A novel approach to the synthesis of biologically active peptides

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Title:
A novel approach to the synthesis of biologically active peptides
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English
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Kayaleh, Roger
Affiliation:
Dr. Dennis Hall
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Subjects

Subjects / Keywords:
Tryptophan
Native Chemical Ligation
Organic Synthesis
Long Range Acyl Migration
N-acyl Migration

Notes

Abstract:
Tryptophan plays a significant role in a living organism. Tryptophan and its derivatives are involved in the formation of peptides, regulation of the immune system, and signaling between neurons. The importance of this amino acid for the synthesis of proteins makes it an important building block for organic synthesis, especially in ligation reactions. Native chemical Ligation (NCL is a method for coupling large peptide fragments and normally requires the use of a cysteine residue at the N-terminal peptidefragment. However, due to the fact that it is such a powerful synthetic technique, there has been a significant amount of research into developing ways to circumvent the need for a cysteine residue. Herein, we report a novel ligation strategy to perform an N- to N- acyl migration using a tryptophan residue on substrates that have cyclic transition states ranging in length from 10 to 18 members. The potency of this methodology as a synthetic route was demonstrated through the use of different targets in the final ligation reaction and high levels of final product that was produced in all but a few reactions.

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University of Florida
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Copyright Roger Kayaleh. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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1 A NOVEL APPROACH TO THE SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES Roger Kayaleh University of Florida Department of Biology June 2014 Advisor: Dr. Dennis Hall ACKNOWLEDGMENTS

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2 I would like to thank Dr. Suvendu Biswas and Mr. Khanh Ha for their support, guidance and encouragement. I would not be where I am today without them. I would also like to thank Dr. Dennis Hall for his role as advisor for my thesis. I would like to thank the late Dr. Alan Katritzky, may his soul rest in peace, for the opportunity to conduct research in his group. Finally, I would like to thank Lauren Wood and my family for keeping me focused and motivated throughout my college career.

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3 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ... 1 LIST OF TABLES ................................ ................................ ................................ ............. 4 LIST OF FIGURES ................................ ................................ ................................ ........... 5 LIST OF SCHEMES ................................ ................................ ................................ ......... 6 LIST OF ABBREVIATIONS ................................ ................................ .............................. 7 INTRODUCTION ................................ ................................ ................................ .............. 9 CHAPTER "# LONG RANGE CHEMICAL LIGATION FROM N N ACYL MIGRATIONS IN TRYPTOPHAN PEPTIDES VIA CYCLIC TRANSITION STATES OF 10 TO 18 MEMBERS link ................................ ................................ .............................. 17 $# Results and Discussion ................................ ................................ ....................... 1 7 2.1 Preliminary Results on N! N Acyl Migrations via 10 12 Membered Cyclic TS ................................ ................................ ................................ ........ 18 2.2 Feasibility of N! N Acyl Migrations via 13 Membered Cyclic TS ................ 19 2.3 Feasibility of N! N Acyl Migrations via 14 Membe red Cyclic TS ................ 21 2.4 Feasibility of N! N Acyl Migrations via 15 Membered Cyclic TS ................ 24 2.5 Feasibility of N! N Acyl Migrations via 16 18 Membered Cyclic TS ........... 25 2.6 Isolation of Ligated Product ................................ ................................ ......... 27 2.7 Competitive Ligation Experiments ................................ ............................... 28 %# Conclusion ................................ ................................ ................................ ........... 30 &# Experimental Section ................................ ................................ .......................... 31 4.1 General Methods ................................ ................................ ......................... 31 4.2 General Procedure for Preparation of Boc Protected Isotetrapeptides 2. 13a i and Isopentapeptides 2. 18a! c ................................ ........................... 31 4.3 General Procedure for Preparation of Unprotected Isotetrapeptides 2. 14a! i of Isopentapeptides 2. 19a! c ................................ ............................. 32 4.4 General Procedure for Chemical Ligation of N Acylisopeptides 2. 14a! i and N Acylisopentapeptides 2. 19a! c in DMF/Piperidine ............................... 32 LIST OF REFERENCES ................................ ................................ ................................ 43

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4 LIST OF TABLES Tab le page Table 1 1: Common sources of tryptophan in a typical diet. ................................ ......... 10 Table 2 1 Chemical ligation of N acyl isopeptide 2. 14a c via 13 membered TS ........... 21 Table 2 2 Chemical ligation of N acyl isopeptide 2.14d f via 14 membered TS ............ 23 Table 2 3 Chemical ligation of N acyl i sopeptide 2.14g i via 15 membered TS ............ 25 Table 2 4 Chemical ligation of N acyl isopeptides 2.19a c via 16 18 membered TS .... 27

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5 LIST OF FIGURES Figure page Figure 1 1: Important pathways for tryptophan metabolism ................................ .......... 11 Figure 2 1 Difference in 1 H spectra of isolated ligated peptide 2. 15e ( left) and starting compound 2. 14e (right) ................................ ................................ .......... 28

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6 LIST OF SCHEMES Scheme page Scheme 1 1 Summary of the synthetic production of EPO by Wang et. Al .................. 14 Scheme 1 2 Synthesis of lymphotactin via NCL 15 ................................ .......................... 15 Scheme 1 3 Native Chemical Ligation ................................ ................................ ........... 15 Scheme 1 4 NCL Derivatives ................................ ................................ ......................... 16 Scheme 2 1 Synthesis of isodipeptide 2.4 and isotripeptides 2.9a c ............................. 18 Scheme 2 2 Chemical ligation of N acyl isopeptides 2.9a c via 10 12 membered TS .. 19 Scheme 2 3 Synthesis of isotetrapeptides 2.14a c for ligation study via 13 membered TS ................................ ................................ ................................ ...... 20 Scheme 2 4 Chemical ligation of N acyl isopeptides 2.14a c via 13 membered TS ..... 21 Scheme 2 5 S ynthesis of isotetrapeptides 2.14d f for ligation study via 14 membered TS ................................ ................................ ................................ ...... 22 Scheme 2 6 Chemical ligation of N acyl isopeptides 2.14d f in via 14 membered TS .. 23 Scheme 2 7 Synthesis of isotetrapept ides 2.14g i for ligation study via 15 membered TS ................................ ................................ ................................ ...... 24 Scheme 2 8 Chemical ligation of N acyl isopeptides 2.14g i via 15 membered TS ...... 25 Scheme 2 9 Synthesis of isopentapeptides 2.19a c for ligation study ........................... 26 Scheme 2 10 Chemical ligation of N acyl isope ptides 2.19a c via 16 18 membered TS ................................ ................................ ................................ ........................ 27 Scheme 2 11 Competitive Chemical ligation of 2.14e in DMF/piperidine ...................... 29

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7 LIST OF ABBREVIATIONS Ala Alanine Cys Cysteine DIPEA N,N diisopropyleth ylamine DMF Dimethylformamide ESI MS Electrospray Ionization Mass Spectrometry Gly Glycine HPLC MS High Performance Liquid Chromotography Mass Specremetry Ile Isoleucine Leu Leucine Lys Lysine MeCN Acetonitrile NAD Nicotinamide Adenine Dinucleotide Pro Proline Thr Threonine Trp Tryptophan TS Transition State Tyr Tyrosine Val Valine

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8 Abstract Tryptoph an plays a significant role in living organism s Tryptophan and its derivatives are involved in the formation of peptides, regulation of the immune system, and signaling between neurons. The importance of this amino acid for the synthesis of proteins makes it an important building block for organic synthesis, especially in ligation reactions. Native chemical ligation (NCL) is a method for coupling large peptide fragments and normally requires the use of a cysteine residue at the N terminal peptide fragment. However, due to the fact t hat NCL is such a powerful synthetic technique, there has been a significant amount of research into developing ways to circumvent the need for a cysteine residue. Herein, we report a novel ligation strategy to perform an N to N acyl migration using a tryptophan residue on substrates that have cyclic transition states ranging in length from 10 to 18 bonds The potency of this methodology as a synthetic route was demonstrated through the use of different targets in the final ligat ion reaction and the amount of final product that was produced in all but a few of th e reactions

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9 CHAPTER 1 INTRODUCTION Each amino acid plays an important role, not just in the ability of the body to synthesize proteins, but also in the availability and production of certain hormones Like other amino acids, only the L isomer of tryptopha n ( L tryptophan) is used in protein synthesis. Tryptophan, however, is exceptional in many ways when compared to the other amino acids. The metabolic pathways that tryptophan follows are complex, with metabolites that are not only highly varied but also so me of the most important molecules found in an organism. Tryptophan is also involved in a myriad of different diseases that affect systems throughout the body. Discovered in the early 1900s by Hopkins and Cole 1 it was quickly realized that tryptophan was an important dietary component. Tryptophan is an essential nutrient in animal diets; however, plants, fungi and bacteria can synthesize it 2 In bacteria and fungi, the biosynthesis of tryptophan is used to ensure an adequate supply for the synthesis of pr oteins while plants use the biosynthetic pathway to provide precursors for molecules that act as regulators for growth, molecules important to pathogen defense, and even agents used to attract pollinators 2 Tryptophan cannot be made by animals and is ther efore considered an essential amino acid in animal diets. Intake in healthy adult humans range from 3.5 mg/kg 3 to 6 mg/kg 4 of body weight per day, however most individuals take in well above the required daily minimum. The average daily intake of tryptopha n for many individuals ranges from 900 to 1000 mg 5 Com mon sources of tryptophan include turkey, chicken, dairy products, chocolate, bread, tuna, and certain fruits (see Table 1 1 ) 5

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10 *The recommended daily allowance for a 79 kg adult is 278 to 476 mg **CAAs=Ile, Leu, Phe, Tyr, and Val, the five large neutral amino acids typically included in the tryptophan/CAA ratio Table 1 1 : Common sources of tryptophan in a typical diet. (Note: The ratio of tryptophan to Competing Amino Acids (CAA) represents the relative abundance of plasma tryptophan to cross the blood brain barrier.) As mentioned earlier, tryptophan is exceptional in that it is the precursor for a myriad of different metabolites. Besides protein synthesis, tryptophan gives rise to serotonin, t ryptamine, NAD, and other biologically i mportant molecules, as seen in F igure 1 1 3

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11 Figure 1 1 : Important pathways for tryptophan metabolism (noteworthy species in red) 3 In the body, t ryptophan i s found either free or bound to albumin However, of the two states, albumin bound tryptophan accounts for about 90% of the tryptophan concentration at equilibrium. However, most tryptophan metabolism occurs in the brain and only the free form can cross the blood brain barrier through a competitive, non specifi c L type amino acid transporter (hence Competitive Amino Acids from Table 1 L-Tryptophan 5-hydroxytryptophan 5-hydroxytryptamine (serotonin) N -acetyl-5-hydroxytryptamine Melatonin Tryptophan Hydroxylase Aromatic amino acid Decarboxylase N -acetyl Transferase 5hydroxyindoleOmethyltransfer ase N -formylKynurenine Kynurenine 3-hydroxyKynurenine 3-hydroxyanthranilic acid Picolinic Acid Xanthurenic Acid Kynurenic Acid Anthranilic Acid Tryptophan2,3-dioxygenase formamidase Hydroxylase Transaminase Kynureninase 2-amino-3-carboxymuconic acid 6-semialdehyde Nicotinamide adenine dinucleotide

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12 1 ) 6 However, tryptophan exhibits a higher affinity for the blood brain barrier than it does for albumin, so about 75% of the bound tryptophan will enter the brain 5 The most imp ortant pathway, the kynurenine pathway, accounts for 90% of tryptophan metabolism. This pathway is named after the first stable tryptophan derivative, kynurenine. Following the pathway, tryptophan is broken down by one of two enzymes, tryptophan 2,3 dioxyg enase (TDO) or indoleamine 2,3 dioxygenase (IDO). While TDO is found mostly in the liver, IDO is the predominant enzyme found elsewhere in the body, including neurons. The ultimate product of this pathway is NAD+, an important molecule in ox idative phospho rylation, but the by products produced along the way all perform important roles. For example, kynurenic acid has been shown to protect the brain against ischemia while 3 hydroxyanthranilic acid plays a role in the regulation of the immune system as well a s being a powerful antioxidant. Picolinic acid prevents the growth an d formation of tumors and exhibits antifungal and antiviral properties. As important as this pathway is and despite the positive effect of many of the products, up regulation of the pathway has been associated with infectious and autoimmune diseases as well as neurological and affective disorders 6 Be sides the kynurenine pathways, t ryptophan is also metab olized into serotonin, and from there into melatonin. Of all the dietary tryptophan, only about 3% is used for the synthesis of serotoni n while only about 1% is used in the brain, due to the difficulties of getting tryptophan across the blood brain barrie r in large concentrations 5 Despite the low rate of synthesis of serotonin, it has been found to be one of the more important neurotransmitters and also to play a role in other parts of the body with about 95% of the serotonin in an individual found in the gut 7 A change from the normal levels

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13 of serotonin can lead to debilitating mental illnesses, for example, an increase in the levels of serotonin during development has been recently suggested to be one of the causes of autism in children 8 while a decreas e can lead to Huntington's disease, schizophrenia, may have a role in the symptoms of Down's Syndrome 8, 9, 10 Serotonin can be converted into melatonin, an important regulator of circadian rhythms. Melatonin is produced in the pineal gland of the brain i n a two step process involving the conversion of serotonin into N acetylserotonin then into melatonin. Melatonin is also produced in the retina, kidneys, and digestive tract 11 Recently, melatonin has been shown to play a role in immunoregulaion. Melatonin is important in regulating hematopoeisis and immune cell production and function as well as exhibiting anti inflammatory properties 11 Tryptophan wa s first synthesized in 1949, but the synthesis was quickly replaced in the 1980s by a fermentation process that incr eased the yield. Because of the increase d yield supplements became widely a vailable. The use however, was linked to an outbreak of eosinophilia myalgia syndrome between 1988 and 1989 5 This disease was characterized by, among other things, cough s, chest pains, fever, and myalgia. Eventually the FDA banned the use of synthetic tryptophan as a supplement. 12 In addition to these functions, tryptophan is also extremely important in the production of proteins. But, despite this importance, and compar ed to other amino acids, tryptophan is the least abundant amino acid in the body. Due to this fact, tryptophan is thought to play a rate limiting step in the synthesis of proteins. As the rate limiting factor in the production of a potential polypeptide, i t can be used to help control diseases that are protein based in nature. Diseases like diabetes mellitus, the 8 th most

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14 common cause of death in the world, are caused by an inability to produce or respond to the peptide hormone insulin. Others, like Zolling er Ellison syndrome are caused by an over production of the peptide hormone gastrin, which can be inhibited by the peptide hormone glucagon 13 Recently, organic synthesis has begun to explore the potential to create hormones using completely synthetic met hods, a previously difficult goal to say the least. Particularly noteworthy was the successful creation of erythropoietin (EPO) using only synthetic methodology by Wang et. Al 14 as seen in S cheme 1 1 Scheme 1 1 Summary of the s ynthetic production of EPO by Wang et. Al NCL has also been used to create the anticoagulant microprotein s, the human neutrophil pro defensin 1, the glycoprotein lymphotactin, and a few others 14 98-124 SEt H + 125-166 H OH R 1) NCL 2) Thz opening 98-166 OH H R 60-97 1) NCL 2) Thz opening 60-166 OH H R 29-59 SEt H R NCL 29-166 H OH R R R R 7) MFD 8) Acm Removal 9) NCL 1-28 SEt H StBu Unfolded Erythropoeitin glycoform Folding Functional Erythropoeitin Notes: R=Acm Synthesis is done with sugars attatched to peptide. For simplicity, the sugars have been excluded from this summary of the scheme.

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15 Scheme 1 2 Synthesis of lymphotactin via NCL 15 NCL is a methodology that was first reported by Wieland et al. and further developed by Kent. 16,17 NCL is a two step process involving the transformation of a thioester bond to a peptide, as seen in scheme 1 3 Scheme 1 3 Native Chemical Ligation NCL is currently the most widely used chemoselective ligation technique 16 The first step of the reaction involves the formation of a thioester between the sulfur of the N terminal cysteine residue and the group to be added to the chain. The entire molecule then undergoes an intramolecular, rapid, S N transfer to form a natural peptide bond as opposed to the thioester present at the beginning. 1-47 + NH 3 O SR + 49-93 CO 2 O S + NH 3 NCL 1-47 + NH 3 O 49-93 CO 2 O SH H N SH SH Disulfide formation 1-47 + NH 3 O 49-93 CO 2 O S H N S 1P SR O + NH 2 S H H N O P2 P1 O NH 2 S H N O P2 NCL 1P O N H H S H N O P2

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16 However, NCL is limited in the fact that it requires a Cys residue at the N terminus resulting in an internal Cys re sidue, only found in about 2.26% of human proteins 19 There have been a number of different techniques developed to address the need for an N terminal Cys residue. Many of these methodologies involve attaching a thiol group to mimic a Cys residue. This can be done with Ala, Val, Leu, Lys, Thr, and Pro. In these processes, once the NCL step has been completed, the thiol group is removed in many cases using metal free desulfurization (MFD ) All this is shown in scheme 1 4 Despite this, there is still an inte nse amount of research into new ligation methods to allow for the creation of different peptides and proteins. Scheme 1 4 NCL Derivatives Until this point, however, the synthesis of a native peptide through an N to N acyl migration has not been explor ed. The Katritzky group recently discovered the first example of a successful migration involving tryptophan containing isopeptides via 10 11 and 12 Membered cyclic transition states. 20 This methodology utilizes none of the previous methods for circum venting the issues involved with NCL but still requires exploration and development through the examination of the following factors: i) the range of cyclic transition states; ii) the best conditions for the ligation step; iii) effects of 1P O N H SH H N O P2 R1 MFD 1P O N H H N O P2 R1 Ala, Val, Leu, Lys,Thr N H N O P2 HS P1 O MFD N H N O P2 P1 O Pro

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17 substituents in t he amino acid residue and rationalization of the relative abundance of the ligated product. In this paper, we examine the structural features controlling the ligation and document a synthetic investigation of N acyl isopeptide ligations to form nat ive pept ides from non terminal t ryptophan residues via 10 to 18 membered cyclic transition states. Herein, we discuss a novel methodology for the coupling of peptide fragments using "Tryptophan ligation" based on native chem ical ligation (NCL) principles. CHAPTE R 2 LONG RANGE CHEMICAL LIGATION FROM N N ACYL MIGRATIONS IN TRYPTOPHAN PEPTIDES VIA CYCLIC TRANSITION STATES OF 10 TO 18 MEMBERS link Results and Discussion Intermediate isodipeptide 2.4 was synthesized and served as the starting material for the investigation of N to N acyl migration via 10 18 membered cyclic transition states. In this study we aim to investigate a novel chemical ligation strategy for N to N acyl transfer by developing a general methodology and a computational model to pred ict the feasibility of N to N acyl migration in longer peptide synthesis. Compound 2.4 was coupled with benzotriazolides of dipeptides 2.12a i and tripeptides 2.17 1 a c of # or $ amino acids to afford isotetrapeptides 2.14a i and isopentapeptides 2.19 a c required for the ligation studies involving 13 18 membered cyclic transition states. To enhance the migration rates, possibly by lowering the steric hindrance at the ligation sites, we placed Gly, # and $ amino acid units within the isopeptides in or der to study the feasibility of N to N acyl migrations in 13 18 membered cyclic transition states. A Reproduced with permission from Chemistry A European Journal 201 4 DOI: 10.1002/chem.201400125 Cop yright 2014 WILEY VCH Verlag GmbH & Co. KGaA, Weinheim.

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18 statistical model was generated using conformational analysis and molecular descriptors. 2.1 Preliminary Results on N! N Acyl Migrations via 10 12 Membered Cyclic TS N Acylation of the indole nitrogen in Trp was challenging, but w as achieved when Boc protected Trp 2. 1 was treated with Cbz Ala Bt 2.2 in MeCN in the presence of strong base (e.g. DBU ) resulting in Boc protected N acylisodipeptide 2. 3 (80%). Subsequent Boc group deprotection was conducted using 4N HCl in dioxane solution to afford the hydrochloride salt of unprotected isodipeptide 2. 4 (91%). Scheme 2 1 Synthesis of isodipeptide 2.4 and isotripeptides 2.9a c In our previous studie s, 20 we tried chemical ligation experiments on 2.4 and observed that chemical ligation via a 7 membered cyclic TS was not favored in either aqueous buffer or basic DMF/piperidine condition. However in longer isopeptides 2.9a c which were prepared by the u sual coupling and deprotection protocol (Scheme 2 1) under DMF/piperidine conditions, N to N acyl migration occurred via 10 12 membered cyclic TS 's (Scheme 2 2) to give Z protected tripeptides 2.10a c ( 44.4%, 71.4%, and 99.1% respectively) as the ligation products. This N to N acyl chemical ligation occurs

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19 in Trp, one of the important natural amino acids present in peptides and proteins. This encouraged us to explore the area of chemical ligation by de veloping a general, high yielding and feasible pathway to the synthesis of Trp containing peptides via ligation techniques. Scheme 2 2 Chemical ligation of N acyl isopeptides 2.9a c via 10 12 membered TS 2.2 Feasibility of N! N Acyl Migrations via 13 Membered Cyclic TS The starting isotetrapeptides 2.14a c for the N to N acyl transfer via 13 membered TS were prepared by a straight forward coupling reaction. N Acylbenzotriazoles are advantageous reagents to construct peptides, peptidomimetics and p eptide conjugates. 24 25 Compound 2.4 and four different Boc protected dipeptide benzotriazolides 2.12a c were first coupled in MeCN/DIPEA to afford Boc protected isotetrapeptides 2.13a c No chromatography was needed and compounds 2.13a c were purified by acidic and basic work ups. Boc deprotection of the Trp containing isotetrapeptides in 4N HCl/dioxane afforded the HCl salt of unprotected isotetrapeptides 2.14a c (Scheme 2 3). The amino acids Gly, Ala, and Pro were chosen for the isotetrapeptide sequence to enable a comparative study on the effect of the substituents at the chemical ligation site for the 13 membered cyclic TS. 50 W, 50 o C, 3 h HN N R O OBn n 2.9a n = 1 2.9b n = 2 2.9c n = 3 O NH 2 O HN N R O OBn n O NH 2 O HN H N OBn n O NH O R O 2.10a n = 1, 44.4% 2.10b n = 2, 71.4% 2.10c n = 3, 99.1% DMF/Piperidine R = Cbz-Ala

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20 Scheme 2 3 Synthesis of isotetrapeptides 2.14a c for ligation study via 13 membered TS Initially, the chemical ligati on experiments were carried out for 2.14a under aqueous conditions (pH 7.4, 1M buffer strength, MW 50 ¡C, 50 W, 3 h) to produce the ligation products. However the expected ligation product was observed in relatively low yield (2%). The ligation experiments were then switched to basic piperidine/DMF condition. This resulted in the expected ligation products, which, in some cases were produced in almost quantitative yield (Table 2 1). In the case of the Pro containing isotetrapeptide 2.14c the relative abund ance of ligated peptides 2.15c was 17%. This result was expected, as it was anticipated that the chemical ligation would be less feasible as a result of the Pro residue inducing a turn in the peptide chain for 2.14d resulting in too large a distance betwe en the reaction sites. HPLC! MS, using (! )ESI MS/MS, confirmed the formation of the ligated products 2.15a! c which produce different MS fragmentation patterns from those of the starting isotetrapeptides 2.14a! c The relative abundance of the crude ligated mixtures were analyzed by HPLC (Table 2 1). 2.4 N H N O + N H OBn O N R O N N 2.12c 82-86% HCl/Dioxane 2.14c 92% O N H R 1 Boc H N O O N H R 1 Boc DIPEA MeCN N H OBn O N R O H N O O + H 3 N R 1 Cl 2.13c 2.4 N H N O + N H OBn O N R O N N 2.12a-b 82% 2.14a-b 92-94% O N Boc H N O O N Boc DIPEA MeCN N H OBn O N R O H N O O + H 2 N Cl 2.13a-b HCl/Dioxane R = Cbz-Ala

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21 Scheme 2 4 Chemical ligation of N acyl isopeptides 2.14a c via 13 membered TS Table 2 1 Chemical ligation of N acyl isopeptide 2. 14a c via 13 membered TS Product characterization by HPLC MS Relative area (%) [a,b] Ligated peptide (LP) React Cyclic TS size Total crude yield (%) of products isolated React (RT) LP (RT) BA (RT) LP [M+H] + found 2.14a 13 87 39.11 (41.3) 60.89 (46.3) 0.00 2.15a 614.3 2.14b 13 88 27.20 (46.3) 72.80 (56.5) 0.00 2.15b 628.3 2.14 c 13 84 83.05 (45.7) 16.95 (54.6) 0.00 2.15 c 654.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time 2.3 Feasibility of N! N Acyl Migrations via 14 Membered Cyclic TS Coupling reactions between 2.4 and four different Boc protected dipeptide benzotriazolides 2.12d f were carried out to study N to N acyl transfer via a 14 membered TS. Boc deprotection of tryptophan tetrapeptides 2.13d f in 4N HCl/dioxane gave the HCl salt of unprotected tetrapeptides 2.14d f which were chosen as potential Piperidine/DMF 50 W, 50 o C, 3 h NH N R O OBn 2.14a R 1 = H 2.14b R 1 = CH 3 2.14c R 1 = CH 2 CH 2 CH 2 for Pro O HN O 2.15a-c 2.16a-c O H 2 N R 1 NH N R O OBn O HN O O H 2 N R 1 O O N H O NH O NH HN OBn R O O N H O NH O NH N OBn R O R R 1 R 1 R = Cbz-Ala

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22 substrates for the ligation study via a 14 membered TS. The amino acids Gly, Ala, Ala, and Pro and other amino acids in the isotetrapeptide sequence were chosen to enable a comparative study on the effect of che mi cal ligation between the 14 membered cyclic TS and the 13 membered cyclic TS. Scheme 2 5 Synthesis of isotetrapeptides 2.14d f for ligation study via 14 membered TS Chemical ligation via a 14 membered cyclic TS was investigated by subjecting isotetrapept ides 2. 14d f to microwave irradiation at 50 ¡C, 50W for 3h using basic piperidine/DMF condition s (Scheme 2 6). The reaction mixtures were cooled, the solvent was removed under reduced pressure, and the ligation mixtures (1.0 mg/mL in methanol) were analyze d by HPLC MS. The NH 2 site of unprotected N acyl isotetrapeptides 2. 14d f was attacked intramolecularly by the amide carbonyl carbon (C=O) linked to indole nitrogen of Trp via a 14 membered cyclic TS (Scheme 2 4) to give ligated peptides 2. 15d f Formatio n of the expected ligation products in the cases of Gly and Ala 2. 15d,e was almost quantitative. However in case the of 2. 14f (Pro at the N terminus) only 2.3% of the ligated product 2. 15f was observed. This is consistent with our findings for 13 membered TS size for ligation products with similar amino 2.4 N H N n O + N H OBn O N R O N N 2.12f HCl/Dioxane 2.14f 94% O N H R 1 Boc H N n O O N H R 1 Boc DIPEA MeCN N H OBn O N R O H N n O O + H 3 N R 1 Cl 2.13f 92% 2.4 N H N n O + N H OBn O N R O N N 2.12d-e 2.14d-e 92-95% O N Boc H N n O O N Boc DIPEA MeCN N H OBn O N R O H N n O O + H 2 N Cl 2.13d-e 84-87% HCl/Dioxane R = Cbz-Ala n = 2

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23 acids. HPLC! MS, using (! )ESI MS/MS, confirmed that the ligated products 2. 15d f each produced different MS fragmentation patterns from tho se of the starting isotetrapeptides 2. 14d f The relative abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 2. Scheme 2 6 Chemical ligation of N acyl isopeptides 2. 14d f in via 14 membered TS Table 2 2 Chemical ligation of N acyl isopeptide 2. 14d f via 14 membered TS Product characterization by HPLC MS Relative area (%) [a,b] Ligated peptide (LP) React Cyclic TS size Total crude yield (%) of products isolated React (RT) LP (RT) BA (RT) LP [M+H] + found 2.14 d 14 83 11.06 (39.3) 88.94 (45.7) 0.00 2.15 d 628.3 2.14 e 14 88 1.75 (48.0) 98.25 (56.5) 0.00 2.15 e 642.3 2.14 f 14 84 97.37 (44.8) 2.63 (55.0) 0.00 2.15 f 668.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time Piperidine/DMF 50 W, 50 o C, 3 h NH N R O OBn 2.14d R 1 = H 2.14e R 1 = CH 3 2.14f R 1 = CH 2 CH 2 CH 2 for Pro O HN O 2.15d-f 2.16d-f O H 2 N R 1 NH N R O OBn O HN O O H 2 N R 1 O O N H O NH O NH HN OBn R O O N H O NH O NH N OBn R O R R 1 R 1 n=2; R = Cbz-Ala n n n n

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24 2.4 Feasibility of N! N Acyl Migrations via 15 Membered Cyclic TS The starting isotetrapeptides for the N to N acyl transfer via a 15 membered TS were prepared following a similar protocol in to that of Scheme 2 5. Four different Boc protected dipeptide benzotriazolides 2. 12g i were first coupled with compound 2. 4 in MeCN/DIPEA to afford Boc protected isotetrapeptides 2. 13g i Boc deprotection in 4N HCl/dioxane gave the HCl salt of free isotetrapeptides 2. 14g i Scheme 2 7 Synthesis of isotetrapeptides 2. 14g i for ligation stud y via 15 membered TS Chemical ligation via a 15 membered cyclic TS was investigated under similar conditions to that of Scheme 2 4. The abundance of the expected ligation products in most cases was low. In the case of proline containing isotetrapeptide 2.14i only 1% of the ligated product 2.15i was observed. It is possible that this result was due to a Pro induced turn in the peptide chain. HPLC! MS using (! )ESI MS/MS, confirmed that the ligated products 2.15g i each produced different MS fragmentation pa tterns from those of the starting isotetrapeptides 2.14g i The relative abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 3. 2.4 N H N n O + N H OBn O N R O N N 2.12i HCl/Dioxane 2.14i 92% O N H R 1 Boc H N n O O N H R 1 Boc DIPEA MeCN N H OBn O N R O H N n O O + H 3 N R 1 Cl 2.13i 86% 2.4 N H N n O + N H OBn O N R O N N 2.12g-h 2.14g-h 90-94% O N Boc H N n O O N Boc N H OBn O N R O H N n O O + H 2 N Cl 2.13g-h 85-87% HCl/Dioxane R = Cbz-Ala n = 3 DIPEA MeCN

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25 Scheme 2 8 Chemical ligation of N acyl isopeptides 2. 14g i via 15 membered TS Table 2 3 Chemical ligation of N acyl isopeptide 2. 14g i via 15 membered TS Product characterization by HPLC MS Relative area (%) [a,b] Ligated peptide (LP) React Cyclic TS size Total crude yield (%) of products isolated React (RT) LP (RT) BA (RT) LP [M+H]+ found 2.14 g 15 85 8.29 (37.7) 91.71 (43.8) 0.00 2.15 g 642.3 2.14 h 15 90 53.60 (45.4) 46.40 (54.1) 0.00 2.15 h 656.3 2.14 i 15 87 99.14 (45.4) 0.86 (55.9) 0.00 2.15 i 682.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product, RT = retention time 2.5 Feasibility of N! N Acyl Migrations via 16 18 Membered Cyclic TS N to N acyl transfer via 16 18 membered TSs would facilitate the synthesis of longer peptides. Coupling reactions between 2.4 and three different sets of Boc protected tripeptide benzotriazolides 2.17a c in Me CN in the presence of 3.0 equiv. of DIPEA at 20 o C gave Boc protected N acylisopentapeptides 2.18a c Compounds 2.18 were purified by acidic and basic workups and no chromatography was required. The Piperidine/DMF 50 W, 50 o C, 3 h NH N R O OBn 2.14g R 1 = H 2.14h R 1 = CH 3 2.14i R 1 = CH 2 CH 2 CH 2 for Pro O HN O 2.15g-i 2.16g-i O H 2 N R 1 NH N R O OBn O HN O O H 2 N R 1 O O N H O NH O NH HN OBn R O O N H O NH O NH N OBn R O R R 1 R 1 n=3; R = Cbz-Ala n n n n

PAGE 26

26 HCl salts of unprotected isopentapeptides 2.19a c were achieved upon Boc deprotection of N acylisopentapetides 2.18a c in 4N HCl/dioxane. Scheme 2 9 Synthesis of isopentapeptides 2.19a c for ligation study The chemical ligation experiments were performed first for 2.19a under basic piperidine/DMF (MW 50¡C, 50W, 3 h) to produce the expected ligation products. Ligation did not occur for 16 membered cyclic transition states (we recovered mainly starting material 2.19a ). Compounds 2.19b and 2.19c were irradiated under microwave conditions in basic piperidi ne/DMF (MW 50¡C, 50 W, 3 h) and the reaction mixtures were analyzed by HPLC MS which showed significant amounts of ligated products. The abundance of the expected ligation products in case of 2.19b was 31% and for 2.19c 21%. HPLC! MS, via (! )ESI MS/MS, con firmed that the ligated products 2.20b,c each produced different MS fragmentation patterns from those of the starting isotetrapeptides 2.19b,c The abundances of the crude ligated mixtures as analyzed by HPLC are shown in Table 2 4. On combining all our ex perimental results, we observed that the intramolecular N to N acyl migration is highly favored for medium ring size cyclic transition states, while for larger ring size cyclic transition states there is a decrease in the ligation products.

PAGE 27

27 Scheme 2 10 Chemical ligation of N acyl isopeptides 2.19a c via 16 18 membered TS Table 2 4 Chemical ligation of N acyl isopeptides 2.19a c via 16 18 membered TS Product characterization by HPLC MS Relative area (%)[a,b] Ligated peptide (LP) React Cyclic TS size Total crude yield (%) of products isolated React (RT) LP (RT) BA (RT) LP [M+H]+ found 2.19a 16 83 100 (49.4) 0 (NA) 0.00 2.20a 2.19b 17 86 69.40 (53.9) 30.60 (66.9) 0.00 2.20b 739.3 2.19c 18 87 79.30 (53.8) 20.70 (67.5) 0.00 2.20c 753.3 [a] Determined by HPLC MS (semi quantitative). The area of ion peak resulting from the sum of the intensities of the [M+H] + and [M+Na] + ions of each compound was integrated (corrected for starting material) [b] LP = ligated peptide, BA = bisacylation product 2.6 Isolation of Ligated Product The formation of ligated product 2.15e from compound 2.14e was further confirmed by isolation via semi preparative HPLC and characterized by 1 H and 13 C NMR spectroscopy, elemental analysis and analytica l HPLC. 1 H NMR spectra showed clear differences in 11.00 7.00 ppm (Figure 2 1). The appearance of new peak in the case of 2.15e at 10.85 ppm in the 1 H NMR (which was absent in 2.14e ) indicated the formation of the desired ligation product. This peak at 10. 85 ppm is a typical NH proton Piperidine/DMF 50W, 50 o C, 3h 2.19a R 1 = CH 3, n=1 2.19b R 1 = H, n=2 2.19c R 1 = H, n=1 R = Cbz-Ala n n n N H OBn O N R O HN O O H 2 N O N R 1 n H N OBn O N R O HN O O H 2 N O N R O O H N O N O NH R 1 O HN NH OBn R 2. 20a-c O O H N O N O NH R 1 O HN N OBn R O R 2. 21a-c

PAGE 28

28 peak from the indole ring in a Trp moiety and clearly confirmed the intramolecular N N acyl migration of Z alanine to the N terminus forming native peptide 2.15e (Scheme 2 6). Figure 2 1 Difference in 1 H spectra of isolated ligated peptide 2. 15e ( left) and starting compound 2. 14e (right) 2.7 Competitive Ligation Experiments To further support the intramolecular nature of the chemical ligation of unprotected isopeptides 2.14a i and 2.19a c we studied the chemical ligation of isotetrapeptide 2.14e in the presence of 20 equiv. of dipeptide 2.22 (H Gly Gly OMe) under same reaction conditions to that of Scheme 2 6. HPLC! MS analysis of the isolated crude product confirmed the formation of 20% of the desired li gation product 2.15e with a retention time at 48.0 min along with 80% of the starting material 2.14e with a retention time at 56.5 min. No bisacylated product 2.16e was observed. It was also observed that there is no Cbz protected tripeptide 2.23 (which is the N acylated product of dipeptide 2.22 via an intermolecular ligation pathway) in the HPLC! MS analysis. This competitive experiment supports the hypothesis that the N to N acylation is intramolecular rather than intermolecular.

PAGE 29

29 Scheme 2 11 Competitive Chemical ligation of 2.14e in DMF/piperidine 2.14e + O H 2 N O H N O 2.15e Ligation LP BA + 2.16e + O N H O H N O O H N O O cross-over product

PAGE 30

30 CHAPTER 3 Conclusion 3. Conclusion The method of synthesis developed herein is a novel and efficient method for the synthesis of tryptophan containing peptides. The intramolecular chemical ligation via an N to N acyl migration was favored through long range 10 to 18 membered cyclic TS 's forming the native peptides. This ligation was achieved without resorting to the use of cysteine, serine, tyrosine, or an auxiliary group at the N terminus of the peptide chain, as is common when using NCL. While there are many efficient methods of synthe sis for the creation of peptides, scientists are devoted to finding more chemoselective methods for the synthesis of modified proteins. With the numerous ligation methods available to researchers, supplemented by the technique discussed herein, it is likel y t hat synthetic proteins will become commonplace Given this, and the amount of research engaged in finding novel methods for the synthesis of long peptides, we believe that the methodology discussed herein to be a significant advancement to the field of organic synthesis.

PAGE 31

31 CHAPTER 4 Experimental Section 4.1 General Methods All commercial materials were used without further purication. All solvents were reagent grade or HPLC grade. Melting points were determined on a capillary point apparatus equipped wi th a digital thermometer and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 DMSO d 6 and CD 3 OD using a 300 MHz and 500 MHz spectrometer (with TMS as an internal standard). All 13 C NMR spectra were recorded with complete proton decoupli ng. All microwave assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. Th e tube was closed with a silicon septum and there action mixture was subjected to microwave irradiation (Discover mode; run time 60 s.; PowerMax cooling mode). HPLC MS analyses were performed on reverse phase gradient Phenomenex Synergi Hydro RP (2.1 # 1 50 mm; 5 $m) + guard column (2 # 4 mm) or Thermoscientic Hypurity C8 (5 $m; 2.1 # 100 mm + guard column) using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; and mass spectrometry was done with electrospray ionization (ESI). 4.2 G eneral Procedure for Preparation of Boc Protected Isotetrapeptides 2. 13a i and Isopentapeptides 2. 18a! c Boc protected benzotriazolides of dipeptides (Boc AA 1 AA 2 Bt) or tripeptides (Boc AA 1 AA 2 AA 3 Bt) (1.0 mmol, 1.0 equiv.) in MeCN (10 mL) were added dro p wise to a solution of 2.4 (1.0 mmol, 1.0 equiv., 0.54 g) and DIPEA (3.0 mmol, 3.0 equiv., 0.52 mL) at room temperature and stirred for 16 h until all the starting material was consumed. MeCN was evaporated and the residue dissolved in EtOAc (50 mL) and

PAGE 32

32 washed with 3N HCl (5 # 50 mL), 5% sodium bicarbonate (2 # 50 mL) and brine (1 # 50 mL). The organic portion was dried over anhydrous Na 2 SO 4 filtered, concentrated and recrystallized from ether to give the corresponding Boc protected isotetra peptides 2.13a i 4.3 General Procedure for Preparation of Unprotected Isotetrapeptides 2. 14a! i of Isopentapeptides 2. 19a! c Boc protected isotetrapeptides 2.13a! i or isopentapeptides 2.19a! c (0.5 mmol) were dissolved in 4 N HCl in 1, 4 dioxane (15mL) at 20 ¡C and stirred for 2 h. The reaction mixture were evaporated, and the residue was recrystallized from diethyl ether to give the corresponding hydrogen chloride salts of unprotected isotetrapeptides 2.14a! i or isopentapeptides 2.19a! c 4.4 General Procedure for Chemical Ligation of N Acylisopeptides 2. 14a! i and N Acylisopentapeptides 2. 19a! c in DMF/Piperidine N Acylisotetrapeptides 2.14a! i or N acylisopentapeptides 2.19a! c as the HCL salts, (0.20 mmol) were each dissolved in a mixture of DMF! piperidine (5 m L/ 1.5 mL), and the mixture was irradiated under microwave (50 ¡C, 50W, 3 h) in a microwave tube. After cooling to room temperature the reaction mixtures were acidified with 2 N HCl to pH = 1. Each mixture was extracted with ethyl acetate (3 # 10 mL), the combined organic extracts were dried over sodium sulfate, and the solvent was removed under reduced pressure. Each ligation mixture was weighed, and then absolution in methanol (1 mg mL 1 ) was analyzed by HPLC! MS. Boc Gly Gly Trp(Z Ala) OBn ( 2.13a ). 0.59 g 86%: mp 90.0! 90.7 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.29 (d, J = 7.5 Hz, 1H), 7.58 7.35 (m, 2H), 7.24 6.88 (m, 12H), 5.08 4.84 (m, 5H), 4.73 4.57 (m, 1H), 3.76 (d, J = 6.6 Hz, 2H), 3.60 (d, J = 7.2 Hz, 2H), 3.18 2.98 (m, 2H), 1.41 1.21 (m, 12H).; 13 C NMR (75 MHz, CDCl 3 ) % 171.9, 171.2,

PAGE 33

33 171.0, 169.5, 156.6, 156.0, 136.3, 136.1, 135.1, 130.6, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 125.8, 124.3, 123.4, 118.9, 117.8, 117.1, 80.4, 67.7, 67.3, 52.3, 49.8, 44.3, 43.4, 28.5, 27.2, 19.7; Anal. Calcd for C 38 H 43 N 5 O 9 : C, 63.94; H, 6.07; N, 9.81. Found: C, 63.74; H, 6.46; N, 9.38 Boc Ala Gly Trp(Z Ala) OBn ( 2.13b ). 0.59 g, 82%: mp 86.0! 88.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.39 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 11.1 Hz, 1H), 7.40 6.96 (m, 15H), 5.94 5.69 (m, 1H), 5.55 5.36 (m, 1H), 5.01 4.87 (m, 6H), 4.19 4.00 (m, 1H), 3.97 3.77 (m, 2H), 3.30 3.15 (m, 2H), 1.53 1.26 (m, 15H).; 13 C NMR (75 MHz, CDCl 3 ) % 174.4, 171.5, 171.1, 169.4, 156.0, 155.6, 136.3, 136.1, 135.0, 130.6, 128.7, 128.7, 128.6, 128.4, 128.4, 128.2, 125.8, 124. 3, 123.3, 118.9, 117.8, 117.1, 80.1, 67.6, 67.3, 52.3, 50.3, 49.8, 43.5, 28.5, 27.4, 19.5, 18.3; Anal. Calcd for C 39 H 45 N 5 O 9 : C, 64.36; H, 6.23; N, 9.62. Found: C, 64.52; H, 6.63; N, 9.38. Boc Pro Gly Trp(Z Ala) OBn (2.13c). 0.62 g, 82%: mp 65.0! 68.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.38 (d, J = 8.4 Hz, 1H), 7.52 7.42 (m, 2H), 7.38 7.23 (m, 13H), 7.20 7.00 (m, 2H), 5.90 5.65 (m, 1H), 5.18 4.92 (m, 6H), 4.23 3.76 (m, 3H), 3.48 3.13 (m, 5H), 1.91 1.63 (m, 3H), 1.42 1.26 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) % 173.8, 171.4, 171.2, 169.8, 156.0, 155.6, 136.4, 136.1, 135.2, 130.6, 128.7, 128.5, 128.3, 128.2, 128.1, 125.8, 124.3, 123.1, 118.9, 118.2, 117.0, 115.2, 80.6, 67.5, 67.3, 60.7, 52.4, 49.8, 47.5, 43.4, 29.8, 28.6, 27.2, 24.7, 19.2; Anal. Calcd for C 41 H 47 N 5 O 9 : C, 65.32; H, 6.28; N, 9.29. Found: C, 65.17; H, 6.62; N, 9.05. Boc Gly BAla Trp(Z Ala) OBn (2.13d). 0.63 g, 86%: mp 88.0! 89.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.29 (d, J = 7.5 Hz, 1H), 7.61 7.35 (m, 2H), 7.24 6.97 (m, 12H), 5.08 4.84 (m, 5H), 4.73 4.57 (m, 1H), 3.54 (d, J = 8.7 Hz, 2H), 3.39 3.26 (m, 2H),

PAGE 34

34 3.14 2.95 (m, 2H), 2.43 2.20 (m, 2H), 1.36 1.21 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) % 171.8, 171.4, 171.1, 169.7, 156.2, 155.8, 136.2, 136.1, 135.1, 130.7, 128.8, 128.7, 128.4, 128.3, 128.2, 126.0, 124.4, 120.5, 1 18.9, 117.1, 114.4, 80.3, 67.7, 67.4, 52.3, 49.7, 44.5, 35.9, 34.5, 28.5, 27.5, 19.4; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 39 H 45 N 5 O 7 Na750.3109; Found 750.3122. Boc Ala Ala Trp(Z Ala) OBn (2.13e). 0.62 g, 84%: mp 96.0! 97.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.29 (d, J = 5.7 Hz, 1H), 7.75 7.59 (m, 2H), 7.50 7.24 (m, 3H), 7.24 6.96 (m, 12H), 6.38 (d, J = 6.9 Hz, 1H), 5.70 (d, J = 7.2 Hz, 1H), 5.06 4.82 (m, 5H), 4.17 3.98 (m, 1H), 3.81 3.23 (m, 2H), 3.22 2.96 (m, 2H), 2.53 2.12 (m, 1H), 1.37 1.10 (m, 15H). 13 C NMR (75 MHz, CDCl 3 ) % 174.1, 172.5, 172.2, 171.3, 156.1, 155.8, 136.4, 136.1, 135.1, 130.5, 128.7, 128.6, 128.2, 128.1, 125.7, 124.2, 122.8, 118.9, 118.5, 118.4, 117.0, 115.1, 79.9, 67.5, 67.2, 52.6, 50.7, 49.7, 36.3, 35.9, 28.4, 27.3, 19.0, 18.5; Anal. Ca lcd for C 40 H 47 N 5 O 9 : C, 64.76; H, 6.39; N, 9.44. Found: C, 64.36; H, 6.56; N, 9.46. Boc Pro Ala Trp(Z Ala) OBn (2.13f). 0.71 g, 92%: mp 77.0! 79.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.36 (d, J = 7.8 Hz, 1H), 7.83 7.72 (m, 1H), 7.52 7.09 (m, 15H), 5.98 (t, J = 10. 8 Hz, 1H), 5.15 4.82 (m, 6H), 4.26 3.94 (m, 1H), 3.94 3.61 (m, 1H), 3.43 2.91 (m, 5H), 2.66 2.07 (m, 2H), 2.03 1.56 (m, 4H), 1.49 1.20 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) % 174.8, 173.5, 172.8, 171.1, 155.8, 155.1, 136.3, 136.1, 135.1, 130.5, 128.8, 128.7, 12 8.4, 128.2, 125.9, 125.7, 124.3, 122.7, 119.0, 117.1, 80.4, 67.6, 67.3, 60.7, 52.8, 49.7, 47.5, 36.8, 36.7, 30.0, 28.6, 27.2, 24.8, 19.5; Anal. Calcd for C 42 H 49 N 5 O 9 : C, 65.69; H, 6.43; N, 9.12. Found: C, 65.47; H, 6.71; N, 9.33.

PAGE 35

35 Boc Gly Gaba Trp(Z Ala) OBn (2.13g). 0.65 g, 87%: mp 124.0! 125.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.28 (d, J = 7.2 Hz, 1H), 7.54 (d, J = 9.9 Hz, 1H), 7.44 (d, J = 6.9 Hz, 1H), 7.24 7.12 (m, 10H), 7.07 7.00 (m, 2H), 5.01 4.84 (m, 5H), 4.12 3.90 (m, 1H), 3.56 (s, 2H), 3.18 2.97 (m, 4H), 2 .27 1.97 (m, 2H), 1.61 (t, J = 7.2 Hz, 2H), 1.36 1.23 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) % 175.8, 173.3, 171.0, 168.9, 156.5, 155.8, 136.2, 135.2, 134.2, 130.6, 128.7, 128.6, 128.4, 128.3, 128.1, 125.9, 124.3, 123.1, 120.3, 119.0, 117.0, 80.4, 68.5, 67.4, 49 .8, 46.3, 45.3, 33.4, 33.3, 28.5, 26.0, 24.3, 19.8; Anal. Calcd for C 40 H 47 N 5 O 9 : C, 64.76; H, 6.39; N, 9.44. Found: C, 64.54; H, 6.42; N, 9.16. Boc Ala Gaba Trp(Z Ala) OBn (2.13h). 0.65 g, 86%: mp 104.0! 106.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.38 (d, J = 8.4 H z, 1H), 7.54 7.39 (m, 2H), 7.34 7.10 (m, 14H), 6.73 (br s, 1H), 6.01 5.62 (m, 1H), 5.10 4.90 (m, 6H), 4.34 3.82 (m, 1H), 3.33 2.95 (m, 4H), 2.42 1.89 (m, 2H), 1.78 1.65 (m, 2H), 1.47 1.24 (m, 15H). 13 C NMR (75 MHz, CDCl 3 ) % 175.3, 174.1, 173.6, 172.0, 156.0, 156.0, 139.1, 136.3, 135.2, 130.6, 128.80 128.7, 128.6, 128.4, 128.2, 125.9, 124.3, 122.7, 119.1, 119.0, 118.6, 117.0, 80.2, 67.5, 67.3, 52.6, 49.7, 45.8, 38.6, 33.1, 30.5, 28.5, 27.5, 19.5, 18.6; Anal. Calcd for C 41 H 49 N 5 O 9 : C,65.15; H, 6.53; N, 9.27. Found: C, 64.83; H, 6.74; N, 8.86. Boc Pro Gaba Trp(Z Ala) OBn (2.13i). 0.67 g, 86%: mp 75.0! 76.0 ¡C 1 H NMR (300 MHz, CDCl 3 ) % 8.38 (d, J = 8.4 Hz, 1H), 7.79 7.42 (m, 2H), 7.40 7.06 (m, 14H), 5.90 5.51 (m, 1H), 5.24 4.67 (m, 5H), 4.33 4.02 (m, 1H), 3.60 2.86 (m, 6H), 2.32 2.06 (m, 3H), 2.03 1.61 (m, 5H), 1.54 1.25 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) % 173.7, 173.4, 172.1, 171.1, 155.9, 155.5, 136.4, 136.1, 135.2, 130.6, 128.7, 128.5, 128.3, 128.2, 125.8, 124.3, 122.8, 119.0, 118.5, 117.0, 80.6, 67.4, 67.2, 60.6, 52.6,

PAGE 36

36 49.8, 47.3, 38.5, 32.9, 29.5, 28.6, 27.5, 26.2, 24.7, 19.4; Anal. Calcd for C 43 H 51 N 5 O 9 : C, 66.05; H, 6.57; N, 8.96. Found: C, 65.68; H, 6.92; N, 8.78. H Gly Gly Trp(Z Ala) OBn ( 2.14a ). 0.31 g, 94%: mp 165.7! 166. 3 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.32 (d, J = 7.5 Hz, 1H), 7.76 7.42 (m, 2H), 7.35 7.05 (m, 12H), 5.26 4.84 (m, 5H), 4.53 4.22 (m, 1H), 3.96 3.75 (m, 2H), 3.62 (d, J = 6.6 Hz, 2H), 3.39 2.24 (m, 2H), 1.33 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) % 171.9 169.9, 168.7, 166.6, 157.1, 136.9, 136.2, 136.0, 129.8, 128.4, 128.3, 128.2, 127.8, 127.5, 125.5, 124.4, 124.0, 118.5, 117.8, 116.7, 66.9, 66.6, 52.7, 49.7, 42.0, 40.4, 26.7, 16.9; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 33 H 36 N 5 O 7 614.2609; Found 614.2614 H Ala Gly Trp(Z Ala) OBn hydrochloride salt (2.14b). 0.30 g, 92%: mp 94.0! 96.0¡C; 1 H NMR (300 MHz, DMSO d 6 ) % 8.85 8.66 (m, 2H), 8.47 8.19 (m, 4H), 8.05 (d, J = 6.6 Hz, 1H), 7.97 7.80 (m, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.44 7.12 (m, 12H), 5.22 4.90 (m, 5 H), 4.81 4.5 (m, 1H), 4.00 3.68 (m, 3H), 3.51 3.29 (m, 2H), 3.29 3.07 (m, 2H), 1.43 1.28 (m, 6H). 13 C NMR (75 MHz, DMSO) % 172.5, 171.8, 170.4, 169.0, 156.5, 137.5, 136.3, 136.0, 130.6, 129.0, 128.6, 128.5, 128.3, 128.1, 125.7, 124.8, 124.3, 119.5, 118.1, 1 16.8, 67.0, 66.3, 53.1, 50.1, 48.8, 42.3, 27.1, 18.1, 17.8; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 34 H 37 N 5 O 7 Na 650.2585; Found 650.2592. H Pro Gly Trp(Z Ala) OBn hydrochloride salt (2.14c). 0.32 g, 92%: mp 65.0! 67.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.35 (d, J = 7.8 Hz, 1H), 7.96 7.55 (m, 1H), 7.72 7.40 (m, 3H), 7.38 7.07 (m, 10H), 5.24 4.89 (m, 6H), 4.41 4.08 (m, 1H), 3.90 (s, 2H), 3.34 3.27 (m, 2H), 3.24 3.10 (m, 2H), 2.52 2.19 (m, 1H), 2.08 1.76 (m, 3H), 1.57 1.31 (m, 3H); 13 C NMR (75 MHz, CD 3 OD) % 173.1, 172.5, 170.9, 170.5, 158.3, 138.2, 137.4, 136.9, 131.7, 129.6, 129.5, 129.4, 129.3, 129.1, 128.7, 126.4,

PAGE 37

37 125.0, 124.8, 119.9, 119.0, 117.7, 68.3, 67.9, 61.1, 53.9, 50.9, 47.6, 43.5, 31.0, 27.9, 25.1, 18.5; Anal. Calcd for C 36 H 40 ClN 5 O 7 : C, 62. 65; H, 5.84; N, 10.15. Found: C, 62.62; H, 6.22; N, 9.98. H Gly Ala Trp(Z Ala) OBn hydrochloride salt (2.14d). 0.31 g, 93%: mp 135.0! 135.6 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.28 (d, J = 8.7 Hz, 1H), 7.59 7.41 (m, 2H), 7.32 6.79 (m, 12H), 5.16 4.84 (m, 5H), 4. 55 4.18 (m, 1H), 3.74 3.42 (m, 2H), 3.39 3.24 (m, 3H), 3.14 2.98 (m, 1H), 2.52 2.21 (m, 2H), 1.31 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) % 172.3, 171.7, 168.7, 166.0, 157.1, 136.9, 136.2, 134.7, 130.5, 128.4, 128.3, 128.0, 127.8, 127.6, 125.5, 125.2, 123.8, 123.2, 118.7, 116.7, 67.0, 66.6, 52.8, 49.7, 40.3, 35.8, 34.9, 26.9, 17.0; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 34 H 38 N 5 O 7 628.2776; Found 628.2777. H Ala Ala Trp(Z Ala) OBn hydrochloride salt (2.14e). 0.33 g, 95%: mp 117.0! 118.0 ¡C; 1 H NMR (300 MHz, DMSO d 6 ) % 8.78 (d, J = 7.5 Hz, 1H), 8.70 8.53 (m, 1H), 8.40 8.27 (m, 3H), 8.08 7.85 (m, 5H), 7.69 7.54 (m, 2H), 7.45 7.16 (m, 11H), 5.24 4.86 (m, 5H), 4.80 4.51 (m, 1H), 3.86 3.68 (m, 1H), 3.40 3.05 (m, 4H), 2.45 2.30 (m, 2H), 1.44 1.23 (m, 6H); 13 C N MR (75 MHz, CDCl 3 ) % 171.8, 171.5, 170.5, 169.4, 155.9, 138.7, 135.7, 135.4, 130.0, 128.4, 127.9, 127.8, 127.7, 127.4, 126.8, 126.4, 125.0, 124.0, 118.9, 117.6, 116.2, 66.4, 65.7, 52.5, 49.5, 48.2, 35.3, 34.7, 26.5, 17.5, 17.3; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 35 H 39 N 5 O 7 Na 664.2742; Found 664.2753. H Pro Ala Trp(Z Ala) OBn hydrochloride salt (2.14f). 0.34 g, 94%: mp 82.0! 84.0 ¡C; 1 H NMR (300 MHz, DMSO d 6 ) % 8.79 8.59 (m, 1H), 8.55 8.23 (m, 2H), 8.18 7.78 (m, 3H), 7.59 (d, J = 6.6 Hz, 1H), 7.48 7.08 (m, 12H), 5.30 4.82 (m, 5H),

PAGE 38

38 4.82 4.51 (m, 2H), 4.20 3.85 (m, 1H), 3.43 2.87 (m, 5H), 2.40 2.07 (m, 2H), 1.89 1.49 (m, 3H), 1.34 (d, J = 6.0 Hz, 3H); 13 C NMR (75 MHz, DMSO) % 171.7, 171.3, 170.3, 167.9, 155.8, 136.8, 135.6, 135.4, 129.9, 128.3, 127.9, 127.8, 127.7, 127.6, 127.3, 125.2, 123.6, 118.8, 117.5, 116.1, 114.8, 66.3, 65.6, 58.5, 52.4, 49.4, 45.4, 35.4, 34.5, 30.6, 29.6, 23.5, 17.3; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 37 H 42 N 5 O 7 668.3040; Found 668.3055. H Gly Gaba Trp(Z Ala) OBn hydrochloride salt (2.14g). 0.32 g, 94%: mp 132.0! 133.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.32 (d, J = 7.8 Hz, 1H), 7.65 7.43 (m, 2H), 7.29 7.03 (m, 12H), 5.12 4.84 (m, 5H), 4.51 4.22 (m, 1H), 3.56 (d, J = 5.4 Hz, 2H), 3.29 ( d, J =6.6 Hz, 2H), 3.18 2.97 (m, 2H), 2.31 1.97 (m, 2H), 1.62 (t, J = 6.2 Hz, 2H), 1.33 (d, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) % 174.2, 172.0, 168.8, 166.0, 157.1, 136.9, 136.3, 135.7, 129.9, 128.4, 128.3, 128.2, 128.1,127.8, 127.6, 125.5, 125.2, 124.5, 124.0, 118.5, 116.6, 68.2, 66.6, 52.7, 49.7, 40.4, 38.6, 32.7, 26.9, 25.4, 17.1;HRMS (ESI TOF) m/z: [M + H] + Calcd for C 35 H 40 N 5 O 7 642.2922; Found 642.2916. H Ala Gaba Trp(Z Ala) OBn hydrochloride salt (2.14h). 0.32 g, 92%: mp 86.0! 88.0 ¡C; 1 H NMR (300 MHz, DMSO d 6 ) % 8.87 (d, J = 11.1 Hz, 1H), 8.72 8.51 (m, 1H), 8.40 8.19 (m, 2H), 8.09 7.74 (m, 3H), 7.74 7.58 (m, 1H), 7.47 7.10 (m, 12H), 5.33 4.72 (m, 5H), 4.69 4.34 (m, 1H), 3.93 3.57 (m, 1H), 3.44 2.78 (m, 4H), 2.65 2.28 (m, 1H), 2.24 1.89 (m, 1H), 1.86 1.46 (m, 2H), 1.46 1.25 (m, 6H); 13 C NMR (75 MHz, DMSO) % 172.7, 172.4, 172.2, 169.9, 156.5, 137.5, 136.4, 136.0, 130.6, 129.0, 128.7, 128.5, 128.4, 128.3, 128.1, 126.0, 125.6, 124.3, 119.6, 118.4, 116.8, 67.0, 66.3, 53.1, 50.1, 48.9, 38.8, 32.9, 28.9, 25.8, 18.0, 17.9; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 36 H 41 N 5 O 7 Na678.2898; Found 678.2897.

PAGE 39

39 H Pro Gaba Trp(Z Ala) OBn hydrochloride salt (2.14i). 0.33 g, 92%: mp 70.0! 71.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.34 (d, J = 7.2 Hz, 1H), 7.97 7.81 (m, 1H), 7.65 7.44 (m, 3H), 7.33 7.15 (m, 10H), 5.20 4.90 (m, 6H), 4.22 4.13 (m, 1H), 3.39 3.28 (m, 4H), 3.19 3.02 (m, 2H), 2.37 2.13 (m, 3H), 2.07 1.86 (m, 3H), 1.78 1.59 (m, 2H), 1.40 (d, J = 6.6 Hz, 3H), 13 C NMR (75 MHz, DMSO) % 172.0, 171.8, 171.5, 167.8, 155.9, 136.8, 135.7, 135.4, 130.0, 12 8.3, 127.9, 127.8, 127.7, 127.4, 125.3, 123.6, 118.9, 117.7, 116.1, 114.9, 66.4, 65.6, 58.8, 52.5, 49.5, 45.5, 38.3, 32.3, 29.7, 26.4, 25.1, 23.6, 17.4; Anal. Calcd for C 32 H 43 N 3 O 7 : C,66.07; H, 7.45; N, 7.22. Found: C, 65.7; H,7.77; N, 7.58; HRMS (ESI TOF) m/z: [M + H] + Calcd for C 38 H 44 N 5 O 7 682.3235; Found 682.3223. Boc Ala Pro Gly Trp(Z Ala) OBn (2.18a). 0.73 g, 87%: mp 70.0! 72.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.93 (br s, 1H), 8.57 8.21 (m, 1H), 7.58 7.36 (m, 2H), 7.33 7.13 (m, 15H), 5.99 5.63 (m, 1H), 5.30 4 .69 (m, 7H), 4.63 3.98 (m, 2H), 3.71 2.90 (m, 5H), 2.30 1.50 (m, 2H), 1.47 0.95 (m, 18H); 13 C NMR (75 MHz, CDCl 3 ) % 174.0, 172.6, 172.1, 171.7, 169.0, 155.9, 155.5, 136.3, 136.1, 135.2, 130.6, 128.8, 128.7, 128.4, 128.3, 128.2, 128.0, 125.8, 124.3, 118.9, 118.3, 117.0, 114.5, 79.8, 67.5, 67.3, 60.6, 52.6, 51.4, 49.9, 48.1, 47.5, 29.8, 28.6, 27.7, 25.4, 19.3, 18.4, 16.9; Anal. Calcd for C 45 H 54 N 6 O 10 : C, 64.42; H, 6.49; N, 10.02. Found: C, 64.42; H, 6.52; N, 9.68. Boc Ala Pro Ala Trp(Z Ala) OBn (2.18b). 0.71 g 85%: mp 136.0! 138.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.46 8.22 (m, 2H), 8.14 7.80 (m, 2H), 7.43 7.30 (m, 5H), 7.19 7.04 (m, 10H), 5.78 (t, J = 7.8 Hz, 1H), 5.31 5.01 (m, 3H), 5.95 4.73 (m, 6H), 4.67 4.32 (m, 2H), 3.58 3.23 (m, 5H), 2.29 1.83 (m, 3H), 1.63 0 .88 (m, 15H); 13 C NMR (75 MHz, CDCl 3 ) % 174.1, 173.9, 173.7, 172.7, 171.1, 156.2, 156.0, 136.3, 136.1,

PAGE 40

40 135.1, 130.0, 128.9, 128.8, 128.7, 128.4, 128.2, 128.0, 125.7, 125.1, 124.4, 119.0, 118.7, 117.1, 79.5, 67.3, 66.1, 53.6, 49.5, 48.9, 47.9, 37.4, 29.6, 2 8.5, 28.2, 26.0, 25.5, 16.6, 15.5; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 45 H 53 N 6 O 10 Na 861.3794; Found 861.3796. Boc Ala Pro Gaba Trp(Z Ala) OBn (2.18c). 0.75 g, 88%: mp 133.0! 134.0 ¡C; 1 H NMR (300 MHz, CDCl 3 ) % 8.42 8.24 (m, 2H), 8.07 7.77 (m, 2H), 7.5 0 7.30 (m, 5H), 7.18 6.96 (m, 10H), 5.85 (t, J = 7.8 Hz, 1H), 5.29 5.02 (m, 3H), 5.00 4.70 (m, 6H), 4.68 4.20 (m, 2H), 3.91 2.89 (m, 5H), 2.13 1.85 (m, 5H), 1.55 1.10 (m, 15H); 13 C NMR (75 MHz, CDCl 3 ) % 174.4, 173.6, 173.0, 172.5, 171.0, 156.0, 155.7, 136 .2, 136.0, 135.2, 130.2, 128.9, 128.7, 128.4, 128.2, 125.9, 124.4, 123.0, 118.7, 118.6, 117.0, 79.5, 67.8, 67.3, 60.3, 52.8, 49.6, 48.6, 47.7, 37.7, 32.2, 29.3, 28.6, 27.5, 25.2, 23.5, 20.1, 17.1; HRMS (ESI TOF) m/z: [M + Na] + Calcd for C 46 H 55 N 6 O 10 Na 875.3950; Found 875.3975. H Ala Pro Ala Trp(Z Ala) OBn hydrochloride salt (2.19a). 0.37 g, 96%: mp 74.0! 76.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.35 (d, J = 7.5 Hz, 1H), 7.76 7.58 (m, 1H), 7.60 7.39 (m, 2H), 7.40 6.89 (m, 11H), 5.25 4.87 (m, 5H), 4.64 4.04 (m, 2H), 3.61 3.37 (m, 3H), 3.35 3.01 (m, 3H), 2.08 1.66 (m, 4H), 1.50 1.25 (m, 9H); 13 C NMR (75 MHz, CD 3 OD) % 174.9, 173.6, 173.3, 172.6, 169.6, 158.3, 138.2, 137.5, 136.9, 131.8, 129.6, 129.5, 129.2, 128.9, 126.5, 125.1, 124.8, 120.0, 119.0, 117.8, 68.3, 67. 9, 61.4, 54.0, 53.7, 51.2, 50.4, 30.9, 26.1, 24.4, 18.4, 18.0, 16.4; Anal. Calcd for C 40 H 47 ClN 6 O 8 : C, 61.97; H, 6.11; N, 10.84. Found: C, 61.61; H, 6.21; N, 10.59. H Ala Pro Ala Trp(Z Ala) OBn hydrochloride salt (2.19b). 0.36 g, 94%: mp 80.0! 81.0 ¡C; 1 H NM R (300 MHz, DMSO d 6 ) % 8.42 8.30 (m, 2H), 8.29 8.22 (m, 1H),

PAGE 41

41 8.08 7.98 (m, 1H), 7.97 7.86 (m, 3H), 7.61 (d, J = 7.2 Hz, 1H), 7.52 7.25 (m, 13H), 4.83 4.52 (m, 5H), 4.70 4.00 (m, 1H), 3.70 3.35 (m, 5H), 3.29 2.90 (m, 3H), 2.37 2.18 (m, 1H), 1.99 1.50 (m, 5H), 1.38 1.25 (m, 6H); 13 C NMR (75 MHz, DMSO) % 171.7, 171.4, 170.8, 170.7, 167.8, 155.8, 138.6, 135.6, 135.4, 129.9, 128.3, 127.7, 127.6, 127.3, 124.9, 123.8, 123.6, 118.8, 117.6, 116.1, 66.3, 65.6, 59.6, 52.3, 49.4, 47.0, 46.7, 34.9, 30.6, 29.1, 26.5, 24.5, 17.3, 15.5; Anal. Calcd for C 40 H 47 ClN 6 O 8 : C, 61.97; H, 6.11; N, 10.84. Found: C, 62.16; H, 6.47; N, 10.54. H Ala Pro Gaba Trp(Z Ala) OBn hydrochloride salt (2.19c). 0.36 g, 92%: mp 130.0! 132.0 ¡C; 1 H NMR (300 MHz, CD 3 OD) % 8.34 (d, J = 7.5 Hz, 1H), 7 .65 7.49 (m, 2H), 7.30 7.09 (m, 12H), 5.31 4.91 (m, 6H), 4.50 4.11 (m, 2H), 3.75 3.46 (m, 2H), 3.26 2.92 (m, 5H), 2.37 2.04 (m, 3H), 1.94 1.56 (m, 4H), 1.46 (d, J = 6.6 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H); 13 C NMR (75 MHz, CD 3 OD) % 175.7, 174.1, 173.1, 173.0, 169.7, 158.4, 138.3, 137.6, 137.0, 131.8, 129.8, 129.6, 129.4, 129.2, 128.9, 128.1, 126.5, 125.1, 124.5, 120.0, 117.9, 68.3, 67.9, 62.1, 54.1, 51.1, 47.4, 47.1, 39.8, 34.1, 31.0, 28.3, 27.3, 26.8, 26.3, 18.4, 16.3; Anal. Calcd for C 41 H 49 ClN 6 O 8 : C, 62.39; H 6.26; N, 10.65. Found: C, 62.15; H, 6.30; N, 10.31. Cbz Ala Ala Bala Trp OBn (2.15e). Compound 2.14e (0.005 mmol, 1.0 equiv., 10 mg) in DMF/piperidine (5 mL) and irradiated in microwave for 3 h at 50 o C. The solvent was evaporated and dried overnight. Th e compound was isolated by HPLC chromatography to give ligated tripeptide Cbz Ala Ala Bala Trp OBn. 8.0mg, 92%: mp 131.0! 132.0 ¡C; 1 H NMR (500 MHz, CD 3 OD) % 10.84 (s, 1H), 8.30 (d, J = 7.5 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.85 7.83 (m, 1H), 7.50 7.44 (m, 2H), 7.35 7.3 (m, 8H), 7.17 6.96 (m, 5H), 5.06 4.98(m, 4H), 4.58 4.53 (m, 1H), 4.26 4.13

PAGE 42

42 (m, 1H), 4.12 3.95(m, 1H), 3.43 2.89 (m, 5H), 2.27 (t, J = 1.5 Hz, 2H), 1.18 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 7.0 Hz, 3H); 13 C NMR (125 MHz, CD 3 OD) % 172.6, 172.4, 171.0, 170.9, 156.3, 137.4, 136.6, 136.3, 128.8, 128.4, 128.3, 128.2, 128.2, 128.1, 127.5, 124.2, 121.5, 118.9, 118.5, 111.9, 109.8, 66.3, 65.9, 53.8, 50.6, 48.6, 40.0, 35.7, 35.3, 27.6, 18.8, 18.4; Anal. Calc d for C 35 H 39 N 5 O 7 : C, 65.51; H, 6.26; N, 10.65. Found: C, 65.32; H, 6.09; N, 10.55.

PAGE 43

43 LIST OF REFERENCES 1. Hopkins, F. G.; Cole, S. W. J. Physiol. 1901 27 418 428. 2. Radwanski, E. R.; Last, R. L. Plant Cell 1995 7 921 934. 3. Sainio, E. L.; Pulkki, K.; Young, S. N. Amino Acids 1996 10 21 47. 4. Young, V. R. Am. Inst. Nutr. 1994 124 1517S 1523S. 5. Richard, D. M.; Dawes, M. A.; Mathias, C. W.; Acheson, A.; Hill Kapturczak, N.; Dougherty, D. M. Int. J. Tryptophan Res. 2009 2 45 60. 6. Chen, Y. ; Guillemin, G. J. Int. J. Tryptophan Res. 2009 2 1 19. 7. Sanger, G. J. Trends Pharmacol. Sci. 2008 29 465 471. 8. Whitaker Azmitia, P. M. Brain Res. Bull. 2001 56 479 485. 9. Blelch, A.; Brown, S. L.; Kahn, R.; van Praag, H. M. Schizophr. Bull. 1988 14 297 315. 10. Reynolds, G. P.; Dalton, C. F.; Tillery, C. L.; Mangiarini, L.; Davies, S. W.; Bates, G. P. J. Neurochem. 2001 72 1773 1776. 11. Szczepanik, M. J. Physiol. Pharmacol. 2007 58 115 124. 12. Lindgren, C. E.; Walker, L. A.; Bolton, P. J. R. Soc. Promot. H ealth 1991 111 29 30. 13. Hansky, J.; Soveny, C.; Korman, M. G. Gut 1973 14 457 461. 14. Wang, P.; Dong, S.; Shieh, J. H.; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S. J. Science 2013 342 1357 1360. 15. Marcaurelle, L. A.; Mizoue, L. S.; Wilken J.; Oldham, L.; Kent, S. B.; Handel, T. M.; Bertozzi, C. R. Chemistry 2001 7 1129 1132. 16. Wilken, J.; Kent, S. B. H. Curr. Opin. Biotechnol. 1998 9 412 426. 17. Dang, B.; Kubota, T.; Mandal, K.; Bezanilla, F.; Kent, S. B. H. J. Am. Chem. Soc. 2013 135 11911 11919. 18. Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem. Int. Ed. Engl. 2008 47 10030 10074. 19. Miseta, A.; Csutora, P. Mol. Biol. Evol. 2000 17 1232 1239. 20. Biswas, S.; Kayaleh, R.; Pillai, G. G.; Seon, C.; Roberts, I.; Popov, V.; Alamry, K. A.; Kat ritzky, A. R. Chemistry 2014 21. Biswas, S.; Abo Dya, N. E.; Oliferenko, A.; Khiabani, A.; Steel, P. J.; Alamry, K. A.; Katritzky, A. R. J. Org. Chem. 2013 78 8502 8509. 22. Abo Dya, N. E.; Biswas, S.; Basak, A.; Avan, I.; Alamry, K. A.; Katritzky, A. R. J. Org. Chem. 2013 78 3541 3552. 23. Biswas, S.; Avan, I.; Basak, A. K.; Abo Dya, N. E.; Asiri, A.; Katritzky, A. R. Amino Acids 2013 45 159 170. 24. Dawson, P. E.; Muir, T. W.; Clark Lewis, I.; Kent, S. B. Science 1994 266 776 779. 25. Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys. Biomol. Struct. 2005 34 91 11 8