pegylated Amino Acids for Site-specific Peptide Incorporation

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Development of Linear and Branched Pre-pegylated Amino Acids for Site-specific PeptideIncorporationParis L. HamiltonClemson University, [email protected]

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DEVELOPMENT OF LINEAR AND BRANCHED PRE-PEGYLATED AMINO

ACIDS FOR SITE-SPECIFIC PEPTIDE INCORPORATION

A Dissertation

Presented to

the Graduate School of

Clemson University

In Partial Fulfillment

of the Requirements for the Degree

Doctor of PhilosophyChemistry

by

Paris L. Hamilton

May 2017

Accepted by:

Brian Dominy, Committee Chair

Modi Wetzler, Co-Chair

William Pennington

Gautam Bhattacharyya

ii

ABSTRACT

The conjugation of poly(ethylene glycol) (PEG) to peptides is known to increase

serum stability of peptides and long polydisperse PEGs have been used for decades as a

strategy to improve pharmacokinetic profiles of protein therapeutics. However, the

effects of PEGylation on the conformational stability and protease resistance of peptides

will unpredictably vary depending on 1) site(s) of PEGylation, 2) conjugation strategy, 3)

chain length, as well as 4) chain branching. Site-specific PEGylation of peptides requires

the use of cumbersome protecting groups and, when performed at lysines, often

transforms the amine into an amide. To facilitate both fundamental and applied studies of

peptide PEGylation, the current toolbox needs to be expanded.

Analogues of glutamine and lysine incorporating a linear PEG or branched di- and

triPEG side chains have been synthesized using several synthetic approaches.

Additionally, biotin and fluorescein have been covalently attached to the N-position of

lysine that is bifunctionalized with a single linear N-PEG chain. Additionally, many of

the synthesized PEG amino acids have been site-specifically incorporated into

biologically relevant peptide sequences.

These PEG-amino acid monomers can be directly incorporated into solid phase

peptide synthesis to study their effects on peptide folding energetics (conformational

stability) and ultimately to further enhance pharmacokinetic properties of PEGylated

peptide drugs. The bulkier nature of the novel branched PEG amino acids can potentially

be used to help solubility in aqueous environments or repel macromolecules from a

iii

peptide cleavage site. Additionally, this strategy should be useful for the generation of

single- and multi-site selectively PEGylated peptides for therapeutic applications.

Due to a growing interest in peptidomimetics for their potential applications in the

pharmaceutical industry, two classes of peptidomimetics were investigated: Freidinger

lactams and peptoids. Freidinger lactams were originally designed as a conformationally

constrained dipeptide that could be used to lock a synthetic peptide into a bioactive

conformation; however, the current synthetic approach is typically low yielding and

limits functionalization. An improved synthetic route to Freidinger lactams was

developed that allows for chirality off the N position. Peptoids, unlike peptides, have

side chains appended to the backbone nitrogen instead of the -carbon; a structural

change that makes them more stable to proteolysis. A small library of antimicrobial

peptoids with varying aliphatic, cationic, and aromatic side chains have been designed

and synthesized.

iv

DEDICATION

This work is dedicated to my family and friends who have supported and

encouraged me throughout my educational career.

v

ACKNOWLEDGMENTS

I would like to take this opportunity to thank my research advisor, Professor Modi

Wetzler, for his guidance, encouragement, and mentoring during the course of this work.

I would also like to thank Dr. Daniel Whitehead for his helpful discussions of

various chemical synthesis procedural suggestions, Dr. Julia Brumaghim for her helpful

suggestions on how to present various results in the most impactful way, as well as Dr.

Brian Dominy for his helpful discussions on peptide/protein folding energetics and

dynamics.

I would also like to thank all members of my committee for their input,

suggestions, and patience during the preparation of this dissertation. Finally, thanks to all

of the undergraduate students who assisted with synthesis, purification, and

characterization of the many small molecules, peptides, and peptoids used in my

research.

vi

TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

DEDICATION ................................................................................................................ iv

ACKNOWLEDGMENTS ............................................................................................... v

LIST OF TABLES .......................................................................................................... ix

LIST OF FIGURES ........................................................................................................ ix

CHAPTER

I. PEPTIDES AS THERAPEUTICS ................................................................. 1

Introduction .............................................................................................. 1

The Role of Natural Peptides in the Body ............................................... 1

Where Peptides fit Between Small Molecules and Proteins .................... 3

Limitation of Peptides as Therapeutics .................................................... 7

Efforts to Overcome Peptide Limitations ................................................ 9

Conclusions ........................................................................................... 16

References ............................................................................................. 18

II. PART I - SYNTHESIS AND INFORPORATION OF LINEAR AND

BRANCHED PEGYLATED GLUTAMINE BUILDING BLOCKS FOR

FMOC SOLID-PHASE SYNTHESIS OF PEPTIDES ......................... 26

Introduction ............................................................................................ 26

Materials and Methods ........................................................................... 30

Retrosynthetic Analysis ......................................................................... 33

Chain Length Selection .......................................................................... 34

Cost Reduction Strategy ........................................................................ 34

Fmoc-Gln(mTEG)1-OH Synthesis ........................................................ 35

Fmoc-Gln(mTEG)2-OH Synthesis ......................................................... 45

Fmoc-Gln(mTEG)3-OH Synthesis ......................................................... 53

vii

II. PART II - STRAIGHTFORWARD SYNTHESIS OF FMOC-PROTECTED

LINEAR AND BRANCHED PEGYLATED LYSINE BUILDING

BLOCKS FOR SITE-SPECIFIC PEPTIDE INCORPORATION ....... 64

Introduction ............................................................................................ 64

Synthesis of Fmoc-Lys(mTEG)1-OH Derivatives and

Fmoc-Lys(mTEG)2-OH .................................................................... 66

Synthesis of N-Fmoc-N-Biotin-mTEG1-Lys-OH and

N-Fmoc-N-Fluorescein-mTEG1-Lys-OH ...................................... 78

Synthesis of N-Fmoc-N-Boc-mTEG1-Lys-OH ................................... 85

Synthesis of Fmoc-Lys(mTEG)3-OH .................................................... 88

II. PART III – SITE-SPECIFIC PEPTIDE INCORPORATION OF LINEAR

AND BRANCHED AMINO ACID MONOMERS .............................. 92

Introduction ............................................................................................ 92

Peptide Synthesis ................................................................................... 93

Conclusions .......................................................................................... 101

References ............................................................................................ 102

III. PROGRESS TOWARDS THE IMPROVED SYNTHESIS OF

FREIDINGER-LIKE LACTAMS FROM CHIRAL AMINES AND

FMOC AMINO ACIDS ....................................................................... 107

Introduction .......................................................................................... 107

Materials and Methods ......................................................................... 113

Results and Discussion ........................................................................ 115

Conclusions .......................................................................................... 132

References ............................................................................................ 133

IV. DESIGN AND DEVELOPMENT OF POTENTIAL ANTIMICROBIAL

PEPTOIDS FOR ENDOSCOPE STERILIZATION ........................... 135

Introduction .......................................................................................... 135

Materials and Methods ......................................................................... 138

Results and Discussion ........................................................................ 141

Conclusions .......................................................................................... 149

References ............................................................................................ 150

V. SUMMARY ............................................................................................... 154

viii

APPENDICES ............................................................................................................. 153

A: PEGylated Amino Acid NMR and MALDI spectra .................................. 156

B: Peptide MALDI and HPLC spectra ........................................................... 223

C: Progress Toward the Improved Synthesis of Freidinger Lactam NMR and IR

spectra ........................................................................................................ 252

D: Antimicrobial Peptoid MALDI and HPLC spectra ................................... 270

LIST OF SYMBOLS AND ABBREVIATIONS ........................................................ 320

ix

LIST OF TABLES

Table Page

2.2.1 Optimization of reductive amination conditions to favor formation of mono-

versus diPEGylated lysine product ........................................................ 79

2.3.1 Sequences of the peptides of interest ........................................................... 93

LIST OF FIGURES

Figure Page

1.1 Select examples of natural peptide hormones within the body...................... 3

1.2 General structures for common half-life extension strategies; PEGylation,

glycosylation, and lipidation ........................................................................ 10

1.3 Select examples of strategies to overcome limitations of peptide

therapeutics .................................................................................................. 13

2.1.1 Structures of natural amino acids; glutamine, asparagine, and lysine ......... 29

2.1.2 Synthetic PEG glutamine targets of interest ................................................ 33

2.1.3 Retrosynthetic strategy to Fmoc-Gln(mTEG)1-OH ..................................... 33

2.1.4 Literature synthetic strategy for monoPEG Fmoc-Gln/Asn ........................ 35

2.1.5 1H NMR comparison of PEG intermediates in the synthesis of monoPEG

amine ............................................................................................................ 37

2.1.6 Alternative PEG-amine synthesis route using cobalt (II) chloride and NMR

comparison to product obtained from reduction of PEG-azide to PEG-amine

using LiAlH4 ................................................................................................ 39

2.1.7 1H NMR spectra comparison of crude Fmoc-Gln(mTEG)1-OPac

synthesized using EDC (top) and CDI (bottom) .......................................... 41

x

List of Figures (Continued)

Figure Page

2.1.8 MALDI-mass spectrum of Fmoc-Gln(mTEG)1-OH .................................... 42

2.1.9 LC-MS comparison for the optimization of phenacyl ester protection ....... 44

2.1.10 Mass spectrum analysis of product formation during the synthesis of di-mTEG

amine ........................................................................................................... 50

2.1.11 1H NMR solvent comparison of Fmoc-Gln(mTEG)2-OPac ........................ 51

2.1.12 Retrosynthetic strategies for Fmoc-Gln(EDA-mTEG3)-OH formation ....... 53

2.1.13 Representative 1H NMR showing the successful synthesis of the desired

intermediate N-boc-protected structure ........................................................ 55

2.1.14 Representative 1H NMR spectrum showing the successful synthesis of the

desired intermediate (Fmoc-Gln(EDA)-OPac) from the use of EDC .......... 56

2.1.15 NMR analysis of crude product from PEGylation via SN2 with mTEG-OMs

….................................................................................................................. 57

2.1.16 MALDI analysis of product formation after PEGylation and subsequent

deprotection.................................................................................................. 59

2.1.17 Proposed effect of any unreacted Tf2O on Fmoc-Gln(EDA)-OPac............. 60

2.1.18 Acid-catalyzed resonance structures of amides ........................................... 61

2.2.1 Current peptide PEGylation strategy using amine conjugation chemistries 64

2.2.2 Typical peptide stabilizing interactions that occur with lysine in its cationic

form .............................................................................................................. 65

2.2.3 Synthetic PEG lysine targets of interest....................................................... 66

2.2.4 Mechanism of monoPEG formation via reductive amination ..................... 66

2.2.5 Aldehyde 1H NMR comparison with literature ........................................... 67

2.2.6 Proposed mechanism in literature for dialkylated product formation ......... 70

xi


Figure Page

2.2.7 Proposed acid-catalyzed mechanisms of mTEG2-Lys formation ................ 71

2.2.8 Synthetic route to mixture of mono-, di-, and triPEG lysine via nucleophilic

addition. MALDI analysis of increasing equivalents of activated PEG to

Fmoc-Lys(NH2)-OPac ................................................................................. 75

2.2.9 Analytical HPLC of crude product mixture containing Fmoc-Lys(mTEG)2-

OPac and Fmoc-Lys(mTEG)3-OPac ............................................................ 76

2.2.10 Alternative strategies for the synthesis of bifunctionalized N-lysine

conjugates .................................................................................................... 83

2.2.11 Salt bridge interactions between glutamic acid and lysine under

physiological conditions .............................................................................. 88

2.2.12 1H NMR and MALDI-MS spectrum showing Fmoc-Lys(mTEG)3-OPac ... 89

2.3.1 GLP-1 bound to extracellular domain of GLP-1 receptor ........................... 95

2.3.2 MALDI-mass spectra of unpurified PEGylated GLP-1 pentamer and

unmodified GLP-1 pentamer showing the feasibility of manual SPPS ....... 96

2.3.3 Analytical HPLC and MALDI-MS spectra for the confirmation of identity

and purity for the site-specifically PEGylated GLP-1 analogues indicated 98

2.3.4 Site-specific peptide PEGylation examples with mass spectrum analysis

highlighting the presence of desired peptide ............................................. 100

2.3.5 Site-specific incorporation of Fmoc-Lys(mTEG)3-OH into varying positions

within a lypressin peptide sequence ........................................................... 101

3.1 Structure of a prototypical Freidinger lactam ............................................ 107

3.2 Select pharmaceutically relevant examples of molecules with Freidinger-like

lactam moiety and chirality off of the -nitrogen .................................. 108

3.3 Original synthetic strategy to Freidinger lactam ........................................ 109

3.4 Proposed approach to Freidinger lactam structures with incorporated chirality

off of N-amine .......................................................................................... 110

xii


Figure Page

3.5 Comparable synthetic routes to gamma-, delta-, and epsilon-lactams....... 111

3.6 Literature study of products formed from diazotization of aliphatic

amines ........................................................................................................ 112

3.7 Literature precedent for the successful diazotization of aliphatic amines . 113

3.8 1H NMR time course analysis of fmoc stability under diazotization

conditions.. ................................................................................................. 116

3.9 Summary of initial diazotization results on non--substituted

aminoalkylcarboxylic acids ...................................................................... 117

3.10 Summary of diazotization attempts on -substituted aminoalkylcarboxylic

acids .......................................................................................................... 119

3.11 NMR analysis of starting material and crude product mixture from attempted

diazotization of Fmoc-Orn(NH2)-OMe ...................................................... 122

3.12 1H NMR spectrum of the unidentified major product after diazotization and

Nspe addition ............................................................................................. 124

3.13 Intramolecular cation-pi interaction ........................................................... 125

3.14 Proposed approach to Freidinger lactam via activation and reduction of

secondary amide to secondary amine ........................................................ 127

3.15 Select examples of literature precedent for reduction of amide to amine.. 128

3.16 Attempted routes to reduce the secondary amide to a secondary amine using

either Tf2O/hydride or Zn-mediated transfer hydrogenation ..................... 129

4.1 Structure of omiganan—a cationic antimicrobial peptide ......................... 136

4.2 Structural difference between the peptide and peptoid backbone ............. 137

4.3 Literature precedent and a general proposed structure for the design of

peptoid trimer library ................................................................................. 138

xiii


Figure Page

4.4 Design and structure of the peptoid trimers synthesized in this work ....... 143

4.5 Two sequences designed to evaluate whether the residue order is important

.............................................................................................................. 144

4.6 Antimicrobial activity of cyclic peptoids found in literature ..................... 145

4.7 Design and structure of the structurally rigid backbone peptoid dimers

synthesized in this work ............................................................................. 146

4.8 Representative structures of peptoid dimers with aromatic ring incorporated

into backbone for increased rigidity .......................................................... 146

4.9 A) Comparison of backbone structures for aromatic backbone peptoids with

traditional peptide and peptoid backbones. B) Synthetic scheme showing that

once the primary amine (R2-NH2) is reacted to form the secondary amine, the

secondary amine will react further with the starting material .................... 147

4.10 Two aromatic backbone peptoid sequences designed to evaluate the

importance of residue order as well as hydrophobicity ............................. 149

LIST OF SCHEMES

Scheme Page

2.1.1 Literature presedent for enzymatic site-selective glutamine side-chain

PEGylation using transglutaminase ............................................................. 29

2.1.2 Synthesis of Fmoc-Gln(mTEG)1-OH .......................................................... 36

2.1.3 Staudinger reduction mechanism ................................................................. 40

2.1.4 Selected literature precedents for phenacyl ester deprotection .................... 43

2.1.5 Proposed synthetic route to Fmoc-Gln(mTEG)2-OH .................................. 45

xiv

List of Schemes (Continued)

Scheme Page

2.1.6 Attempted synthetic routes to (mTEG)2-NH ............................................... 46

2.1.7 Potential alternative route to diPEG-amine ................................................. 47

2.1.8 Additional attempted synthetic routes to (mTEG)2-NH .............................. 49

2.1.9 Synthesis of Fmoc-Gln(mTEG)2-OH .......................................................... 52

2.1.10 Optimization of coupling strategy towards Fmoc-Gln(mTEG)3-OH ......... 54

2.1.11 Summary of experimental observations during the PEGylation step of

triPEG-Gln synthesis ................................................................................... 58

2.1.12 Attempted 2nd generation strategy leading to Fmoc-Gln(mTEG)3-OH ...... 63

2.2.1 1H NMR of crude product from reductive alkylation conditions on Fmoc-

Lys(NH2)-OPac suggests monoPEG product (Fmoc-Lys(mTEG)1-OPac) . 68

2.2.2 1H NMR of purified Fmoc-Lys(mTEG)2-OH from reductive alkylation of

Fmoc-Lys(NH2)-OH .................................................................................... 69

2.2.3 Top - literature precedent for a radical reaction to form imine from a benzylic

alcohol and benzylic amine. Bottom – proposed reaction using literature

conditions to form monoPEGylated lysine .................................................. 73

2.2.4 Synthesis of Fmoc-Lys(mTEG)1-OPac and Fmoc-Lys(mTEG)2-OPac via

nucleophilic addition .................................................................................... 77

2.2.5 Proposed route to N-Fmoc-N-(Biotin/Boc/Fluoresein)-mTEG1-Lys-OH

analogues...................................................................................................... 78

2.2.6 Top - Synthetic route to Fmoc-Lys(mTEG)1-OAll through a TEMPO

activated radical reaction. Bottom – Synthetic route to N-Fmoc-N-

Fluorescein-Lys(mTEG)1-OAll ................................................................... 80

2.2.7 Synthetic route to NHS-activated biotin and fluorescein ............................ 81

2.2.8 Attempted route to N-Fmoc-N-Biotin-mTEG1-Lys-OH and N-Fmoc-N-

Fluorescein-mTEG1-Lys-OH via reductive alkylation product ................... 82

xv


Scheme Page

2.2.9 Attempted route to N-Fmoc-N-Biotin-mTEG1-Lys-OH (top) and

N-Fmoc-N-Fluorescein-mTEG1-Lys-OH (bottom) via nucleophilic

alkylation product ........................................................................................ 84

2.2.10 Attempted synthetic route to N-Boc-N-Fmoc-Lys(mTEG)1-OH ............. 85

2.2.11 Synthetic routes to N-Boc-N-Fmoc-Lys(mTEG)1-OH ............................. 86

2.2.12 A – Literature examples of selective oxidation of proline to 5-hydroxyproline

(top) and piperidine to 6-hydroxypiperidine (bottom) using iron catalyst. B –

Literature example of proline oxidation to open-chain monomethoxy ornithine

derivative. C – Iron catalyst structure. D – Possible proposed future synthetic

route towards Fmoc-Lys(mTEG)1-OH ........................................................ 87

2.2.13 Synthetic route to Fmoc-Lys(mTEG)3-OH and MALDI-MS of Fmoc-

Lys(mTEG)3-OH.......................................................................................... 90

2.2.14 Synthetic route to Fmoc-Lys(mTEG)3-OH using O-Allyl ester and a

representative MALDI-MS showing the presence of desired Fmoc-

Lys(mTEG)3-OAll ....................................................................................... 91

2.3.1 Solid Phase Peptide Synthesis steps ............................................................ 92

3.1 Reaction of 5-bromovaleric acid with (S)-(-)-1-phenylethylamine ........... 118

3.2 Small-scale test reaction performed to help elucidate whether the bromoacid

starting material was formed through diazotization................................... 120

3.3 Attempted synthetic route to the lactam product ....................................... 123

3.4 Diazotization of ornithine leads to a mixture of products.......................... 126

3.5 Attempted synthetic route to desired Freidinger lactam via Fmoc-Glu(OtBu)-

OH using O-Allyl ester protection and reduction of amide to amine ........ 130

3.6 Cyclization of Fmoc-Orn(Nspe)-OAll to the desired -Freidinger-like

lactam ......................................................................................................... 131

4.1 Ronald Zuckermann’s submonomer approach to peptoid synthesis.......... 141

xvi


Scheme Page

4.2 Synthesis of structurally rigid-backbone peptoid trimers .......................... 148

1

CHAPTER ONE

PEPTIDES AS THERAPEUTICS

The role of natural peptides in the body.

Over 7000 natural peptides have been identified thus far in the human body—

exhibiting a wide array of biological activities. The major role of peptides within biological

systems is as signaling molecules; typically binding to G protein-coupled receptors (GPCR’s)

and triggering a cascade of reactions. Some of the broad classifications of these peptides

include hormones, metabolic peptides, and antimicrobial peptides.

Early research on small-molecule and protein hormones, such as testosterone and

growth hormone, respectively, sparked knowledge of the endocrine system that progressively

led to studies identifying peptide hormones. The pituitary gland within the brain was

correctly thought to be highly important to hormone production. A major role of the pituitary

gland is to regulate the amount of hormones within the body: ensuring that not too much (or

too little) is in circulation at a time. By the 1980’s it was understood that peptide hormones

are very important to human physiology.1

Peptide hormones (Figure 1.1), such as glucagon and growth hormone-releasing

hormone, are secreted primarily from various organs within the neuroendocrine system, but

can also be secreted by the heart and gastrointestinal tract. The peptide hormones are released

only in response to some extracellular stimuli; for a hormone, such as insulin, the stimulus is

a high blood-glucose level. Upon their release into the plasma, these peptide hormones allow

for the maintenance of homeostasis and the regulation of countless cellular functions. In

2

contrast to steroid-type hormones that are more lipid soluble and act on intracellular targets,

peptide hormones typically act on cell surfaces.

Within this group of peptide hormones are subclasses, such as incretin hormones like

glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) that

stimulate a decrease in blood glucose in response to the biological environment. Additionally,

the human body has peptide hormones like ghrelin to help regulate our appetite. Ghrelin is

considered “the hunger hormone” because it makes us feel hungry, while GLP-1 has

opposingly demonstrated the ability to decrease gastric emptying and make us feel full.

Peptide hormones, like atrial and brain natriuretic peptides, have major roles in

cardiovascular health as well as metabolism regulation.2 Similarly, a peptide such as

vasopressin, or antidiuretic hormone, has shown versatile physiological effects with not only

its cardiovascular role of increasing vasoconstriction, but also in the regulation of water

retention via acting on the kidney.

Antimicrobial peptides (AMPs), increasingly called host defense peptides (HDPs) due

to their immunomodulatory effects, are an important component of our human immune

system.3 Typically, AMP/HDPs are cationic and exist primarily in two major classes:

defensins (cysteine-rich; ß-sheet) and cathelicidins (non-cysteine-rich; a -helix).4 These

natural peptides have evolved in vivo to offer broad-spectrum protection against microbial

infection.5, 6 With antimicrobial agents, there is always the possibility of bacteria developing

resistance; AMPs are no exception.7

Nature has optimized peptides to exhibit a high selectivity for their native receptors,

which leads to less adverse side reactions. With over 600 proteases existing in the human

body, peptides are readily degraded. Therefore, peptides exhibit low accumulation in various

3

tissues within biological systems and their degradation pathways are well understood.8 In

many ways, beyond their high selectivity for native receptors, peptides are within a

goldilocks zone between small molecule and protein therapeutics.

Figure 1.1. Select examples of natural peptide hormones within the body.

Where peptide therapeutics fit in the spectrum between small molecules and proteins.

In the late 19th century and very early 20th century, diabetes was being recognized as a

devastating killer of the population with no treatment options in sight. Around 1920 it was

suggested that diabetes resulted from a lack of a pancreatic secretion/hormone. Dr. Frederick

Banting, a physician, and his lab assistant Charles Best, a medical student, surgically

removed the pancreas of stray dogs—later from calves at a slaughterhouse—to extract the

4

hormone from pancreatic islets without destroying the peptide. This work led them to the

isolation and discovery of insulin in 1921. In 1922, Leonard Thompson—on the brink of

death—was recorded as the first individual to receive an insulin injection for the successful

treatment of type-I diabetes. Banting went on to receive the 1923 Nobel Prize in Physiology

or Medicine for his discovery. The turnaround time between isolation of insulin and the first

patient treatment was incredibly fast and is no longer representative of the time to market

found in today’s drug discovery pipeline.

The discovery and medical usage of insulin was an early example of the power

peptides could have in the treatment of illness. Standard conventions identify peptides as

amino acids linked through amide bonds between 2 and 100 residues in length before being

considered a protein. However, the pharmaceutical industry blurs the lines even further by

classifying peptides under ~1300 Daltons (Da) as small molecules; gonadorelin (FactrelTM) is

one such peptide therapeutic that falls in that category. Alternatively, protein therapeutics are

typically classified as having molecular weights over 10,000 Da. Peptides usually violate

Lipinski’s rule of five which predicts oral bioavailability;9 While the rule of five was derived

for oral availability via passive diffusion across the intestinal membranes, peptides are

usually orally not bioavailable due to proteolytic digestion in the gut. Despite this handicap

they are definitely a highly sought-after drug class for targeted therapies with minimal side

effects.

The current global peptide therapeutic market is ~$19 billion (US) as of 2015 and is

expected to grow to over $23 billion (US) by 2020.10 To date, 63 peptide drugs have been US

Food and Drug Administration (FDA) approved and with over 700 clinical trials currently

studying peptide therapeutics, the field will likely grow in the number of approved drugs for

5

years to come. Nevertheless, amongst large pharmaceutical companies, peptide molecules are

still the minority of drugs being developed.

Peptides of varying length have found usefulness within diverse therapeutic

indications from osteoporosis (teriparatide) to cancer (degarelix) and even type-II diabetes

(liraglutide) and obesity.11 Because of high specificity to their native receptors due to their

high surface area, peptides exhibit minimal off-target effects that would traditionally produce

side effects in patients. Given the increasing concerns about toxicity (off-target effects) and

drug-drug interactions in the pharmaceutical industry, coupled with the higher relative costs

of producting protein therapeutics that also inherently mitigate these concerns, one would

expect peptide therapeutics to grow for the forseeable future.

Short natural peptides like oxytocin and vancomycin (a glycopeptide) are approved

for usage in various indications, such as labor induction and the treatment of infection,

respectively. GLP-1 analogues continue to exert a dominance within the type-II diabetes

treatment market with brands such as exenatide (BydureonTM) and liraglutide (VictozaTM).

Additonally, a 32-amino acid peptide, Nesiritide, is used for the treatment of congestive heart

failure.

It has been known for over a decade that there are peptides able to trigger cellular

apoptosis through caspase activation.12, 13 Research in the area of aptoptosis is associated with

a growing number of human diseases such as cancer, neurodegenerative disease and

autoimmune disorders. Short peptides such as Leuprolide have been approved for the

treatment of prostate cancer.

Fields such as Alzheimers research with amyloid- peptides—disruption of plaque

formation or disruption of amyloid precursor protein fragmentation—continue to be a

6

challenge,14, 15 with notable phase III clinical trials in 2016 of Aß plaque removal agents, such

as Eli Lilly’s Solanezumab, that successfully cleared plaques but did not improve function.16

There are, however, some FDA-approved small-molecule drugs for the treatment of

symptoms, such as rivastigmine (ExelonTM) and memantine (NamendaTM).17

Natural peptides have some very noteworthy benefits when it comes to their usage as

therapeutics. Amongst these benefits, they have a potentially improved safety margin, in

addition to potentially higher and more efficacious dosing. Therapeutics are rated in

accordance with their safety margin—a measure of the difference between the minimum

toxic dosage and minimum therapeutic dosage of a drug.18

A majority of the biologically relevant protein-protein interactions occur within the cell as

opposed to outside of the cell (or on the cell surface). However, current strategies involving

small molecules or antibodies are not equipped to handle these intracellular interactions. In

the late 1980’s, it was discovered that certain proteins could cross the cellular membrane and

subsequently, that only a portion of the protein was necessary for crossing the membrane.19,

20, 21 It is known that cell permeability falls off at higher molecular weights.22, 23 So, large

molecules, such as proteins, have encountered difficulties when it comes to crossing the

cellular membrane, where potential drug targets may reside. Therefore, researchers are

looking into the possibility of targeted delivery through conjugation of small molecules and

biologics with cell-penetrating peptides (CPP). In order for this strategy to work, the

cytostolic delivery efficiency is a metric that needs to be improved. If a CPP has low delivery

efficiency, then large dosages must be used to get enough of the drug into the cell. Large

dosages, however, tend to lead to increased toxicity. Some of the earliest examples showed

poor cytostolic delivery efficiencies (e.g. 1-5%).21 The stabilization of peptides that can cross

7

into intracellular space has been explored using a variety of strategies in order to optimize

peptides for disrupting protein-protein interactions.24

Therefore, even though peptides may offer advantages where their protein and small

molecule counterparts have limitations, the characteristic shortcomings of natural peptides

need to be overcome before they can be useful as therapeutic agents. However, exceptions do

occur as in the case of corticotropin and, formerly, insulin.

The limitations of peptides as therapeutics.

Peptides (i.e. mainly linear peptides) have some notable shortcomings when it comes

to their potential usage as therapeutics.25 Economically, peptides generally have higher costs

associated with their production compared to small molecules. For peptides synthesized

through biological methods, the manufacturing process is more complex, and thus more

expensive, than a chemical synthesis production.26 For example, the production cost of a 500

Da molecular weight small-molecule is 10-fold lower than that of a 5000 Da peptide.27

However, for smaller peptides, the cost should be more on par with that of small molecules.

The solid-phase synthesis of batch amounts of peptides is faster and cheaper than the

recombinant methods used for some longer sequences especially when economy of scale is

taken into consideration; yielding the ability to reduce production costs to < $1 (USD) per

gram per amino acid residue.27, 28, 29 Furthermore, peptides obtained from SPPS have better

purity profiles than those from recombinant methods.30

Most peptides tend to exhibit poor metabolic stability, membrane permeability, and oral

bioavailability. A notable exclusion to these downfalls is the peptide therapeutic enfuvirtide

(FuzeonTM), used in the treatment of HIV-1 infection; which exhibits an 84% bioavailability

8

and an impressive 3.8-hour biological half-life. The half-life of many native peptides is on the

order of minutes in blood. The poor stability of peptides to native proteases and peptidases

leads to inactivation as well as rapid clearance—typically, requiring constant dosing for

beneficial pharmaceutical effects. The practical effectiveness of constant dosing relys heavily

on patient compliance—the ability of the patient to follow the advice of a medical

professional. Realistically, the frequent dosings necessary with most peptide drugs often lead

to low patient compliance. Peptide therapeutics are usually injected with only a few orally

available peptides to date, such as cyclosporine (NeoralTM) and desmopressin (MiniriniTM).

The route of administration is known to have a significant effect on the pharmacokinetic and

pharmacodynamic profile of peptides.31 An increase in the availability of orally administered

peptide therapies is a highly sought-after goal amongst researchers in the field because it

would lead to improved patient compliance. However, due to the charge and polarity of

peptides, they tend to exhibit low permeability across gut membranes. Companies, such as

Protagonist Therapeutics, are aiming to develop oral peptide therapeutics for gastrointestinal

disorders and diseases—they currently have one peptide entering phase IIb trials and another

oral candidate entering phase I.

Small molecule therapeutics are typically favored when a binding pocket is targeted

for (de)activation deep within a protein whereas biologics are better for the targeting and

disruption of protein-protein interactions.32, 33 Peptides are preferred when traditional small

molecule approaches fail to produce a successful clinical candidate for a particular

therapeutic indication. For reasons such as those mentioned, peptides are ripe to fill a void

left by a mounting interest in targets that are not effectively drugged by small molecules or

antibodies.

9

A major concern in the peptide therapeutic area is the possibility of peptide

immunogenicity—the ability of peptides to provoke unwanted immune responses. There has

been debatable views on assessing a products immunogenicity which has led to nonuniform

assessements and descriptions; however recent effort have attempted to standardize the type

of data collection necessary to report on immunogenicity of products.34 If there remains a

perception of immunogenicity amongst pharma/biotech investors, then the full potential of

peptide therapeutics won’t be realized. There is, however, potential for epitopes to be

chemically attached to peptides for usage as vaccines.35

The growth of cardiovascular and metabolic diseases is likely to induce a rise in

peptide therapeutic research due to high overlap with natural peptide hormone involvement in

the regulation of these biological systems. Additionally, peptide therapeutics are increasingly

being studied for their ability to treat gastrointestinal diseases and disorders, such as the

recent (2012) FDA approval of linaclotide (LinzessTM), a 14-amino acid peptide guanylate

cyclase 2C agonist.36, 37 Various technological advances over the past decades have renewed

interest in the usage of peptides as potential pharmaceutical agents.

Efforts to overcome peptide limitations.

The challenges to using peptides as therapeutics can be overcome through various

chemical modification strategies such as (but not limited to) half-life extension (HLE)

strategies.38 Some common peptide HLE strategies include cyclization, PEGylation (Figure

1.2), O-glycosylation (Figure 1.2), and backbone modifications (e.g., D-amino acids, N-

methylation, etc.). The HLE strategies are extremely important, in the case of peptides versus

proteins especially, because metabolic degradation is known to increase with a decrease in

10

size.39 The discussion below focuses on chemical modifications at the expense of other valid

and successful HLE strategies such as formulation development.

Figure 1.2. General structures for common half-life extension strategies; PEGylation,

glycosylation, and lipidation.

Direct chemical modification (post-synthetic) strategies happen later in the peptide

therapeutic optimization process, which can often lead to a reduction in potency for lead

compounds; sending researchers back to the proverbial square one. Selective glycosylation—

the addition of saccharide units—is one such strategy that has been demonstrated on

interferon -1a to improve various pharmacologically relevant parameters such as half-life,

solubility, and stability.40 This strategy capitalizes on a method which is thought to be a

major route of protein elimination from circulation, as well as the importance of sugars in

various cellular processes.

Another strategy that employs the conjugation of a known polymeric unit is selective

PEGylation. PEGylation refers to the attachment of polyethylene glycol (PEG) units to a

target molecule. These PEG units typically exist as large polymer chains and have found

considerable usage within the pharmaceutical industry, as laxatives, and within cosmetics due

11

to their water soluble, hydro- and lipophilic, nontoxic nature. Lysine residues are by far

amongst the most PEGylated site on peptides, however this can typically reduce efficacy;

especially if the original lysine was part of a salt-bridge, binding pocket, or active site. As an

alternative, the site-specific, long-chain PEGylation of glutamine has been achieved

successfully on a range of peptides (and proteins) by several groups.41, 42, 43, 44 Recent

research has identified that a non-neglible percentage of healthy individuals likely have some

antibodies to PEG, which would effectively diminish the effectiveness of any PEGylated

therapeutics used on these individuals.45, 46 However, there is also research that suggests these

anti-PEG antibodies bind to larger PEGs with higher affinity than smaller ones.47 It is also

unclear how effectively the anti-PEG antibodies recognize and bind to PEG chains that are

terminated in hydroxyls versus methoxy, as well as the antibody’s ability to distinguish

between linear versus branched PEG chain modifications. This suggests that peptide

PEGylation with short-chains could be an effective future strategy for consideration in the

peptide therapeutic arena.

For the pharmaceutical industry to minimize financial risks associated with failed

drug candidates late in the development pipeline it is crucial to move certain modification

strategies and testing to earlier in the drug development process. The incorporation of

unnatural amino acids directly into SPPS procedures is a more facile and straightforward

modification strategy than post-SPPS derivitization. This pre-synthetic modification method

has allowed researchers to introduce various functionalities and modifications such as D-

amino acids, N-methylated backbones, lipidation (Figure 1.2), alkene side-chains for peptide

stapling, and more recently short PEG-chains.

12

Peptide stapling is used to stabilize secondary structure and improve protease

resistance, yet there have been no clinically successful candidates with this modification.

Recent studies out of MIT have introduced a N-arylation method capable of macrocyclizing

unprotected peptides; leading to improved proteolytic stability and improved binding

affinity.48, 49, 50, 51 Strategies like N-methylation/alkylation and D-amino acid incorporation

are often used in combination with other strategies in order to achieve the maximum

beneficial effects, such as in the widely used cyclosporine A and recently approved

etelcalcetide (ParsabivTM, Figure 1.3). Sometimes, however, swapping out L-amino acids for

their D- counterparts can cause local kinks in structure which then affect native activity.

Lipid conjugation is one practical approach that has circumvented some of the delivery

issues associated with unfavorable peptide pharmacokinetics. The rationale for this approach

is that the lipid chain increases the association with albumin, which gives the therapeutic

agent increased protection from blood-circulating peptidases and proteases. This strategy has

led to two-lipid-peptide conjugates currently on the market; recombinant insulin detemir

(LevemirTM) and liraglutide (VictozaTM), a blockbuster peptide drug. Only the latter is formed

through direct chemical modification with a C16 fatty acid chain after expression of

recombinant DNA in Saccharomyces cerevisiae. The C14 fatty acid chain of insulin detemir

is installed at a lysine residue during its complete synthesis within yeast cells (all insulins are

at least largely biosynthesized due to their complex disulfide pattern).52

13

NN

HN

HN N

HN

O

N

O

O

OO

O

ON

O

N

O

N

O

HN O

Cyclosporin

H2NNH

HN

NH

HN

NH

HN

NH

SNHHN

HN

NH

HN

NH

HN

NH

S

S

O

O

O

O

O

OO

OO

O

O

O

O

O

O OH

H2NH2N

O

HO

ONH S

OH

O

H2N

NHH2N

NH

S

HOHN

NH

HN

NH

HN

NH

O

O

O

O

O

O

NH

O

NH2

H2N O

S

OHS

NH

H2N

NH

OH

HO

O

Zinconotide

Cyclic, N-methylation

SS

HN

O

O

NH

O

HN

HN

NH

NH

HNS

S HNOH

HN

NH

O

OO

O

O

OH

HO

NH2

O

O

H2N

O

N OH

OO

HO

O

O

O

OH

H

OH

OH

NH2

Octreotide-doxorubicin

HN NH

HN

NH

HN

NH

HN

NH

SS O

NH2

OH

NH2

Etelcalcetide

O

O

O

O

O

O HN OO NH2

NH

NH2

HN

H2N

HN

NH

HN

NHH2N

(D)

(L)(D)

(D)

(D)

(D)

(D)

(D)

D-amino acids, Disulfide bond

Cyclic, Disulfide bridgesCyclic, Disulfide bridges

Figure 1.3. Select examples of strategies to overcome limitations of peptide therapeutics.

Cyclic Peptides have proven to be a great strategy for half-life extension of short

peptide sequences such as octreotide (SandostatinTM) and lanreotide (SomatulineTM), both of

which are long-acting analogs of somatostatin. As is the case with linaclotide, the

incorporation of multiple disulfide bonds has demonstrated the ability to improve metabolic

resistance. Research into this area of multiple disulfide bonds can be traced back to the work

of David J. Craik and coworkers for their late 20th century work on plant peptides known as

cyclotides; especially kalata B1.53, 54 Despite over whelming skepticism in the pharmaceutical

industry at the time, their group was able to demonstrate that peptides with this structural

14

motif were highly stable to extreme conditions.55 In the traditional fashion of scientific

progress, Craik et al. built their work on the accomplishments of scientific predecessors;

namely, Lorents Gran who originally discovered and named kalata B1 without any structural

elucidation.56, 57 Spawning from these early discoveries, pharmaceutical researchers

worldwide have gained increasing interest in trying to modify these (and other) cyclic peptide

structures in order to overcome stability limitations. Alanine scans and lysine scans have been

investigated to identify potential sites important for bioactivity as well as for optimization.58,

59 The chemical synthesis of these cysteine knot motifs has been investigated with current

SPPS technology and other strategies,60, 61, 62, 63, 64, 65, 66, 67, 68 but promising biological

synthesis approaches have also been developed.69, 70, 71, 72 Cyclotides have a diverse array of

biological activities from antimicrobial to antitumor, which could lend inspiration to current

and future research within the peptide therapeutics community.73

Ziconotide (PrialtTM, Figure 1.3), a conotoxin derived from a cone snail (Conus

Magnus), is similar to cyclotides such as kalata B1 in that it has multiple cysteine bridges.

This particular cyclic peptide has a biological half-life of several hours compared to the

typical minutes of uncyclized peptides. Cyclosporin (Figure 1.3) is another cyclic peptide

that is approved for multiple indications, however it’s backbone is also N-methylated.

Research has also suggested that ester bonds in the backbone of cyclic peptides, such as

jasplankinolide, are stable to protease resistance.74 Stereochemistry of the side-chains on

peptide macrocycles is also known to affect physiochemical properties like cellular

permeability.

More recently, work in groups such as the Pei laboratory has combined strategies of

cyclization with CPPs to improve proteolytic stability as well as cytostolic delivery

15

efficiency.75, 76, 77 His group has demonstrated the ability for cyclic CPPs to deliver small

molecule cargos as well as proteins and short peptides as well.75, 76

The attachment of PEG to biologics has been well studied in recent decades for its ability

to improve the pharmacologic profile of therapeutically relevant biologic compounds.

PEGylated biologics account for 17 FDA approvals to date, representing a mature technology

for overcoming various peptide limitations. The PEG molecule itself is generally regarded as

safe by the FDA. PEGylated amino acid monomers have been synthesized and reported in the

literature and allow for easy incorporation to SPPS.78 Joshua Price and coworkers

significantly advanced the understanding of beneficial effects of short-chain PEGylation of

peptides. Studies on their model peptide showed that PEGylation thermally stabilized

conformation better than N-glycosylation and that the increased conformational stability leads

to higher proteolytic stability.79 Thus showing that conformational stability could be a

predictive trait for researchers to screen for in efforts to overcome peptide limitations with

PEGylation. Subsequently, it was elucidated that 3- to 4-ether linkage PEGs are the

minimally effective length for achieving conformational stability.80 Not only was it observed

that optimal PEG positioning within a peptide was important, but conjugation strategy and

branching also played a role in affecting PEG benefits.79, 81 Evolution of this technique to

more amino acid side chains, such as serine, could allow for the easy scanning of PEGylation

benefits at various peptide sites; giving rise to potential PEG-scans analogous to alanine

scans which produce analogs of peptides where residues are replaced by alanines to identify

which side chains are necessary for activity.

Peptide-drug conjugates are a method for introducing multiple functionalities within the

same molecule. For instance, one molecule may be highly specific for a desired receptor,

16

whereas another molecule is toxic to a cell so bridging the two—through a linker molecule—

yields a molecule with potentially synergistic effects. These conjugates have found usefulness

as targeted delivery systems82 for research areas such as targeted tumor therapy.48

Octreotide-doxorubicin (Figure 1.3) is one such conjugate for the tumor-targeted delivery.83

A future research area for peptide therapeutics could likely be with bicyclic peptides—a

currently underdeveloped class that displays potential in the form of very high affinity,

relatively small size (1.5-2 kDa), and easy tunability.84, 85, 86 The bicyclic peptides, as a

platform, have a lot of potential for multiple indications although most research at the

moment is been directed towards tumor-targeting.87, 88, 89 Bicyclic peptides have even

displayed the ability to interrupt protein-protein interactions.90

Conclusion

In summary, the usage of peptides as therapeutics is a market full of growth potential.

Historically, peptides have flown under the radar in terms of therapeutic applications, but as

technology has improved, peptide limitations are becoming more manageable. Owing to their

great receptor specificity and binding affinity, peptide therapeutics will continue to gain

growing importance within the pharmaceutical industry; especially as human life expectancy

increases and individuals are taking progressively more medications for chronic illnesses.

There is no one best peptide modification strategy that will overcome the issues associated

with using peptides as therapeutics, but a mixture of strategies could lead to improved clinical

outcomes for drug developers. Yet, for the peptide therapeutic market to be more effectively

mined: 1) new scaffolds need to be elucidated from natural sources, 2) the threat of peptide

immunogenicity needs to be addressed, 3) chemical modification strategies need to be

expanded and SARs evaluated for peptides at earlier stages of the drug development process,

17

4) the cost of large-scale peptide manufacturing will need to decrease as technology

improves.

18

References

1. Niall, H. D. (1982) The evolution of peptide hormones. Ann. Rev. Physiol. 44, 615-624.

2. Suzuki, T., Yamazaki, T., and Yazaki, Y. (2001) The role of natriuretic peptides in the

cardiovascular system. Cardiovasc. Res. 51, 489-494.

3. Wiesner, J., and Vilcinskas, A. (2010) Antimicrobial peptides: The ancient arm of the

human immune system. Virulence. 1, 440-464.

4. Guani-Guerra, E., Santos-Mendoza, T., Lugo-Reyes, S. O., and Teran, L. M. (2010)

Antimicrobial pepties: General overview and clinical implications in human health and

disease. Clinic. Immunol. 135, 1-11.

5. Gallo, R. L., Murakami, M., Ohtake, T., and Zaiou, M. (2002) Biology and clinical

relevance of naturally occurring antimicrobial peptides. J. Allergy Clin. Immunol. 110, 823-

831.

6. Diamond, G., Beckloff, N., Weinberg, A., and Kisich, K. O. (2009) The roles of

antimicrobial peptides in innate host defense. Curr. Pharm. Des. 15, 2377-2392.

7. Gruenheid, S., and Le Moual, H. (2012) Resistance to antimicrobial peptides in gram-

negative bacteria. FEMS Microbiol. Lett. 330, 81-89.

8. Lopez-Otin, C., and Matrisian, L. M. (2007) Emerging roles of proteases in tumour

suppression. Nat Rev Cancer. 7, 800-808.

9. Lipinski, C. A., Lombardo, F., Dominy, B. W., and Feeney, P. J. (1997) Experimental and

computational approaches to estimate solubility and permeability in drug discovery and

development settings. Adv. Drug Deliv. Rev. 23, 3-25.

10. Transparency Market Research. (2016) Peptide therapeutics market - increasing demand

for peptide therapeutics in cancer and diabetes treatment to boost sales; global industry

analysis, size, share, growth, trends and forecast 2020: TMR.

https://globenewswire.com/news-release/2016/09/12/871199/0/en/Peptide-Therapeutics-

Market-Increasing-Demand-for-Peptide-Therapeutics-in-Cancer-and-Diabetes-Treatment-to-

Boost-Sales-Global-Industry-Analysis-Size-Share-Growth-Trends-and-Forecas.html ed.,

Globe Newswire, Internet.

11. Thundimadathil, J. (2012) Cancer treatment using peptides: Current therapies and future

prospects. J. Amino Acids. 2012, 967347.

19

12. Buckley, C. D., Pilling, D., Henriquez, N. V., Parsonage, G., Threlfall, K., Scheel-

Toellner, D., Simmons, D. L., Akbar, A. N., Lord, J. M., and Salmon, M. (1999) RGD

peptides induce apoptosis by direct caspase-3 activation. Nature. 397, 534-539.

13. Philchenkov, A. (2004) Caspases: Potential targets for regulating cell death. J. Cell Mol.

Med. 8, 432-444.

14. Jacobson, S. A., and Sabbagh, M. N. (2011) Investigational drugs for the treatment of

AD: What can we learn from negative trials? Alzheimer's Res. Ther. 3, 14.

15. Mullane, K., and Williams, M. (2013) Alzheimer's therapeutics: Continued clinical

failures question the validity of the amyloid hypothesis-but what lies beyond? Biochem.

Pharmacol. 85, 289-305.

16. Siemers, E. R., Sundell, K. L., Carlson, C., Case, M., Sethuraman, G., Liu-Seifert, H.,

Dowsett, S. A., Pontecorvo, M. J., Dean, R. A., and Demattos, R. (2016) Solanezumab trials:

Secondary outcomes in mild alzheimer's disease patients. Alzheimers Dement. 12, 110-120.

17. Hyde, C., Peters, J., Bond, M., Rogers, G., Hoyle, M., Anderson, R., Jeffreys, M., Davis,

S., Thokala, P., and Moxham, T. (2013) Evolution of the evidence on the effectiveness and

cost-effectiveness of acetylcholinesterase inhibitors and memantine for alzheimer's disease:

Systematic review and economic model. Age Ageing. 42, 14-20.

18. Fosgerau, K., and Hoffmann, T. (2015) Peptide therapeutics: Current status and future

directions. Drug Discov. Today. 20, 122-128.

19. Green, M., and Loewenstein, P. M. (1988) Autonomous functional domains of chemically

synthesized human immunodeficiency virus tat trans-activator protein. Cell. 55, 1179-1188.

20. Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the tat protein from human

immunodeficiency virus. Cell. 55, 1189-1193.

21. La Rochelle, J. R., Cobb, G. B., Steinauer, A., Rhoades, E., and Schepartz, A. (2015)

Fluorescence correlation spectroscopy reveals highly efficient cytosolic delivery of certain

penta-arg proteins and stapled peptides. J. Am. Chem. Soc. 137, 2536-2541.

22. Villar, E. A., Beglov, D., Chennamadhavuni, S., Porco, J. A. J., Kozakov, D., Vajda, S.,

and Whitty, A. (2014) How proteins bind macrocycles. Nat. Chem. Bio. 10, 723-731.

23. Matsson, P., and Kihlberg, J. (2017) How big is too big for cell permeability? J. Med.

Chem. 60, 1662-1664.

24. Tsomaia, N. (2015) Peptide therapeutics: Targeting the undruggable space. Eur. J. Med.

Chem. 94, 459-470.

20

25. Otvos, L., and Wade, J. D. (2014) Current challenges in peptide-based drug discovery.

Front. Chem. 2, 1-4.

26. Rozek, R. P. (2013) Economic aspects of small and large molecule pharmaceutical

technologies. Adv. Econom. Bus. 1, 258-269.

27. Bray, B. L. (2003) Large-scale manufacture of peptide therapeutics by chemical

synthesis. Rev. Drug Discov. 2, 587-593.

28. Otvos, L. (2014) Peptide-based drug research and development: Relative costs,

comparative value. Pharm. Outsourcing. 15, 16-20.

29. Lax, R. (2010) The future of peptide development in the pharmaceutical industry.

PharMa. Int. Pept. Rev., 10-15.

30. Guzman, F., Barberis, S., and Illanes, A. (2007) Peptide synthesis: Chemical or

enzymatic. J. Biotechnol. 10, 279-314.

31. Grant, M., and Leone-Bay, A. (2012) Peptide therapeutics: It’s all in the delivery. Ther.

Deliv. 3, 981-996.

32. Loregian, A., and Palù, G. (2005) Disruption of protein–protein interactions: Towards

new targets for chemotherapy. J. Cell. Physiol. 204, 750-762.

33. Wójcik, P. (2016) Peptide-based inhibitors of protein-protein interactions. Bioorg. Med.

Chem. Lett. 26, 707-713.

34. Shankar, G., Arkin, S., Cocea, L., Devanarayan, V., Kirshner, S., Kromminga, A.,

Quarmby, V., Richards, S., Schneider, C. K., Subramanyam, M., Swanson, S., Verthelyi, D.,

and Yim, S. (2014) Assessment and reporting of the clinical immunogenicity of therapeutic

proteins and Peptides—Harmonized terminology and tactical recommendations. AAPS J. 16,

658-673.

35. Dudek, N. L., Perlmutter, P., Aguilar, M. -., Croft, N. P., and Purcell, A. W. (2010)

Epitope discovery and their use in peptide based vaccines. Curr. Pharm. Des. 16, 3149-3157.

36. Love, B. L., Johnson, A., and Smith, L. S. (2014) Linaclotide: A novel agent for chronic

constipation and irritable bowel syndrome. Am. J. Health Syst. Pharm. 71, 1081-1091.

37. Yu, S. W., and Rao, S. S. (2014) Advances in the management of constipation-

predominant irritable bowel syndrome: The role of linaclotide. Therap. Adv. Gastroenterol. 7,

193-205.

21

38. Gunnoo, S. B., and Madder, A. (2016) Bioconjugation - using selective chemistry to

enhance the properties of proteins and peptides as therapeutics and carriers. Org. Biomol.

Chem. 14, 8002-2013.

39. Meijer, D., and Ziegler, K. (1993) Biological Barriers to Protein Delivery. Plenium Press,

New York.

40. Song, K., Yoon, I. S., Kim, N. A., Kim, D. H., Lee, J., Lee, H. J., Lee, S., Choi, S., Choi,

M. K., Kim, H. H., Jeong, S. H., Son, W. S., Kim, D. D., and Shin, Y. K. (2014)

Glycoengineering of interferon-β 1a improves its biophysical and pharmacokinetic properties.

PLoS One. 9, e96967/1-e96967/14.

41. Fabio, S., Silvia, S., Rodolfo, S., Giancarlo, T., Gaetano, O., Riccardo, S., Simona, D.,

Pierluigi, O., and Stefano, G. (2012) Enzymatic mono-pegylation of glucagon-like peptide 1

towards long lasting treatment of type 2 diabetes. Results Pharma. Sci. 2, 58-65.

42. Sato, H. (2002) Enzymatic procedure for site-specific PEGylation of proteins. Adv. Drug

Deliv. Rev. 54, 487-504.

43. Fontana, A., Spolaore, B., Mero, A., and Veronese, F. M. (2008) Site-specific

modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv.

Drug Deliver. Rev. 60, 13-28.

44. Mero, A., Spolaore, B., Veronese, F. M., and Fontana, A. (2009) Transglutaminase-

mediated PEGylation of proteins: Direct identification of the sites of protein modification by

mass spectrometry using a novel monodisperse PEG. Bioconjug. Chem. 20, 384-389.

45. Yang, Q., and Lai, S. K. (2015) Anti-PEG immunity: Emergence, characteristics, and

unaddressed questions. WIREs Nanomed. Nanobiotechnol. 7, 655-677.

46. Lubich, C., Allacher, P., De la Rosa, M., Bauer, A., Prenninger, T., Horling, F. M.,

Siekmann, J., Oldenburg, J., Scheiflinger, F., and Reipert, B. M. (2016) The mystery of

antibodies against polyethylene glycol (PEG) - what do we know? Pharm. Res. 33, 2239-

2249.

47. Saifer, M. G. P., Williams, L. D., Sobczyk, M. A., Michaels, S. J., and Sherman, M. R.

(2014) Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies

induced by methoxyPEG-proteins. Mol. Immunol. 57, 236-246.

48. Böhme, D., and Beck-Sickinger, A. G. (2015) Drug delivery and release systems for

targeted tumor therapy. J. Pept. Sci. 21, 186-200.

49. Lautrette, G., Toute, F., Lee, G. G., Dai, P., and Pentelute, B. L. (2016) Nitrogen

arylation for macrocyclization of unprotected peptides. J. Am. Chem. Soc. 138, 8340-8343.

22

50. Nahrwold, M., Weib, C., Bogner, T., Mertink, F., Conradi, J., Sammet, B., Palmisano, R.,

Royo Gracia, S., Preube, T., and Sewald, N. (2013) Conjugates of modified cryptophycins

and RGD-peptides enter target cells by endocytosis. J. Med. Chem. 56, 1853-1864.

51. Zwanziger, D., Irfan, U. K., Neundorf, I., Sieger, S., Lehmann, L., Friebe, M.,

Dinkelborg, L., and Beck-Sickinger, A. G. (2008) Novel chemically modified analogues of

neuropeptide Y for tumor targeting. Bioconjug. Chem. 19, 1430-1438.

52. Bockus, A. T., Lexa, K. W., Pye, C. R., Kalgutkar, A. S., Gardner, J. W., Hund, K. C. R.,

Hewitt, W. M., Schwochert, J. A., Glassey, E., Price, D. A., Mathiowetz, A. M., Liras, S.,

Jacobson, M. P., and Lokey, R. S. (2015) Probing the physicochemical boundaries of cell

permeability and oral bioavailability in lipophilic macrocycles inspired by natural products. J.

Med. Chem. 58, 4581-4589.

53. Saether, O., Craik, D. J., Campbell, I. D., Sletten, K., Juul, J., and Norman, D. G. (1995)

Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide

kalata B1. Biochemistry. 34, 4147-4158.

54. Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) Plant cyclotides: A unique

family of cyclic and knotted proteins that defines the cyclic cysteine knot structural motif. J.

Mol. Bio. 294, 1327-1336.

55. Colgrave, M. L., and Craik, D. J. (2004) Thermal, chemical, and enzymatic stability of

the cyclotide kalata B1: The importance of the cyclic cysteine knot. Biochemistry. 43, 5965-

5975.

56. Gran, L. (1973) Oxytocic principles of oldenlandia affinis. Lloydia. 36, 174-178.

57. Sletten, K., and Gran, L. (1973) Some molecular properties of kalata peptide B1: A

uterotonic polypeptide isolated from oldenlandia affinis DC. Medd. Nor. Farm. Selsk. 7-8,

69-82.

58. Simonsen, S. M., Sando, L., Rosengren, K. J., Wang, C. K., Colgrave, M. L., Daly, N. L.,

and Craik, D. J. (2008) Alanine scanning mutagenesis of the prototypic cyclotide reveals a

cluster of residues essential for bioactivity. J. Biol. Chem. 283, 9805-9813.

59. Huang, Y. H., Colgrave, M. L., Clark, R. J., Kotze, A. C., and Craik, D. J. (2010) Lysine-

scanning mutagenesis reveals a previously unidentified amendable face of the cyclotide

kalata B1 for the optimization of nematocidal activity. J. Biol. Chem. 285, 10797-10805.

60. Tam, J. P., and Lu, Y. A. (1997) Synthesis of large cyclic cystine-knot peptide by

orthogonal coupling strategy using unprotected peptide precursors. Tetrahedron Lett. 38,

5599-5602.

23

61. Tam, J. P., and Lu, Y. A. (1998) A biomimetic strategy in the synthesis and fragmentation

of cyclic protein. Protein Sci. 7, 1583-1592.

62. Daly, N. L., Love, S., Alewood, P. F., and Craik, D. J. (1999) Chemical synthesis and

folding pathways of large cyclic polypeptides: Studies of the cystine knot polypeptide kalata

B1. . Biochemistry. 38, 10606-10614.

63. Tam, J. P., Lu, Y. A., and Yu, Q. (1999) Thia zip reaction for synthesis of large cyclic

peptides: Mechanisms and applications. J. Am. Chem. Soc. 121, 4316-4324.

64. Thongyoo, P., Tate, E. W., and Leatherbarrow, R. J. (2006) Total synthesis of the

macrocyclic cysteine knot microprotein MCoTI-II. Chem. Commun. (Camb. ). , 2848-2850.

65. Avrutina, O., Schmoldt, H. U., Gabrijelcic-Geiger, D., Wentzel, A., Frauendorf, H.,

Sommerhoff, C. P., Diederichsen, U., and Kolmar, H. (2008) Head-to-tail cyclized cystine-

knot peptides by a combined recombinant and chemical route of synthesis. ChemBioChem. 9,

33-37.

66. Cemazar, M., and Craik, D. J. (2008) Microwave-assisted boc-solid phase peptide

synthesis of cyclic cysteine-rich peptides. J. Pept. Sci. 14, 683-689.

67. Zheng, J. S., Chang, H. N., Shi, J., and Liu, L. (2012) Chemical synthesis of a cyclotide

via intramolecular cyclization of peptide O-esters. Sci. China. Chem. 55, 64-69.

68. Zheng, J. S., Tang, S., Guo, Y., Chang, H. N., and Liu, L. (2012) Synthesis of cyclic

peptides and cyclic proteins via ligation of peptide hydrazides. ChemBioChem. 13, 542-546.

69. Kimura, R. H., Tran, A. T., and Camarero, J. A. (2006) Biosynthesis of the cyclotide

kalata B1 by using protein splicing. Angew. Chem. Int. Ed. 118, 987-990.

70. Camarero, J. A., Kimura, R. H., Woo, Y. H., Cantor, J., and Steenblock, E. (2007) A cell

based approach for the biosynthesis/screening of cyclic peptide libraries against bacterial

toxins. Chim. Oggi. 25, 20-23.

71. Camarero, J. A., Kimura, R. H., Woo, Y. H., Shekhtman, A., and Cantor, J. (2007)

Biosynthesis of a fully functional cyclotide inside living bacterial cells. ChemBioChem. 8,

1363-1366.

72. Aboye, T. L., and Camarero, J. A. (2012) Biological synthesis of circular polypeptides. J.

Biol. Chem. 287, 27026-27032.

73. Craik, D. J. (2013) Joseph rudinger memorial lecture: Discovery and applications of

cyclotides. J. Pept. Sci. 19, 393-407.

24

74. Bockus, A. T., McEwen, C. M., and Lokey, R. S. (2013) Form and function in cyclic

peptide natural products: A pharmacokinetic perspective. Curr. Top. Med. Chem. 13, 821-

836.

75. Qian, Z., Liu, T., Liu, Y. Y., Briesewitz, R., Barrios, A. M., Jhiang, S. M., and Pei, D.

(2013) Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs.

ACS Chem. Biol. 8, 423-431.

76. Qian, Z., LaRochelle, J. R., Jiang, B., Lian, W., Hard, R. L., Selner, N. G.,

Luechapanichkul, R., Barrios, A. M., and Pei, D. (2014) Early endosomal escape of a cyclic

cell-penetrating peptide allows effective cytosolic cargo delivery. Biochemistry. 53, 4034-

4046.

77. Qian, Z., Martyna, A., Hard, R. L., Wang, J., Appiah-Kubi, G., Coss, C., Phelps, M. A.,

Rossman, J. S., and Pei, D. (2016) Discovery and mechanism of highly efficient cyclic cell-

penetrating peptides. Biochemistry. 55, 2601-2612.

78. Price, J. L., Powers, E. T., and Kelly, J. W. (2011) N-PEGylation of a reverse turn is

stabilizing in multiple sequence contexts, unlike N-GlcNAcylation. ACS Chem. Biol. 6, 1188-

1192.

79. Lawrence, P. B., Gavrilov, Y., Matthews, S. S., Langlois, M. I., Shental-Bechor, D.,

Greenblatt, H. M., Pandev, B. K., Smith, M. S., Paxman, R., Torgerson, C. D., Merrell, J. P.,

Ritz, C. C., Prigozhin, M. B., Levy, Y., and Price, J. L. (2014) Criteria for selecting

PEGylation sites on proteins for higher thermodynamic and proteolytic stability. J. Am.

Chem. Soc. 136, 17547-17560.

80. Pandey, B. K., Smith, M. S., Torgerson, C., Lawrence, P. B., Matthews, S. S., Watkins,

E., Groves, M. L., Prigozhin, M. B., and Price, J. L. (2013) Impact of site-specific

PEGylation on the conformational stability and folding rate of the pin WW domain depends

strongly on PEG oligomer length. Bioconjug. Chem. 24, 796-802.

81. Lawrence, P. B., Billings, W. M., Miller, M. B., Pandey, B. K., Stephens, A. R., Langlois,

M. I., and Price, J. L. (2016) Conjugation strategy strongly impacts the conformational

stability of a PEG-protein conjugate. ACS Chem. Biol. 11, 1805-1809.

82. Wang, Y., Cheetham, A. G., Angacian, G., Su, H., Xie, L., and Cui, H. (2016) Peptide-

drug conjugates as effective prodrug strategies for targeted delivery. Adv. Drug Deliv. Rev.,

Ahead of print.

83. Lelle, M., Kaloyanova, S., Freidel, C., Theodoropoulou, M., Musheev, M., Niehrs, C.,

Stalla, G., and Peneva, K. (2015) Octreotide-mediated tumor-targeted drug delivery via a

cleavable Doxorubicin–Peptide conjugate. Mol. Pharm. 12, 4290-4300.

25

84. Heinis, C., Rutherford, T., Freund, S., and Winter, G. (2009) Phage-encoded

combinatorial chemical libraries based on bicyclic peptides. Nature Chem. Bio. 5, 502-507.

85. Baeriswyl, V., Rapley, H., Pollaro, L., Stace, C., Teufel, D., Walker, E., Chen, S., Winter,

G., Tite, J., and Heinis, C. (2012) Bicyclic peptides with optimized ring size inhibit human

plasma kallikrein and its orthologues while sparing paralogous proteases. Chem. Med. Chem.

7, 1173-1176.

86. Chen, S., Bertoldo, D., Angelini, A., Pojer, F., and Heinis, C. (2014) Peptide ligands

stabilized by small molecules. Angew. Chem. Int. Ed. 53, 1602-1606.

87. Trinh, T. B., Upadhyaya, P., Qian, Z., and Pei, D. (2016) Discovery of a direct ras

inhibitor by screening a combinatorial library of cell-permeable bicyclic peptides. ACS

Comb. Sci. 18, 75-85.

88. Qian, Z., Rhodes, C. A., McCroskey, L. C., Wen, J., Appiah-Kubi, G., Wang, D. J.,

Guttridge, D. C., and Pei, D. (2016) Enhancing the cell permeability and metabolic stability

of peptidyl drugs by reversible bicyclization. Angew. Chem. Int. Ed. 55, 1-6.

89. Pollaro, L., Raghunathan, S., Morales-Sanfrutos, J., Angelini, A., Kontos, S., and Heinis,

C. (2015) Bicyclic peptide conjugated to an albumin-binding tag diffuse efficiently into solid

tumors. Mol. Cancer Ther. 14, 151-161.

90. Lian, W., Upadhyaya, P., Rhodes, C. A., Liu, Y., and Pei, D. (2013) Protein-protein

interaction inhibitors: Discovery of a tumor necrosis factor-α antagonist. J. Am. Chem. Soc.

135, 11990-11995.

26

CHAPTER TWO

PART I

SYNTHESIS OF LINEAR AND BRANCHED PEGYLATED GLUTAMINE BUILDING

BLOCKS FOR FMOC SOLID-PHASE SYNTHESIS OF PEPTIDES

Introduction

Peptides have great potential as therapeutics because of their high selectivity for

native receptors.1, 2 However, peptide therapeutics suffer from short half-lives due to protease

degradation, poor solubility, and low oral bioavailability.1

Many techniques such as glycosylation, N-methylation, peptide stapling, D-amino

acid incorporation, and others, have been used to mitigate the pitfalls of using peptide

therapeutics,3, 4, 5, 6 but these efforts have found limited clinical success.

The poly(ethylene glycol) polymer has demonstrated numerous benefits when

conjugated to peptides and proteins, including increased half-life due to protease stability and

improved solubility.7 The PEG molecule is generally regarded as safe by the FDA and

conjugation of PEG to proteins has resulted in 15 FDA approvals to date.

There is growing debate about the potential disadvanages of peptide PEGylation,8, 9, 10,

11 but the concerns focus about long, polydisperse PEGs instead of short, monodisperse PEGs

as utilized here.

Peptides are typically PEGylated at the N-terminus or on a lysine or cysteine

residue,12 typically requiring cumbersome orthogonal protecting groups (Nvoc, Dde,

27

ivDde).13 Deprotection of the residue of interest, after peptide synthesis, liberates the desired

modification site where reactivity can be directed specifically through introduction of an

activated PEG (typically NHS esters). These modifications can have substantial

consequences on the peptide, such as by replacing the naturally positively charged lysine

amine side chain with a neutral amide. Unsurprisingly, conjugation strategy has a strong

impact on the conformational stability of the resulting conjugate.14 Therefore, a post-

synthetic modification, which is how PEG is typically attached to peptides, can diminish

biological activity if not conjugated in an optimal fashion and demonstrates a need to test for

PEGylation benefits earlier in the drug discovery process. Synthesizing amino acids already

incorporating short, discrete PEGs would advantageously enable direct incorporation of

PEGylated amino acids during peptide synthesis and obviate the need for orthogonal

protecting strategies. A pre-PEGylated amino acid strategy could give rise to a PEG-scan

capability where each amino acid is systematically replaced by a PEGylated amino acid,

allowing for a screen of PEGylation benefits from an earlier stage of the lead optimization

process.

Site-specific PEGylation of peptides has been achieved by researchers using varying

methods and has been reviewed by others in the literature.15, 16 The bulk of these PEGylation

methods involve a post-synthetic peptide modification that specifically targets the N-

terminus, incorporated non-natural amino acid side-chains (e.g. azides, alkynes), or the

nucleophilic nature of thiols. Short monodisperse PEG attachments to peptides, like

neuropeptide Y, galanin, GIP, as well as proteins have recently displayed improved

pharmacokinetic properties.17, 18, 19, 20 Furthermore, we hypothesize that, our synthetic

28

strategy enables facile incorporation of PEG topologies (e.g. triPEGylated lysine) never

before attempted.

The amino acid building block method is the simplist way to incorporate

modifications into a peptide and has been explored with a variety of side-chain

modifications.21, 22, 23, 24, 25, 26, 27, 28, 29, 30 While short PEGylated (linear, 4-ether linkages)

asparagine has been reported previously by others,10, 31 we believe it highly relevant to

expand the building blocks to glutamine and lysine, as they are amongst the most solvent

accessible amino acids found in protein structures.32 To this aim, we have adapted a

straightforward strategy entailing the conversion of commercially available Fmoc-Lys(Boc)-

OH and Fmoc-Glu(OtBu)-OH into PEGylated Fmoc-amino acids suitable for solid-phase

peptide synthesis (SPPS). Thus, we have developed (1) a scalable strategy for pre-PEGylation

that does not require orthogonal protecting groups, and (2) Fmoc-protected amino acids with

unprecedented degrees of branched PEGs.

Typically, to achieve site-selective PEGylation at glutamine within a peptide

sequence, transglutaminase would need to be used in the presence of peptide and the desired

modification with amine functionality (Figure 2.1.1).33 This method has been used to

specifically PEGylate the GLP-1 (7-36 amide)34 as well as proteins such as apomyoglobin

(apoMb), α-lactalbumin (α-LA), human growth hormone (hGH), human granulocyte colony-

stimulating factor (hG-CSF) and human interlukin-2 (hIL-2).35, 36

29

Scheme 2.1.1. Literature presedent for enzymatic site-selective glutamine side-chain

PEGylation using transglutaminase. R = peptide.33

The ideal sites for this proposed PEGylation of peptides would be residues that are

solvent exposed in peptides and if modified will potentially not interfere with the peptides’

structure or function. Glutamines (Gln), asparagines (Asn), and lysines (Lys) (Figure 2.1.1)

are most often found on the solvent exposed portion of peptide drugs and provide

solubility.32, 37 Since Gln and Asn do not participate in salt bridges or hydrophobic

interactions, they are ideal for direct PEG modification. Although lys does often participate in

salt bridges, our proposed PEGylation should not significantly affect its pKa and ability to

participate in salt bridges. Our proposed PEGylation method is in contrast with using

orthogonal protecting groups to couple lysines with PEG carboxylic acids, which removes the

positive charge required for salt bridges.

Glutamine (Gln) Asparagine (Asn) Lysine (Lys)

Figure 2.1.1. Structures of natural amino acids; glutamine, asparagine, and lysine.

30

Folding of the peptide will have a substantial effect on the amount of protease

protection that this PEG side chain will provide. If a peptide has a helical fold, then there is

potential to have protease protection up to 3 or 4 residues away from the site of PEG

modification. Increasing peptide conformational stability through short-chain PEGylation

has demonstrated that it can be an effective strategy for increasing proteolytic stability.31, 38

Additionally, short-chain PEG branching on a peptide has shown to be more effective at

stabilizing peptide conformation as compared to short linear PEG chains.14 Recent molecular

dynamic simulations support the experimental results by suggesting PEG increases

conformational stability through shielding of a peptide’s backbone hydrogen bonds.11

MATERIALS AND METHODS

Chemicals

All Fmoc-protected amino acids were obtained from Aapptec (Louisville, KY) and

used without further purification. Triethylene glycol monomethyl ether (TEGME or mTEG)

obtained from TCI America (Portland, OR). Trifluoroacetic acid, trifluoromethanesulfonic

anhydride were obtained from Oakwood Chemicals (Estill, SC). 1-Propylphosphonic acid

cyclic anhydride obtained from Alfa Aesar (Haverhill, MA). N,N’-Diisopropylethyl amine

and triisopropylethyl amine were obtained from Chem-Impex International (Wood Dale, IL).

Methanol, dichloromethane, acetone, sodium bicarbonate (NaHCO3) were obtained from

VWR International (Radnor, PA). Acetonitrile obtained from Sigma-Aldrich (St. Louis, MO).

All chemicals were used without further purification from suppliers.

31

Instrumentation

NMR. 1H and 13C NMR spectra were collected on a Bruker 500 MHz FT-NMR

spectrometer. All collected spectra for intermediates and final products can be found in

Appendix A.

Peptide Synthesis. Coupling of Amino Acids. The peptides reported here were

synthesized manually using standard Fmoc-SPPS methods on Rink Amide resin. Rink Amide

MBHA resin (100 mg) with 0.51 mmol/g loading was swollen in DMF (1 mL, 2 x 1 min)

with stirring. Resin was deprotecting by stirring with 20% 4-methylpiperidine in DMF (1 mL,

2 x 1 min). N-Fmoc protected amino acids (4 equiv) were then coupled to resin in DMF in

the precence of HCTU (16 equiv), 6-Cl-HOBt (8 equiv) and DIEA (16 equiv) for 5 min (2 x).

Resin was washed extensively with DMF (3 x 1 mL) between reactions. Only the N-termini

of model -helical peptides (containing i+4 E-K) were acetylated with acetic anhydride, the

resin was washed with DCM (2 x 1 mL). All peptides were cleaved from the Rink Amide

resin as peptide amides with a 95:2.5:2.5 trifluoroacetic acid/triisopropyl silane/water

mixture.

Coupling of PEGylated Amino Acids. After the desired amino acid couplings had been

achieved, resin was deprotected with 20% 4-methylpiperidine in DMF (1 mL, 2 x 1 min) and

washed with DMF (3 x 1 mL). The resin was mixed with the desired PEGylated amino acid

(4 equiv) in DMF in the presence of HCTU (16 equiv), 6-Cl-HOBt (8 equiv), and DIEA (16

equiv) for 10 minutes (2 x). After coupling, resin was washed with DMF (3 x 1 mL) followed

by 20% 4-methylpiperidine in DMF (1 mL, 2 x 1 min). The remainder of the amino acids

were then coupled to the resin normally as described above.

32

MALDI-MS. MALDI-MS spectra were collected using a Bruker Omniflex MALDI-

TOF mass spectrometer. All collected spectra for small molecule intermediates and final

products can be found in Appendix A. All collected spectra for peptide products can be found

in Appendix B.

HPLC Purification. The peptides were purified by C18 reverse-phased

chromatography, and peptide identity was confirmed by MALDI-mass spectrometry. The

peptides were purified on a Shumadzu HPLC instrument equipped with HASIL 100 RP-C18

5μm column. The column dimensions are 250 x 10 mm. Pore size 80 Å. Purification/analysis

was done in the following elution sequence― Solvents: A, Acetonitrile (with 0.1% TFA); B,

H2O (with 0.1% TFA). The solvent gradient used for compound purification was: 0-1 min,

5% A in B; 1-20 min, 5-95% A in B; 20-23 min, 95% A in B; 23-25 min, 5% A in B at a flow

rate of 1.0 mL/min. Peptide purity was assessed by analytical C18 reverse-phase

chromatography using the Shumadzu HPLC system. All collected spectra for peptide

products can be found in Appendix B.

33

RESULTS AND DISCUSSION

Retrosynthetic Analysis. The proposed synthetic mTEG-glutamine building blocks are

shown in Figure 2.1.2. The desired mTEG modification for glutamine (and asparagine)

derivatives would be easily accessible through the amide bond formation of an mTEG-amine and

the free side-chain carboxylic acid of an α-COOH–protected glutamic acid (Figure 2.1.3).

PEGylated amines of varying lengths could be synthesized and coupled to the protected glutamic

acid intermediate by starting with the appropriate, commercially available monomethoxy-PEG-

alcohol.

FmocHNOH

O

NH

OO

OO

FmocHN

O

OH

NHO

N

FmocHNOH

O

N OO

O

O

OO

O

OOO

O

OO

O

O

O

1 2 3

Figure 2.1.2. Synthetic PEG glutamine targets of interest. 1 – mTEG1 glutamine. 2 –

mTEG2 glutamine. 3 – mTEG3 glutamine.

Figure 2.1.3. Retrosynthetic strategy to Fmoc-Gln(mTEG)1-OH.

34

Chain Length Selection. We decided to use a 3-ether linkage for the initial PEG chain

attachment. C-C bonds are around 1.5 Å long and a C-O bond is not much different (1.4 Å).

Having 10 bonds, the equivalent of a 3-ether linkage, should theoretically be able to reach

between 10-15 Å. This could interfere with protease binding near neighboring residues no

matter the peptide folding. However, if the peptide is folded, protection is possible further away

from the site of PEGylation. In an alpha helix, there are 3.4 residues per turn and it is 5 Å from

one loop in an alpha helix to the next loop so this is the theoretical basis upon which our

proposed PEG chain length was chosen.

Additionally, in recent literature, PEG chains of 3-4 ether linkages have demonstrated to

be the minimally useful length in improving peptide conformational stability and improving

protease resistance.31

Cost Reduction of Strategy. Other researchers have synthesized a longer PEG Gln

analog from the -OtBu-protected glutamic acid starting material, but this route isn’t the most

scalable due to the high costs of starting materials (Figure 2.1.4). Our route would be more

economically feasible for many researchers especially those who want to make these PEGylated

amino acids on a larger scale for synthesizing and testing multiple peptide analogs.

35

Figure 2.1.4. Top – Literature synthetic strategy for monoPEG Fmoc-Gln/Asn. Bottom – Cost

of starting materials for literature route (left) versus our proposed route (right).

Fmoc-Gln(mTEG)1-OH Synthesis. PEGylated glutamine, 1, was identified as an initial

target due to the prevalence of glutamine in many biologically relevant peptides, and the

proposed retrosynthetic route for amino acid 1 (Scheme 2.1.4) requires an orthogonal protecting

group. Selection of the appropriate carboxylic acid protecting group is essential in this synthetic

strategy with the following restraints: (1) The free α-carboxylic acid needs to be protected under

conditions that will not cleave the other protecting groups; (2) The existing side chain protecting

groups need to be cleaved without affecting the α-carboxylic acid protecting group under acidic

hydrolysis (pH < 1); and (3) the α-carboxylic acid protecting group needs to be removed under

conditions that would not deprotect the N-α-Fmoc or racemize the alpha carbon. Phenacyl ester

(OPac) was chosen as a protecting group since it can be installed and removed using orthogonal

36

chemistries without racemizing the alpha-carbon by utilizing Mg, MeOH/DMF, and AcOH.39

Alternatives choices include using either an allyl ester or 2,2,2-trichloroethyl ester.

The PEG-amine (mTEG-NH2), was synthesized from the inexpensive commercially

available tris(ethylene glycol)monomethyl ether (Scheme 2.1.2).40, 41 The alcohol was converted

into a better leaving group by a reaction with methanesulfonyl chloride (MsCl). A subsequent

reaction with sodium azide displaced the mesylate with an azide. These reactions have repeatedly

been performed at >10g scales. A reduction of the azide (mTEG-N3) with lithium aluminum

hydride (LiAlH4) afforded the desired mTEG-NH2. The intermediates and final product can be

easily followed by NMR analysis as shown in Figure 2.1.6.

Scheme 2.1.2. Synthesis of 1. Reagents and conditions: a) Bromoacetophenone, Na2CO3,

N(Bu)4Br, DCM, r.t., 24-72 hr, 82-95%. b) 3:1 (v/v) TFA:DCM, r.t., 2h, 92%-quant. c) CDI,

DCM (or dry MeCN), r.t., 4 hr, 65-81%. d) Mg, AcOH, MeOH, DMF, r.t., 90%-quant. or Zn

dust, acetylacetone, pyridine, EtOAc, 45 oC, 4 hr. e) MsCl, DIEA, DCM, 0 oC, 2 hr, quant. f)

NaN3, H2O, MeOH, reflux, 4 hr, 56%. g) LiAlH4, THF (or ether), 2 hr, r.t., quant.

37

There are alternatives to the chosen route, but LiAlH4 reduction of azide was selected as

most efficient and safest for our purposes. The Mitsunobu reaction wasn’t used to generate the

azide since (1) it uses azidic acid (HN3) which is explosive, and (2) triphenyl phosphine oxide

byproduct and unreacted triphenyl phosphine are water insoluble and often chromatography is

required for the separation of products which is difficult even after chromatography.

If the mesyl ester was reacted with CN- and then reduced with NaBH4 to avoid azide, this

path would add an additional carbon atom; however, there is no practical or strategic reason that

we would not want an extra carbon. It is a plausible alternative route to the amine. Additionally,

the use of cyanide comes with its own problems. If it is used and becomes acidic, forming HCN,

that’s extremely toxic. Even though if you have CN- then of course that is not under acidic

Figure 2.1.5. 1H NMR comparison of mTEG intermediates in the synthesis of monoPEGylated

amine (mTEG-NH2). The mTEG-amine has been previously reported in the literature.40, 41

38

conditions, but you would have to be very careful with even the waste produced. The azide is

potentially explosive and should be worked with cautiously. A general rule is to have at least 3

carbons per azide so ours should theoretically not be explosive and has proven to be stable and

easy to handle. Lithium aluminum hydride is potentially dangerous with fire hazards, but it can

be managed with careful lab technique and reaction conditions (e.g. THF instead of Et2O).

In 2000, there was a publication from a group in Italy where an azide reduction that

utilizes Cobalt (II) chloride and NaBH4 in water was used and that is a potentially safer route that

holds some promise.42 This route was examined experimentally for its feasibility and practicality

and forms the monoPEG-amine as observed by NMR analysis (Figure 2.1.7). There is more than

one correct answer for this synthesis.

39

Figure 2.1.6. Alternative mTEG-amine synthesis route using cobalt (II) chloride42 and NMR

comparison to product obtained from reduction of mTEG-azide to mTEG-amine using

LiAlH4.

Another alternative method to obtaining monoPEG-amine from the azide precursor

without using LiAlH4 could be the Staudinger reduction (shown below, Scheme 2.1.3). The

azide can also be reduced via Pd-catalyzed hydrogenation conditions. Neither of these two routes

were examined because the LiAlH4 route works cleanly and in high yield and is more atom

economical.

40

Scheme 2.1.3. Staudinger reduction mechanism.

The synthesis of intermediates Fmoc-Glu(OtBu)-OPac and Fmoc-Glu(OH)-OPac was

carried out as shown (Scheme 2.1.2) in >10g scales, and required only optimization of the

TFA:H2O ratios to ensure full cleavage of the γ-carboxylate t-butyl protection group. Coupling

of the mTEG-NH2 with Fmoc-Glu(OH)-OPac (Figure 2.1.7) required some screening of the

coupling reagent to ensure high yields. The coupling was originally screened using three

carbodiimides to activate the carboxylic acid: diisopropylcarbodiimide (DIC),

dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

For DIC and DCC, it was thought that the urea side product would precipitate from the reaction

solvent (DCM) and leave only product in solution. For EDC, the urea product that forms is

water-soluble and an aqueous wash in the work up should theoretically yield a cleaner product.

However, in all cases 1H NMR revealed poor separation of the urea products and low coupling

yields, possibly due to hydrogen bonding between the urea and PEG. The final coupling reagent

tested was carbodiimidazole (CDI) and its reaction by-products are CO2(g) and a water-soluble

imidazole which, following aqueous separation, yielded a purer product than the use of all other

coupling reagents (Figure 2.1.7). It should also be noted that it was observed that upon addition

of only 1 equivalent of coupling reagent, the best yield obtained was 50%, which was likely due

to the formation of a symmetric anhydride species in solution. This can be overcome by the

addition of more coupling reagent 30 minutes to 1 hour after the initial addition of 1 equivalent

41

of coupling reagent which increases product yield to 65-81% after purification. A subsequent

deprotection of the α-phenacyl ester produced 1 in great yield (Scheme 2.1.2).

Figure 2.1.7. 1H NMR spectra comparison of crude Fmoc-Gln(mTEG)1-OPac

synthesized using EDC (top) and CDI (bottom).

42

Initial usage of the Mg/AcOH cleavage conditions39 allowed for the clean deprotection of

OPac from the monoPEGylated glutamine derivative (Figure 2.1.8), however in the synthesis of

later mPEG-amino acid derivatives, the removal of residual AcOH (or Mg(OAc)2) from the

product was quite difficult.

Magnesium is used in the literature39 instead of zinc because it would create less acidic

salts upon phenacyl ester deprotection. Zinc is, in some respects, chemically similar to

magnesium, because its ion is of similar size and its only common oxidation state is +2. The zinc

would be oxidized, forming hydrogen gas and dissolving the zinc:

Zn(s) + 2 CH3COOH(aq) ---> 2 CH3COO-(aq) + Zn2+(aq) + H2(g)

0.0

0.2

0.4

0.6

0.8

1.0

4x10

Inte

ns. [a

.u.]

500 510 520 530 540 550 560 570 580 590

m /z

[M+Na]+

[M+H]+

515.414

537.347

510 520 530 540 550 560 570

0.2

580 590 m/z

0.0

0.4

0.6

0.8

1.0

x104

550 560500

MW=514.57

Figure 2.1.8. MALDI-mass spectrum of 1.

http://en.wikipedia.org/wiki/Magnesium

http://en.wikipedia.org/wiki/Ion

43

The presence of AcOH in solution with a desired Fmoc-amino acid during the solid phase

peptide synthesis (SPPS) results in the coupling of AcOH to the growing peptide chain;

effectively halting the peptide growth at that stage. Therefore, in some cases, it was preferrable

to use the Zn/acetylacetone(acac)/pyridine deprotection conditions43 (Scheme 2.1.4) to yield the

desired products. Although the Zn deprotection is reported to work at 35 oC between 60-90 min,

I routinely needed to elevate the temperature to 55-70 oC (Figure 2.1.9). Additionally, the

solvent can be changed from DMF to ACN without hindering the product yield. This solvent

change is favorable due to the difficulty in removing DMF from the final PEGylated amino

acids.

Scheme 2.1.4. Selected literature precedents for phenacyl ester deprotection.39, 43

44

Alternative protecting groups and cleavage conditions are plausible. Instead of phenacyl

ester a trichloroethyl ester protecting group would also be stable under similar conditions.

Phenacyl ester however has been cleaved from amino acids in literature and has been reported to

have no racemization of the alpha carbon,39 whereas the trichloroethyl ester has not. Allyl ester is

most often used to protect carboxylic acids. Palladium is used in allyl ester removal, which is

typically unrecoverable and on the large scale that I will be trying this method would not be cost

effective.

Figure 2.1.9. LC-MS comparison for the optimization of phenacyl ester protection.

45

Fmoc-Gln(mTEG)2-OH Synthesis.

The proposed synthetic route towards Fmoc-Gln(mTEG)2-OH was relatively

straightforward once diPEG-amine was obtained (Scheme 2.1.5). Utilizing the previously

synthesized Fmoc-Glu(OH)-OPac as a starting point, a diPEGylated amine needed to be

synthesized and coupled to the free carboxylic acid. While some diPEGylated amines are

commercially available, the typical price range can be as low as $700-1000 per gram, making the

synthesis from cheaper starting materials an attractive first step. Subsequent deprotection of the

-phenacyl ester from N-Fmoc-N-OPac-Gln(mTEG)2 would yield the desired Fmoc-protected

product.

FmocHNO

O

OH

O

O

FmocHNO

O

O

O

N

O O

HN

O O

O O

OO

nn

n = 3Gln

FmocHNOH

O

O

N

O Onn

CouplingReagent

n = 3Gln

need to synthesize

Deprotection

Scheme 2.1.5. Proposed synthetic route to Fmoc-Gln(mTEG)2-OH (2).

There are various ways that diPEG-amine could be synthesized so it was a matter of

determining which route was most efficient in practice and which would better fit our needs as

far as undesired side products.

It was decided that the first attempts would be to synthesize the diPEG amine by

exploiting an E1cb mechanism, as seen in Scheme 2.1.6, because in theory this route should

minimize the number of steps to the desired product and it should progress in high yield as long

46

as the appropriate leaving group is chosen correctly (i.e. styrene or isobutylene). Those leaving

groups would also provide convenient NMR peaks to integrate and assess the completion of the

reactions.

NH2

HN

OO

O O

O

O

NH2

HN

O O

O O

O

O

HN

O

O

O

O

OOE1cb

E1cbOH

HN

OH

Cl

HN

Cl

+

+

a

a

b

b

cd

Scheme 2.1.6. Attempted synthetic routes to (mTEG)2-NH. The two synthetic routes on the

left assume an E1cb mechanism. Reagents and conditions: a) TEGME, Tf2O, DIEA, DCM, 0

oC. b) TFA. c) SOCl2, DCM, 0 oC. d) NaHMDS, Diethylene glycol monomethyl ether.

TEGME was initially reacted with trifluoromethane sulfonic anhydride in DCM before

the slow addition to a solution of DIEA and either t-butyl amine or 1-phenylethyl amine in

DCM. The reaction progress was monitored by MALDI mass spectra; however, E1cb mechanism

never seemed to go to completion; showing desired product as well as the diPEGylated

intermediates.

Alternatively, a synthetic route was attempted that aimed to PEGylate the alkyl groups of

diethanol amine (Scheme 2.1.6). The conversion of the hydroxyls on diethanol amine into

chlorides was accomplished with thionyl chloride, however the subsequent attempt at displacing

the chloride with the sodium alkoxide salt of diethyleneglycol monomethyl ether (DEGME) was

unsuccessful. NMR of the reaction products suggested unreacted DEGME. Even if the

47

aziridinium ion were to form, the desired product would have been obtained from nucleophilic

attack and ring opening, suggesting the chloride may not have been present when alkoxide was

added. If the reaction was in the presence of water from either the atmosphere, solvent or in one

of the reagents, the chloride could have converted back into diethanol amine. Some possibilities

for the presence of water are: 1) the atmospheric absorption of water over time by DEGME; and

2) water condensation inside of the open reaction vessel due to the reaction being at low

temperature. The reactions were not rigorously performed under water free conditions.

At this point, alternative methods we sought and evaluated for their synthetic feasibility

and efficiency. One literature route44 that was considered is shown below in Scheme 2.1.7. This

route first involves trityl protecting diethanol amine by way of SN1 in a polar protic solvent.

Next, the free hydroxyls are deprotonated and serve as nucleophiles in the displacement of

tosylate from di(ethylene glycol) monomethyl ether (DEGME). A subsequent trityl deprotection

under acidic conditions would yield the desired product in relatively good yields according to

literature. This particular set of reactions was not very trustworthy due to the high possibility that

hydroxyl ions could react with tosylated DEGME and significantly reduce the product yield.

Cl N

OHHO

OTs

O

O

N

OO

OO

OO

HN

OO

OO

OO

HCl

96%KOH(s)/THF85%

or50 NaOH(s)/THF/PTC

60%

HN

OHHO

Scheme 2.1.7. Potential alternative route to mTEG2-amine.44

48

Alternatively, a similar approach could be taken, where the free hydroxyl on DEGME

can be converted into a leaving group and reacted with an N-boc-protected diethanol amine

under basic conditions (Scheme 2.1.8A-B). A subsequent deprotection of the boc under acidic

conditions (TFA/DCM or HCl/Dioxane) would yield the desired product and have more easily

separable by-products, CO2 (g) and t-BuOH. A similar route can be envisioned where an N-

benzyl-protected diethanol amine is also reacted with an activated DEGME under basic

conditions, followed by debenzylation under Pd-catalyzed hydrogenation (Scheme 2.1.8C).

These proposed routes did not work successfully in liberating the desired mTEG2-amine due to

failed attempts at synthesizing, isolating, and characterizing the N-boc or N-benzyl protected

mTEG intermediates. In a separate project, we have also seen near-quantitative deprotection of

boc during intramolecular generation of acid from diethanol amine even under basic conditions.

49

OH

HN

OH

OH

BocN

OH

N

O O

O O

OO

Boc

X

O

O

HN

O O

O O

OOX = OTs or I

OH

O

O

Cl

O

O

I

O

O

OTs

O

O

CO2(g)tBuOH

24%

68.5%Unattempted

OH

HN

OH

OH

BnN

OH

N

O O

O O

OO

Cl

O

O

HN

O O

O O

OO

B

C

A a b c

d e f

g hi

N

O O

O O

OO

OMs

O

O

HN

O O

O O

OO

NH2

O

37-69% 29%

Dj k

Scheme 2.1.8. A – synthetic route to diethylene glycol monomethyl ether (DEGME)

derivatives with varying leaving groups. B – proposed synthetic route to (mTEG)2-amine via

reaction between N-boc-diethanolamine and activated DEGME. C – proposed synthetic route

to (mTEG)2-amine via reaction between N-benzyl-diethanolamine and activated DEGME.

Reagents and conditions: a) tosyl chloride. b) SOCl2, DCM. c) NaI, acetone, reflux. d) Boc2O.

e) NaH, ACN. f) TFA:DCM (3:1, v/v). g) benzyl bromide, K2CO3, MeOH, reflux. h) NaH,

THF, 65 oC. i) H2, Pd/C, MeOH, 4h, r.t. j) Benzylamine, Na2CO3, ACN, reflux, 18 hr, 37-

69%. k) H2, Pd/C, MeOH, r.t., 4h, 29%-quant.

50

The final route that was selected involved the synthesis of the benzyl protected diPEG

amine through reacting benzyl amine with excess mesylated TEGME under reflux overnight.

This reaction yielded the desired benzyl protected diPEG amine in decent yields. The subsequent

benzyl deprotection was performed by Pd-catalyzed hydrogenation and was successful in

converting all starting material into desired product as judged through MALDI analysis of the

reaction (Figure 2.1.10) and 1H NMR.

39

8.9

49

N-benzyl-(mTEG)2-amine

0

500

1000

1500

2000

Inte

ns.

[a

.u.]

30

9.3

16

33

1.1

79

(mTEG)2-amine

0

1000

2000

3000

4000

5000

6000

Inte

ns.

[a

.u.]

225 250 275 300 325 350 375 400 425 450m/z

MW=399.53

MW=309.40

[M+H

]+

[M+Na]+

[M+H]+

N

O O

O O

OO

OMs

O

Oreflux18hrs

ACN

Na2CO3

NH2

O

37-69%

N

O O

O O

OO

HN

O O

O O

OO

H2

Pd/C

MeOH4 hrsr.t.

29%

Figure 2.1.10. Mass spectrum analysis of product formation during the synthesis of di-mTEG

amine.

When coupling di-mTEG amine to Fmoc-Glu(OH)-OPac, the reaction worked best with

propylphosphonic anhydride (T3P) as the coupling reagent, compared to using CDI, EDC, or

DCC. Additionally, it’s important to note that when di-mTEG amine was stirred for 19 hrs in the

51

presence of Fmoc-Glu(OH)-OPac, CDI, in a mix of dry ACN/DCM (1:1), MALDI analysis

showed not only some desired product, but also the presence of Fmoc-deprotected product at m/z

558.45. This is hypothetically due to the fact that the PEGylated amine being coupled is a

secondary amine and Fmoc is usually deprotected with piperidine which is also a secondary

amine with a pKa ~11. The imidazole that would be created during the reaction would have a

pKa ~ 6.95 as seen in literature and, as such, should not be basic enough to deprotonate and

deprotect Fmoc.

Figure 2.1.11. 1H NMR solvent comparison of Fmoc-Gln(mTEG)2-OPac. Top trace is Fmoc-

Gln(mTEG)2-OPac sample in CDCl3. Bottom trace is the same sample in d-acetone.

52

One potential reason for the observed line broadening in chloroform (Figure 2.1.11)

includes possible molecular aggregation. There could be intermolecular pi-stacking occurring in

solution or maybe even a type of micelle formation with a hydrophilic tail (PEG chains) and

hydrophobic head group (Fmoc and OPac protecting groups). This hypothesis could be tested

with NMR diffusion experiments that are outside the scope of the current work. It is important to

note that the OPac deprotected final product (subsequent synthetic step) shows sharp lines in

CDCl3 during NMR analysis.

FmocHNO

OHO

FmocHNO

O

N

O O

HN

O O

O O

OOnn

n = 3Gln

FmocHNOH

O

N

O Onn

n = 3Gln

a bO

O

O

O

O

Scheme 2.1.9. Synthesis of Fmoc-Gln(mTEG)2-OH. Reagents and conditions: a) T3P, DIEA,

MeCN, r.t., 18 h, 43%. b) Zn dust, acetylacetone, pyridine, EtOAc, 45 oC, 4 h, 40%.

The deprotection of the phenacyl ester under Zn-catalyzed acidic reduction conditions

yielded the desired Fmoc-Gln(mTEG)2-OH product in 40% yield (Scheme 2.1.9).

53

Fmoc-Gln(mTEG)3-OH Synthesis.

FmocHNO

OPac

OHO

X

O

O

O

FmocHNO

OPac

NHO

NH2

FmocHNO

OH

NHO

N

n = 0, Aspn = 1, Glu

nn n

n = 0, Asnn = 1, Gln


NH2

NPEGPEG

PEG

X = Leaving Group

PEG

PEG

PEG

Figure 2.1.12. Retrosynthetic strategies for Fmoc-Gln(EDA-mTEG3)-OH (3) formation.

The desired Fmoc-Gln(mTEG)3-OH product can theoretically be synthesized via two

pathways arising from either C(O)-N (amide bond) or C-N bond disconnections as shown above

(Figure 2.1.12). The first attempted route was to couple a N-Boc-ethylene diamine (N-Boc-

EDA) to N-Fmoc-Glu(OH)-OPac, followed by the PEGylation of the free amine through

nucleophic additions. The second-generation strategy that was attempted involved

prePEGylating the diamine linker and subsequently coupling that amine to glutamic acid. Both

routes have their benefits and pitfalls as will be discussed.

54

Scheme 2.1.10. Optimization of coupling strategy towards Fmoc-Gln(mTEG)3-OH.

The coupling strategy of N-Boc-ethylenediamine to Fmoc-Glu(OH)-OPac needed to be

optimized because the conditions used for coupling monoPEG-amine gave low yields (~20%) in

this system. Therefore, a range of coupling reagents was tested to elucidate which would provide

the cleanest crude product and best yields after purification (Scheme 2.1.10).

Attempted coupling with PyBOP and carbodiimides such as CDI, DCC, DIC, and EDC,

gave low yields of desired product (<12-38%). If there was any trace water present during the

reaction, the PyBop reagent, which contains a dehydrating agent along with HOBt, should have

yielded more product than the carbodiimides. However, T3P coupling to form the desired amide

bond gave consistently higher yields (50-70%) of the desired product (Figure 2.1.13).

55

Figure 2.1.13. Representative 1H NMR showing the successful synthesis of the desired

intermediate N-boc-protected structure.

The attempted coupling of N-Boc-ethylenediamine to Fmoc-Glu(OH)-OPac with EDC-

HCl in the absence of base yielded a boc-deprotected desired product (Figure 2.1.14) in good

yield upon purification. One of the side products isolated was the EDC-activated ester of Fmoc-

Glu(OH)-OPac by 1H NMR analysis, which is generally thought to be unstable. To potentially

drive the reaction to completion and avoid any isolation of the activated ester, 6-Cl-HOBt was

added to the coupling reaction and the activated ester was no longer observed as a side product

upon purification and the reaction yield was increased.

56

Figure 2.1.14. Representative 1H NMR spectrum showing the successful synthesis of the

desired intermediate (Fmoc-Gln(EDA)-OPac) from the use of EDC, which deprotected the N-

Boc-protecting group and eliminated the subsequent step.

The initial reaction of Fmoc-Gln(EDA)-OPac with mTEG-OMs at room temperature in

the presence of DIEA slowly but exclusively led to the diPEGylated product as seen in Figure

2.1.15.

57

From these results, various changes were considered for increasing the yield of the

desired triPEGylated product; 1) increasing the reaction temperature, 2) adding more base, and

3) using an activated PEG with a better leaving group (i.e. triflate or iodide). Attempts are

summarized below in Scheme 2.1.11.

Figure 2.1.15. NMR analysis of crude product from PEGylation via SN2 with mTEG-OMs.

58

The reaction of Fmoc-Gln(EDA)-OPac with mTEG-OTf in the presence of DIEA at room

temperature favorably produced the triPEGylated product. Alternatively, a reaction using mTEG-

OMs as the activated PEG yielded only diPEGylated product and when potassium iodide and

heat were added to promote the reaction progress, the reaction products were neither desired

products nor starting material. When the diPEG product was obtained from either route, it is

important to note that small scale reaction attempts at converting Fmoc-Gln(EDA-mTEG2)-OPac

into Fmoc-Gln(EDA-mTEG3)-OPac by subjecting this molecule to a further PEGylation using a

triflate leaving group did show success via MALDI mass spectral analysis, however the

conversion of Fmoc-Gln(EDA-mTEG2)-OPac to Fmoc-Gln(EDA-mTEG3)-OPac was not

TEGME-OTf

Scheme 2.1.11. Summary of experimental observations during the PEGylation step of

triPEG-Gln synthesis. 1 represents Fmoc-Gln(EDA-mTEG3)-OPac. 2 represents Fmoc-

Gln(EDA-mTEG2)-OPac.

59

reattempted on a large scale with an objective of obtaining a percent yield due to the success of

the mTEG-Oms route which was concurrently investigated.

Upon deprotection of Fmoc-Gln(EDA-mTEG3)-OPac, there was no starting material

observed in MALDI mass spectroscopic analysis at m/z 955. It is unclear what the [M-18]+ could

be that is observed at m/z 818 (Figure 2.1.16). However, this sequence of reactions was

attempted on both the asparagine (n = 0) and glutamine derivatives (n = 1), showing the same

observation. Upon purification, the product and the [M-18]+ peak are observed to be two

different products that are separable via silica column chromatography.

DCM

FmocHN

O

OH

NHO

NmTEG

mTEG

mTEG

n = 0 or 1

m/z = 836.45

Zn, acac, pyFmocHN

O

O

NHO

NmTEG

mTEG

mTEG

Onn

ACN

70 oC, 5 hr43% (2 steps, n=0)<5% (2 steps, n=1)

TfO

O

O

O

(mTEG-OTf)

n = 0 or 1

m/z = 955.14

FmocHN

O

O

NHO

NH2

O

n

Figure 2.1.16. MALDI analysis of product formation after PEGylation and subsequent

deprotection.

60

The mass difference of the products being 18 is very close to an OH loss, which a

conversion from COOH to C(O)H could be imagined. However, 1H NMR analysis does not

reveal an aldehyde proton. Further analysis of the 1H NMR shows that the Fmoc and PEG are

attached to the molecule so the major side product is structurally similar to the desired product.

Literature analysis suggests that under conditions that introduce trifluoromethane sulfonic

anhydride (Tf2O) or zinc-catalysis, a chemoselective reduction of an amide to an imine or amine

is plausible (Figure 2.1.17). Additionally, it has been observed in literature that the presence of

electron withdrawing groups near the amide increases the speed of reactivity.

Figure 2.1.17. Proposed effect of any unreacted Tf2O on Fmoc-Gln(EDA)-OPac. a Synlett.

2010, 1829-1832. J. Am. Chem. Soc. 2010, 132, 12817-12819. b J. Am. Chem. Soc. 2010, 132,

1770-1771.

61

We propose that under our PEGylation conditions, the secondary amide is being reduced

to the secondary aldimine and not further reduced to the secondary amine. Since there is only a

slight excess of Tf2O in these reaction conditions yet the side product predominates over the

desired product, perhaps triflic acid (CF3SO3H) also promotes the amide reduction. Under acidic

conditions, the amide is preferentially protonated at the carbonyl oxygen (Figure 2.1.18).45, 46, 47,

48 It is believed that the reductive conditions, Mg/AcOH or Zn/pyridine/acetylacetone, are not an

efficient system for the reduction of an imine to an amine. The proposed OTf-intermediate is

very similar to the POCl3-activated amide intermediate from a Vilsmeier reaction. Imines are

typically stable under anhydrous conditions, but unstable to hydrolysis under aqueous conditions;

especially under acid catalysis. It is believed that the aliphatic alkyl substituients on both the

imine nitrogen and carbon atoms adds to the stability of the imine. Due to this undesired

reduction, the focus shifted to the other strategy illustrated in Figure 2.1.12.

Figure 2.1.18. Acid-catalyzed resonance structures of amides.

62

2nd Generation Strategy. In a one pot-like reaction, the free amine of N-Boc-ethylene

diamine can be alkylated to a quaternary ammonium in the presence of mTEG-OTf as long as

there is an excess of base present (6 equiv.). Multiple attempts suggest that even in the presence

of the smallest amount of triflic acid (TfOH), a side product of the reaction, can deprotect the N-

Boc and lead to the undesired alkylation of both amines. Additionally, the base selection and

solvent are important. K2CO3(s) worked well in a 1:4 mixture of MeCN:DCM, however

Na2CO3(s) and TEA were unsuccessful at formation of the triPEGylated intermediate. Upon

confirmation of N-Boc-protected triPEGylated product from mass spectral analysis, the reaction

mixture is partitioned between ice water and DCM, before collecting the organic layer and

concentrating it down. A TFA:DCM (3/1, v/v) deprotection of N-Boc for 1 h to liberate the free

amine, evaporation of solvent, followed by the amide bond formation between the crude

triPEGylated amine and Fmoc-Glu(OH)-OPac, employing T3P as the coupling agent, yielded the

desired N-Fmoc and –OPac protected triPEGylated product (Scheme 2.1.12) by NMR and

mass spectral analysis.

63

FmocHN

O

OPac

OHO

FmocHN

O

OPac

NHO

NTEGMETEGME

TEGME

FmocHN

O

OH

NHO

NTEGMETEGME

TEGME

n

nn

Step 3

Step 1 Step 2

Step 4


H2N

NHBoc

N

NHBoc

TEGME

TEGMETEGME N

NH2

TEGME

TEGMETEGME


66-73%

+

a b

c

d

Scheme 2.1.12. Attempted 2nd generation strategy leading to Fmoc-Gln(mTEG)3-OH.

Reagents and conditions: a) K2CO3. b) TFA:DCM (3:1, v/v). c) T3P, ACN, 2h, r.t. d) Mg,

MeOH:DMF (8:2, v/v), AcOH, 1h.

The subsequent phenacylester deprotection with Mg/AcOH of the Fmoc-Gln(mTEG)3-

OPac material did not yield product nor starting material by MALDI ([M-51]+, [M-51+Na]+, [M-

51+K]+) nor NMR analysis. The sequence of reactions has not been duplicated for optimization.

64

PART II

SCALABLE SYNTHESIS OF FMOC-PROTECTED LINEAR AND BRANCHED

PEGYLATED LYSINE BUILDING BLOCKS FOR SITE-SPECIFIC PEPTIDE

INCORPORATION

INTRODUCTION

R' NH

ON

n

OR

R' NH

OO

n

H2N-O-RH2N

Oxime Formation

Aldehyde or ketone

NaCNBH3

H2NR

OReductive Amination

R

N

R

HN

NH2

Amide BondR O

O

N

O

O

HN

R

O

Figure 2.2.1. Current peptide PEGylation strategy using amine conjugation chemistries. R =

PEG chain. Black circles = amino acids chemically linked through amide bonds into a

peptide.

The PEGylation of lysine is one of the most extensively used strategies for peptide

incorporation due to the accessibility of lysines on the solvent-exposed surface. The current

PEGylation strategy for researchers typicaly falls into one of few categories (Figure 2.2.1): 1)

reductive amination of a free amine with a PEG-aldehyde. This route allows for improved

selectivity over the usage of PEG-NHS esters, but is typically performed on the N-terminus due

to the pKa difference between the N and N amine, 6.8-8 and 10.8, respectively; 2) amide bond

formation using a PEG-NHS ester. This pathway, typically used on lysine’s side-chain amine

within peptide sequences, loses the typical charged nature of lysine residues in solution; 3)

65

oxime formation specifically on the N-terminus. To date, there are no examples of PEGylated

lysine monomers available for direct usage in SPPS.

It is known that the strategy of PEG conjugation to a peptide has a strong impact on the

conformational stability.49 Amide bond coupling can lose the charged nature of the amine when

under biological conditions meaning the lysine side chain is less likely to participate in any

favorable salt-bridges or pi-cation interactions with other residues (Figure 2.2.2). Thus, the

design and synthesis of PEG-lysine conjugates that do not convert the side-chain amine into an

amide would be highly relevant and useful tools for the study of peptide PEGylation benefits.

Figure 2.2.2. Typical peptide stabilizing interactions that occur with lysine in its cationic

form.

It has already been mentioned that one reason for the selection of lysine was due to its

presence as one of the more solvent accessible amino acid residues present in peptides and

proteins because of its role in aiding solubility. Therefore, we propose various PEGylated

analogues of lysine (Figure 2.2.3) and have synthesized them to demonstrate the feasibility of

synthesis and their potential in site-specific peptide incorporation.

66

NHHN

S

O

H

H

FmocHN

O

OH

N

O

O

O

O

FmocHN

O

OH

N

O

O

O

FmocHN

O

OH

N

O

O

O

O

O

O

HO

FmocHN

O

OH

BocN

O

O

O O

O

O

FmocHN

O

OH

N

O

O

OO

O

O

O O

O

1 2 3

4 5

Figure 2.2.3. Synthetic mTEG lysine targets of interest. 1 – N-Fmoc-mTEG1 lysine. 2 –

N-Fmoc-mTEG2 lysine. 3 – N-Fmoc-mTEG3 lysine. 4 – N-Fmoc-mTEG1 lysine (N-

biotin). 5 – N-Fmoc-mTEG1 lysine (N-fluorescein).


Synthesis of Fmoc-Lys(mTEG1)-OH Derivatives & Fmoc-Lys(mTEG2)-OH

Figure 2.2.4. Mechanism of monoPEG formation via reductive amination. R = PEG. R’ =

Lysine.

67

The selected route towards monoPEGylating lysine is shown mechanistically by Figure

2.2.4 and involves performing a reductive amination between N-Fmoc-Lys(NH2)-OPac and a

mTEG-aldehyde. Being that mTEG-aldehyde has been synthesized in literature,50 this provided

us with a 1H NMR to compare our product to (Figure 2.2.5).

Figure 2.2.5. Aldehyde NMR comparison with literature50 to demonstrate that aldehyde can be

used without further purification for reductive amination.1996 spectrum was reported after

column purification.

After successful synthesis of mTEG-aldehyde, the crude material was reacted with the

free side-chain amine on N-Fmoc-Lys(NH2)-OPac prior to reducing the intermediate imine with

NaCNBH3. The initial 1H NMR of the crude product suggested successful synthesis of desired

product (Scheme 2.2.1). However, upon purification, NMR analysis suggested phenacyl ester

68

deprotection. If the carboxylic acid is deprotected at this stage in the proposed reaction sequence,

the subsequent reactivity of the secondary amine with a biotin or fluorescein carboxylic acid in

the presence of a coupling reagent would yield a mixture of inter- and intramolecular reactivity

products.

20

PEG

19

4,5,78

33,43,1

Fmoc,OPac

1H NMR of Crude Product

HOO

OO

FmocHN

O

O

NH2

OO

OO

"swern"

OO

OO

NaCNBH3

MeCN

FmocHN

O

O

HN

O

O

O

OO

23%-Quant.

(1 equiv.)

(1.2 equiv.)

Scheme 2.2.1. 1H NMR of crude product from reductive alkylation conditions on Fmoc-

Lys(NH2)-OPac suggests monoPEG product (Fmoc-Lys(mTEG)1-OPac).

It was determined that a protecting group was not necessary to obtain the desired final

product, Fmoc-Lys(mTEG)1-OH, from the reductive amination to attach PEG. Reactions were

performed where the starting material no longer contained a phenacyl ester group during the

reductive amination protocol because the carboxylic acid would not be reduced under such

conditions. Resulting spectra from isolated purification fractions suggested diPEGylation of the

lysine side chain (Scheme 2.2.2), which was surprising when thinking about the mechanism of

reactivity. This result, however, was duplicated in multiple experiments and observed in

69

fractions after multiple column purifications, producing the possibility that excess PEG was

streaking along the silica column and eluting with desired product. It was also found that Fmoc-

Lys(mTEG)2-OH is water soluble while Fmoc-Lys(mTEG)1-OH is not. This difference in

solubility is useful when separating reaction products after reductive amination. 46, 45, 43, 42, 22

FmocHN

O

OH

HN

O

O

O

FmocHN

O

OH

NH2

NaCNBH3

HO

O

O

O

O

O

O

O

(1 equiv.)

(1.2 equiv)

FmocHN

O

OH

N

O

O

OO

O

O

"swern"

[O] Dry MeCN

DM

SO

FmocHN

O

OH

HN

O

O

O

FmocHN

O

OH

NH2

NaCNBH3

HO

O

O

O

O

O

O

O

(1 equiv.)

(1.2 equiv)

FmocHN

O

OH

N

O

O

OO

O

O

"swern"

[O] Dry MeCN

39%

OCH3

Fmoc Fmoc

(CH & CH2)

&

α-CH

O

Aliphatic

-CH2-CH2-CH2--CH2-N

Scheme 2.2.2. 1H NMR of purified Fmoc-Lys(mTEG)2-OH from reductive alkylation of Fmoc-

Lys(NH2)-OH.

This type of diPEGylation, while not commonly observed in literature, has nonetheless

been reported by researchers at Johnson and Johnson with certain types of aldehydes, such as

straight chain aldehydes, and with primary amines from - and -amino esters.17,18,47,20 The

researchers found their desired monoalkylated product in the presence of 15-20% dialkylated

product and proposed a plausible mechanism as seen below in Figure 2.2.6. They believed that

the reduction of the imine was occurring slowly in solution, leaving the secondary amine to react

70

with the imine. They then proceeded to perform a reaction with an imine and a secondary amine

under reducing conditions to show that the dialkylated product is indeed formed.

Figure 2.2.6. Top – proposed mechanism for dialkylated product formation. Bottom –

reaction performed by the same researchers to confirm that an imine reacting with an amine

in the presence of reducing conditions will form dialkylated amine. Synlett 1994, 1, 81-83.

I hypothesize that the initial formation of the dialkylated product differs from the

proposed mechanism found in literature due to reproducible mass spectral evidence that shows

the formation of dialkylated product prior to the addition of borohydride. Under my experimental

conditions, it is not a matter of how fast the imine is reduced because the dialkylated product is

forming under slightly acidic conditions. However, it is plausible that the subsequent addition of

a borohydride reagent, such as NaBH4 or NaCNBH3, could potentially lead to the formation of

additional dialkylated product.

71

Figure 2.2.7. Proposed acid-catalyzed mechanisms of mTEG2-Lys formation.

The use of NaCNBH3 is favorable under biological conditions because it is stable to

aqueous and slightly acidic conditions. These reasons make the formation of an imine crosslink

plausible in the presence of NaCNBH3 with a slow reduction of imine. Biologically, similar

imine crosslinking reactions occur as imine intermediates from the oxidation of lysine side

chains by lysyl oxidase react with N-amines from lysine.

Two different crosslinking mechanisms could be occurring in solution. The imine

crosslinking mechanism (Figure 2.2.7), which is similar to the literature proposed mechanism,

begins with the protonation of the imine nitrogen to the iminium salt, making the imine carbon

position more electrophilic. After a nucleophilic attack by an N-amine and a proton transfer,

72

there is an expulsion of a primary amine upon imine formation. The nucleophilic addition of the

hydride leads to the reduced diPEGylated amine.

It is not surprising that aldol-like reactivity is observed from the reductive amination of

the aliphatic amine with a linear aldehyde. The aldol-like crosslinking mechanism proceeds

under acid-catalyzed conditions and in the absence of any reduced amine to react with the imine.

Initially the imine tautomerizes into an enamine which can proceed to do an intermolecular

nucleophilic attack on the imine carbon (C=N). The subsequent loss of an amine is driven by the

formation of a conjugated pi-system.

As results suggest, there is a mass corresponding to diPEGylated lysine prior to the

addition of borohydride reagent making it plausible to think the aldol-like crosslink is the

predominant linkage. However, if NaCNBH3 is then added and it is observed that additional

diPEG lysine is formed, then it is also hypothetically plausible to think that there may be a

mixture of the two products which may be very difficult to separate via column chromatography.

Analysis of the TLC does show two very close spots, which could be supporting evidence for a

mixture of both diPEGylated products. Since the aldol-like crosslink leads to a secondary amine

which could form an amide, whereas the imine crosslink would form a tertiary amine incapable

of forming an amide, a reactivity test could help confirm the prescence of both analogs. NMR

proof of the aldol-like cross-link would be difficult to obtain even from a pure sample due to the

overlap of all the PEG proton signals. A test reaction of purified diPEG lysine product, from

reductive alkylation, in the presence of excess HCTU and 6-Cl-HOBt resulted in a mixture of

cylized product and unreacted starting material by mass analysis. This result supports the

presence of both structural isomers of diPEG lysine being formed.

73

Upon searching the literature, a new potential route was selected in an attempt to

minimize any diPEGylated product and maximize the monoPEG. This route involves a radical

reaction and was shown to work in various cases.48 However, when attempted multiple times,

there was only minimal conversion (< 5 %) of N-Fmoc-Lys(NH2)-OAll into desired

monoPEGylated product. This was further confirmed by subsequent reactivity with activated

fluorescein and detection of minimal Nε-mTEG-Nε-fluorescein-labeled product by MALDI-MS.

Literature Precedent:

Application of Lit. Procedure to Our System:

Adv. Synth. Catal. 2012, 354, 2671-2677.

FmocHN

O

O

NH2

FmocHN

O

O

HN

O

O

O

R

TEMPO, CuI, Bipy

OH

O

O

O

R = H orR

<10%

Scheme 2.2.3. Top - literature precedent for a radical reaction to form imine from a benzylic

alcohol and benzylic amine. Bottom – proposed reaction using literature conditions to form

monoPEGylated lysine.

Literature from other researchers gives possible explaination to our poor yielding

attempts at converting tri(ethylene)glycol monomethyl ether into tri(ethylene)glycal monomethyl

ether.49 There are some potential limitations of using the proposed radical reaction route within

our proposed chemical system: (1) it has been noted by authors that in some cases, alcohols

containing a vicinal heteroatom, such as an ether, underwent slow or incomplete oxidation,49 (2)

74

if COOH is present and unprotected during the reaction, uncontrolled reactivity can, not

surprisingly, occur with this functional group. Additonally, the presence of the acidic O-H has

been theorized to potentially prevent the formation of a reactive Cu-alkoxide species.49

The other potential route to PEGylating lysine is via nucleophilic addition. However, as

expected, the reactivity was difficult to control unless triPEG was the desired product; creating

mixtures of either starting amine and mono-PEG or mono- and diPEG, depending on the reaction

conditions and the amount of activated mTEG and/or base added. The product mixture from this

reaction is much more sensitive to the amount of base added than the amount of activated PEG

(TfO-mTEG).

75

FmocHN

O

O

N

O

O

O

O

O

O

O

FmocHN

O

O

HN

O

O

O

O

FmocHN

O

O

N

O

O

OO

O

O

OO

O

O+ +FmocHN

O

O

NH2

O

TfO

O

O

O

DCM

DIEA

485.5

05

STARTING MATERIAL

0

500

1000

1500

Inte

ns.

[a.u

.]

485.5

47

631.3

03

777.1

83

1 EQUIV DIEA

0

1000

2000

Inte

ns.

[a.u

.]

777.3

44

631.4

62

923.3

14

2 EQUIV DIEA

0

500

1000

1500

Inte

ns.

[a.u

.]

777.0

50

922.9

51

631.2

07

2.5 EQUIV DIEA

0.0

0.5

1.0

4x10

Inte

ns.

[a.u

.]

922.9

91

835.0

45

791.0

78

3 EQUIV DIEA

0.0

0.5

1.0

4x10

Inte

ns.

[a.u

.]

400 500 600 700 800 900 1000m/z

Figure 2.2.8. Synthetic route to mixture of mono-, di-, and triPEG lysine via nucleophilic

addition. MALDI analysis of increasing equivalents of activated PEG to Fmoc-Lys(NH2)-OPac.

While mono- or diPEG products cannot be produced exclusively in a controlled fashion

via this route, the mixture obtained can be controlled (Figure 2.2.8). The separation of products

(Fmoc-Lys(mTEG)1-OPac and Fmoc-Lys(mTEG)2-OPac) via silica chromatography is tedious

but allows for the isolation of mono- or diPEG products in good yield (~50%) (Scheme 2.2.1).

However, if Fmoc-Lys(mTEG)2-OPac is the desired product, then performing the alkylation

reaction very carefully to get a mixture of the desired product with Fmoc-Lys(mTEG)3-OPac

76

would be optimal because it is easier to separate compared to a mixture of mono- and diPEG.

The separation of di- and triPEGylated lysine is optimal when the -COOH is protected as the

phenacyl ester (Figure 2.2.9); upon deprotection, the di- and triPEGylated lysine products

overlap using RP-HPLC.

Figure 2.2.9. Analytical HPLC of crude product mixture containing Fmoc-Lys(mTEG)2-

OPac and Fmoc-Lys(mTEG)3-OPac.

The Zn-catalyzed removal of OPac from diPEGylated lysine results in the isolation of the

desired Fmoc-Lys(mTEG)2-OH (Scheme 2.2.1).

77

FmocHN

O

O

HN

O

O

O

O

FmocHN

O

O

N

O

O

O

O

O

O

O

FmocHN

O

O

NH2

O

FmocHN

O

O

NH2

O

TfO

O

O

O

TfO

O

O

O

DCM

DCM

DIEA(1 eq)

DIEA(2 eq)

53%

50%

FmocHN

O

OH

N

O

O

OO

O

O

Mg

AcOH< 10%

*(isolatedfromHPLC)

*

Scheme 2.2.4. Synthesis of Fmoc-Lys(mTEG)1-OPac and Fmoc-Lys(mTEG)2-OPac via

nucleophilic addition.

78

N-Fmoc-N-Biotin-mTEG1-Lys-OH and N-Fmoc-N-Fluorescein-mTEG1-Lys-OH Synthesis. HO

OO

O

FmocHN

O

OH

NH2

OO

OO

“Swern"

OO

OO

NaCNBH3

MeCN

FmocHN

O

OH

HN

O

O

O

FmocHN

O

OH

N

O

O

O

R

R = Boc, Biotin, or Fluorescein

23%-Quant.

Scheme 2.2.5. Proposed route to N-Fmoc-N-(Biotin/Boc/Fluoresein)-mTEG1-Lys-OH

analogues.

Orthogonally protected lysines are often used to functionalize the side chain with a

fluorescent moiety or biotin which are highly hydrophobic. Therefore, a lysine pre-

functionalized with one of those moieties and a PEG for solubility would be doubly

advantageous. The most straight-forward way of difunctionalizing the N-amine of lysine with

PEG and either boc, biotin, or fluorescein would be through the reductive alkylation of the free

amine (Scheme 2.2.5) with the appropriate mPEG-aldehyde and a subsequent amide bond

formation. While mono PEGylation of N-Fmoc-lysine can be accomplished with a protecting

group on the -COOH through nucleophilic addition, the product yield often suffers as a result

of multiple alkylation. While the reductive alkylation route has also shown to produce a mixture

of mono- and diPEGylated species, various conditions were attempted to tune the product

mixture to the desired monoPEGylated product (Table 2.2.1).

79

The conditions tested for their ability to preferentially form mono- versus diPEGylated

lysine were: 1) order of reagent addition; 2) reducing agent; 3) solvent selected; 4) length of time

for reaction; and 5) the presence of the -phenacyl ester. When the phenacyl ester was present as

the protecting group for the -COOH, it consistently deprotected under the reaction conditions.

However, surprisingly, the presence of the phenacyl ester led to the formation of more

monoPEGyated lysine product. It’s noteworthy to mention that when MeOH was used as the

reaction solvent, there was often formation of a significant amount of methyl ester which

lowered the amount of desired product recovered. While it was seen that the amount of

PEGylation could be controlled under reaction conditions, there was no observable benefit over

the nucleophilic addition of PEG to Fmoc-Lys(NH2)-OPac.

Table 2.2.1. Optimization of reductive amination conditions to favor formation of mono-

versus diPEGylated lysine product.

O

O

O

O

FmocHN

O

OR

(i) mix aldehyde and amine, stir 3 hrs, add reducing agent. Stir o.n.(ii) mix aldehyde, amine, and reducing agent. Stir o.n.(iii) mix aldehyde and amine, stir 3 hrs, add reducing agent and stir for 10 mins. (iv) mix aldehyde, amine, and reducing agent. Stir 3 hrs.

a

NH2

FmocHN

O

OH

HN

O

O

O

FmocHN

O

OH

N

O

O

OO

O

O

+

R Conditions Major ProductSolvent

H

CH2C(O)Ph

MeOH

MeCN

Reducing Agent

NaCNBH3

NaBH4(iii) B

H

H

(ii)

(i) NaCNBH3

NaCNBH3

MeCN

B

B

CH2C(O)Ph NaBH4(iii)

(ii) MeCN

MeOH A

A

CH2C(O)Ph (iv) NaCNBH3 MeCN A

A B

80

OH

O

O

O

HO

FmocHN

O

O

NH2

FmocHN

O

O

N

O

O

O

TEMPO, CuI, Bipy, TEGME.

FmocHN

O

O

N

O

O

O

O

O

O

HO

FmocHN

O

O

HN

O

O

O

T3P

DIEAACN

NaCNBH3

FmocHN

O

O

HN

O

O

OACN <10%

(after purification)

Scheme 2.2.6. Top - Synthetic route to Fmoc-Lys(mTEG)1-OAll through a TEMPO-

activated radical reaction. Bottom – Synthetic route to N-Fmoc-N-Fluorescein-

Lys(mTEG)1-OAll.

The initial attempts at synthesizing Fmoc-Lys(mTEG)1-OAll through a TEMPO-

activated radical reaction (Scheme 2.2.6) generated poor product yield after purification (as

mentioned earlier) even though there were a few spots observed in the TLC analysis and the

separation was not difficult via silica chromatography.

A small-scale (45 mg) test coupling reaction was attempted with the purified Fmoc-

Lys(mTEG)1-OAll product from the radical TEMPO-activated PEGylation procedure. MALDI

analysis revealed that the desired product was formed in the reaction mixture and the 1H NMR

analysis also suggested the desired product (with impurities) upon comparison with the predicted

spectrum. No starting material (Fmoc-Lys(mTEG)1-OAll) was observed in the MALDI analysis

of the product, indicating that it had all been consumed during the reaction.

81

Further attempts to synthesize N-Fmoc-N-Fluorescein-Lys(mTEG)1-OAll through this

route gave similar results, however MALDI analysis indicated that not only was there some

desired product present, but there was also N-Fmoc-N-Fluorescein-Lys-OAll. So the fact that

the PEG chain was absent in some of the product, confirms that the radical reaction was not a

successful strategy to employ for this molecular system as discussed earlier.

Scheme 2.2.7. Synthetic route to NHS-activated biotin and fluorescein.

An alternative functionalization strategy was then sought that activated the biotin and

fluorescein carboxylic acids into N-hydroxy succinimide (NHS)-esters (Scheme 2.2.7). The

initial attempt at synthesizing this NHS-activated biotin with EDC, instead of DCC, at room

temperature (22-25 oC) for 18 h, produced very poor results with a mixture of desired product

and unreacted starting material present by MALDI analysis. After isolation of desired fractions

from purification, NMR analysis did not support product formation because the spectra did not

match spectra reported in literature.

Literature precedent for NHS-activated fluorescein and biotin conjugates were followed

to synthesize the desired activated esters in good to great yields as a red solid and a white solid,

respectively.

82

mTEG1-Lys(fromreductivealkylation)

NHHN

S

O

H

H

FmocHN

O

OH

N

O

O

O

O

FmocHN

O

OH

HNO

OO

NHHN

S

O

HH

O

OR

R = NHS

DMF

DIEA

<5%

FmocHN

O

OH

N

O

O

O

O

O

O

HO

FmocHN

O

OH

HNO

OO

O

O

O

HO

OR

R = NHS

DIEA

DMF

<5%

Scheme 2.2.8. Attempted route to N-Fmoc-N-Biotin-mTEG1-Lys-OH and N-Fmoc-N-

Fluorescein-mTEG1-Lys-OH via reductive alkylation product.

Amide bond formation was attempted between the N-amine of Fmoc-Lys(mTEG)1-OH

from reductive alkylation and the NHS-esters of biotin and fluorescein (Scheme 2.2.8). Upon

purification of the reaction, the starting materials were obtained unreacted with only trace

amounts of desired products present by MALDI analysis. One hypothesis for the limited

reactivity is that the reaction needed more time to react because NHS esters react significantly

slower with secondary amines than they do for primary amines. Another hypothesis for the

limited reactivity could be that the NHS-esters could have limited solubility in the DMF. This is

plausible especially in the case of biotin-NHS because upon isolation from silica

chromatography, it was only soluble in d6-DMSO as opposed to d4-MeOD, CDCl3, D2O, nor d6-

83

acetone. To test this hypothesis, DMSO was used as a reaction solvent and the reaction was

carried out in the presence of Cs2CO3.

Figure 2.2.10. Alternative strategies for the synthesis of bifunctionalized N-lysine

conjugates.

Alternative hypotheses for the limited reaction of NHS-esters with Fmoc-Lys(mTEG)1-

OH are that: 1) some minor side products from the reductive alkylation process carried through

the purification and are interfering with reactivity; 2) Having a vicinal oxygen atom neighboring

the N-amine is decreasing the nucleophilicity of the nitrogen lone pair; or 3) The -carboxylic

acid is causing some intermolecular interference with the N-amines nucleophilicity through

hydrogen bonding.

84

To test these hypotheses, OPac-protected monoPEGylated lysine was synthesized from a

nucleophilic alkylation reaction and reacted with biotin-NHS and fluorescein-NHS in DMSO

with Cs2CO3.

NHHN

S

O

H

H

FmocHN

O

OH

N

O

O

O

O

FmocHN

O

O

HNO

OO

NHHN

S

O

HH

O

OR

R = NHS

2) Zn, acac, py, 60oC

1) Base (DIEA or Cs2CO3), Solvent (DMSO or DMF)

FmocHN

O

OH

N

O

O

O

O

O

O

HO

FmocHN

O

O

HNO

OO

O

O

O

HO

OR

R = NHS

O

O

2) Zn, acac, py, 60oC

1) Base (DIEA or Cs2CO3), Solvent (DMSO or DMF)

mTEG1-Lys(from SN2 alkylation)

Scheme 2.2.9. Attempted route to N-Fmoc-N-Biotin-mTEG1-Lys-OH (top) and N-Fmoc-N-

Fluorescein-mTEG1-Lys-OH (bottom) via nucleophilic alkylation product.

The reaction of Fmoc-Lys(mTEG)-OPac with Fluorescein-NHS in the presence of base

(either DIEA or Cs2CO3) (Scheme 2.2.9) repeatedly yielded a product with a m/z ratio of 959,

however the desired product has a molecular weight of 947 g/mol. It is unclear as to what could

yield the [M+12]+ peak that is observable in MALDI-MS. Analysis of the purified material by 1H

NMR suggested that the fluorescein was attached to the fmoc and phenacyl ester protected

85

lysine. Upon phenacyl ester deprotection with Mg/AcOH, the desired N-Fmoc-N-Fluorescein-

mTEG1-Lys-OH would have a m/z ratio of 828.92, however the m/z observed in the product is

845.14 [M+16]+.

N-Fmoc-N-Boc-mTEG1-Lys-OH Synthesis.

A test reaction on the N-Boc-protected material showed that the boc was not stable to the

mild acidic reduction conditions, yielding complete OPac and N-Boc deprotection after 4 h at

room temperature (Scheme 2.2.10).

FmocHN

O

O

BocN

O

O

O

O

FmocHN

O

OH

BocN

O

O

O

FmocHN

O

O

HN

O

O

O

O

Boc2O

DIEA< 5%

Mg

AcOH

Scheme 2.2.10. Attempted synthetic route to N-Boc-N-Fmoc-Lys(mTEG)1-OH.

Therefore, the phenacyl ester needs to be deprotected prior to N-Boc-protection. The

attempted N-Boc-protection of monoPEG lysine material from reductive alkylation conditions

resulted in low isolated yield (Scheme 2.2.11 – Top). To rule out the usage of hydrolyzed boc

anhydride (Boc2O), NMR analysis supported that the reagent was not decomposed to tert-

butanol. Alternatively, the Fmoc-Lys(mTEG)1-OPac obtained from nucleophilic addition was

deprotected and subsequently reacted with boc anhydride to produce the desired N-Boc-N-

Fmoc-Lys(mTEG)1-OH (Scheme 2.2.11 – Bottom). This sequence of reactions resulted in

86

phenacyl ester deprotected material after stirring with Mg/AcOH, but after a workup and

subsequent reaction with boc anhydride in THF/tBuOH there was no bocylated product was

observed by mass spectral analysis.

NH

OO

O

FmocHN

O

OH

Boc2O

DIEATHF

5-10%

NBoc

OO

O

FmocHN

O

OH

NH

OO

O

FmocHN

O

OPac

1) Mg, AcOH

2) Boc2ODIEATHF N

Boc

OO

O

FmocHN

O

OH

Scheme 2.2.11. Synthetic routes to N-Boc-N-Fmoc-Lys(mTEG)1-OH.

Potential Future Synthetic Route. Very recently, it has been observed that proline and

piperidine derivatives can be selectively oxidized to the 5- and 6-hydroxy compounds,

respectively, using iron catalysis (Figure 2.2.12A and C). This versatile hydroxy intermediate

can subsequently be reacted with an amine and reducing agent to open up the ring and form a

methoxyethyl amine linkage while retaining the correct stereochemistry (Figure 2.2.12B). A

possible future route towards Fmoc-Lys(mTEG)1-OH, could be envisioned (Figure 2.2.12D) by

beginning with the appropriate piperidine analog and oxidizing to the hydroxy compound prior

to reaction with a mPEG-amine of desired length and reducing agent such as NaCNBH3. This

route would first require the synthesis and isolation of the iron catalyst. According to literature,

the Fmoc protecting group is stable to peroxyacid, and from experimentation, it is known that it

is stable to metal (Mg and Zn)/AcOH conditions. However, the Fmoc stability under these

specific conditions would first need to be ascertained.

87

Scheme 2.2.12. A – Literature examples of selective oxidation of proline to 5-hydroxyproline

(top) and piperidine to 6-hydroxypiperidine (bottom) using iron catalyst.50 B – Literature

example of proline oxidation to open-chain monomethoxy ornithine derivative. C – Iron

catalyst structure. D – Possible proposed future synthetic route towards Fmoc-Lys(mTEG)1-

OH.

88

Fmoc-Lys(mTEG3)-OH Synthesis

The lysine side-chain is typically protonated under physiological conditions and

participates in salt bridges (Figure 2.2.11). A salt bridge is actually a combination of two

noncovalent interactions: hydrogen bonding and electrostatic interactions. This is most

commonly observed to contribute stability (~3-4 kcal/mol up to 8 kcal/mol if buried in a protein

interior) to the folded conformation of proteins. Although noncovalent interactions are known to

be relatively weak interactions, small stabilizing interactions can add up to make an important

contribution to the overall stability of a conformer.

Figure 2.2.11. Salt bridge interactions between glutamic acid and lysine under

physiological conditions.

Originally, the plan was to synthesize diPEG-lysine via the nucleophilic addition route.

However, what we not surprisingly observed through experimentation is that we were

consistently not only making diPEG, but also tri-PEG in solution due to the great leaving group

ability of the triflate. As mentioned earlier, the two products are separable from each other by

89

column chromatography because the N-Fmoc-Lys(mTEG)3-OPac (Rf ~ 0.18 in 5% MeOH in

DCM), N-Fmoc-Lys(mTEG)2-OPac (Rf ~ 0.58 in 5% MeOH in DCM). Additionally, if any N-

Fmoc-Lys(mTEG)1-OPac is present, it can also be isolated from the reaction; having an Rf ~

0.39 in 5% MeOH in DCM. When Fmoc-Lys(NH2)-OPac is used in the presence of TfO-mTEG

and excess DIEA, the major product is Fmoc-Lys(mTEG)3-OPac (Figure 2.2.12) with no

residual mono- or diPEGylated product.

H2O

O

H H

Solvent

OCH3

Fmoc & OPac Fmoc (CH & CH2)

&

α-CH

[M]+

FmocHN

O

O

NmTEG

mTEG

mTEG

O

FmocHN

O

O

NH2

O

TfO

O

O

O(mTEG-OTf)

DIEADCM73%

O

Figure 2.2.12. 1H NMR and MALDI-MS spectrum showing Fmoc-Lys(mTEG)3-OPac.

The mild Mg-catalyzed acidic deprotection of phenacyl ester to liberate the carboxylic

acid that is present in the final product went to completion as monitored by mass spectrum

analysis (Scheme 2.2.13). However, there was a tedious purification that was necessary to obtain

pure Fmoc-Lys(mTEG)3-OH. The desired product was very polar and purification via normal

90

silica gel is not the optimal method to be used. Alternatively, it would likely be better to do

purification via RP-HPLC.

FmocHN

O

OH

NHBoc

FmocHN

O

O

NHBoc

FmocHN

O

O

NH2

FmocHN

O

O

NmTEGmTEG

mTEG

FmocHN

O

OH

NmTEGmTEG

mTEG

a b c d

OOOStep 1 Step 2 Step 3 Step 4

Scheme 2.2.13. Synthetic route to Fmoc-Lys(mTEG)3-OH using OPac ester and MALDI-MS

spectrum showing the presence of Fmoc-Lys(mTEG)3-OH. Reagents and conditions: a)

Bromoacetophenone, Na2CO3, N(Bu)4Br, DCM, r.t., 24-72 h, 95-98%. b) 3:1 (v/v) TFA:H2O,

r.t., 2 h, quant. c) Triethylene glycol monomethylether (mTEG), (Tf)2O, 0 oC, 1 h, 73%. d) Mg,

AcOH, MeOH, DMF, r.t., 63%-quant.

An alternative protecting group was selected for attempting to synthesize the

triPEGylated lysine derivative due to the original difficulty in trying to remove all of the AcOH

from the desired product. Any residual AcOH is a problem for SPPS conditions because AcOH

will couple to a peptide sequence instead of the desired PEGylated lysine. The phenacyl ester

deprotection conditions of Fmoc-Lys(mTEG)3-OPac using Zn catalysis worked at higher

91

temperatures (~70-75 oC), but the isolated yield was slightly lower than deprotection with

Mg/AcOH. Allyl ester deprotection can be done under Pd-mediated conditions in the absence of

AcOH and would potentially produce an easier mixture for purification.

FmocHN

O

O

NH2

FmocHN

O

OH

N

O

O

OO

O

O

OO

O

FmocHN

O

O

N

O

O

OO

O

O

OO

O

DCM

TfO

O

O

O

DIEA

Pd(PPh3)4

N-methylmorpholine

dry THF, r.t., 24 hr

49% (over 2 steps)

[M]+ =847.5

Scheme 2.2.14. Synthetic route to Fmoc-Lys(mTEG)3-OH using O-Allyl ester and a

representative MALDI-MS showing the presence of desired Fmoc-Lys(mTEG)3-OAll.

The reaction of Fmoc-Lys(NH2)-OAll with mTEG-OTf (3 equiv.) in the presence of

excess DIEA allows for the formation of the desired triPEG-product (Scheme 2.2.14). The Pd-

catalyzed deprotection of the allyl ester leads to the desired final product in good yield (49%)

over two steps, but there was no significant advantage of this method over deprotecting the OPac

under Zn-catalyzed acidic reduction.

92

PART III

Site-Specific Peptide Incorporation of Linear and Branched Amino Acid Monomers

Upon sysnthesis of desired Fmoc-protected amino acid building blocks, they were

incorporated into traditional Fmoc solid-phase peptide synthesis (SPPS) (Scheme 2.3.1) to

demonstrate that they could be selectively placed anywhere within a peptide sequence.

The resin used was dependent on the type of C-terminus needed in the desired peptide

sequence of interest. Wang or 2-chlorotrityl is used for incorporation of a carboxylic acid at the

C-terminus. Rink amide was utilized to incorporate an amide at the C-terminus. On wang resin,

coupling of the first residue takes the longest due to the need for a symmetric anhydride to

couple the first residue to the resin. The cleavage conditions for trityl resins are mild enough that

the side chain protecting groups can be retained. These various factors were taken into

consideration when selecting the proper resin for each peptide sequcnce.

The coupling reagents used were HCTU and 6-Cl-HOBt. The cleavage conditions used

were 95% TFA, 2.5% H2O, and 2.5% Triisopropylsilane. Some other cleavage conditions that

are used by researchers for Wang and rink amide resins are (1) 95% TFA, 2.5% H2O, 2.5%

Triisopropylsilane and (2) 95% TFA, 2.5% H2O, 2.5% Thiol. (3) For 2-chlorotrityl resin;

hexafluoroisopropanol (HFIP).

Scheme 2.3.1. Solid Phase Peptide Synthesis steps. HCTU= 2-(6-Chloro-1H-benzotriazole-1-

93

yl)-1,1,3,3-tetramethylaminium hexafluorophosphate

Peptide Synthesis. Regulation of appetite is critically important, both in its suppression

for the treatment and prevention of obesity and diabetes, as well as for inducing hunger in

patients suffering from severe morbidities, such as heart failure, chronic obstructive pulmonary

disease, and chemotherapy-induced cachexia. Almost all of the relevant signaling hormones are

peptides with molecular weights that hinder mimicry or blocking of their actions with small

molecule drugs.

Table 2.3.1. Sequences of the peptides of interest. Amino acids in bold represent

potential modification sites of interest.

Sample Sequence

Human GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2

Exenatide HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

Liraglutide HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-NH2

Lypressin CYFQNCPKG-NH2 (Disulfide bridge: C1-C6)

Sup35 NDFQKQQKQA (127-136)

Glucagon-like-peptide 1 (GLP-1) is one of the most important peptide hormones

controlling appetite (Table 2.3.1). GLP-1 is the primary hormone responsible for appetite

suppression and also beneficially increases glucose-dependent insulin production and restores

insulin sensitivity.51, 52 Since GLP-1 restores important natural functionality that is lost in

diabetes patients, longer-lasting versions of GLP-1 are desirable. Unsurprisingly, five important

diabetes drugs (exenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide) are GLP-1

analogs. GLP-1 is degraded by the ubiquitous catabolic enzyme dipeptidyl peptidase IV (DPP4)

that cleaves off an essential N-terminal dipeptide moiety.53, 54 This cleavage is the major route of

94

elimination of GLP-1 in the body leading to its short half-life (1.5-2.1 mins),55 and several

effective treatments for diabetes are small molecule DPP4 inhibitors (saxagliptin, sitagliptin,

vildagliptin, and linagliptin). Although natural GLP-1 has highly beneficial anti-diabetic effects,

native GLP-1 is so short lived so as to be therapeutically useless (hypothetically requiring

continuous infusion to exert the beneficial effect). Proteolytic degradation is also a problem for

the GLP-1 analog drugs, such as exenatide and liraglutide, thus demonstrating the need for

effective PEGylation strategies for GLP-1 (analogs).

Given the crystal structure (PDB ID: 3IOL) of GLP-1 bound to the extracellular domain

of GLP-1 receptor (Figure 2.3.1), it is theoretically possible to predict which modification sites

won’t disrupt the receptor binding of the PEG-modified peptides. Additionally, potential site

selection benefits take into account the studies in literature that have evaluated the beneficial

effects of PEGylating GLP-1 at positions Lys26, Lys34 and Gln23 with chain lengths varying from

1kDa-20kDa.33, 56, 57

95

Figure 2.3.1. GLP-1 bound to extracellular domain of GLP-1 receptor. (GLP1R) PDB ID:

3IOL. H - His - Ala - Glu - Gly - Thr - Phe - Thr - Ser - Asp - Val - Ser - Ser - Tyr - Leu -

Glu - Gly - Gln - Ala - Ala - Lys - Glu - Phe - Ile - Ala - Trp - Leu - Val - Lys - Gly - Arg -

NH2

96

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Inte

ns. [

a.u.

]

300 350 400 450 500 550 600 650 700 750

m /z

[2 α-HCCA+H]

[M+H]

[M+Na]

[M+K]

513.610

379.549

HisAla Glu Gly Thr

MW=512.52

535.600

1010

.490

273.

942

335.

816

213.

062

864.

241

379.

724

102.

000

922.

308

572.

699

0

1000

2000

3000

4000

Inte

ns. [

a.u.

]

200 400 600 800 1000 1200 1400 1600 1800 2000

m /z

1010.490

Leu Val Lys Gly ArgmTEG3

MW=1010.29

H7AEGTFTSD15VSSYLEGQAA25KEFIAWLVKGR36

1000

0

2000

3000

4000Intens.[a.u.]

0.5

0

1.0

1.5

2.0

Intens.[a.u.]

2.5

x104

300 350 400 450 500 550 600 650 700

m/z200 400 600 800 1000 1200 1400 1600 1800 2000

750 m/z

27

3.9

42

335.8

16

864.2

41

379.7

24

213.0

62

102.0

00

H7AEGTFTSD15VSSYLEGQAA25KEFIAWLVKGR36

Figure 2.3.2. MALDI-mass spectra of unpurified PEGylated GLP-1 pentamer and unmodified

GLP-1 pentamer showing the feasibility of manual SPPS.

97

The preliminary results presented above (Figure 2.3.2) show that Fmoc-Lys(mTEG)3-

OH can be incorporated into GLP-1 (7-36 amide) even though the full-length sequence has not

been synthesized.

There was initially an issue in the case of coupling the (mTEG)1-Gln to a growing

peptide chain, which could have been the water molecules that are intimately associated with the

PEG chain. The 1H NMR of the (mTEG)1-Gln showed at least 3-4 equivalents of water per

amino acid, which could disrupt the amide bond formation in coupling. To remedy this issue, I

attempted to use an EDC/HOBt mixture when coupling the PEG amino acid as well as the

subsequent amino acid. EDC was used because of its dehydrating nature as opposed to

originally using HCTU, but this method was unsuccessful.

The next attempt was to increase the reaction time for the coupling of the PEGylated

glutamine. The 1H NMR shows the presence of the polar solvent DMF with the product due to

the deprotection conditions used. While polar solvents solvate polar reagents and make SN2

reactions proceed slower, it is better to have a slow reaction than no reaction at all. After two 5-

minute couplings of (mTEG)1-Gln, a very small peak at the desired m/z ratio was observed

amongst other products.

Subsequent attempts at incorporating (mTEG)1-Gln into the synthesis of PEGylated GLP-

1 analogues yielded site-specifically modified analogues at positions Q9 and Q23 as shown

below (Figure 2.3.3). These following attempts utilized reacting 4 equivalents of PEGylated

amino acid with the resin in the presence of HCTU for 10 minutes, twice. The next amino acid

was also reacted with the resin for two 10 minute rounds with HCTU.

98

Figure 2.3.3. Analytical HPLC and MALDI-MS spectra for the confirmation of identity and

purity for the site-specifically PEGylated GLP-1 analogues indicated.

99

After GLP-1 analogues were synthesized, lypressin analogues were attempted with the

PEGylated amino acid monomers previously discussed in earlier sections of this dissertation.

Lypressin is an analogue of Vasopressin, which is a first line treatment option for a complication

of liver disease known as gastroesophageal bleeding as well as for septic shock. To our

knowledge, short chain PEGylation has not been reported for either sequence (GLP-1 nor

Lypressin). Our PEGylation attempts were successful in producing the desired lypressin products

(Figures 2.3.4 and 2.3.5). The production of (mTEG)1-Gln4-Lypressin, shows another product

that is 191 mass units lower than the desired product at 1011.552. This observed lower mass is

too low to reflect a missing PEG-glutamine and the closest attempts at matching the weight

resulted in a potentially missing proline and cysteine; however, a missing proline and cysteine

lead to a lower mass of 199 instead of 191.

100

Figure 2.3.4. Site-specific peptide PEGylation examples with mass spectrum analysis

highlighting the presence of desired peptide.

101

Figure 2.3.5. Site-specific incorporation of Fmoc-Lys(mTEG)3-OH into varying positions within

a lypressin peptide sequence.

Conclusion

In summary, we have developed an efficient and straightforward synthesis of peptides

containing short PEG chains via PEG amino acid building block incorporation into standard

Fmoc SPPS. The general availability of these building blocks gives access to any desired

sequence containing mPEGylation of varying lengths to target improved pharmacokinetic

properties.

102

References

1. Fosgerau, K., and Hoffmann, T. (2015) Peptide therapeutics: Current status and future

directions. Drug Discov. Today. 20, 122-128.

2. Tsomaia, N. (2015) Peptide therapeutics: Targeting the undruggable space. Eur. J. Med.

Chem. 94, 459-470.

3. Jones, E. M., and Polt, R. (2015) CNS active O-linked glycopeptides. Front. Chem. 3, 1-9.

4. Chatterjee, J., Rechenmacher, F., and Kessler, H. (2013) N-methylation of peptides and

proteins: An important element for modulating biological functions. Angew. Chem. Int. Ed. 52,

254-269.

5. Wójcik, P. (2016) Peptide-based inhibitors of protein-protein interactions. Bioorg. Med.

Chem. Lett. 26, 707-713.

6. Gunnoo, S. B., and Madder, A. (2016) Bioconjugation - using selective chemistry to enhance

the properties of proteins and peptides as therapeutics and carriers. Org. Biomol. Chem. 14,

8002-2013.

7. Saifer, M. G. P., Williams, L. D., Sobczyk, M. A., Michaels, S. J., and Sherman, M. R. (2014)

Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies induced by

methoxyPEG-proteins. Mol. Immunol. 57, 236-246.

8. Lubich, C., Allacher, P., De la Rosa, M., Bauer, A., Prenninger, T., Horling, F. M., Siekmann,

J., Oldenburg, J., Scheiflinger, F., and Reipert, B. M. (2016) The mystery of antibodies against

polyethylene glycol (PEG) - what do we know? Pharm. Res. 33, 2239-2249.

9. Price, J. L., Powers, E. T., and Kelly, J. W. (2011) N-PEGylation of a reverse turn is

stabilizing in multiple sequence contexts, unlike N-GlcNAcylation. ACS Chem. Biol. 6, 1188-

1192.

10. DeBenedictis, E. P., Hamed, E., and Keten, S. (2016) Mechanical reinforcement of proteins

with polymer conjugation. ACS Nano. 10, 2259-2267.

11. Veronese, F. M., and Pasut, G. (2005) PEGylation, successful approach to drug delivery.

Drug Discov. Today. 10, 1451-1458.

12. Isidro-Llobet, A., Alvarez, M., and Albericio, F. (2009) Amino acid-protecting groups.

Chem. Rev. 109, 2455-2504.

13. Lawrence, P. B., and Price, J. L. (2016) How PEGylation influences protein conformational

stability. Curr. Opin. Chem. Biol. 34, 88-94.

103

14. Nischan, N., and Hackenberger, C. P. R. (2014) Site-specific PEGylation of proteins: Recent

developments. J. Org. Chem. 79, 10727-10733.

15. Dozier, J. K., and Distefano, M. D. (2015) Site-specific PEGylation of therapeutic proteins.

Int. J. Mol. Sci. 16, 25831-25864.

16. Zhang, L., Klein, B. D., Metcalf, C. S., Smith, M. D., McDougle, D. R., Lee, H. -., White, H.

S., and Bulaj, G. (2013) Incorporation of monodisperse oligoethyleneglycol amino acids into

anticonvulsant analogues of galanin and neuropeptide Y provides peripherally acting analgesics.

Mol. Pharmaceutics. 10, 574-585.

17. Li, L., Crow, D., Turatti, F., Bading, J. R., Anderson, A. -., Poku, E., Yazaki, P. J.,

Carmichael, J., Leong, D., Wheatcroft, M. P., Raubitschek, A. A., Hudson, P. J., Colcher, D., and

Shively, J. E. (2011) Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated

diabody reveals the effect of increasing PEG size on kidney clearance and tumor uptake with

improved 64-copper PEG imaging. Bioconjug. Chem. 22, 709-716.

18. Gault, V. A., Kerr, B. D., Irwin, N., and Flatt, P. R. (2008) C-terminal mini-PEGylation of

glucose-dependent insulinotropic polypeptide exhibits metabolic stability and improved glucose

homeostasis in dietary-induced diabetes. Biochem. Pharmacol. 75, 2325-2333.

19. Zang, Q., Tada, S., Uzawa, T., Kiga, D., Yamamura, M., and Ito, Y. (2015) Two site genetic

incorporation of varying length polyethylene glycol into the backbone of one peptide. Chem.

Comm. 51, 14385-14388.

20. Yu, F., McConnell, M. S., and Nguyen, H. M. (2015) Scalable synthesis of fmoc-protected

GalNAc-threonine amino acid and TN antigen via nickel catalysis. Org. Lett. 17, 2018-2021.

21. Comegna, D., de Paola, I., Saviano, M., Del Gatto, A., and Vaccaro, L. (2015)

Straightforward entry to S-glycosylated fmoc-amino acids and their application to solid phase

synthesis of glycopeptides and glycopeptidomimetics. Org. Lett. 17, 640-643.

22. Albers, M. F., and Hedberg, C. (2013) Amino acid building blocks for fmoc solid-phase

synthesis of peptides phosphocholinated at serine, threonine, and tyrosine. J. Org. Chem. 78,

2715-2719.

23. Eerland, M. F., and Hedberg, C. (2012) Design and synthesis of an fmoc-SPPS-compatible

amino acid building block mimicking the transition state of phosphohistidine phosphatase. J.

Org. Chem. 77, 2047-2052.

24. Albers, M. F., van Vliet, B., and Hedberg, C. (2011) Amino acid building blocks for efficient

fmoc solid-phase synthesis of peptides adenylylated at serine or threonine. Org. Lett. 13, 6014-

6017.

104

25. van der Heden van Noort, G.J., van der Horst, M. G., Overkleeft, H. S., van der Marel, G. A.,

and Filippov, D. V. (2010) Synthesis of mono-ADP-ribosylated oligopeptides using ribosylated

amino acid building blocks. J. Am. Chem. Soc. 132, 5236-5240.

26. Rush, J., and Bertozzi, C. R. (2006) An alpha-formylglycine building block for fmoc-based

solid-phase peptide synthesis. Org. Lett. 8, 131-134.

27. Bejugam, M., and Flitsch, S. L. (2004) An efficient synthetic route to glycoamino acid

building blocks for glycopeptide synthesis. Org. Lett. 6, 4001-4004.

28. Rothman, D. M., Vazquez, M. E., Vogel, E. M., and Imperiali, B. (2003) Caged phospho-

amino acid building blocks for solid-phase peptide synthesis. J. Org. Chem. 68, 6795-6798.

29. Byk, G., and Scherman, D. (1996) Synthesis of novel (N-farnesyl)amino acids and their

incorporation into peptides. Int. J. Peptide Protein Res. 47, 333-339.

30. Pandey, B. K., Smith, M. S., Torgerson, C., Lawrence, P. B., Matthews, S. S., Watkins, E.,

Groves, M. L., Prigozhin, M. B., and Price, J. L. (2013) Impact of site-specific PEGylation on

the conformational stability and folding rate of the pin WW domain depends strongly on PEG

oligomer length. Bioconjug. Chem. 24, 796-802.

31. Trevino, S. R., Scholtz, J. M., and Pace, C. N. (2007) Amino acid contribution to protein

solubility: Asp, glu, and ser contribute more favorably than the other hydrophilic amino acids in

RNase sa. J. Mol. Bio. 366, 449-460.

32. Sato, H. (2002) Enzymatic procedure for site-specific PEGylation of proteins. Adv. Drug

Deliver. Rev. 54, 487-504.

33. Selis, F., Schrepfer, R., Sanna, R., Scaramuzza, S., Tonon, G., Dedoni, S., Onali, P., Orsini,

G., and Genovese, S. (2012) Enzymatic mono-pegylation of glucagon-like peptide 1 towards

long lasting treatment of type 2 diabetes. Results Pharma. Sci. 2, 58-65.

34. Fontana, A., Spolaore, B., Mero, A., and Veronese, F. M. (2008) Site-specific modification

and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv. Drug Deliver.

Rev. 60, 13-28.

35. Mero, A., Spolaore, B., Veronese, F. M., and Fontana, A. (2009) Transglutaminase-mediated

PEGylation of proteins: Direct identification of the sites of protein modification by mass

spectrometry using a novel monodisperse PEG. Bioconjug. Chem. 20, 384-389.

36. Pintar, A., Carugo, O., and Pongor, S. (2003) Atom depth in protein structure and function.

Trends Biochem. Sci. 28, 593-597.

37. Lawrence, P. B., Gavrilov, Y., Matthews, S. S., Langlois, M. I., Shental-Bechor, D.,

Greenblatt, H. M., Pandev, B. K., Smith, M. S., Paxman, R., Torgerson, C. D., Merrell, J. P.,

Ritz, C. C., Prigozhin, M. B., Levy, Y., and Price, J. L. (2014) Criteria for selecting PEGylation

105

sites on proteins for higher thermodynamic and proteolytic stability. J. Am. Chem. Soc. 136,

17547-17560.

38. Kokinaki, S., Leondiadis, L., and Ferderigos, N. (2005) A novel and efficient method for

cleavage of phenacylesters by magnesium reduction with acetic acid. Org. Lett. 7, 1723-1724.

39. McFarland, J. M., and Francis, M. B. (2005) Reductive alkylation of proteins using iridium

catalyzed transfer hydrogenation. J. Am. Chem. Soc. 127, 13490-13491.

40. Schmidt, M., Amstutz, R., Crass, G., and Seebach, D. (1980) Preparation of some chiral

aminodiols from tartaric acid. chiral lithium aluminates for asymmetric hydrogenations. Chem.

Ber. 113, 1691-1707.

41. Fringuelli, F., Pizzo, F., and Vaccaro, L. (2000) Cobalt(II) chloride-catalyzed chemoselective

sodium borohydride reductions of azides in water. Synthesis. 5, 646-650.

42. Hagiwara, D., Neya, M., and Hashimoto, M. (1990) A novel and efficient method for

cleavage of phenacyl esters by zinc reduction with acetylacetone and pyridine. Tetrahedron Lett.

31, 6539-6542.

43. Selve, C., Ravey, J. C., Stebe, M. J., El Moudjahid, C., Moumni, E. M., and Delpuech, J. I.

(1991) Monodisperse perfluoro-polyethoxylated amphiphilic compounds with two-chain polar

head - preparation and properties. Tetrahedron. 47, 411-428.

44. Lawrence, P. B., Billings, W. M., Miller, M. B., Pandey, B. K., Stephens, A. R., Langlois, M.

I., and Price, J. L. (2016) Conjugation strategy strongly impacts the conformational stability of a

PEG-protein conjugate. ACS Chem. Biol. 11, 1805-1809.

45. Da Ros, T., Prato, M., Novello, F., Maggini, M., and Banfi, E. (1996) Easy access to water

soluble fullerene derivatives via 1,3-dipolar cycloadditions of azomethine ylides to C60. J. Org.

Chem. 61, 9070-9072.

46. Abdel-Magid, A. F., Harris, B. D., and Maryanoff, C. A. (1994) A reductive

Amination/Lactamization procedure using borohydride reagents. Synlett. 1, 81-83.

47. Abdel-Magid, A. F., Carson, K. G., Harris, B. D., Maryanoff, C. A., and Shah, R. D. (1996)

Reductive amination of aldehyde and ketones with sodium triacetoxyborohydride. studies on

direct and indirect reductive amination procedures. J. Org. Chem. 61, 3849-3862.

48. Tian, H., Yu, X., Li, Q., Wang, J., and Xu, Q. (2012) General, green, and scalable synthesis

of imines from alcohols and amines by a mild and efficient copper-catalyzed aerobic oxidative

reaction in open air at room temperature. Adv. Synth. Catal. 354, 2671-2677.

49. Hoover, J. M., Steves, J. E., and Stahl, S. S. (2012) Copper(I)/TEMPO-catalyzed aerobic

oxidation of primary alcohols to aldehydes with ambient air. Nat. Protoc. 7, 1161-1166.

106

50. Osberger, T. J., Rogness, D. C., Kohrt, J. T., Stepan, A. F., and White, M. C. (2016)

Oxidative diversification of amino acids and peptides by small-molecule iron catalysis. Nature.

537, 214-219.

51. Drucker, D. J. (2001) Development of glucagon-like peptide-1-based pharmaceuticals as

therapeutic agents for the treatment of diabetes. Curr. Pharm. Des. 7, 1399-1412.

52. Drucker, D. J. (2006) The biology of incretin hormones. Cell Metab. 3, 153-165.

53. Mentlein, R., Gallwitz, B., and Schmidt, W. E. (1993) Dipeptidyl-peptidase IV hydrolyzes

gastric inhibitory polypeptide, glucagon-like peptide-1(7-36) amide, peptide histidine methionine

and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829-835.

54. Gallwitz, B., Witt, M., Paetzold, G., Morys-Wortmann, C., Zimmerman, B., Eckart, K.,

Folsch, U. R., and Schmidt, W. E. (1994) Structure/activity characterization of glucagon-like

peptide-1. Eur. J. Biochem. 225, 1151-1156.

55. Vilsboll, T., Agerso, H., Krarup, T., and Holst, J. J. (2003) Similar elimination rates of

glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects. J. Clin.

Endocrinol. Metab. 88, 220-224.

56. Chae, S. Y., Chun, Y. G., Lee, S., Jin, C. -., Lee, E. S., Lee, K. C., and Youn, Y. S. (2009)

Pharmacokinetic and pharmacodynamic evaluation of site-specific PEGylated glucagon-like

peptide-1 analogs as flexible postprandial-glucose controllers. J. Pharm. Sci. 4, 1556-1567.

57. Lee, S. -., Lee, S., Youn, Y. S., Na, D. H., Chae, S. Y., Byun, Y., and Lee, K. C. (2005)

Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1.

Bioconjug. Chem. 16, 377-382.

58. Klok, H. -., Vandermeulen, G. W. M., Nuhn, H., Rosler, A., Hamley, I. W., Castelletto, V.,

Xu, H., and Sheiko, S. S. (2005) Peptide mediated formation of hierarchically organized solution

and solid state polymer nanostructures. Faraday Discuss. 128, 29-41.

107

CHAPTER THREE

PROGRESS TOWARDS THE IMPROVED SYNTHESIS OF FREIDINGER-LIKE LACTAMS

FROM CHIRAL AMINES AND FMOC AMINO ACIDS

Introduction

N

O

HN

R

OH

O

N

O

R1HN

R2

Useful FrameworkPrototypical Freidinger Lactam

A B

Figure 3.1. A) Structure of a prototypical Freidinger lactam. B) Generic Freidinger-like lactam

framework that is particularly useful given the number of literature references.

Freidinger lactams are an important molecular framework in pharmaceutical

development. On the top left is a general structure of a Freidinger lactam. On the right is a

generalized substructure that has greater than 2000 patent references. The original synthetic

approach involved a one-pot protecting group removal and reductive amination of the N-Cbz

protected amine.1 Subsequently, the N-amine was cyclized with the -carboxylic acid to yield

the desired product. Typically, this route requires juggling of protecting groups and there is

typically no stereochemistry incorporated off of the N-amine position (R1 in Figure 1B). The

number of patent references on this generalized structure demonstrates the pharmaceutical

usefulness of this lactam backbone. One sought-after usage of Freidinger lactams, beyond its use

as a small molecule scaffold, is its potential use in developing conformationally constrained

108

peptidomimetic compounds.2 In the early 1980’s, Freidinger incorporated his lactam scaffold

into luteinizing hormone-releasing hormone to produce a conformationally constrained mimic,

which displayed biological activity better than the parent compound.3 The team found that the

conformational constraint mimicked the beta turn of the endogenous luteinizing hormone-

releasing hormone when bound to its receptor. This result was an early example that sparked

interest in development of conformationally constrained peptides. More recently, other

researchers have designed peptidomimetic compounds containing Freidinger lactams that

demonstrate potential within the pharmaceutical industry.4

N

OHN

HO

N

O

H

N

Palonosetron (AloxiTM)

Prevention & Treatment of Chemotherapy-induced nausea and

vomiting

O

HN

HN

NH2

O

NO

Br

O

O

NH

O

O

O

NH

OHN

O

O NH2

Symplocamide ANatural Product (Discovered 2008)

Potent Cytotoxin - shown potential in usages for lung cancer, neuroblastoma,

infectious diseases (HIV, HCV)

Figure 3.2. Select pharmaceutically relevant examples of molecules with Freidinger-like -

lactam moiety and chirality off the -nitrogen.

109

Symplocamide A, (Figure 3.2) discovered in 2008, has displayed high selectivity for

inhibition of serine proteases and potent activity against cancer cells and neuroblastoma cells.5

Palonosetron (Figure 3.2), used in the prevention and treatment of chemotherapy-induced

nausea and vomiting, is also around a -lactam moiety.

OH

O

BocHN

CbzHN

OH

O

BocHN

HN

N

O

BocHN

MeOH, H2O, AcOH

H2, Pd/CCOOH

COOH

OHC-COOH

dipeptide

55oC, DMFor

heat, CH3CNTEA

a

bg

d

a

dbg

Figure 3.3. Original synthetic strategy to Freidinger lactam.1

The current synthesis of Freidinger lactams (Figure 3.3) currently has low yield and

requires intermediate purification. The current strategy also limits the functionality of the lactam

ring because the functionalization of the N-amine is performed from reductive amination with

an aldehyde that cannot be chiral at the carbon that becomes bonded to the N-amine (the carbon

that was once the carbonyl carbon).

110

OUR PROPOSED APPROACH

OH

O

FmocHN

H2N

OH

O

FmocHN

Br

OH

O

FmocHN

NH

N

O

FmocHN

N

O

HN

R3

1 2 3

4

5

H2SO4

NaBrHBr

NaNO2

Key StepR2

R1

R2

R1

R2

R1

*

**

Figure 3.4. Proposed approach to Freidinger lactam structures with incorporated chirality off N-

amine.

Our propsed approach (Figure 3.4) begins with commercially available Fmoc-ornithine

and converts the side-chain amine into a bromide utilizing diazotization conditions and having

the bromide nucleophile in solution simultaneously to displace the unstable aliphatic diazonium.

From the bromide intermediate (2), (S)-5-Bromo-2-[9H-fluoren-9-

yl)methoxycarbonylamino]valeric acid, any chiral amine could be used to displace the bromide

and a subsequent cyclization leads to the desired lactam ring scaffold. From there, the Fmoc

protecting group could be removed if reacting that nitrogen is sought after, but further

derivatization beyond the N-Fmoc-protected N-lactam is beyond the scope of the work

presented here. However, the strategies presented herein could be utilized to develop larger

molecules such as symplocamide A (Figure 3.2). Furthermore, our proposed approach allows for

incorporation of chirality on the lactam N carbon and increases the versatility of the framework

in pharmaceutical applications.

111

COMPARABLE APPROACHES (LIT.)

Figure 3.5. Comparable synthetic routes to gamma-, delta-, and epsilon-lactams.6, 7, 8, 9 A)

Thioether route from Int. J. Pept. Protein Res. 1990, 35, 481-494. B) Photoredox route from J.

Org. Chem. 1997, 62, 654-63. C) Reductive amination route from J. Org. Chem. 2005, 70, 5946-

5953. D) Reduction to alcohol and subsequent conversion to bromide route from Org. Lett. 2013,

15, 448-451.

There are other routes depicted in literature (Figure 3.5) that form lactam rings of

varying size (5 to 10-membered rings). The reductive amination route to attaching an amine to

the side-chain prior to cyclization is still in use today.10 However, there are other notable

attempts that have not gained wide acceptance, such as using ring-closing olefin metathesis,

112

which leaves an unsaturated double bond within the newly formed lactam ring (a potentially

useful functionality for further ring functionalization).11 There has also been interest by others in

improving the synthetic route to Freidinger lactams using a combination of microwave

irradiation and solid-phase synthesis (Wang resin).12 Nevertheless, this solid-phase synthesis

approach would be very atom uneconomic for larger scale synthesis.

DIAZOTIZATION OF ALIPHATIC AMINES (LIT.)

Figure 3.6. Literature study of products formed from diazotization of aliphatic amines

Reference table: J. Am. Chem. Soc. 1965, 87, 5790-5791.13

Literature precedent shows that under diazotization conditions, aliphatic amines produce

an unstable diazonium salt that either quickly degrades into an alkene or is converted into an

alkyl halide or alcohol through nucleophilic displacement (Figure 3.6). These results

demonstrate that in some cases a haloacid, such as HCl, is enough of a halogen source to allow

for substitution. Furthermore, solvent selection plays an important role in the product outcome.

113

Additional literature precedent provides us with examples that diazotization is in fact

possible in the presence of an adjacent carboxylic acid (Figure 3.7).14, 15 However, in our

designed route, our amine of interest is not on the -carbon, but instead on the -carbon position.

And surprisingly, even under the highly acidic reaction conditions, there is even retention of a

boc-protecting groups on amines and t-butyl protecting groups on carboxylic acids.14, 15

Figure 3.7. Literature precedent for the successful diazotization of aliphatic amines.


General

Diazotization procedure. Desired aminocarboxylic acid (1 equiv.) was dissolved in HBr

(16-48%) with sodium bromide (7.5 equiv) and cooled to 0 oC. A solution of sodium nitrite (3

equiv.) and sodium bromide (2.5 equiv.) in water was added dropwise to the reaction at 0 oC.

The reaction mixture stirred for 1-18 h. The resulting oil was extracted with DCM (3x). The

DCM extracts were combined, washed with water (1x), saturated aqueous sodium chloride

solution (3x), dried over sodium sulfate, and concentrated.

114

Column chromatography was performed on silica gel 60, particle size 0.040-0.063 mm.

1H and 13C NMR spectra were recorded on either a Bruker or JEOL spectrometer (500 MHz). All

collected MALDI mass spectra for this chapter can be found in Appendix C.

Materials

Trifluoroacetic acid (TFA), zinc acetate (Zn(OAc)2), acetonitrile (ACN) were purchased

from Sigma (St. Louis, MO). Concentrated hydrochloric acid (HCl) obtained from Fisher

scientific. Granular sodium nitrite (NaNO2) obtained from Macron chemicals (Center Valley,

PA). Hydrobromic acid 48% (HBr) obtained from EMD Millipore (Billerica, MA). Methanol

(MeOH) and sodium sulfate (Na2SO4) obtained from VWR International (Radnor, PA). Sodium

bromide (NaBr), sulfuric acid (H2SO4) obtained from J. T. Baker (Center Valley, PA). 4-

methylmorpholine, sodium borohydride 98% (NaBH4) obtained from Alfa Aesar (Haverhill,

MA). Sodium cyanoborohydride (NaCNBH3) obtained from AK Scientific.

Tetrakis(triphenylphosphine) palladium (0) 99% ((Ph3P)4Pd) obtained from Strem Chemicals

(Newburyport, MA). (S)-(-)-1-phenylethylamine obtained from TCI America (Portland, OR).

N,N’-carbonyldiimidazole (CDI), triflic anhydride (Tf2O) obtained from Oakwood Chemicals

(Estill, SC). Thionyl chloride (SOCl2) obtained from TCI America (Portland, OR). Fmoc-

Glu(OtBu)-OH obtained from Aapptec (Louisville, KY). N,N’-diisopropylethylamine (DIEA),

Nα-Fmoc-Nδ-Boc-L-ornithine obtained from Chem-Impex International (Wood Dale, IL). All

Fmoc-protected amino acids were used without further purification. All reagents and organic

solvents used were at least ACS grade.

115

MALDI-MS

Molecular weight was obtained from MALDI mass spectrometry using Bruker

Daltonics/Omniflex MALDI mass spectrometer. Samples were prepared by mixing 1.5 μL of

aliquot with 1.5 μL of the matrix solution, a saturated solution of α-cyanohydroxycinnamic acid

in acetone with 0.3% TFA. One microliter of the sample mixture was spotted unto the sample

plate and dried by vacuum prior to mass spectrometry. Data for 2 ns pulses of the 337 nm

nitrogen laser were averaged for each spectrum in a linear mode, and positive ion TOF detection

was performed using an accelerating voltage of 25 kV. All collected MALDI mass spectra for

this chapter can be found in Appendix C.


If the proposed route is to be successful, one of the first vital questions that needed to be

understood was whether the Fmoc-protected amine was stable to diazotization conditions. This

was tested by placing Fmoc-Ala-OH in diazotization conditions (NaNO2, HBr, NaBr, 0 oC) for

two hours and monitoring the starting material by 1H NMR after 20 minutes, 1 hour, 2 hours, and

24 hours (Figure 3.8). The conclusion of the experiment was that the Fmoc is stable under these

conditions for at least 2 hours because there was no presence of the distinctive CH2 singlet from

the side product.

116

CH2

&

Figure 3.8. 1H NMR time course analysis of fmoc stability under diazotization conditions.

Reagents and conditions: a) NaNO2, NaBr, 16% HBr, 0 oC.

The next potential synthetic problem that needed investigation was two-fold: 1) would

the bromide displace the unstable diazonium that is formed to yield the alkyl bromide; and 2)

how stable is the diazoalkylcarboxylic acid and/or the subsequently formed bromoalkyl

carboxylic acid. These issues were tested by taking linear amino acids with varying carbon chain

lengths (4-6) and exposing them to diazotization conditions to see whether or not intramolecular

cyclization to the lactone occurs or if the bromine replaces the diazo group.

117

entry aminoacida bromoacid lactone ratio(%)b

1 32:68

2 60:40

3 93.5:6.5

4 98:0

aAminoacidscommerciallyavailable.bRatioofbromoacid:lactonedeterminedbyNMR.

Table1.

NH2HO

O

n

n = 1, 2, 3, 4

BrHO

O

n

vsO

O

nn = 1, 2, 3, 4

reagents and conditions: a) NaNO2, NaBr, 16% HBr, H2O, 0 oC.

a

Figure 3.9. Summary of initial diazotization results. Reagents and conditions: a) NaNO2, NaBr,

16% HBr, 0 oC.

Upon exposure of 6-aminoheptanoic acid to diazotization conditions (Figure 3.9, entry

4), 1H NMR spectrum of the product mixture showed 6-bromohexanoic acid as the major

product. There was a minor –O-CH2 triplet peak that gave indication of a small amount of ε-

caprolactone formation. Diazotization of 5-aminovaleric acid was accomplished in the presence

of high [Br-]; 10 equivalents compared to the 5 equivalents previously used (Figure 3.9, entry

3). The lactone was only 6.5% of the product mixture so there was 93.5% bromovaleric acid

formation. The hypothesis moving forward was that increasing the concentration of reagents

118

would aid formation of the bromoacid. The γ-aminobutyric acid was exposed to diazotization

conditions (Figure 3.9, entry 2) and almost equal amounts of 4-bromo-n-butyric acid and

lactone were observed in the product mixture, 60:40 percent product mixture respectively. In the

case of β-alanine (Figure 3.9, entry 1), β-propiolactone was observed as the major product in

the product mixture with 3-bromopropionic acid as the minor product, which is likely due to the

close proximity of the short-lived diazo group to the carboxylic acid position. This close

proximity may make it more likely to form the ring even though the 4-membered ring would be

higher in energy to form and therefore a less stable product.

HO

O

Br N

ONH2

a

Scheme 3.1. Reaction of 5-bromovaleric acid with (S)-(-)-1-phenylethylamine. Reagents and

conditions: a) ACN, Na2SO4, 18-72 h, r.t.

A reaction of the bromovaleric acid with (S)-(-)-phenylethylamine (Nspe) yields the

desired -lactam structure as shown in Scheme 3.1. This reactivity supports the presence of the

bromoacid as the intermediate that was formed from the diazotization.

Since the diazotization was successfully performed on simple aliphatic aminoacids

(Figure 3.9) and the conversion of the bromoalkyl carboxylates to the desired lactams was

demonstrated, the next step toward the desired Freidinger-like lactams was to test -

aminocarboxylic acids that have a protected -amine substituent. Fmoc-protected amino acids,

such as Nα-Fmoc-Nδ-Boc-L-ornithine, are commercially available and provide an excellent

starting point for the formation of -lactam rings via our proposed diazotization route.

119

Since the diazotization occurs under acidic conditions, it was hypothesized that the Nδ-

Boc could be removed in one-pot with the desired subsequent diazotization. Surprisingly, the

attempted removal of N-Boc from Nα-Fmoc-Nδ-Boc-L-ornithine in situ while performing

diazotization yielded no removal of Boc and no desired product and the Boc-protected starting

material was recovered from 16% HBr (aq).

entry R1 R2 Product % Yield

5a* not bromide -

5b not bromide -

5c not bromide -

5d not bromide -

Figure 3.10. Diazotization attempts yielded no desired product. Reagents and conditions: a) 5 eq

NaNO2, 20 eq NaBr, 48% HBr, 0 oC. *This reaction was tested under a variety of conditions

(e.g. NaNO2, H2SO4, NaBr, THF:H2O, -80 oC; NaNO2, TsOH, NaBr, THF:H2O, -40 oC.) and

yielded similar results under each.

Fmoc-Orn(NH2)-OH, 5a, obtained from Boc deprotection of Nα-Fmoc-Nδ-Boc-L-

ornithine under 3:1 (v/v) TFA:H2O conditions was difficult to redissolve in MeOH, acetone, and

D2O. However, 1H NMR of the sample was obtained in D2O. A reaction of Fmoc-Orn(NH2)-OH

120

under diazotization conditions (Figure 3.10, entry 5a) yielded an unclear and crowded 1H NMR

of the crude reaction mixture. MALDI-mass spectral analysis of the reaction mixture was

complex and indicated that multiple species were present, yet closer analysis suggested that the

lactone product formed (m/z 337) in competition with the desired bromide (MW = 418.28, m/z

438 [M+H]+ and 440– [M+Na]+ for bromine isotopes 79 and 81). The diazotization reaction

initially yielded promise, but replication and optimization efforts were unsuccessful. Further

attempts at forming the brominated product gave MALDI-mass spectrum peaks around the m/z

ratio expected for the product if two water molecules were closely associated with the desired

product (m/z 453.7 and 455.7 – for bromine isotopes 79 and 81). Unfortunately, however, the

bromide nor the lactone were the major product of the initial reaction and were present in

negligible amounts after purification of the reaction mixture. Further NMR analysis did not help

in elucidating that the desired product was the major component.

HO

O

NHFmoc

Br OFmocHN

ODIEA

DCM

Molecular Weight: 337.38Molecular Weight: 418.29

Scheme 3.2. Small-scale test reaction performed to help elucidate whether the bromoacid

starting material was formed through diazotization.

A subsequent attempt at the diazotization of the amine showed the presence of a

brominated product (amongst other peaks) in the MALDI spectrum, with the correct isotopic

distribution of peak m/z ratios to what is expected for the desired product containing a bromine

atom. A small-scale test reaction of the material from that reaction in the presence of a base, such

as DIEA, should cyclize to form the lactone (Scheme 3.2) and the change in mass along with the

121

difference in isotopic distribution would be detectable by MALDI-mass spectral analysis. The

result of this test reaction suggested that detectable amounts of the desired product were present,

however they were overshadowed by a [M+60]+ product peak which support the notion that the

desired product was not the major component of the diazotization reaction.

With the knowledge that there are detectable amounts of lactone occurring in the

reaction, the hypothesis moving forward was that protecting the carboxylic acid as a methyl ester

will increase the stability of the bromoacid product and decrease any potential cyclization

(Figure 3.10, entry 5b). Methyl ester formation and Nδ-Boc deprotection of Nα-Fmoc-Nδ-Boc-

L-ornithine proceeded in excellent yield using thionyl chloride in MeOH. The liberated amine

was subjected to the optimized diazotization conditions and the product mixture was analyzed

for formation of desired bromide.

122

Figure 3.11. NMR analysis of starting material (bottom) and crude product mixture (top) from

attempted diazotization of Fmoc-Orn(NH2)-OMe. CH2 peak shift observed in crude product.

Multiple attempts suggest a shift in peak position. The chemical structure on the top spectrum is

that of the desired product.

NMR analysis of the starting material and product (Figure 3.11) showed a downfield

shift in the CH2 peak adjacent to the side-chain amine suggesting an electron withdrawing group

was formed. While it was unclear from NMR and a MALDI-mass spectrum analysis if the

product was the result of NH2 being converted into Br, a subsequent reaction with a nucleophilic

amine to displace the bromide would support the presence of product from a successful

diazotization; thus, (S)-(-)-phenylethylamine was used (Scheme 3.3). However, upon analysis of

123

this test reaction, the desired lactam product was not conclusively seen. If the desired -lactam

were present, it looked like an impurity (by NMR) in a different product. These results suggest

that the product after diazotization is not likely the desired bromide and that the CH2-NH2 shift

observed by 1H NMR was likely caused by some other (as of yet unidentified) reactivity of the

aliphatic amine.

Scheme 3.3. Attempted synthetic route to the lactam product. reagents and conditions: a) SOCl2,

MeOH, Quant. b) 5 eq NaNO2, 20 eq NaBr, 48% HBr (aq), H2O, 0 oC. c) (S)-(-)-

phenylethylamine, DIEA, 75 oC, 4 hr or no DIEA.

Instead of the desired product being formed from the reaction of (S)-(-)-phenylethylamine

with the diazotization product (Scheme 3.3, reaction c), the major product displays distinct 1H

NMR (Figure 3.12) and IR spectra that has yet to be matched to a molecular structure. This

result indicates the presence of an unknown major product that would be the result from an

unknown sequence of chemical reactions. Elucidation of this unknown product would shed light

on the type of reactivity occurring under these chemical conditions and could potentially lead to

the development of a new synthetic reaction. Further investigation of the structure of this

unidentified product were outside the scope of this project and were not attempted beyond trying

to crystallize the product in an effort to obtain an X-ray crystal structure.

124

Figure 3.12. 1H NMR spectrum of the unidentified major product after diazotization and Nspe

addition.

Crystallization attempts: Various methods were attempted to form crystals of the

purified unknown product that was formed from the reaction: 1) slow evaporation from

dichloromethane alone at room temperature. This resulted in a powder along the walls of the

culture tube used; 2) layering hexane atop product dissolved in dichloromethane at room

temperature. This resulted in some powder substance along the walls of the culture tube and most

of the powder at the bottom of the tube; 3) from a slow evaporation of a DCM/MeOH solvent

mixture at room temperature. This resulted in the same powder formation as that from

125

dichloromethane alone; 4) slow evaporation from chloroform with a top layer of hexanes at room

temperature.

NH

O

NNH

O

OO

Folded Extended

uncharged analogueindole-3-acetic acid choline ester

Figure 3.13. Intramolecular cation-pi interaction. Adapted from reference: K. Aoki, et al., J.

Chem. Soc. Chem. Commun. 1995, 2221-2222.16

One potential issue (amongst other possibilities) could be the presence of a pi-cation

interaction (Figure 3.13) in solution or potentially nucleophilic carbonyl adjacent to N in fmoc

that interferes with the diazotization process. One potential way to test if the diazotization is

disrupted by an interaction with the pi-system of the Fmoc-protecting group is to use a different

protecting group on the alpha amine. So, the hypothesis for subsequent reactions (Figure 3.10,

entries 5c-5d) was that if the presence of Fmoc is hindering the diazotization reaction, then

replacing Fmoc (or NH-C(O)-) should restore the desired reactivity to what was observed when

there was no alpha substituent.

If the pi-cation interaction was the hinderance for the reaction, then replacing Fmoc

would remove the aromatic rings and allow the reaction to proceed as it did with 5-aminovaleric

acid. However, when the substitution on the N-amine was changed to mimic a piperidine ring

(Figure 3.10, entry 5c), the resulting product mixture under the conditions tested, did not

display the desired promine product. To remove the lone pair of electrons from consideration as

126

a possible source of interference, the N-amine was trimethylated to the ammonium (Figure

3.10, entry 5d) yet the result remained unchanged; no desired bromine product was evident in

the product mixture. It remains unclear as to why the N-aliphatic amine diazotization and

subsequent displacement with bromide worked in the absence but not the presence of an N-

amine.

Scheme 3.4. Diazotization of ornithine leads to a mixture of products.17

In the late 1980’s, it was observed that one of the potential products from subjecting

ornithine to diazotization conditions is the amine being substituted for an alcohol (Scheme

3.4).17 This would arise from the displacement of the diazonium by a water molecule, which is in

high concentration as the reaction solvent. This hydroxyl substitution could theoretically be one

of the products observed in the reactions tested within this chapter, however efforts were

attempted to increase the bromide ion concentration to compete with water for nucleophilic

displacement of the diazonium. It should also be noted that if a hydroxyl was formed from the

diazotization of the N-amine, then this would also cause a 1H NMR shift in the CH2 protons

(Figure 3.11) due to the increased electronegativity of the oxygen atom compared to the

nitrogen. However, if the alcohol was formed, the subsequent reaction with (S)-(-)-

phenylethylamine would not explain the 1H NMR that was observed for the major product

(Figure 3.12).

127

ALTERNATIVE SYNTHETIC ROUTE (AMIDE TO AMINE REDUCTION)

OHFmocHN

O

O O

OAllFmocHN

O

HN

R1 R2

O

1. Allyl bromide2. TFA:DCM3. CDI, amine

1) Tf2O

2) [R]

OHFmocHN

O

HN

R1 R2

NFmocHN

O

R2

R1

*CDI

**

viaActivation and Reduction of

Secondary Amide to Amine Key Step

78

10 113) Deprotection

Figure 3.14. Proposed approach to Freidinger lactam via activation and reduction of secondary

amide to secondary amine.

Based on the unsuccessful results observed from the attempted aliphatic amine

diazotization, an alternative route was theorized for further investigation to the desired lactam

framework (Figure 3.14). Starting from commercially available N-Fmoc-Glu(OtBu)-OH, an

allyl protection of the -carboxylic acid and subsequent acidic deprotection of the side-chain

tert-butyl liberates the side-chain -carboxylic acid to form N-Fmoc-Glu(OH)-OAll. N-Fmoc-

Glu(OH)-OAll in the presence of CDI and a chiral amine forms the desired amide bond. A

reduction of the secondary amide to the secondary amine allows for the eventual cyclization to

form the -lactam with chirality off of the N-amine. The key step in this procedure is the

reduction of the secondary amide to the secondary amine because traditional chemistry is not

suitable to perfom this transformation on our substrate selectively in the presence of a carbamate

and ester/carboxylic acid.

128

Figure 3.15. Select examples of literature precedent for reduction of amide to amine.18, 19, 20 Top

– Reduction of secondary amides via Tf2O activation and subsequent reduction. Bottom –

Reduction of tertiary amides via zinc acetate-catalysis.

Traditionally, researchers have used strong reducing agents such as LiAlH4 in order to

reduce amides to amines. However, this reduction approach would not only reduce the amide and

ester functional groups, but it would also deprotect the Fmoc amine, making this synthetic route

implausible for our goals. More recently, there have been literature examples of amides being

reduced in one pot under more mild conditions (Figure 3.15) with either: 1) triflic anhydride

activation and NaBH4;18, 19, 21 or 2) transition metal-catalyzed.20, 21 Therefore, various conditions

were attempted in order to determine the feasibility of this synthetic approach to the Freidinger-

like lactams.

129

Figure 3.16. Attempted routes to reduce the secondary amide to a secondary amine using either

Tf2O/hydride or Zn-mediated transfer hydrogenation.

The reaction conditions that were selected to test for secondary amide reduction (Figure

3.16) were conditions that had been shown to work in other chemical systems within the

literature. Three of the routes attempted utilize Tf2O to first activate the secondary amide into the

presumed imidol triflate intermediate prior to reduction with a hydride source. These reactions

have been reported in other systems to proceed well at 0 oC to room temperature.18 The two

small-scale reaction routes that introduced DIEA with Tf2O for the activation of the amide led

mainly to unidentified products with higher mass than that of starting material and product. The

130

route that did not have a base present during the activation step proceeded cleanly to the desired

amine reduction product. The Tf2O/NaBH4 amide reduction reaction demonstrated

chemoselectivity for the amide in the presence of an ester and carbamate.

A fourth set of conditions was chosen that utilized Zn-catalysis in the presence of a

trialkyl silane. Literature precedent of similar reaction conditions utilized Zn(OAc)2 as the zinc

source and triethoxysilane.20 Researchers did note that at room temperature, other silanes did not

react, but some silanes (PhSiH3, Ph2SiH2, and (EtO)2MeSiH gave excellent yields for the

reduction of their model system (N,N-dimethylbenzamide) at 65 oC. I chose to utilize

triisopropylsilane (TIPS) because 1) to my knowledge others have not reported on its feasibility

under these reaction conditions and 2) it was readily accessible. Unfortunately, there was no

reactivity at all under these Zn/TIPS conditions as assessed by MALDI-mass spectral analysis

and the starting material was recovered. This result shows that TIPS has similar reactivity to

triethylsilane which was tested in literature.18

Scheme 3.5. Attempted synthetic route to desired Freidinger lactam via Fmoc-Glu(OtBu)-OH

using O-Allyl ester protection and reduction of amide to amine. Reagents and conditions: a)

allylbromide, DIEA, reflux, 1h. b) TFA:DCM 3:1 (v/v), 1.5 h, r.t. c) (s)-(-)-1-Phenylethylamine,

CDI, DIEA, DCM. d) Tf2O, DCM, 0oC, 30 mins, NaBH4, THF:DCM (2:1), r.t., 1 h. e)

131

(Ph3P)4Pd, N-methylmorpholine, THF (dry), 18h. This reaction was unsuccessful—failing to

produce the desired product. f) CDI, DCM, 2h. This reaction was unattempted.

Once it was shown that the amide could be reduced efficiently to the amine, the material

still needed to be cyclized to form the desired lactam product (Scheme 3.5). Cyclization could

potentially occur via two different pathways: 1) the allyl ester is deprotected to liberate the

carboxylic acid prior to using a coupling reagent to form an amide bond with the side-chain

amine; or 2) the amine could potentially displace the O-allyl in the presence of base with or

without heating. The side-chain secondary amine is known, from the original Freidinger lactam

paper, to form 5- or 6-membered rings via the displacement of an ester.1

NFmocHN

O

*

FmocHN

HN

O

O

*

MW = 498.62[M+H]+= 499.62

MW = 440.54[M+H]+= 441.54

DIEA, NaHCO3

DMF, 90-100 oC8h

132

Scheme 3.6. Cyclization of Fmoc-Orn(Nspe)-OAll to the desired -Freidinger-like lactam.

Reagents and conditions: DIEA, NaHCO3, DMF, 90-100 oC, 8h.

Alternative reaction conditions were tested on a small scale to go from the Fmoc-

Orn(Nspe)-OAll to the -lactam that avoided the usage of Pd-catalysis (Scheme 3.6). These

initial efforts demonstrated that in the presence of DIEA and NaHCO3, Fmoc-Orn(Nspe)-OAll

could be cyclized without first deprotecting the allyl ester to the free carboxylic acid and

subsequently utilizing a coupling reagent as initially proposed.

Conclusions

In summary, the current synthetic route to Freidinger lactams is typically low yielding

and very limited in functionalization. We sought a standard reagent that could be readily used to

create Freidinger lactams incorporating Nδ-chirality. Instead of needing to synthesize a novel

reagent (2) ((S)-5-Bromo-2-[9H-fluoren-9-yl)methoxycarbonylamino]valeric acid), we have

instead demonstrated the more easily accessible Fmoc-Glu(OH)-OAll to function in that

capacity. The application of amide to amine methodology that was developed herein could be

useful in forming Freidinger lactams with defined stereochemistry as a building block to more

complex small-molecules or even find usage in the design of conformationally constrained

peptidomimetics. The strategy that was developed here proceeds in very good yields and allows

for functionalization off the -nitrogen with the usage of any chiral or non-chiral amine.

133

References:

1. Freidinger, R. M., Perlow, D. S., and Veber, D. F. (1982) Protected lactam-bridged dipeptides

for use as conformational constraints in peptides. J. Org. Chem. 47, 104-109.

2. Perdih, A., and Kikelj, D. (2006) The application of freidinger lactams and their analogs in the

design of conformationally constrained peptidomimetics. Curr. Med. Chem. 13, 1525-1556.

3. Freidinger, R. M., Veber, D. F., Perlow, D. S., Brooks, J. R., and Saperstein, R. (1980)

Bioactive conformation of luteinizing hormone-releasing hormone: Evidence from a

conformationally constrained analog. Science. 210, 656-658.

4. Gomez, C., Bai, L., Zhang, J., Nikolovska-Coleska, Z., Chen, J., Yi, H., and Wang, S. (2009)

Design, synthesis, and evaluation of peptidomimetics containing freidinger lactams as STAT3

inhibitors. Bioorg. Med. Chem. Lett. 19, 1733-1736.

5. Linington, R. G., Edwards, D. J., Shuman, C. F., McPhail, K. L., Matainaho, T., and Gerwick,

W. H. (2008) Symplocamide A, a potent cytotoxin and chymotrypsin inhibitor from the marine

cyanobacterium symploca sp. J. Nat. Prod. 71, 22-27.

6. Lee, J. P., Dunlap, B., and Rich, D. H. (1990) Synthesis and immunosuppressive activities of

conformationally restricted cyclosporin lactam analogs. Int. J. Pept. Protein Res. 35, 481-494.

7. Wolfe, M. S., Dutta, D., and Aubé, J. (1997) Stereoselective synthesis of freidinger lactams

using oxaziridines derived from amino acids. J. Org. Chem. 62, 654-663.

8. Kumar, S., Flamant-Robin, C., Wang, Q., Chiaroni, A., and Sasaki, N. A. (2005) Synthesis of

4-substituted-3-aminopiperidin-2-ones: Application to the synthesis of a conformationally

constrained tetrapeptide -acetyl-ser-asp-lys-pro. J. Org. Chem. 70, 5946-5953.

9. Ottersbach, P. A., Schmitz, J., Schnakenburg, G., and Guetschow, M. (2013) An access to aza-

freidinger lactams and E-locked analogs. Org. Lett. 15, 448-451.

10. Durham, T. B., Toth, J. L., Klimkowski, V. J., Cao, J. X. C., Siesky, A. M., Alexander-

Chacko, J., Wu, G. Y., Dixon, J. T., McGee, J. E., Wang, Y., Guo, S. Y., Cavitt, R. N.,

Schindler, J., Thibodeaux, S. J., Calvert, N. A., Coghlan, M. J., Sindelar, D. K., Christe, M.,

Kiselyov, V. V., Michael, M. D., and Sloop, K. W. (2015) Dual exosite-binding inhibitors of

insulin-degrading enzyme challenge its role as the primary mediator of insulin clearance in vivo.

J. Biol. Chem. 290, 20044-20059.

11. Hoffman, T., Waibel, R., and Gmeiner, P. (2003) A general approach to dehydro-freidinger

lactams: Ex-chiral pool synthesis and spectroscopic evaluation as potential reverse turn inducers.

J. Org. Chem. 68, 62-69.

134

12. Lama, T., Campiglia, P., Carotenuto, A., Auriemma, L., Gomez-Monterrey, I., Novellino, E.,

and Grieco, P. (2005) A novel route to synthesize freidinger lactams by microwave irradiation. J.

Pept. Res. 66, 231-235.

13. Bayless, J. H., Mendicino, F. D., and Friedman, L. (1965) Aprotic diazotization of aliphatic

amines--intra- and intermolecular reactions of poorly solvated cations. J. Am. Chem. Soc. 87,

5790-5791.

14. Moumne, R., Lavielle, S., and Karoyan, P. (2006) Efficient synthesis of β2-amino acid by

homologation of α-amino acids involving the reformatsky reaction and mannich-type iminium

electrophile. J. Org. Chem. 71, 3332-3334.

15. Souers, A. J., Schürer, S., Kwack, H., Virgilio, A. A., and Ellman, J. A. (1999) Preparation of

enantioenriched a-bromo acids incorporating diverse functionality. Synthesis. 4, 583-585.

16. Aoki, K., Murayama, K., and Nishiyama, H. (1995) Cation-π interaction between the

trimethylammonium moiety and the aromatic ring within indole-3-acetic acid choline ester, a

model compound for molecular recognition between acetylcholine and its esterase: An x-ray

study. J. Chem. Soc. Chem. Commun. , 2221-2222.

17. Gouesnard, J. P. (1989) Reactivity of sodium nitrite. V. action on amino acids, peptides, and

proteins. Bull. Soc. Chim. Fr. 1, 88-94.

18. Xiang, S. H., Xu, J., Yuan, H. Q., and Huang, P. Q. (2010) Amide activation by Tf2O:

Reduction of amides to amines by NaBH4 under mild conditions. Synlett. , 1829-1832.

19. Pelletier, G., Bechara, W. S., and Charette, A. B. (2010) Controlled and chemoselective

reduction of secondary amides. J. Am. Chem. Soc. 132, 12817-12819.

20. Das, S., Addis, D., Zhou, S., Junge, K., and Beller, M. (2010) Zinc-catalyzed reduction of

amides: Unprecedented selectivity and functional group tolerance. J. Am. Chem. Soc. 132, 1770-

1771.

21. Huang, P. Q., and Geng, H. (2015) Simple, versatile, and chemoselective reduction of

secondary amides and lactams to amines with the tf2O-NaBH4 or cp2ZrHCl-NaBH4 system. Org.

Chem. Front. 2, 150-158.

135

CHAPTER FOUR

DESIGN AND DEVELOPMENT OF POTENTIAL ANTIMICROBIAL PEPTOIDS FOR

ENDOSCOPE STERILIZATION

Introduction

Endoscopes come in various types and are used in 700,000 operations annually.1 At a

cost of $40,000-$80,000 per endoscope,1 they need to be rapidly cleaned between patients.

However, the FDA issued a report saying that even under duodenoscope manufacturer

reprocessing instructions, infections could still occur.2

The emergence of MDR bacterial strains such as MRSA, which have led to post-op

infections due to the inability to effectively sterilize surgical instruments, such as

duodenoscopes, between procedures has become a major problem for health systems across the

United States.3, 4 Additionally, outbreaks of carbapenem-resistant enterobacteriaceae (CRE),

such as Escherichia coli and Klebsiella pneumoniae, related to duodenoscopes have resulted in

patient infections and even deaths.5

The traditional FDA-approved sterilization method for endoscopes involve automated

endoscope reprocessors (AER). The first step in sterilizing an endoscope centers on submerging

the device in a liquid basin with a high-level disinfection solution (HLD), containing chemicals

such as glutaraldehyde, hydrogen peroxide, or peracetic acid, for example. The endoscope

channels are connected to the AER and the HLD is circulated under pressure through the device

for roughly 30 minutes. The AER then rinses the scopes with water, removing the toxic HLD

solution, and washed again with ethyl or isopropyl alcohol and dried with air.6 Other methods are

also used to sterilize endoscopes including ethylene oxide (EO), ozone, and gamma/electron

beam irradiation (extremely rare). EO sterilization is an effective way to sterilize endoscopes,7

136

but has been found to be hazardous to patients, staff, and the environment. There is significant

risk associated with handling and exposure to the highly flammable gas. EO sterilization has also

been shown to be a relatively expensive process for health systems requiring significant

infrastructure investment.8 Gamma and electron irradiation has been shown to damage some

medical devices.9

AERs are used to clean duodenoscopes—complex endoscopes with many working parts

that are used to treat problems in the pancreas and bile ducts. Recently, the FDA reported

infections occurring in patients who underwent procedures with duodenoscopes that were

reportedly sterilized with AERs using HLD solutions before the procedures began.10 Bacterial

species can become resistant to the solutions used in AERs. For example, it was shown that

certain strains of Mycobacteria spp. developed a resistance to aldehyde-based HLDs11 and

enzymes like catalase, possessed by many different bacterial species enable a resistance to H2O2-

based HLDs.

H2N

HN

NH

HN

O

N

O

O

HN NH2

NH

O

NH

HN

OO

NH

O

N

NH

HN

OO

HN

NH

O

NH

NH2

NH

HN NH2

NH

HN

O

NH2

O

NH2

NH

Figure 4.1. Structure of omiganan—a cationic antimicrobial peptide.

The short half-life and poor oral bioavailability of antimicrobial peptides are major

challenges for why they tend to fail late-stage clinical trials. While there are examples of

137

antimicrobial peptides that have made it far in clinical trials, such as the cationic peptide

omiganan (Figure 4.1), they have not effectively shown the ability to meet clinical endpoints.

Peptoids (N-substituted glycine oligomers)—a class of peptidomimetics—are similar to

α-peptides in structure (Figure 4.2), but the side chains are attached to an Nα-amide nitrogen

backbone as opposed to the Cα-carbon, making them highly resistant to protease activity and

potentially suitable to succeed where antimicrobial peptides are currently failing.12, 13

Figure 4.2. Structural difference between the peptide and peptoid backbone.

Multiple groups have shown that peptoids are powerful antimicrobial agents in vitro, with

potential to combat multi-drug resistant (MDR) bacterial strains.14, 15, 16 There are multiple ways

peptoids act to disrupt bacterial cells, but they are generally thought to penetrate the bacterial cell

wall and membrane, leading to pore formation and leakage of the cell.16 Similar to antimicrobial

peptides,17 the combination of amphiphilic, cationic, and hydrophobic side chains are thought to

play a role in this disruption by interacting with the anionic bacterial membrane.18, 19 Peptoids

also appear to be resistant to proteolytic degradation due to their abiotic backbone structure.20

138

HNN

NN

R1 O

O

O

R2

R3

R4

N

O

NH2

OR5

HNN

OR1

R2

N

O

NH2

OR3

Antimicrob. Agents Chemother. 2011, 55(1), 417-420

no chirality of backbone

no hydrogen bonding

N

NH3

N N

n

n = 1, Npentn = 6, Ndecn = 9, Ntridec

NH3

NH3

NH3

Literature Precedent - Antimicrobial Peptoids:

Our Proposed Antimicrobial Library Structures:

n

Figure 4.3. Literature precedent and a general proposed structure for the design of peptoid trimer

library.

Here, we synthesize a potentially potent class of novel antimicrobial peptoids in a way

that is facile and economically feasible—with the expectation that, based on literature

precedence of similar molecular structures (Figure 4.3), they will effectively sterilize sensitive

surgical instruments, like duodenoscopes. Both peptoid trimers and dimers were synthesized.


General

Column chromatography of 27-33 was performed on silica gel 60, particle size 0.040-

0.063 mm. 1H and 13C NMR spectra of 27-33 were recorded on either a Bruker or JEOL

spectrometer (500 MHz). All characterization spectra can be found in Appendix D.

Materials

Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Sigma (St.

Louis, MO). Bromoacetic acid, N,N’-diisopropylethylamine (DIEA) was obtained from Chem-

Impex International (Wood Dale, IL). Dimethylformamide (DMF), 2-Phenylethylamine, 1-

139

hexylamine, 1-octylamine, 1-dodecylamine, N,N-diethylenediamine, 1,3-diaminopropane, and

diethylenetriamine were obtained from Alfa Aesar (Haverhill, MA). 3,3-diphenylpropylamine

was obtained from AK Scientific. 4-Chloromethylbenzoyl chloride was obtained from TCI

America (Portland, OR). Rink Amide Resin was obtained from Aapptec (Louisville, KY). All

reagents and organic solvents used were at least ACS grade.

Peptoid Synthesis

Solid-Phase Synthesis. There are several ways to synthesize peptoids in lab. One solid-

phase synthesis of peptoids utilizes reductive alkylation of glycine with an aldehyde or ketone to

create an N-alkylated glycine derivative;21 the monomer approach, which features the sequential

coupling of typically Fmoc-protected N-substituted glycine molecules using standard peptide

synthesis technique;22 and the sub-monomer approach,12 which is most similar to our approach

shown here. Peptoid synthesis was done on 100 mg Rink amide resin. Swelling and deprotection

of the Rink amide resin begins with the addition of dimethylformamide (DMF) followed by

stirring for 1 minute. 20 % 4-methylpiperdine is then added for initial deprotection and the

solution stirs for 1 minute. Following the addition of each substance, 3 washes with 1 mL of

DMF were performed. Once the initial deprotection is performed, residues can be added.

Addition of residues to the resin follows a two-step process: acylation followed by an SN2

displacement reaction. Prior to adding the first amine, an acylation reaction occurs by adding 938

µL of 0.4 M BrAA in combination with 62 μL of diisopropylcarbodiimide (DIC) (to a final

concentration of 0.4 M DIC). Displacement then occurs as the amine of choice (R2—NH2, 0.5 M

in DMF) displaces the halide (—Br) to form the N-substituted glycine residue (SN2 reaction).

The process can be completed as many times as is necessary to achieve a peptoid of the desired

length with the appropriate structure. Here, we performed the reaction 3 times to create a peptoid

140

trimer and also synthesized peptoid dimers containing an aromatic residue using slightly

modified procedures. Once the trimer is created, the peptoid must be cleaved from the resin. To

do this, a 95% trifluoroacetic acid (TFA):2.5% triisopropylsilane (TIPS):2.5% H2O solution was

used to cleave the peptoid from the resin. The solution was then vortexed for 30 minutes and

dried using N2 gas. Following the drying, the samples were re-dissolved in a 50:50 mixture of

acetonitrile and water and were analyzed using MALDI and HPLC.

Solution-Phase.

Synthesis of p-(chloromethyl)phenyl][2-(diethylamino)ethylamino]formaldehyde (26). A

stirring solution of chloromethylbenzoyl chloride (3.00 g, 15.9 mmol) in DCM was cooled to 0

oC prior to adding N,N-diethylethylenediamine (2.24 mL, 15.9 mmol). The reaction was allowed

to warm to room temperature and stirred for 1 h before concentrating under vacuum to obtain

crude 26 (quant.). NMR analysis of the crude material revealed it was pure enough to use in the

subsequent step without further purification.

General Procedure for the Synthesis of (27-31). A solution of 26 (1 equiv.) and desired

amine (0.5 equiv.) was made in ACN with stirring at room temperature. DIEA (1 equiv.) was

added to the reaction mixture. DMF and KI (1 equiv.) which was then stirred 18 h. The reaction

was quenched with excess H2O and the product extracted with DCM (3 x). All products were

purified using silica via column chromatography with a DCM:MeOH gradient as the mobile

phase. Product yields over 2 steps were: 27 (17%); 28 (47%); 29 (43%); 30 (33%); 31 (58%).

141

MALDI-MS

Molecular weight was obtained from MALDI mass spectrometry using a Bruker

Daltonics/Omniflex MALDI mass spectrometer. Samples were prepared by mixing 1.5 μL of

aliquot with 1.5 μL of the matrix solution, a saturated solution of α-cyanohydroxycinnamic acid

in acetone with 0.3% TFA. One microliter of the sample mixture was spotted into a cell of the

sample plate and dried prior to mass spectrometry. Data for 2 ns pulses of the 337 nm nitrogen

laser were averaged for each spectrum in a linear mode, and positive ion TOF detection was

performed using an accelerating voltage of 25 kV.

Results and Discussion

Scheme 4.1. Ronald Zuckermann’s submonomer approach to peptoid synthesis. Elongation

entails iterative haloacetylation (with a haloacetic acid) and nucleophilic displacement (with a

primary amine, R-NH2) steps. The varying of R-groups on the amine allow for incorporation of

chemical diversity into the peptoid chain.

In the 1990’s, Ronald Zuckermann developed a submonomer approach to peptoid

synthesis that is still in use today (Scheme 4.1).12 These traditional peptoid synthesis methods

utilize coupling times of 1.5-3 hours per residue.12, 23 However, our lab has developed a synthetic

142

approach to peptoid oligomers that utilizes 1 minute peptoid couplings that drastically reduce the

time necessary for the synthesis of these molecules.24

Section 4.1: Trimers with aliphatic backbone.

Herein we designed the peptoid trimers (Figure 4.4) with 3 different amine-based side-

chains (cationic, aliphatic, aromatic). The cationic chain was used due to its potential as a

bacterial cell membrane-disrupter. The aliphatic and aromatic structures were used in order to

increase the lipophilicity of the peptoid molecules. Notable differences in toxicity have been

noted in the literature depending on whether hydrophobicity results from aliphatic or aromatic

side chains.15 We created many different structures, each one with a different combination of

cationic, aliphatic, and aromatic side chains. The sidechains could be further fine-tuned if

necessary, but the overall experimental goal is to bracket potential extremes, primarily in

hydrophobicity, in a Design-of-Experiemtns-type approach. These specifc structures or second-

generation ones derived from them could potentially act alone or in combination as potent agents

against the wide variety of membrane-containing targets that need to be sterilized from

endoscopes-human cell, yeast, gram-positive and –negative bacteria, spores, HIV, and

hepatitis—eventually leading to the development of a new solution for the sterilization of

endoscopes with the use of AERs.

143

entry R1(Cationic) R2(Aromatic) R3(Aliphatic)

1 NH2 Ph C6

2 NH2 Ph C8

3 NH2 Ph C12

4 2NH2 Ph C6

5 2NH2 Ph C8

6 2NH2 Ph C12

7 NH2 2Ph C6

8 NH2 2Ph C8

9 NH2 2Ph C12

10 2NH2 2Ph C6

11 2NH2 2Ph C8

12 2NH2 2Ph C12

Trimers (12 compounds):

R2: R3:

R1N

NNH2

OO

OR2

R3

R1:

HN

NH2

N

H2N

H2N

(C6) (C8) (C12)(Ph) (2Ph)(NH2) (2NH2)

Figure 4.4. Design and structure of the peptoid trimers synthesized in this work.

To test whether changing the order of the residues matters, the two control sequences

below were also synthesized (Figure 4.5). If the reverse-order sequences 13 and 14 have

markedly different activity than their analogs 4 and 12, respectively, then additional sequences

altering the residue order will be synthesized and tested.

144

n = 1, C6

n

NN

O

N

O

NH2

O

NH2

H2N

and

n = 7, C12

NN

O

N

O

NH2

O

NH2

H2N

n

13 14

Figure 4.5. Two sequences designed to evaluate whether the residue order is important (13 is an

analog of 4, 14 is an analog of 12).

There have been some recent examples where increasing the hydrophobicity of cyclic

antimicrobial peptoids have resulted in lower MIC’s (Figure 4.6).25 It was observed that when

the propyldiphenyl was used versus a benzyl in a comparable structure, the peptoid was much

more potent (Figure 4.6). Similarly, when the cationic chain was one carbon shorter, the

diphenylethyl substitution was a more potent antimicrobial agent than the phenylmethyl (Figure

4.6). These results suggest that amongst the structures synthesized in our work, the

diphenylpropyl analogues may display higher antimicrobial activity than the monophenyl

counterparts.

N

N

N

NN

N

R

R

R

O

O

O

O

O

O

NH2

NH2

H2N

m

n

nn

R

3.9

>500

p

MIC (ug/mL)

n = 1, m = 1

n = 0, m = 0 < 8

n = 1, p = 0

n = 0, p = 1 128

-

-

-

-

a

a

b

b

145

Figure 4.6. Antimicrobial activity of cyclic peptoids found in literature. aEur. J. Org. Chem.

2013, 3560-3566.b Biopolymers 2014, 103, 227-236.

Researchers have shown more recently that by taking a linear peptoid sequence and

cyclizing it, that the potency of the antimicrobial peptoid is increased.26 Therefore, cyclization of

the most potent trimers developed herein could potentially improve their activity although

cyclization of peptoid trimers is somewhat challenging.27

Section 4.2: Dimers with aromatic backbone.

There is literature precedent that linear dimer peptoids can display low micromolar

antimicrobial activity against a range of bacterial strains.14 Therefore, a range of peptoid dimers

were designed (Figure 4.7) using similar side chains as those used with trimer synthesis.

Cationic, aliphatic and aromatic residues were added to a similar backbone. However, here we

are looking at whether an aromatic component of the backbone increases the lipophilicity and

potency of the peptoid. The cationic portion of each dimer structure also has either: 1) a primary

and secondary amine; or 2) two primary and a tertiary amine. The resulting antimicrobial activity

of this peptoid class will also shed light on the importance of multiple positively charged groups

on smaller structures.

146

entry R1(Aliphatic/Aromatic) R2(Cationic)

15 C6 NH2

16 C8 NH2

17 C12 NH2

18 Ph NH2

19 2Ph NH2

20 C6 2NH2

21 C8 2NH2

22 C12 2NH2

23 Ph 2NH2

24 2Ph 2NH2

N

O

R2

Dimers (10 compounds):

R1 O

NH2

R1:R2:

HN

NH2

N

H2N

H2N

(C6) (C8) (C12)(Ph) (2Ph)(NH2) (2NH2)

Figure 4.7. Design and structure of the structurally rigid backbone peptoid dimers synthesized in

this work.

To test whether the positioning of the aliphatic chain—in relation to the aromatic

backbone—plays an important role in antimicrobial activity, peptoid 25 was also synthesized

(Figure 4.8).

O

NH2

N

O

N

O

N

NH2

H2N

O

NH2

vs

N

H2N

H2N20 25

Figure 4.8. Representative structures of peptoid dimers with aromatic ring incorporated into

147

backbone for increased rigidity.

Section 4.3: Peptoid Trimers with Aromatic Backbone via Solution-Phase Synthesis.

Hypothetically, a monomeric peptoid or aromatic peptoid structure could also be

envisioned, illustrated with an aromatic residue in Figure 4.9A. Preventing dialkylation is

difficult, however, and therefore choosing stoichiometry to intentionally select for dialkylation

was decided as the most optimal approach (Figure 4.9B). These structures also enable densely

concentrating substantial hydrophobicity and cationic charge without the need for a large portion

of the molecular weight to contain a peptidic backbone which is not thought to contribute to the

membrane-lysing effects. Additionally, in contrast to all published examples of antimicrobial

peptoids which utilize primary amines as cationic side chains, these peptoids utilize tertiary

amines exclusively. Therefore, they would be compatible with existing aldehyde-based AER

disinfectants.

Figure 4.9. A) Comparison of backbone structures for aromatic backbone peptoids with

148

traditional peptide and peptoid backbones. B) Synthetic scheme showing that once the primary

amine (R2-NH2) is reacted to form the secondary amine, the secondary amine will react further

with the starting material.

These aromatic backbone peptoid trimers were synthesized as described above in the

methods section, starting from commercially available chloromethylbenzoyl chloride and N,N-

diethylethylenediamine to make 26. The subsequent addition of 0.5 equivalents of the desired

amine in the presence of 1 equivalent of both 26 and KI, yielded the desired products 27-31 in

decent yields. The synthetic route to these structures was not optimized to achieve better yield

because the main goal was to produce sufficient material to proceed with structural analysis and

antimicrobial testing.

N

O

HN

O

NH

NN

N

O

HN

O

NH

NN

N

O

HN

O

NH

NN

N

O

HN

O

NH

NN

N

O

HN

O

NH

NN

C6

C8 C12

Cl

O

HN

N

26

27

28

2930

31

a

aa

aa

Scheme 4.2. Synthesis of structurally rigid-backbone peptoid trimers. Reagents and conditions:

a) amine, KI, DMF. General synthetic method provided above.

149

Two additional peptoids were synthesized, p-[({[p-

(hexylamino)carbonylphenyl]methyl}[2-

(diethylamino)ethyl]amino)methyl]phenyl}formaldehyde (32) and p-[({[p-

(phenethylamino)carbonylphenyl]methyl}[2-

(diethylamino)ethyl]amino)methyl]phenyl}formaldehyde (33) (Figure 4.10), for comparison

with the other structurally rigid peptoids (Scheme 4.2). 32 is an analog of 27, 33 is an analog of

30.

N

O

NHHN

O N

N

O

NHHN

O N

3233

Figure 4.10. Two aromatic backbone peptoid sequences designed to evaluate the importance of

residue order as well as hydrophobicity (32 is an analog of 27, 33 is an analog of 30).

Conclusions

In summary, the rise of multi-drug resistant bacteria is an alarming issue and

antimicrobial peptoids provide an ideal scaffold for the enhancement of activity against various

bacterial strains. The experiments performed here have demonstrated that it is possible to achieve

successful peptoid coupling using 1-minute coupling times versus the traditional 30 minutes to 3

hours used by other researchers. Additionally, the rational design of these structures will allow

for the further investigation of the impact of side-chain selection, peptoid length, and backbone

rigidity on the effectiveness of short, linear antimicrobial peptoids. The ease of synthesis for

150

these short peptoid structures will enable the quantitative structure activity relationship analysis

to develop predictive models for improved activity.

References

1. Drugwatch. (2017 Mar 6) ERCP and duodenoscopes. https://www.drugwatch.com/ercp/ ed.,

Drugwatch, Internet.

2. U.S. Food and Drug Administration. (2015 Mar 4) Design and endoscopic retrograde

cholangiopancreatography (ERCP) duodenoscopes may impede effective cleaning: FDA safety

communication. https://www.fda.gov/medicaldevices/safety/alertsandnotices/ucm434871.htm

ed., U.S. Food and Drug Administration, Internet.

3. Kallen, A. J., Mu, Y., Bulens, S., Reingold, A., Petit, S., Gershman, K., Ray, S. M., Harrison,

L. H., Lynfield, R., Dumyati, G., Townes, J. M., Schaffner, W., Patel, P. R., and Fridkin, S. K.

(2010) Health Care–Associated invasive MRSA infections, 2005-2008. JAMA. 304, 641-648.

4. Ha, J., and Son, B. K. (2015) Current issues in duodenoscope-associated infections: Now is

the time to take action. Clin. Endosc. 48, 361-363.

5. Rubin, Z. A., and Murthy, R. K. (2016) Outbreaks associated with duodenoscopes: New

challenges and controversies. Curr. Opin. Infect. Dis. 29, 407-414.

6. Parsi, M. A., Sullivan, S. A., Goodman, A., Manfredi, M., Navaneethan, U., Pannala, R.,

Smith, Z. L., Thosani, N., Banerjee, S., and Maple, J. T. (2016) Automated endoscope

reprocessors. Gastroentest. Endosc. 84, 885-892.

7. Naryzhny, I., Silas, D., and Chi, K. (2016) Impact of ethylene oxide gas sterilization of

duodenoscopes after a carbapenem-resistant enterobacteriaceae outbreak. Gastroentest. Endosc.

84, 259-262.

8. Mendes, G. C. C., Brandao, T. R. S., and Silva, C. L. M. (2007) Ethylene oxide sterilization of

medical devices: A review. Am. J. Infect. Control. 35, 574-581.

9. Burg, K. J. L., and Shalaby, S. W. (1996) Radiation sterilization of medical devices and

pharmaceuticals. ACS Symp. Ser. 620, 240-245.

10. U.S. Food and Drug Administration. (2017 Jan 1) Infections associated with reprocessed

duodenoscopes.

https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ReprocessingofReusableM

edicalDevices/ucm454630.htm ed., U.S. Food and Drug Administration, Internet.

151

11. Fisher, C. W., Fiorello, A., Shaffer, D., Jackson, M., and McDonnell, G. E. (2012) Aldehyde-

resistant mycobacteria bacteria associated with the use of endoscope reprocessing systems. Am.

J. Infect. Control. 40, 880-882.

12. Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., and Moos, W. H. (1992) Efficient method for

the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase

synthesis. J. Am. Chem. Soc. 114, 10646-10647.

13. Miller, S. M., Simon, R. J., Ng, S., Zuckermann, R. N., Kerr, J. M., and Moos, W. H. (1995)

Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid, and

N-substituted glycine peptide and peptoid oligomers. Drug Dev. Res. 35, 20-32.

14. Goodson, B., Ehrhardt, A., Ng, S., Nuss, J., Johnson, K., Giedlin, M., Yamamoto, R., Moos,

W. H., Kreier, A., Ladner, M., Giacona, M. B., Vitt, C., and Winter, J. (1999) Characterization of

novel antimicrobial peptoids. Antimicrob. Agents Chemother. 43, 1429-1434.

15. Patch, J. A., and Barron, A. E. (2003) Helical peptoid mimics of magainin-2 amide. J. Am.

Chem. Soc. 125, 12092-12093.

16. Chongsiriwatana, N. P., Patch, J. A., Czyzewski, A. M., Dohm, M. T., Ivankin, A.,

Gidalevitz, D., Zuckermann, R. N., and Barron, A. E. (2008) Peptoids that mimic the structure,

function, and mechanism of helical antimicrobial peptides. Proc. Natl. Acad. Sci. USA. 105,

2794-2799.

17. Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature. 415, 389-395.

18. Mendez-Samperio, P. (2014) Peptidomimetics as a new generation of antimicrobial agents:

Current progress. Infect. Drug Resist. 7, 229-237.

19. Schuerholz, T., Doemming, S., Hornef, M., Dupont, A., Kowalski, I., Kaconis, Y.,

Heinbockel, L., Andrae, J., Garidel, P., and Gutsmann, T. (2012) Bacterial cell wall compounds

as promising targets of antimicrobial agents II. immunological and clinical aspects. Curr. Drug

Targets. 13, 1131-1137.

20. Huang, M. L., Benson, M. A., Shin, S. B. Y., Torres, V. J., and Kirshenbaum, K. (2013)

Amphiphilic cyclic peptoids that exhibit antimicrobial activity by disrupting staphylococcus

aureus membranes. Eur. J. Med. Chem. 2013, 3560-3566.

21. Tal-Gan, Y., Freeman, N. S., Klein, S., Levitzki, A., and Gilon, C. (2010) Synthesis and

structure-activity relationship studies of peptidomimetic PKB/akt inhibitors: The significance of

backbone interactions. Bioorg. Med. Chem. 18, 2976-2985.

22. Simon, R. J., Kania, R. S., Zuckermann, R. N., Huebner, V. D., Jewell, D. A., Banville, S.,

Ng, S., Wang, L., Rosenberg, S., and Marlowe, C. K. (1992) Peptoids: a modular approach to

drug discovery. Proc. Natl. Acad. Sci. USA. 89, 9367-9371.

152

23. Culf, A. S., and Ouellette, R. J. (2010) Solid-phase synthesis of N-substituted glycine

oligomers (α-peptoids) and derivatives. Molecules. 15, 5282-5335.

24. Dong, D. Y. (2016) Part 1: One minute peptoid synthesis, from peptoid oligomers to peptoid

polymers. part 2: Synthesis of creatine derivatives. Masters ed., Clemson University, Clemson,

SC.

25. Huang, M. L., Benson, M. A., Shin, S. B. Y., Torres, V. J., and Kirshenbaum, K. (2013)

Amphiphilic cyclic peptoids that exhibit antimicrobial activity by disrupting staphylococcus

aureus membranes. Eur. J. Org. Chem. 2013, 3560-3566.

26. Andreev, K., Michael, W., Martynowycz, M. W., Ivankin, A., Huang, M. L., Kuzmenko, I.,

Meron, M., Lin, B., Kirshenbaum, K., and Gidalevitz, D. (2016) Cyclization improves

membrane permeation by antimicrobial peptoids. Langmuir. 32, 12905-12913.

27. Culf, A. S., Cuperlovic-Culf, M., Leger, D. A., and Decken, A. (2014) Small head-to-tail

macrocyclic α-peptoids. Org. Lett. 16, 2780-2783.

153

CHAPTER FIVE

SUMMARY

The field of peptidomimetics is important in medicinal chemistry and aims to develop

peptide analogues that retain (or enhance) biological activity yet possess more drug-like

properties such as increased protease stability, improved bioavailability, enhanced membrane

permeability, and other pharmacological parameters. Peptidomimetics encompass various

modifications such as the incorporation of unnatural amino acids (e.g. PEG-amino acids),

backbone modifications (e.g. peptoids), proline-mimetics as turn inducers (e.g. Freidinger

lactams), and many other variations. The work presented in this dissertation builds upon the

existing knowledge in the peptidomimetic field by: i) introducing pre-PEGylated amino acids

(linear and branched) that can be easily incorporated into SPPS to yield peptides with site-

specific PEGylation; ii) the development of a novel synthetic route to obtain Freidinger-like

lactams with incorporated chirality off the -nitrogen; and iii) the development of a library of

short peptoids with potential antimicrobial activities.

The usage of the pre-PEGylated amino acids, such as those developed herein (i.e. chapter

two), will enable researchers the ability to screen for the most optimal site of PEGylation within

any sequence; using a PEG-scan approach—similar to how an alanine scan is used to determine

residues important for peptide activity. These pre-PEGylated amino acids (glutamine,

asparagine, and lysine) can be incorporated site-specifically as demonstrated in this work (i.e.

chapter three) and provide novel conjugation strategies for PEG attachment to the side-chain of

amino acids. The lysine PEGylation strategy developed in this work allows for the side-chain

amine (N) to become positively charged under physiological conditions as opposed to the

traditional PEGylation strategy that converts amines into amides; a strategy that does not allow

154

the original side-chain amine to continue participation in structure stabilizing salt-bridges or

potential pi-cation interactions.

The novel synthetic route to Freidinger-like lactams that was developed in this work (i.e.

chapter three) will expand the toolbox of reactions used by synthetic and medicinal chemistry

researchers to get to these desired frameworks. If chiral amino acids (e.g. - or -amino acids,

such as L-alanine or a chiral unnatural amino acid) are used in the synthesis instead of the model

chiral amine used here, (S)-(-)-phenylethylamine, then the lactam product can be incorporated

into a synthetic peptide of interest to introduce a conformational constraint. The ability to

incorporate a variety of chiral groups off the -nitrogen can have an unpredictable effect on the

modulation of pharmacological parameters and will be dependent on factors such as (but not

limited to): i) the R-group that is attached at the chiral carbon adjacent to the -nitrogen; ii) any

intramolecular interactions the R-group has with neighboring residues; and iii) the sequence and

solution conformation(s) of the peptide.

The library of rationally-designed short peptoids developed herein (i.e. chapter four) need

screening against various bacterial strains before structure activity relationships can be

established. However, a display of low micromolar or better (i.e. nanomolar) antimicrobial

activity by any of the peptoid structures against bacterial strains will generate lead compounds

that can be further optimized.

155

APPENDICES

156

APPENDIX A

(PREPEGYLATED AMINO ACIDS)

Data shown in this section were collected to help identify the desired products and purity of

samples.

NMR and MALDI Spectra

General. Column chromatography was performed on silica gel 60, particle size 0.040-0.063

mm. 1H and 13C NMR spectra were recorded on either a Bruker (300 MHz or 500 MHz) or

JEOL spectrometer (500 MHz). Thin-layer chromatography (TLC) was performed using silica

gel precoated aluminum backed plates (200 m thickness). 1H NMR and 13C NMR were

recorded in either CDCl3, MeOH-d4, (CH3)3CO-d6, or (CH3)3SO-d6. 1H NMR spectra are

reported as in units of parts per million (ppm) with the solvent resonance as the internal

standard. 13C NMR spectra are reported as in units of ppm relative to the signal of CDCl3,

MeOH-d4, (CH3)3CO-d6, or (CH3)3SO-d6. 13C NMR spectra were recorded with complete proton

decoupling. Mass of samples were analyzed by either MALDI mass spectrometry or high-

resolution mass spectrometry using ESI.

Synthesis of Nα-Fmoc-N-mTEG1-L-Glutamine (Fmoc-Glu(mTEG)1-OH).

157

(Fmoc-Glu(OtBu)-OPac)

Procedure

To a stirring solution of Fmoc-Glu(OtBu)-OH (4 g, 9.401 mmol) and Na2CO3 (4.98 g,

47.00 mmol) in DCM (50 mL) was added 2-bromoacetophenone (2.06 g, 10.34 mmol).

Reaction mixture stirred for 18-70 h at room temperature (22-27 oC). Reaction diluted with

water and organic layer isolated. Organic layer washed with saturated sodium carbonate (2 x)

followed by a brine wash. Organic layer dried over sodium sulfate, filtered, and concentrated.

Desired product purified by flash chromatography using silica powder. Dichloromethane (200

mL), 1.5% MeOH/DCM (400 mL), and 5% MeOH/DCM (200 mL) used as eluents. Product

isolated as a solid (4.626 g, 90.5% yield).

Analytical Data –

1H NMR (300 MHz, Chloroform-d) δ 7.95 – 7.85 (m, 1H), 7.81 – 7.71 (m, 1H), 7.68 – 7.56 (m,

2H), 7.55 – 7.23 (m, 4H), 5.66 (d, J = 8.1 Hz, 1H), 5.53 (d, J = 16.4 Hz, 1H), 5.31 (d, J = 16.3

Hz, 1H), 4.58 (td, J = 8.1, 4.7 Hz, 1H), 4.41 (td, J = 11.4, 11.0, 7.2 Hz, 1H), 4.39 – 4.18 (m, 1H),

2.49 (td, J = 7.0, 6.5, 4.0 Hz, 1H), 2.46 – 2.27 (m, 0H), 2.15 (dq, J = 14.6, 7.3 Hz, 1H), 1.47 (s,

6H).

158

Figure A-1. 1H NMR spectrum for Fmoc-Glu(OtBu)-OPac.

13C NMR (126 MHz, CHLOROFORM-D) δ 191.38, 172.47, 171.72, 156.15, 144.04, 143.84,

141.40, 141.38, 134.14, 134.06, 129.03, 127.84, 127.79, 127.78, 127.18, 125.28, 125.23, 120.06,

80.94, 77.37, 77.32, 77.11, 76.86, 67.23, 66.70, 53.73, 53.51, 47.25, 31.52, 28.19, 27.51.

159

Figure A-2. 13C NMR spectrum for Fmoc-Glu(OtBu)-OPac.

160

58

0.2

66

56

4.3

28

0

500

1000

1500

2000

Inte

ns.

[a.

u.]

500 525 550 575 600 625m/z

Figure A-3. MALDI-MS spectrum for Fmoc-Glu(OtBu)-OPac.

(Fmoc-Glu(OH)-OPac)

Procedure

Fmoc-Glu(OtBu)-OPac dissolved in TFA:H2O (3:1) v/v mixture and stirred 3 h at room

temperature (22-27 oC). Saturated aqueous sodium bicarbonate added to neutralize the acid. The

desired product was vacuum filtered from solution and allowed to dry (quant. yield)

161

Analytical Data –

1H NMR (500 MHz, DMSO-d6) δ 8.63 (d, J = 6.8 Hz, 1H), 7.97 (d, J = 7.7 Hz, 2H), 7.89 (d, J =

7.5 Hz, 2H), 7.75 – 7.60 (m, 3H), 7.56 (t, J = 7.7 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.34 (td, J =

7.4, 4.1 Hz, 2H), 5.56 (d, J = 16.9 Hz, 1H), 5.46 (d, J = 16.8 Hz, 1H), 4.27 – 4.16 (m, 4H), 2.19

(t, J = 7.6 Hz, 2H), 2.15 – 2.05 (m, 1H), 1.97 – 1.85 (m, 1H).

Figure A-4. 1H NMR spectrum for Fmoc-Glu(OH)-OPac

162

13C NMR (500 MHz, DMSO) δ 192.97, 174.23, 172.26, 156.59, 144.25, 144.19, 141.17, 134.49,

134.20, 129.39, 128.26, 128.11, 127.55, 125.71, 120.59, 67.32, 66.22, 53.46, 47.07, 40.78, 40.50,

40.23, 39.95, 39.67, 39.39, 39.22, 39.11, 30.30, 26.67.

Figure A-5. 13C NMR spectrum for Fmoc-Glu(OH)-OPac

163

50

8.4

67

52

4.4

01

0

200

400

600

800

1000

Inte

ns.

[a.

u.]

400 450 500 550 600 650m/z

Figure A-6. MALDI-MS spectrum for Fmoc-Glu(OH)-OPac

(TEGME-N3)

HOO

OO

MsOO

OO

N3

OO

ONaN3MsCl

DIEA

Procedure

A stirring solution of tri(ethylene glycol)monomethyl ether (0.65 mL, 4.5 mmol) and N,

N-diisopropylethylamine (0.86 mL, 4.95 mmol) in DCM (12 mL) was cooled to 0 oC. To the

stirring solution was added methane sulfonyl chloride (0.38 mL, 4.95 mmol) via syringe. The

reaction mixture was stirred for 1.5 h at 0 oC. The reaction transferred to a separatory funnel and

washed with brine (100 mL). Organic phase concentrated under reduced pressure. The residue

164

was partitioned between hexanes and H2O in separatory funnel. Aqueous phase was isolated and

NaCl (2 g) was added and agitated vigorously to dissolve the salt. The solution was extracted

with DCM (50 mL x 3). The organic layers were combined, dried over Na2SO4, filtered, and

concentrated under reduced pressure to yield a yellow oil (1.08 g, quantitative yield).

A solution of TEGME-OMs (1 g, 4.38 mmol) and sodium azide (3.42 g, 52.56 mmol) in

H2O (14 mL) and MeOH (5 mL) was stirred in a glass vial at reflux for 4 h, during which time

the methanol was evaporated. The aqueous phase was extracted with DCM (8 mL x 4). Organic

layers combined, dried over MgSO4, and concentrated to yield crude TEGME-N3 (430 mg,

56.1%) as a yellow oil.

165

Analytical Data

Figure A-7. 1H NMR spectrum for TEGME-N3. Solvent: CDCl3.

13C NMR (126 MHz, CHLOROFORM-D) δ 71.75, 70.50, 70.45, 70.37, 69.87, 58.78, 50.51.

166

Figure A-8. 13C NMR spectrum for TEGME-N3.

(TEGME-NH2)

N3

OO

OH2N

OO

OLiAlH4

Procedure

TEGME-N3 (5 g, 28.5 mmol) was dissolved in dry THF (100 mL). This solution was

added dropwise to a stirring suspension of LiAlH4 (2.37 g, 62.5 mmol) in THF (50 mL). The

reaction was stirred for 2 h at room temperature under N2 (g). The reaction was cooled to 0 oC

and a stream of N2 (g) was placed over the reaction mixture. A small amount of MeOH was

added very slowly, followed by the addition of small portions of H2O. Solvent was vacuum

167

filtered from the solid inorganic byproducts. An additional filtration through filter paper yielded

crude TEGME-NH2 (4.31 g, quantitative yield).

Analytical Data

1H NMR (500 MHz, Chloroform-d) δ 3.32 (pd, J = 5.4, 4.6, 2.6 Hz, 6H), 3.25 – 3.21 (m, 4H),

3.18 (s, 3H), 3.05 (s, 3H), 2.57 (t, J = 5.2 Hz, 1H).

Figure A-9. 1H NMR spectrum for TEGME-NH2.

13C NMR (126 MHz, CHLOROFORM-D) δ 72.44, 71.53, 71.37, 69.97, 69.84, 69.65, 58.33,

40.58.

168

Figure A-10. 13C NMR spectrum for TEGME-NH2.

169

(Fmoc-Gln(mTEG)1-OPac)

Procedure

Carbonyldiimidazole (CDI) (0.458 g, 2.822 mmol) was added to a suspension of Fmoc-

Glu(OH)-OPac in DCM (5 mL). Solution was then poured slowly into a roundbottom containing

TEGME-NH2 (0.421 g, 2.822 mmol) in DCM. The reaction allowed to stir for 1.5 h. The

organic layer washed with saturated sodium bicarbonate (1 x), 0.10 M HCl (1 x), H2O (1 x), and

brine (1 x). The organic layer dried over sodium sulfate, decanted, and concentrated to yield

desired product (1.177 g, 98.9% yield).

Analytical Data

1H NMR (500 MHz, Chloroform-d) δ 7.88 (d, J = 7.7 Hz, 3H), 7.74 (d, J = 7.5 Hz, 3H), 7.65 –

7.56 (m, 4H), 7.47 (t, J = 7.7 Hz, 3H), 7.41 – 7.35 (m, 3H), 7.30 (tt, J = 7.5, 1.5 Hz, 3H), 5.67 (d,

J = 8.2 Hz, 1H), 5.51 (d, J = 16.3 Hz, 1H), 5.29 (d, J = 16.3 Hz, 1H), 4.60 (t, J = 6.8 Hz, 1H),

4.39 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.0 Hz, 1H), 3.82 – 3.44 (m, 10H), 3.37 (s, 2H), 2.60 (t, J =

7.3 Hz, 2H), 2.39 (dd, J = 14.5, 7.2 Hz, 1H), 2.17 (dd, J = 14.4, 7.3 Hz, 1H).

170

Figure A-11. 1H NMR spectrum for Fmoc-Gln(mTEG)1-OPac.

13C NMR (126 MHz, CHLOROFORM-D) δ 191.43, 171.58, 156.22, 143.98, 141.42,

134.22, 127.86, 125.26, 77.38, 76.90, 71.98, 70.66, 67.26, 66.82, 53.42, 47.22, 29.84,

27.54.

171

Figure A-12. 13C NMR spectrum for Fmoc-Gln(mTEG)1-OPac.

172

631.1

69

669.0

62

653.1

14

645.1

60

0

200

400

600

800

1000

1200

Inte

ns.

[a.u

.]

500 550 600 650 700 750m/z

Figure A-13. MALDI-MS spectrum for Fmoc-Gln(mTEG)1-OPac. Calculated m/z =

632.71. Observed m/z = 631.169 [M+H]+, 653.114 [M+Na]+, 669.062 [M+K]+.

173

(Fmoc-Gln(mTEG)1-OH)

Procedure

Fmoc-Glu(mTEG)1-OPac was dissolved in MeOH with stirring at room temperature. Mg

granules (7 equiv.) were added, followed by AcOH (12 equiv.). The reaction mixture allowed to

stir for 16 h. The reaction was concentrated in the presence of silica powder and dry loaded onto

a silica column. The crude product was purified by column chromatography on silica gel using

dichloromethane-methanol (0-20% methanol in dichloromethane) as eluent to obtain desired

product. Desired fractions combined and concentrated to yield desired product.

Analytical Data

1H NMR (500 MHz, Chloroform-d) δ 7.71 (dd, J = 15.5, 8.2 Hz, 2H), 7.58 (q, J = 10.7, 9.1 Hz,

2H), 7.44 – 7.17 (m, 5H), 4.40 – 3.99 (m, 3H), 3.69 – 3.30 (m, 12H), 3.26 (d, J = 16.1 Hz, 3H),

2.24 (d, J = 48.8 Hz, 2H), 2.00 (s, 2H), 1.72 – 1.59 (m, 1H), 1.43 (q, J = 7.4 Hz, 0H), 1.37 – 1.20

(m, 1H).

174

Figure A-14. 1H NMR spectrum for Fmoc-Gln(mTEG)1-OH.

13C NMR (126 MHz, CDCl3) δ 174.00, 156.58, 144.15, 143.88, 141.14, 127.58, 127.10, 125.30,

119.79, 77.29, 77.23, 77.16, 77.03, 76.78, 71.78, 71.67, 70.21, 70.08, 69.86, 69.61, 66.77, 58.85,

47.10, 39.25, 32.38, 29.71, 23.97, 19.72, 13.65.

175

Figure A-15. 13C NMR spectrum for Fmoc-Gln(mTEG)1-OH.

176

0.0

0.2

0.4

0.6

0.8

1.0

4x10In

tens

. [a.u

.]

500 510 520 530 540 550 560 570 580 590

m /z

[M+Na]+

[M+H]+

515.414

537.347

510 520 530 540 550 560 570

0.2

580 590 m/z

0.0

0.4

0.6

0.8

1.0

x104

550 560500

MW=514.57

Figure A-16. MALDI-MS spectrum for Fmoc-Gln(mTEG)1-OH. Calculated m/z = 515.57

[M+H]+. Observed m/z = 515.414 [M+H]+, 537.347 [M+Na]+.

177

Synthesis of Nα-Fmoc-N-mTEG2-L-Glutamine.

(N-Benzyl-(mTEG)2-Amine)

N

O O

O O

OO

Procedure

To a solution of TEGME-OMs (12 g, 49.527 mmol) in dry ACN (40 mL) was added benzyl

amine (2.16 mL, 19.811 mmol). Sodium carbonate (6.3 g, 59.432 mmol) dissolved in water (10

mL) and added to the reaction mixture. The reaction was heated to reflux and stirred for 72 h.

The organic layer was concentrated and the crude product was purified by column

chromatography on silica gel using dichloromethane-methanol (0-20% methanol in

dichloromethane) as eluent to obtain desired product. Desired fractions combined and

concentrated to yield desired product (5.456 g, 68.9%).

178

Analytical Data

39

8.9

49

26

5.5

39

0

500

1000

1500

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550m/z

Figure A-17. MALDI-MS spectrum for N-Benzyl-(mTEG)2-Amine. Calculated m/z =

401.51 [M+H]+. Observed m/z = 398.949 [M+H]+.

((mTEG)2-Amine)

HN

O O

O O

OO

179

Procedure

To a solution of N-Benzyl-(mTEG)2-Amine (1.21 g, 3.021 mmol) in MeOH (50 mL) was added

Pd/C (0.161 g, 1.511 mmol) with stirring. The roundbottom was evacuated and subsequently

filled with H2(g). The reaction was stirred at room temperature for 18 h. The reaction mixture

was filtered through a bed of celite before concentrating the solvent to yield crude product (0.892

mg, 95.5%). The crude material was used as is for subsequent coupling to Fmoc-Glu(OH)-OPac.

Analytical Data

30

9.3

16

33

1.1

79

0

1000

2000

3000

4000

5000

Inte

ns.

[a

.u.]

100 150 200 250 300 350 400 450m/z

Figure A-18. MALDI-MS spectrum for (mTEG)2-Amine. Calculated m/z = 310.22

[M+H]+, 332.22 [M+Na]+. Observed m/z = 309.316 [M+H]+, 331.179 [M+Na]+.

180

NH2

HN

OO

O O

O

O

TFATfOTEGME

DIEA (2eq)

DCM

0oC

NH2

HN

O O

O O

O

O

HN

O

O

O

O

OO

TFA

E1cb

E1cb+

+

Figure A-19. A representative MALDI-MS analysis of crude product mixture from E1cb

reaction mixture.

181

Procedure

Propylphosphonic anhydride (T3P) was added to a solution of Fmoc-Glu(OH)-OPac in a mixture

of DCM and ACN. To the solution was added (mTEG)2-NH in DCM. The reaction was stirred

for 1.5 h. The organic layer was concentrated and the crude product was purified by column



concentrated to yield desired product.

Analytical Data

1H NMR (500 MHz, Acetone-d6) δ 8.01 (d, J = 7.7 Hz, 2H), 7.85 (d, J = 7.5 Hz, 2H), 7.70 (dt, J

= 23.9, 7.3 Hz, 3H), 7.56 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H),

5.62 (d, J = 16.6 Hz, 1H), 5.47 (d, J = 16.6 Hz, 1H), 4.50 (dd, J = 9.2, 5.1 Hz, 1H), 4.34 (dt, J =

7.0, 3.2 Hz, 2H), 4.24 (t, J = 7.2 Hz, 1H), 4.01 – 3.35 (m, 23H), 3.32 – 3.23 (m, 6H), 3.14 (s,

1H), 2.62 (d, J = 7.7 Hz, 2H), 2.39 – 2.30 (m, 1H), 2.14 (dt, J = 14.4, 7.4 Hz, 1H), 2.10 – 2.03

(m, 10H), 1.40 (s, 2H), 0.97 (t, J = 7.3 Hz, 1H).

182

Figure A-20. 1H NMR spectrum for Fmoc-Gln(mTEG)2-OPac.

13C NMR (126 MHz, ACETONE-D6) δ 205.47, 205.33, 205.32, 205.31, 205.16, 192.00, 141.29,

133.86, 128.95, 127.84, 127.72, 127.16, 125.37, 119.99, 71.87, 70.31, 70.24, 70.16, 66.71, 66.47,

57.99, 54.34, 47.19, 29.77, 29.62, 29.58, 29.48, 29.42, 29.39, 29.33, 29.27, 29.17, 29.14, 29.12,

29.09, 29.02, 28.97, 28.96, 28.94, 28.93, 28.87, 28.82, 28.78, 28.71, 28.56, 27.00.

183

Figure A-21. 13C NMR spectrum for Fmoc-Gln(mTEG)2-OPac.

184

79

9.2

29

81

5.1

99

77

1.1

13

82

1.2

60

77

7.2

76

84

3.2

33

83

8.9

01

78

5.4

23

0

1000

2000

3000

4000

Inte

ns.

[a

.u.]

780 800 820 840 860m/z

Figure A-22. MALDI-MS spectrum for Fmoc-Gln(mTEG)2-OPac. Calculated m/z =

779.70 [M+H]+, 801.70 [M+Na]+, 817.70 [M+K]+. Observed m/z = 799.22 [M+Na]+,

815.19 [M+K]+.

(Fmoc-Gln(mTEG)2-OH)

Procedure

185

Fmoc-Glu(mTEG)2-OPac was dissolved in AcOH with stirring at room temperature. Mg

granules (7 equiv.) was added. The reaction mixture allowed to stir for 16 h. The reaction was

concentrated and dry loaded onto silica column. The crude product was purified by column



concentrated to yield desired product.

Analytical Data

1H NMR (500 MHz, Methanol-d4) δ 7.82 – 7.77 (m, 2H), 7.67 (p, J = 5.5, 4.4 Hz, 2H), 7.38 (tt, J

= 6.4, 2.4 Hz, 2H), 7.31 (tdt, J = 7.4, 3.6, 1.8 Hz, 2H), 4.36 (dtd, J = 20.7, 6.8, 4.4 Hz, 2H), 4.26

– 4.18 (m, 1H), 3.64 – 3.44 (m, 27H), 3.37 – 3.33 (m, 5H), 2.62 – 2.51 (m, 1H), 2.38 (dt, J =

23.6, 7.6 Hz, 1H), 2.17 (dt, J = 13.3, 7.0 Hz, 1H), 1.97 – 1.85 (m, 1H), 1.35 (dt, J = 7.3, 3.8 Hz,

2H).

186

Figure A-23. 1H NMR spectrum for Fmoc-Gln(mTEG)2-OH.

13C NMR (126 MHz, Methanol-d4) δ 175.07, 174.06, 173.83, 157.37, 143.87, 141.29, 127.49,

126.88, 124.98, 119.64, 72.34, 71.59, 70.21, 70.03, 69.91, 69.18, 68.86, 66.69, 66.57, 60.90,

57.75, 54.52, 48.79, 45.92, 42.38, 39.15, 39.10, 29.92, 29.21, 26.97, 26.60, 15.86, 11.84.

187

Figure A-24. 13C NMR spectrum for Fmoc-Gln(mTEG)2-OH.

188

Figure A-25. MALDI-MS spectrum for Fmoc-Gln(mTEG)2-OH. Calculated m/z =

663.76 [M+Na]+, 699.76 [M+K]+. Observed m/z = 682.73 [M+Na]+, 699.35 [M+K]+.

189

Synthesis of Nα-Fmoc-N-mTEG3-L-Glutamine.

(Fmoc-Gln(NH-CH2CH2-NHBoc)-OPac)

Procedure

To a stirring solution of Fmoc-Glu(OH)-OPac and H2N-CH2CH2-NHBoc in ACN was

added T3P and DIEA. The reaction was stirred for 16 h at room temperature (22-27 oC). The

crude product was purified by column chromatography on silica gel using dichloromethane-

methanol (0-5% methanol in dichloromethane) as eluent to obtain desired product.

190

Analytical Data

1H NMR (300 MHz, Chloroform-d) δ 7.97 – 7.88 (m, 2H), 7.78 (d, J = 7.5 Hz, 2H), 7.64 (dd, J =

7.6, 3.5 Hz, 3H), 7.53 (t, J = 7.5 Hz, 2H), 7.47 – 7.24 (m, 6H), 6.63 (s, 1H), 5.95 (s, 1H), 5.62 (d,

J = 16.5 Hz, 1H), 5.39 – 5.26 (m, 2H), 5.17 (s, 1H), 4.54 (d, J = 6.7 Hz, 1H), 4.44 (d, J = 7.1 Hz,

2H), 4.25 (t, J = 6.9 Hz, 1H), 3.30 (s, 3H), 2.43 (s, 2H), 2.22 (d, J = 6.8 Hz, 1H), 1.71 (s, 1H),

1.39 (s, 11H).

Figure A-26. 1H NMR spectrum for Fmoc-Gln(NH-CH2CH2-NHBoc)-OPac.

191

(Fmoc-Gln(NH-CH2CH2-NH2)-OPac)

Procedure

To a stirring solution of Fmoc-Glu(OH)-OPac and H2N-CH2CH2-NHBoc in DMF was

added EDC and DIEA. The reaction was stirred for 18 h at room temperature (22-27 oC). The



Analytical Data

1H NMR (500 MHz, Chloroform-d) δ 7.87 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.59

(ddt, J = 7.1, 5.1, 2.5 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.41 – 7.32 (m, 1H), 7.29 (ddd, J = 7.7,

4.7, 1.5 Hz, 1H), 5.84 (d, J = 8.1 Hz, 0H), 5.49 (d, J = 16.3 Hz, 0H), 5.29 (d, J = 16.3 Hz, 0H),

4.58 (q, J = 7.6 Hz, 0H), 4.36 (d, J = 7.2 Hz, 1H), 4.21 (t, J = 7.3 Hz, 1H), 3.86 (s, 5H), 3.19 –

3.08 (m, 1H), 2.70 (d, J = 14.9 Hz, 1H), 2.63 – 2.53 (m, 1H), 2.37 (dd, J = 13.9, 6.6 Hz, 0H),

2.17 (dt, J = 14.4, 7.3 Hz, 0H).

192

Figure A-27. 1H NMR spectrum for Fmoc-Gln(NH-CH2CH2-NH2)-OPac.

193

Synthesis of Nα-Fmoc-N-mTEG3-L-Lysine.

(Fmoc-Lys(NHBoc)-OPac)

Procedure

To a solution of Nα-Fmoc-Nε-Boc-Lys-OH and bromoacetophenone in DCM was added a

solution of sodium carbonate and tetrabutylammonium bromide in H2O and reaction was stirred

at room temperature (22-27 oC) for 16-72 h. Organic layer washed with water, and then

concentrated. Desired product purified by flash chromatography on silica gel using

dichloromethane-methanol (0-20% methanol in dichloromethane) as eluent to obtain desired

product.

Analytical Data –

194

1H NMR (500 MHz, Chloroform-d) δ 7.93 – 7.87 (m, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.66 – 7.57

(m, 3H), 7.48 (t, J = 7.8 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 7.4, 1.3 Hz, 2H), 5.58 –

5.51 (m, 1H), 5.26 (d, J = 16.3 Hz, 1H), 4.90 (s, 1H), 4.54 (td, J = 7.8, 5.2 Hz, 1H), 4.40 (qd, J =

10.6, 7.1 Hz, 2H), 4.23 (t, J = 7.1 Hz, 1H), 3.17 (d, J = 8.3 Hz, 2H), 2.03 (ddd, J = 15.0, 9.4, 5.1

Hz, 1H), 1.89 (s, 1H), 1.60 – 1.48 (m, 3H), 1.43 (s, 9H).

Figure A-28. 1H NMR spectrum for Fmoc-Lys(NHBoc)-OPac.

13C NMR (126 MHz, Chloroform-d) δ 191.67, 172.11, 156.30, 156.12, 144.02, 143.85, 141.40,

141.38, 134.22, 133.98, 130.08, 129.03, 127.88, 127.80, 127.18, 125.24, 120.08, 120.06, 79.13,

77.41, 77.16, 76.91, 67.16, 66.55, 53.90, 47.26, 39.96, 32.05, 29.60, 28.55, 24.21, 22.12, 19.85,

13.78.

195

Figure A-29. 13C NMR spectrum for Fmoc-Lys(NHBoc)-OPac.

196

Figure A-30. MALDI-MS spectrum for Fmoc-Lys(NHBoc)-OPac. Calculated m/z =

586.69 [M+H]+, 609.67 [M+Na]+. Observed m/z = 609.64 [M+Na]+.

(Fmoc-Lys(MTEG)3-OPac)

Procedure

Fmoc-Lys(NHBoc)-OPac dissolved in TFA:DCM (3:1 v/v) and stirred at room

temperature (22-27 oC) for 2 h. The solvent was evaporated under a stream of N2 (g) to yield

197

crude Fmoc-Lys(NH2)-OPac. Complete conversion to product monitored by MALDI mass

spectroscopy.

To a stirring solution of tri(ethyleneglycol)monomethyl ether (TEGME or mTEG) in

DCM at 0 oC was added trifluorosulfonic anhydride ((Tf)2O) dropwise. Solution stirred for 5-10

mins before adding it to the crude solution of Fmoc-Lys(Boc)-OPac in DCM at 0 oC. The

reaction was stirred for 1 h at 0 oC before evaporating the solvent under a stream of N2 (g). The



Analytical Data –

1H NMR (300 MHz, DMSO-d6) δ 8.01 – 7.94 (m, 2H), 7.90 (dd, J = 8.0, 4.2 Hz, 4H), 7.72 (dt, J

= 10.6, 5.3 Hz, 5H), 7.57 (t, J = 7.5 Hz, 3H), 7.47 – 7.38 (m, 5H), 7.37 – 7.30 (m, 4H), 5.71 –

5.40 (m, 1H), 4.50 – 4.04 (m, 2H), 3.85 – 3.40 (m, 36H), 3.22 (s, 9H), 1.97 (d, J = 9.4 Hz, 0H),

1.72 (s, 5H), 1.42 (s, 7H).

198

Figure A-31. 1H NMR spectrum for Fmoc-Lys(mTEG)3-OPac.

13C NMR (75 MHz, DMSO) δ 192.91, 172.38, 156.63, 144.25, 144.17, 141.20, 134.54, 134.18,

129.40, 128.24, 128.11, 127.52, 125.68, 120.60, 79.86, 79.42, 78.98, 71.72, 70.18, 69.99, 69.94,

67.23, 66.17, 64.13, 60.39, 59.23, 58.50, 55.36, 54.04, 47.10, 40.80, 40.69, 40.53, 40.25, 39.97,

39.69, 39.41, 39.23, 39.13, 30.99, 22.77.

199

Figure A-32. 13C NMR spectrum for Fmoc-Lys(mTEG)3-OPac.

200

92

5.4

84

0

250

500

750

1000

1250

Inte

ns.

[a.

u.]

850 900 950 1000 1050m/z

Figure A-33. MALDI-MS spectrum for Fmoc-Lys(mTEG)3-OPac. Calculated m/z =

925.51 [M]+. Observed m/z = 925.484 [M]+.

(Fmoc-Lys(mTEG)3-OH)

Procedure

Fmoc-Lys(mTEG)3-OPac was dissolved in AcOH with stirring at room temperature

followed by the addition of Mg granules. The reaction was stirred for 1 h. Magnesium was

201

filtered from the reaction mixture and the AcOH evaporated under vacuum. The crude product

was purified by column chromatography on silica gel using dichloromethane-methanol (0-20%

methanol in dichloromethane) as eluent to obtain desired product.

Analytical Data

1H NMR (300 MHz, DMSO-d6) δ 7.88 (d, J = 7.4 Hz, 2H), 7.69 (s, 2H), 7.41 (t, J = 7.4 Hz, 3H),

7.31 (t, J = 7.4 Hz, 3H), 4.43 – 4.09 (m, 1H), 4.03 (s, 0H), 3.76 (s, 2H), 3.61 (s, 3H), 3.53 – 3.34

(m, 18H), 3.20 (q, J = 2.8, 1.7 Hz, 8H), 1.63 (s, 2H), 1.21 (s, 2H).

Figure A-34. 1H NMR spectrum for Fmoc-Lys(mTEG)3-OH.

202

13C NMR (126 MHz, Acetone) δ 205.45, 205.29, 205.13, 204.94, 137.95, 128.86, 127.20,

121.13, 119.77, 107.98, 71.78, 70.05, 69.98, 64.45, 59.82, 59.70, 57.96, 33.02, 29.70, 29.61,

29.51, 29.41, 29.36, 29.26, 29.20, 29.11, 29.05, 28.95, 28.89, 28.80, 28.71, 28.65, 28.52, 28.49.

Figure A-35. 13C NMR spectrum for Fmoc-Lys(mTEG)3-OH.

203

807.64

2

719.58

9

661.54

9

0

250

500

750

1000

1250

Intens

.[a.u.]

400 600 800 1000 1200 1400 1600m/z

67

Exact Mass = 807.99

807

.64

2

PEG3-Lysine (K)

Figure A-36. MALDI-MS spectrum for Fmoc-Lys(mTEG)3-OH. Calculated m/z =

807.99 [M]+. Observed m/z = 807.642 [M]+.

204

Synthesis of Nα-Fmoc-N-mTEG2-L-Lysine (Isomer 1)

FmocHNO

OH

NHO

OO

OO

O


FmocHNO

OH

NHO

OO

OO

O

Procedure

Fmoc-Lys(NHBoc)-OH was dissolved in TFA:DCM (3:1) v/v with stirring at room

temperature (22-27 oC). The reaction mixture was stirred for 2 h. The reaction was diluted with

DCM and neutralized with saturated aqueous sodium carbonate. The organic layer was isolated

and concentrated.

A solution of oxalyl chloride (0.24 mL, 2.714 mmol) in dry DCM (20 mL) prepared and

cooled in dry ice/acetone under N2 (g) and stirred for 15 mins. DMSO (0.29 mL, 4.071 mmol) in

dry DCM was added and stirred at -78 oC for 20 mins. TEGME (0.062 mL, 0.385 mmol) was

205

added slowly and the reaction mixture stirred for 30 mins. Triethylamine (1.14 mL, 8.142

mmol) added slowly over 1 min and the reaction mixture was stirred for 30 mins at -78 oC before

warming to room temperature (22-27 oC) slowly. The reaction mixture was concentrated under a

stream of N2 (g).

A solution of Fmoc-Lys(NH2)-OH (600 mg, 1.629 mmol) with crude TEGME-aldehyde

was prepared in ACN (20 mL) and stirred. Solid NaCNBH3 (0.256 g, 4.071 mmol) was added to

the stirring reaction and allowed to stir for 18 h at room temperature (22-27 oC). The reaction

was quenched with 0.012 M HCl, followed by the addition of brine, and DCM. The organic

layer was separated and concentrated to yield 1.95 g of crude material. The crude product was

purified by column chromatography on silica gel using dichloromethane-methanol (0-10%

methanol in dichloromethane) as eluent to obtain desired product.

Analytical Data

1H NMR (500 MHz, CDCl3) 1H NMR (500 MHz, Chloroform-d) δ 9.30 (s, 1H), 7.65 (d, J = 7.4

Hz, 3H), 7.55 (t, J = 8.0 Hz, 2H), 7.31 – 7.26 (m, 3H), 7.24 – 7.18 (m, 2H), 4.31 – 4.16 (m, 3H),

4.12 (t, J = 7.2 Hz, 1H), 3.78 (t, J = 5.1 Hz, 2H), 3.67 – 3.37 (m, 20H), 3.29 – 3.25 (m, 2H), 3.24

(s, 4H), 3.12 – 3.06 (m, 6H), 2.92 (s, 2H), 1.39 (dt, J = 17.3, 5.6 Hz, 2H).

206



127.73, 127.23, 127.15, 125.32, 125.28, 124.90, 119.93, 85.23, 77.56, 77.30, 77.05, 72.56, 72.55,

71.93, 71.85, 71.83, 71.79, 71.61, 71.05, 71.02, 70.93, 70.77, 70.69, 70.66, 70.51, 70.40, 70.38,

70.35, 70.21, 70.20, 70.16, 70.14, 70.05, 66.98, 65.93, 61.52, 61.46, 58.96, 58.88, 58.87, 53.83,

52.46, 47.58, 47.18, 47.13, 46.16, 46.02, 45.88, 40.90, 31.42, 26.81, 25.40, 22.60, 22.51, 8.85,

8.78, 8.75, 8.72, 8.67, 8.65, 8.65, 8.64, 8.63, 8.62, 8.61, 8.60, 8.56, 8.55.

207


208

69

9.5

43

68

2.9

12

69

2.9

82

0

250

500

750

1000

1250

Inte

ns.

[a.

u.]

620 640 660 680 700m/z

Figure A-39. MALDI-MS spectrum for Fmoc-Lys(mTEG)2-OH.

209

Synthesis of Nα-Fmoc-N-mTEG2-L-Lysine (Isomer 2)

(Fmoc-Lys(mTEG)2-OPac)

FmocHN

O

O

N

O

O

OO

O

O

O

Procedure

To a solution of TEGME (0.87 mL, 5.427 mmol) in DCM at 0 oC was added

trifluoromethanesulfonic anhydride (0.93 mL, 5.562 mmol) and allowed to stir for 3 mins. The

reaction mixture was transferred portion-wise to a solution of Fmoc-Lys(NH2)-OPac (1.32 g,

2.713 mmol) and DIEA (0.47 mL, 2.713 mmol) in DCM (20 mL) at 0 oC and stirred for 20 mins.

The reaction mixture was concentrated under reduced pressure to yield crude material. The crude

product was purified by column chromatography on silica gel using dichloromethane-methanol

(0-6% methanol in dichloromethane) as eluent to obtain product. The desired fractions were

combined and concentrated to yield the desired product (1.05 g, 50% yield).

210

Analytical Data

Figure A-40. 1H NMR spectrum for Fmoc-Lys(mTEG)2-OPac.


127.80, 127.18, 125.16, 120.05, 71.89, 71.89, 70.43, 70.30, 66.60, 65.99, 58.98, 53.84, 52.72,

47.25, 33.54, 32.01, 29.79, 59.51, 23.37, 22.77, 22.45, 19.85, 18.51, 17.90, 14.30, 0.48, -0.15.

211

Figure A-41. 13C NMR spectrum for Fmoc-Lys(mTEG)2-OPac.

77

7.0

82

79

9.0

67

65

9.2

48

64

3.2

21

68

9.1

81

81

5.0

31

0.0

0.2

0.4

0.6

0.8

1.0

4x10

Inte

ns.

[a

.u.]

450 500 550 600 650 700 750 800 850 900 950m/z

212

Figure A-42. MALDI-MS spectrum for Fmoc-Lys(mTEG)2-OPac.


Procedure

A solution of Fmoc-Lys(mTEG)2-OPac (628 mg, 0.806 mmol) was dissolved in 4%

pyridine in acetonitrile (50 mL) and stirred. Zn dust (420 mg, 6.419 mmol) and acetylacetone

(1.33 mL, 12.84 mmol) added to the stirring reaction and heated to 85 oC. The reaction mixture

was stirred for 5 h at 85 oC. The reaction was filtered to removed undissolved Zn dust. The

filtrate was concentrated under reduced pressure to yield crude material. The crude product was

purified by column chromatography on silica gel using dichloromethane-methanol (0-10%

methanol in dichloromethane) as eluent to obtain product. The desired fractions were combined

and concentrated to yield the desired product (213 mg, 40% yield).

Analytical Data

213

Figure A-43. 1H NMR spectrum for Fmoc-Lys(mTEG)2-OH. PH6011.


125.27, 124.42, 120.40, 120.01, 71.83, 70.41, 70.27, 66.99, 65.46, 58.96, 58.87, 54.20, 52.78,

47.22, 31.33, 23.90, 22.69, 21.81, 19.70, 13.59.


214

65

9.3

05

68

1.2

60

0

500

1000

1500

2000

2500

Inte

ns.

[a

.u.]

450 500 550 600 650 700 750 800 850 900 950m/z

Figure A-45. MALDI-MS spectrum for Fmoc-Lys(mTEG)2-OH.

215

Synthesis of Nα-Fmoc-N-Boc-N-mTEG1-L-Lysine.

Analytical Data


Procedure

Fmoc-Lys(NHBoc)-OH (0.763 g, 1.629 mmol) was dissolved in TFA:DCM (3:1) v/v

with stirring at room temperature (22-27 oC). The reaction mixture was stirred for 2 h. The

reaction was diluted with DCM and neutralized with saturated aqueous sodium carbonate. The

organic layer was isolated and concentrated to yield crude Fmoc-Lys(NH2)-OH.

Method A: A solution of oxalyl chloride (0.24 mL, 2.714 mmol) in dry DCM (20 mL)

prepared and cooled in dry ice/acetone under N2 (g) and stirred for 15 mins. DMSO (0.29 mL,

4.071 mmol) in dry DCM was added and stirred at -78 oC for 20 mins. TEGME (0.062 mL,

0.385 mmol) was added slowly and the reaction mixture stirred for 30 mins. Triethylamine (1.14

mL, 8.142 mmol) added slowly over 1 min and the reaction mixture was stirred for 30 mins at -

216

78 oC before warming to room temperature (22-27 oC) slowly. The reaction mixture was

concentrated under a stream of N2 (g).

A solution of crude Fmoc-Lys(NH2)-OH (600 mg, 1.629 mmol) with crude TEGME-

aldehyde was prepared in ACN (20 mL) and stirred. Solid NaCNBH3 (0.256 g, 4.071 mmol)

was added to the stirring reaction and allowed to stir for 18 h at room temperature (22-27 oC).

The reaction was quenched with 0.012 M HCl, followed by the addition of brine, and DCM. The

organic layer was separated and concentrated to yield 1.95 g of crude material. The crude

product was purified by column chromatography on silica gel using dichloromethane-methanol

(0-10% methanol in dichloromethane) as eluent to obtain desired product.

Analytical Data

1H NMR (500 MHz, Chloroform-d) δ 11.27 (s, 1H), 9.33 (s, 1H), 7.68 (d, J = 7.4 Hz, 2H), 7.57

(t, J = 8.0 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.25 (dt, J = 7.5, 3.8 Hz, 2H), 6.11 (d, J = 8.0 Hz,

1H), 4.35 – 4.19 (m, 3H), 4.15 (t, J = 7.2 Hz, 1H), 3.81 (t, J = 5.0 Hz, 2H), 3.61 – 3.50 (m, 8H),

3.44 (q, J = 6.3, 3.9 Hz, 3H), 3.31 – 3.28 (m, 1H), 3.26 (s, 3H), 2.94 (q, J = 7.3, 6.8 Hz, 2H),

1.90 – 1.63 (m, 4H), 1.46 – 1.37 (m, 2H).

217



127.65, 127.09, 125.28, 125.24, 119.85, 77.70, 77.44, 77.21, 77.19, 72.48, 72.46, 71.84, 71.74,

71.53, 70.41, 70.24, 70.13, 70.06, 70.05, 69.99, 66.84, 65.83, 61.34, 61.29, 58.78, 58.72, 53.69,

52.33, 47.47, 47.07, 40.74, 31.23, 25.33, 22.57, 22.30.

218


219

51

3.5

53

53

5.4

82

55

1.4

19

0

1000

2000

3000

4000

Inte

ns.

[a.

u.]

450 500 550 600m/z

Figure A-48. MALDI-MS spectrum for Fmoc-Lys(mTEG)1-OH. Calculated m/z =

515.62 [M+H]+. Observed m/z = 513.55 [M+H]+, 535.48 [M+Na]+, 551.41 [M+K]+.

(Nα-Fmoc-Nε-Boc-Lys(mTEG)1-OH)

Procedure

To a stirring solution of Fmoc-Lys(mTEG)1-OH (200 mg, 0.388 mmol) in tBuOH (10

mL) was added DIEA (0.35 mL, 1.943 mmol). Subsequently, Boc2O (0.53 mL, 2.332 mmol) was

added and the reaction mixture was stirred at room temperature (22-27 oC) for 72 h. The solvent

220

was evaporated under reduced pressure. The crude product was purified by column

chromatography on silica gel using 30% EtOAc (in hexane), 60% EtOAc (in hexane), then

dichloromethane-methanol (0-2% methanol in dichloromethane) as eluent to obtain product. The

desired fraction was concentrated to yield the desired product (10 mg, 4% yield).

Analytical Data

H2O

Chloroform

Figure A-49. 1H NMR spectrum for Nα-Fmoc-Nε-Boc-Lys(mTEG)1-OH.


127.72, 127.09, 125.12, 120.00, 79.12, 71.92, 70.61, 70.58, 70.55, 68.89, 67.02, 66.97, 64.40,

64.24, 59.02, 53.75, 52.46, 47.19, 40.10, 32.21, 29.63, 29.56, 29.34, 28.45, 28.36, 28.04, 22.33.

221

Figure A-50. 13C NMR spectrum for Nα-Fmoc-Nε-Boc-Lys(mTEG)1-OH.

222

65

1.2

92

63

5.3

42

54

8.8

33

59

5.3

20

0

200

400

600

800

1000

Inte

ns.

[a

.u.]

550 600 650 700 750m/z

Figure A-51. MALDI-MS spectrum for Nα-Fmoc-Nε-Boc-Lys(mTEG)1-OH. Calculated

m/z = 635.74 [M+Na]+. Observed m/z = 635.34 [M+Na]+.

223

APPENDIX B

(PEPTIDES)

Data shown in this section (HPLC, MALDI-MS, CD) were collected to help identify the desired

products, purity of samples, and/or -helical content.

General

Synthesis. Manual synthesis was used to couple Fmoc amino acids (8 equiv), applying

2-chlorotrityl chloride resin (1.11 mmol/g loading) or rink amide resin (0.5 mmol/g loading) and

standard DIC/6-Cl-HOBt activation. Coupling of mTEGylated amino acids to a growing peptide

sequence was extended from our typical 5-minute coupling time up to 10 minutes.

Sample calculation for amount of PEG amino acid (AA) to use during Fmoc SPPS (8

equiv. PEG AA): 0.1 g (amount of resin) * 0.5 mmol/g (resin loading) * 1 mol/1000 mmol

(conversion factor) * 8 mol AA/1 mol resin * 514.58 g/mol (MW of Fmoc-Gln(mTEG)1-OH) =

206 mg needed for peptide synthesis.

Cleavage. Cleavage of the peptides from the resin and global deprotection was

performed in trifluoroacetic acid/triisopropylsilane/water (90:5:5). Subsequently, concentration

in vacuo, precipitation with diethyl ether, and purification by preparative reversed-phase (C18)

HPLC yielded the mPEGylated peptides in pure form. We did not observe -elimination products

of the peptides, nor any truncations due to insufficient coupling of building blocks 12, 13, 16,

and 19.

224

Yield Calculation. Theoretical yield of peptides were calculated following the following

formula: Theoretical yield = resin loading capacity (mmol/g) * amount of resin used (g) *

conversion factor (1 mol/1000 mmol) * molecular weight of peptide (g/mol). Sample calculation:

0.5 mmol/g * 0.1 g * 1053 g/mol * 1 mol/1000 mmol = 0.0526 g = 52.6 mg (100% theoretical

yield). A 20% yield of peptide from this sample calculation would yield 21 mg.

Peptide Concentration Measurements. Concentrations of stock solutions of peptides

were determined by UV absorbance. The UV absorbance method involved measuring the

absorbance of peptides containing tryptophan in 0.15 M NaCl, pH 7.0, and then calculating the

molar concentration from 280nm = 5590 M-1 cm-1 for tryptophan peptides.

Circular Dichroism Measurements. CD measurements were made using an JASCO

J810 spectropolarimeter. Measurements were made in a 1.0-cm quartz cell in 0.15 M NaCl, 0 oC,

pH 7. CD measurements are reported as mean residue ellipticity at a given wavelength of peptide

samples and as molar ellipticity for tryptophan model compounds; all measurements are reported

in degrees’ centimeter squared per decimole.

225

Peptide Synthesis and Analysis

Figure B-1. MALDI spectrum of Lypressin (L8VP). Cys-Tyr-Phe-Gln-Asn-Cys-Pro-

Lys-Gly-NH2. Calculated m/z = 1057.23 [M+H]+. Observed m/z = 1058.21 [M+H]+.

226

Figure B-2. Analytical HPLC data for crude Lypressin (L8VP). Sample was injected

onto a C18 analytical column (4.6 mm x 10 cm) and eluted using isocratic solution of 5%

acetonitrile in water for 2 minutes, followed by a linear gradient of 5-95% acetonitrile in

water (constant 0.1% TFA) over 25 minutes. Desired product elutes at ~6 minutes. Cys-

Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2.

227

228

12

02

.04

9

12

22

.04

1

12

40

.09

1

0

50

100

150

200

250

300In

ten

s. [

a.u

.]

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z

Figure B-3. MALDI spectrum of mTEG1 Gln4-L8VP. Cys-Tyr-Phe-Gln(PEG)1-Asn-Cys-Pro-

Lys-Gly-NH2. Calculated m/z = 1203.42 [M+H]+. Observed m/z (crude sample) = 1204.87

[M+H]+. Observed m/z (purified sample) = 1202.05 [M+H]+.

229

Figure B-4.1. Analytical HPLC data for purified mTEG1 Gln4-L8VP. Sample was

injected onto a HASIL C18 analytical column (100 mm x 4.6 cm) and eluted using

isocratic solution of 5% acetonitrile in water for 2 minutes, followed by a linear gradient

of 5-20% acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2

minutes, a linear gradient of 20-40% over 5 minutes, 40% for 2 minutes, a linear gradient

of 40-95% over 8 minutes. Desired product elutes at ~11.5 minutes. Cys-Tyr-Phe-

Gln(mTEG)1-Asn-Cys-Pro-Lys-Gly-NH2.

Datafile Name:TH21 24m frac1.lcdSample Name:TH21 24m frac Sample ID:TH21 24m frac

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 min

0

10

20

30

40

50

60

70

80

90

100

110

120

130

mV

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

psi(x10,000)B.ConcDetector A 220nm

230

Figure B-4.2. Analytical HPLC data for purified mTEG1 Gln4-L8VP. Sample was

injected onto a C18 analytical column (250 x 10 mm) and eluted using isocratic solution

of 5% acetonitrile in water for 3 minutes, followed by a linear gradient of 5-20%

acetonitrile in water (constant 0.1% TFA) over 12 minutes, 20% acetonitrile for 4

minutes, a linear gradient of 20-40% over 10 minutes, 40% for 4 minutes, a linear

gradient of 40-95% over 16 minutes. Desired product elutes at ~27.6 minutes. Cys-Tyr-

Phe-Gln(mTEG)1-Asn-Cys-Pro-Lys-Gly-NH2.

231

Figure B-5. MALDI spectrum of PEG2 Gln4-L8VP. Cys-Tyr-Phe-Gln(mTEG)2-Asn-

Cys-Pro-Lys-Gly-NH2. Calculated m/z = 1349.60 [M+H]+. Observed m/z = 1350.84

[M+H]+, 1372.29 [M+Na]+, 1388.24 [M+K]+.

232

Datafile Name:TH14 29.5m frac 1.lcdSample Name:TH14 29.5m frac Sample ID:TH14 29.5m frac

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

mV

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4


Figure B-6. Analytical HPLC data for purified PEG2 Gln4-L8VP. Sample was injected

onto a C18 analytical column (250 x 10 mm) and eluted using isocratic solution of 5%


water (constant 0.1% TFA) over 12 minutes, 20% acetonitrile for 4 minutes, a linear

gradient of 20-40% over 10 minutes, 40% for 4 minutes, a linear gradient of 40-95% over

16 minutes. Desired product elutes at ~30.2 minutes. Cys-Tyr-Phe-Gln(mTEG)2-Asn-

Cys-Pro-Lys-Gly-NH2.

233

Figure B-7. MALDI spectrum of PEG Lys4-L8VP. Cys-Tyr-Phe-Lys(mTEG)3-Asn-

Cys-Pro-Lys-Gly-NH2. Calculated m/z (with disulfide bond) = 1495.83 [M]+. Calculated

m/z (without disulfide bond) = 1497.85 [M]+. Observed m/z = 1497.85 [M]+.

234

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0mV

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%

B.Conc.(Method)Detector A:260nm

Figure B-8. PEG Lys4-L8VP. Cys-Tyr-Phe-Lys(mTEG)3-Asn-Cys-Pro-Lys-Gly-NH2.

Description: HAISIL 100 C18 5 m. Dimensions: 100 x 4.6 mm. Particle size: 5 m.

Pore size: 80angstroms. Mobile phase 5-95% Acetonitrile in Water. Flow Rate: 1

mL/min. Temp: 25 oC. Injection: 10 L. Desired products elutes at ~2.5-3.5 minutes.

Peptide was in 75% water/25%ACN before injection.

235

Figure B-9. MALDI spectrum of mTEG Lys8-L8VP. Cys-Tyr-Phe-Gln-Asn-Cys-Pro-

Lys(mTEG)3-Gly-NH2. Calculated m/z (with disulfide bond) = 1494.78 [M]+. Calculated

m/z (without disulfide bond) = 1496.80 [M]+. Observed m/z = 1497.37 [M]+.

236

Datafile Name:TH10 30.1m frac 1.lcdSample Name:TH10 30.1m frac Sample ID:TH10 30.1m frac

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 min

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mV

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4


Figure B-10. Analytical HPLC data for purified mTEG3 Lys8-L8VP. Sample was





gradient of 40-95% over 16 minutes. Desired product elutes at ~30.8 minutes. Cys-Tyr-

Phe-Gln-Asn-Cys-Pro-Lys(mTEG)3-Gly-NH2.

237

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min-10.0

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0

42.5

45.0

mV

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5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%


Figure B-11. mTEG Lys8-L8VP. Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys(mTEG)3-Gly-

NH2. Description: HAISIL 100 C18 5 m. Dimensions: 100 x 4.6 mm. Particle size: 5

m. Pore size: 80 angstroms. Mobile phase 5-95% Acetonitrile in Water. Flow Rate: 1

mL/min. Temp: 55 oC. Injection: 10 L. Desired product elutes at ~8 min. Peptide was in

95% water/5%ACN before injection.

238

Figure B-12. MALDI spectrum of (mTEG)3-Asn5-Lypressin. Cys-Tyr-Phe-Gln-Asn

(mTEG)3-Cys-Pro-Lys-Gly-NH2. Calculated m/z (with disulfide bond)= 1537.76 [M]+.

Observed m/z = 1537.41 [M]+.

239

Figure B-13. MALDI-MS of NDFQKQQKQA (Sup35...127-136). Non-human amyloid

sequence.

240

Figure B-14. MALDI-MS of crude peptide.

HAEGTFTSDVSSYLEGQ(mTEG)AAKEFIAWLVKGR. Calculated m/z = 3444.88.

Observed m/z = 3446.757.

Figure B-15. RP-HPLC profile of crude GLP-1(Q23) 7-36 amide.

HAEGTFTSDVSSYLEGQ(mTEG)AAKEFIAWLVKGR. Wavelength = 254 nm.

241

Injection volume = 20 μL. 0-100% ACN over 10 mins. 100% ACN from 10-20 mins.

Purity = 54.7%

Figure B-16. MALDI-MS of GLP-1(Q9) 7-36 amide.

HAQ(mTEG)GTFTSDVSSYLEGQAAKEFIAWLVKGR. Calculated m/z = 3443.89.

Observed m/z = 3446.261.

242

Figure B-17. RP-HPLC profile of GLP-1(Q9) 7-36 amide.

HAQ(mTEG)GTFTSDVSSYLEGQAAKEFIAWLVKGR. Wavelength = 254 nm.

Injection volume = 15 μL. 0-100% ACN over 10 mins. 100% ACN from 10-20 mins.

Purity = 74.6%

243

Figure B-18. RP-HPLC of control solution. Wavelength = 254 nm. Injection volume =

20 μL. 0-100% ACN over 10 mins. 100% ACN from 10-20 mins

17

83

.26

3

17

11

.22

6

15

82

.18

1

16

53

.20

4

0

200

400

600

800

Inte

ns.

[a

.u.]

1400 1450 1500 1550 1600 1650 1700 1750 1800m/z

Figure B-19. MALDI-MS of crude peptide. Ac-WG(EAAAK)3A-NH2. Calculated m/z

= 1784.99. Observed m/z = 1783.263.

244

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

mV

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%


Figure B-20. Analytical HPLC of crude peptide. Ac-WG(EAAAK)3A-NH2. Calculated

m/z = 1784.99. Observed m/z = 1783.263. Sample was injected onto a C18 analytical

column (150 mm x 4.6 mm) and eluted using isocratic solution of 5% acetonitrile in

water for 2 minutes, followed by a linear gradient of 5-20% acetonitrile in water (constant

0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a linear gradient of 20-40%

over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8 minutes. Desired

product elutes at ~11 minutes.

245

22

25

.80

9

22

52

.88

60

100

200

300

Inte

ns.

[a

.u.]

1950 2000 2050 2100 2150 2200 2250 2300 2350m/z

Figure B-21. MALDI-MS of crude peptide. Ac-

WGEAAAKEAAAK(mTEG)3EAAAKA-NH2. Calculated m/z = 2224.56. Observed m/z

[M+H]+= 2225.81.

246

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-25

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500mV

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%


Figure B-22. Analytical HPLC of crude peptide. Ac-


= 2221.769. Sample was injected onto a C4 analytical column (150 mm x 2.1 cm) and

eluted using isocratic solution of 5% acetonitrile in water for 2 minutes, followed by a

linear gradient of 5-20% acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20%

acetonitrile for 2 minutes, a linear gradient of 20-40% over 5 minutes, 40% for 2 minutes,

a linear gradient of 40-95% over 8 minutes.

247

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

600

625

mV

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%




= 2221.769. Sample was injected onto a C18 analytical column (150 mm x 4.6 mm) and




a linear gradient of 40-95% over 8 minutes. Desired product elutes at ~14.5 minutes.

248

20

78

.59

7

21

06

.99

4

0

200

400

600

800

Inte

ns.

[a

.u.]

1800 1850 1900 1950 2000 2050 2100 2150 2200 2250m/z



= 2078.60.

249

19

29

.28

2

0

200

400

600

800In

ten

s. [

a.u

.]

1800 1850 1900 1950 2000 2050 2100 2150 2200 2250m/z



= 1929.28.

250

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

mV

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

%




= 1928.993. Sample was injected onto a C18 analytical column (150 mm x 4.6 mm) and




a linear gradient of 40-95% over 8 minutes. Desired product elutes at ~13.3-13.5 minutes.

251

Figure B-27. Preliminary dichroic spectra of the peptides

AcWGEAAAKEAAAKEAAAKA-NH2 (TH23 unmodified, 72.4% -helix),

AcWGEAAAKEAAAK(mTEG)1EAAAKA-NH2 (TH25 monoPEG, 2.1% -helix),

AcWGEAAAKEAAAK(mTEG)2EAAAKA-NH2 (TH25 diPEG, 31.9% -helix), and

AcWGEAAAKEAAAK(mTEG)3EAAAKA-NH2 (TH24 triPEG, 19.3% -helix). Peptide

concentrations were 30 M (except diPEG 6.7M). All solvents contained 150 mM NaCl,

pH 7.0. Spectra recorded at 2 oC. Spectra collected on each sequence after first HPLC

purification.

252

APPENDIX C

(LACTAM SYNTHESIS)

Data shown in this section were collected to help identify the desired products and purity of

samples.

NMR and MALDI Spectra

General. Column chromatography was performed on silica gel 60, particle size 0.040-0.063

mm. 1H and 13C NMR spectra were recorded on either a Bruker (300 MHz or 500 MHz) or

JEOL spectrometer (500 MHz).

Figure C-1. 1H NMR comparison of crude product mixture from diazotization attempt on 5-

aminovalericacid at 5 equiv NaBr/2 equiv NaNO2 (Top) and 10 equiv NaBr/5 equiv NaNO2

(Bottom). Reagents and conditions: a) NaNO2, NaBr, 16% HBr, 0oC, 1 h.

253

Figure C-2. 1H NMR of crude product mixture from diazotization of -alanine at 5 equiv

NaBr/2 equiv NaNO2 (Top) and 10 equiv NaBr/5 equiv NaNO2 (Bottom). Reagents and

conditions: a) NaNO2, NaBr, 16% HBr, 0oC, 1 h.

Scheme C-1. Attempt to remove Boc and perform diazotization yielded no removal of Boc and

no desired product. Starting material recovered. (PH0019, PH0021) reagents and conditions: a)

5 eq NaNO2, 20 eq NaBr, 48% HBr,TFA, -6 oC to r.t. or 3 eq NaNO2, 10 eq NaBr, 2.5M H2SO4,

254

0 oC-r.t, 66 hrs.

Synthesis of Product X (Unknown product from attempted diazotization and subsequent

reaction with Nspe).

Procedure

Fmoc-Orn(NH2)-OH and NaBr were dissolved in 16-48% HBr with stirring at 0 oC. A

solution of NaNO2 was added slowly to the stirring reaction mixture. The reaction was stirred for

1 h. The reaction was diluted with EtOAc and the organic layer separated. The aqueous layer

was extracted twice more with EtOAc and the organic layers were combined, dried over sodium

sulfate, and concentrated.

The crude product was dissolved in dry ACN and Nspe was added. The reaction mixture

allowed to stir for 18 h at room temperature. The solvent was concentrated and the crude

product purified by flash chromatography using silica gel.

Scheme C-2. Proposed synthetic route with protection of carboxylic acid as a methyl ester.

Reagents and conditions: a) T3P, MeOH, 27.3%. b) 3:1 (v/v) TFA:DCM. c) 5 eq NaNO2, 20 eq

NaBr, 48% HBr, 0 oC.

255

Analytical Data

63

2

66

56

99

75

07

63

80

98

53

88

49

05

10

00

10

19

10

59

10

78

11

25

11

78

12

69

13

31

13

97

14

15

14

29

14

48

14

75

14

91

15

09

15

25

15

43

15

60

15

98

16

27

16

38

16

55

16

86

17

01

17

19

17

35

17

51

17

74

18

13

18

46

18

71

23

43

23

63

28

52

29

22

33

8535

28

35

47

35

68

35

88

36

19

36

30

36

50

80

82

84

86

88

90

92

94

96

98

100

%T

1000 1500 2000 2500 3000 3500

Wav enumbers (cm-1)

Figure C-3. IR spectrum of Product X.

1H NMR (500 MHz, Chloroform-d) δ 7.81 – 7.77 (m, 1H), 7.66 – 7.57 (m, 3H), 7.53 (ddd, J =

8.1, 3.1, 1.8 Hz, 2H), 7.42 – 7.33 (m, 2H), 7.29 (dtd, J = 8.6, 4.1, 1.8 Hz, 3H), 3.92 (t, J = 6.3

Hz, 1H), 3.81 (q, J = 6.6 Hz, 1H), 3.08 (dd, J = 12.2, 5.8 Hz, 1H), 2.86 (dd, J = 12.2, 6.8 Hz,

1H), 1.34 (d, J = 6.6 Hz, 3H).

256

Figure C-4. 1H NMR spectrum for Product X. Top – full spectrum. Bottom – expanded region

of the 1H NMR.

257

13C NMR (126 MHz, CDCl3) δ 147.83, 147.78, 145.34, 139.31, 139.21, 133.13, 130.54, 130.48,

128.70, 128.60, 128.52, 128.47, 128.35, 128.14, 128.07, 127.05, 126.71, 125.90, 121.24, 121.17,

121.10, 77.47, 77.35, 77.30, 77.10, 76.84, 58.33, 50.39, 49.97, 48.19, 26.67, 24.70.

Figure C-5. 13C NMR spectrum for Product X.

258

Figure C-6. COSY for Product X.

259

Predicted Spectra.

Figure C-7. Predicted spectrum for desired final product.

260

Figure C-8. Predicted 1H NMR spectrum for the structures shown on spectrum.

261

Figure C-9. Expanded region of predicted 1H NMR.

262

Synthesis of (1R)-3-[(R)-1-Phenylethyl]-2-oxocyclohexylamino (9H-fluoren-9-yl)acetate.

NFmocHN

O

Vinyl-(R)-5-[(S)-1-phenylethylamino]-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-5-

oxovalerate (Fmoc-Gln(Nspe)-OAll)

FmocHN

OHN

O

O

Procedure

To a stirring solution of Fmoc-Glu(OtBu)-OH (2.0 g, 4.7 mmol) and DIEA (1.64 mL, 9.4

mmol) in allyl bromide (15 mL) was prepared. The reaction mixture was heated to reflux for 1 h.

Upon cooling, the resulting reaction mixture was poured into EtOAc (200 mL) and the

precipitated DIEA-HCl was filtered. The filtrate was washed with 0.01 M HCl (1 x), saturated

aqueous sodium bicarbonate (1 x), and then with water (1 x). The organic layer containing crude

Fmoc-Glu(OtBu)-OAll was isolated dried over sodium sulfate, filtered, and concentrated.

The crude product was dissolved in TFA:DCM (20 mL, 1:1, v/v) with stirring at room

temperature for 1.5 h. The reaction was concentrated to yield crude Fmoc-Glu(OH)-OAll.

The crude Fmoc-Glu(OH)-OAll was dissolved in DCM (50 mL) and DIEA (0.55 g)

added. A subsequent addition of CDI (1.03 g) was made and the reaction mixture was stirred for

263

10 min. (S)-(-)-phenylethylamine was added and the reaction mixture was stirred for 30 min. The

reaction mixture was concentrated under reduced pressure.

Desired product purified by flash chromatography on silica gel using dichloromethane-

methanol (0-5% methanol in dichloromethane) as eluent to obtain desired product. The desired

fractions were combined and concentrated. The product was isolated as a pale yellow solid (1.87

g, 77.6% yield over 3 steps).

Analytical Data –

Figure C-10. 1H NMR spectrum for Fmoc-Gln(Nspe)-OAll.


141.41, 141.38, 131.49, 128.76, 127.82, 127.46, 127.18, 126.31, 125.86, 125.21, 120.08, 119.42,

119.22, 67.11, 66.28, 55.39, 53.59, 50.26, 49.05, 47.27, 32.62, 29.21, 28.79, 24.89, 23.47, 21.93.

264

Figure C-11. 13C NMR spectrum for Fmoc-Gln(Nspe)-OAll.

51

1.5

20

54

9.3

79

53

3.4

47

48

5.5

84

0

500

1000

1500

2000

2500

3000

Inte

ns.

[a

.u.]

400 450 500 550 600 650m/z

Figure C-12. MALDI-MS spectrum for Fmoc-Gln(Nspe)-OAll.

265

266

Vinyl-(R)-5-[(S)-1-phenylethylamino]-2-[(9H-fluoren-9-yl)methoxycarbonylamino]valerate

(Fmoc-Orn(Nspe)-OAll)

FmocHN

HN

O

O

Procedure

To a stirring solution of Fmoc-Glu(Nspe)-OAll (50 mg, 0.10 mmol) in DCM (3 mL) at 0

oC was added trifluoromethanefulfonic anhydride (0.02 mL, 0.11 mmol). The reaction mixture

stirred for 30 min. The reaction was brought to room temperature (20 oC) before diluting with

THF (6 mL). To the stirring reaction mixture was added NaBH4 (5 mg, 0.13 mmol). The reaction

was stirred for 1 h at room temperature before quenching with water. The desired product

(Fmoc-Orn(Nspe)-OAll) was not purified and isolated.

267

Analytical Data –

49

7.3

91

44

0.5

77

41

8.6

64

0

100

200

300

400

500

Inte

ns.

[a

.u.]

400 420 440 460 480 500 520 540 560 580m/z

Figure C-13. MALDI-MS spectrum for Fmoc-Orn(Nspe)-OAll.

268

(R)-3-[(R)-1-Phenylethyl]-2-oxocyclohexylamino (9H-fluoren-9-yl)acetate.

NFmocHN

O

Procedure

A solution of Fmoc-Orn(Nspe)-OAll (0.57 g, 1.143 mmol), DIEA (0.25 mL, 2.286

mmol), and NaHCO3 (0.066 g, 0.057 mmol) in DMF (20 mL). The reaction mixture was

brought to 90 oC and stirred for 18 h. The reaction was diluted with water and DCM. The organic

layer was isolated and concentrated under reduced pressure. Product not purified for 1H and 13C

NMR.

269

Analytical Data –

44

3.5

12

42

1.6

09

37

1.0

07

51

0.3

55

0

200

400

600

800

1000

Inte

ns.

[a

.u.]

350 400 450 500 550 600m/z

Figure C-14. MALDI-MS spectrum for (R)-3-[(R)-1-Phenylethyl]-2-

oxocyclohexylamino (9H-fluoren-9-yl)acetate from preliminary text reaction. Calculated

m/z = 441.54 [M+H]+. Observed m/z = 443.51 [M+H]+.

270

APPENDIX D

(PEPTOIDS)

Data shown in this section (HPLC, MALDI-MS, NMR) were collected to help identify the

desired products and purity of peptoid samples.

MALDI and HPLC Spectra

HNN

O

N

O

NH2

O

H2N

271

43

2.7

58

55

8.4

95

51

6.5

43

45

4.6

56

37

7.7

49

47

0.5

78

65

0.2

95

59

5.3

51

32

8.8

02

43

9.9

97

0

2000

4000

6000

8000In

ten

s. [

a.u

.]

350 400 450 500 550 600 650 700m/z

Figure D-1. Top - MALDI spectrum of 1. Calculated m/z = 434.00 [M+H]+. Observed m/z

= 432.75 [M+H]+. Bottom – Analytical HPLC of crude 1. Sample was injected onto a C4

analytical column (150 mm x 2.1 cm) and eluted using isocratic solution of 5% acetonitrile

in water for 2 minutes, followed by a linear gradient of 5-20% acetonitrile in water

272

(constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a linear gradient of

20-40% over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8 minutes.

Desired product elutes at ~12 minutes.

HNN

O

N

O

NH2

O

H2N n

n = 3, C8

46

0.7

82

58

6.5

70

48

2.6

93

57

2.5

76

37

7.8

31

49

8.6

24

67

8.4

01

65

8.2

98

62

3.4

54

34

7.1

41

63

8.4

43

0

1000

2000

3000

4000

5000

6000

Inte

ns.

[a

.u.]

350 400 450 500 550 600 650 700m/z

273

Figure D-2. Top - MALDI spectrum of 2. Calculated m/z = 461.65. Observed m/z = 460.782

[M+K]+. Bottom – Analytical HPLC of crude 2. Sample was injected onto a C4 analytical

column (150 mm x 2.1 cm) and eluted using isocratic solution of 5% acetonitrile in water for 2

minutes, followed by a linear gradient of 5-20% acetonitrile in water (constant 0.1% TFA) over 3

minutes, 20% acetonitrile for 2 minutes, a linear gradient of 20-40% over 5 minutes, 40% for 2

minutes, a linear gradient of 40-95% over 8 minutes. Desired product elutes at ~13.5 minutes.

HNN

O

N

O

NH2

O

H2N n

n = 7, C12

274

51

6.5

90

37

7.7

91

22

7.6

56

62

0.3

61

55

4.4

48

21

1.8

22

53

8.5

18

68

4.3

50

24

4.6

71

26

2.5

44

0

500

1000

1500

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600 650m/z

Figure D-3. Top - MALDI spectrum of 3. Calculated m/z = 517.76, 557 [M+K]+.

Observed m/z = 516.59 [M+H]+. MC26. Bottom – Analytical HPLC of crude 3. Sample

275

was injected onto a C4 analytical column (150 mm x 2.1 cm) and eluted using isocratic

solution of 5% acetonitrile in water for 2 minutes, followed by a linear gradient of 5-20%

acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a

linear gradient of 20-40% over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95%

over 8 minutes. Desired product elutes at ~17 minutes.

NN

O

N

O

NH2

O

NH2

H2N

46

1.6

88

30

3.3

54

48

3.5

99

51

6.5

52

37

7.7

58

22

7.6

01

21

1.7

65

58

7.4

64

62

4.3

35

0

1000

2000

3000

4000

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600 650m/z

276

Figure D-4. MW = 462.64. Top - MALDI spectrum of 4. Calculated m/z = 463.64

[M+H]+. Observed m/z = 461.68 [M+H]+. Bottom – Analytical HPLC of crude 4. Sample

was injected onto a C4 analytical column (150 mm x 2.1 cm) and eluted using isocratic


acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes,

a linear gradient of 20-40% over 5 minutes, 40% for 2 minutes, a linear gradient of 40-

95% over 8 minutes. Desired product elutes at ~11 minutes.

NN

O

N

O

NH2

O

NH2 n

n = 3, C8

H2N

277

48

9.6

97

57

2.5

54

51

1.6

21

61

5.5

24

0

1000

2000

3000

Inte

ns.

[a

.u.]

350 400 450 500 550 600 650 700m/z

Figure D-5. Top - MALDI spectrum of 5. Calculated m/z = 491.69 [M+H]+. Observed

m/z = 489.697 [M+H]+. Bottom – Analytical HPLC of crude 5. Sample was injected onto

a C4 analytical column (150 mm x 2.1 cm) and eluted using isocratic solution of 5%

278



gradient of 20-40% over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8

minutes. Desired product elutes at ~13.5 minutes.

NN

O

N

O

NH2

O

NH2 n

n = 7, C12

H2N

54

5.6

34

56

7.5

75

68

4.5

14

58

3.5

25

0

500

1000

1500

2000

Inte

ns.

[a

.u.]

350 400 450 500 550 600 650 700m/z

279

Figure D-6. Top - MALDI spectrum of 6. Calculated m/z = 547.80 [M+H]+. Observed m/z

= 545.63 [M+H]+. Bottom – Analytical HPLC of crude 6. Analytical HPLC data for crude

peptoid. Sample was injected onto a C4 analytical column (150 mm x 2.1 cm) and eluted

using isocratic solution of 5% acetonitrile in water for 2 minutes, followed by a linear

gradient of 5-20% acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20%


a linear gradient of 40-95% over 8 minutes. Desired product elutes at ~17.5 minutes.

HNN

O

N

O

NH2

O

H2N

280

52

2.6

23

60

6.4

97

54

4.5

49

64

8.4

78

56

0.4

85

37

7.8

26

62

8.4

38

53

6.6

01

65

8.2

85

40

8.8

92

0

1000

2000

3000

4000In

ten

s. [

a.u

.]

350 400 450 500 550 600 650 700m/z

Figure D-7. Top - MALDI spectrum of 7. Calculated m/z = 524.73 [M+H]+, 562.73 [M+K]+.

Observed m/z = 522.623 [M+H]+, 560.485 [M+K]+. Bottom – Analytical HPLC of crude 7.

Sample was injected onto a C4 analytical column (150 mm x 2.1 cm) and eluted using isocratic

281


acetonitrile in water (constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a

linear gradient of 20-40% over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8


HNN

O

N

O

NH2

O

H2N

n = 3, C8

n

55

0.5

49

30

3.3

90

0

200

400

600

800

1000

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600 650m/z

282

Figure D-8. MW = 551.78. Top - MALDI spectrum of 8. Calculated m/z = 552.78 [M+H]+.

Observed m/z = 550.54 [M+H]+. Bottom – Analytical HPLC of crude 8. Sample was injected

onto a C4 analytical column (150 mm x 2.1 cm) and eluted using isocratic solution of 5%

acetonitrile in water for 2 minutes, followed by a linear gradient of 5-20% acetonitrile in water

(constant 0.1% TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a linear gradient of 20-40%

over 5 minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8 minutes. Desired product

elutes at ~16 minutes.

HNN

O

N

O

NH2

O

H2N

n = 7, C12

n

283

60

6.4

92

30

3.3

90

0

100

200

300

400In

ten

s. [

a.u

.]

200 250 300 350 400 450 500 550 600 650m/z

Figure D-9. MW = 607.46. Top - MALDI spectrum of 9. Calculated m/z = 608.46 [M+H]+.



284




elutes at ~19.5 minutes.

NN

O

N

O

NH2

O

NH2

H2N

285

Figure D-10. Top - MALDI spectrum of 10. Calculated m/z = 553.77 [M+H]+. 575.7 [M+Na]+.






elutes at ~5 minutes.

286

n = 3, C8

n

NN

O

N

O

NH2

O

NH2

H2N

57

9.5

32

60

1.4

67

66

2.4

38

37

7.8

18

21

1.7

99

61

7.4

12

30

3.4

03

22

7.6

44

0

1000

2000

3000

4000

5000

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600 650m/z

287







minutes. Desired product elutes at ~16 minutes.

n = 7, C12

n

NN

O

N

O

NH2

O

NH2

H2N

288

63

5.4

51

65

7.3

94

30

3.3

86

0

500

1000

1500

Inte

ns.

[a

.u.]

200 300 400 500 600 700m/z




289





NH

O

N

NH2

H2N

O

C6

19

8.9

59

15

9.4

68

34

8.0

97

21

1.7

87

17

2.2

05

43

1.7

98

19

0.0

17

37

7.7

89

28

5.3

81

22

7.6

22

29

9.2

26

33

1.1

20

23

2.7

41

36

9.9

71

35

8.0

35

20

4.9

80

24

9.4

29

21

8.8

59

27

2.4

68

47

7.2

13

18

1.1

92

45

3.7

11

0

500

1000

1500

2000

2500

Inte

ns.

[a

.u.]

150 200 250 300 350 400 450m/z

290

Datafile Name:MC37 25.7m frac 1.lcdSample Name:MC37 25.7m frac Sample ID:MC37 25.7m frac

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 min

0

5

10

15

20

25

30

35

40

45

50

mV

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4


Figure D-13. Top - MALDI spectrum of crude 15. Calculated m/z = 349.49 [M+H]+.

Observed m/z = 348.09 [M+H]+. Bottom – Analytical HPLC of purified 15. Sample was





gradient of 40-95% over 16 minutes. Desired product elutes at ~27.4 minutes.

NH

O

N

NH2

H2N

O

C8

291

19

8.9

80

18

7.1

97

37

6.0

50

29

9.2

66

21

1.8

10

22

7.6

51

28

5.4

17

48

7.7

46

17

2.2

18

24

9.4

60

33

1.1

64

23

2.7

67

39

7.9

40

26

5.3

19

20

5.0

04

38

5.9

87

41

3.8

52

35

9.9

92

21

8.8

75

32

1.1

76

16

3.3

61

0

1000

2000

3000

4000

5000

Inte

ns.

[a

.u.]

150 200 250 300 350 400 450m/z



a C18 analytical column (250 x 10 mm) and eluted using isocratic solution of 5%

292




16 minutes. Desired product elutes at ~31.1 minutes.

NH

O

N

NH2

H2N

O

C12

293

28

5.4

04

24

2.7

55

19

8.9

66

43

1.8

75

33

1.1

48

21

1.8

02

19

1.1

22

23

2.7

55

17

2.2

16

29

9.2

46

21

8.8

64

20

4.9

92

44

1.8

21

45

6.8

07

27

2.4

91

37

7.8

19

41

6.6

11

35

5.0

27

0

500

1000

1500Inte

ns.

[a

.u.]

150 200 250 300 350 400 450m/z




294




8 minutes. Desired product elutes at ~21 minutes.

NH

O

N

NH2

H2N

O

50

0.6

40

34

0.1

19

51

0.5

85

63

3.4

07

37

7.9

54

27

7.4

61

79

4.2

89

47

2.6

68

35

0.0

58

29

9.2

60

77

8.9

94

36

8.0

20

67

1.3

55

48

5.3

53

31

1.2

41

52

2.5

54

61

8.1

20

65

5.3

53

25

5.6

79

38

6.9

95

41

5.9

21

0

1000

2000

3000

4000

Inte

ns.

[a

.u.]

300 400 500 600 700 800m/z

295


m/z = 368.02 [M+H]+. Bottom – Analytical HPLC of crude 18. Sample was injected onto a

C18 analytical column (250 x 10 mm) and eluted using isocratic solution of 5% acetonitrile

in water for 3 minutes, followed by a linear gradient of 5-20% acetonitrile in water

(constant 0.1% TFA) over 12 minutes, 20% acetonitrile for 4 minutes, a linear gradient of

20-40% over 10 minutes, 40% for 4 minutes, a linear gradient of 40-95% over 16 minutes.

Desired product elutes at ~27.7 minutes.

NH

O

N

NH2

H2N

O

296

28

5.4

22

37

7.8

14

33

1.1

52

45

7.6

81

46

7.6

30

30

3.3

30

40

2.7

56

0

500

1000

1500

2000

2500

3000

Inte

ns.

[a

.u.]

300 350 400 450 500 550m/z



297

a C18 analytical column (250 x 10 mm) and eluted using isocratic solution of 5%





N

O

N

NH2

H2N

O

NH2 C6

37

7.7

72

21

1.7

77

22

7.6

12

28

1.4

80

24

9.4

19

33

7.1

88

0

250

500

750

1000

1250

1500

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600m/z

298








N

O

N

NH2

H2N

O

NH2 C8

299

21

1.8

18

22

7.6

55

37

7.8

41

33

7.1

97

40

3.9

35

24

9.4

66

28

1.5

34

54

8.8

44

26

5.3

24

42

6.8

60

24

1.8

20

0

500

1000

1500

2000

2500

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600m/z

Analy

tical HPLC

Figure D-19. Top - MALDI spectrum of 21. Calculated m/z = 406.59 [M+H]+. Observed m/z =

300

403.93 [M+H]+. Bottom – Analytical HPLC of crude 21. Sample was injected onto a C4

analytical column (150 mm x 2.1 cm) and eluted using isocratic solution of 5% acetonitrile in

water for 2 minutes, followed by a linear gradient of 5-20% acetonitrile in water (constant 0.1%

TFA) over 3 minutes, 20% acetonitrile for 2 minutes, a linear gradient of 20-40% over 5

minutes, 40% for 2 minutes, a linear gradient of 40-95% over 8 minutes. Desired product elutes

at ~10 minutes.

N

O

N

NH2

H2N

O

NH2 C12

301

21

1.8

00

46

0.8

09

22

7.6

38

37

7.8

25

24

2.7

54

48

2.7

26

33

7.1

76

28

5.4

04

56

4.5

63

41

5.8

08

0

250

500

750

1000

1250

Inte

ns.

[a

.u.]

200 250 300 350 400 450 500 550 600m/z



302






N

O

N

NH2

H2N

O

NH2

28

5.4

13

36

8.9

82

52

9.4

91

33

1.1

47

29

9.2

55

66

2.2

45

39

6.8

81

50

1.5

38

47

1.6

10

33

7.2

57

31

1.2

35

55

1.4

19

41

8.7

86

69

0.2

31

0

1000

2000

3000

4000

Inte

ns.

[a

.u.]

300 350 400 450 500 550 600 650 700m/z

303


m/z = 396.88 [M+H]+. Bottom – Analytical HPLC of purified 23. Sample was injected

onto a C18 analytical column (250 x 10 mm) and eluted using isocratic solution of 5%





N

O

N

NH2

H2N

O

NH2

304

50

1.4

77

48

5.5

25

40

8.7

84

38

8.9

08

56

1.3

40

47

2.5

43

39

7.7

91

0

50

100

150

200

250

300

Inte

ns.

[a

.u.]

400 425 450 475 500 525 550 575 600m/z



305






N

O

HN

O

NH

NN

C6 5

64

.62

9

25

0.6

50

33

3.2

51

37

7.8

44

69

8.4

29

28

1.5

39

58

6.5

71

0

1000

2000

3000

4000

5000

Inte

ns.

[a

.u.]

200 300 400 500 600 700 800m/z

306

Figure D-23. MALDI spectrum of 27. Calculated m/z = 566.85 [M+H]+. Observed m/z =

564.62 [M+H]+.

N

O

HN

O

NH

NN

C8

59

2.5

90

50

9.6

31

25

0.6

20

13

0.8

77

61

4.5

26

36

1.1

17

27

8.4

24

21

1.7

96

72

6.4

11

23

3.7

50

64

3.3

91

49

3.6

88

19

0.0

10

0

500

1000

1500

2000

Inte

ns.

[a

.u.]

100 200 300 400 500 600 700 800m/z

Figure D-24. MALDI spectrum of 31. Calculated m/z = 594.90 [M+H]+. Observed m/z =

592.50 [M+H]+.

307

N

O

HN

O

NH

NN

C12

64

8.5

72

67

0.5

19

21

1.8

09

25

0.6

38

41

6.9

78

44

3.5

45

14

1.7

01

37

7.8

82

39

9.7

30

28

1.5

35

0

2000

4000

6000

8000

Inte

ns.

[a

.u.]

100 200 300 400 500 600 700 800m/z

308


m/z = 648.57 [M+H]+. Bottom – 1H NMR of 28.

N

O

HN

O

NH

NN

309

21

1.7

53

28

5.3

43

33

7.1

92

37

7.7

43

23

3.5

43

35

9.0

63

58

4.4

04

26

2.4

84

40

5.7

96

30

3.2

87

44

3.4

28

60

6.3

35

64

7.9

12

51

0.5

01

0

1000

2000

3000

4000

5000Inte

ns.

[a

.u.]

200 300 400 500 600 700 800m/z



310

N

O

HN

O

NH

NN

67

4.3

62

21

1.7

92

28

5.3

90

69

6.3

17

33

7.2

38

57

6.4

33

37

7.8

00

22

7.6

22

0

250

500

750

1000

1250

Inte

ns.

[a

.u.]

200 300 400 500 600 700 800m/z

311



O

NH2

N

O

N

H2N

H2N

312

31

1.2

64

50

9.6

25

64

2.3

79

37

7.0

40

53

7.5

47

40

4.9

18

28

5.4

38

29

9.2

84

31

8.8

27

39

2.9

31

0

1000

2000

3000

4000

5000

6000Inte

ns.

[a

.u.]

300 350 400 450 500 550 600 650 700m/z


m/z = 377.04 [M+H]+. Bottom – Analytical HPLC of crude 25.

313

n = 1, C6

n

NN

O

N

O

NH2

O

NH2

H2N

46

1.7

68

37

7.8

46

28

5.4

40

48

3.6

82

53

6.5

55

21

1.8

48

24

4.7

08

50

1.6

74

29

5.3

25

0.0

0.5

1.0

1.5

4x10

Inte

ns.

[a

.u.]

200 300 400 500 600 700m/z

314








n = 7, C12

NN

O

N

O

NH2

O

NH2

H2N

n

315

63

5.4

94

28

5.4

47

37

7.8

57

65

7.4

38

67

5.4

58

21

1.8

46

0

500

1000

1500

Inte

ns.

[a

.u.]

200 300 400 500 600 700m/z



316






N

O

NHHN

O N

317

53

4.6

36

54

9.6

07

58

5.4

43

0

200

400

600

800

1000In

ten

s. [

a.u

.]

475 500 525 550 575 600 625 650m/z


318


N

O

NHHN

O N

58

9.4

06

65

2.2

46

61

1.3

46

51

6.5

06

0

1000

2000

3000

4000

5000

6000

Inte

ns.

[a

.u.]

400 450 500 550 600 650m/z

319


m/z = 589.41 [M+H]+.Bottom – 1H NMR of 33.

320

LIST OF SYMBOLS AND ABBREVIATIONS

SPPS Solid phase peptide synthesis

ΔG* Gibbs free energy

NMR Nuclear magnetic resonance

PPM Parts per million

TEGME (mTEG) Tri(ethyleneglycol) monomethyl ether

COSY Correlation spectroscopy

NOESY Nuclear Overhauser effect spectroscopy

EDA Ethylene diamine

NOE Nuclear Overhauser effect

TLC Thin layer chromatography

DCM Dichloromethane

AcOEt Ethyl acetate

MeOH Methanol

AcOH Acetic Acid

AcAc Acetylacetone

Py Pyridine

Lys Lysine

Gln Glutamine

Glu Glutamic acid

Asn Asparagine

Asp Aspartic acid

T3P Propylphosphonic anhydride

321

NHS N-hydroxysuccinimide

TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl

Bipy 2,2’-Bipyridine

DIEA Diisopropylethyl amine

DMF N,N-Dimethyl formamide

OPac Phenacyl ester

NaCNBH3 Sodium cyanoborohydride

NaBH4 Sodium borohydride

CDI Carbonyldiimidazole

DCC N,N’-Dicyclohexylcarbodiimide

DIC N,N’-Diisopropylcarbodiimide

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

6-Cl-HOBt 1-Hydroxy-6-chloro-benzotriazole

HCTU O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium hexafluorophosphate

TFA Trifluoroacetic acid

Tf2O Triflic anhydride

Date post:	25-Apr-2023
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pegylated Amino Acids for Site-specific Peptide Incorporation

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