roger kayaleh university of florida department of biology

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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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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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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

1. LONG RANGE CHEMICAL LIGATION FROM N→N ACYL MIGRATIONS IN TRYPTOPHAN PEPTIDES VIA CYCLIC TRANSITION STATES OF 10- TO 18- MEMBERS link.............................................................................................. 17

2. Results and Discussion ....................................................................................... 17

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 Membered 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

3. Conclusion........................................................................................................... 30

4. 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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LIST OF TABLES

Table 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 isopeptide 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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LIST OF FIGURES

Figure page Figure 1-1: Important pathways for tryptophan metabolism .......................................... 11

Figure 2-1 Difference in 1H spectra of isolated ligated peptide 2.15e (left) and starting compound 2.14e (right) .......................................................................... 28

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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 NCL15.......................................................... 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 Synthesis 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 isotetrapeptides 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 isopeptides 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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LIST OF ABBREVIATIONS

Ala Alanine

Cys Cysteine

DIPEA N,N-diisopropylethylamine

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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Abstract

Tryptophan plays a significant role in living organisms. 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 that 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 ligation reaction and the amount of final product that was produced in all but a few of the reactions.

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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 tryptophan (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 some 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 proteins 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 therefore considered an essential

amino acid in animal diets. Intake in healthy adult humans range from 3.5 mg/kg3 to 6

mg/kg4 of body weight per day, however most individuals take in well above the

required daily minimum. The average daily intake of tryptophan for many individuals

ranges from 900 to 1000 mg.5 Common sources of tryptophan include turkey, chicken,

dairy products, chocolate, bread, tuna, and certain fruits (see Table 1-1). 5

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*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, tryptamine, NAD, and other biologically important molecules, as seen in

Figure 1-1.3

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Figure 1-1: Important pathways for tryptophan metabolism (noteworthy species in red)3

In the body, tryptophan is 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-

specific 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-acetylTransferase

5-hydroxyindole-

O-methyltransfer

ase

N-formyl-Kynurenine

Kynurenine

3-hydroxy-Kynurenine

3-hydroxy-anthranilic

acid

Picolinic Acid

XanthurenicAcid

KynurenicAcid

Anthranilic Acid

Tryptophan-2,3-dioxygenase

formamidase

Hydroxylase

TransaminaseKynureninase

2-amino-3-carboxy-muconic acid

6-semialdehyde

Nicotinamide adenine

dinucleotide

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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 important 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-dioxygenase (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 oxidative phosphorylation, 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 as being a powerful antioxidant. Picolinic acid prevents

the growth and 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

Besides the kynurenine pathways, tryptophan is also metabolized into serotonin,

and from there into melatonin. Of all the dietary tryptophan, only about 3% is used for

the synthesis of serotonin, while only about 1% is used in the brain, due to the

difficulties of getting tryptophan across the blood-brain barrier 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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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 children8 while a decrease 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 in 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 was first synthesized in 1949, but the synthesis was quickly replaced

in the 1980s by a fermentation process that increased the yield. Because of the

increased yield, supplements became widely available. 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, coughs, 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 compared 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, it can be used to help control

diseases that are protein based in nature. Diseases like diabetes mellitus, the 8th most

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common cause of death in the world, are caused by an inability to produce or respond

to the peptide hormone insulin. Others, like Zollinger-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 methods, 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 Scheme 1-1.

Scheme 1-1 Summary of the synthetic production of EPO by Wang et. Al

NCL has also been used to create the anticoagulant microprotein s, the human

neutrophil pro α-defensin-1, the glycoprotein lymphotactin, and a few others.14

98-124 SEtH

+

125-166H OH

R1) NCL2) Thz opening 98-166 OHH

R 60-971) NCL

2) Thz opening 60-166 OHH

R

29-59 SEtH

R

NCL

29-166H OH

R R R

R

7) MFD8) Acm Removal9) NCL

1-28 SEtH

StBuUnfolded Erythropoeitin glycoform

Folding

Functional Erythropoeitin

Notes:R=AcmSynthesis is done with sugars attatched to peptide. For simplicity, the sugars have been excluded from this summary of the scheme.

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Scheme 1-2 Synthesis of lymphotactin via NCL15

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+NH3O

SR+

49-93 CO2-

O

S-

+NH3

NCL

1-47+NH3O

49-93 CO2-

O

SHHN

SH

SH

Disulfide formation

1-47+NH3O

49-93 CO2-

OS

HN

S

1P SR

O+NH2

SHHN

OP2

P1O NH2S

HN

OP2

NCL

1P

O

NH

HSHN

OP2

16

However, NCL is limited in the fact that it requires a Cys residue at the N-

terminus, resulting in an internal Cys residue, only found in about 2.26% of human

proteins19. 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 intense 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 explored. 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 circumventing 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

NH

SHHN

OP2

R1 MFD

1P

O

NH

HN

OP2

R1

Ala, Val, Leu, Lys,Thr

NHN

OP2

HS

P1O

MFD

NHN

OP2

P1O

Pro

17

substituents in the 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

native peptides from non-terminal tryptophan-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 chemical ligation (NCL)

principles.

CHAPTER 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 predict 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 2.171a-c of

α-, β- or γ-amino acids to afford isotetrapeptides 2.14a-i and isopentapeptides 2.19a-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, β-, and γ-amino acid units within the isopeptides in order 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 2014, DOI: 10.1002/chem.201400125 Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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 was 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 studies, 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 usual 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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in Trp, one of the important natural amino acids present in peptides and proteins. This

encouraged us to explore the area of chemical ligation by developing a general, high

yielding and feasible pathway to the synthesis of 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 peptide 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 oC, 3 hHN

N

RO

OBn

n

2.9a n = 12.9b n = 22.9c n = 3

O

NH2

OHN

N

RO

OBn

n

O

NH2

O

HN

HN

OBn

n

O

NH

O

RO

2.10a n = 1, 44.4%2.10b n = 2, 71.4%2.10c n = 3, 99.1%

DMF/Piperidine

R = Cbz-Ala

20

Scheme 2-3 Synthesis of isotetrapeptides 2.14a-c for ligation study via 13 membered TS

Initially, the chemical ligation experiments were carried out for 2.14a under

aqueous conditions (pH 7.4, 1M buffer strength, MW 50 °C, 50 W, 3 h) to produce the

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 abundance 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 between 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

NHN

O

+ NH

OBn

O

NR

ON

N

2.12c

82-86%

HCl/Dioxane

2.14c 92%

ONH

R1

BocHN

O

ONH

R1

BocDIPEA

MeCN NH

OBn

O

NR

OHN

O

O

+H3N

R1

Cl-

2.13c

2.4N

HN

O

+ NH

OBn

O

NR

ON

N

2.12a-b

82%

2.14a-b 92-94%

ON

Boc HN

O

ON

BocDIPEA

MeCNNH

OBn

O

NR

OHN

O

O

+H2NCl-

2.13a-b

HCl/Dioxane

R = Cbz-Ala

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

2.14c 13 84 83.05 (45.7)

16.95 (54.6)

0.00

2.15c 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


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 oC, 3 hNH

N

RO

OBn2.14a R1 = H2.14b R1 = CH32.14c R1 = CH2CH2CH2 for Pro

O

HN

O

2.15a-c

2.16a-c

O

H2NR1

NH

N

RO

OBn

O

HN

O

O

H2NR1

O

ONH

ONH

O

NH

HN OBn

R

O

ONH

ONH

O

NH

N OBn

R

OR

R1

R1

R = Cbz-Ala

22

substrates for the ligation study via a 14 membered TS. The amino acids Gly, Ala, β-

Ala, and Pro and other amino acids in the isotetrapeptide sequence were chosen to

enable a comparative study on the effect of chemical ligation between the 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

isotetrapeptides 2.14d-f to microwave irradiation at 50 °C, 50W for 3h using basic

piperidine/DMF conditions (Scheme 2-6). The reaction mixtures were cooled, the

solvent was removed under reduced pressure, and the ligation mixtures (1.0 mg/mL in

methanol) were analyzed by HPLC-MS. The NH2- 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. Formation 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

NHN

n

O

+ NH

OBn

O

NR

ONN

2.12f

HCl/Dioxane

2.14f 94%

ONH

R1

BocHN

n

O

ONH

R1

BocDIPEA

MeCN NH

OBn

O

NR

OHN

n

O

O

+H3N

R1

Cl-

2.13f 92%

2.4N

HN

n

O

+ NH

OBn

O

NR

ONN

2.12d-e2.14d-e 92-95%

ON

BocHN

n

O

ON

BocDIPEA

MeCN NH

OBn

O

NR

OHN

n

O

O

+H2NCl-

2.13d-e 84-87%

HCl/Dioxane

R = Cbz-Ala n = 2

23

acids. HPLC−MS, using (−)ESI-MS/MS, confirmed that the ligated products 2.15d-f

each produced different MS fragmentation patterns from those 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



(RT) LP (RT) BA (RT) LP [M+H]+

found 2.14d 14 83 11.06

(39.3) 88.94 (45.7)

0.00

2.15d 628.3

2.14e 14 88 1.75 (48.0)

98.25 (56.5)

0.00

2.15e 642.3

2.14f 14 84 97.37 (44.8)

2.63 (55.0)

0.00

2.15f 668.3


Piperidine/DMF

50 W, 50 oC, 3 hNH

N

RO

OBn2.14d R1 = H2.14e R1 = CH32.14f R1 = CH2CH2CH2 for Pro

O

HN

O

2.15d-f

2.16d-f

O

H2NR1

NH

N

RO

OBn

O

HN

O

O

H2NR1

O

ONH

ONH

O

NH

HN OBn

R

O

ONH

ONH

O

NH

N OBn

R

OR

R1

R1

n=2; R = Cbz-Ala

n n

n

n

24


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 study 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 patterns 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.

2.4N

HN

n

O

+NH

OBn

O

NR

ONN

2.12i

HCl/Dioxane

2.14i 92%

ONH

R1

BocHN

n

O

ONH

R1

BocDIPEAMeCN N

HOBn

O

NR

OHN

n

O

O

+H3N

R1

Cl-

2.13i 86%

2.4N

HN

n

O

+NH

OBn

O

NR

ONN

2.12g-h2.14g-h 90-94%

ON

BocHN

n

O

ON

BocNH

OBn

O

NR

OHN

n

O

O

+H2NCl-

2.13g-h 85-87%

HCl/Dioxane

R = Cbz-Alan = 3

DIPEAMeCN

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]





(RT) LP (RT) BA

(RT) LP [M+H]+ found

2.14g 15 85 8.29 (37.7)

91.71 (43.8)

0.00

2.15g 642.3

2.14h 15 90 53.60 (45.4)

46.40 (54.1)

0.00

2.15h 656.3

2.14i 15 87 99.14 (45.4)

0.86 (55.9)

0.00

2.15i 682.3


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 MeCN in the presence of 3.0 equiv. of

DIPEA at 20 oC 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 oC, 3 hNH

N

RO

OBn2.14g R1 = H2.14h R1 = CH32.14i R1 = CH2CH2CH2 for Pro

O

HN

O

2.15g-i

2.16g-i

O

H2NR1

NH

N

RO

OBn

O

HN

O

O

H2NR1

O

ONH

ONH

O

NH

HN OBn

R

O

ONH

ONH

O

NH

N OBn

R

OR

R1

R1

n=3; R = Cbz-Ala

n n

n

n

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 piperidine/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, confirmed 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 experimental results, we observed that the

intramolecular N- to N-acyl migration is highly favored for medium ring size cyclic

transition states, while for larger ring size cyclic transition states there is a decrease in

the ligation products.

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]



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 1H and 13C

NMR spectroscopy, elemental analysis and analytical HPLC. 1H 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 1H 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, 50oC, 3h

2.19a R1 = CH3, n=12.19b R1 = H, n=22.19c R1 = H, n=1

R = Cbz-Ala

n

n

n NH

OBn

O

NR

OHN

O

OH2N

ON

R1

n HN

OBn

O

NR

OHN

O

OH2N

ON

R

O

OHN

O

N

ONH

R1

O

HN

NHOBn

R 2.20a-c

O

OHN

O

N

ONH

R1

O

HN

NOBn

R

OR

2.21a-c

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 1H 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 ligation 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.

29

Scheme 2-11 Competitive Chemical ligation of 2.14e in DMF/piperidine

2.14e +

OH2N

OHN O 2.15e

Ligation

LP BA+ 2.16e +

ONH

OHN O

OHN

O

O

cross-over product

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 synthesis 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 likely that 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.

31

CHAPTER 4 Experimental Section

4.1 General Methods

All commercial materials were used without further purification. All solvents were

reagent grade or HPLC grade. Melting points were determined on a capillary point

apparatus equipped with a digital thermometer and are uncorrected. 1H NMR and 13C

NMR spectra were recorded in CDCl3, DMSO-d6, and CD3OD using a 300 MHz and 500

MHz spectrometer (with TMS as an internal standard). All 13C NMR spectra were

recorded with complete proton decoupling. All microwave assisted reactions were

carried out with a single mode cavity Discover Microwave Synthesizer (CEM

Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure

microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon

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 × 150 mm; 5 µm) +

guard column (2 × 4 mm) or Thermoscientific Hypurity C8 (5 µm; 2.1 × 100 mm + guard

column) using 0.2% acetic acid in H2O/methanol as mobile phases; wavelength = 254

nm; and mass spectrometry was done with electrospray ionization (ESI).

4.2 General Procedure for Preparation of Boc- Protected Isotetrapeptides 2.13a-i and Isopentapeptides 2.18a−c

Boc protected benzotriazolides of dipeptides (Boc-AA1-AA2–Bt) or tripeptides

(Boc-AA1-AA2-AA3-Bt) (1.0 mmol, 1.0 equiv.) in MeCN (10 mL) were added drop wise to

a solution of 2.4 (1.0 mmol, 1.0 equiv., 0.54 g) and DIPEA (3.0 mmol, 3.0 equiv., 0.52

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

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 Na2SO4, filtered, concentrated and

recrystallized from ether to give the corresponding Boc-protected isotetrapeptides

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 mL/ 1.5 mL),

and the mixture was irradiated under microwave (50 °C, 50W, 3 h) in a microwave tube.

After cooling to room temperature the reaction mixtures were acidified with 2 N HCl to

pH = 1. Each mixture was extracted with ethyl acetate (3 × 10 mL), the combined

organic extracts were dried over sodium sulfate, and the solvent was removed under

reduced pressure. Each ligation mixture was weighed, and then absolution in methanol

(1 mg mL−1) was analyzed by HPLC−MS.

Boc–Gly–Gly–Trp(Z-Ala)-OBn (2.13a). 0.59 g, 86%: mp 90.0−90.7 °C; 1H NMR

(300 MHz, CD3OD) δ 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).; 13C NMR (75 MHz, CDCl3) δ 171.9, 171.2,

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 C38H43N5O9: 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; 1H NMR

(300 MHz, CDCl3) δ 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).; 13C NMR (75 MHz, CDCl3)

δ 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 C39H45N5O9: 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; 1H NMR

(300 MHz, CDCl3) δ 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); 13C NMR (75 MHz, CDCl3) δ 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 C41H47N5O9: 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; 1H NMR


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),

34

3.14–2.95 (m, 2H), 2.43–2.20 (m, 2H), 1.36–1.21 (m, 12H); 13C NMR (75 MHz, CDCl3) δ

171.8, 171.4, 171.1, 169.7, 156.2, 155.8, 136.2, 136.1, 135.1, 130.7, 128.8, 128.7,

128.4, 128.3, 128.2, 126.0, 124.4, 120.5, 118.9, 117.1, 114.4, 80.3, 67.7, 67.4, 52.3,

49.7, 44.5, 35.9, 34.5, 28.5, 27.5, 19.4; HRMS (ESI–TOF) m/z: [M + Na]+ Calcd for

C39H45N5O7Na750.3109; Found 750.3122.

Boc–Ala–βAla–Trp(Z-Ala)-OBn (2.13e).0.62 g, 84%: mp 96.0−97.0 °C; 1H NMR


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). 13C NMR (75 MHz, CDCl3) δ 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. Calcd for C40H47N5O9: C, 64.76; H, 6.39; N, 9.44. Found: C,

64.36; H, 6.56; N, 9.46.

Boc–Pro–βAla–Trp(Z-Ala)-OBn (2.13f). 0.71 g, 92%: mp 77.0−79.0 °C; 1H NMR


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);13C NMR

(75 MHz, CDCl3) δ 174.8, 173.5, 172.8, 171.1, 155.8, 155.1, 136.3, 136.1, 135.1, 130.5,

128.8, 128.7, 128.4, 128.2, 125.9, 125.7, 124.3, 122.7, 119.0, 117.1, 80.4, 67.6, 67.3,

60.7, 52.8, 49.7, 47.5, 36.8, 36.7, 30.0, 28.6, 27.2, 24.8, 19.5; Anal. Calcd for

C42H49N5O9: C, 65.69; H, 6.43; N, 9.12. Found: C, 65.47; H, 6.71; N, 9.33.

35

Boc–Gly–Gaba–Trp(Z-Ala)-OBn (2.13g). 0.65 g, 87%: mp 124.0−125.0 °C; 1H

NMR (300 MHz, CD3OD) δ 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);13C NMR (75 MHz, CDCl3) δ 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 C40H47N5O9: 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; 1H

NMR (300 MHz, CDCl3) δ 8.38 (d, J = 8.4 Hz, 1H), 7.54–7.39 (m, 2H), 7.34–7.10 (m,

14H), 6.73 (br s, 1H), 6.01–5.62 (m, 1H), 5.10–4.90 (m, 6H), 4.34–3.82 (m, 1H), 3.33–

2.95 (m, 4H), 2.42–1.89 (m, 2H), 1.78–1.65 (m, 2H), 1.47–1.24 (m, 15H).13C NMR (75

MHz, CDCl3) δ 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 C41H49N5O9: 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 °C1H NMR


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); 13C NMR (75 MHz, CDCl3) δ

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,

36

49.8, 47.3, 38.5, 32.9, 29.5, 28.6, 27.5, 26.2, 24.7, 19.4; Anal. Calcd for C43H51N5O9: 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; 1H NMR


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); 13C NMR (75 MHz, CD3OD) δ 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 C33H36N5O7614.2609; Found 614.2614.

H–Ala–Gly–Trp(Z-Ala)-OBn hydrochloride salt (2.14b). 0.30 g, 92%: mp

94.0−96.0°C; 1H NMR (300 MHz, DMSO–d6) δ 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, 5H), 4.81–4.5 (m, 1H), 4.00–3.68 (m, 3H), 3.51–3.29 (m, 2H),

3.29–3.07 (m, 2H), 1.43–1.28 (m, 6H).13C NMR (75 MHz, DMSO) δ 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, 116.8, 67.0, 66.3, 53.1, 50.1, 48.8, 42.3, 27.1, 18.1, 17.8;

HRMS (ESI–TOF) m/z: [M + Na]+ Calcd for C34H37N5O7Na 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; 1H NMR (300 MHz, CD3OD) δ 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); 13C NMR (75 MHz, CD3OD) δ 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,

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 C36H40ClN5O7: C, 62.65; H, 5.84; N, 10.15. Found: C, 62.62;

H, 6.22; N, 9.98.

H–Gly–βAla–Trp(Z-Ala)-OBn hydrochloride salt (2.14d). 0.31 g, 93%: mp


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);13C

NMR (75 MHz, CD3OD) δ 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

C34H38N5O7 628.2776; Found 628.2777.

H–Ala–βAla–Trp(Z-Ala)-OBn hydrochloride salt (2.14e). 0.33 g, 95%: mp

117.0−118.0 °C; 1H NMR (300 MHz, DMSO–d6) δ 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);13C NMR (75 MHz, CDCl3) δ 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 C35H39N5O7Na 664.2742; Found

664.2753.

H–Pro–βAla–Trp(Z-Ala)-OBn hydrochloride salt (2.14f).0.34 g, 94%: mp

82.0−84.0 °C; 1H NMR (300 MHz, DMSO–d6) δ 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),

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); 13C NMR (75 MHz, DMSO) δ 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 C37H42N5O7 668.3040;

Found 668.3055.

H–Gly–Gaba–Trp(Z-Ala)-OBn hydrochloride salt (2.14g). 0.32 g, 94%: mp


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);13C NMR (75 MHz, CDCl3) δ 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 C35H40N5O7 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; 1H NMR (300 MHz, DMSO–d6) δ 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);13C NMR (75 MHz,

DMSO) δ 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

C36H41N5O7Na678.2898; Found 678.2897.

39

H–Pro–Gaba–Trp(Z-Ala)-OBn hydrochloride salt (2.14i). 0.33 g, 92%: mp


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), 13C NMR (75 MHz, DMSO) δ 172.0, 171.8,

171.5, 167.8, 155.9, 136.8, 135.7, 135.4, 130.0, 128.3, 127.9, 127.8, 127.7, 127.4,

125.3, 123.6, 118.9, 117.7, 116.1, 114.9, 66.4, 65.6, 58.8, 52.5, 49.5, 45.5, 38.3, 32.3,

29.7, 26.4, 25.1, 23.6, 17.4; Anal. Calcd for C32H43N3O7: 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 C38H44N5O7

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; 1H

NMR (300 MHz, CDCl3) δ 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); 13C NMR (75 MHz, CDCl3) δ 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

C45H54N6O10: C, 64.42; H, 6.49; N, 10.02. Found: C, 64.42; H, 6.52; N, 9.68.

Boc–Ala–Pro–βAla–Trp(Z-Ala)-OBn (2.18b).0.71 g, 85%: mp 136.0−138.0 °C; 1H

NMR (300 MHz, CDCl3) δ 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); 13C

NMR (75 MHz, CDCl3) δ 174.1, 173.9, 173.7, 172.7, 171.1, 156.2, 156.0, 136.3, 136.1,

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, 28.5, 28.2, 26.0, 25.5,

16.6, 15.5; HRMS (ESI–TOF) m/z: [M + Na]+ Calcd for C45H53N6O10Na 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;

1H NMR (300 MHz, CDCl3) δ 8.42–8.24 (m, 2H), 8.07–7.77 (m, 2H), 7.50–7.30 (m, 5H),

7.18–6.96 (m, 10H), 5.85 (t, J = 7.8 Hz, 1H), 5.29–5.02 (m, 3H), 5.00–4.70 (m, 6H),

4.68–4.20 (m, 2H), 3.91–2.89 (m, 5H), 2.13–1.85 (m, 5H), 1.55–1.10 (m, 15H); 13C

NMR (75 MHz, CDCl3) δ 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 C46H55N6O10Na 875.3950; Found

875.3975.

H–Ala–Pro–Ala–Trp(Z-Ala)-OBn hydrochloride salt (2.19a). 0.37 g, 96%: mp


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); 13C NMR

(75 MHz, CD3OD) δ 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

C40H47ClN6O8: C, 61.97; H, 6.11; N, 10.84. Found: C, 61.61; H, 6.21; N, 10.59.

H–Ala–Pro–βAla–Trp(Z-Ala)-OBn hydrochloride salt (2.19b).0.36 g, 94%: mp

80.0−81.0 °C; 1H NMR (300 MHz, DMSO–d6) δ 8.42–8.30 (m, 2H), 8.29–8.22 (m, 1H),

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); 13C NMR (75 MHz, DMSO) δ 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 C40H47ClN6O8: 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


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); 13C NMR (75 MHz, CD3OD) δ 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 C41H49ClN6O8: 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 oC. The solvent

was evaporated and dried overnight. The compound was isolated by HPLC

chromatography to give ligated tripeptide Cbz-Ala-Ala-Bala-Trp-OBn. 8.0mg, 92%: mp

131.0−132.0 °C;1H NMR (500 MHz, CD3OD) δ 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

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); 13C NMR (125 MHz, CD3OD) δ 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. Calcd for C35H39N5O7: C, 65.51; H, 6.26; N, 10.65. Found: C,

65.32; H, 6.09; N, 10.55.

43

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roger kayaleh university of florida department of biology

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