18 HPLC/UV/(+)ESI MSn. Mobile Phases: A = 0.2% HOAc in H 2 O (B&J) B = 0.2% HOAc in Methanol (B&J ). Acetic Acid (glacial, ACS Cerified Plus; Fisher Scientific) Gradient: @ 0.15 mL/min: A:B(min) = 100:0(0)=>5:95(45 60). Cyclo (D,L Phe D Pro) 2 6 7 c MW 488: A number of m/z 489 and m/z 511 ion peaks were detected and examined. MW 488 isomers: There appeared to be at least two isomers present due to combinations of diastereomers with RT 43.96 and RT 67.37. MW 488:

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171 There was a co e luting compound which produced an m/z 488.2 ion peak which did not correlate to the m/z 489 ion p eak of MW 488. [M+Na]+, m/z 511 RT 49.42 min. C yclo ( D,L Phe D Pro ) 2 (6.7c) was purified by gradient chromatography DCM:Ether 1:9 to 1:1. Colorless oil, 75 m g, 0. 15 mmol 61% yield. 1 H NMR ( CDCl 3 300 1.76 1.96 (m, 6H), 2.02 2.18 (m, 2H), 2.62 2.76 (m, 4H), 3.45 3.58 (m, 4H), 4.53 (dd, J = 8.1, 4.0 Hz, 2H), 4.77 (dd, J = 13.2, 2.7 Hz, 2H), 5.89 (br s, 2H), 7.05 7.38 (m, 10H). 13 C NMR ( CDCl 3 75 MHz): 22.9, 23.6, 28.8, 29.9, 43.9, 46.9, 56.9, 59.8, 126.0, 126.4, 128.6, 128.8, 129.0, 129.5, 142.0, 169.1, 170.2 HRMS (ESI) calcd for C 28 H 32 N 4 O 4 Na [M + Na] + 511.2316, found 511.2303. Cyclo ( D,L Phe D Pro) 4 6.9 c MW 998. The m/z 977 ion ith m/z 500 ion peaks. In addition, MS/MS of the m/z 997 ion peaks produced m/z 500 via loss of 477 u. These are MW 477 compounds which produced m/z 500 [M+Na]+ and m/z 997 [M+Na+M]+ ions Cyclizations of dipeptide Cbz Ala 3 Aze Bt (6.5e) Cyclo oligomeriz ation of Cbz Ala 3 Aze Bt 6 .5e 0.1 mM of 6.5e was treated with conditions stated in general procedure. HPLC: Agilent (Palo Alto, CA) 1100 series binary pump. C olumn: waters XTerra MS C18 3.5 um(2.1 x 150 mm; S/N=T03401K); with Phenomenex C18 Security Guard Column (2 x 4 mm). Mobile Phases: MP A: 0.2% HOAc in H 2 O ; MP B: 0.2% HOAc in Methanol H 2 O (Optima; LCMS grade; Fisher Scientific). Methanol (MeOH; Optima; LCMS grade; Fisher Scientific). Acetic acid, glacial: Certified ACS Plus (Fisher Scientific). Gradient: @ 0.17 mL/min: A:B(min) = 95:5(0)=>65:35(10)> 5:95(65 80)

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172 Only cyclo[ Ala 3 Aze] 2 was detected with MW 308 RT 38.7. The (+)ESI MS was dominated by the m/z 331 ion, a possible [M+Na]+ ion. The m/z 331 was dissociated to m/z 275, 208, 180 and several other product ions. Cyclizations of dipeptide Cbz Ala L 2 Aze Bt (6.5 f) Cyclo oligomerization of Cbz Ala L 2 Aze Bt (6.5f). 15 mM of 6.5f was treated Ala L 2 Aze] 3 was detected. HPLC: Agilent (Palo Alto, CA) 1100 series binary pump. Column : Waters XTerra MS C18 3.5 um(2.1 x 150 mm; S/N=T03401K); with Phenomenex C18 Security Guard Column (2 x 4 mm). Mobile Phases: MP A: 0.2% HOAc in H2O ; MP B: 0.2% HOAc in Methanol H 2 O (Optima; LCMS grade; Fisher Scientific). Methanol (MeOH; Optima; LCMS grade; Fisher Sc ientific). Acetic acid, glacial: Certified ACS Plus (Fisher Scientific). Gradient: @ 0.17 mL/min: A:B(min) = 95:5(0)=>65:35(10)> 5:95(65 80). Cyclo [ Ala L 2 Aze] 3 MW 462, RT 67.75: At RT 67.7 were matching m/z 485 and 463 ion peaks. MW 462 RT 67.7 min: While the (+)ESI MS (top) contains the expected m/z 463 and m/z 485 ions (along with m/z 507) indicative of a MW 462, the m/z 463 ion was relative ly stable towards CID as m/z 463 wa s still the most abundant ion.

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173 CHAPTER 7 C ONFORMATIONALLY ASSISTED LACTAMIZATIONS FOR THE SYNTHESIS OF BIS 2,5 DIKETOPIPERAZINES Introduction 2,5 D iketopiperazines (2,5 DKPs) occur in numerous natural products often as such, but also embedded in larger, more complex molecular architectures in a variety of natural products from fungi, bacteria, the plant kingdom, and mammals (Figure 7 1). 77 125 2,5 DPKs have the ability to bind to a wide range of receptors together with several characteristics, a ttractive in scaffolds for drug discovery. 76 DKPs are small, conformationally constrained heterocyclic molecules stable to proteolysis D iversity can be introduced at up to six positions and stereochemistry con trolled at up to four positions. 77 126 127 Recent advances in solid phase methodology have increased their availability for combinatorial drug discovery 126 128 In sharp contrast to numero us studies dedicated to the synthesis and biological properties of DKPs, relatively few bis DKPs have been reported 77 129 130 They have, however, shown considerable biological ac tivity: (i) (+) WIN 64821, f rom Aspergillus flavus cultures is a potent competitive P antagonist with submicromolar potency for both the human neurokinin 1 and the cholecystokinin B receptors; 131 (ii) dimeric diketopiperazine ( ) ditryptophenaline alkaloids and ( ) N1 (2 phenylethylene)ditryptophenaline inh ibit the former receptor; 132 (iii) (+) dideoxyverticillin A is a tyrosine kinase inhibitor with potent antitumor activity ; 132 (iv), (v) Reproduced in part with permission from Ha, K ; Lebedyeva, I ; Li, Z ; Martin, K ; Williams, B ; Faby, E ; Nasajpour, A ; Pillai, G G.; Al Youbi, A. O.; Katritzky, A R. The Journal of Organic Chemistry 2013 78 8510 85 23. Copyright 2013 American Chemical Society and Nsengiyumva, O ; Hamedzadeh, S ; McDaniel, J ; Macho, J ; Simpson, G ; Panda, S S.; Ha, K ; Lebedyeva, I ; Faidallah, H M.; AL Mohammadi, M M ; Hall C. D .; Katritz ky, A R Organic & Biomolecular Chemistry 2015 13 4399 4403 Copyright 2015 The Royal Society of Chemistry

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174 n aturally occurring bis DPKs chaetocin and chetomin are in hibitors of HIF p300/CBP interaction, although the inhibition mechanism remains unclear. 133 Comparative analysis has shown that bis DKPs impact highly on the expression level of hy poxia inducible genes and have more genome wide effects than DPKs ; 134 (vi) X ray crys t allographic studies show that xylylene linked bis 2,5 DKPs obtained by direct C 3 alkylation of the N substituted 2,5 DKP core via carbanion chemistry, can adopt open and closed conformations, which enable them to serve as building block s for metallo supramolecular assemblies, metal organic polygons and other metal organic materials. 135 Figure 7 1. Representative of bioactive natural products c ontaining DKP Head to tail condensation between the N and C termini of the corresponding linear peptides represents the most straightforward synthesis for bis 2,5 DKPs (Figure 7 2) 136 137 However, head to tail condensation may require harsh conditions which causes partial epimerization, other side reactions, and low yields. D imeric DKPs can

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175 also be obtained by radical dimerizatio n (Figure 7 3) 129 138 139 or direct modificat ion of the N alkylated DKP core via carbanion chemistry (Figure 7 4), 134 135 but these procedures are challenging because multiple protection and deprotection steps limit the methodology to specific peptide sequences. Figure 7 2. Synthesis of bis DKP via h ead to tail condensatio n between the N and C termini Figure 7 3. Radica l dimerization in synthesis of bis DKP Figure 7 4. Synthesis of bis DKP using carbanion chemistry

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176 This chapter describes a method for the synthesis of both symmetrical and unsymmetrical bis 2,5 DKP s by triethylamine prom oted macrolactamization from peptidoyl benzotriazol ide s containing proline as a turn introducer (Figure 7 5) P roline has high tendency to induce reverse turns in polypeptides because it can accommodat e both the cis and the trans conformer s of a tertiary Xaa Pro amide bond (where Xaa represents any l amino acid). 5 It has therefore been utilized to introduce reverse turn s to achiev e short end to end distance in peptide chains. 140 Figure 7 5 Ring construction strategy to form DKPs Results and Discussion Synthesis of S ymmetrical B is DPKs Dimeric DKP 7.5a was sy nthesized in 4 steps (Figure 7 6 ): (i) N a N a bis Cbz L cystine (7.1a) was converted to benzotriazolide 7.2a in 86% yield; (ii) reaction of l Cbz L cystinyl benzotriazole 7.2a with D proline according to our previously reported procedure, 141 142 was complete within 3 h at 20 o C and produced dipeptide dimer bis Cbz L Cys D Pro OH 7.3a; (iii) the reaction of 7.3a with BtS(O)Bt, generated in situ at 20 o C in dry THF, led without epimerization to the Cbz N protected dipeptidoyl benzotriazolides 7.4a (82%); (iv) macrocyclization of 7.4a formed bis (Cbz L Cys D Pro) 7.5a ( Figure 7 6 Table 7 1). Optimum p reparative conditions co reagents and solvents for cyclization of 7 .4a were examined: little reaction was observed when compound 7. 4a was treated under

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177 microwave in acetonitrile without additive for 3 h and conversion reache d just 10% after 18 h under reflux in acetonitrile. Addition of triethylamine ( 2.2 equiv.), however gave the desired novel cyclic bis (Cbz L Cys D Pro) 7.5a in 76% yield ( Figure 7 6 Table 1) The presence of water (MeCN/H 2 O, 9 : 1) in the reaction mixture cause d minimal ( <5 %) hydrolysis of 7.4a Figure 7 6 C yclization of bis Cbz dip eptidoyl benzotriazoles 7.4a e No epimerization of 7.5a was detected by HPLC. Thus, HPLC analysis [chirobiotic T column (250 mm x 4.6 mm), detectio n at 2 54 nm, flow rate 5 mL min 1 MeOH] showed a single peak, retention time 12.0 min on 7 .5a which confirmed the absence of diastereomers in the lactamization product bis 2,5 DKP 7. 5a. To provide further racemization free evidence during Bt mediated lac tamization, dimeric 2,5 DKP bis (Cbz L Cys D,L Pro) 7 .5b was synthesized (Figure 7 6 Table 7 1) HPLC analysis [chirobiotic T column (250 mm 4.6 mm), de tection at 230 nm, flow rate 0.25 mL/min,

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178 MeOH] on 7. 5a (single peak, retention time 19.0 min) and 7. 5b (two equal peaks, retention times 18.9 and 20.6 min) confirmed that product 7. 5a is enantiomerically pure Compounds 7.5a,b w ere characterized by 1 H and 13 C NMR and HRMS ( Table 7 1 ) Table 7 1. En route to symmetrical bis DKPs Entry Time (h) HMRS (M+Na + ) Product Yield (%) Calcd. Found 1 48 689.1710 689.1731 76 2 50 689.1710 689.1724 78 3 58 717.2023 717.2034 83 4 62 833.2860 833.2862 84 5 64 861.3173 861.3175 88 A similar protocol was used to synthesize bis 2,5 DPK 7.5c bis (Cbz Homo D,L Cys D Pro) (83%) by cyclization of its precursor 7. 4c ( Figur e 7 6 Table 7 1). Compound 7.5c was purified by semi preparative HPLC and fully characterized by 1 H and 13 C NMR spectroscopy, HRESI MS, and analytical HPLC. 13 C NMR spectra indicated formation of

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179 desired lactamization product 7. 5c (appearance of two more up field amide carbon signals), and was corroborated by 1 H NMR data. Similarly, bis 2,5 DPK bis (Cbz L Cys L Hyp(OtBu) ) 7.5d and bis 2,5 DPK bis (Cbz Homo D,L Cys L Hyp(OtBu) ) 7.5e were synthesized in 84 % and 88% respectively ( Figure 7 6 Table 7 1 ). F urth er D evelopment of the Method for S yn thesis of Symmetrical B is 2,5 DPKs To broaden the utility of our method, we applied the procedure to the cyclization of open chain peptidoyl benzotriazolide s 7. 10a c for the synthesis of symmetrical dimeric DPKs 7. 11a c (Table 7 2) with aliphat 7 and 7 8, Table 7 2). Figure 7 7 Synthesis of bis benzotriazoles 7.10a c Figure 7 8 2 ,5 DKPs 7.11a c Bis DKP derivative ICRF 159 with an aliphatic linker between the DKP units showed unique preclinical properties: chelation with divalent cations, potential amelioration of anthracycline cardiac toxicity and possible antimetastatic effects. 143 144 Dicarboxylic acids: 3,3 dimethylglutaric and trans 1,4 cyclohexanedicarboxylic acids were chosen as linker precusors for the DKPs units To study the macrolactamization

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180 reaction to form symmetrical bis DKPs 7. 11a c the starting materials 7. 10a c were obtained in a six step procedure starting from commercially available bis benzotriazolides 7. 6a,b. Ta ble 7 2 En route to symmetrical bis DKPs 7.11a c Entry Time (h) HMRS (M+Na + ) Product Yield (%) Calcd. Found 1 78 865.4107 865.4125 91 2 82 827.2391 827.2371 85 3 84 827.2391 827.2398 81 The symmetrical N Cbz protected tripeptide derivatives 7. 7a b were prepared in yields of 72 78% by coupling bis benzotriazolides 7. 6a b with N Cbz L lysine or N C bz L cysteine Compounds 7. 7a b were then converted into N (Cbz aminoacyl) benzotriazoles 7. 8a b (78% 83% ) which w ere subsequently coupled with D proline or L proline to afford the side chain linked tetrapeptides 7. 9a c Treatment o f N Cbz tetra peptides 7. 9a c with eight equi valents of BtH and two equivalent s of thionyl chloride in tetrahydrofuran at 45 C for 6 h gave N protected tetrapeptidoyl benzotriazolides 7 .10a c in yield s 68 88 % (Figure 7 7 and 7 8 Table 7 2).

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181 Cyclizatio n of bis benzotriazolides 7. 10a c was carried out in dry acetonitrile in the presence of 3.5 equivalents of triethylamine. The macrolactamization mixture was stirred at room temperature until the TLC revealed complete reaction Open chain N Cbz protected p eptidoyl benzotriazolides 7. 10a c were converted into symmetrical bis 2,5 diketopiperazines 7. 11a c in yields 91%, 85% and 81% correspondingly by our cyclization strategy (Figure 7 8 Table 7 2). Synth esis of Unsymmetrical B is 2,5 DKPs L iterature examples of unsymmetrical bis DPKs include compounds endowed with antagonist and anticancer activity 77 145 146 We therefore target ed bis DKPs with unsymmetrical linkers t o broaden the scope of our methodology Our strategy included three key transformations: (i) preparation of unsymmetrical linkers (7. 15a e) derived from amino dicarboxylic acids (7. 12a,b ) to provide functional groups in one of the amino acid cons tituents of the DKPs (Figure 7 9 ); (ii) coupling of linkers (7. 15a e ) at both C termini with a turn introducer proline unit as a second amino acid that forms a DPK unit forming intermediates ( 7. 17a e ) (Figure 7 10 ); (iii) lactamization of ( 7 .17a e) to form the designed bis 2,5 DKPs ( 7. 19a e ). Cbz N protected L aspartic and L glutamic acids were chosen as linker precursors. Figure 7 9 shows coupling reactions with side chain functional groups in the link ers. Cbz L As p OH (7. 12a ) and Cbz L Glu OH (7. 12b ) were each converted into N (Cbz aminoacyl) benzotriazolides 7. 13a,b (86% and 92% respectively). C oupling of these benzotriazole derivatives 7. 13a,b with functional groups of the side chain s in Cbz N prot ected aminoacids 7. 14a c in the presence of diisopropylethylamine (for 7. 15a,b ) or triethylamine (for 7. 15c e ) gave 7. 15a e ( 88 93 %) (F igure 7 9 )

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182 Figure 7 9 Preparation of 7. 15a e Figu re 7 10 Synthesis of bis DKPs 7. 19a e with unsymmetrical linkers

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183 Table 7 3. En route to unsymmetrical bis DKPs 7.19a e Entry Time (h) HMRS (M+Na + ) Product Yield (%) Calcd. Found 1 48 890.2855 890.2828 72 3 52 904.3012 904.3021 88 2 54 922.2398 922.2382 76 4 48 936.2555 936.2562 86 5 52 972.4114 972.4115 88 Peptides 7. 15a e were converted into the corresponding benzotriazolides 7. 16a e (68 78%, Figure 7 10 ). Coupling 7. 16a e with Cbz protected L proline or D proline in acetonitrile water (3:1) in the presence of triethylamine provided peptides 7. 17a e which were subsequently treated with in situ generated BtS(O)Bt to afford the peptide benzotriazolides 7. 18a e in 62 72% yields. C yclization of 7. 18a e occurred under

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184 conditions similar to those used for the macrocyclization of 7. 4a c and gave the desired bis 2,5 DPKs 7. 19a e in yield s of 72 88 % (Figure 7 10 Table 7 3). S amples 7.19a e (Table 7 3) showed the advantage of our base assisted lactamization of open chain N Cbz protected peptidoyl benzotriazolides for synthesis of DKPs derivatives since direct m acrolactamization of N protected dipetides to form DKPs using peptide coupling reagents often requires harsh conditions, 77 147 148 and the deprotection/cyclization strategy can lead to extended procedures and lower yields 77 149 Conclusion I n summary, we have developed novel straightforward and versatile Bt mediated macrolactamizations for the synthesis of bis 2,5 DKPs with both symmetrical and unsymmetrical linkers. The methodology was utilized to ring clos e a series of p eptidoyl benzotriazolides yielding a small library of bis DKPs with novel features which could not be prepared efficiently using previously reported methods. T he approach described here in should provide a convenient entry to the design and synthesis of a variety of bis DKPs with potential utility in drug discovery, biological catalysis and materials chemistry Our Bt assisted macrocyclization offers the following advantages: (i) macrolactamization at room temperature; (ii) formation of bis 2,5 DKPs in good yields with no detectable racemization; (i ii ) the use of commercially available and inexpensive reagents, and (iv) easy purification Given that there are an increasing number of studies involving the synthesis of symmetrical and unsymmetrical DKPs to eva luate their biological activities, we believe this new approach represents a significant development in the field.

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185 Experimental Section General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 acetone d 6 or CD 3 OD using a 300 or 500 MHz spectrometer (with TMS as an internal standard). The following abbreviations are used to describe spin multiplicity: s = singlet, d = doublet, t = triplet q = quartet, m = multiplet, br s = broad singlet, dd = doublet of doublets, ddd = doublet of doublets of doublets and dt = doublet of triplets 3,3 DMG refers to 3,3 dimethyl glutarate, and trans 1,4 CHD refers to trans cyclohexane 1,4 dicarboxylate. HP LC MS analyses were performed on a reverse phase gradient using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; mass spectrometry was done with electrospray ionization (ESI) matrix assisted laser desorption/ionisation time of fligh t (MALDI TOF) or a tmospheric pressure chemical ionization ( APCI ). Ether refers to diethyl ether. General P rocedure I for the P reparation of B is Benzotriazolides 7.2a b and 7.13a b A stirred solution of 1 H benzotriazole (BtH) ( 8 equiv. ) in dry tetrahydrofur an (T HF) (10 mL/1 g) was treated at 20 C with thionyl chloride (SOCl 2 ) ( 2 equiv. ). After 20 minutes, a solution of 7. 1 a b or 7.12 a b ( 1 equiv. ) in dry THF (10 mL /1 g) was added drop wise and the resulting solution was then stirred for 2 h at 20 C. Upon c ompletion, the mixture was filtered, and THF was removed under reduced pressure. The residue was dissolved by dichloromethane (CH 2 Cl 2 50 mL/1 g of 7. 2a b or 7.13a b) and washed successively with HCl (4N, 2 1 mL/1 mL CH 2 Cl 2 ), aq. Na 2 CO 3 (10%, 2 1 mL/1 m L CH 2 Cl 2 ) and brine (1 mL/1 mL ). The organic layer was dried over magnesium sulfate

PAGE 186

186 ( MgSO 4 ) filtered and evaporated to give the crude product. The solid was recrystallized from CH 2 Cl 2 /hexanes to yield bis benzotriazolides 7. 2a b and 7. 13a b (Cbz L Cys Bt ) 2 ( 7. 2a). The compound was prepared according to general procedure I for preparation of bis bentrotriazolides from (Cbz Cys OH) 2 (3.17 g, 6.25 mmol) BtH (5.9 6 g, 50.0 mmol) and SOCl 2 (0.9 1 mL 12.5 mmol). White microcrystals, 3.82 g, 5.38 mmol, 86% yield ; mp 152 156 C. 1 H NMR (CDCl 3 3.2 0 3.3 7 (m, 2H), 3. 38 3.50 (m, 2H), 5.11 ( br s, 4H), 5.91 (d, J = 7. 2 Hz, 2H), 5.97 6.07 (m, 2H), 7.26 7.38 (m 10H), 7.51 (t, J = 7.3 Hz, 2H), 7.65 (t, J = 7.2 Hz 2H), 8.05 (d, J = 8.2 Hz, 2H), 8.19 (d, J = 8.2 Hz, 2H) 13 C NMR (CDCl 3 75 41.0, 54.3, 67.7, 114.5, 120.6, 126.9, 128.4, 128.7, 131.2, 136.1, 146.2, 155.9, 169.4 Anal. Calcd for C 34 H 30 N 8 O 6 S 2 : C 57.45, H 4.25, N 15.76. Found: C 57.66, H 4.09, N 15.40 (Cbz Homo D L Cys Bt) 2 ( 7. 2b). The compound was prepared according to general procedure I for preparation of symmetrical bis benzotriazolides from BtH (3.56 g, 29.8 mmo l), SOCl 2 (0.54 mL, 7.5 mmol) and (Cbz D,L Homo Cys OH) 2 (2.0 g, 3.7 mmol) White microcry stals, 1.86 g, 2.5 mmol, 68% yield; mp 98 104 C. 1 H NMR ( CDCl 3 300 2.21 2.32 (m, 2H), 2.48 2.62 (m, 2H), 2.85 2.96 (m, 4H), 5.15 (br s, 4H), 5.87 5.95 (m, 2H), 6.00 6.13 (m, 2H), 7.16 (br s, 2H), 7.28 7.42 (m, 8H), 7.50 7.57 (m, 2H), 7.66 7.70 (m, 2H), 8.09 8.15 (m, 2 H), 8.26 (t, J = 7.6 Hz, 2 H) 13 C NMR ( CDCl 3 33.1, 34.7 54.2, 67.6, 114.5, 120.6, 126.8, 128.5 128.7, 131.1, 131.3, 136.1, 146.2, 156.3, 171.3 HRMS (ESI) calcd for C 36 H 34 N 8 O 6 S 2 Na [M + Na] + 761.1935 found 761.1949. Cbz L Asp (Bt) Bt ( 7. 13a). The compound was prepared according to genera l procedure I for preparation of symmetrical bis benzotriazolides BtH (3.39 g, 28.5 mmol)

PAGE 187

187 SOCl 2 (0.52 mL, 7.1 mmol) and Cbz L Asp OH 7. 12a (4.00 g, 3.74 mmol). White microcrystals, 1.51 g 3.22 mmol, 86 % yield ; mp 132 134 C 1 H NMR ( CDCl 3 300 4. 35 4.54 (m, 2H), 5.13 (br s, 2H), 6.08 6.13 (m, 1H), 6.19 6.26 (m, 1H), 7.26 7.36 (m, 5H), 7.46 7.71 (m, 4H), 8.07 8.19 (m, 3H), 8.26 (d, J = 8.5 Hz 1H) Cbz L Glu (Bt) Bt ( 7. 13b). The compound was prepared according to general procedure I for preparation of symmetrical bis benzotriazolides from BtH (13.56 g, 113.84 mmol) SOCl 2 (2.07 mL, 26.46 mmol) and Cbz L Glu OH 7. 12b ( 4.00 g, 14.23 mmol) White microcrystals, 6.33 g, 13.09 mmol, 92% yield; mp 156 157 C. 1 H NMR ( CDCl 3 2.47 2.61 (m, 1H), 2.73 2.87 (m, 1H), 3.72 (t, J = 7.1 Hz, 2H), 5.10 (br s, 2H), 5.80 6.04 (m, 2H), 7.20 7.38 (m, 5H), 7.4 5 7.71 (m, 4H), 8.07 8.26 (m, 4H) 13 C NMR ( CDCl 3 27.5, 31.9, 54.3, 67.7, 114.5, 120.4, 120.7, 126.5, 126.9, 128.4, 128.7, 130.7, 131.1, 131.2, 136.0, 146.3, 156.1, 171.1, 171.3 Anal. Calcd for C 25 H 21 N 7 O 4 : C 62.11 H 4.38 N 20.28 Found: C 62.15 H 4.26 N 20.56. General P rocedure II for the P reparation of Di sulfide Dipeptides 7.3a e Di sulfide dipeptide benz otriazolides 7. 2a b ( 1 equiv.) were each suspended in acetonitrile/water (3:1 ) (25 mL/1 g) and a solution of D proline, D ,L proline or Hyp (OtBu) ( 2 equiv.) in water (10 mL/1 g of proline) containing triethyl amine ( 2 2.2 equiv. ) was added slowly. The mixtu re s w ere stirred at 20 C for 15 h until TLC revealed consumption of the starting materials. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate. Th e solution was washed with 4 N HCl (3 1 5 mL /1 mL of ethyl acetat e) and brine (1 mL /1 mL of ethyl acetate ). Recry stallization from ethyl acetate/ hexanes yielded di sulfide dipeptides 7.3a e (Cbz L Cys D Pro OH) 2 (7.3a). The compou nd was prepared according to general procedure II for preparation of di sulfide dipeptides from benzotriazolide 7. 2a

PAGE 188

188 ( Cbz L Cys Bt) 2 ( 3.00 g, 4.22 mmol) and D proline (0.97 g, 8.44 mmol) White microcrystals 2.22 g, 3.17 mmol, 75% yield; mp 78 80 C. 1 H NMR (DMSO d 6 300 MHz 1.96 (m, 6H), 2.08 2.18 (m, 2H), 2.85 (dd, J = 13.9, 9.9 Hz, 2H), 3.02 (dd, J = 13.7, 3.9 Hz, 2H), 3.56 3.68 (m, 4H), 4.24 (dd, J = 8.8, 3.8 Hz, 2H), 4.48 4.62 (m, 2H), 5.0 3 (br s, 4H), 7.28 7.51 (m, 10H), 7.78 (d, J = 8.2 Hz, 2H), 12.37 (br s, 2H). 13 C NMR (CD 3 .0, 129.1, 129 .6, 138.2, 158.6, 171.5, 175.3. HRMS (ESI) calcd for C 32 H 37 N 4 O 10 S 2 [M H] 701.1945 found 701.1959 (Cbz L Cys D,L Pro OH) 2 (7.3b). The compound was prepared according to general procedure II for preparation of di sulfide dipeptide from ben zotriazolide 7 .2a (Cbz L Cys Bt) 2 (2.00 g, 2.82 mmol) and D,L proline (0.65 g, 5.63 mmol). White microcrystals, 1.54 g, 2.20 mmol, 78% yield; mp 68 72 C. 1 H and 13 C NMR were identical to 7. 3a. (Cbz D,L Homo Cys D Pro OH) 2 ( 7. 3c). The compound was prepared according to general procedure II for preparation of di sulfide dipeptides from (Cbz D L Homo Cys Bt) 2 7. 2c (1.0 g, 1.35 mmol) and D proline ( 0.31 g, 2.70 mmol) Sticky gel 0.77 g, 1.05 mmol, 78% yield. 1 H NMR ( DMSO d 6 1.80 2.00 (m. 8H), 2.02 2.24 (m, 4H), 2.62 2.82 (m, 4H), 3.64 (br s, 4H), 4.14 4.27 (m, 2 H), 5.01 (t, J = 3.4 Hz, 2H), 5.04 (br s, 4H), 7.24 7.40 (m, 10H), 7.81 (d, J = 8.3 Hz, 2H) 13 C NMR ( CD 3 OD 26.0, 30.2, 32.2 35.6, 53.0 54. 1, 67.8 127.2, 128.9, 129.1, 129.5, 138.1, 158.7, 174.2. HRMS (ESI) calcd for C 34 H 4 1 N 4 O 10 S 2 [M H] 729.2259 found 729.226 8 Bis Cbz L Cys L Hyp(O t Bu) OH (7.3d). The compound was prepared according to general procedure II for preparation of di sulfide dipeptides from (Cbz L Cys Bt) 2 7. 2c

PAGE 189

189 and Hyp(O t Bu ). Sticky oil 1.47 g, 1.73 mmol, 82 % yield. 1 H NMR ( CD 3 OD 300 MHz): 7.42 7.21 (m, 10H), 5.09 (br s, 4H), 5.15 5.03 (m, 4H), 4.80 (dd, 2H), 4.54 4.32 (m, 4H), 3.92 (dd, J = 10.7, 5.5 Hz, 2H), 3.66 (dd, 2H), 3.17 (dd, J = 14.1, 4.8 Hz, 2H), 2.89 (dd, J = 14.3, 9.2 Hz, 2H), 2.25 1.98 (m, 4H), 1.19 (br s, 18H) 13 C NMR ( CD 3 OD 171.5, 158.5, 138.1, 130.0, 129.6, 129.4, 129.1, 129.0, 128.9, 75.6, 71.1, 68.4, 68.0, 59.6, 55. 5, 53.5, 41.0, 38. 2, 29.1, 28.7 HRMS (ESI): calcd for C 40 H 53 N 4 O 12 S 2 [M H] 845.3095 found 845.3091. Bis Cbz Homo D,L Cys L Hyp(O t Bu) OH (7.3e). The compound was prepared according to general procedure II for preparation of di sulfide dipeptides from ( Cbz Homo D,L Cys ) 2 7. 2 b and Hyp(O t Bu ). White solid 0.24 g, 0.65 mmol, 77% yield; mp 79.0 83.0 C 1 H NMR ( CD 3 OD 7.38 7.26 (m, 10H), 5.18 5.04 (m, 4H), 4.66 4.58 (m, 2H), 4.49 (t, J = 6.7 Hz, 2H), 4.46 4.29 (m, 2H), 3.92 3.82 (m, 2H), 3.74 3.62 (m, 2H), 2.87 2.63 (m, 4H), 2.23 2.13 (m, 4H), 2.13 2.05 (m, 2H), 2.05 1.9 1 (m, 2H), 1.33 1.11 (m, 18H) 13 C NMR ( CD 3 OD 175.5, 172.6, 158.5, 138.3, 129.6, 129.2, 129.1, 129.0, 110.2, 75.7, 71.2, 69.3, 67.9, 67.8, 59.5, 59.3, 55.7, 54.2, 52.6, 38.4, 38.3, 35.6, 35.0, 32.5, 28.74, 28.71 HRMS (ESI): calcd for C 42 H 57 N 4 O 12 S 2 [M H] 873.3408 found 873.3401. General P rocedure III for the P reparation of Di Sulfide Dipeptidoyl Bentrotriazolides 7.4a e A stirred solution of BtH ( 8 equiv. ) in dry tetrahydrofuran (THF) (15 mL/1 g) was treated at 2 0 C with SOCl 2 ( 1 equiv. ). After 20 minu tes, a solution of 7. 3a e ( 1 equiv. ) in dry THF (15 mL /1 g 7.3a e ) was added drop wise and the resulting solution s were then stirred for 1.5 h at 20 C. The ice bath was then removed and the reaction mixture was stirred for an additional 1.5 h at room tem perature. The mixture was filtered and

PAGE 190

190 THF was removed under reduced pressure. The residue was dissolved CH 2 Cl 2 ( 10 0 mL /1 g 7. 3a c ) and washed successively with HCl (4N, 2 0.7 mL /1 mL of CH 2 Cl 2 ), Na 2 CO 3 10 wt. % in water (2 1 mL /1 mL of CH 2 Cl 2 ) and bri ne (1 30 mL). The organic layer was dried over magnesium s ulfate, filtered and evaporated. The crude product was then recrystallized from CH 2 Cl 2 /hexanes to yield di sulfide dipeptidoyl bentrotriazolides 7. 4a e (Cbz L Cys D Pro Bt) 2 ( 7. 4a). The compound was prepared according to general procedure III for preparation of di sulfide benzotriazolide from 7. 3a ( Cbz L Cys D Pro OH) 2 (1.50 g, 2.13 mmol) BtH (2.03 g, 17.07 mmol) in and SOCl 2 (0.31 mL, 4.27 mmol). White microcrystals 1.58 g, 1.75 mmol 82% yield ; mp 119 124 C. 1 H NMR (DMSO d 6 2.02 2.26 (m, 6H), 2.38 2.47 (m, 2H), 2.89 (dd, J = 13.7, 8.8 Hz, 2H), 3.14 (dd, J = 13.7, 5.1 Hz, 2H), 3.74 3.95 (m, 4H), 4.72 4.82 (m, 2H), 5.09 (br s, 4H), 5.68 5.78 (m, 2H), 7.28 7.42 (m, 10H), 7.64 (dt, J = 8.3, 1.2 Hz, 2H), 7.81 (dt, J = 8.3 1.1 Hz, 2 H), 7.84 (d, J = 8.6 Hz, 2H), 8.22 (d, J = 8.3 Hz, 2H), 8.29 (d, J = 8.3 Hz, 2H) 13 C NMR ( CDCl 3 75 25.2, 29.9, 41.2, 48.0, 52.1, 60.0, 67.3, 114.7, 120.4, 126.6, 128.2, 128.3, 128.7, 130.7, 131.3, 136.3, 146.2, 155.9, 169.0, 170.3 HRMS (ESI) calcd for C 44 H 44 N 10 O 8 S 2 Na [M + Na] + 927.2677, found 927.26 81 (Cbz L Cys D,L Pro Bt)2 (7.4b). The compound was prepared according to general procedure III for the preparation of di sulfide benzotriazolide from 7.3b (Cbz L Cys D,L Pro OH)2 (1.0 0 g, 1.42 mmol), BtH (1.36 g, 11.39 mmol) and SOCl 2 (0.21 mL, 2.84 mmol). White microcrystals, 1.09 g, 1.21 mmol, 85% yield; mp 111 114 C. 1H and 13 C NMR were identical to 7. 4a.

PAGE 191

191 (Cbz D,L Homo Cys D Pro Bt) 2 (7.4c). The compound was prepared according to g eneral procedure III for preparation of di sulfide dipeptidoyl benzotriazolides from BtH (5.95 g, 50. 0 mmol), SOCl 2 (0.90 mL, 12.5mmol) and (Cbz D L Homo Cys D Pro OH) 2 7. 3c (4.57 g, 6.25 mmol). White microcrystals, 5.32 g, 5.7 mmol 91% yield; mp 83 84 C. 1 H NMR (DMSO d 6 1.78 2.00 (m, 4H), 2.01 2.26 (m, 8H), 2.66 2.86 (m, 4H), 3.49 4.29 (m, 4H), 4.34 4.74 (m, 2H), 5.03 (br s, 4H), 5.21 5.32 (m, 1H), 5.60 5.80 (m, 1H), 7.24 7.46 (m, 12H), 7.56 7.84 (m, 4H), 8.14 8.24 (m, 2H), 8.26 8.34 (m, 2 H) 13 C NMR ( CDCl 3 25.2, 25.5, 29.9, 32.4, 34.1, 47.9, 51.5, 59.9, 67.2, 114.6, 120.4, 126.4, 126.6, 128.3, 128.7, 128.9, 130.8, 136.3, 146.1, 156.3, 170.2. HRMS (ESI) calcd for C 46 H 48 N 10 O 8 S 2 Na [M + Na] + 955.2990, found 955.299 6 ( Cbz L Cys L Hyp(O t Bu) Bt) 2 ( 7.4 d ). The compound was prepared according to general procedure III for preparation of di sulfide dipeptidoyl benzotriazolides from BtH, SOCl 2 and (Cbz L Cys L Hyp(O t Bu) OH) 2 7. 3 d). White solid 0.84 g, 0.80 mmol, 68 % yield; mp 74.0 80.0 C 1 H NMR ( CDCl 3 3 8.25 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 8.4 Hz, 2H), 7.71 7.57 (m, 2H), 7.56 7.46 (m, 2H), 7.45 7.21 (m, 10H), 6.12 5.92 (m, 2H), 5.87 (d, J = 8.9 Hz, 2H), 5.20 5.03 (m, 4H), 5.03 4.82 (m, 2H), 4.73 4.19 (m, 2H), 4.18 3.86 (m, 2H) 3.75 (t, J = 12.9 Hz, 2H), 3.41 2.71 (m, 4H), 2.60 2.40 (m, 2H), 2.3 7 2.19 (m, 2H), 1.19 (s, 18H) 13 C NMR ( CDCl 3 170.4, 169.4, 166.7, 156.1, 152.5, 146.2, 136.3, 131.3, 130.7, 128.8, 128.7, 128.3, 128.2, 128.1, 127.7, 126.6, 120.4, 114 .7, 74.8, 74.6, 69.9, 69.4, 67.6, 67.3, 67.1, 58.9, 58.8, 55.0, 54.0, 52.2, 42.1, 41.4, 39.3, 38.5, 37.8, 2 9.9, 28.4 HRMS (ESI): calcd for C 52 H 60 N 10 O 10 S 2 Na [M + Na] + 1071.3827 found 1071.3825.

PAGE 192

192 ( Cbz D,L Homo Cys L Hyp(O t Bu) Bt) 2 ( 7.4e ). ). The compound was prepared according to general procedure III for preparation of di sulfide dipeptidoyl benzotriazolides from BtH, SOCl 2 and (Cbz D,L Homo Cys L Hyp(O t Bu) OH) 2 7. 3 e White solid, 0.94 g, 0.87 mmol, 76 % yield ; mp 77.0 82.0 C 1 H NMR (CDCl 3 300 8.31 8.19 (m, 2H), 8.18 8.05 (m, 2H), 7.74 7.57 (m, 2H), 7.57 7.44 (m, 2H), 7.40 7.27 (m, 10H), 6.13 5.95 (m, 2H), 5.93 5.58 (m, 2H), 5.19 5.00 (m, 4H), 4.86 4.71 (m, 2H), 4.55 4.40 (m, 2H), 4.15 3.89 (m, 2H), 3.79 3.5 6 (m, 2H), 2.88 2.65 (m, 4H), 2.62 2.43 (m, 2H), 2.34 2.21 (m, 6H ), 1.25 1.12 (m, 18H) 13 C NMR (CDCl 3 170.5, 170.4, 156.3, 146.3, 136.4, 131.3, 130.8, 128.9, 128.7, 128.4, 128.3, 128.2, 126.6, 126.3, 120.5, 114.7, 74.9, 70.0, 69.7, 67. 2, 58.6, 54.7, 51.5, 37.6, 32.5, 29.9, 28.4 28.3. HRMS (ESI): calcd for C 54 H 64 N 10 O 10 S 2 Na [M + Na] + 1099.4140 found 1099.4135. General P rocedure IV for the Cyclization of Di Sulfide Dipeptidoyl Bentrotriazolides 7.4a c to Form Symmetrical B is DKPs 7.5a e A solution of di sulfide dipeptidoyl bentrotriazolides 7. 4a e (1 equiv. ) and triethylamine ( 2.2 equiv. ) in dry acetonitrile ( 15 mL /1 g ) was stirred at room temperature until the TLC revealed complet ion of the reaction. T he mixture was then concentrated un der vacuum and the residue was dissolved in ethyl acetate T he organic layer was washed with 4 N HCl (3 1 mL /1 mL of ethyl acetate) and Na 2 CO 3 10 wt. % in water (3 1 mL /1 mL of ethyl acetate ), and purified by column chromatography (hexanes/ethyl acetate gradient) to give the corresponding symmetrical b is DKPs 7. 5a e. Bis [cyclo (Cbz L Cys D Pro)] (7.5a). The compound was prepared according to general procedure IV for preparation of symmetrical bis DKPs from (Cbz L Cys D Pro Bt) 2 7.4a (0.90 g, 1.00 mmol) White microcrystals 0. 51 g, 0. 76 mmol 76 % yield; mp

PAGE 193

193 1 16 1 20 C. 1 H NMR ( CDCl 3 1.86 1.94 (m, 2H), 1.97 2.11 (m, 4H), 2. 38 2.46 (m, 2H), 3.23 (d, J = 6.1 Hz, 2H), 3.42 3.63 (m, 6H), 4. 39 (dd J = 9.1, 6.9 Hz, 2H), 5.04 ( dd J = 5.8, 5.8 Hz, 2 H), 5.27 (d, J = 7.5 Hz, 2H), 5.31 ( d J = 7.5 Hz, 2 H), 7.31 7.41 (m, 10H) 13 C NMR ( CDCl 3 22.5, 29.7, 40.6, 45.8, 60.0, 60.7, 69.7, 128.6, 128.9, 134.7, 152.1, 163.1, 167.3 HRMS (ESI) calcd for C 32 H 34 N 4 O 8 S 2 Na [M + Na] + 689.1710 found 689.173 1 Bis [cyclo (Cbz L Cys D,L Pro)] (7.5b). The compound was prepared according to general procedure IV for the preparation of symmetrical bis DKPs from (Cbz L Cys D,L Pro Bt) 2 7.4b (0.75 g, 0.83 mmol) Isolated as mixture of diastereomers White microcrystals, 0.43 g, 0.65 mmol, 78% yield; mp 96 101 C. 1 H and 13 C NMR were identical to 7. 5a HRMS (ESI) calcd for C 32 H 34 N 4 O 8 S 2 Na [M + Na] + 689.1710 found 689 .1724. Bis [cyclo (Cbz Homo D,L Cys D Pro)] (7.5c). The compound was prepared according to general procedure IV for preparation of symmetrical bis DKPs from 7 .4c (Cbz D,L Homo Cys D Pro Bt) 2 (0.90 g, 0.5 0 mmol) White mic rocrystals, 0.29 g, 0.42 mmol 83% yield; mp 85 86 C. 1 H NMR ( CDCl 3 1.86 2.24 (m, 10 H), 2.35 2.48 (m, 2H), 2.64 2.78 (m, 4H), 3.48 3.59 (m, 4H), 4.26 (t, J = 7.6 Hz, 2H), 4.85 (t, J = 7.7 Hz, 2H), 5.29 (br s, 4H) 7.26 7.44 (m, 10H) 13 C NMR ( CDCl 3 23.1, 29.6, 32.0, 34.4, 46.0, 59.9, 60.7, 69.8, 1 28.8, 129.0, 129.1, 135.0, 152.5, 164.9, 167.7 HRMS (ESI) calcd for C 34 H 38 N 4 O 8 S 2 Na [M + Na] + 717.2023 found 717.2034 C yclo [Bis Cbz L Cys L Hyp(O t Bu)] (7.5d) The compound was prepared according to general procedure IV for preparation of symmetrical bi s DKPs from 7 5d ( Cbz L Cys L Hyp(O t Bu) Bt) 2 Sticky oil, 227 mg, 0.28 mmol, 8 4% yield. 1 H NMR

PAGE 194

194 ( CD 3 OD 7.51 7.22 (m, 10H), 5.48 (dd, J = 4.0, 2.0 Hz, 2H), 5.34 (s, 4H), 4.59 (dd, J = 5.5, 4.1 Hz, 2H), 3.78 (dd, J = 11.6, 5.6 Hz, 2H), 3.59 (dd, J = 11.8, 4.1 Hz, 2H), 3.47 (dd, J = 14.3, 4.5 Hz, 2H), 3.35 (dd, J = 4.2, 0.9 Hz, 2H), 2.88 (dd, J = 14.2, 5.2, 1.0 Hz, 2H), 2.33 (dd, J = 14.2, 5.8 Hz, 2H), 2.2 3 2.20 (m, 2H), 1.21 (s, 18H). 13 C NMR ( CD 3 OD 168.6, 167.3, 166.3, 164.7, 152.5, 136.7, 136.6, 131.6, 130.1, 130.0, 129.7, 129.6, 129.4, 127.2, 75.9, 71.6, 70.6, 69.2, 60.1, 59.2, 57.0, 55.5, 52.7 39.8, 38.6, 34.0, 32.4, 28.7. HRMS (ESI): calcd for C 40 H 50 N 4 O 10 S 2 Na [M + Na] + 833.2860 found 833.2862 C yclo [Bis Cbz Homo D,L Cys L Hyp(O t Bu)] ( 7.5e ) The compound was prepared according to general procedure IV for preparation of symm etrical bis DKPs from 7 5e ( Cbz Cbz Homo D,L Cys L Hyp(O t Bu) Bt) 2 Sticky oil, 240 mg, 0.2 9 mmol, 88 % yield. 1 H NMR ( CD 3 OD 5 7.49 7.25 (m, 10H), 5.35 5.24 (m, 4H), 4.98 4.87 (m, 2H), 4.72 4.50 (m, 2H), 4.48 4.33 (m, 2H), 3.80 3.69 (m, 2H), 3.60 3.38 (m, 2H), 2.83 2.65 (m, 4H), 2.53 2.43 (m, 2H), 2.34 2.18 (m, 4H), 2.17 1.9 5 (m, 2H), 1.25 1.16 ( m, 18H). 13 C NMR ( CD 3 OD 172.1, 169.6, 169.0, 166.7, 153.5, 136.7, 136.6, 130.0, 129.8, 129.7, 129.6, 129.4, 129.2, 129.0, 121.2, 76.0, 75.6, 71.2, 70.4, 69.4, 68.9, 68.7, 67.7, 61.4, 60.2, 59.6, 59.0, 55.5, 53.9, 53.4, 44.8, 39.0, 37.8, 35.4, 3 5.3, 32.3, 30.9 28.7, 28.6. HRMS (ESI): calcd for C 42 H 54 N 4 O 10 S 2 Na [M + Na] + 861.3173 found 861.3175 General P rocedure V for the O S and N Acylations of B is Benzotriazolides 7.6a b and 7.13a b for Preparation of Compounds 7. 7a b and 7. 15a e Bis benzo triazolides 7. 6a b or 7.13a b (1 equiv.) were each suspended in acetonitrile /water (3:1) (20 mL/1 g) (for S and N acylations ) or acetonitrile (for O acylation ) and a solution of Cbz L Cys OH (2 2.1 equiv. ) or Cbz L Lys OH (2 2.1 equiv.)

PAGE 195

195 in water (10 mL/1 g ) cont aining triethylamine (2.2 equiv. ) or Cbz L Ser OH (2 2.1 equiv.) in acetonitrile (10 mL/1 g) cont aining diisopropylamine (DIPEA) (6.5 equiv. ) w as added slowly. The mixture s were stirred at 20 C for 16 72 h until the TLC revealed consumption of the s tarting materials. The solvent was removed under reduced pressure and the residue wa s dissolved in ethyl acetate (30 mL /1 g of 7. 6a b 7. 13a b ). T he organic layer was washed with 4 N HCl (3 1 mL /1 mL of ethyl acetate) evaporated and the products were recr ystallized from ethyl acetate /hexanes to yield 7. 7a b or 7. 15a e. 3,3 DMG (Cbz L Lys OH) 2 (7.7a). The compound was prepared by N acylation of 3,3 DMG (Bt) 2 7. 6a (3.94 g, 10.87 mmol) by Cbz L Lys OH (6.09 g, 21.73 mmol) according to general procedure V. Sti cky gel, 5.36 g, 7.83 mmol, 72% yield. 1 H NMR (CD 3 1.08 (s, 6H), 1.36 1.61 (m, 8H), 1.64 1.75 (m, 2H), 1.80 1.92 (m, 2H), 2.25 (s, 2H), 2.35 (s, 2H), 3.18 ( t, J = 6.8 Hz 2 H), 3.26 ( t, J = 6.8 Hz 2 H), 4.09 4.17 (m, 2H), 5.09 (s, 4H), 7.27 7.38 (m, 10H) 13 C NMR (CD 3 24.4, 2 8.7, 30.0, 32.5, 34.1 40.2 47.0 55.3, 67.7 128.9, 129.1, 129.6 138.2, 158.7, 174.4, 176.0. HRMS (ESI) calcd for C 35 H 47 N 4 O 10 [M H] 683.328 7 found 683.32 93. Trans 1,4 CHD (Cbz L Cys OH) 2 ( 7. 7b). The compound was prepared by S acylation of t rans 1,4 CHD (Bt) 2 7. 6b ( 1.12 g, 2.99 mmol) a ccording to general procedure V Sticky gel, 1.5 1 g, 2.33 mmol, 78% yield. 1 H NMR (CD 3 1.52 1.75 (m, 4H), 1.78 2.06 (m, 4H), 2.40 2.68 (m, 2H), 3.13 (dd, J = 13.7, 8.9 Hz, 2H), 3.50 (dd, J = 13.8, 4.5 Hz, 2H), 4.36 (dd, J = 8.6 Hz, 4.4 Hz, 2H), 5.18 5.01 (m, 4H), 7.21 7.41 (m, 10H). 13 C NMR (CDCl 3 49.3, 53.7, 67.4,

PAGE 196

196 128.3, 128.3 128.7, 136.1, 156.2, 173.8, 201.6. Compound 7. 7b was characterized by 1 H and 13 C NMR Cbz L Asp (Cbz L Ser OH) Cbz L Ser OH ( 7. 15a). The compound was prepared by O acylation of Cbz L Asp (Bt) Bt 7.13a ( 4.0 0 g, 8.52 mmol) according to general procedure V Sticky gel, 5.32 g, 7.50 mmol, 88% yield. 1 H NMR ( CD 3 OD 2.7 9 (dd J = 16.9, 7.0 Hz, 1 H), 2. 87 (dd J = 16.9, 7.0 Hz, 1 H) 3. 79 (ddd J = 18.0, 11.4, 4.5 Hz, 1H), 4.26 4.42 (m, 2H), 4.45 4.63 (m, 4H), 5.07 (br s, 6H), 7.26 7.40 ( m, 15H) 13 C NMR ( CD 3 OD 37.2, 51.9, 54.6, 57.8, 63.2, 65.6, 66.0, 68.0, 68.1, 127.2, 128.9, 129 .1, 129.3, 1 29.5, 137.9, 138.0, 158.4, 158.5, 171.7, 172.0, 172.4, 172.6. Anal. Calcd for C 34 H 35 N 3 O 14 : C 57.54 H 4.97 N 5.92 Found: C 57.37, H 5.02, N 5.96. Cbz L Glu (Cbz L Ser OH) Cbz L Ser OH (7.15b). The compound was prepared by O acylation of Cbz L Glu (Bt) B t 7.13b ( 4.0 g, 8.28 mmol) according to general procedure V. Sticky gel, 5.51 g, 7.62 mmol, 92% yield 1 H NMR (DMSO d 6 1.93 2.04 (m, 1H), 2.21 2.40 ( m, 2H), 2.42 2.47 (m, 1 H), 3.66 (d, J = 4.8 Hz, 1H), 4.00 4.12 (m, 1H), 4.20 (dd, J = 10. 5, 6.9 Hz, 1H), 4.31 4.42 (m, 2H), 4.48 (dd, J = 10.8, 4.5 Hz, 1H), 4.70 (dd, J = 9.8 2 .6 Hz, 1H), 4.99 5.07 (m, 4H), 5. 15 5.26 (m 2H) 7.24 7.46 (m, 15H), 7.62 7.80 (m, 2H), 7.91 (br s, 1H) 13 C NMR (DMSO d 6 75 MHz): 20.9, 30.6, 52.7, 56.6, 58.3, 61.3, 64.0, 65.4, 65.7, 67.3, 127.5, 127.7, 128.1, 128.3, 135.4, 136.8, 150.5, 156.0, 170.5, 170.9, 172.0, 172.8 HRMS (ESI) c alcd for C 35 H 36 N 3 O 14 [M H ] 722.2192 found 722.21 81. Cbz L Asp (Cbz L Cys OH) Cbz L Cys OH (7.15c). The compound was prepared by S acylation of Cbz L Asp (Bt) Bt 7.13a (0.97 g, 2.07 mmol) according to

PAGE 197

197 general procedure V. Sticky gel, 1.38 g, 1.86 m mol, 90% yield 1 H NMR ( DMSO d 6 300 3.19 (m, 6H), 4.02 4.19 (m, 2H), 4. 42 4.68 (m, 1H), 5.0 3 (br s, 4H), 5.07 (br s, 2H), 7.22 7.41 (m, 15H), 7.75 (d, J = 9 .0 Hz 2H), 8.18 (t, J = 9.0 Hz 1H). 13 C NMR (CD 3 OD, 75 MHz): 128.9 129.1, 12 9.6, 138.2, 158.5, 165.0, 173.4, 196.7 HRMS (ESI) calcd for C 34 H 34 N 3 O 12 S 2 [M H] 740.1578, found 740.15 94. Cbz L Glu (Cbz L Cys OH) Cbz L Cys OH (7.15d). The compound was prepared by S acylation of Cbz Glu (Bt) Bt 7.13b ( 4.0 0 g, 14.2 mmol) according to general procedure V. Sticky gel, 9.65 g, 12.78 m mol, 90% yield 1 H NMR ( CDCl 3 300 1. 94 2.08 (m, 1H), 2.14 2.49 (m, 2H), 2.50 2.71 (m, 1H), 3.05 3.38 (m, 3H), 3 .43 3.58 (m, 1H), 4.47 4.80 (m, 3H), 4.99 5 .26 (m, 6H), 5.56 (d, J = 9.0 Hz, 1H), 5.87 (d, J = 9.0 Hz, 1H), 7.23 7.40 (m, 14H), 7.53 (br s, 2 H) 13 C NMR ( CDCl 3 75 M 22.5, 30.8, 53.3, 65. 3 67.5, 68.8, 128.3, 128.4, 128.7, 134.8, 136.0, 150.8, 156.2, 173.1, 173.7, 198.3 HRMS (ESI) calcd for C 35 H 36 N 3 O 12 S 2 [M H] 754.1735 found 754.17 16. Cbz L Asp (Cbz L Lys OH) Cbz L Lys OH (7.15e). The compound was prepared by N acylation of Cbz L Asp (Bt) Bt 7.7b (4.0 g, 8.52 mmol) by Cbz L Lys OH (4.78 g, 17.05 mmol) according to general procedure V Sticky gel, 6.27g, 7.92 mmol, 93% yield. 1 H NMR (CD 3 OD 1.37 1.45 (m, 5 H ), 1.57 1.72 (m, 5H), 1.80 1.90 (m, 2H), 2.64 (dd, J = 5.7, 5.7 Hz, 1 H ), 2.67 (dd, J = 5.6, 5.6 Hz, 1H), 3.00 (dd, J = 10.7, 5.6 Hz, 1H) 3.52 (t, J = 3.9 Hz, 3H), 4.111 4.15 (m, 2H), 4.32 4.39 (m, 1H), 5.06 (s, 2H), 5.08 (s, 4H), 7.25 7.31 (m, 3H), 7.32 7.38 (m, 12H). 13 C NMR (CD 3 OD 23.8, 27.9, 29.7, 32.1, 36.1, 39.4, 51.1, 51.2, 55.1, 67.6, 67.9, 68.0, 128.7, 128.9, 129.1,

PAGE 198

198 129.4, 137.8, 138.1, 158.1, 158.6, 175.8, 176.9, 178.3. Anal. Calcd for C 40 H 49 N 5 O 12 : C 60.67 H 6.24 N 8.84 Found: C 60.61, H 6.27, N 9.91. General P rocedure VI for the Preparation of B is Benzotriazolides 7.8a b and 7.16a e A stirred solution of BtH (8 equiv.) in dry THF (10 mL/1 g) was treated at 20 C by SOCl2 (2 equiv.). After 20 minutes, a solution of 7.8a b or 7.15a e (1 equiv.) in dry THF (15 mL/1 g) was add ed drop wise 20 C and each resulting solution was stirred for 2 h at 20 C. The ice bath was removed and each reaction mixture was stirred for additional 2 h at room temperature. The mixtures were filtered, and THF was removed under reduced pressure. Ea ch residue was dissolved in ethyl acetate (50 mL/1 g of 7.7a b or 7.15a e) and washed successively with HCl (4N, 2 1.5 mL/1 mL of ethyl acetate), Na2CO3 10 wt. % in water (2 1.5 mL/1 mL of ethyl acetate) and brine (1 1 mL/1 mL of ethyl acetate). The organic layers were dried over sodium sulfate, filtered, evaporated, and recrystallized from CH2Cl2/hexane to give the corresponding bis benzotriazolides 7.8a b or 7.16a e. 3,3 DMG (Cbz L Lys Bt)2 (7.8a). The compound was prepared accoding to general proce dure VI for preparation of bis benzotriazolid e from BtH. Sticky gel 1.01 g, 1.14 mmol, 78% yield. 1 H NMR (DMSO d 6 1.50 (m, 8H), 1.79 1.96 (m, 4H), 2.25 (s, 4H), 2.92 3.06 (m, 4H), 5.03 ( br s, 4H), 5.28 5.52 (m, 2H), 7.28 7.38 (m, 10H), 7.53 7.66 (m, 2 H), 7.74 7.81 (m, 2H), 7.85 7.92 (m, 2H), 8.16 8.29 (m, 4 H). 13 C NMR ( CDCl 3 75 MHz): 30.9, 32.6, 34.1, 44.4, 69.4, 114.6, 120.1, 125.9, 126.3, 127.7, 128.5, 130.5, 131.1, 138.9, 146.1, 156.5, 170.9, 172.4. HRMS (ESI) calcd for C 47 H 54 N 10 O 8 Na [M + Na ] + 909.4018 found 909.4 038.

PAGE 199

199 Trans 1,4 CHD (Cbz L Cys Bt)2 (7.8b). The compound was prepared according to general procedure VI for preparation of bis benzotriazolides from BtH Sticky gel, 0.70 g, 0.83 mmol, 83% yield. 1 H NMR (CDCl 3 1.83 (m, 8H), 2.42 2.58 (m, 2H), 3.51 (dd, J = 14.6, 4.7 Hz, 2H), 3.76 (dd, J = 14.6, 5.3 Hz, 2H), 5.08 5.17 (m 4H), 5.87 6.20 (m, 4H), 7.20 7.42 (m, 10H), 7.51 (t, J = 7.8 Hz, 2H), 7.64 (t, J = 7.8 Hz, 2H), 8.11 8.24 (m, 4H). 13 C NMR (CDCl 3 67.4, 114.3, 120.4, 126.6, 128.2, 128.2, 1 28.5, 130.9, 131.0, 146.1, 155.6, 168.8, 200.9. HRMS (ESI) calcd for C 42 H 40 N 8 O 8 S 2 Na [M + Na] + 871.2303 found 871.232 2 Cbz L Asp (Cbz L Ser Bt) Cbz L Ser Bt (7.16a). The compound was prepared according to general procedure VI from BtH (3.5 g, 29.6 mmol) SOCl 2 (0.5 4 mL, 7.5 mmol) and Cbz L Asp (Cbz L Ser OH) Cbz L Ser OH 7.15a (2.00 g, 3.7 mmol) White microcrystals, 1.86 g, 2.5 mmol 68%; mp 80 83 C 1 H NMR (CDCl 3 2.76 (dd, J = 17.0, 4.4 Hz, 1H), 2.87 (dd, J = 17.4, 4.5 Hz, 1H), 4.50 4.59 (m, 2H), 4.65 4.73 ( m, 1H), 4.77 4.96 (m, 2H), 5.02 5.17 (m, 6H), 5.79 5.90 ( m, 1H), 5.94 6.60 ( m 2H), 6.24 (t, J = 10.2 Hz, 2H), 7.20 7.37 (m, 15H), 7.43 7.59 (m, 4 H), 8.05 8.14 (m, 4H). 13 C NMR ( DMSO d 6 35.5 50.1, 53.6 63.1, 63.6 65.7, 66.11, 113.9, 120.22 126.8, 127. 7, 127. 9 128. 4 130.6, 130. 9 131. 2, 136.5, 136.6 136.8, 14 5.3 155.7 156.1 168.4, 168.5, 169.5 170.3. HRMS (ESI) calcd for C 46 H 41 N 9 O 12 Na [M + Na]+ 934.2767 found 934.27 56 Cbz L Glu (Cbz L Ser Bt) Cbz L Ser Bt (7.16b). The compound was prepared according to general procedure VI for preparation of bis benzotriazolides from BtH (3.56 g, 29.8 mmol) SOCl 2 (2.17 mL, 7.5 mmol) and Cbz L Glu ( Cbz L Ser OH) Cbz L Ser OH 7.15b ( 2.00 g, 3.7 mmol) W hite microcrystals, 1.86 g, 2.5 mmol, 68% yield; mp 71

PAGE 200

200 75 C. 1 H NMR ( CDCl 3 1.97 2.13 (m, 1H), 2.22 2.37 (m, 1H), 2.39 2.66 (m, 2H), 4.44 (dd, J = 12.3, 5.1 Hz, 1H), 4.66 ( dd J = 9.3, 2.9 Hz, 1 H), 4.68 ( dd J = 9.3, 2.3 Hz, 1 H) 4.78 4.86 (m, 2H), 5.04 5.20 (m, 4H), 5.21 5.34 (m, 2H), 5.81 (d, J = 7.5 Hz, 1H), 5.94 6.08 (m, 2H), 6.09 6.1 9 (m, 1H), 6.39 6.52 (m, 1H), 7.28 7. 42 (m, 15H), 7.48 7.60 (m, 2H), 7.61 7.7 0 (m, 2H), 8.09 8. 32 (m, 4 H) 13 C NMR ( CDCl 3 21.8, 21.9, 31.2, 54.9, 58.8, 59.0, 65.3, 67.8, 69.0 114.4, 115.1, 120.7, 126.3, 127.1, 128.4, 128. 5, 128.7, 131.1, 131.3, 135.0, 136.0, 138.7, 146.2, 151.5, 156.1, 168.0 170.8, 172.7, 173.6 HRMS (ESI ) calcd for C 47 H 43 N 9 O 12 Na [M + Na] + 948.2923, found 948.29 38. Cbz L Asp (Cbz L Cys Bt) Cbz L Cys Bt (7.16c). The compound was prepared according to gene ral procedure VI for the preparation of bis benzotriazolides. Sticky gel, 0.99 g, 1.05 mmol, 78% yield. 1 H NMR (CDCl 3 3.71 (m, 4H), 3.76 4.00 (m, 2H), 4.50 4.96 (m, 1H), 5.15 (br s, 6H), 5.40 5.80 (m, 2H), 5.80 6.20 (m, 3H), 7.24 7.40 (m, 15H), 7.42 7.59 (m, 3H), 7.60 7.78 (m, 1H), 7.85 8.28 (m, 4H). 13 C NMR (CDCl 3 .2, 57.4, 67.9, 115.1, 120.6, 126.3, 126.9, 128.3, 128.8, 131.2, 136.0, 138.8, 146.2, 155.9, 168.9, 196.6. HRMS (ESI) calcd for C 46 H 41 N 9 O 10 S 2 Na [M + Na] + 966.2310, found 966.2304. Cbz L Glu (Cbz L Cys Bt) Cbz L Cys Bt (7.16d). The compound was prepared by general procedure VI for preparation of bis benzotriazolides. Sticky gel, 0.93 g, 0.97 m mol, 68% yield. 1 H NMR ( CD 3 OD 1.9 2 2.04 (m, 1 H), 2.28 2.50 (m, 1H), 2.51 2.81 (m, 2H), 3. 47 3.70 (m, 4H), 4.18 4.46 (m, 1H), 5.07 5.17 (m, 5H), 5.19 5.29 (m, 1H), 5.88 5.99 (m, 2H), 7.27 7.40 (m, 1 5 H), 7.57 7.6 9 (m, 2H), 7.71 7.8 5 (m, 2H), 8.15 8.22 (m, 2H), 8.23 8.28 (m, 2H) 13 C NMR ( CDCl 3 22.6, 27.5, 30.7,

PAGE 201

201 31.6, 39.3, 54.0, 60. 0 65.1, 67.5, 68.7, 114.3, 114.4, 120.4, 120.5, 125.9, 126.7, 126.9, 128.2, 128.6, 131.0, 136.0, 146.0, 155.8, 168.6, 168.9, 197.9, 198.5 HRMS (ESI) calcd for C 47 H 43 N 9 O 10 S 2 Na [M + Na ] + 980.2466, found 980.24 82. Cbz L Asp (Cbz L Lys Bt) Cbz L Lys Bt (7.16e). The compound was prepared acco r ding to general procedure VI for preparation of bis benzotriazolide from BtH (2.41 g, 20.22 mmol) SOCl 2 (0.37 mL, 5.05 mmol) and Cbz L Asp (Cbz L L ys OH) Cbz L Lys OH 7.15e (2.00 g, 2.53 mmol) Sticky gel 1.86 g, 1.87 mmol 74% yield 1 H NMR (CDCl 3 1.44 1.71 (m, 7 H ), 1.72 1.83 (m, 1H), 1.85 1.98 (m, 2H), 2.05 2.16 (m, 2H), 2.70 2.88 (m, 1H) 2.99 3.06 (m, 2H), 3.51 3.62 (m, 3H), 4.21 4.32 (m, 1H), 5.04 5.13 (m, 7H), 5.64 5.77 (m, 3H), 5.79 5.85 (m, 1H), 6.00 (br s, 1H), 7.10 (br s, 1H), 7.26 7.38 (m, 15H), 7.52 (t, J = 4.7 Hz, 2 H ), 7.66 (t, J = 4.4 Hz, 2H), 8.12 (d, J = 5.1 Hz, 2H), 8.25 (d, J = 4.8 Hz, 2H). 13 C NMR (CDCl 3 22.3, 26.6, 32.1, 35.6, 38.3, 50.1, 54.5, 67.2, 114.4, 120.3, 126.5, 128.1, 128.2, 128.3, 128.5, 130.8, 131.1, 135.8, 136.1, 145.9, 156.0, 156.3, 171.7, 174.6, 176.1. HRMS (ESI ) calcd for C 52 H 55 N 11 O 10 Na [M + Na] + 1016.4026 found 1016.40 33. General P rocedure VII for the Coupling of B is Benzotriazolides 8a b and 16a e with Proline to Prepare Compounds 7.9a c and 7.17a e Bis benzotriazolides 7. 8a b or 7. 17a e (1 equiv.) w ere each suspended in acetonitrile/water (3:1) (15 mL/1 g) and a solution of D or L proline ( 2 2.1 equiv. ) in water (10 mL/1 g) containing triethyl amine ( 2 2.2 equiv. ) was added slowly. The mi xture s were stirred at 20 C for up to 16 h until the TLC revealed consumption of the starting materials. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate ( 30 mL /1 g of 8a b or 16a e ). T h e organic layer was washed w ith 4 N HCl (3 1 mL /1 mL of ethyl acetate ) and Na 2 CO 3 10 wt. % in water (3 1 mL /1

PAGE 202

202 mL of ethyl acetate), and purified by column chromatography (ethyl acetate / hexanes gradient ) to yield compounds 7. 9a c or 7.17a e. 3,3 DMG (Cbz L Lys D Pro OH)2 (7.9a). T he compound was prepared by coupling of bis benzotriazolide 3,3 DMG (Cbz L Lys Bt) 2 7.8a (2.00 g, 2.30 mmol) w ith D proline (0.52 g, 4.6 mmol) according to general procedure VII. Stick y gel, 1.67 g, 1.89 mmol, 84% yield. 1 H NMR (CD 3 6H), 1.28 1.40 (m, 4H), 1.45 1.59 (m, 4H), 1.60 1.77 (m, 4H), 1.78 2.12 (m, 5H), 2.13 2.31 (m, 3H), 2.52 (s, 4H), 3.14 3.25 (m, 1H), 3.4 1 3.55 (m 1H), 3.58 3.69 (m, 1H), 3.68 3.77 (m, 4H), 3.80 3.87 (m, 1H), 4.00 4.22 ( m, 2H), 4.31 4.53 (m, 2H), 5.03 5.1 5 (m, 4H),7.28 7.37 (m, 10H). 13 C NMR (CD 3 24.0, 24.4, 25.8, 27.8, 28.5, 30.1 32.2, 40.0, 47.0, 54.0, 55.3, 60.8 67.8, 127.2, 128.9, 129.5, 140.1, 158.2 174.2, 175.4, 176.0. HRMS (ESI) calcd for C 45 H 61 N 6 O 12 [M H] 877.434 2 found 877.43 61. Trans 1,4 CHD (Cbz L Cys D Pro OH)2 (7 .9b). The compound was prepared by coupling of bis benzotriazolide t rans 1,4 CHD (Cbz L Cys Bt) 2 7.8b (1.25 g, 1.47 mmol) with D proline (0.34 g, 2.95 mmol) according to general procedure VII. Sticky gel, 0.99 g, 1.18 mmol, 80% yield. 1 H NMR ( DMSO d 6 300 1.99 (m, 14H), 2.01 2.23 (m, 2 H), 2.58 2.74 (m, 2H), 2.91 (dd J = 13.3 9.3 Hz 2H) 3. 18 (dd, J = 13.7 5.0 Hz, 2H), 3 .51 3.72 (m, 4H ), 4.19 (dd, J = 9.1, 3.7 Hz 2H), 4.41 4.54 (m, 1H), 4.95 5.12 (m, 5H), 7.23 7.42 (m, 10H), 7.60 7.76 ( m, 2H ), 12.45 (br s, 2H). 13 C NMR (DMSO d 6 75 29.8, 46.2, 48.2, 51.2, 58.4, 65.2, 127.2, 12 7.4, 127.9, 136.6, 155.4, 156.6, 167.7, 171.4, 172.6, 200.6, 201.2. Compound 7.9b was characterized by 1 H and 13 C NMR.

PAGE 203

203 Trans 1,4 CHD (Cbz L Cy s L Pro OH)2 (7.9c). The compound was prepared by coupling of bis benzotriazolide t rans 1,4 CHD (Cbz L Cys Bt) 2 7.8b (1.25 g, 1.47 mmol) with L proline (0.34 g, 2.95 mmol) according to general procedure VII. Sticky gel, 0.93 g, 1.11 mmol, 75 % yield 1 H and 13 C NMR were identical to 7.9b. Cbz L Asp (Cbz L Ser L Pro OH) Cbz L Ser L Pro OH (7.17a). The compound was prepared by coupling of bis benzotriazolide Cbz L Asp (Cbz L Ser Bt) Cbz L Ser Bt 7.16a (4.0 g, 4.44 mmol) with L proline (1.40 g, 8.77 mmol) accor ding to general procedure VI I White microcrystals, 3.69 g, 4.08 mmol, 92% yield; Sticky gel. 1 H NMR (DMSO d 6 1.62 1.94 (m, 6H), 1.98 2.20 (m, 2H), 2.72 (dd, J = 16.5, 8.0 Hz, 1H), 2.84 (dd, J = 16.8, 4.7 Hz, 1H), 3.56 3.66 (m, 3H), 3.93 4.05 (m, 2H), 4.22 4.27 (m, 2H), 4.30 4.37 (m, 2H), 4.44 4.53 (m, 2H), 4.57 4.65 (m, 2H) 4.93 5.06 (m, 6H), 7.24 7.37 (m, 15H), 7.71 (d, J = 8.3Hz, 1 H), 7.80 (t, J = 7.6 Hz, 2 H) 13 C NMR ( CD 3 OD 25.8, 30.1, 37.1, 51.8, 53.1, 60.6, 64.9, 65.2, 68.0, 115.7, 127.2, 128.7, 128.9, 129.1, 129.5, 138.0, 140.0, 158.3, 169.8, 171.8, 172.2, 175.2 HRMS ( ESI ) calcd for C 44 H 48 N 5 O 16 [M H ] 902.3091, found 902.3099. Cbz L Glu (Cbz L Ser D Pro OH) Cbz L Ser D Pro OH (7.17b). The compound was prepared by coupling Cbz L Glu ( Cbz L Ser Bt ) Cbz L Ser Bt 7.16b (1.0 g, 1.08 mmol) and D proline (0.25 g, 2.16 mmol) according to general procedure VII. Sticky gel 0.81 g, 0.89 mmol, 82% yield. 1 H NMR ( CD 3 OD 1.78 2.10 (m, 6H), 2.10 2.32 (m, 3H), 2.34 2.46 (m, 2H), 2.48 2.62 (m, 1H), 3.41 3.78 (m, 1 H), 3.57 3.75 (m, 2H), 4.03 4.18 (m, 1H), 4.18 4.46 (m, 3H), 4.48 4.60 (m, 1H), 4.62 4.73 (m, 2H), 4.76 4.89 (m 1H), 5.04 5 .11 (m, 4H), 5.12 5.30 ( m, 2H), 7.23 7.38 (m, 15H) 13 C NMR ( CD 3 OD 22.8, 26.2, 30.7 32.3, 53.1, 60.7, 61.1, 66.0, 68.4, 69.8,

PAGE 204

204 116.1, 127.6, 129.4, 129.5, 129.7, 130.0, 130.1, 137.1, 138.5, 140.5, 152.6, 158.6, 169.7, 173.0, 175.7, 176.4 HRMS (ESI) calcd for C 45 H 50 N 5 O 16 [M H ] 916.3247 found 916.32 66. Cbz L Asp (Cbz L Cys L Pro OH) Cbz L Cys L Pro OH (7.17c). The compound was prepared by coupling of bis benzotriazolide Cbz L Asp (Cbz L Cys Bt) Cbz L Cys Bt 7.16c (1.04 g, 1.10 mmol) with D Pro OH (0.25 g, 2.20 mmol) a ccording to general procedure VII. Sticky gel, 0.89 g, 0.95 mmol, 86% yield. 1 H NMR (DMSO d6, 300 MHz): 1.50 2.02 (m, 5H), 2.02 2.35 (m, 2H), 2.62 3.20 (m, 7H), 3.48 3.73 (m, 3H), 3.95 4.14 (m, 1H), 4.14 4.29 (m, 2H), 4.39 4.74 (m, 2H), 4.93 5.18 (m, 7H), 7.25 7.42 (m, 15H), 7.73 (d, J = 8.1 Hz, 2H), 7.80 8.21 (m, 1H). 13 C NMR ( CD 3 OD 3 00 MHz): 30.4, 31.9, 45.9, 53.2, 54.9, 59.2, 60.8, 68.0, 128.9, 129.1, 129.6, 138.2, 158.2, 158.5, 170.6, 173.5, 175.4, 196.7. HRMS (ESI) calcd for C 44 H 48 N 5 O 14 S 2 [M H] 934.2634 found 934.26 49. Cbz L Glu (Cbz L Cys D Pro OH) Cbz L Cys D Pro OH (7.17d). The compound was prepared by coupling of bis benzotriazolide Cbz L Glu (Cbz L Cys Bt) Cbz L Cys Bt 7. 16d ( 1.0 g, 1.04 mmol) with D Pro OH (0.24 g, 2.09 mmol) a ccording to general procedure VII. Sticky gel, 0.81 g, 0.89 mmol, 82% yield. 1 H NMR ( CD 3 OD 1.75 2.06 (m, 8H), 2.12 2.31 (m, 3H), 2.32 2.74 (m, 3H), 2.99 3.18 (m, 2H), 3. 31 3. 62 (m, 2H), 3.6 3 3.78 ( m 2H), 4.2 4 4.48 ( m 3 H), 4. 54 4.74 (m, 1H), 5.0 2 5.1 2 (m, 5H), 5.1 3 5.26 (m, 2H), 7.23 7.4 8 (m, 15H) 13 C NMR ( CD 3 OD 23.4, 25.8, 30.4, 3 1 7, 32.1, 40.7, 48.0, 52.8, 53.4 60.8, 61.7 66.8, 68.0, 69.5, 128.9, 129.2, 129.3 129.4, 129.6, 136.6, 138.2, 152.1, 158.3, 158.4, 170.4, 170.7 175.4, 176.1, 200.4. HRMS (ESI ) calcd for C 45 H 50 N 5 O 14 S 2 [M H] 948.2790 found 948.27 72.

PAGE 205

205 Cbz L Asp (Cbz L Lys D Pro OH) Cbz L Lys D Pro OH (7.17e). The compound was prepared by coupling of bis benzotriazolide Cbz L Asp (Cbz L Lys Bt) Cbz L Lys Bt 7.16e (2 .0 g, 2.01 mmol) w ith D proline (0.46 g, 4.03 mmol) according to general procedure VII. Stic ky gel, 0.77 g, 1.53 mmol, 76% yiel d. 1 H NMR (CD 3 OD 1.22 1.52 (m, 6 H), 1.53 1.80 (m, 7H), 1.82 1.94 (m, 2H), 1.95 2.08 (m, 3H), 2.16 2.32 (m, 2H), 2.63 (dd, J = 5.9, 5.9 Hz, 1H), 2.66 (dd, J = 5.7, 5.7 Hz, 1H), 3.00 (dd, J = 10.8, 5.4 Hz, 1H), 3.08 3.19 (m, 1H), 3.46 3.55 (m, 4H), 3.56 3.67 (m, 1H), 3.78 3.85 (m, 1H), 4.32 4.42 (m, 3H), 4.43 4.48 (m, 1H), 5.05 5.10 (m, 7H), 7.27 7.40 (m, 15H). 13 C NMR (CDCl 3 21.8, 22.8, 24.7, 26.9, 28.8, 31.6, 35.6, 28.4, 47.3, 50.0, 52.2, 53.6, 59. 5, 66.9, 67.2, 76.8, 77.3, 77.7, 136.0, 136.3, 156.2, 156.5, 171.5, 174.0, 175.0, 176.5. HRMS (ESI) calcd for C 50 H 62 N 7 O 14 [M H] 984.4349 found 984.43 62 General P rocedure VIII for the Preparation of Cyclization Precursors 7.10a c and 7.18a e A stirred solution of BtH ( 8 equiv. ) in dry THF (10 mL / 1 g) was treated at 0 C with SOCl 2 ( 2 equiv. ). After 20 minutes, the reaction mixture was cooled to 45 o C in acetone/dry ice bath and a solution of 7. 9a c or 7.17a e ( 1 equiv. ) in dry THF (15 mL /1 g ) was adde d drop wise The resulting solution was stirred for 6 h at 45.0 C, after which THF was removed under reduced pressure. The residue w as dissolved by ethyl acetate (2 0 mL /1 g of 9a c or 18a e ) and washed with brine (2 1.5 mL /1 mL ), HCl (4 N 2 1 mL /1 mL ethyl acetate ), Na 2 CO 3 10 wt. % in water (2 1 mL /1 mL ethyl acetate ). The organic layer was dried over sodium su lfate, filtered and evaporated. The residue was recrystallized with CH 2 Cl 2 /hexane to yield compounds 7. 10a c or 7. 1 8 a e. 3,3 DMG (Cbz L Lys D Pro Bt) 2 (7.10a). The compound was prepare by general procedure VIII for preparation of bis benzotriazolide cyclization precursor from BtH (2.17

PAGE 206

206 g, 18.2 mmol), SOCl 2 (0.33 mL, 4.6 mmol) and 3,3 DMG (Cbz L Lys D Pro OH) 2 7.9a (2.0 g, 2.30 mmol). Sticky ge l, 1.67 g, 1.55 mmol, 68% yield. 1 H NMR (CDCl 3 300 1.31 (m, 6H), 1.33 1.87 (m, 10H), 2.10 2.31 (m, 4H), 2.50 (br s, 4H), 3.26 (br s, 2H), 3.52 3.58 (m, 1H), 3.68 3.85 (m, 4H), 3.90 4.08 (m, 1H), 4.56 4.70 (m, 2H), 5.03 5.18 (m, 4H), 5.51 5.67 (m, 2H), 5.68 5.82 (m, 2H), 5.88 5.98 (m, 2H), 7.27 7.42 (m, 10H), 7.52 (t, J = 7.2 Hz, 2H), 7.66 (t, J = 7.7 Hz, 2H), 8.13 (d, J = 8.1 Hz, 2H), 8.27 (d, J = 8.4 Hz, 2H). 13 C NMR (CDCl 3 25.2, 27.9, 28.9, 29.3, 29.8, 32.3, 39.0, 46.6, 47.7, 52.5, 59.8, 67.1, 114.7, 1 20.4, 126.5, 128.1, 128.7, 130.7, 131.4, 136.6, 146.2, 156.0, 170.3, 170.7, 172.2. HRMS (ESI) calcd for C 57 H 68 N 12 O 10 Na [M + Na] + 1103.5074 found 1103.5096. Trans 1,4 CHD (Cbz L Cys D Pro Bt) 2 (7.10b). The compound was prepared by general procedure VIII fr om BtH (0.95 g, 8.00 mmol), SOCl 2 (0.15 mL, 2.00 mmol) and t rans 1,4 CHD ( Cbz L Cys D Pro OH ) 2 7.9b (0.84 g, 1.00 mmol). Sticky gel, 0. 72 g, 0.85 mmol, 85% yield. 1 H NMR (DMSO d 6 300 MHz): 1.52 (m, 5H), 2.05 2 .78 (m, 5H), 2.06 2.31 (m, 6H), 2.47 2.71 (m, 2H), 3.30 (dd, J = 14.1, 4.2 Hz, 2H), 3.38 (dd, J = 13.5 6.6Hz, 2H), 3.64 4.09 (m, 4H), 4.8 4 4.80 (m 2 H), 5.03 5.18 (m, 4H), 5.70 (dd, J = 14.2 Hz, 8.0 Hz, 2H), 5.84 (dd, J = 8.4 Hz 3 .9 Hz, 2H), 7.26 7.42 (m, 10 H), 7.46 7.56 (m, 2H), 7.59 7.69 (m, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H). 13 C NMR (CDCl 3 120.2, 126.4, 127.9, 128.0, 128.5, 130.5, 1 31.2, 136.4, 146.0, 155.8, 168.4, 170.0, 201.6 HRMS (ESI ) calcd for C 52 H 54 N 10 O 10 S 2 Na [M + Na] + 1065.3358 found 1065.33 72. Trans 1,4 CHD (Cbz L Cys l Pro Bt) 2 (7.10c). The compound was prepared by general procedure VIII from BtH (1.00 g, 8.42 mmol), SOCl 2 (0.1 6 mL, 2.11 mmol) and

PAGE 207

207 t rans 1,4 CHD ( Cbz L Cys L Pro OH ) 2 7.9c (0.88 g, 1.05 mmol). Sticky gel, 0. 78 g, 0.92 mmol, 88 % yield. 1 H and 13 C NMR were identical to 7.10b. Cbz L Asp (Cbz L Ser L Pro Bt) Cbz L Ser L Pro Bt (7.18a). T he compound was prepared according to general procedure V II I from BtH ( 0. 528 g, 4.43 mmol) SOCl 2 (0.16 mL, 2.21 mmol) and Cbz L Asp (Cbz L Ser L Pro OH) Cbz L Ser L Pro OH 7.17a ( 0.5 g, 0.553 mmol) Sticky gel, 0.44 g, 0.40 mmol, 72% yield; 1 H NMR ( DMSO d 6 1. 64 1.98 (m, 2 H), 1.98 2.30 (m, 6H), 2.68 2.96 (m, 2H), 3.72 3.90 (m, 3H), 3.95 4.17 (m, 2H), 4.28 4.58, (m, 4H), 4.64 4.80 (m, 2 H), 5. 02 (br s 6H), 5.68 5.84 (m, 2 H), 7.24 7.42 (m, 15H), 7. 56 7. 6 8 (m, 3 H), 7. 68 7.85 (m, 4 H), 8.0 8 8. 30 (m, 4 H) 13 C NMR (CDCl 3 75 25.3, 29.8, 36.7, 47.9, 50.9, 51.8, 60.1, 67.3, 114.5, 120.3, 126.5, 127.7, 128.2, 128.6, 130.8, 131.2, 136.3, 146.0, 156.2, 167.6, 169.9, 170.4. HRMS (ESI) calcd for C 56 H 55 N 11 O 14 Na [M + Na] + 1128.3828 found 1128.3845 Cbz L Glu (Cbz L Ser D Pro B t) Cbz L Ser D Pro Bt (7.18b). The compound was prepared according to general procedure VIII from BtH (1.56 g, 13.07 mmol) SOCl 2 (0.24 mL, 3.27 mmol) and Cbz L Glu (Cbz L Ser D Pro OH) Cbz L Ser D Pro OH 7.17b (1.50 g, 1.63 mmol) White microcrystals 1.2 4 g, 1.11 mmol 68% yield; mp 85 89 C. 1 H NMR ( CDCl 3 1.88 2.10 (m, 6H), 2.1 4 2.2 3 (m, 2H), 2.28 2.48 (m, 2H), 2.4 8 2.76 (m, 2H), 3.45 3.6 8 (m, 3 H), 3.82 3.9 4 (m, 1 H), 4.19 4.4 2 (m, 3 H), 4.48 4.66 (m, 3H), 4.92 5.08 (m, 2 H), 5.12 5.17 (m 1H), 5.20 5.35 (m, 4H), 5.74 (d, J = 8.2 Hz, 2 H), 5.8 6 5. 9 3 (m, 3H), 7.28 7.40 (m, 15H), 7.50 7.56 (m, 2H), 7.6 4 7.70 (m, 2H) 8.13 8.16 (m, 2H), 8.2 8.27 (m, 2H) 13 C NMR ( CDCl 3 21.9, 22.3, 25.2, 29.6 29.9, 31.2, 45.8, 52.1, 58.8, 59.8, 60.1, 64.8, 67.4, 68.7, 67.0, 69.9, 114.6,

PAGE 208

208 115.2, 120.5, 125.9 126.7, 128.2, 1 28.3, 128.5, 128.7, 128.9, 130.8, 134.6, 135.0, 136.3, 146.2, 151.3 152.2, 156.0, 162.2, 167.3, 170.6, 172.9 HRMS (ESI) calcd for C 57 H 57 N 11 O 14 Na [M + Na] + 1142.3979 found 1142.39 95. Cbz L Asp (Cbz L Cys L Pro Bt) Cbz L Cys L Pro Bt (7. 18c). The compound w as prepared according to general procedure VIII from BtH (2.00g, 16.8 mmol), SOCl 2 (0.30 mL, 4.2 mmol) and Cbz L Asp (Cbz L Cys D Pro OH) Cbz L Cys D Pro OH (2.00 g, 2.1 mmol). Sticky gel, 1.60 g, 1.40 mmol, 67% yield. 1 H NMR (CDCl 3 30 1.89 2.32 (m, 8H), 2.40 2.72 (m, 2H), 2.78 3.45 (m, 4H), 3.48 3.82 (m, 3H), 3.82 4.08 (m, 2H), 4.3 4 4. 94 (m, 2H), 4.95 5.28 (m, 6H), 5.46 5.78 (m, 1H), 5.78 5.98 (m, 2H), 5.98 6.12 (m, 1H), 6.18 6.50 (m, 1H), 7.15 7.44 (m, 15H), 7.50 7.62 (m, 2H) 7.62 7.74 (m, 2H), 8.08 8.20 (m, 2H), 8.20 8.32 (m, 2H). 13 C NMR (CDCl3, 75 24.9, 28.6, 29.6, 31.3, 40.6, 44.7, 47.6, 51.5, 51.9, 57.4, 59.5, 59.8, 67.1, 67.3, 114.3, 114.8, 120.1, 120.2, 125.7, 126.3, 126.5, 127.9, 130.5, 130.9, 131.0, 135.9, 13 6.1, 138.5, 145.8, 155.9, 168.7, 169.7, 195.9, 199.5. HRMS ( ESI ) calcd for C 56 H 55 N 11 O 12 S 2 Na [M + Na] + 1160.3365, found 1160.33 55. Cbz L Glu (Cbz L Cys D Pro Bt) Cbz L Cys D Pro Bt (7.18d). The compound was prepared by general procedure VIII from BtH ( 2.00 g, 16.8 mmol) SOCl 2 ( 0.30 mL, 4.2 mmol) and Cbz L Glu (Cbz L Cys D Pro OH) Cbz L Cys D Pro OH (2.0 g, 2.1 mmol) White microcrystals, 1.50 g, 1.3 mmol 62% yield; mp 93 98 C 1 H NMR ( CDCl 3 1.80 2.09 (m, 1H), 2.1 0 2.3 4 (m, 6H), 2.36 2.78 (m, 5H), 3.1 0 3. 40 (m, 3H), 3.4 0 3.6 5 (m, 2H), 3. 88 4.44 (m 3 H), 4.77 (dd, J = 9. 5 2. 3 Hz, 1H), 4. 7 8 4.94 (m, 1H), 4.98 5.1 6 (m, 5H), 5.2 0 5. 38 (m, 2H), 5.73 (d, J = 9 Hz, 2H), 5.7 6 5.8 8 (m, 3 H), 7.33 7.45 (m, 15H), 7.4 6 7.5 5 (m, 2 H), 7.6 0 7.68 (m, 2H), 8. 00 8.1 5 (m, 2H), 8.2 0

PAGE 209

209 8.26 (m, 2 H) 13 C NMR ( CDCl 3 22.6, 25.2, 29.9, 31.0, 31.7, 47.7, 51.3, 60.0, 60.3, 65.3, 67.2, 68.7, 114.6, 120.4, 125.8, 126.6, 128.3, 128.7, 130.8, 131.3, 1 35.1, 136.3, 146.1, 151.1, 155 .9, 168.0, 170.1, 173.2, 199.0. HRMS (ESI) calcd for C 57 H 57 N 11 O 12 S 2 Na [M + Na] + 1174.3522 found 1174.35 41. Cbz L Asp (Cbz L Lys D Pro Bt) Cbz L Lys D Pro Bt (7.18e). T he compound was prepare d by general procedure VIII for pr eparation of bis benzotriazolide cyclization precursor from BtH (0.97 g, 8.12 mmol) SOCl 2 (0.15 mL, 2.03 mmol). and Cbz L Asp (Cbz L Lys D Pro OH) Cbz L Lys D Pro OH 7.17e (1.00 g, 1.01 mmol) Sticky gel, 0.83 g, 0.70 mmol, 69% yield. 1 H NMR (CDCl 3 300 M 1.28 1.42 (m, 5 H), 1.56 1.64 (m, 5H), 1.64 1.84 (m, 5H), 2.12 2.26 (m, 5H), 2.50 2.66 (m, 2H), 3.03 (dd, J = 10.8, 5.4 Hz, 2 H), 3.50 3.66 (m, 3H), 3.68 3.74 (m, 2H), 3.92 3.98 (m, 1H), 4.36 4.48 (m, 2H), 4.55 4.61 (m, 1H), 5.03 5.13 (m, 7H ), 5.60 5. 68 (m, 2H), 5.92 (dd, J = 5.4, 2.1 Hz, 2H), 5.98 (d, J = 3.0 Hz, 1H ), 7.27 7.36 (m, 17H), 7.48 7.54 (m, 2H), 7.65 (t, J = 4.4 Hz, 2H), 8.11 8.14 (m, 2H), 8.22 8.27 (m, 2H) 13 C NMR (CDCl 3 21.8, 21.9, 25.1, 26.9, 29.7, 32.4, 35.9, 38.5, 47.6, 50 .1, 52.1, 59.7, 67.0, 67.4, 114.7, 120.4, 126.6, 127.9, 128.1, 128.2, 128.3, 128.4, 128.7, 130.7, 131.3, 136.4, 146.1, 156.2, 156.3, 170.3, 170.7, 174.7, 176.1. HRMS (ESI) calcd for C 62 H 69 N 13 O 12 Na [M + Na] + 1210.5081 found 1210.99. General P rocedure IX fo r the Cyclization of Precursor 7.10a c to Form Symmetrical B is DKPs 7.11a c and 7.18a e to Form Unsymmetrical B is DKPs 7.19a e A solution of 7 .10a c or 7.18a e ( 1 equiv. ) and triethylamine ( 2 2.2 equiv. ) in dry acetonitrile (100 mL) was stirred for 48 84 h until TLC revealed cyclization was complete. Each mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate ( 20 mL /1 g of 7 .11a c or 7.19a e). T he organic layer was washed with

PAGE 210

210 4N HCl (3 1 mL /1 mL ethyl acetate ) and Na 2 CO 3 10 wt. % in water (3 1 mL /1 mL ethyl acetate ), and purified by column chrom atography ( CH 2 Cl 2 /hexanes gradient) to give the corresponding symmetrical bis DKPs 7 .11a c or unsymmetrical bis DKPs 7.19a e. 3,3 DMG cyclo (Cbz L Lys D Pro)] 2 (7.11a). The unsymmetr ical bis DKP was prepared by macro cyclization of 3,3 DMG (Cbz L Lys D Pro Bt) 2 7.10a (0.50 g, 0.46 mmol) according to general procedure IX. Sticky gel, 0.35 g, 0.42 mmol, 91% yield. 1 H NMR (CD 3 0.98 (br s, 6H), 1.22 1.34 (m, 8H), 1.42 1.63 (m, 6H), 1. 7 8 2.00 (m, 4H), 2.13 2.24 (m, 1H), 2.28 2.37 (m, 1H), 2.47 (br s, 4 H), 3.07 3.19 (m, 1H), 3.37 3.52 (m, 2H), 3.54 3.61 (m, 1H), 3.62 3.73 (m, 3H), 3.73 3.83 (m, 1H), 4.02 4. 38 (m, 2H), 4.40 4.82 (m, 2H), 4.99 5.27 (m, 4 H), 7.21 7.36 (m, 8H), 7. 37 7.44 (m, 2H) 13 C NMR (CDCl 3 22.8, 24.8, 27.8, 29.4, 29.9, 31.4, 38.6, 46.5, 52.5, 59.6, 61.5, 67.1, 69.9, 128.1, 128.7, 129.0, 134.9, 156.1, 165.3, 167.9, 172.3 HRMS (ESI) calcd C 45 H 58 N 6 O 10 Na [M + Na ] + 865.4107 found 865.41 25 Trans 1,4 CHD [ cyclo (Cbz L Cys D Pro)] 2 ( 7.11b). The compound was prepared by macro cyclization of bis benzotriazolide precursor t rans 1,4 CHD (Cbz L Cys D Pro Bt) 2 7. 10b (1.05 g, 1. 24 mmol) according to general procedure IX. Sticky gel, 0. 85 g, 1.05 mmol, 85 % yield. 1 H NMR (CD 3 49 1.67 (m, 3H), 1.74 2.12 (m, 12H), 2.34 2.46 (m, 2H), 2.5 0 2.58 (m, 1H), 3.34 3.59 (m, 8H), 4.66 (dd, J = 8.0, 8.0 Hz, 2H), 4.95 (dd, J = 7.1, 7.1 Hz, 2H), 5.22 5.36 (m, 4H), 7.22 7.45 (m, 10H). 13 C NMR (CD 3 .0, 61.3, 61.7, 70.4, 129.6, 129.7, 129.8, 136.7, 153.6, 165.4, 169.0, 202. 3. HRMS (ESI) calcd for C 40 H 44 N 4 O 10 Na [M + Na] + 827.2391 found 827.2371

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211 Trans 1,4 CHD [cyclo (Cbz L Cys l Pro)] 2 (7.11c). The compound was prepared by macro cyclization of bis ben zotriazolide precursor trans 1,4 CHD (Cbz L Cys L Pro Bt) 2 7.10c ( 0.78 g, 0 93 mmol) according to general procedure IX. Sticky gel, 0. 61 g, 0.75 mmol, 81 % yield 1 H and 13 C NMR were identical to 7.11b. HRMS (ESI) calcd for C 40 H 44 N 4 O 10 Na [M + Na] + 827.2391 f ound 827.2398 Cbz L Asp [cyclo (Cbz L Ser L Pro)] cyclo (Cbz L Ser L Pro) (7.19a). The compound was prepared by ma c r o cyclization of bis benzotriazolide precursor Cbz L Asp (Cbz L Ser L Pro Bt) Cbz L Ser L Pro Bt 7.18a (1 0 g, 0.9 mmol) according to gener al procedure IX. Sticky gel, 0.56 g, 0.65 mmol, 72% yield. 1 H NMR (DMSO d 6 300 1.69 1.98 (m, 6H), 2.0 2 2.24 (m, 2H), 2.54 2.7 8 (m, 2H), 3.54 3.62 (m, 4H), 3.68 4.06 (m, 2H), 4. 2 3 4.80 (m, 6H), 4.82 4.92 (m, 1H), 4.94 5.06 (m, 4H), 5.20 5.29 (m, 2H), 7.24 7.43 (m, 15H), 7.68 7.70 (m, 1H) 13 C NMR ( CDCl 3 22.4, 22.8, 29.6, 31.8, 36.1, 45.8, 50.3, 60.0, 64.0, 65.2, 66.0, 67.3, 67.5, 69.8, 128.3, 128.5, 128.7, 128.8, 134.6, 136.0, 152.2, 156.0, 162.5, 167.3, 170.3. HRMS (ESI) calcd for C 44 H 45 N 5 O 14 Na [M + Na] + 890.2855 found 890.2828 Cbz L Glu [cyclo (Cbz L Ser D Pro)] cyclo (Cbz L Ser D Pro) (7.19b). The bis DKP was prepared by macro cyclization of Cbz L Glu (Cbz L Ser D Pro Bt) Cbz L Ser D Pro Bt 7.18b (0.25 g, 0. 23 mmol) according to general procedure IX. White microcrystals, 178.4 mg, 0.20 mmol, 88% yield; mp 70 72 C. 1 H NMR (CD 3 OD 300 1.91 2.10 (m, 6H), 2.26 2.45 (m, 4H), 2.46 2.60 (m, 2H), 3.41 3.62 (m, 4H), 4.36 4.44 (m, 2H), 4. 49 (dd, J = 12.0, 4.5 Hz, 2H), 4.57 (dd, J = 11.7, 4.2 Hz, 2H), 4.73 (dd, J = 9.5, 2.9 Hz, 1H),5.01 (t, J = 4.2 Hz, 2H), 5.13 5.30 ( m, 2H), 5.31 (b r s 4H), 7.28 7.39 (m, 12H), 7.43 7.4 7 (m, 3H) 13 C NMR (CD 3 OD 22.6, 23.2, 26.7,

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212 27.1, 30.4, 32.0, 44.4, 46.9, 60.3, 61.1, 61.6, 66.1, 68.0, 69.6, 70.5, 129.0, 129.2, 129.5, 129.6, 129.7, 136.6, 136.7, 152.5, 153.5, 1 64.2, 168.9, 172.3, 175.6 HRMS ( ESI ) calcd for C 45 H 47 N 5 O 14 Na [M + Na] + 904. 3012, found 904.3021. Cbz L Asp [cyclo (Cbz L Ser L Pro)] cyclo (Cbz L Ser L Pro) (7.19c). The compound was prepared by macro cyclization of bis benzotriazolide precursor Cbz L Asp (Cbz L Cys L Pro Bt) Cbz L Cys L Pro Bt 7.18c (1.14 g, 1.00 mmol) according to general procedure IX Sticky gel, 0.69 g, 0.76 mmol, 76% yield. 1 H NMR (DMSO d 6 300 MHz ): 1.72 2.04 (m, 8H), 2.18 2.24 (m, 2H), 3.21 3.30 (m, 2H), 3.38 3.42 (m, 6H), 4.60 (dd, J = 9.8, 5.6 Hz, 2H), 4.76 4.83 (m, 1H), 4.91 (t, J = 4.4 Hz, 1H), 4.98 5.12 (m, 3H), 5.20 5.34 (m, 4H), 7.22 7.51 (m, 16H). 13 C NMR (DMSO d 6 75 MHz): 22.2, 28.6, 29 .4, 37.9, 45.3, 59.0, 59.5, 65.6, 66.0, 68.4, 127.7, 127.9, 128.2, 135.2, 136.6, 136.9, 151.7, 151.9, 155.8, 162.3, 162.4, 166.9, 167.3, 199.6, 199.9. HRMS (ESI) calcd for C 44 H 45 N 5 O 12 S 2 Na [M + Na] + 922.2398 found 922.2382. Cbz L Glu [cyclo (Cbz L Cys D Pr o)] cyclo (Cbz L Cys D Pro) (7.19d). The compound was prepared by macro cyclization of bis benzotriazolide precursor Glu (Cbz L Cys D Pro Bt) Cbz L Cys D Pro Bt 7.18d (0.25 g, 0.22 mmol) according to general procedure IX Sticky gel, 0.17 g, 0.19 mmol, 86% yield. 1 H NMR ( CDCl 3 300 1.88 2.37 (m, 9H), 2.38 2.52 (m, 3H), 2.53 2.67 (m, 1H), 3.2 9 (dd J = 14.1, 5.8 Hz, 2 H), 3.24 3.43 (m, 3H), 3.46 3.66 (m, 4 H), 4.50 (dd, J = 9.3, 7.2 Hz, 2 H), 4.78 (dd, J = 9.0 2. 4 Hz, 1H), 4.93 (dd, J = 8.7, 5.7 Hz, 1H ), 5.04 5.16 (m, 1H), 5.18 5.37 (m, 6H), 7.25 7.45 ( m, 16 H) 13 C NMR ( CDCl 3 22.5, 22.6, 28.9, 29.4, 30.9, 45.7, 59.7, 60.3, 65.1, 68.7, 69.8, 128.3, 128.4, 128.5, 128.7, 128.9, 134.6, 135.0,

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213 152.2, 162.7, 167.2, 172.9, 197.7 HRMS ( ESI) calcd f or C 45 H 47 N 5 O 12 S 2 Na [M + Na] + 936.255 5 found 936.25 62. Cbz L Asp [cyclo (Cbz L Lys D Pro)] cyclo (Cbz L Lys D Pro) (7.19e). The unsymmetrical bis DKP was prepared by macro cyclization of Cbz L Asp (Cbz L Lys D Pro Bt) Cbz L Lys D Pro Bt 7.18e ( 0.35 g, 0.2 9 mmol) according to general procedure IX. Sticky gel, 0.24 g, 0.26 mmol, 88% yield. 1 H NMR (CD 3 OD 1.34 1.44 (m, 4H), 1.49 1.68 (m, 3H), 1.77 1.89 (m, 2H), 1.90 2.01 (m, 5H), 2.03 2.12 (m, 2H), 2.33 3.39 (m, 2H), 2.62 (dd, J = 5.0, 5.0 Hz, 1H), 2.66 (dd, J = 5.0, 5.0 Hz, 1H), 2.95 3.03 (m, 2H), 3.43 3.53 (m, 6H), 4.33 4.40 (m, 2H), 4.47 4. 54 (m, 1H), 4.68 4.75 (m, 2H), 5.08 (br s, 4H), 5.26 5.32 (m, 2H), 7.25 7.38 (m, 13H), 7.42 7.46 (m, 2H). 13 C NMR (CDCl 3 21.9, 22.6, 22.7, 26.3, 29.2, 30.4, 36.1, 38.1, 45.8, 50.0, 59.4, 60.8, 67.4, 69.4, 128.3, 128.5, 128.7, 128.8, 134.8, 136. 1, 152.1, 156.3, 165.1, 167.6, 174.9, 176.5 HRMS ( MALDI TOF) calcd for C 50 H 59 N 7 O 12 Na [M + Na] + 972.4114 found 972.4115

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214 CHAPTER 8 SYNTHESIS OF POLYKETIDE FRAGMENT OF APRATOXINS Introduction Apratoxins 150 151 152 (Figure 8 1) are marine natural products of mixed biogenetic origin. Apratoxins were isolated from cyanobacterial Lyngbya spp. (now known as Moor ea bouillonii ) collected in Guam and Palau. 32 153 154 They are cyclodepsipeptides that embody both polypeptide and polyketide domains. In contrast to most known potent anticancer natural products, the cellular a nd molecular basis of apratoxin act ion is the inhibition of cotranslational translocation 155 Figure 8 1. Structures of natural a pratoxins The first member, apratoxin A, was isolated in 2001 by Moore, Paul and co workers 31 from the marine cyanobacterium Lyngbya majuscula ( Fin ger's Reef, Apra Harbor, Guam ) Apratoxin A exhibited potent in vitro cytotoxicity against KB and LoVo cancer cell lines, with IC 50 values of 0.52 nM and 0.38 nM, respectively. A pratoxin A is composed of discrete polyketide and polypeptide domains, joined via amide and ester

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215 linkage s (Figure 8 1). The polypeptide domain contains three methylated L amino acid residues ( O methyltyrosine, N methyl alanine, N methylisoleucine), one L proline residue, a modified D cysteine residue; and the polyketide domain cont ains four stereogenic centers. T he slight structural differences in the apratoxin family showed mixed biogenetic origins This variation is common among cyanobacteria, which often produce more than one member of a certain class of compounds due to relaxed specificity of biosyn thetic enzymes during biosynthesis of precursor Apratoxin A was rigorously profiled and shown to possess broad spectrum but differential in vitro activity. 32 Because of their biological activity and intriguing structures, they have been subject to several total syntheses and SAR studies. Recently apratoxins were found to prevent cotranslational translocation 155 and thereby downregulate various receptors, including r eceptor tyrosine kinases (RTKs). They inhibit trafficking of ot her secretory molecules, including growth factors that act on RTKs. 32 Figur e 8 2. Activities of synthetic a pratoxins A novel more potent apratoxin S4 was synthesized, a fold improved in vitro activity, even though the new analog still ha d the potential to be deactivated by dehydration. However, the new apratoxin was found to be stable during repurifications and stability tests. 32 I t is important to develop synthetic approaches to the novel apratoxin derivatives to further study their biological applications.

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216 The synthesis of apratoxin A was first described by Forsyth. 156 Their strategy for constructing the sensitive t hiazoline moiety was a one pot Staudinger reduction/intramolecular aza Wittig reaction. Other routes toward apratoxin A have also been reported by Takahashi, 157 Doi 158 and Ma. 159 As shown in Figure 8 3, apratoxin A could be disconnected to yield tetrapeptide and the polyketide fragment s containing a thiazoline ring (Figure 8 3, S trategy I) Figure 8 3. Retrosynthetic analysis of apratoxin A. The most common and effective strategy toward total synthesis of apratoxin A is based on condensation of the pept ide and polyketide domains with the proline residue (Figure 8 3, Strategy II) This strategy was used with a higher yielding amide formation between the isoleucine carboxylate of tripeptide and the proline amine moiety. A useful strategy in the syntheses o f apratoxin A is also the late stage assembly of the thiazoline moiety, which is oxidatively sensitive and potentially prone to unwanted side reactions. On the other hand, tripeptide fragments can theoretically be accessed in a relatively

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217 straightforward w ay using standard pept ide synthesis procedures. T he polyketide and thiazoline portions of the structure present a significant synthetic challenge, which necessitated the development of new synthetic strategies. Figure 8 4 Detailed r etro synthetic analysis of synthetic a pratoxin S4 Figure 8 4 shows detailed retro synthetic analysis of apratoxin S4 ( an apratoxin A/E hybrid, with improved the antitumor activity and tolerability in vivo ). T he key step for the synthesis of apra toxin S4 is the formation of the thiazoline ring in the intermediate from the open chain cyclization precursor (Figure 8 4). Experiments showed that different degrees of methylation at C 34 or different configuration at C30 affected the ring formation. Comp ound 8. 3 (Figure 8 4) can be disconnected into a modified cysteine an d

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218 proline ester based carboxylic acid 8. 4 (Figure 8 4 ), which in turn can be synthesized from aldehyde 8. 5. This chapter describes the large scale synthesis of aldehyde 8. 5 and its asymme tric crotylation A ldehyde 8. 5 can be used for synthesis of modified a pratoxins for further biological studies. Result s and Discussion Multigram Synthesis of the Key Aldehyde The synthesis of aldehyde 8. 5 has been reported using different strate gies; 160 but the scale of preparation was typically only a few grams. 32 A ldehyde 8. 5 can be prepared from alcohol 8. 6 (Figure 8 5) via a 6 step procedure. However, its preparation on a large scale is a challenging undertaking because the alcohol substrate 8. 2 and d iene ester 8. 8 have low molecular weights and boiling points. T he preparations involve highly reactive bases which can ignite spontaneously if exposed to air. Moreover, the ring closing metathesis of diene 8. 8 leading to lactone 8. 9 requires high dilution ring closing conditions (Figure 8 5). Figure 8 5. Synthesis of a ldehyde 8. 5 The synthesis of compound 8. 5 can be achieved from alcohol 8. 6 which can be synthesized from pivalaldehyde 8. 13 (F igure 8 6 ) To install the C 40 stereocenter,

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219 h ydroxy ketone 8. 14 was prepared via a D proline catalyzed aldol reaction of pivalaldehyde 8. 13 with acetone. K etone 8. 14 then underwent p rotection with TBS followed by reduction with NaBH 4 and elimination through mesylate to form 155 g of TBS protected alkenyl alcohol 8. 17. TBS deprotection of 8. 17 afforded allyl alcohol 8. 6, which is the key step in the preparation of 8. 6 due to its high volatile property. The solvent (Et 2 O and THF) can be removed by distillation using a Vigreux fractiona tion column in small scale reactions H owever, for large scale reactions, this was not feasible and we used a combination of a cooling method and Vigreux fraction which gave 78 g of 8.6 in 96% yield. Figure 8 6. Synthesis of alcohol 8.6 Alcohol 8. 6 was then treated with acryloyl chloride 8.7 in the presence of TEA to form diene ester 8. 8 (Figure 8 5). Ring closing metathesis (RCM) of diene 8. 8 to form lactone 8. 9 ca n be carried out in the presence of first generation 160 or second generation of Grubbs' catalyst s ( Figure 8 7). 154 In addition, titanium tetra isopropoxide can be added in catalytic amount to increase the yield of the RCM product. 161 The RCM reaction of dien e ester 8. 8 also require highly diluted conditions to favor the intramolecular cyclization product. High dilution conditions can be challenging to carry out on a large scale reaction

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220 Figure 8 7. Grubb catalysts T he RCM of the diene ester 8. 8 was carried out under several condition s that are presented in Table 8 1. Under similar conditions, second generation of Grubbs' catalyst proved to be more efficient (Table 8 1 entri es 1, 2). Higher substrate concentration resulted in an unsatisfactory yield of the desired compound (Table 8 1, entry 3). Table 8 1. Conditions for ring closing metathesis of diene 8. 8 Entry Catalyst, mol% Amount of 8. 8 (g) Ti(OiPr) 4 (mol%) Concentratio n of substrate 8. 8 (mM) Reaction time (h) Yield, (%) 1 2 nd gr, 10% 0. 523 NA 2 6 71 2 1 st gr, 15% 0.523 NA 2 6 63 3 1 st gr, 15% 0.523 NA 3 6 47 4 2 nd gr, 15% 0.523 20 2 6 79 5 2 nd gr, 15% 6 20 2 4 75 6 2 nd gr, 15% 6 NA 2 4 69 When the diene ester 8. 8 was converted to the corresponding lactone 8. 9 in the presence of 20 mol% Ti(OiPr) 4 and 15 mol% of second generation of Grubbs' catalyst the RCM reaction gave lactone 8. 9 in higher yields (Table 8 1, entry 4). Using second generation of Grubbs' cata lyst with Ti(OiPr) 4 additive, however, the reaction gave the product 8. 9 in similar yields (Table 8 1, entries 4 and 5). Considering the price of

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221 reagents for a scale up preparation and the product yield, we chose the coupling conditions of entry 4 for th e improved RCM process of ester 8. 8 (Table 8 2). Table 8 2: Ring closing metathesis of compound 8. 5 Entry Amount of 8. 8 (g) Ti(OiPr) 4 (mL) Solvent (DCM, L) Reaction times (h) Yield (%) 1 15 6.2 4.5 5.5 78 2 16 6.4 4.8 5 74 3 16 6.3 4.7 4.8 75 4 12 6.4 4.5 5.6 Lactone 8. 9 was then subjected to a conjugate addition with methyllithium and copper (I) cyanide to provide the methyl substituted lactone 8. 1 0 as the only detectable stereo isomer (Figure 8 8, Table 8 3). Figure 8 8. 1,4 Addition to lactone 8. 8 in the presence of Cu (I ) salt The stereochemical outcome resulted from the mixed higher order cyanocuprate mediated 1,4 conjugated addition. The kinetically controlled conjugate addition to a conjugated enon e takes place preferentially by way of the chair like enolate, which resulted from the more stable half chair conformer. As shown in Figure 8 9, attack on the top face of the more stable conformation gives the chairlike enolate ion, which then in turn prod uced 8. 10. The sterically hindered t butyl group enhanced the selectivity via formation of the most stable conformer.

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222 Figure 8 9. Stereoselectivity in 1,4 addition of lactone 8. 9 Table 8 3. Copper (I) mediated 1,4 addition unsaturated lactone 8. 9 Entry Amount of lactone 8. 9 (g) Amount of CuCN (g), 1.6 M in diethyl ether MeLi (ml) Reaction time (h) Yield (g,%) 1 3 2.1, 30 10 2.81, 86% 2 6 4.3, 62 11 5.83, 88% 3 8 5.6, 85 14 7.24, 82% 4 8 5.6, 88 12 17, 87% 5 7.3 5.6, 88 12.5 Ring opening of the lactone 8. 10 with N,O dimethyl hydroxylamine gave the corresponding alcohol 8. 11 (Figure 8 1). p Methoxybenzyl bromide (PMB) protection of

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223 alcohol 8. 11 was achieved using PMBBr 162 163 or PMBOC(NH)CCl 3 164 (Figure 8 10, Table 8 4) Figure 8 10. PMB protection of alcohol 8. 1 1 Entry Protecting reagent Base, equiv Catalyst, mol% Conversion (%) Yield (%) 1 PMB Br NaH, 1 TBAI, 15 0 ND 2 PMB Br NaH, 1.5 TBAI,15 <20% ND 3 PMB Br NaH,2 TBAI, 15 <30% ND 4 PMBOC( NH )CCl 3 None TfOH, 1 75% 92% 5 PMBOC( NH )CCl 3 None TfOH, 1. 5 80% 91 % 6 PMBOC(NH)CCl 3 None Sc(TfO) 3 10 50% ND 7 PMBOC(NH)CCl 3 None La(TfO) 3 10 56% ND The conversion was low (<30%) when PMBBr was used to protect the hydroxy group of 8. 11 in the presence of NaH/tetra n butyl ammonium iodide (TBAI) in THF. PMBOC(NH)CCl 3 /TfOH was effective to convert alcohol 8. 11 into corresponding PMB ether in 80% conversion and 91 %yield. Weinreb amide 8.12 was obtained in 32 g using

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224 optimized conditions. Amide 8.12 was readily converted into the corresponding aldehyde 8. 12 by treatment with DIBAL H (Figure 8 5) Anti Crotylation of the C35 44 Aldehyde Fragment Asymmetric aldehyde c rotylation reactions are widely used for the synthesis of natural product containing a polyketide fragments. 165 166 167 168 169 170 Because of their important applications, many crotylating reagents have been developed (F igure 8 11). 171 172 173 174 175 176 Figure 8 11 Contempo rary crotylating reagents for anti addition of aldehyde The most widely used reagents are crotylboranes Despite several important con ceptual and/or practical advances, since the development of Brown's 177 a nd Roush's 178 crotylboronates the development of an asymmetric aldehyde crotylation method that is characterized by ( i ) wide scope and generality with respect to the aldehyde substrate, ( ii ) access to either diastereomer and (3) sustainable, safe, inexpensive, and scalable procedures remains a major problem. To construct the C34(Me) C35(OH) chiral unit of the key polyketide fragment (Figures 8 1 and 8 4), t here are only two anti selective allylation methods that have

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225 shown to be effective the Brown protocol 179 180 and the recently r eported EZ CrotylMix silicon based methodology (Figure 8 12 and 8 13). 32 Figure 8 12. Asymmetric crotylation of the key a ldehyde 8. 5 The Brown protocol requires the metallation of butene under cryogenic conditions, and quite often suffers from di fficulties in product isolation. By contrast, the EZ CrotylMixes is a highly attractive option for large scale reactions and in particular for complex aldehydes 181 Therefore, Leighton reagent CrotylMixes were chosen. Figure 8 1 3. Transition s tate model in diastereoselection in r eactions of EZ CrotylMix Ne xt, we set our goal to synthesize the crotylating reagent. The preparation of the EZ CrotylMixes 8. 18 involves the reaction of the requisite enantiomer of the diamine ligand 8. 22 with trans crotyltrichlorosilane and DBU, followed by

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226 concentra tion, trituration and f iltration of the DBUHCl salts w ith pentane recrystallization of the crotylsilane reagent, and mixing with the Sc(OTf) 3 catalyst all under strictly anhydrous conditions ( Figure 8 14). 182 183 184 Figure 8 14. Synthesis of Leighton crotylating reagent Although, reagent 8.18 (Figure 8 13) was obtained in 80% crude yield and 1 H spectra analysis clearly showed the formation of the compound, we were unable to purify the product. Indeed, all attempts led to decompos ition due to extended exposure to air and moisture. Other asymmetric methods for crotylation reported in the literature suffer from one or more drawbacks such as air or moisture sensitivity, the need for a multistep preparation of chiral reagents, unrepro ductibility or inco mpatibility with aldehydes. This prompted us to develop an efficient alternative to replace the capricious crotylation r eaction for the preparation of the polyketide fragment For this purpose, we

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227 investigated a simple proline mediated a ldolizatione / homologation synthetic sequence, which does not require the handling of sensitive reactants or the use of complex catalyst (Figure 8 15 ). Figure 8 15. Synthesis of polyketide fragment of apratoxins In order to evaluate this strategy, the synthesis of the alcohol 8. 6 was studied in the course of a two step procedure. U sing the L proline catalyzed cross aldol asymmetric reaction 185 between aldehyde 8. 5 and propionaldehyde afforded the intermediate 8. 23. Aldehyde 8. 23 was then used as a crude substrate for homologation conditions 186 to furnish the expected homoallylic alcohol 8.19 without loss of stereoselectivity and excellent con trol of the stereoselectivity No other diastereomer was detected by NMR and alcohol 8.19 was obtained in 52 % yield from crude intermediate 8.23 Conclusion A large scale preparation route to syntheses of the key aldehyde leading to polyketide fragments o f modified cyclic peptide apratoxins has been developed Advancing apratoxins or analogs thereof into further applications will require more efficient synthetic routes that are sustainable, safe, inexpensive, technically simple to perform, and readily sca lable. As a result of optimizing the reaction conditions and improving efficiency, this route is suitable for a large scale preparation. Key steps in volve asymmetric allylation, RCM, and asymmet ric conjugate d additions.

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228 Enantioselective aldehyde crotylation of the key a ldehyde is the major step in the total synthesis of apratoxins, but despite many co nceptual advances over the years, most of the current crotylating methods are elusive. W e developed a novel enantioselective aldol/olefin homologation reaction, which may be a valuable alternative to classical stereoselective crotylation methods. Experimen tal General Methods All commercial reagents were used without further purification unless otherwise noted. Tetrahydrofuran (THF) and diethyl ether (Et 2 O) were distilled from sodium chips in the presence of a small amount of benzophenone; CH 2 Cl 2 and toluene were distilled from CaH 2 ; MeCN, N,N dimethylformamide (DMF) were dried with 4 molecular sieves (MS) and MeOH dried with 3 MS; 4 M hydrochloric acid (HCl) solution in ethyl acetate was prepared by dissolving HCl gas (yielding by dropping aqueous hydroc hloric acid (34%) to concentrated sulfuric acid (98%)) to ethyl acetate. All reactions were performed in heat gun dried flasks (400 C under reduced pressure) under an inert atmosphere of anhydrous Ar unless otherwise noted. Thin layer chromatography was p erformed on EMD silica gel 60 F254 glass plates and preparative thin layer chromatography was performed on Whatman silica gel 60 F254 glass plates (layer mesh silica gel. Nuc lear magnetic resonance (NMR) spectra were recorded on a Varian Mercury 400 MHz. Chemical shifts for proton nuclear magnetic resonance ( 1 H NMR) spectra are reported in parts per million relative to the signal residual CDCl 3 at 7.26 ppm. Chemicals shifts fo r carbon nuclear magnetic resonance ( 13 C NMR) spectra are reported in parts per million relative to the center line of the CDCl 3 triplet at 77.16 ppm.

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229 The abbreviations s, d, dd, ddd, dddd, t, q, br, and m stand for the resonance multiplicity singlet, doub let, doublet of doublets, doublet of doublet of doublets, doublet of doublet of doublet of doublets, triplet, quartet, broad and multiplet, respectively. LR MS data was obtained using a 3200 QTrap triple quadrupole mass spectrometer and detection by electr ospray ionization MS in the positive ion mode. Preparation of C ompounds 8.6, 8.14 17 (S) 5,5 dimethyl 4 hydroxyhexan 2 one (8.14). 156 D proline (18.94, 164.53 mmol) was added to the mixture of acetone (0.8 L) and DMSO (3.2 L) at room temperature and the mixture was stirred at the same temperature f or 1h before pivalaldehyde (61 mL, 0.548 mol) was added. After stirring at room temperature for 4 days, the mixture was cooled to 0 C with an ice water bath and saturated aqueous NH 4 Cl solution ( 2 L) was added to quench this reaction. The mixture was extr acted with ethyl acetate (1 .5 L 3) and the extract was concentrated under reduced pressure to remove most of the ethyl acetate and acetone. Then the concentrated mixture was diluted with another 800 mL of ethyl acetate and washed with small portions of wa ter (200 mL 5) to remove most of the DMSO. The organic layer was dried with anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (8 20% ethyl acetate in hexane) to give product 8.14 ( 65 g, 82%) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ): 3.72 (ddd, J = 10.8, 3.6, 2.0 Hz, 1H), 2.85 (m, 1H), 2.63 (dd, J = 17.6, 2.4 Hz, 1H), 2.48 (dd, J = 17.2, 10.8 Hz, 1H), 2.20 (s, 3H), 0.90 (s, 9H). (S) 5,5 Dimethyl 4 (tert butyldimethylsilyloxy)hex an 2 one (8.15). 32 3,4 tert Butyldimethylsilyl chloride (TBS Cl) ( 102 g, 0.68 mol ) and imidazole ( 89.65 g, 1.32 mol) were added to the solution of compound 8.14 (62.66 g, 0.434 mol) in DMF (100 mL). After stirring at room temperature for 24 h under Ar, the reaction was quenched by

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230 addition of 150 mL methanol and 1.5 L water The mixture was extracted with ethyl acetate (1.5 L 3 ), the combined organic layers were concentr ated in vacuo and purified by column chromatography on silica gel (5% ethyl acetate in hexane) to give product 8.15 (95.5, 85%) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ): 3.95 (dd, J = 6.0, 4.0 Hz, 1H), 2.61 (dd, J = 17.2 Hz, 1H), 2.49 (dd, J = 17.6, 6.4 Hz, 1H), 2.15 (s, 3H), 0.86 (s, 9H), 0.84 (s, 9H), 0.06 (s, 3H), 0.06 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ): 207.9, 75.1, 48.2, 35.7, 31.43, 26.2, 26.0, 18.4, 4.0, 4.8. ( S) tert Butyl (1 tert butylbut 3 enyloxy)dimethylsilane (8.16). 32 NaBH 4 (95 g, 2.5 mol) was added to the solution of compound 8. 15 (324 g, 1.25 mol) in MeOH (3.5 L) at 0 C. After stirred at the 0 C for 40 min, the reaction was concentrated first and then 300 mL water was added at 0 C. The mixture was extracted with ethyl acetate (3 L 3), washed sequentially with brine (3L 2) and water (2 L 2), dried with anhydrous MgSO 4 evaporated in vacuo to give the crude alcohol 10 which was used in the next step without further purification. (S) tert butyl((2,2 dimethylhex 5 en 3 yl)oxy)dimethylsilane (8.17). 179 Et 3 N (99 ml, 715 mmol) and MsCl (55.4 ml, 715 mmol) were added to the solution of the crude 8.16 in dry CH 2 Cl 2 (1 L) at 0 C under Ar atmosphere. After stirring at the same temperature for 2.5 h, this reaction was quenched by brine (0.5 L). The organic layer was separated and water layer was extracted with CH 2 Cl 2 (300 mL 2). The combined CH 2 Cl 2 fractions were washed with water (300 mL 2), dried with anhydrous MgSO 4 and evaporated in vacuo to g ive the crude mesylate. t BuOK (132.3 g, 1.18 mol) was added to the solution of the above crude mesylate in dried toluene (1.2 L). The suspended mixture was heated to reflux for 1 h,

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231 cooled to room temperature and 0.6 L water was added. The organic layer w as separated and the water layer was extracted with heptane (0.9 L 3). The combined toluene and heptane layer was washed with brine (0.6 L 3) and water (0.6 L 2), dried with anhydrous MgSO 4 evaporated in vacuo and purified by column chromatography ( 100% hexane) to give product (80 g, 90 % 3 steps) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ): 5.88 (dddd, J = 16.8, 9.6, 7.2, 7.2 Hz, 1H), 5.00 (m, 1H), 3.32 (dd, J = 8.0, 4.0 Hz, 1H), 2.35 (m,1H), 2.13 (m, 1H), 0.90 (s, 9H), 0.87 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ): 137.9, 115.7, 80.2, 38.5, 36.3, 26.7, 26.3, 18.5, 3 .1, 4.1 (S) 2,2 Dimethyl 5 hexen 3 ol (8.6). 159 The solution of tetra n butylammonium floride trihydrate (TBAF) (82.07 g, 260.116 mmol) in THF (220 mL) was added to the mixture of compound 8.11 (160.3 g. 21.0 g, 0.662 mol) and 4 molecular sieves (pre dried at 450 C under reduced pressure 1,5 h) in anhydrous THF (2.3 L) at 0 C. Then the reaction mixture was stirred at room temperature overnight and filtered through a small pad of Celite (washed with diethyl ether). The f iltrate was quenched with 1.5 L of water, extracted with diethyl ether (2.2 L 3), washed with brine (2.2 L 2), dried over anhydrous MgSO 4 concentrated with cooling/condensing fraction distillation system under moderate vacuum, and further concentrated by Vigreux fraction distillation column. The concentrated mixture was purified by column chromatography eluted by 3 5% diethyl ether in pentane. The eluted product fractions were also concentrated by cooling/condensing fraction distillation system under m oderate vacuum to give product 8.6 and further distilled by Vigreux fraction distillation column to provide product 8.12 ( 78 g g 92 %). 1 H NMR (400 MHz, CDCl 3 ): 5.86 (dddd, J = 14.4, 10.4, 8.8, 6.0 Hz,

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232 1H), 5.14 (m, 2H), 3.25 (dd, J = 10.4, 2.0 Hz, 1H), 2.39 2.33 (m, 1H), 1.98 (ddd, J = 13.6, 9.6, 9.6 Hz, 1H), 0.91 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ): 136.7, 117.9, 78.2, 36.7, 34.7, 25.9. Synthesis of K ey Aldehyde 8. 5 S) 2,2 dimethylhex 5 en 3 yl acrylate (8.8). 159 180 Acryl oyl chloride (62 mL, 0.76 mol) and triethylamine (242 mL, 1.756 mol) were added sequentially to the solution of compound 8.6 (75, 0.585 mol) in anhydrous diethyl ether (2.5 L) at 0 C. The reaction was warmed to room temperature and stirred at room temperature for 4 h, then poured into cold water (2 L) and extracted with diethyl ether (2 L 4). The organic layer of extractions was washed with saturated NaHCO 3 (2 L 2), saturated NH 4 Cl (2 L), brine (2 L), dried with anhydrous MgSO 4 concentrated (first by cooling/condensing fraction distillation system under moderate vacuum and further by Vigreux fraction distillation column) and purified by column chromatography to give product 8.8 (101 g, 95%). Because the compound can easily evaporates along with solvent, the product fraction of column was also concentrated first by cooling/condensing fraction distillation system 187 188 under moderate vacuum and further by Vigreux fraction distillation column. General procedure for RCM Procedure A: 32 Ti(OiPr) 4 (20 mol% to substrate 8.8) was added to the solution of diene 8 in CH 2 Cl 2 (resulted in 2 or 3 mM of substrate 8.8) under Ar. The resulting second generation catalyst (15mol %) in CH 2 Cl 2 (degassed) was added under refluxing. The reaction mixture continued to reflux for another 4 6 h, was then cooled to room temperature, evaporated in vacuo and purified by column chromatography (eluted by diet hyl ether/pentane 2:7) to give product as a colorless oil.

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233 Procedure B: 160 Ar gas wa s passed through to the solution of diene 8.8 in CH 2 Cl 2 (resulted in 2 or 3 mM of substrate 8.8) for 1 generation catalyst (10 or 15 mol %) in CH 2 Cl 2 (degassed) was added under refluxing. The reaction mixture continued t o reflux for another 4 6 h, was then cooled to room temperature, evaporated in vacuo and purified by column chromatography (eluted by diethyl ether/pentane 2:7) to give product as a colorless oil. (6 S ) 6 tert Butyl 5,6 dihydro pyran 2 one (8.9). Colorless oil (65 g, 75%). 1 H J = 11.0, 6.4, 2.8 Hz, 1H), 6.01 (m, 1H), 4.06 (dd, J = 12.0, 4.4 Hz, 1H), 2.39 2.24 (m, 1H), 1.00 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ): 165.1, 145.7, 121.3, 85.4, 34.0, 25.6, 24.7. Procedure for c onjugate d methyl a dditi on Methyllithium (1.6 M in diethyl ether, 10 120 mL, 2.6 equiv) was added slowly (over 1 h) to the suspension of CuCN (1.2 equiv) in diethyl ether at 78 C. After the mixture was stirred at the same temperature for 40 min, it was moved to an ice bath for another 40 min, then re cooled to 78 C before compound lactone 8.9 (0.5 8g mmol) was added slowly (over 1 h) in dried diethyl ether (15 mL/ 1g). The reaction mixture was kept at 78 C 40 min, warmed to 50 40 C 40 min, 20 C 1 h, was then quenc hed with 5% FeCl 3 extracted with diethyl ether, washed with brine, dried with anhydrous MgSO 4 concentrated in vacuo purified by column chromatography (eluted by diethyl ether/pentane 1:4) to give product 8.10 as a colorless oil. 160 (4R,6S) 6 tert Butyl 4 methyl tetrahydro pyran 2 one (8.10). Colorless oil (67 g, 85%). 1 H NMR (400 MHz, C DCl 3 ): 3.97 (dd, J = 11.8, 3.6 Hz, 1H), 2.53 2.46 (m, 1H), 2.21 2.16 (m, 1H), 1.80 (dddd, J = 14.0, 11.6, 7.2 Hz, 1H),1.49 (ddd, J = 14.4, 3.2, 3.2 Hz, 1H), 1.09 (d, J = 6.4, 3H), 0.95 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 ): 173.4,

PAGE 234

234 83.8, 37.1, 34.1, 29. 9, 25.6, 24.1. MS ( m/z ): [M+H] + calcd for C 10 H 19 O 2 171.1, found 171.1. Procedure for ring opening of lactone 8. 10 Trimethylaluminum ((CH 3 ) 3 Al) (2M in hexane, 3 equiv) was added to the solution of N,Odimethylhydroxylamine hydrochloride (3 equiv. ) in CH 2 Cl 2 (1.5 mL/1g) at 78 C, then warmed to room temperature and kept at room temperature overnight. The solution of compound 10 ( Table 8 3, 1 8g) in CH 2 Cl 2 (2.5 mL/1 g of 8.10) was added slowly to the above solution over 1 h at 0 C. The reaction mixture was stirred at 0 C for 40 min and room temperature for another 5 h, was then concentrated to about 100 mL, acetate, washed with brine, dried with anhydrous MgSO 4 and purified by column chro matography (eluted by ethyl acetate/hexane 1:2) to give product 8.11 159 (3R,5S) 5 Hydroxy 3,6,6 trimethyl heptanoic acid methoxy methyl amide (8.11). Thick colorless oil (72 g, 90%). 1 H NMR (400 MHz, CDCl 3 ): 3.68 (s, 3H), 3.18 (s, 3H),3.13 (dd, J = 10.4, 2.4 Hz, 1H), 2.71 (br, 1H), 2.47 2.41 (m, 1H), 2.31 2.28 (m, 2H), 1.43 1.29 (m, 2H), 1.02 (d, J = 6.4 Hz, 3H), 0.87 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ): 175.0, 76.5, 61.3, 39.6, 38.3, 34.8, 32.4, 26.4, 26.0, 22.5 General procedure for PMB protection Procedure A 164 163 using PMBOC(NH)CCl 3 4 Methoxybenzyl 2,2,2 trichloroacetimidate (2 equiv) and trifluoromethane sulfonic acid (TfOH) (1 or 1.5 mol%) or Sc(TfO) 3 (10 mol%) or La (TfO) 3 (10 mol%) was added sequentially to the solution of 8.11 (025 8g) in THF (1.5 mL/1g) at 0 C. The resulting mixt ure was stirred at room temperature overnight and was then diluted with ethyl acetate, quenched with saturated NaHCO 3 extracted with ethyl acetate, dried with anhydrous MgSO 4 and evaporated in

PAGE 235

235 vacuo Hexane was added to the residue, which resulted in the precipitation of a white solid (2,2,2 trichloroacetimidate). The solid was filtered off, and the filtrate was concentrated and purified by column chromatography (eluted by 20 50% ethyl acetate in hexane) to give product 12 (50 80% conversion) (recovered s tarting material is used in next batch). Procedure B using PMBBr. 162 To a suspension of NaH (1 2 equiv ) in THF at 0 C were added solutions of amide 8.11, TBAI (15 mol%) dissolving in THF PMB Br (1 1.1 equiv) in THF via a cannula. The reaction mixture was st irred at room temperature for 16 h and then quenched with cold water. Volatiles were removed under reduced pressure and the residue was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous solution of NH 4 Cl, brine, a nd dried over MgSO 4 Conversion was determined by proton NMR analysis. (3R,5S) 5 (4 Methoxy benzyloxy) 3,6,6 trimethylheptanoic acid methoxy methyl amide (8.12). Colorless oil (32 g, 85%). 1 H NMR (400 MHz, CDCl 3 ): 7.31 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 4.60 (d, J = 10.4 Hz, 1H), 4.49 (d, J = 10.4, 1H), 3.78 (s, 3H), 3.67 (s, 3H), 3.19 (s, 3H), 3.07 (dd, J = 8.4, 2.8 Hz, 1H), 2.52 2.50 (m, 1H), 2.32 2.22 (m, 2H), 1.54 1.40 (m, 2H), 1.01 (d, J = 6.4 Hz, 3H), 0.93 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 61.3, 55.3, 39.1, 38.7, 36.3, 32.2, 27.8, 26.6, 21.7. MS ( m/z ): [M+H] + calcd for C 20 H 34 NO 4 352.2 found 352.1.

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236 Procedure for reduction of W ein reb amide Diisobutylaluminum hydride (1M in toluene, 8.27, 28.4 mmol) was added dropwise to the solution of 8.12 (4g, 11.34 mmol) in THF (240 mL) at 78 C. The ether (40 0 mL) when stirred at 78 C for 30 min. After the two phase mixture had been stirred vigorously at room temperature for 2 h, it was extracted with ethyl acetate (400 mL 3), washed with brine (180 mL 3), dried with anhydrous MgSO 4 concentrated in vacuo and purified by column chromatography (eluted by 8% ethyl acetate in hexane) to give product 2.8 g (85%) as a colorless oil. 32 (3R,5S) 5 (4 Methoxy benzyloxy) 3,6,6 tri methylheptanal (8.5). 1 H NMR (400 MHz, CDCl 3 ): 9.68 (s, 1H), 7.28 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 4.57 (d, J = 10.8 Hz, 1H), 4.48 (d, J = 10.8, 1H), 3.79 (s, 3H), 2.45 2.49 (m, 1H), 2.21 2.10 (m, 2H), 1.50 1.38 (m, 2H), 1.00 (d, J = 6.4 Hz, 3H), 0.94 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 25.7, 21.7. Synthesis of Leighton Anti Crotylating Reagent (1E,1'E) N,N' ((1S,2S) cyclohexane 1,2 diyl)bis(1 (4 bromophenyl) methanimine) (8.21). 184 To (1S,2S ) (+) 1,2 diaminocyclohexane L tartrate (4.8 g, 18.2 mmol, 1.0 equiv.) in water (85 mL) and EtOH (42.5 mL) was added K 2 CO 3 (5.02 g, 36.3 mmol, 2.0 equiv.) followed by a solution of 4 bromobenzaldehyde (6.72 g, 36.3 mmol, 2.0 equiv.) and methanesulfonic acid (141 L, 2.18 mmol, 0.12 equiv.) in DCM (85 mL) via dropping funnel over the course of 1 hour. The mixture was stirred at room temperature for 15 hours and then at reflux for 1 hour. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to a volume of ca. 50 mL.

PAGE 237

237 Then water (50 mL) was added and the mixture was filtered. The collected solid was dried under v acumn t o give the title diimin. White solid (6.92 g, 85%). 1 H NMR (4 00 MHz, CDCl 3 ppm): 8.11 (s, 2H), 7.45 (s, 8H), 3.41 3.34 (m, 2H), 1.91 1.74 (m, 6H), 1.54 1.44 (m, 2H). 13 C NMR (300 MHz, CDCl 3 ppm): 159.7, 135.1, 131.6, 129.3, 124.6, 73.7, 32.8, 24.4. 8, 1400, 1376, 1341, 1295, 1068, 1010. (1S,2S) N1,N2 bis(4 bromobenzyl)cyclohexane 1,2 diamine (8.22). 189 The diimin solid 21 (6.9 g, 15.39 mmol) was suspended in MeOH (30 mL) and cooled to 0 C befo re NaBH4 (1.71 g, 45.4 mmol, 2.95 equiv.) was added in portions. After bubbling had ceased (ca. 2 hou rs), the reaction was heated at reflux temperature for 90 minutes. It was then concentrated under reduced pressure, 1M NaOH (50 mL) and hexane/EtOAc 1:1 (50 mL) were added, the phases were separated, and the aqueous layer was extracted with hexane/EtOAc 1: 1 (twice 50 mL). The combined organic layers were washed with brine (15 mL), dried over MgSO 4 filtered and concentrated under reduced pressure. Diamine 8.22 (6.5 g, 14 .3 6 mmol, 92 %) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCl 3 ): 7.44 7.38 (m, 4H ), 7.20 7.14 (m, 4H), 3.83 (d, J = 13.4, 2H), 3.59 (d, J = 13.4 Hz, 2H), 2.25 2.16 (m, 2H), 2.16 2.07 (m, 2H), 1.82 (br s, 2H), 1.77 1.65 (m, 2H), 1.31 1.12 (m, 2H), 1.09 0.92 (m, 2H); 13 C NMR (100 MHz, CDCl 3 ): 140.2, 131.5, 129.9, 120.6, 61.0, 50.4, 31. 7, 25.1 (3a S ,7a S ) 1,3 Bis(4 bromobenzyl) 2 ((E) but 2 enyl) 2 chloro octahydro 1H benzo[d] [1,3,2] diazasilole (8.23) 190 181 191 To a solution of ((E) but 2 enyl) trichlorosilane (2 mL, 13.2 mmol, 1.1 equiv) in dry CH 2 Cl 2 (24 mL) at 0 C was added DBU (3.9 mL, 26.4 mmol, 2.2 equiv). A solution of (1S,2S) N,N0 bis (4 bromobenzyl) cyclohexane 1,2 diamine (5.43 g, 12 mmol, 1 equiv) in CH 2 Cl 2 (12 mL) was then added

PAGE 238

238 slowly. The reaction mixture was allowed to warm to room temperature. After 12 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was diluted in pentane (36 mL) and vigorously stirred overnight to ensure complete precipitation of DBU salts. The solution of pentane with desired reagent was then t ransferred to another flash using a cannula The solution was concentrated and washed with pentane to furnish the expected crude product as a yellow powder ( 2.34 g, 35 %). 1 H NMR (300 MHz, CDCl 3 ) 7.50 7.23 (m, 8H), 5.32 5.17 (m, 2H), 4.15 3.96 (m, 2H), 3.86 3.70 (m, 2H), 2.80 2.66 (m, 2H), 1.71 1.50 (m, 9H), 1.34 0.85 (m, 4H) Synthesis of Compound 8.19 (3R,4S,6S,8S) 8 ((4 methoxybenzyl)oxy) 3,6,9,9 tetramethyldec 1 en 4 ol (8.19) To a solution o f aldehyde 8.5 (0.3 g, 10.25 mmol, 1 equiv) and (L) proline (16 mg, 0.14 mmol, 0.2 equiv) in DMF (0.7 mL) at 0 C was added slowly a solution of propionaldehyde (0.1 mL, 1.5 mmol, 2 equiv) in DMF (0.5 mL). After 72 h the solvent was partially removed unde r reduced pressure the reaction was quenched with water and extracted with EtOAc/toluene 2:1. The organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude intermediate 8.23 was used without purification. Yellow oil (216 mg, 0.616 mmol, 60%) 185 T he crude product 8.23 186 (0.5 M in toluene, 0.9 mL, 1.5 equiv) was then added at 0 C. After 1 h, the reaction mixture was quenched with an aqueous solution of 10% NaOH, diluted in diethyl ether, and filtered through Celite. After removal of the solvents, the residue was purified by flash column chromatography (EtOAc/ Hexanes 10:90) to fu rnish the expected produc t as a colorless oil (111.6 mg, 52 %) 1 H NMR(400 MHz, CDCl 3 ): 7.30 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H),5.75 (m, 1H), 5.10 5.14 (m, 2H), 4.63 (d, J = 10.8 Hz, 1H),

PAGE 239

239 4.51 (d, J =10.8 Hz, 1H), 3.79 (s, 3 H), 3.50 (m, 1H), 3 .11 (dd, J = 9.3, 2.9 Hz, 1H),2.17 (m, 1H), 1.96 (m, 1H), 1.57 (ddd, J = 13.7, 10.7, 2.9 Hz, 1H), 1.47(ddd, J = 14.2, 8.8, 3.9 Hz, 1H), 1.35 (ddd, J = 14.2, 9.3, 2.4 Hz, 1H), 1.12(ddd, J = 13.7, 9.3, 2.4 Hz, 1H), 1.03 (d, J = 7.3 Hz, 3H), 0.96 (d, J =6.8 Hz 3H), 0.93 (s, 9H).

PAGE 240

240 APPENDIX A NMR SPECTRA Figure A 1. 1 H NMR spectrum of compound 8.14 in CDCl 3 (400 MHz)

PAGE 241

241 Figure A 2 1 3 C NMR spectrum of compound 8.14 in CDCl 3 (1 00 MHz)

PAGE 242

242 Figure A 3 1 H NMR spectrum of compound 8.6 in CDCl 3 (400 MHz)

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243 Figure A 4 1 H NMR spectrum of compound 8.8 in CDCl 3 (400 MHz)

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244 Figur e A 5 1 H NMR spectrum of co mpound 8. 9 in CDCl 3 (400 MHz)

PAGE 245

245 Figure A 6 13 C NMR spectrum of compound 8.9 in CDCl 3 (1 00 MHz)

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246 Figure A 7 1 H NMR spectrum of compound 8. 10 in CDCl 3 (400 MHz)

PAGE 247

247 Figure A 8 1 3 C NMR spectrum of compound 8.10 in CDCl3 (1 00 MHz)

PAGE 248

248 Figure A 9 1 H NMR spectrum of compound 8.11 in CDCl3 (400 MHz)

PAGE 249

249 Figure A 1 0 1 3 C NMR spectrum of compound 8.1 1 in CDCl 3 (1 00 MHz)

PAGE 250

250 Figure A 1 1 1 H NMR spectrum of compound 8. 12 in CDCl 3 (400 MHz)

PAGE 251

251 Figure A 1 2 1 3 C NMR spectrum of compound 8. 12 in CDCl 3 (1 00 MHz)

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252 Figure A 1 3 1 H NMR spectrum of compound 8. 5 in CDCl 3 (400 MHz)

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253 Figure A 1 4 1 3 C NMR spectrum of compound 8. 5 in CDCl 3 (1 00 MHz)

PAGE 254

254 Figure A 1 5 1 H NMR spectrum of PMB Br in CDCl 3 (400 MHz)

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255 Figure A 1 6 1 3 C NMR spectrum of PMB Br in CDCl 3 (1 00 MHz)

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256 Figure A 1 7 1 H NMR spectrum of PMB trichloroacetamide in CDCl 3 (400 MHz)

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257 Figure A 1 8 13 C NMR spectrum of PMB trichloroacetamide in CDCl 3 (1 00 MHz)

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258 Figure A 1 9 1 H NMR spectrum of compound 8.21 in CDCl 3 (400 MHz)

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259 Figure A 20 1 3 C NMR spectrum of compound 8. 21 in CDCl 3 (1 00 MHz)

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260 Figure A 2 1. 1 H NMR spectrum of compound 8. 22 in CDCl 3 (400 MHz)

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261 Figure A 22 1 3 C NMR spectrum of compound 8.22 in CDCl 3 (1 00 MHz)

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262 Figure A 23 1 H NMR spectrum of tricloro( E crotyl ) silane in CDCl 3 (400 MHz)

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263 Figu re A 24 1 H NMR spectrum of compound 8. 18 in CDCl 3 (400 MHz)

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264 Figure A 25 1 H NMR spectrum of compound 8. 18 in CDCl 3 (1 00 MHz)

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265 Figure A 26 1 H NMR spectrum of compound 8. 19 in CDCl 3 (400 MHz)

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275 BIOGRAPHICAL SKETCH Khanh Ha was born in Thai Nguyen, Vietn am. In 2002, after graduating from Thai Nguyen High Shool for Gif ted Students, he was awarded Fu ll Academic Scholarship by the Ministry of Training and Education of Vietnam to study Chemical Engineering at Irkutsk State Technical University in Russia. In 2006, he started his undergraduate research at the Favorsky Irkutsk Institute of Chemistry, a Sib erian Branch of the Russian Academy of Sciences under supervision of Professor Galina Levkovskaya as a Favorsky Scholar. His diploma thesis was appreciated by the State Board of Examiners, and he was awarded the Young Scientist certificate and the Bachelor Degree with Highest Honor in 2009. In 2010, he began his graduate studies at Chemistry Department under the supervision of Professor Alan Katritzky. In 2014, he moved to Dr. Center for Natural Products, Drug Discovery and Development t o finish up his thesis research focuses on methodologies for cyclization and coupling of peptides and synthesis of a pratoxin polyketide fragments which remains one of the main problems of contemporary organic chemistry. D r. Ha has published seven papers during his stay at the University of Florida. In addition he has participated in several internationally renowned conferences and delivered either a poster or an oral presentation. Khanh was recognized with the Certificate of Outstanding Achieveme nt, the Proctor & Gamble Award for Research Excellence, the Graduate Student Mentoring Award t he Alec Courtelis Award for excellent academic performance the Science for Life Graduate Student Award Presidential Service Award Synfacts Prize and recently the Leadership Development Award from The Younger Chemists Committee of the American Chemical Society Dr. Ha has been serving in reviewer p anel for journal s of the Royal Society of Chemistry since 2012