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Novel synthesis of β-glycosides for SPPS of GLCNAC glycoproteins and study of their site-specific biochemical and biophysical consequences

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Novel synthesis of β-glycosides for SPPS of GLCNAC glycoproteins and study of their site-specific biochemical and biophysical consequences

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NOVEL SYNTHESIS OF β-GLYCOSIDES FOR SPPS OF GLCNAC
GLYCOPROTEINS AND STUDY OF THEIR SITE-SPECIFIC BIOCHEMICAL
AND BIOPHYSICAL CONSEQUENCES

by

Cesar Augusto De Leon

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirement for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2018

Copyright 2018 Cesar Augusto De Leon

ii

Acknowledgements
To my wife, Berenice, for your endless support, unconditional love, and willingness to
sacrifice your education so that I could pursue mine. You are the cornerstone of my success
and the foundation to my happiness. Despite the ups and downs in our relationship, you
never gave up. I still think back to the day I saw you standing at the bus stop with your
brother. I just knew you were the one even before I met you. I love you a lot and want to
thank you for teaching me the meaning of being a true friend, soulmate, and husband.

To my three sons Tezcatlipoca, Tlalollin, and Tonatiuh. Words cannot describe the joy you
three bring into my life. I love coming home to you and teaching you guys about science,
math, and soccer. You three bring meaning and purpose to my life. You guys are the fuel
to my ambition and the reason I strive for academic excellence. I hope that you guys take
the best of both me and your mom and never give up in the pursuit of your dreams.

To my mom, Sara, for being my biggest cheerleader. You are one of kind. Your love has
no boundaries. Thank you for all the times you kept me out of trouble. I am not sure there
are too many mothers that would drive around the neighborhood for hours honking and
knocking on every door to find their son. Nor many that would financially support their
son’s children for five years, so that he and his wife could pursue their education. Although
I doubt that I am the savior of the world, I strive hard to be your John Connor. Thank you
for always believing in me. I hope I make you proud.

To my dad, Albert, for all the extraordinary experiences we shared together when I was kid
and continue to share now. Despite your absence during my early teens, you came through
when I most needed you. You are a great father and grandfather. Thank you for showing
me and my family love and compassion despite our different bloodline. I very much cherish
the times we discuss life and science over some tequila. I look forward to spending time
with you the way you do with my grandpa Antonio.

To Matt, thank you for seeing something in me and taking me under your wing. Not only
have you guided me throughout my journey in graduate school, but you entrusted me with
the freedom to explore my own curiosities. You are beyond just my advisor, you are my
friend. Thank you for helping to stay out of jail and motivating me to not leave with a
masters. I hope our communication extends beyond my time at USC and that my future
scientific success makes you proud.

iii

Table of Contents
Acknowledgments

List of Figures

List of Schemes

List of Tables

Abstract

Chapter 1. Stereoselective Glycosylations of N-Acetyl
Glucosamine
Introduction
Biological Significance of N-Acetyl Glucosamine
Chemical Glycosylation
Chemical Strategies for the Synthesis of β-glycosides
Conclusion and Future Outlook
Chapter One References

Chapter 2. Democratizing The Synthesis of β-N-acetyl-glycosides
For The Preparation of O- And S-GlcAcylated Peptides
And Proteins
Introduction
Results
Discussion
Materials and Methods
Chapter Two References

Chapter 3. O-GlcNAcylation of α-synuclein at serine 87 reduces
aggregation without affecting membrane binding
Introduction
Results
Discussion
Materials and Methods
Chapter Three References

Chapter 4. The sulfur-linked analog of O-GlcNAc (S-GlcNAc) is
an enzymatically stable and a reasonable structural-
surrogate for O-GlcNAc at the peptide and protein
levels
Introduction
Results
Discussion
ii

v

ix

x

xi

1

1
2
8
12
18
20

25

25
28
38
40
51

55

55
58
76
78
88

93

93
97
119

iv

Materials and Methods
Chapter Four References

Chapter 5. O-GlcNAc modification inhibits the calpain-mediated
cleavage of α-synuclein
Introduction
Results
Discussion
Materials and Methods
Chapter Five References

References

Appendices
Appendix A: NMR Spectra

122
131

137

137
141
145
148
152

159

178
178

v

List of Figures
Figure 1-1: GlcNAc is found as a structural unit in several
biologically relevant carbohydrates.

Figure 1-2: O-GlcNAcylation: the addition of a single unit of
GlcNAc to serine/threonine residues of intracellular
proteins.

Figure 1-3: Synthetic strategies resolve the difficulty in obtaining
homogenous samples of GlcNAcylated proteins for
functional studies.

Figure 1-4: Chemical glycosylation and general mechanistic
considerations.

Figure 1-5: Arming and disarming effect of protecting groups on
the reactivity of the leaving group and stability of
glycosyl carbocation.

Figure 1-6: Neighboring group participation.

Figure 1-7: Anomeric effect.

Figure 1-8: Koenigs-Knorr reaction.

Figure 1-9: Monovalent N-Protecting groups.

Figure 1-10: Bivalent N-Protecting groups.

Figure 1-11: Diversity in the type of leaving groups used in the
synthesis of β-glycosides.

Figure 1-12: Copper catalyzed activation of methyloxazoline for the
efficient synthesis of β-glycosides.

Figure 2-1: Increasing the sugar donor equivalents improves the
product conversion.

Figure 2-2: Lowering the catalyst loading improves the product
conversion and minimizes.

4

5

7

9

10

11

12

13

15

15

16

19

31

32

vi

Figure 2-3: Varying the reaction solvent has significant effect on
the product conversion.

Figure 2-4: Indium bromide can activate both α- and β-
Ac
4
GlcNAc.

Figure 2-5: Indium bromide can catalyze the glycosylation of the
2-methyl oxazoline.

Figure 3-1: O-GlcNAcylation and α-synuclein.

Figure 3-2: Synthesis and characterization of unmodified and O-
GlcNAcylated α-synuclein (α-synuclein(gS87).

Figure 3-3: Characterization of α-synuclein protein fragments.

Figure 3-4: Ligation of peptide 2 and protein 4 and the subsequent
deprotection to give protein fragment 5.

Figure 3-5: Ligation of peptide 3 and protein 4 and the subsequent
deprotection to give protein fragment 6.

Figure 3-6: Ligation of proteins 5 or 6 and protein-thioester 1 to
give proteins 7 or 8.

Figure 3-7: Desulfurization of proteins 7 or 8.

Figure 3-8: Structural characterization of α-synuclein(gS87) and
α-synuclein(S87E).

Figure 3-9: O-GlcNAcylation of α-synuclein at S87 inhibits
protein aggregation.

Figure 3-10: Expression and characterization of α-synuclein(S87E).

Figure 3-11: Structural characterization of synthetic and
recombinant unmodified α-synuclein using circular
dichroism (CD) and dynamic light scattering (DLS).

Figure 3-12: Transmission electron microscopy (TEM) images of
the from the α-synuclein aggregation reactions.

33

34

39

56

59

60

61

62

63

64

65

67

68

68

69

vii

Figure 3-13: O-GlcNAcylation at S87 inhibits α-synuclein
aggregation without affecting membrane binding.

Figure 3-14: Characterization of the additional α-synuclein mutants
S87A, S87D, S87W, and S87K.

Figure 3-15: Analysis of mutant α-synuclein aggregation and
membrane binding.

Figure 4-1: O-GlcNAcylation and the corresponding S-GlcNAc
analog.

Figure 4-2: Synthetic routes to S-GlcNAcylated amino acids for
solid phase peptide synthesis.

Figure 4-3: Characterization of O-GlcNAcylated and S-
GlcNAcylated model peptides.

Figure 4-4: O-GlcNAcylation and S-GlcNAcylation have similar
small effects on peptide structure by CD.

Figure 4-5: S-GlcNAc is a good structural mimic of O-GlcNAc.

Figure 4-6: ab initio folding of O vs. S GlcNAcylated model
peptides.

Figure 4-7: QM optimized models of extended and β-hairpin
conformations of O- and S-GlcNAcylated tetra-
peptides.

Figure 4-8: In the context of a peptide, S-GlcNAc is completely
stable against human OGA enzymatic deglycosylation.

Figure 4-9: S-GlcNAcylation in a peptide is resistant to O-
GlcNAcase (OGA) mediated deglycosylation.

Figure 4-10: Semisynthesis of S-GlcNAcylated α-synuclein.

Figure 4-11: Characterization of S-GlcNAcylated thioester peptide
9.

Figure 4-12: Ligation of peptide 9 and protein 10 and the
subsequent deprotection to give the corresponding α-
synuclein fragment.

72

74

75

94

98

101

102

103

105

106

107

108

110

111

112

viii

Figure 4-13: Ligation with protein thioester 8 to yield full-length S-
GlcNAcylated α-synuclein.

Figure 4-14: Desulfurization of S-GlcNAcylated α-synuclein.

Figure 4-15: S-GlcNAcylation of α-synuclein is enzymatically
stable.

Figure 4-16: Neither O-GlcNAcylation or S-GlcNAcylation induces
secondary structure in α-synuclein.

Figure 4-17: S-GlcNAcylation has identical effects as O-
GlcNAcylation on the membrane binding and
aggregation of α-synuclein.

Figure 5-1: O-GlcNAc modification and calpain cleavage of α-
synuclein.

Figure 5-2: Synthesis of α-synuclein bearing site-specific O-
GlcNAcylation.

Figure 5-3: O-GlcNAcylation blocks the cleavage of α-synuclein
by calpain.

Figure 5-4: Identification of the α-synuclein-derived fragments
after calpain cleavage.

113

113

115

117

118

139

142

144

147

ix

List of Schemes
Scheme 2-1: Previous Methods for β-Ac
3
GlcNAc Amino Acids.

Scheme 2-2: Postulating Indium Bromide Mediated Glycosylation.

26

27

x

List of Tables
Table 2-1: Screening Conditions for Glycosylation of
Fmoc-Ser-OH.

Table 2-2: Evaluating Substrate Scope With Optimized
Conditions.

Table 2-3: Evaluating Substrate Scope With Optimized
Conditions.

29

35

37

xi
Abstract

O-GlcNAcylation is a post-translational modification that involves the β-linkage of a single
unit of N-acetyl-D-glucosamine (GlcNAc) to the alcoholic residues (serine/threonine) of
nuclear, cytosolic, and mitochondrial proteins. Despite the identification of thousands of
O-GlcNAcylated proteins involved in several important cellular processes and diseases,
the functional outcome of this site-specific modification on the protein’s biophysical and
biochemical properties remains difficult to evaluate. This is in part due to the difficulty in
isolating homogenous samples of O-GlcNAcylated proteins from natural sources and the
limiting number of mutational and chemical strategies available to study this modification.
The advent of solid-phase peptide synthesis (SPPS) has revolutionized the field of O-
GlcNAcylation by enabling the chemical synthesis of peptides bearing site-specific O-
GlcNAc units. These well-defined glycopeptides have in-turn facilitated the preparation of
homogeneously O-GlcNAcylated proteins suitable for in vitro biological studies. However,
the chemical synthesis of the glycosylated amino acids required for SPPS still remains a
challenge. Described here is the facile synthesis of GlcNAc β-glycosides and the
application of these building blocks for the construction of homogenous site-specifically
modified proteins for in vitro functional studies. Particularly, we focus on the semi-
synthetic preparation of the site-specifically O-GlcNAcylated neuronal protein, α-
synuclein. This protein has been demonstrated to play a causative role in synucleinopathies
such as Parkinson’s disease, where it aggregates to form toxic protein deposits known as
Lewy bodies. Using a variety of in vitro assays, we demonstrate that site-specific O-
GlcNAcylation of α-synuclein inhibits its propensity to aggregate.
1

Chapter 1. Stereoselective Glycosylations of N-Acetyl Glucosamine

Introduction
Carbohydrates are the most abundant biomacromolecules in all living systems. The term
carbohydrate refers to biological polymers composed of individual units called
monosaccharides (Ghazarian et al., 2011). These units can be found individually or linked
together via glycosidic bonds formed by carbohydrate-specific proteins called glycosyl
transferases to form disaccharides, oligosaccharides, and polysaccharides. Unlike the
structural units of other important biological polymers such as nucleic acids and proteins,
monosaccharides contain more than one site through which extension can occur. Thus,
carbohydrates are not restricted to form linear polymers and can form highly diverse
structures with several branching points (Ghazarian et al., 2011). The complexity of
carbohydrates is further exacerbated by the stereochemistry of the bond linking the
monosaccharides, which can either be axial (alpha, denoted by α) or equatorial (beta,
denoted by β) and depends entirely on the stereochemical preference of the corresponding
glycosyl transferase (Werz et al., 2007). Additionally, carbohydrates can be further
elaborated by glycosyl hydrolases, enzymes that remove monosaccharides. Interestingly,
the structure of carbohydrates is not template driven as is the structure of nucleic acids and
proteins, whose sequence is encoded in DNA. The structure of carbohydrates depends
solely on the activity of glycosyl transferases and hydrolases. Carbohydrates are commonly
found in nature as glycoconjugates, in which they are attached to proteins, lipids, or both
lipids and proteins through an oxygen (O-linked) or nitrogen atom (N-linked) via glycosyl
2

transferases. Interestingly, carbohydrates are major constituents of every cell surface where
they have been demonstrated to play significant roles as signaling molecules or receptors
in several important biological events such as host cell communication, cell-cell adhesion,
host-cell recognition, host-pathogen infection, and immune response (Ashry and Aly,
2007). Additionally, carbohydrates play significant roles within cells such as in the
production of energy from their metabolism (Glycolysis) or through their conjugation to
other biomolecules (Glycosylation) in order to alter their stability, shape, localization,
activity, or function.

Biological Significance Of N-acetyl glucosamine
A recent statistical analysis of a glycoprotein databank revealed that the most abundant
mammalian monosaccharide is N-acetylglucosamine (GlcNAc) (Werz et al., 2007). Not
surprisingly, GlcNAc is found to be incorporated in a myriad of extracellular
oligosaccharides, polysaccharides, and cell-surface glycoproteins (Figure 1-1) (Ashry and
Aly, 2007). For example, human milk oligosaccharides (HMOs) are unbound
oligosaccharides that contain GlcNAc units embedded within (Figure 1-1). These HMOs
are found in breast milk and play significant roles in the infant’s brain development and
early defense against pathological microorganisms (Ashry and Aly, 2007). Similarly,
GlcNAc can be found within the linear polysaccharide hyaluronic acid (HA) which is a
major structural component in the extracellular matrix and whose interactions with other
proteins can modulate cell mobility and adhesion (Figure 1-1) (DeAngelis, 1999).
Moreover, the glycoproteins that decorate the cell-surface of red blood cells and that are
3

responsible for determining the blood group of an individual contain GlcNAc units either
within or at their terminal non-reducing ends (Figure 1-1) (Watkins, 1980). Aside from the
physiological role of these cell-surface glycoproteins, some GlcNAc containing
glycoconjugates are involved in pathophysiological events as well. For example, malignant
tumor cells have been demonstrated to heavily express cell-surface glycolipids that contain
a specific oligosaccharide moiety known as epitopes (Figure 1-1) (e.g. Sialyl-Lewis A).
These GlcNAc containing epitopes are recognized by membrane-bound proteins known as
selectins that are found on the host cells. Adhesion of tumor cells to the host cell via the
interaction of these epitopes and selectins is believed to be the mechanism by which cancer
spreads and avoids detection by the immune system (Ugorski and Laskowska, 2002).

As previously detailed, carbohydrates can also be linked to their conjugates via a nitrogen
atom. These glycoconjugates are generally classified as N-linked glycoproteins. In
eukaryotes, N-glycosylation involves the addition of a common tetradecasaccharide,
Glc
3
Man
9
GlcNAc
2
, that contains GlcNAc at its reducing end to asparagine residues of
nascent polypeptides (Figure 1-1) (Aebi, 2013). Interestingly, all protein substrates for N-
glycosylation have the consensus sequence of asparagine-X-Serine/Threonine (N-X-S/T),
where X can be any amino acid except proline (Schwarz and Aebi, 2011). These glycans
help proteins properly fold as they are being synthesized, assume the proper conformation
after translation, and serve as signals to sort out misfolded proteins (Imperiali and
O’Connor, 1999). Following protein synthesis in the endoplasmic reticulum, this common
core is then further elaborated in the Golgi in a cell-type and protein dependent fashion
4

Figure 1-1. GlcNAc is found as a structural unit in several biologically relevant
carbohydrates.

through the sequential action of a myriad of glycosyl transferases and hydrolases, thus
resulting in the highly diverse structures that are observed for N-glycosylation (Schwarz
and Aebi, 2011). These N-linked glycoproteins are then secreted to the cell-surface where
they play a diverse set of functions.
O
O
O
HO
OH
HO
AcHN
OH
OH
O
O
OH
OH
OH
OH
O
OH
OH
OH
O
HO
Lacto-N-tetraose
(Backbone of Human Milk Oligosaccharides)
HO
O
O
AcHN
OH
O
O
OH
OH
OH
O
OH
OH
OH
O
HO
O
AcHN
OH
HO
HO
O
O
OH
HO
O
O
Me
OH
OH
OH
Blood Group Antigen
(Cell Surface of Red Blood Cells)
O
O
O
O
OH
O
AcHN
OH
OH
O
OH
O
Me
OH
OH
OH
O
HO
AcHN
HO
OH
OH
HO O
Sialyl-Lewis A
(sLe
a
Epitope)
HO
O
O
AcHN
OH
HO
O
O
AcHN
OH
H
N
O
HO
O
HO
OH
OH
O
HO
O
HO
OH
O
O
O
HO
OH
O
O
O
HO
OH
HO
O
HO
HO O
HO
O
HO
HOHO
HO
O
HO
HO O
HO
O
HO
HO O
O
O
HO
HOHO
O
O
HO
HO
OH
HO
O
HO
HO
OH
HO
O
HO
O
OH
Glc
3
Man
9
GlcNAc
2
(Core of Eukaryotic N-Linked Glycoproteins)
O
O
O
HO
OH
O
OH
O
HO
AcHN
OH
n
Hyaluronic Acid
(Extracellular Polysaccharide)
O
O
HO
AcHN
OH
O
5

In all of the examples above, GlcNAc is an important constituent of the oligosaccharide,
polysaccharide, or glycan of the glycoconjugates. However, there exist a unique form of
glycosylation in which GlcNAc is the only monosaccharide that is covalently attached to
intracellular proteins. This form of glycosylation is called O-GlcNAcylation (Figure 1-2).
It involves the dynamic addition and removal of a single unit of GlcNAc to the alcoholic
residues (serine/threonine) of nuclear, cytosolic, and mitochondrial proteins (Zachara and
Hart, 2002). Unlike the myriad of enzymes involved in the glycosylation of secreted
proteins, only two enzymes are connected with O-GlcNAcylation. O-GlcNAc transferase
(OGT) is responsible for the β-linkage of GlcNAc to the hydroxyl group of serine and

Figure 1-2. O-GlcNAcylation: the addition of a single unit of GlcNAc to serine/threonine
residues of intracellular proteins.

HO
H(Me)
HO
O
HO
AcHN
OH
O
H(Me)
PROTEIN PROTEIN
OGT
OGA
HO
O
HO
AcHN
OH
OH
HO
O
HO
AcHN
OH
O
P
O
P
O
O
OHOH
N
NH
O
O
O O
OH OH
6

threonine, while O-GlcNAcase (OGA) mediates the cleavage of the modification (Figure
1-2) (Bond and Hanover, 2015). Through the combined activity of OGT and OGA, this
post-translational modification (PTM) cycles rapidly on and off protein substrates multiple
times throughout the lifetime of the protein. Moreover, the sites of O-GlcNAcylation are
often the sites or near the sites of other PTMs such as phosphorylation and ubiquitination.
Thus, this modification can modulate the downstream effects of other PTMs by competing
for the sites of modification. The substrate for OGT is the nucleotide activated sugar, UDP-
GlcNAc, which is derived from the metabolism of glucose via the hexosamine biosynthetic
pathway (Yang and Qian, 2017). To date, a consensus sequence for OGT substrate
recognition remains elusive and yet thousands of proteins are O-GlcNAc modified in a cell,
substrate, and context-dependent manner. Structural and biochemical evidence suggest that
OGT uses certain structural motifs, such as its TPR domains, and other adapter proteins to
discriminate against protein substrates (Yang and Qian, 2017). Moreover, the sites of
modification are often loops and terminal domains which indicate OGT’s strong preference
for intrinsically disordered regions. Additionally, the expression, activity, and substrate
specificity of OGT is sensitive to the levels of intracellular UDP-GlcNAc and other
external cues (e.g. heat shock), thus allowing O-GlcNAcylation to act as a sensor capable
of signaling the status of the cellular environment through modification of substrate
proteins and cross-talk with other PTMs (Hardiville and Hart, 2014). The dynamic nature,
diverse substrate scope, interplay, and sensitivity of O-GlcNAcylation places this
modification at the nexus of several important cellular processes such as transcription,
translation, organelle biogenesis, protein homeostasis, and metabolism (Hardiville and
7

Hart, 2014). Not surprisingly, proper levels of O-GlcNAcylation are required for cellular
and organismal survival and the misregulation of this modification has been implicated in
the etiopathogenesis of several diseases such as diabetes, cancer, and neurodegenerative
disorders (Slawson and Hart, 2011; Yang et al., 2008; Zhu et al., Shan, 2014). The role of
O-GlcNAcylation in neurodegenerative disorders, particularly Parkinson’s disease, is a
major focus of this thesis.

Figure 1-3. Synthetic strategies resolve the difficulty in obtaining homogenous samples of
GlcNAcylated proteins for functional studies.

The isolation of GlcNAc containing carbohydrates and site-specifically O-GlcNAc
modified glycoproteins from natural sources or in vitro enzymatic techniques for structural
studies, functional studies, and therapeutic applications is quite challenging. The difficulty
arises from the previously alluded to fact that the structure and site of carbohydrate
modifications is not template driven, but rather dependent on the activity of glycosyl
transferases and hydrolases. Thus, a given protein substrate may exist in different isoforms
Glycoprotein
Glycoprotein
Glycoprotein
Different Carbohydrate Multiple Sites Of Modification
Different Site
Glycoprotein
Functional Site Of Interest
Heterogenous Mixture Of A Glycoprotein
Natural Sources
Glycoprotein
Functional Site Of Interest
Highly Pure And Homogenous
Sample Of A Glycoprotein
Synthetic Strategies
8

(glycoforms) that differ in the number of modifications, the site of modification, or the
structure of the carbohydrate (Figure 1-3). This microheterogeneity makes it difficult to
separate complex mixtures of a given glycoprotein and associate its carbohydrate structure
or site of modification with a functional outcome. Thus, synthetic strategies that can readily
afford highly pure and homogenous samples of a given glycoprotein is of great importance.
The following sections outline the development of chemical glycosylation strategies for
the synthesis of N-acetyl glucosamine β-glycosides: building blocks used in the preparation
of complex GlcNAc-containing carbohydrates and site-specifically modified O-
GlcNAcylated proteins.

Chemical Glycosylation
For decades, chemist have long sought to mimic the regio- and stereo-selective
glycosylation that is catalyzed by glycosyl transferases. In nature, the enzymes active site
confers substrate preference and modulates the substrates conformation, thus leading to
regioselectivity and limiting the stereochemical outcome to one anomer, α or β. In a
laboratory setting, however, the absence of an enzyme’s microenvironment makes it far
more difficult to control and predict the stereochemical outcome of a glycosylation
reaction. Chemical glycosylation can be viewed as a nucleophilic substitution reaction
between a glycosyl donor (electrophile) and a glycosyl acceptor (nucleophile) (Figure 1-
4). Generally, the glycosyl donor is fully protected at the C-2, C-3, C-4, and C-6 positions
9

Figure 1-4. Chemical glycosylation and general mechanistic considerations.

and pre-activated with a leaving group at the anomeric position via an oxygen exchange
reaction involving acid catalysis or base promoted alkylation/acylation (Zhu and Schmidt,
2009). The leaving group is then activated for departure by either a promotor or catalyst to
generate an unstable anomeric carbocation, that is rapidly stabilized by the C-5 ring oxygen
to form an oxocarbenium ion. Upon resonance stabilization, the C-1 anomeric carbon
hybridizes to Sp2 character, thereby conferring a significant amount of planarity at the site
of nucleophilic substitution. As a result, nucleophilic attack by the glycosyl acceptor can
occur from either the axial or equatorial face to produce α-glycosides or β-glycosides,
respectively.

The rate limiting step in any glycosylation reaction is the departure of the leaving group to
generate the oxocarbenium ion, thus the reaction rate is dependent on the reactivity of the
leaving group and the stability of the oxocarbenium ion (Mydock and Demchenko, 2010).
The rate of leaving group activation rests on the choice of promoter or catalyst and can be
influenced by the protecting groups on the other positions of the glycosyl donor
(arming/disarming effect) (Fraser-Reid and López, 2010). For example, electron
O
LG
PGO
O
PGO
O
PGO
O
PGO
OR
OR
Promoter
Rate
Determining
Step
Glycosyl Donor
Reactivity
Oxocarbenium
Stability
ROH
Glycosyl Acceptor
Kinetic
vs
Thermodynamic
α-glycoside β-glycoside
Stereoselectivity
+
Nucleophilic Substitution
Electrophile
Nucleophile
O
PGO
Carbocation
10

withdrawing substituents such as esters can drastically reduce the reactivity of the glycosyl
donor by reducing the electron density of the leaving group (Figure 1-5). On the contrary,
electron donating substituents such as ethers can increase the reactivity of the glycosyl
donor by increasing the nucleophilicity of the leaving group, thus enhancing its activation
(Figure 1-5). Similarly, these protecting groups can either stabilize or destabilize the
carbocation that is generated upon departure of the leaving group and hence affect the
formation and stability of the oxocarbenium (Mydock and Demchenko, 2010).

Figure 1-5. Arming and disarming effect of protecting groups on the reactivity of the leaving
group and stability of glycosyl carbocation.

The stereochemical outcome of the reaction can rely on either the facial preference (kinetic)
of the glycosyl acceptor upon nucleophilic attack or the stability of the products that are
formed (thermodynamic). The facial preference of the glycosyl acceptor can be modulated
by the protecting groups on the glycosyl donor, particularly, the protecting group at the C-
2 position can sterically or covalently block the nucleophile’s approach from one face to
yield exclusively the β-glycoside (neighboring group participation, Figure 1-6) (Enugala
O
LG
O
R
O
O
LG
O
R
O
O
R
O
O
O
R
Ester Protecting Groups
(Electron withdrawing)
Ether Protecting Groups
(Electron Donating)
Glycosyl Donor
“Disarmed”
Glycosyl Donor
“Armed”
Carbocation
Destabilized
Carbocation
Stabilized
11

et al., 2012). Unfortunately, the absence of a participating group at the C-2 position does
not guarantee the exclusive formation of α-glycosides. In such cases, the reaction
conditions can have a drastic effect on the stereochemical outcome of the glycosylation.
For example, the use of acetonitrile favors the formation of β-glycosides (nitrile effect)
(Crich and Patel, 2006). Still, a more common outcome in the absence of participating
groups is the production of anomeric mixtures that are extremely difficult to separate using
traditional purification techniques. Thus, the stereoselective synthesis of α-glycosides is a
bit more challenging that the corresponding β-glycosides, which greatly benefit from
neighboring group participation.

Figure 1-6. Neighboring group participation.

The difference in stability between the synthetic products (α and β-glycosides) is attributed
to the anomeric effect. According to the anomeric effect, α-glycosides are inherently more
stable than β-glycosides on the grounds of electrostatics and hyperconjugation (Das and
Mukhopadhyay, 2016). For example, the dipole of the equatorial bond in β-glycosides is
parallel to the dipole of the lone pair electrons on the C-5 oxygen, thereby producing
considerable repulsion and destabilization (Figure 1-7). This unfavorable dipole-dipole
interaction is minimized when the anomeric substituent lies on the axial position (α-
O
LG
PGO
X
O
R
O
PGO
X
R
O
O
PGO
X
O
X = O (Ester)
X = N (Amide/Carbamate)
X = O (Acyloxonium)
X = N (Oxazolinium)
ROH
O
OR
PGO
X
O
R
β-glycoside
Oxocarbenium
Blocks α-face from attack
12

glycosides). Moreover, the anti-bonding orbital of the axial bond can benefit from the
hyperconjugation of the lone pair electrons on the C-5 oxygen, which further strengthen
and stabilize the α-glycosides (Figure 1-7). This stabilization is absent in β-glycosides due
to the fact that the equatorial anti-bonding orbital lies on a different plane than the lone pair
electrons of the C-5 oxygen. The stereochemical outcome of the reaction is further
complicated by the fact that the kinetic products can often undergo anomerization to the
more stable thermodynamic products.

Figure 1-7. Anomeric effect.

Chemical Strategies for the Synthesis of β-glycosides
N-acetyl glucosamine is predominantly linked in nature as the β-anomer. As a
consequence, synthetic strategies for GlcNAc glycosides must be highly stereospecific.
Given that the N-acetyl functionality is a C-2 protecting group capable of neighboring
group participation, the use of GlcNAc donors represents the most direct approach to β-
glycosides. The first glycosylation reaction involving an N-acetyl glucosamine donor was
O
OR
PGO
O
PGO
OR
O OR O
OR
Axial
(Stabilized)
Equatorial
(Destabilized)
O
PGO
O
PGO
X
Equatorial
(No Hyperconjugation)
Axial
(Hyperconjugation)
Hyperconjugation:
Electrostatics:
13

reported in 1901 by Koenigs and Knorr (Figure 1-8) (Koenigs and Knorr, 1901). In their
studies, GlcNAc was conjugated to alcoholic acceptors via the activation of GlcNAc α-
halides with silver salts to form exclusively the β-glycosides. While their method allowed
for the direct access of GlcNAc β-glycosides, the reaction only worked well with reactive
nucleophiles such as primary alcohols. With more sterically hindered alcohols, the major
product was a stable methyloxazoline that resisted glycosylation and thereby reduced the

Figure 1-8. Koenigs-Knorr reaction.

rate and yield of the reaction. This intermediate was a consequence of neighboring group
participation and resulted from the proton abstraction of the initially formed
methyloxazolinium. Additionally, their reaction conditions required stoichiometric or even
excess amount of the promoter for the glycosylation to work efficiently. Another limitation
to their synthetic approach, was the use of glycosyl halides that are highly prone to
hydrolysis and rather difficult to prepare. Nevertheless, the Koenigs-Knorr reaction led to
AcO
O
AcO
AcHN
OAc
Cl
AcO
O
AcO
OAc
HN
O
AcO
O
AcO
AcHN
OAc
OR
Methyloxazolinium
GlcNAc
β-glycoside
Major Anomer
Halophilic
Promoter
Reactive
Alcohol
Neighboring
Group
Participation
β-Directed
Attack
AcO
O
AcO
OAc
N
O
Methyloxazoline
Stable Intermediate
Major Product
Sterically
Hindered
Alcohol
Proton
Abstraction
Poor Reactivity
14

the development of many new glucosamine N-protecting groups that were designed to
address the inherent limitations of the methyloxazoline, while maintaining the high
stereoselectivity conferred by the neighboring group participation (Enugala et al., 2012;
Aly and El Sayed, 2016). These protecting groups can be generally classified into two
categories: monovalent and bivalent. Monovalent protecting groups are still susceptible to
proton abstraction but contain additional electronegative atoms that increase the reactivity
of the oxazoline generated from proton abstraction (Figure 1-9). Thereby, overcoming the
difficulties associated with the reactivity of the methyloxazoline derived from N-acetyl
donors. On the other hand, bivalent protecting groups do not contain a proton that can be
abstracted and yet can still participate to control the stereochemical outcome of the reaction
through the formation of a bicyclic unstable intermediate (Figure 1-10). Hence, bivalent
protecting groups simultaneously bypass the poor reactivity and formation of the stable
methyloxazoline. Obvious requirements in the use of β-directing monovalent or bivalent
N-protecting groups is the need for their regioselective installment, stability to various
chemistry methods, and susceptibility to facile deprotection. Moreover, the final
deprotected free amine must be acetylated to afford the naturally occurring N-acetyl
protecting group. The regioselective installment for the majority of these protecting groups
can be easily achieved under basic conditions starting from commercially available D-
glucosamine (Enugala et al., 2012). However, commonly encountered deprotection
strategies involve strong acidic, basic, or reductive conditions that hinder their general
applicability.

15

Figure 1-9. Monovalent N-Protecting groups.

Figure 1-10. Bivalent N-Protecting groups.

AcO
O
AcO
NH
OAc
AcO
O
AcO
OAc
N
O
X
AcO
O
AcO
NH
OAc
OR
Oxazoline Major Anomer
Glycosyl
Acceptor
β-Directed
Attack
LG
O
X
Reactive Intermediate
Neighboring
Group
Participation
Proton
Abstraction
N-Deprotection
N-Acetylation
O
X
AcO
O
AcO
AcHN
OAc
OR
GlcNAc
β-glycoside
N-Protected
β-glycoside
AcO
O
AcO
NH
OAc
LG
O
O
Cl
3
C
AcO
O
AcO
NH
OAc
LG
O
O
AcO
O
AcO
NH
OAc
LG
O
O
AcO
O
AcO
NH
OAc
LG
O
Cl
3
C
N-Trichloroethoxycarbonyl
(N-Troc)
N-Allyloxycarbonyl
(N-Alloc)
N-Carboxybenzyl
(N-Cbz)
N-Trichloroacetyl
(N-TCA)
Representative Examples:
AcO
O
AcO
N
OAc
AcO
O
AcO
OAc
N-Protected
β-glycoside
Exclusive Anomer
Promoter
Glycosyl
Acceptor
Neighboring
Group
Participation
β-Directed
Attack
LG
O O
Y X
N O
X
O
Y
Unstable Intermediate
AcO
O
AcO
N
OAc
OR
O O
Y X
N-Deprotection
N-Acetylation
AcO
O
AcO
AcHN
OAc
OR
GlcNAc
β-glycoside
AcO
O
AcO
N
OAc
LG
AcO
O
AcO
N
OAc
LG
AcO
O
AcO
N
OAc
LG
AcO
O
AcO
N
OAc
LG
Representative Examples:
O
O
Cl
Cl
Cl
Cl
O
O
S
S
O
O
O
O
N-Phthaloyl
(N-Phth)
N-Tetrachlorophthaloyl
(N-TCP)
N,N-Dithiasuccinoyl
(N-DTs)
N-Diphenylmaloyl
(N-DPM)
16

Figure 1-11. Diversity in the type of leaving groups used in the synthesis of β-glycosides.

With the emergence of new N-protecting groups, developments in the type of leaving
groups used has also increased (Figure 1-11). While the Koenigs-Knorr reaction made use
of glycosyl halides, new chemical glycosylation strategies for GlcNAc β-glycosides more
commonly employ anomeric esters, imidates, thioglycosides, and thioimidates. The
O
LG
O
S
O
S
O
S
O
S
O
N
O
O
O
O
O
O
O
O
HN
CH
3
O
O
HN
CCl
3
O
O P OBu
O
OBu
O
I
O
F
O
Br
Thioglycosides
Thioimidates
Esters
Imidates
Phosphates
Halides
S
N
17

advantage of these leaving groups over halides is their ease of preparation and stability to
other chemical transformations, thereby allowing the construction of complex glycosyl
donors. A limitation to these leaving groups, with the exception of imidates, is the need for
stoichiometric or even excess amounts of toxic promoters (Ranade and Demchenko, 2013).
For example, anomeric acetates are typically activated with 1-4 equivalents of BF
3
.
Et
2
O.
Similarly, thioglycosides and thioimidates require excess amounts of thiophilic promoter
systems such as NIS/TMSOTf or AgOTf. On the other hand, imidates such as the
commonly used trichloroacetimidates can be activated with catalytic amounts of TMSOTf.

Although both the development of new N-protecting groups and anomeric leaving groups
have addressed the limitations of the Koenigs-Knorr reaction, they inherently complicate
the synthesis of GlcNAc β-glycosides by extending the number of synthetic
transformations, which lower the overall synthetic yield. Alternatively, other groups have
focused on developing chemical methods to activate the stable methyloxazoline that results
from using the native N-acetyl group. This approach has the conceptual advantage that the
methyloxazoline is easily prepared and no additional transformations are needed after the
glycosylation event. The first method was developed by Khorlin et al. and it involved the
use of p-TsOH as the promoter in refluxing CH
3
NO
2
/tolune (Khorlin, Shul'man, Zurabyan,
Privalova, & Kopaevich, n.d.). Although great yields were obtained for β-glycosides, the
reaction only worked well with small reactive alcohols. When more sterically hindered
alcohols were used, a common byproduct in their reactions was a 2-acetamido-D-glycal.
Furthermore, the strong acidic and temperature conditions restricted its application to
18

acceptors with acid-labile protecting groups. The change from protic acid promoters to
metal-based Lewis acids, such as FeCl
3
, by Kiso et al. led to milder conditions, but was
still not amenable to less reactive alcohols (Kiso and Anderson, 1985). Given the high
affinity of copper(II) for nitrogen-based ligands, Wittmann et al. postulated that copper(II)
salts, such as CuCl
2
, could activate the methyloxazoline through coordination of the
nitrogen heteroatom to generate an activated methyloxazolinium complex (Figure 1-12).
As expected, treatment of the methyloxazoline with CuCl
2
in the presence of small or
sterically hindered alcohols led to the exclusive formation of β-glycosides in great yields
(Wittmann and Lennartz, 2002). Moreover, acid-labile groups were not affected by the
metal-based Lewis acid. The only drawback to the use of copper(II) salts was the need for
excess amounts and prolonged reaction times. These limitations were surpassed through
the use of Yb(OTf)
3
. This lanthanide promoter could be used in catalytic amounts (5 mol%)
to yield exclusively β-glycosides in high yield with shorter reaction times (Crasto and
Jones, 2004).

Conclusions and Future Outlook
Given the various aspects that control the reaction rate, regioselectivity, and
stereoselectivity of chemical glycosylation, this reaction is considered as one of the most
difficult to control. As is evident from the previous sections, the ongoing theme in the field
of carbohydrate chemistry has been in the optimization of earlier studies, with a major
focus on protecting group and leaving group manipulations on the glycosyl donor. This is
no surprise as these two aspects play a significant role in the rate and stereoselectivity of
19

the chemical glycosylation. To this date, no universal glycosylation reaction exists and may
never be obtained. Instead, the design of a synthetic route to any oligosaccharide,
polysaccharide, or glycoconjugate will stem from either in-depth knowledge gained from
experience or trial and error. Despite the several improvements to the Koenigs-Knorr
reaction, particularly the activation of the methyloxazoline which minimizes the overall
synthetic steps, no chemical method exist that utilizes commercially available GlcNAc
donors with catalytic amounts of a non-toxic promoter to afford β-glycosides in great
yields. The development of such a reaction should expedite the synthesis of β-glycosides
for functional studies without the need of extensive experience in carbohydrate chemistry.

Figure 1-12. Copper catalyzed activation of methyloxazoline for the efficient synthesis of β-
glycosides.

AcO
O
AcO
OAc
N
O
CuCl
2
(1.5 - 4.0 eq.)
CHCl
3
AcO
O
AcO
OAc
N
O
Cu
Cl
Cl
ROH
AcO
O
AcO
AcHN
OAc
OR
GlcNAc
β-glycoside
Methyloxazoline
Stable Intermediate
Activated
Copper Complex
Representative Alcohols (ROH):
O
OH
O
O
O
O
O
OBz
O
O
OH
N
3
OH
FmocHN
O
O
OH
20

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25

Chapter 2. Democratizing The Synthesis of β-N-acetyl-glycosides For
The Preparation of O- And S-GlcAcylated Peptides And Proteins
‡

Introduction
O-GlcNAcylation is a dynamic post-translational modification (PTM) of intracellular
proteins, in which the single monosaccharide N-acetylglucosamine (GlcNAc) is β-linked
to serine or threonine residues of substrate proteins (Bond and Hanover, 2015; Yang and
Qian, 2017).

This highly abundant PTM is critical for several biological processes and its
misregulation has important implications in mammalian development, survival, and
disease. Currently, the only approach to decipher the biochemical and biophysical function
of site-specific O-GlcNAcylation is through the semi-synthetic preparation of the
glycosylated target protein, which requires site-specifically modified glycopeptides (Chuh
et al., 2016). Given the heterogeneity that arises from in vitro enzymatic glycosylation
(Yuzwa et al., 2010) and the racemization observed with post-translational mutagenesis
(Wright et al., 2016; Yang et al., 2016), these glycopeptides must be prepared using solid-
phase peptide synthesis (SPPS). Additionally, the key O-GlcNAcylated amino acids
required for SPPS must also be chemically synthesized, thereby rendering site-specifically
modified proteins out of reach for many non-specialists interested in studying the function
of O-GlcNAcylation. The majority of published routes for the O-GlcNAcylated amino
acids require the prior synthetic preparation of complex glycosyl halides, acetates, trichloro

‡
Geoffrey Lang and Marcos I. Saavedra (University of Southern California) contributed to the work
presented in this chapter.
26

Scheme 2-1. Previous Methods for β-Ac 3GlcNAc Amino Acids.

acetimidates, or thioglycosides that may contain special protecting groups, such as
trichloroethoxycarbonyl (Troc), at the C-2 amino group of glucosamine (Scheme 2-1)
(Dullenkopf et al., 1996; Enugala et al., 2012; Marotta et al., 2012; Meinjohanns et al.,
1995; Mitchell et al., 2001; Saha and Schmidt, 1997). These protecting groups are
introduced in order to enable neighboring group participation (NGP), which results in the
formation of an intermediate oxazolinium that restricts the nucleophile’s approach from the
α-face and thus favors the formation of β-glycosides. Theoretically, the same
stereochemical outcome can be achieved with the use of the native acetyl group at C-2
amino group of glucosamine, but experimentally the glycosylation of this charged
O AcO
AcO
OAc
N O
O Cl
3
C
H
O AcO
AcO
NH
OAc
SPh
O
O
Cl
3
C
O AcO
AcO
NH
OAc
OAc
O
O
Cl
3
C
O AcO
AcO
HN
OAc
O
O
Cl
3
C
O
CCl
3
NH
O AcO
AcO
HN
OAc
O
O
Cl
3
C
Br
2-Troc Oxazolinium α-Bromide α-Trichloroacetimidate
β-Thioglycoside β-Acetate
O AcO
AcO
AcHN
OAc
O Me
FmocHN
OW
O
β-Ac
3
GlcNAc Threonine
X H(Me)
FmocHN
OW
O
N-Troc Deprotection
&
N-Acetylation
X = OH, SH
W= H, Bn, Pfp
BF
3
.
Et
2
O
TMSOTf AgOTf
NIS
TfOH
3 Steps 2 Steps
3 Steps 3 Steps
O AcO
AcO
AcHN
OAc
O H
FmocHN
OW
O
β-Ac
3
GlcNAc Serine
O AcO
AcO
AcHN
OAc
S H
FmocHN
OW
O
β-Ac
3
GlcNAc Cysteine
NGP
27

intermediate has proven to be quite challenging owing to its poor reactivity and inclination
to form a stable 2-methyl oxazoline that results from proton abstraction (Scheme 2-2)
(Arsequell et al., 1994). As noted above, the acetyl group is therefore typically replaced by
the more reactive carbamates that contain additional electronegative atoms. Even so, the
major drawbacks to the use of these intricate donor sugars is the need for stoichiometric
amounts of toxic promoters (e.g. 3 equivalents of BF
3
.
OEt
2
), extremely dry conditions (e.g.
molecular sieves), and the need for additional chemical transformations after the
glycosylation event (e.g. N-Troc deprotection/N-Acetylation). It is therefore imperative to
develop an operationally-simple synthetic route to O-GlcNAc modified amino acids in
order to expedite the generation of glycopeptides for both biological and functional studies.

Scheme 2-2. Postulating Indium Bromide Mediated Glycosylation.

Recently, Polt and coworkers demonstrated the ability of indium bromide (InBr
3
) to
mediate the activation of glucose anomeric acetates and facilitate the formation of an
acyloxonium ion that results from the NGP of the C-2 acetate (Coss et al., 2012; Lefever
et al., 2012; Szabó et al., 2016). This reactive intermediate was suggested to be responsible
for the exclusive formation of β-glycosides. Thus, we hypothesized that InBr
3
may also be
able to mediate the activation of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-D-
O AcO
AcO
NH
OAc
OAc
O AcO
AcO
OAc
N O
H
2-Methyl
Oxazolinium
Poor Reactivity
β-Ac
4
GlcNAc
O
O AcO
AcO
OAc
N O
2-Methyl
Oxazoline
Stable
-H
+H
O AcO
AcO
AcHN
OAc
X H(Me)
FmocHN
OH
O
β-Ac
3
GlcNAc Amino Acids
InBr
3
Anomeric Activation
InBr
3
Fmoc-Amino Acid
Ring-Opening
Neighboring Group Participation
Step 2 Step 1
28

glucopyranose (β-Ac
4
GlcNAc) and favor β-glycosides through the NGP of the C-2 amide
(Scheme 2-2, step 1). As previously detailed, the only major concern was that the poor
reactivity of the 2-methyl oxazolinium and the stability of its neutral counterpart (2-methyl
oxazoline) that are expected to be generated in situ could hinder the glycosylation event.
However, in support of our hypothesis Braga and coworkers have demonstrated the ability
of indium (III) salts to effect the cleavage of simple oxazolines to yield β-seleno amides
through coordination of the nitrogen heteroatom (Braga et al., 2007). Thus, it seemed
plausible that InBr
3
could also promote the ring-opening of the oxazoline derived from β-
Ac
4
GlcNAc (Scheme 2-2, step 2), which is generally considered an unreactive byproduct
of glycosylation reactions.

Results
InBr
3
can mediate the glycosylation of Fmoc-serine.
Using Fmoc-Ser-OH as a model acceptor (Table 2-1), we initially attempted the
glycosylation reaction at an analytical scale with stoichiometric amounts of InBr
3
and β-
Ac
4
GlcNAc in dichloroethane (DCE) (Entry 1). After heating the reaction vessel at 80 °C
for 16 hours, analysis of the crude mixture by high performance liquid chromatography
(RP-HPLC) revealed a very complex mixture of signals (Figure 2-1). Using a previously
synthesized standard, the product signal for β-Ac
3
GlcNAc serine (compound 2.1) was
identified from the crude trace and verified using mass spectroscopy. Despite the very low
conversion of 29%, these remarkable results encouraged us to further optimize the reaction
through the manipulation of sugar donor equivalents, amount of Lewis acid, concentration,
29

Entry
Donor
(eq.)
InBr 3
(mol%)

Solvent
Concentration
(mM)
Conversion
a

(%)
1 1 100 DCE 200 29
2 2 100 DCE 200 30
3 3 100 DCE 200 63
4 3 5 DCE 200 49
5 3 10 DCE 200 64
6 3 20 DCE 200 75
7 3 50 DCE 200 77
8 3 20
THF
b

200 2
9 3 20 Toluene 200 64
10 3 20 CH 3CN 200 61
11 3 20
CHCl 3
b

200 22
12 3 20 Dioxane 200 2
13 3
c
20 DCE 200 77

Table 2-1. Screening Conditions for Glycosylation of Fmoc-Ser-OH. Unless otherwise noted,
reactions were ran at 80 °C.
a
Determined from HPLC chromatogram.
b
Reaction was ran at 60 °C.
c
Used α-Ac 4GlcNAc.

and the reaction solvent. As indicated in Table 2-1, we found that increasing the equivalents
of β-Ac
4
GlcNAc improves the product conversion (Entries 1-3), but doesn’t prevent the
formation of impurities (Figure 2-1). We postulated that stoichiometric InBr
3
, together with
the high temperature and acidity of the reaction, facilitated the caramelization of the sugar
donor. This was supported by the presence of high molecular weight products and the dark
O
AcO
AcO
AcHN
OAc
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
OAc
HO
FmocHN
OH
O
InBr
3
Thermoshaker
16 hours β-Ac
3
GlcNAc Serine
β-Ac
4
GlcNAc
Fmoc-Ser-OH
30

brown color of the reactions. Although the caramelization temperature of sugars is typically
high, it is well known that strong acidic or basic conditions can accelerate the process
(Hong and Betti, 2016; Hrynets et al., 2015). With that in mind, we hypothesized that
lowering the amount of InBr
3
could resolve the formation of impurities. Excitingly,
lowering the loading of InBr
3
not only minimized the formation of side products as evident
by the HPLC traces (Figure 2-2), but also improved the product conversion (Entries 4-9).
At lower loadings, we found there to be a linear relationship between the amount of Lewis
acid used and the overall product conversion. However, the conversion did not improve
notably after 20 mol%, indicating that catalytic amounts of InBr
3
are optimal to achieve
maximal conversion and minimal side product formation. Interestingly, changing the
solvent had a significant effect on the reaction (Figure 2-3). Higher conversions were
obtained with dichloroethane than with chloroform (CHCl
3
) and other non-halogenated
solvents such as toluene, acetonitrile (CH
3
CN), tetrahydrofuran (THF), and 1,4-dioxane
(Entries 8-12). To evaluate whether the use of the more reactive β-anomer of the glycosyl
donor is key to this transformation, we tested the reaction with α-Ac
4
GlcNAc (Entry 13).
Interestingly, both anomers display similar reactivity and result in near identical product
conversions (Figure 2-4). However, given the crystalline nature of the β-anomer, we
decided to use β-Ac
4
GlcNAc for the remainder of the paper.

31

Figure 2-1. Increasing the sugar donor equivalents improves the product conversion. The
glycosylation of Fmoc-Ser-OH with various equivalents of β-Ac 4GlcNAc at 100 mol% InBr 3 in
dichloroethane (80 °C)(200 mM) for 16 hours was investigated. Initial conditions employed one
equivalent of β-Ac 4GlcNAc and resulted in a low product conversion. The increase to either two
or three equivalents improved the product conversion with the latter displaying the greatest
improvement. However, the higher product conversion was accompanied by an increase in side
product formation. Both the peaks for the starting material and product are indicated by the dotted
lines and are labeled accordingly. RP-HPLC conditions were 0−100% buffer B over 15 min; buffer
A consisted of 0.1% TFA in H 2O, and buffer B consisted of 0.1% TFA and 90% ACN in H 2O.

Absorbance (mAu)
100
225
350
475
600
No Sugar Donor
Absorbance (mAu)
100
225
350
475
600
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
1 equiv.
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
3 equiv.
2 equiv. Fmoc-Ser-OH
Ac3GlcNAc Serine Ac3GlcNAc Serine
Fmoc-Ser-OH
Time (minutes)
32

Figure 2-2. Lowering the catalyst loading improves the product conversion and minimizes
side product formation. The glycosylation of Fmoc-Ser-OH (1 eq.) with β-Ac 4GlcNAc (3 eq.) at
various loadings of InBr 3 (mol% are with respect to β-Ac 4GlcNAc) in dichloroethane (80 °C)(200
mM) for 16 hours was investigated. The use of 20 mol% catalyst yielded the greatest conversion
and minimized the side products that are formed at higher catalyst loadings. All crude samples were
analyzed by RP-HPLC. Conditions were 0−100% buffer B over 15 min; buffer A consisted of 0.1%
TFA in H 2O, and buffer B consisted of 0.1% TFA and 90% ACN in H 2O. Both the peaks for the
starting material and product are indicated by the dotted lines and are labeled accordingly.

Absorbance (mAu)
100
225
350
475
600
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
20 mol% Catalyst
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
100 mol% Catalyst
Absorbance (mAu)
100
225
350
475
600
5 mol% Catalyst 50 mol% Catalyst
Ac3GlcNAc Serine
Fmoc-Ser-OH
Ac3GlcNAc Serine
Fmoc-Ser-OH
Time (minutes)
33

Figure 2-3. Varying the reaction solvent has significant effect on the product conversion. The
glycosylation of Fmoc-Ser-OH with 3 eq. of β-Ac 4GlcNAc at 20 mol% InBr 3 in various solvents
(200 mM) for 16 hours was investigated. The use of dichloroethane yielded the greatest product
conversion. Both the peaks for the starting material and product are indicated by the dotted lines
and are labeled accordingly. RP-HPLC conditions were 0−100% buffer B over 15 min; buffer A
consisted of 0.1% TFA in H 2O, and buffer B consisted of 0.1% TFA and 90% ACN in H 2O.

Solven
Title
Absorbance (mAu)
100
225
350
475
600
CH3CN
Absorbance (mAu)
100
225
350
475
600
Toluene
Tetrahydrofuran
Chloroform
Absorbance (mAu)
100
225
350
475
600
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
1,4-Dioxane
Time (minutes)
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
Dichloroethane
Ac3GlcNAc Serine
Ac3GlcNAc Serine
Fmoc-Ser-OH
Fmoc-Ser-OH
34

Figure 2-4. Indium bromide can activate both α- and β-Ac 4GlcNAc. The glycosylation of
Fmoc-Ser-OH with 3 eq. of either α- or β-Ac 4GlcNAc at 20 mol% InBr 3 in dichloroethane (100
mM) for 16 hours was investigated. The HPLC chromatogram of the α-anomer (left side) displays
a similar peak distribution and product conversion to the glycosylation of Fmoc-Ser-OH with β-
Ac 4GlcNAc (right side). These results suggest that InBr 3 can activate either anomer. Both the peaks
for the starting material and product are indicated by the dotted lines and are labeled accordingly.
RP-HPLC conditions were 0−100% buffer B over 15 min; buffer A consisted of 0.1% TFA in H 2O,
and buffer B consisted of 0.1% TFA and 90% ACN in H 2O.

Optimal conditions can tolerate other amino acid substrates and do not require a strict
anhydrous environment.
Having optimized the conditions to maximize product conversion, we proceeded to run the
reaction at a larger scale with a refluxing temperature of 84 °C for 16 hours (Table 2-2).
Following purification, Ac
3
GlcNAc serine was isolated in 80% yield (Entry 1) and
exclusively as the β-anomer as determined by nuclear magnetic resonance (NMR).
Extension of the same conditions to the secondary alcohol of Fmoc-Thr-OH yielded β-
Ac
3
GlcNAc threonine (compound 2.3) in 77% yield (Entry 2). Notably, these yields were
obtained without the need of dry solvents or special handling of the catalyst, suggesting a
resilience to moisture. In order to investigate the effect of water on the catalyst, we
purchased the monohydrate of Fmoc-Thr-OH and submitted it to the optimized conditions.
100
225
350
475
600
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
alpha-Ac4GlcNAc
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
beta-Ac4GlcNAc
Ac3GlcNAc Serine
Fmoc-Ser-OH
Ac3GlcNAc Serine
Fmoc-Ser-OH
Time (minutes)
35

Table 2-2. Evaluating Substrate Scope With Optimized Conditions.
a
Isolated Yield.
b
Determined by
1
H NMR.
c
Monohydrate.

Surprisingly, an equivalent of water had only a very small effect as β-Ac
3
GlcNAc threonine
(compound 2.3) was isolated in a 71% yield (Entry 3). Thus, highlighting the ability of the
reaction to tolerate the presence of water. Given that we previously demonstrated the
enzymatic stability of S-GlcNAc (β-Ac
3
GlcNAc cysteine) and its potential utility for in
vivo studies, we decided to further extend the reaction to Fmoc-Cys-OH (De Leon et al.,
2017). Unfortunately, attempts with the commercial hydrate afforded β-Ac
3
GlcNAc
cysteine (compound 2.5) in 50% yield (Entry 4). We attributed the lower yield to the
oxidation of cysteine in the presence of stoichiometric amounts of water. Attempts to
O
AcO
AcO
AcHN
OAc
O
AcO
AcO
AcHN
OAc
X
FmocHN
OH
O
OAc
β-Ac
3
GlcNAc Amino Acids
β-Ac
4
GlcNAc
Fmoc-Amino Acids
20 mol% InBr
3
DCE, Reflux
16 hours
H(Me)
X = Oxygen
X = Sulfur
Entry
(Fmoc-Amino Acid)

X

Product
Yield
a

(%)

α:β
b

1 Fmoc-Ser-OH O β-Ac 3GlcNAc Serine (2.1) 80 β
2 Fmoc-Thr-OH O β-Ac 3GlcNAc Threonine (2.3) 77 β
3 Fmoc-Thr-OH
c

O β-Ac 3GlcNAc Threonine (2.3) 71 β
4 Fmoc-Cys-OH
c

S β-Ac 3GlcNAc Cysteine (2.5) 50 β
36

generate anhydrous Fmoc-Cys-OH from commercial derivatives proved problematic as
full reduction could not be achieved and residual disulfides hampered the glycosylation.

In situ deprotection strategy overcomes the difficulty with cysteine glycosylation.
A commercial cysteine building block that is typically used in SPPS is the triphenylmethyl
(trityl, Trt) protected cysteine pentafluorophenyl ester (Fmoc-Cys(Trt)-OPfp). Trityl ethers
are routinely deprotected using acidic conditions in the presence of cation scavengers such
as triisopropylsilane (TIPS) in organic solvents. Thus, we postulated that the presence of a
cation scavenger combined with the acidic conditions of the glycosylation reaction could
facilitate the in situ deprotection to unmask the thiol for nucleophilic attack (Table 2-3). As
foreseen, treatment of Fmoc-Cys(Trt)-OPfp with the optimized reaction conditions and
excess TIPS led to the formation of β-Ac
3
GlcNAc cysteine Pfp ester (compound 2.6) in
73% yield (Entry 1). Since it is common practice to activate the c-terminal acids prior to
SPPS, the in situ trityl deprotection and glycosylation of Pfp activated cysteine
conveniently kills two birds with one stone. Granted that the trityl ether of Fmoc-serine is
also commercially available, we next investigated the in situ deprotection and
glycosylation of the corresponding Pfp ester. Disappointingly, the glycosylation of the in
situ deprotected alcoholic side chain did not proceed to any significant extent (Entry 2).
The successful glycosylation with the cysteine c-terminal ester is probably due to the thiol’s
larger atom and lone pair orbital size that allow it to overcome the sterics imposed by the
ester. Nevertheless, the c-terminal free acids of serine and threonine generated according
37

to Table 2-2 can be readily activated for SPPS as their Pfp ester in great yields, compound
2.2 and compound 2.4, respectively.

Table 2-3. Evaluating Substrate Scope With Optimized Conditions.
a
Isolated Yield.
b
Determined by
1
H NMR.

X XH
TIPS
O
AcO
AcO
AcHN
OAc
O
AcO
AcO
AcHN
OAc
X
FmocHN
OPfp
O
OAc
TrtX
FmocHN
OPfp
O
20 mol% InBr
3
Triisopropylsilane (TIPS)
DCE, Reflux β-Ac
4
GlcNAc
Acetic Acid
Activation
Glycosylation
In Situ Deprotection
X
1
= Oxygen, Sulfur

Entry

X

Product
Yield
a

(%)

α:β
b

1 S β-Ac 3GlcNAc Cys-OPfp (2.6) 73 β
2 O β-Ac 3GlcNAc Ser-OPfp (2.2) 10 β
38

InBr
3
is a dual catalyst in these reactions.
Lastly, in light of the exclusive formation of β-glycosides using various acceptors, we
reasoned that NGP of the C-2 amide to generate the 2-methyl oxazoline must be occurring
as postulated in Scheme 2-2 (step 1). In support of this, we were able to detect formation
of the 2-methyl oxazoline during the course of the reactions by mass spectroscopy,
however, its isolation from the crude mixtures proved very difficult. To test whether InBr
3

could also catalyze the ring-opening of the 2-methyl oxazoline as hypothesized in Scheme
2-2 (step 2), we synthesized the 2-methyl oxazoline (compound 2.7) according to the
published literature procedure (Nakabayashi et al., 1986) and submitted it to the optimized
conditions using Fmoc-Ser-OH as the amino acid. HPLC analysis of the crude reaction
revealed an almost identical chromatogram to the reaction with β-Ac
4
GlcNAc (Figure 2-
5) and NMR confirmed the exclusive formation of the β-anomer. Importantly, no product
formation was observed in the absence of InBr
3
. Hence, our data provides evidence for the
2-methyl oxazoline as a key intermediate and suggest that InBr
3
is a dual catalyst in these
reactions with roles in activating the anomeric acetate and catalyzing the ring-opening of
the 2-methyl oxazoline.

Discussion
Herein, we report the facile synthesis of serine, threonine, and cysteine β-glycosides using
commercially available β-Ac
4
GlcNAc and catalytic amounts of InBr
3
. The present route
yields exclusively the β-glycosides in comparable yields to published methods without the
need of several chemical transformations. Unlike other Lewis acids used in glycosylation
39

Figure 2-5. Indium bromide can catalyze the glycosylation of the 2-methyl oxazoline. The
glycosylation of Fmoc-Ser-OH with 3 eq. of 2-Methyl Oxazoline at 20 mol% InBr 3 in
dichloroethane (100 mM) for 16 hours was investigated. The HPLC chromatogram of the 2-methyl
oxazoline reaction (right side) displays a similar peak distribution to the glycosylation of Fmoc-
Ser-OH with β-Ac 4GlcNAc (left side). Glycosylation with the oxazoline, however, gave a lower
product conversion. Both the peaks for the starting material and product are indicated by the dotted
lines and are labeled accordingly. RP-HPLC conditions were 0−100% buffer B over 15 min; buffer
A consisted of 0.1% TFA in H 2O, and buffer B consisted of 0.1% TFA and 90% ACN in H 2O.

reactions, indium can tolerate moderate levels of water, as exemplified by the glycosylation
of amino acid hydrates. Additionally, the complication of thiol oxidation in the
glycosylation of cysteine hydrate was overcome through the development of an in situ trityl
deprotection strategy. Lastly, we provide evidence that supports the neighboring group
participation of the C-2 amide and demonstrate the dual catalyst feature of InBr
3
in the
activation and glycosylation event. Overall, this synthetic route represents a far safer
alternative to current procedures and does not require extensive experience in carbohydrate
chemistry. We believe these convenient features will make this synthetic route the standard
choice in the preparation of β-Ac
3
GlcNAc amino acids for use in SPPS.

9.60
10.20
10.80
11.40
12.00
12.60
13.20
13.80
2-Methyl Oxazoline Rxn
Absorbance (mAu)
100
225
350
475
600
9.60
10.20
10.80
11.40
12.00
12.60
13.20
Ac4GlcNAc Rxn
Ac3GlcNAc Serine
Fmoc-Ser-OH
Ac3GlcNAc Serine
Time (minutes)
Fmoc-Ser-OH
40

Materials and Methods
All solvents and reagents were purchased from commercial sources and used without any
further purification. All silica gel column chromatography was performed using 60 Å silica
gel (EMD) and all thin-layer chromatography (TLC) was performed using 60 Å, F254
silica gel plates (EMD) with detection by ultraviolet light and staining with either ceric
ammonium molybdate (CAM) or Ninhydrin. Reverse-phase high-performance liquid
chromatography (RP-HPLC) was performed using an Agilent Technologies 1200 Series
HPLC instrument with a diode array detector. Unless otherwise stated, the following HPLC
buffers were used: buffer A, 0.1% TFA in H
2
O; buffer B, 0.1% TFA and 90% ACN in H
2
O.
Mass spectra were acquired on an API 3000 LC/MS-MS system (Applied
Biosystems/MDS SCIEX).
1
H and
13
C NMR spectra were acquired on a Varian Mercury
500 MHz or -600 MHz magnetic resonance spectrometer. Chemical shifts are quoted in
parts per million from residual solvent peak (CDCl
3
:
1
H - 7.26 ppm and
13
C - 77.16 ppm
and CD
3
OD:
1
H - 4.78, 3.31 ppm and
13
C - 49.15 ppm) and coupling constants (J) given in
Hertz. Multiplicities are abbreviated as: b (broad), s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet) or combinations thereof.

Chemical Synthesis.
Compound 2.1 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-serine (β-
Ac
3
GlcNAc Serine). Commercially available Ac
4
GlcNAc (356
mg, 0.9165 mmol), InBr
3
(65 mg, 0.1833 mmol) (20 mol% with respect to donor), and
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
41

Fmoc-Ser-OH (100 mg, 0.3055 mmol) were added to a round bottom flask containing a
stir bar under open atmosphere. Mixture was then suspended in 1,2-dichloroethane (200
mM). A reflux condenser was added and reaction vessel was submerged in an oil bath set
at 60 °C. A nitrogen balloon was added and the reaction was heated to reflux (>84 °C) for
16 hours under nitrogen. After the reaction no longer progressed by TLC (7:2:1: EtOAc:
MeOH: H
2
O), the reaction was allowed to cool to room temperature and concentrated under
vacuo. The black residue was azeotroped several times with toluene under high vacuum.
The fluffy black solid was then resuspended in dichloromethane (DCM) and applied to
flash column chromatography (5% MeOH in DCM (0.1% AcOH)). Fractions containing
the product were combined and concentrated in vacuo. The acidic dark brown syrup was
then diluted in EtOAc and transferred to a separatory funnel. Organic layer was made basic
by the addition of aqueous saturated sodium bicarbonate. After gently mixing the two
layers (no inversion), the aqueous layer was collected and the organic was again made
basic. This process was repeated until no more product remained in the organic layer as
determined by TLC. The basic aqueous fractions were then combined and washed with
fresh EtOAc. The basic layer was then collected in a glass beaker and acidified gently with
5M HCl while stirring. The solid was then extracted from the aqueous solution using
dichloromethane. The organic layer was collected and dried using sodium sulfate.
Following filtration, the organic layer was concentrated in vacuo to afford β-Ac
3
GlcNAc
Serine as a white or light brown foam (160 mg, 80% yield).
1
H NMR (600 MHz, CD
3
OD)
δ 7.77 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 6.1 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.5
Hz, 2H), 5.20 (t, J = 9.3 Hz, 1H), 4.95 (t, J = 9.7 Hz, 1H), 4.68 (d, J = 8.4 Hz, 1H), 4.40
42

(dd, J = 10.6, 6.9 Hz, 1H), 4.33 – 4.27 (m, 2H), 4.24 (dd, J = 12.3, 4.6 Hz, 1H), 4.20 (t, J =
6.8 Hz, 1H), 4.13 – 4.10 (m, 1H), 4.10 – 4.07 (m, 1H), 3.88 (dd, J = 10.8, 3.0 Hz, 1H), 3.80
(dd, J = 10.3, 8.7 Hz, 1H), 3.75 (ddd, J = 10.1, 4.5, 2.4 Hz, 1H), 2.00, 1.98, 1.95, 1.82 (4 x
s, 12H).
13
C NMR (151 MHz, CD
3
OD) δ 172.31, 170.91, 170.37, 169.83, 156.87, 143.86,
143.78, 141.14, 127.38, 126.79, 124.81, 124.77, 119.51, 100.47, 72.54, 71.55, 69.09,
68.70, 66.59, 61.78, 53.95, 48.42, 46.94, 21.47, 19.21, 19.16, 19.12. ESI-MS calculated for
(M - H)
-
m/z 655.21, found m/z 655.20. Characterization data was in agreement with
previously reported data (Mitchell et al., 2001).

Compound 2.2 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Serine Pfp
Ester (β-Ac
3
GlcNAc Ser-OPfp). To a stirring solution of 2.1 (272
mg, 0.420 mmol) in anhydrous dimethylformamide (4 mL) under N
2
was added anhydrous
pyridine (135 µL, 1.68 mmol). To this solution was added dropwise pentafluorophenyl
trifluoroacetate (216 µL, 1.26 mmol). The reaction was allowed to stir at room temperature
overnight. Reaction progress was monitored by TLC (4:6 EtOAc: Hexane). Once the
reaction was complete, the mixture was concentrated in vacuo and azeotroped with toluene
multiple times. The residue was then suspended in DCM and purified by flash
chromatography (30% Acetone in Hexane) and concentrated in vacuo to afford β-
Ac
3
GlcNAc Ser-OPfp as an off-white solid (345 mg, 87% yield).
1
H NMR (600 MHz,
CDCl
3
) δ 7.76 (d, J = 7.7 Hz, 2H), 7.65 (d, J = 7.5 Hz, 2H), 7.41 – 7.36 (m, 2H), 7.32 –
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
43

7.29 (m, 2H), 6.10 (d, J = 8.7 Hz, 1H), 5.58 (d, J = 8.1 Hz, 1H), 5.28 (t, J = 10.0 Hz, 1H),
5.05 (t, J = 9.6 Hz, 1H), 4.88 – 4.85 (m, 1H), 4.84 (d, J = 8.2 Hz, 1H), 4.50 (dd, J = 10.7,
7.0 Hz, 1H), 4.45 – 4.38 (m, 2H), 4.24 (t, J = 7.0 Hz, 1H), 4.21 (dd, J = 12.3, 4.8 Hz, 1H),
4.12 (dd, J = 11.9, 2.6 Hz, 1H), 3.97 (dd, J = 10.7, 3.2 Hz, 1H), 3.74 (dt, J = 10.5, 8.1 Hz,
1H), 3.69 (ddd, J = 10.1, 4.9, 2.5 Hz, 1H), 2.04 (bs, 6H), 2.03, 1.88 (2 x s, 6H).
13
C NMR
(151 MHz, CDCl
3
) δ 170.91, 170.86, 170.59, 169.32, 166.25, 156.02, 143.70, 143.60,
141.25, 127.75, 127.10, 125.13, 119.95, 100.58, 72.13, 71.90, 68.36, 68.26, 67.21, 61.91,
54.94, 54.30, 47.12, 23.21, 20.58. ESI-MS calculated for (M) m/z 822.21, found m/z
822.80. Characterization data was in agreement with previously reported data (Saha and
Schmidt, 1997).

Compound 2.3 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-threonine
(β-Ac
3
GlcNAc Threonine). Commercially available Ac
4
GlcNAc
(300 mg, 0.7704 mmol), InBr
3
(55 mg, 0.1541 mmol) (20 mol% with respect to donor),
and Fmoc-Thr-OH (100 mg, 0.2568 mmol) were added to a round bottom flask containing
a stir bar under open atmosphere. Mixture was then suspended in 1,2-dichloroethane (200
mM). A reflux condenser was added and reaction vessel was submerged in an oil bath set
at 60 °C. A nitrogen balloon was added and the reaction was heated to reflux (>84 °C) for
16 hours under nitrogen. After the reaction no longer progressed by TLC (7:2:1: EtOAc:
MeOH: H
2
O), the reaction was allowed to cool to room temperature and concentrated under
vacuo. The black residue was azeotroped several times with toluene under high vacuum.
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
44

The fluffy black solid was then resuspended in dichloromethane (DCM) and applied to
flash column chromatography (5% MeOH in DCM (0.1% AcOH)). Fractions containing
the product were combined and concentrated in vacuo. The acidic dark brown syrup was
then diluted in EtOAc and transferred to a separatory funnel. Organic layer was made basic
by the addition of aqueous saturated sodium bicarbonate. After gently mixing the two
layers (no inversion), the aqueous layer was collected and the organic was again made
basic. This process was repeated until no more product remained in the organic layer as
determined by TLC. The basic aqueous fractions were then combined and washed with
fresh EtOAc. The basic layer was then collected in a glass beaker and acidified gently with
5M HCl while stirring. The solid was then extracted from the aqueous solution using
dichloromethane. The organic layer was collected and dried using sodium sulfate.
Following filtration, the organic layer was concentrated in vacuo to afford β-Ac
3
GlcNAc
Threonine as a white or light brown foam (132 mg, 77% yield).
1
H NMR (600 MHz,
CD
3
OD) δ 7.78 (d, J = 7.6 Hz, 2H), 7.67 (t, J = 7.9 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.30
(t, J = 7.4 Hz, 2H), 5.21 (t, J = 9.7 Hz, 1H), 4.97 (t, J = 9.7 Hz, 1H), 4.67 (d, J = 8.5 Hz,
1H), 4.41 (qd, J = 6.3, 2.8 Hz, 1H), 4.35 (d, J = 7.1 Hz, 2H), 4.27 (dd, J = 12.3, 4.4 Hz,
1H), 4.24 (d, J = 7.1 Hz, 1H), 4.22 (d, J = 2.7 Hz, 1H), 4.09 (dd, J = 12.2, 2.5 Hz, 1H), 3.79
(dd, J = 10.5, 8.6 Hz, 1H), 3.73 (ddd, J = 10.0, 4.3, 2.6 Hz, 1H), 2.00, 1.98, 1.97, 1.90 (4 x
s, 12H), 1.18 (d, J = 6.4 Hz, 3H).
13
C NMR (151 MHz, CD
3
OD) δ 172.28, 171.02, 170.37,
169.81, 157.56, 143.86, 141.13, 127.36, 126.74, 124.86, 119.49, 99.49, 75.31, 72.40,
71.36, 68.72, 66.78, 61.77, 58.45, 54.10, 48.42, 21.41, 19.25, 19.15, 19.11, 16.60. ESI-MS
45

calculated for (M - H)
-
m/z 669.23, found m/z 669.20. Characterization data was in
agreement with previously reported data (Mitchell et al., 2001).

Compound 2.4 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Threonine
Pfp Ester (β-Ac
3
GlcNAc Thr-OPfp). To a stirring solution of 2.3
(200 mg, 0.2982 mmol) in anhydrous dimethylformamide (3 mL) under N
2
was added
anhydrous pyridine (96 µL, 1.193 mmol). To this solution was added dropwise
pentafluorophenyl trifluoroacetate (154 µL, 0.8946 mmol). The reaction was allowed to
stir at room temperature overnight. Reaction progress was monitored by TLC (4:6 EtOAc:
Hexane). Once the reaction was complete, the mixture was concentrated in vacuo and
azeotroped with toluene multiple times. The residue was then suspended in DCM and
purified by flash chromatography (30% Acetone in Hexane) and concentrated in vacuo to
afford β-Ac
3
GlcNAc Thr-OPfp as an off-white solid (185 mg, 74% yield).
1
H NMR (600
MHz, CDCl
3
) δ 7.74 (d, J = 7.5 Hz, 2H), 7.64 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H),
7.29 (t, J = 7.4 Hz, 2H), 6.03 (d, J = 8.8 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 5.27 (t, J = 9.8
Hz, 1H), 5.05 (t, J = 9.7 Hz, 1H), 4.73 (d, J = 8.2 Hz, 1H), 4.68 (dd, J = 8.9, 2.9 Hz, 1H),
4.55 (dd, J = 6.3, 2.9 Hz, 1H), 4.43 (ddd, J = 35.0, 10.6, 7.3 Hz, 3H), 4.24 (t, J = 7.2 Hz,
1H), 4.18 (dd, J = 12.3, 4.8 Hz, 1H), 4.04 (dd, J = 12.2, 2.2 Hz, 1H), 3.81 – 3.75 (m, 1H),
3.67 (ddd, J = 10.0, 4.7, 2.5 Hz, 1H), 2.03, 2.01, 1.98 , 1.93 (4 x s, 12H), 1.28 (d, J = 6.3
Hz, 3H). 13C NMR (151 MHz, CDCl
3
) δ 171.09, 170.60, 170.46, 169.27, 166.46, 156.62,
143.82, 143.57, 141.25, 127.72, 127.03, 125.16, 119.92, 98.18, 73.20, 71.87, 71.72, 68.40,
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
46

67.35, 61.86, 58.69, 55.09, 47.11, 23.34, 20.65, 20.56, 20.41, 16.49. ESI-MS calculated for
(M) m/z 836.22, found m/z 836.90. Characterization data was in agreement with previously
reported data (Saha and Schmidt, 1997).

Compound 2.5 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
(β-Ac
3
GlcNAc Cysteine). Commercially available Ac
4
GlcNAc
(970 mg, 2.49 mmol), InBr
3
(176 mg, 0.498 mmol) (20 mol% with respect to donor), and
Fmoc-Cys-OH Hydrate (300 mg, 0.830 mmol) were added to a round bottom flask
containing a stir bar under open atmosphere. Mixture was then suspended in 1,2-
dichloroethane (200 mM). A reflux condenser was added and reaction vessel was
submerged in an oil bath set at 60 °C. A nitrogen balloon was added and the reaction was
heated to reflux (>84 °C) for 16 hours under nitrogen. After the reaction no longer
progressed by TLC (7:2:1: EtOAc: MeOH: H
2
O), the reaction was allowed to cool to room
temperature and concentrated under vacuo. The black residue was azeotroped several times
with toluene under high vacuum. The fluffy black solid was then resuspended in
dichloromethane (DCM) and applied to flash column chromatography (5% MeOH in DCM
(0.1% AcOH)). Fractions containing the product were combined and concentrated in
vacuo. The acidic dark brown syrup was then diluted in EtOAc and transferred to a
separatory funnel. Organic layer was made basic by the addition of aqueous saturated
sodium bicarbonate. After gently mixing the two layers (no inversion), the aqueous layer
O
AcO
AcO
AcHN
OAc
S
FmocHN
OH
O
47

was collected and the organic was again made basic. This process was repeated until no
more product remained in the organic layer as determined by TLC. The basic aqueous
fractions were then combined and washed with fresh EtOAc. The basic layer was then
collected in a glass beaker and acidified gently with 5M HCl while stirring. The solid was
then extracted from the aqueous solution using dichloromethane. The organic layer was
collected and dried using sodium sulfate. Following filtration, the organic layer was
concentrated in vacuo to afford β-Ac
3
GlcNAc Cysteine as a white or light brown foam
(279 mg, 50% yield).
1
H NMR (600 MHz, CD
3
OD) δ 7.78 (d, J = 7.6 Hz, 2H), 7.67 (t, J =
7.8 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.32 – 7.29 (m, 2H), 5.18 (t, 1H), 4.98 (t, J = 9.7 Hz,
1H), 4.76 (d, J = 10.4 Hz, 1H), 4.42 (dd, J = 8.8, 4.2 Hz, 1H), 4.38 – 4.31 (m, 2H), 4.24 (t,
J = 7.0 Hz, 1H), 4.16 (dd, J = 12.3, 5.2 Hz, 1H), 4.12 – 4.06 (m, 1H), 3.98 (t, J = 10.3 Hz,
1H), 3.76 (ddd, J = 10.1, 5.2, 2.4 Hz, 1H), 3.34 (dd, J = 14.3, 4.2 Hz, 1H), 2.86 (dd, J =
14.3, 8.9 Hz, 1H), 1.99, 1.99, 1.96, 1.82 (4 x s, 12H).
13
C NMR (151 MHz, CD
3
OD) δ
171.90, 171.00, 170.36, 169.77, 156.99, 143.80, 143.76, 141.13, 127.39, 126.81, 126.79,
124.85, 119.51, 83.14, 75.58, 73.67, 68.68, 66.74, 62.06, 52.80, 47.99, 47.85, 47.70, 47.56,
47.42, 47.28, 47.14, 46.91, 31.19, 21.26, 19.21, 19.13, 19.10.ESI-MS calcd for (M - H)
-
m/z 671.19, found m/z 671.20. Characterization data was in agreement with previously
reported data (Ohnishi et al., 2000).

48

Compound 2.6 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
Pfp Ester (β-Ac
3
GlcNAc Cys-OPfp). InBr
3
(566 mg, 1.59 mmol)
and Fmoc-Cys(Trt)-OPfp (6.0 mg, 7.98 mmol) were added to a round bottom flask
containing a stir bar under open atmosphere. Mixture was then suspended in 1,2-
dichloroethane (80 mL) and triisopropylsilane (5.4 mL, 26.33 mmol) was added dropwise.
Solution was allowed to stir at room temperature for 10 minutes under open atmosphere.
Ac
4
GlcNAc (3.73 g, 9.58 mmol) was then added. A reflux condenser was added and
reaction vessel was submerged in an oil bath set at 60 °C. A nitrogen balloon was added
and reaction was heated to reflux for 16 hours under nitrogen. After the reaction no longer
progressed by TLC (7:3 EtOAc: Hexane), the reaction was allowed to cool to room
temperature and concentrated under vacuum to afford an off-white solid. The solid was
then resuspended in dichloromethane (DCM) and applied to flash column chromatography
(40-70% EtOAc in Hexane). Fractions containing the product were combined and
concentrated in vacuo to afford β-Ac
3
GlcNAc Cys-OPfp as a white solid (4.9 g, 73%).
1
H
NMR (500 MHz, CDCl
3
) δ 7.78 (d, J = 7.5 Hz, 2H), 7.64 (t, J = 7.3 Hz, 2H), 7.41 (t, J =
7.5 Hz, 2H), 7.33 (t, J = 7.0 Hz, 2H), 6.32 (d, J = 7.6 Hz, 1H), 5.60 (d, J = 8.9 Hz, 1H),
5.18 (t, J = 9.7 Hz, 1H), 5.0 9 (t, J = 9.6 Hz, 1H), 4.87 (td, J = 7.9, 3.9 Hz, 1H), 4.64 (d, J
= 10.4 Hz, 1H), 4.55 (dd, J = 10.4, 6.9 Hz, 1H), 4.44 (dd, J = 10.3, 7.0 Hz, 1H), 4.27 (t, J
= 6.8 Hz, 1H), 4.16 – 4.10 (m, 2H), 4.07 (dd, J = 12.7, 5.6 Hz, 1H), 3.69 (dt, J = 6.7, 4.1
Hz, 1H), 3.49 (dd, J = 14.6, 3.4 Hz, 1H), 3.11 (dd, J = 14.4, 8.4 Hz, 1H), 2.06, 2.06, 2.00,
1.96 (4 x s, 12H).
13
C NMR (151 MHz, CDCl
3
) δ 171.09, 170.73, 170.57, 169.18, 166.98,
O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
49

156.04, 143.66, 143.56, 141.28, 141.26, 127.74, 127.71, 127.11, 127.08, 125.05, 124.95,
120.00, 119.98, 83.82, 77.19, 76.98, 76.77, 76.36, 73.34, 68.12, 67.20, 62.11, 53.73, 52.93,
47.10, 31.28, 29.67, 23.12, 20.61, 20.54, 20.48. ESI-MS calculated for (M) m/z 838.18,
found m/z 838.80. Characterization data was in agreement with previously reported data
(De Leon et al., 2017).

Compound 2.7 2-Methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-
glucopyrano)-[2,1-d]-2-oxazoline (2-Methyl Oxazoline). To a solution of
commercially available β-Ac
4
GlcNAc (500 mg, 1.284 mmol) in dichloroethane (13 mL)
was added dropwise trimethylsilyl triflate (255 µL, 1.412 mmol) under nitrogen
atmosphere. The reaction was then heated at 50 °C. Reaction progress was monitored by
TLC using 5% MeOH in CH
2
Cl
2
. Upon completion, reaction was allowed to cool to room
temperature and made slightly basic by the addition of triethylamine (520 µL, 3.744 mmol).
The reaction was then diluted with DCM and transferred to a separatory funnel and washed
with water (2X) and brine. Organic layer was dried using sodium sulfate, filtered, and
concentrated in vacuo. Residue was dissolved in 100:200:1 Toluene: EtOAc: triethylamine
and purified by column chromatography using the same solvent system. Fractions
containing the product were collected and concentrated in vacuo to afford the 2-methyl
oxazoline as a light-yellow oil in 94% yield (400 mg).
1
H NMR (400 MHz, CDCl
3
) δ 5.96
(d, J = 7.4 Hz, 1H), 5.27 – 5.25 (m, 1H), 4.93 (ddd, J = 9.3, 2.1, 1.3 Hz, 1H), 4.18 (s, 1H),
4.17 (d, J = 1.6 Hz, 1H), 4.13 (dddd, J = 7.7, 3.7, 3.1, 1.8 Hz, 1H), 3.63 – 3.58 (m, 1H),
O
AcO
AcO
OAc
N
O
50

2.11 (s, 3H), 2.10 (s, 3H), 2.09 (d, J = 1.8 Hz, 3H), 2.08 (s, 3H). Spectroscopic data was in
agreement with previously reported data (Srivastava, 1982).

Glycosylation for HPLC Analysis. Respective amounts of donor, InBr
3
, and amino acid
were added to a 1.5 mL Eppendorf tube. Mixture was suspended in the appropriate solvent
and at the determined concentration of 200 mM. The Eppendorf tube was capped, vortexed
for one minute, and heated at 60 or 80 °C with constant agitation (1000 rpm) in a
Thermomixer F1.5 (Eppendorf) for 16 hours. The reaction was aliquoted and diluted with
methanol for HPLC analysis (0 - 100% buffer B over 15 minutes).

51

Chapter 2 References
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solid-phase glycopeptide synthesis: 2-acetamido-2-deoxy-β- D-glycosides of FmocSerOH
and FmocThrOH. J. Chem. Soc. Chem. Comm. 20, 2383–2384.
Bond, M. R., and Hanover, J. A. (2015). A little sugar goes a long way: The cell biology
of O-GlcNAc. J. Cell Biol. 208, 869–880.
Braga, A. L., Vargas, F., Galetto, F. Z., Paixão, M. W., Schwab, R. S., and Taube, P. S.
(2007). One-Pot Indium Iodide Mediated Synthesis of Chiral β-Seleno Amides and
Selenocysteine Derivatives by Ring-Opening Reaction of 2-Oxazolines. Eur. J. Org. Chem.
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Chuh, K. N., Batt, A. R., and Pratt, M. R. (2016). Chemical Methods for Encoding and
Decoding of Posttranslational Modifications. Cell Chem. Biol. 23, 86–107.
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Competent Lewis Acid Catalysts: Indium(III) and Bismuth(III) Salts Produce
Rhamnosides (=6-Deoxymannosides) in High Yield and Purity. Helv. Chim. Acta 95,
2652–2659.
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Analogue of O-GlcNAc (S-GlcNAc) Is an Enzymatically Stable and Reasonable Structural
Surrogate for O-GlcNAc at the Peptide and Protein Levels. Biochemistry 56, 3507–3517.
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Trichloroethoxycarbonyl-glucosamine derivatives as glycosyl donors. Carbohyd. Res. 296,
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GlcNAc: a potential molecular probe for the functional study of O-GlcNAc. Bioorg. Med.
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D- glucopyranosyl)-serine and -threonine building blocks for glycopeptide formation. J.
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O-GlcNAc tau antibody. Amino Acids 40, 857–868.

55

Chapter 3. O-GlcNAcylation of α-synuclein at serine 87 reduces
aggregation without affecting membrane binding
†

Introduction
The addition of the monosaccharide N-acetyl-glucosamine, or O-GlcNAc modification
(Figure 3-1A), occurs in plants and animals and has been found on hundreds of proteins in
the cytosol, nucleus, and mitochondria (Bond and Hanover, 2015; Hardiville and Hart,
2014). The sugar is added to the side chain hydroxyl groups of serine and threonine residues
of substrate proteins by the enzyme O-GlcNAc transferase (OGT) and can be rendered
dynamic through subsequent removal by O-GlcNAcase (OGA). There are several lines of
evidence that implicate the misregulation of O-GlcNAcylation in neurodegenerative
diseases (Yuzwa and Vocadlo, 2014; Zhu et. al., 2014). Mice with neuron-specific deletion
of OGT have hyperphosphorylated tau and suffer from locomotor defects before dying
within 10 days of birth (O'Donnell et al., 2004). Decreased global levels of O-
GlcNAcylation has also been observed in the brains of patients who succumbed to
Alzheimer's disease (AD) (Liu et al., 2004). Furthermore, increasing the levels of O-
GlcNAcylation in a mouse model of AD with a small-molecule inhibitor of OGA slowed
the pace of neurodegeneration (Yuzwa et al., 2012), and the same small-molecule reduced
the amount of hyperphosphorylated tau in healthy rats (Yuzwa et al., 2008). Notably,
several of the proteins that form the toxic aggregates that are hallmarks of

†
Yuka E. Lewis, Ana Galesic, Paul M. Levine, Natalie Lamiri, and Caroline K. Brennan (University of
Southern California) contributed to the work presented in this chapter.
56

neurodegenerative diseases are directly modified by O-GlcNAcylation, which can result in
inhibition of protein aggregation. For example, Vocadlo and co-workers enzymatically O-
GlcNAcylated the AD-associated protein tau in a heterogeneous fashion and showed that
this modified protein had a decreased propensity for aggregation (Yuzwa et al., 2012). We
previously used synthetic strategies to build site-specifically O-GlcNAcylated α-synuclein
peptides and the full-length protein, the major aggregating protein in Parkinson’s disease
(PD), and found that the modification completely blocks its aggregation (Marotta et al.,
2012; Marotta et al., 2015).

Figure 3-1. O-GlcNAcylation and α-synuclein. (A) O-GlcNAcylation is the addition of N-acetyl-
glucosamine to serine and threonine residues of intracellular proteins. The modification is added
by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). (B) Various proteomics
experiments on rodent brain samples and human erythrocytes have identified nine different O-
GlcNAcylation sites on α-synuclein.

α-Synuclein is a small (140 amino acids) protein that is highly enriched in pre-synaptic
neurons of the central nervous system (Lashuel et al., 2013), where it appears to be involved
in vesicle remodeling and trafficking (Emanuele and Chieregatti, 2015). When in contact
with membranes, the protein forms an extended α-helix that can induce membrane bending
57

(Jao et al., 2008; Mizuno et al., 2012; Varkey et al., 2010), while it exists as predominantly
an unstructured monomer in solution and the cytosol. In PD and other synucleinopathies,
however, α-synuclein is found in aggregates that have the features of the β-sheet rich fibers
that are common to all amyloid proteins (Fink, 2006). Solid-state NMR and EPR
experiments have defined the region of α-synuclein that forms the core of these aggregates
to be approximately residues 61-95 (Chen et al., 2007; Tuttle et al., 2016; Vilar et al., 2008).
Several proteomics studies from both neurons and erythrocytes, which also have high
concentrations of α-synuclein, have identified nine different in vivo sites of O-
GlcNAcylation (Figure 3-1B) (Alfaro et al., 2012; Morris et al., 2015; Wang et al., 2009;
Wang et al., 2010). This modification being present at many sites is not necessarily
surprising as OGT prefers to modify unstructured regions of proteins. As mentioned above,
we previously used a synthetic protein chemistry strategy to investigate the consequences
of O-GlcNAcylation at α-synuclein T72 (Marotta et al., 2015). We found that modification
at T72 completely blocks in vitro the first step of the aggregation process, termed
nucleation, and had a slight inhibitory effect on the second elongation step that generates
larger fiber structures. Here, we continue to explore the effects of O-GlcNAcylation on the
biophysical properties of α-synuclein. More specifically, we chose to focus on
modification at residue S87. Notably, this serine is mutated to glutamine in rodent α-
synucleins, and mouse synuclein was recently found to inhibit the aggregation of the
human protein (Fares et al., 2016). Furthermore, S87 is also a site of phosphorylation, and
both the enzymatically phosphorylated protein and the phosphomimetic mutation S87E
inhibit α-synuclein aggregation, although phosphorylation at this site also inhibits
58

membrane binding (Paleologou et al., 2010). In a similar strategy to our previous effort,
we used chemical ligation to prepare α-synuclein with O-GlcNAcylation at S87 and found
that this modification also inhibits protein aggregation, albeit to a lesser extent than O-
GlcNAcylation at T72 or the S87E mutation. However, in contrast to phosphorylation, O-
GlcNAcylation had no observable effect on the membrane binding properties of α-
synuclein. These results add additional support for the targeting of OGA to raise O-
GlcNAcylation levels as a treatment strategy for PD. Furthermore, they demonstrate
biophysical differences between O-GlcNAcylation and phosphorylation at the same site,
encouraging the further study of the effects of sterics versus charge in the modulation of
protein aggregation.

Results
In order to directly test the effect of O-GlcNAcylation at S87 on α-synuclein aggregation
and membrane binding, we set out to first prepare the modified protein using expressed
protein ligation (EPL). Traditional EPL takes advantage of cysteine residues at the ligation
sites through a transthioesterification reaction followed by an S to N acyl shift. α-Synuclein
contains no native cysteines, so we decided to introduce these residues at positions 76 and
91 in the primary sequence, which are alanine residues in the native protein. These cysteine
residues then enable us to retrosynthetically deconstruct α-synuclein (Figure 3-2A) into a
recombinant protein thioester (1, residues 1-75), a synthetic peptide (2 or 3, residues 76-
90), and a recombinant C-terminal fragment (4, residues 91-140). The recombinant protein
thioester 1 was prepared by expression of the corresponding intein fusion in E. coli
59

Figure 3-2. Synthesis and characterization of unmodified and O-GlcNAcylated α-synuclein
(α-synuclein(gS87). (A) α-Synuclein can be retrosynthetically deconstructed into an N-terminal
protein thioester (1), a synthetic peptide (2 or 3), and a C-terminal recombinant protein (4). (B)
Characterization of both synthetic proteins by RP-HPLC and ESI-MS: unmodified α-synuclein
expected mass is 14,460 Da, and the observed mass was 14,461 ± 2 Da; α-synuclein(gS87)
expected mass is 14,663 Da, and the observed mass was 14,666 ± 2 Da.

followed by thiolysis. The peptides 2 (unmodified) or 3 (O-GlcNAcylated at S87) were
synthesized as the corresponding thioesters by Fmoc-based solid phase peptide synthesis
using the Dawson aminobenzyol resin (Blanco-Canosa and Dawson, 2008). Finally,
fragment 4 was prepared by expression in E. coli where the critical N-terminal cysteine
residue was generated by the action of an endogenous methionine aminopeptidase. All of
these peptide and protein fragments were then purified by RP-HPLC and characterized by
ESI-MS (Figure 3-3). Incubation of either peptide 2 or 3 and fragment 4 resulted in
formation of the ligation product (Figures 3-4 and 3-5). At this time, the pH of the buffer
was reduced and the N-terminal cysteine protecting-group was removed using
60

methoxylamine. The resulting proteins 5 and 6 were purified by RP-HPLC and
characterized by ESI-MS (Figures 3-4 and 3-5). These proteins were then incubated
separately with the recombinant protein thioester 1 to yield the corresponding unmodified
or O-GlcNAcylated proteins 7 and 8 (Figure 3-6). In order to generate the α-synuclein
proteins with no mutations, the cysteine residues at the ligation sites were then transformed
to the native alanine residues using radical-mediated desulfurization (Figure 3-7). The final
products 9 and 10 were then purified by RP-HPLC and characterized by ESI-MS (Figure
3-2B).

Figure 3-3. Characterization of α-synuclein protein fragments. Recombinant protein-thioester
(1) and the recombinant C-terminus (4) were characterized by RP-HPLC and ESI-MS. 1 expected
mass is 7,686 Da, and the observed mass was 7,686 ± 1 Da; 4 expected mass is 5,593 Da, and the
observed mass was 5,595 ± 2 Da.

61

Figure 3-4. Ligation of peptide 2 and protein 4 and the subsequent deprotection to give
protein fragment 5. The ligation reaction was followed by RP-HPLC and the identity of
intermediate product C was confirmed by ESI-MS. This was followed by deprotection in the same
pot to give the product D, which again was confirmed by ESI-MS. C expected mass is 6,992 Da,
and the observed mass was 6,993 ± 1 Da; D expected mass is 6,980 Da, and the observed mass was
6,979 ± 1 Da.

62

Figure 3-5. Ligation of peptide 3 and protein 4 and the subsequent deprotection to give
protein fragment 6. The ligation reaction was followed by RP-HPLC and the identity of
intermediate product C was confirmed by ESI-MS. This was followed by deprotection in the same
pot to give the product D, which again was confirmed by ESI-MS. C expected mass is 7,195 Da,
and the observed mass was 7,195 ± 1 Da; D expected mass is 7,184 Da, and the observed mass was
7,184 ± 1 Da.

63

Figure 3-6. Ligation of proteins 5 or 6 and protein-thioester 1 to give proteins 7 or 8. The
ligation reactions were followed by RP-HPLC and the identities of the products C were confirmed
by ESI-MS. Unmodified C expected mass is 15,524 Da, and the observed mass was 15,525 ± 2 Da;
O-GlcNAcylated C expected mass is 14,728 ± 2 Da, and the observed mass was 14,728 ± 2 Da.

64

Figure 3-7. Desulfurization of proteins 7 or 8. The desulfurization reactions were followed by
RP-HPLC and the identities of the products B were confirmed by ESI-MS. Unmodified B expected
mass is 14,460 Da, and the observed mass was 14,461 ± 1 Da; O-GlcNAcylated B expected mass
is 14,663 Da, and the observed mass was 14,666 ± 2 Da.

65

Figure 3-8. Structural characterization of α-synuclein(gS87) and α-synuclein(S87E) using
circular dichroism (CD) and dynamic light scattering (DLS). (A) α-Synuclein(gS87) and α-
synuclein(S87E) are unstructured in solution. CD spectra were collected for freshly dissolved
proteins at 7.5 μM concentration. (B) Both proteins were monomeric in nature with Stoke’s Radii
of approximately 4 nm, and no significant, larger peaks. The indicated proteins were analyzed using
DLS at 50 μM concentration.

With the synthetic proteins in hand, we first compared the unmodified, synthetic α-
synuclein to fully recombinant protein. Analysis by circular dichroism (CD) spectroscopy
showed that both proteins were unstructured in solution (Figure 3-8A) and dynamic light
scattering (DLS) analysis demonstrated that both proteins were monomeric in nature
(Figure 3-8B). These two proteins were then subjected in triplicate to aggregation
conditions at a concentration of 50 μM for 7 days. Reaction aliquots were removed after
72, 120, and 168 h for visualization of any fiber formation using thioflavin T (ThT)
fluorescence (Figure 3-9A), which demonstrated that synthetic and recombinant α-
synuclein aggregated with essentially the same kinetics. To visualize the structure of the
fibers that formed, aliquots from the end of the aggregation assay were collected and
transmission electron microscopy (TEM) was performed, and both the recombinant and
synthetic proteins formed α-synuclein fibers that are consistent with amyloid structures
(Figure 3-9B). These data demonstrate that our synthetic preparation of α-synuclein did
66

not affect its biophysical properties. Next, we set out to determine the consequences of O-
GlcNAcylation at S87 using our synthetic protein, termed α-synuclein(gS87). As
mentioned above S87 is also a known site of phosphorylation, so we also expressed the
S87E mutant of α-synuclein (α-synuclein(S87E), Figure 3-10, as this glutamic acid mutant
has been shown to recapitulate some of the effects of phosphorylation at the same site. CD
spectroscopy showed that neither O-GlcNAcylation or the S87E mutation induced any
secondary structure in α-synuclein (Figure 3-11A), and both proteins were monomeric in
nature as determined by DLS (Figure 3-11B). We next simultaneously examined the
aggregation of unmodified α-synuclein, α-synuclein(gS87), and α-synuclein(S87E) using
a combination of ThT fluorescence, SDS-PAGE analysis, and TEM. First, aggregation
reactions at a concentration of 50 μM were again initiated in triplicate. After 48, 72, 120,
and 168 hours aliquots were removed and analyzed by ThT fluorescence (Figure 3-9C).
These data showed that α-synuclein(gS87) still aggregated but with slower kinetics than
the unmodified protein, and that α-synuclein(S87E) was essentially totally resistant to
aggregation, consistent with previously published results. Next, the structure of any
aggregates that formed after 168 h was visualized using TEM (Figure 3-9D and Figure 3-
12). As expected, the unmodified α-synuclein formed large, regular fibers. In contrast, α-
synuclein(S87E) formed only amorphous structures that are consistent with protein
precipitation during the preparation of the TEM grids and what could be small, irregular
structures. α-Synuclein(gS87) formed both shorter fibers and small structures that are
similar to those formed by protein precipitation during TEM preparation. As a
complementary assay, identical aliquots were first subjected to centrifugation to separate
67

Figure 3-9. O-GlcNAcylation of α-synuclein at S87 inhibits protein aggregation. (A)
Synthetic, unmodified α-synuclein aggregates in the same fashion as recombinant protein.
Recombinant or unmodified, synthetic α-synuclein were incubated under aggregation conditions
(50 μM concentration and agitation at 37 °C) for the indicated lengths of time before analysis by
ThT fluorescence (λ ex = 450 nm, λ em = 482 nm). The y-axis shows the fold-increase of fluorescence
compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from the mean of
biological replicates (n = 3). (B) The structures formed in the same aggregation reactions were
visualized by TEM after 168 h. Scale bars, 500 nm. (C) O-GlcNAcylation and a phosphomimetic
mutation at S87 inhibit aggregation. Recombinant α-synuclein, α-synuclein(gS87), or α-
synuclein(S87E) were subjected to aggregation conditions (50 μM concentration and agitation at
37 °C) for the indicated lengths of time before analysis by ThT fluorescence (λ ex = 450 nm, λ em =
482 nm). The y-axis shows the fold-increase of fluorescence compared with the corresponding
protein at t = 0. Error bars represent ±s.e.m from the mean of biological replicates (n = 3), and
statistical significance was calculated using a two-tailed Student’s t-test. (D) The same reactions
were analyzed by TEM after 168 h. (E) Aliquots from the same reactions were collected and the
soluble fractions collected by centrifugation. These soluble proteins were then separated by SDS-
PAGE and visualized by Coomassie blue staining. The data is representative of two biological
experiments.

68

Figure 3-10. Expression and characterization of α-synuclein(S87E). Recombinant α-
synuclein(S87E) was characterized by RP-HPLC and ESI-MS. Expected mass is 14,502 Da and
the observed mass was 14,504 ± 1 Da.

Figure 3-11. Structural characterization of synthetic and recombinant unmodified α-
synuclein using circular dichroism (CD) and dynamic light scattering (DLS). (A) Both
recombinant and synthetic α-synuclein are unstructured in solution. CD spectra were collected for
freshly dissolved proteins at 7.5 μM concentration. (B) Both proteins were monomeric in nature
with Stoke’s Radii of approximately 4 nm, and no significant, larger peaks. The indicated proteins
were analyzed using DLS at 50 μM concentration.

69

Figure 3-12. Transmission electron microscopy (TEM) images of the from the α-synuclein
aggregation reactions. Unmodified α-synuclein, α-synuclein(gS87), or α-synuclein(S87E) were
subjected to aggregation conditions (50 μM concentration and agitation at 37 °C) for 168 h before
visualization of any aggregate structures by TEM. Scale bars, 500 nm.

the aggregates from soluble material. The soluble fraction was then concentrated by
lyophilization and then subjected to SDS-PAGE and visualized by Coomassie staining
(Figure 3-9E). Consistent with the ThT fluorescence the unmodified protein was lost from
the soluble fraction to a much higher extent than either α-synuclein(gS87) or α-
synuclein(S87E), further demonstrating that both O-GlcNAcylation and the
phosphomimetic mutation inhibit aggregation. These data support a model where both O-
GlcNAcylation and phosphorylation at S87 have a similar effect on the formation of fibers,
namely the inhibition of the formation of fibers and the formation of small, irregular
structures. However, the phosphomimetic is more strongly inhibitory compared to O-
GlcNAcylation at the same site. The aggregation of α-synuclein is known to be
70

concentration dependent. Therefore, we next asked if the inhibitory capacity of O-
GlcNAcylation at S87 is dependent on the concentration of the protein, and aggregation
reactions were performed at a concentration of 25 μM. As expected, analysis of protein
aggregation by ThT fluorescence (Figure 3-13A) showed that aggregation of unmodified
α-synuclein over the same timeframe was reduced by ~5-fold. Again, aggregation mixtures
with α-synuclein(S87E) did not give any ThT signal over the course of the reaction.
Interestingly, the consequences of O-GlcNAcylation were more significant, suggesting that
it could have a reasonable inhibitory effect in neurons where the concentration of α-
synuclein has been estimated to be between 10 and 100 μM.

We next set out to determine the effects of O-GlcNAcylation on the endogenous function
of α-synuclein. As mentioned above, α-synuclein forms an extended α-helix when it comes
into contact with negatively charged membranes, and α-synuclein can remodel membranes
in isolation and can collaborate with other proteins to alter vesicle trafficking. Notably, α-
synuclein that has been enzymatically phosphorylated at S87 has reduced affinity for
membranes and therefore may inhibit these functions; however, the phosphomimetic
mutation S87E had no observable effect (Paleologou et al., 2010). To determine if O-
GlcNAcylation at S87 has consequences on membrane binding, unmodified α-synuclein,
α-synuclein(gS87), or α-synuclein(S87E) was incubated with an excess of vesicles for 20
min before measuring the induction of any secondary structure by CD spectroscopy (Figure
3-13B). As expected from previous results, unmodified protein and α-synuclein(S87E)
formed α-helical structures in the presence of the negatively charged lipid vesicles made
71

up of POPG or POPS, and this binding to POPS vesicles can be reduced by in introduction
of the zwitterionic lipid POPC. Importantly, these same vesicles have been used
extensively for the analysis of α-synuclein membrane interactions (Auluck et al., 2010;
Pfefferkorn et al., 2012). We also observed no major difference upon O-GlcNAcylation of
α-synuclein at S87, indicating that this modification does not have notable effect on the
affinity or binding mode of α-synuclein to membranes.

72

Figure 3-13. O-GlcNAcylation at S87 inhibits α-synuclein aggregation without affecting
membrane binding. (A) O-GlcNAcylation at S87 is more inhibitory towards aggregation at lower
protein concentrations. Recombinant α-synuclein, α-synuclein(gS87), or α-synuclein(S87E) were
subjected to aggregation conditions (25 μM concentration and agitation at 37 °C) for the indicated
lengths of time before analysis by ThT fluorescence (λ ex = 450 nm, λ em = 482 nm). The y-axis shows
the fold-increase of fluorescence compared with the corresponding protein at t = 0. Error bars
represent ±s.e.m from the mean of biological replicates (n = 3), and statistical significance was
calculated using a two-tailed Student’s t-test. (B) O-GlcNAcylation at S87 has no effect on α-
synuclein membrane binding. Recombinant α-synuclein, α-synuclein(gS87), or α-synuclein(S87E)
were incubated with a 100-fold excess of the indicated, preformed vesicles and analyzed using
circular dichroism (CD). In the presence of negatively charged vesicles (POPG or POPS), all of the
proteins gave essentially indistinguishable CD spectra consistent with the formation of an extended
α-helix. The introduction of a zwitterionic lipid (POPC) reduced the α-helix formation equally for
both proteins. POPG = 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)]; POPS =
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine.
73

Finally, we set out to explore the effect of different substitutions at residue 87 in more
detail. More specifically, we chose a further set of mutations (S87A, S87D, S87W, and
S87K) and expressed and purified the corresponding α-synuclein proteins (Figure 3-14).
We chose S87A as it represents the loss-of-function mutant that cannot be modified and
has been used in the past to understand the effect of phosphorylation at this position
(Oueslati et al., 2012; Paleologou et al., 2010). α-Synuclein(S87D) was chosen as it also
contains a negative charge, the exact position of that charge with respect to the protein
backbone is altered compared to S87E. The S87W mutation increases the steric bulk of this
position similarly to O-GlcNAcylation; however, unlike the carbohydrate tryptophan is
largely hydrophobic in nature. Finally, we chose S87K to reverse the charge at this position
compared to S87E. Wild-type α-synuclein and each of these mutants were then subjected
to aggregation conditions at 25 μM concentration before analysis by ThT fluorescence
(Figure 3-15A). In the case of S87A, we found that this mutation was slightly inhibitory to
aggregation, while S87D was also inhibitory to a larger extent. In contrast, the S87W and
S87K mutations increased the aggregation, with S87K having a larger effect. Together
these data indicate that inhibition of aggregation by O-GlcNAcylation and phosphorylation
(or the pseudo-phosphate S87E) at this position is not driven by sterics alone but rather by
hydrophilicity or negative charge, respectively. Next, we also examined the effect(s) of
these mutations on α-synuclein membrane binding by incubating the proteins in excess of
POPG vesicles for 20 min before analysis by CD spectroscopy (Figure 3-15B).
Interestingly, all of the mutations besides S87D had very little effect on the induction of
the α-synuclein α-helix, similar to O-GlcNAcylation and S87E. In contrast, α-
74

synuclein(S87D) showed less membrane binding that is quite similar in magnitude to
phosphorylation at this residue (Paleologou et al., 2010), suggesting that α-synuclein is
very sensitive to the exact position of the negative charge at serine 87.

Figure 3-14. Characterization of the additional α-synuclein mutants S87A, S87D, S87W, and
S87K. The indicated recombinant α-synuclein mutants were characterized by RP-HPLC and ESI-
MS.Expected mass of α-synuclein(S87A) is 14,435 Da, and observed mass was 14,435 ± 1 Da.
Expected mass of α-synuclein(S87D) is 14,488 Da, and observed mass was 14,489 ± 1 Da.
Expected mass of α-synuclein(S87W) is 14,560 Da, and observed mass was 14,562 ± 2.5 Da.
Expected mass of α-synuclein(S87K) is 14,501 Da, and observed mass was 14,503 ± 1 Da.

75

Figure 3-15. Analysis of mutant α-synuclein aggregation and membrane binding. (A)
Mutations of α-synuclein at S87 have different effects on protein aggregation. Recombinant α-
synuclein or the indicated α-synuclein mutants were individually subjected to aggregation
conditions (25 μM concentration and agitation at 37 °C) for the indicated lengths of time before
analysis by ThT fluorescence (λ ex = 450 nm, λ em = 482 nm). The y-axis shows the fold-increase of
fluorescence compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from
the mean of biological replicates (n = 3), and statistical significance was calculated using a two-
tailed Student’s t-test. (B) Mutation of S87 to D inhibits α-synuclein membrane binding.
Recombinant α-synuclein or the indicated α-synuclein mutants were incubated with a 100-fold
excess of the indicated, preformed vesicles and analyzed using circular dichroism (CD). In the
presence of negatively charged vesicles (POPG), most of the proteins gave essentially
indistinguishable CD spectra consistent with the formation of an extended α-helix. However, α-
synuclein(S87D) showed diminished signal corresponding to less membrane binding and α-helix
formation. POPG = 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)].

76

Discussion
Here, we have reported the synthesis of α-synuclein with site-specific O-GlcNAcylation at
residue 87 and show that this modification inhibits protein aggregation. However, this
modification is less inhibitory when compared to O-GlcNAcylation at T72, which we
previously prepared and investigated. More specifically, α-synuclein(gT72) is completely
resistant to aggregation at 50 μM concentration, while α-synuclein(gS87) shows
significant aggregation at this concentration and even some aggregation at 25 μM. We
speculate that this observation can be explained by the effects of O-GlcNAcylation on the
equilibrium between monomeric α-synuclein in solution and protofiber structures that will
seed a rapid extension reaction that forms mature fibers. In the case of O-GlcNAcylation
at T72 the equilibrium strongly favors monomeric protein, while modification at S87 has
a smaller effect that can be overcome at higher monomer concentrations. The site-specific
difference are also consistent with a recent NMR structure of the α-synuclein fiber (Tuttle
et al., 2016), as well as previous structural models that have been developed using EPR
and NMR spectroscopy (Chen et al., 2007; Vilar et al., 2008), as well as cryo-electron
microscopy (Rodriguez et al., 2015). More specifically, T72 lies in the middle of a β-strand
in the core structure that makes up the individual monomers in the fiber, while S87 is
towards the edge of this core in a potentially more flexible turn between two β-strands that
may more readily accommodate the steric bulk of the O-GlcNAcylation. Interestingly, we
also find that O-GlcNAcylation is less inhibitory than the pseudo-phosphate S87E
mutation. Importantly, our data showing that α-synuclein(S87E) completely blocks
aggregation at these concentrations is totally consistent with previous reports (Paleologou
77

et al., 2010). Other mutations at S87 ranged from partial inhibition, like S87D, to
potentiation (S87W and S87K) of protein aggregation. Again, these observations can be
rationalized by the NMR structure (Tuttle et al., 2016). The residues that surround S87 in
the fiber structure all mostly hydrophobic in nature except for a single glutamic acid at
residue 82. Therefore, the negative charge(s) on S87E, S87D, and phosphorylated S87
would strongly promote interactions this area with bulk water, while the uncharged
carbohydrate could be more easily desolvated to allow for the formation of the hydrophobic
core of the fiber and suggest that the increased steric bulk of O-GlcNAc at S87 is not
sufficient to completely block aggregation. Furthermore, the relatively hydrophobic bulk
of tryptophan in S87W is well tolerated in the aggregate-formation process. Interestingly,
the S87K mutation shows the most aggregation, which might be explained through a
favorable interaction with the glutamic acid at residue 82 in α-synuclein. This result means
that we cannot rule out a charge repulsion to explain the strong inhibition of the longer
S87E compared to S87D, as it may be in closer physical proximity to E82.

In contrast to the effects on protein aggregation, we find that O-GlcNAcylation at S87 has
essentially no effect on the ability of α-synuclein to interact with lipid vesicles. This stands
in contrast to the observations that have been made with α-synuclein that has been
enzymatically phosphorylated at S87 (Paleologou et al., 2010). Again, these data fit with
previous the structural characterization of the α-synuclein extended helix using EPR
spectroscopy (Jao et al., 2008). According to the model developed from these data, S87
lies towards the end of the α-helical structure (residue 90) and close to the lipid-solvent
78

interface near the phosphate headgroups. Therefore, it is not surprising that the doubly-
charged phosphorylated S87 would experience more electrostatic repulsion compared to
either S87E or O-GlcNAcylation at this position. Notably, however, α-synuclein(S87D)
also shows less membrane binding, demonstrating that the exact position of the negative
charge may be equally important. This suggests that O-GlcNAcylation may have a smaller
effect on the healthy functions of α-synuclein when compared to phosphorylation at the
same site. In total, these data add further support for increasing O-GlcNAcylation as a
potential therapeutic strategy in PD and highlight the utility of synthetic approaches for
testing the consequences of protein posttranslational modifications.

Materials and Methods
All chemicals and solvents were purchased from commercial venders (Fluka, EMD,
Novagen, Sigmα-Aldrich, etc) and used without any additional purification. Growth media
(Luria-Bertani, Miller) were prepared and sterilized according to the manufacture protocol.
Antibiotics (Kanamycin sulfate, EMD, and Ampicillin sodium salt, EMD) were prepared
as stock solution (50 mg mL
-1
and 100 mg mL
-1
respectively), and stored at -20 ℃.
Analytical thin-layer chromatography was performed on 60 Å F254 silica plates with
detection by UV light and/or ceric ammonium molybdate (CAM). Agilent Technologies
1200 Series HPLC with Diode Array Detector was used for reverse phase high performance
liquid chromatography (RP-HPLC). Unless otherwise noted, the HPLC buffers were,
buffer A: water with 0.1% TFA, buffer B: 90% acetonitrile, 10% water with 0.1% TFA.
Mass spectra were obtained using API 150EX (Applied Biosystems/MDS SCIEX).
79

Expression of recombinant wild type α-synuclein and mutant α-synuclein proteins.
BL21(DE3) E. coli transformed with pRK172 construct containing human wild-type α-
synuclein or α-synuclein(S87E) was grown until its OD
600
was above 0.6. The culture was
induced by addition of 0.5 mM IPTG and incubation for 20 h at 25 ℃. The culture was
pelleted by centrifugation at 6,000 rpm. The pellet was lysed by 3 times of freeze and thaw
cycle. The resulting lysate was resuspended in lysis buffer (500 mM NaCl, 100 mM Tris,
10 mM beta-mercaptoethanol, 1 mM EDTA, pH 8.0), and heated at 80 ℃ for 10 min. The
lysate was allowed to cool down to room temperature before the addition of protease
inhibitor cocktail (mini complete EDTA free, Roche). The resulting solution was incubated
in ice for 30 min, and the cell debris was pelleted by centrifugation at 15,000 rpm for 30
min at 4 ℃. The pH of the supernatant was adjusted to 3.5 with 1M HCl, and the resulting
solution was incubated on ice for additional 30 min. The lysate was cleared by
centrifugation (15,000 rpm, 30 min, 4 ℃), and then dialyzed against 3 X 1 L of a degassed
1% acetic acid solution. The dialyzed solution was cleared by centrifugation (6,000 rpm,
15 min, 4 ℃). α-Synuclein was purified on RP-HPLC (40-60% B over 60 min), and
lyophilized. The purified protein was characterized by RP-HPLC and ESI-MS. Expected
mass of wild type α-synuclein is 14,460 Da, and the observed mass was 14,460 ± 3 Da.
Expected mass of α-synuclein(S87E) is 14,502 Da, and observed mass was 14,504 ± 1 Da.
Expected mass of α-synuclein(S87A) is 14,435 Da, and observed mass was 14,435 ± 1 Da.
Expected mass of α-synuclein(S87D) is 14,488 Da, and observed mass was 14,489 ± 1 Da.
80

Expected mass of α-synuclein(S87W) is 14,560 Da, and observed mass was 14,562 ± 2.5
Da. Expected mass of α-synuclein(S87K) is 14,501 Da, and observed mass was 14,503 ±
1 Da.

Expression and purification of α-synuclein C-terminal fragment. Transformed
BL21(DE3) E. coli with pET42b plasmid containing α-synuclein(C91-140) was expressed
and semi-purified by HCl treatment and centrifugation as described above for full-length
α-synuclein. α-Synuclein(C91-140) was then further purified on RP-HPLC (10-45% B
linear gradient over 60 min). The purified fragment was characterized by RP-HPLC and
ESI-MS. Expected mass is 5,593 Da, and the observed mass was 5,595 ± 2 Da.

Expression and purification of α-synuclein N-terminal thioester. Transformed
BL21(DE3) E. coli with pTXB1 plasmid containing α-synuclein(1-75)-AvaDnaE-Histag
was grown to OD
600
above 0.6. The induction of culture was conducted by addition of 0.5
mM IPTG and incubation for 17 h at 25 ℃. The culture was centrifuged (6,000 rpm, 30
min, 4 ℃) and resuspended with lysis buffer (50 mM phosphate, 5 mM imidazole, 300
mM NaCl, pH 8.0). The cells were lysed by tip sonication (30s/30s ON/OFF cycle, 6 min
total, 4 ℃). The lysate was centrifuged (15,000 rpm, 30 min, 4 ℃), and the supernatant
was loaded on HisTrap column (GE healthcare). The protein was bound to the column by
washing with 5 column volumes (CVs) of buffer A (50 mM phosphate, 300 mM NaCl, 20
mM imidazole, pH 8.0), and eluted with 5 CVs of buffer B (50 mM phosphate, 300 mM
NaCl, 250 mM imidazole, pH 8.0). Elution fractions were pooled and dialyzed against 3X
81

1 L of degassed buffer C (100 mM phosphate, 150 mM NaCl, 1 mM TCEP, 1mM EDTA
pH 7.5). The dialyzed solution was incubated with fresh TCEP (2 mM final concentration)
and mercaptoethane sulfonate (MesNa, 200 mM final concentration) for 3 d to generate C-
terminal thioester group. The resulting solution was purified on C4 semiprep RP-HPLC
(35-55% B over 60 min). The expected mass is 7,686 Da, and the observed mass was 7,686
± 1 Da.

Solid phase synthesis of peptide thioesters. All peptide synthesis was performed manually
using protected Dawson linker resin (Millipore). Commercially available side chain and
N-Fmoc protected amino acids (10 equivalents) were activated by the incubation with
DIEA (20 equivalents) and HBTU (10 equivalents) for 15 min and coupled for 1 h 30 min
with nitrogen gas agitation. The completion of reaction was confirmed using Kaiser test. If
needed, a second coupling was performed by incubating 10 equivalents amino acids, 10
equivalents HOBt, and 12 equivalents DCC for 2 h with nitrogen gas agitation. For
incorporation of O-GlcNAcylated serine, pentafluorophenyl (PFP) activated O-GlcNAc
Fmoc-Serine was synthesized and purified as described previously (Marotta et al., 2012).
Two equivalents of this amino-acid were incubated with the peptide resin overnight.
Terminal N-Fmoc protecting groups were removed by 20% piperidine (in DMF) incubation
for 5 min, followed by 20 min incubation with flesh 20% piperidine. For O-GlcNAc
modified peptide, the O-acetyl groups were removed after the completion of the peptide
synthesis by treatment of hydrazine hydrate (80% in MeOH) for 30 min twice on resin.
Before the cleavage of peptides, the Dawson linker was activated with treatment of para-
82

nitrophenyl chloroformate (5 equivalents in CH
2
Cl
2
) for 1 h, followed by incubation with
excess DIEA (5 equivalents in DMF) for 30 min. The peptides were cleaved from resin by
incubation in cleavage cocktail (95% TFA, 2.5% water, 2.5% TIS) for 4 h, and precipitated
in ice-cold diethyl ether overnight. The pellet was then collected by centrifugation (6,000
x g, 30 min, 4 ℃), and resuspended with water/acetonitrile mixture. The solution was
lyophilized, resolubilized, and incubated in thiolysis buffer (6 M guanidine-HCl, 200 mM
phosphate, 100 mM MesNa, pH 7.5) for 4 h. The desired peptide-thioester was purified on
C18 semiprep RP-HPLC (0-30% B over 60 min). Purified peptides were characterized by
ESI-MS. The expected mass for unmodified peptide is 1,540 Da, and the observed mass
was 1,541 Da. The expected mass for O-GlcNAc modified peptide is 1,743 Da, and the
observed mass was 1,742 Da.

Unmodified α-synuclein synthesis. Lyophilized peptide thioester (4 mg, 1 equivalent, 4
mM) and C-terminal fragment (26 mg, 2 equivalents) were solubilized in ligation buffer (6
M guanidine-HCl, 300 mM phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5) and rocked
at room temperature. The reaction was monitored by RP-HPLC (10-45% B over 60 min).
Once the completion of the ligation reaction was confirmed by RP-HPLC and ESI-MS, the
reaction mixture was diluted to 2 mM and acidified to pH 4 with HCl. Methoxyamine (100
mM final concentration) was added, and the resulting solution was incubated at room
temperature for additional 4 h. The deprotection of thioproline was confirmed by ESI-MS.
The product was purified on C18 semiprep RP-HPLC, and lyophilized. Subsequently, the
purified and lyophilized product (1 equivalent, 2 mM) and N-terminal thioester (2
83

equivalents) were resuspended in the same ligation buffer as above. The reaction was
rocked at 25 ℃ and monitored by RP-HPLC (25-60% B over 60 min). Once the reaction
was completed, the product was purified by C4 semiprep RP-HPLC and lyophilized.
Radical catalyzed desulfurization was performed by solubilizing the full-length protein at
the concentration of 0.75 mg ml
-1
in buffer (6 M guanidine-HCl, 300 mM phosphate, 300
mM TCEP, 2.5% v/v ethanethiol, 10% v/v tertbutylthiol, pH 7.0) and addition of radical
initiator, VΑ-061 (200 mM in MeOH, 2 mM final concentration). The reaction was
incubated at 37 ℃ with constant agitation for 16 h. The product was purified with C4
analytical RP-HPLC (25-60% B over 60 min). The purified unmodified α-synuclein was
characterized by RP-HPLC (0-70% B over 60 min), ESI-MS. The expected mass is 14,460
Da, and the observed mass was 14,461 ± 2 Da.

Synthesis of O-GlcNAc modified α-synuclein. Purified O-GlcNAc modified peptide
thioester (4 mM, 1 equivalent) and α-synuclein C-terminal fragment (2 equivalents) were
resuspended in the ligation buffer (6 M guanidine-HCl, 300 mM phosphate, 30 mM TCEP,
30 mM MPAA, pH 7.5), and the resulting solution was rocked for 15 h at 25 ℃. The
reaction was monitored by RP-HPLC (10-45% B over 60 min). Once the completion of the
ligation reaction was confirmed by RP-HPLC and ESI-MS, the reaction mixture was
diluted to 2 mM and acidified to pH 4 by addition of HCl. Methoxyamine (100 mM final
concentration) was added to the solution and incubated for additional 4 h. Deprotection of
thioproline was observed by ESI-MS. The product was purified by RP-HPLC (10-45% B
over 60 min) and lyophilized. Subsequently, the lyophilized product (1 equivalent, 2 mM)
84

was dissolved in the ligation buffer with N-terminal thioester (2 equivalents), and rocked
at room temperature for 30 h. The reaction was monitored by RP-HPLC. Once the reaction
was completed, the product was isolated on C4 analytical RP-HPLC (25-60% B over 60
min). The product fractions were pooled and lyophilized. Finally, radical-catalyzed
desulfurization was performed by resuspending full length O-GlcNAc modified α-
synuclein with cysteines in desulfurization buffer (6 M guanidine-HCl, 200 mM phosphate,
300 mM TCEP, 2.5% ethanethiol, 10% tertbutylthiol, pH 7.0). The reaction was initiated
with the addition of radical initiator, VΑ-061 (200 mM in MeOH, 2 mM final
concentration), and the reaction solution was incubated at 37 ℃ with constant agitation in
inert gas for 12 h. Desulfurized product was purified on C4 analytical RP-HPLC (25-60%
B over 60 min). The purified O-GlcNAc modified α-synuclein was characterized by RP-
HPLC and ESI-MS. The expected mass is 14,663 Da, and the observed mass was 14,666
± 2 Da.

Aggregation reaction. Synthetic or recombinant protein was dissolved with bath
sonication in a reaction buffer (10 mM phosphate, 0.05% sodium azide, pH 7.4) to make
its final concentration at either 50 µM or 25 µM. The solution was centrifuged at 15,000
rpm for 15 min at 4 ℃ to remove any debris, and the supernatant was aliquoted into
triplicate reactions. The samples were incubated at 37 ℃ with constant agitation (1,000
rpm) in a Thermomixer F1.5 (Eppendorf) for 7 d. At each time point, solution was aliquoted
for ThT analysis.

85

Circular dichroism (CD) spectroscopy. All circular dichroism (CD) spectra were taken
with Jasco-J-815 spectrometer at room temperature. Sample aliquots were diluted to 7.5
µM with the aggregation reaction buffer without sodium azide in a 1 mm path length quartz
cuvette at 25 ℃. The far UV spectra (195 nm-250 nm) were obtained by averaging three
scans with 50 nm min
-1
scanning speed, 1 nm bandwidth, a 0.1 nm step size, data integral
speed of 4. The buffer readings were subtracted for all samples, and the data were converted
into mean residue ellipticity.

Dynamic light scattering (DLS). Dynamic light scattering data were obtained with Wyatt
Technologies Dynastar. All samples were at t = 0 h of aggregation reaction (50 µM). For
all data, an average of 10 scans at 25 ℃ was obtained with laser power adjusted to intensity
of 2.6E
6
counts sec
-1
. To calculate radii, Raleigh sphere approximation was used.

Thioflavin T (ThT) fluorescence. α-Synuclein aggregation progression was quantified by
ThT fluorescence. Samples from the aggregation reaction were diluted to 1.25 µM protein
concentration with 20 µM ThT dye in the reaction buffer, followed by brief vortex and
incubation for 2 min. Samples in 10 mm path length quartz cuvette were analyzed using
NanoLog spectrofluorometer (Horiba), λ
ex
at 450 nm with 4 nm slit, λ
em
at 482 nm with 4
nm slit, data integration time of 0.1 sec, averaging 3 scans. Data were measured in triplicate
for all aggregation reaction conditions.

86

Transmission electron microscopy (TEM). At the end of the aggregation reactions, protein
solution was diluted to 15 µM by adding the reaction buffer, and the diluted solution (10
µL) was incubated with a formvar coated copper grid (150 mesh, Electron Microscopy
Science) for 5 min. Subsequently, the grid was negatively stained with 1% uranyl acetate
for 2 min and washed three times with 1% uranyl acetate. Each time excess liquid was
removed with filter paper. The grid was dried for 48 h. Grids were visualized with a JOEL
JEM-2100F transmission electron microscope operated at 200 kV, 600,000x
magnification, and an Orius Pre-GIF CCD.

SDS-PAGE Analysis. At each time point, 10 uL of aggregation reaction sample was
aliquoted, centrifuged at 20,000 x g for 1 h at 25 ℃. The supernatant was transferred into
a new tube and lyophilized to dryness. The lyophilized sample was solubilized in fresh 8M
urea 20 mM HEPES buffer (pH 8.0) with subsequent bath sonication for 20 min. The
sample was boiled for 10 min with 4X SDS loading buffer and loaded on 4-20% Criterion
precast gel (BioRad) and separated by SDS-PAGE at 195V. The gel was stained with
Coomassie brilliant blue for 30 min, and destained with 1:4:5 acetic acid/water/methanol
solution overnight.

Circular dichroism (CD) of α-synuclein with lipids. All circular dichroism (CD) spectra
were collected with Jasco-J-815 spectrometer at room temperature. Samples were prepared
by mixing 1:100 ratio of a protein and desired lipid mixture and incubated at room
temperature for 20 min. Lipid vesicles were prepared with 1-palmitoyl-2-oleoyl-sn-
87

glycero-3-[phospho-RAC-(1-glycerol)] (POPG), or by mixing different ratio of 1-
palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine (POPC). Dried lipid films were solubilized in 10 mM phosphate
buffer at pH 7.4 by vortexing. All spectra (190 nm-250 nm) were collected with scan rate
of 50 nm min
-1
, band width of 1 nm, data integration time of 8 sec, and a 0.1 nm step
resolution. Appropriate buffer spectra were subtracted from the final spectra.

88

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93

Chapter 4. The sulfur-linked analog of O-GlcNAc (S-GlcNAc) is an
enzymatically stable and a reasonable structural-surrogate for
O-GlcNAc at the peptide and protein levels
*

Introduction
The addition of the single monosaccharide β-N-acetyl glucosamine (O-GlcNAc
modification) to the hydroxyl side chains of serine/threonine residues of nuclear, cytosolic,
and mitochondrial proteins is an abundant intracellular posttranslational modification
(PTM) in plants and animals (Figure 4-1A) (Bond and Hanover, 2015). The installation of
this PTM is catalyzed by O-GlcNAc transferase (OGT) and can be subsequently removed
by the glycosidase O-GlcNAcase (OGA) (Vocadlo, 2012). The proper maintenance of O-
GlcNAcylation is required for development in both mice and Drosophila, as genetic
knockouts of OGT are lethal, and conditional knockouts at the cellular level result in cell-
cycle arrest and death (Gambetta et al., 2009; O'Donnell et al., 2004; Shafi et al., 2000;
Sinclair et al., 2009). Moreover, misregulation of O-GlcNAcylation is an important feature
of human disease (Ma and Vosseller, 2013; Yuzwa and Vocadlo, 2014; Zhu et al., 2014).
For example, all types of cancer to be examined have higher overall levels of O-
GlcNAcylation compared to healthy tissue, and this increase has been shown to be critical
for cancer cell survival and tumorigenesis (Ma and Vosseller, 2013). In direct contrast, the

*
Paul M. Levine (University of Southern California) and Timothy W. Craven (University of Washington)
contributed to the work presented in this chapter.
94

global amounts of O-GlcNAcylation are reduced in the brains of Alzheimer’s disease
patients (Liu et al., 2004), and a small molecule inhibitor of OGA that elevates the levels
of the modification reduces the progression of neurodegeneration in an Alzheimer’s disease
mouse model (Yuzwa et al., 2012). Despite the identification of over one thousand
potentially O-GlcNAcylated proteins (Ma and Hart, 2014), the biochemical consequences
of most of these events are unknown, and the exact molecular roles for O-GlcNAcylation
in both basic biology and disease are far from being completely characterized.

Figure 4-1. O-GlcNAcylation and the corresponding S-GlcNAc analog. A) O-GlcNAcylation
is the addition of N-acetyl-glucosamine to serine and threonine residues of intracellular proteins. It
is added by the enzyme O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). B)
S-GlcNAc analogs of O-GlcNAc could be used as enzymatically stable analogs in synthetic
proteins but whether human OGA can remove these modifications was an open question.

The preparation of site-specifically modified proteins using synthetic proteins has enabled
the direct characterization of the effects of several PTMs in an in vitro setting. For example,
we previously utilized the technique of expressed protein ligation (EPL) to prepare the
Parkinson’s disease associated protein α-synuclein with an O-GlcNAc modification at two
95

different sites. Biochemical and biophysical analysis then revealed that O-GlcNAcylation
inhibits α-synuclein aggregation without having a pronounced effect on its ability to bind
membranes (Lewis et al., 2017; Marotta et al., 2015), a key feature of its physiological
roles. However, extension of these studies with synthetic proteins to the context of cell
culture or an in vivo model is limited by the lability of the O-GlcNAc modification to the
enzymatic activity of endogenous OGA. This barrier could be overcome by the use of
enzymatically-stable analogs of the modification, as long as they faithfully mimic its
biophysical characteristics. For example, stable analogs of ubiquitin can be made by
inserting mutations at its C-terminus that render it resistant to deubiquitinases, which
recently enabled the chemical installation of ubiquitin onto histones in nucleo (David et
al., 2015). Similarly, the thioglycoside analog of O-GlcNAc, S-linked-β-N-acetyl
glucosamine (S-GlcNAc, Figure 4-1B) attached to a cysteine residue instead of serine, was
used to examine the effect of O-GlcNAcylation on casein kinase II using microinjection
into living cells (Tarrant et al., 2012), as many thioglycosides have been shown to be stable
against removal by glycosidases (Driguez, 2001). However, Vocadlo et al. demonstrated
that human OGA (hOGA) is a proficient bifunctional catalyst capable of cleaving both
oxygen and and small-molecule thioglycosides (Macauley et al., 2005). While these
thioglycosides were aryl and therefore more activated as leaving groups, this result still
raises the possibility that it can remove S-GlcNAc from peptides and proteins.

Notably, S-GlcNAcylation on cysteine residues of proteins in mice and humans was
recently identified using electron transfer dissociation mass spectroscopy (ETD), and the
same study biochemically confirmed that recombinant OGT can transfer to cysteine
96

residues to generate S-GlcNAcylation (Maynard et al., 2016). Taken together, these
previous results highlight the need to determine both the structural effects of S-
GlcNAcylation and its stability against hOGA, as they are critical to both its use as a O-
GlcNAc surrogate in synthetic proteins and its investigation as an endogenous cysteine
PTM. Here, we developed a new synthetic route to a cysteine S-GlcNAc amino acid for
solid phase peptides synthesis, which complements other methods liked disulfide
(Bernardes et al., 2008) and dehydroalanine tagging (Lercher et al., 2015). We then
generated S-GlcNAcylated and O-GlcNAcylated peptides corresponding to the N-terminal
region of the mouse β-estrogen receptor, as the O-GlcNAc modified peptide had been used
previously to demonstrate that this modification stabilizes a β-turn in the peptide (Chen et
al., 2006). We demonstrate that both the S- and O-modifications induce similar structures
in these model peptides by using a combination of 2D-NMR and computational modeling.
We then use EPL to generate α-synuclein with S-GlcNAcylation at physiologically
relevant residue 87 and use recombinant hOGA to show that this modification is
completely enzymatically stable at both the peptide and protein level. Finally, we show
that S-GlcNAcylation has identical effects on α-synuclein aggregation and membrane
binding when compared to the site-specifically O-GlcNAcylated protein. Together these
data indicate that S-GlcNAc will have similar structural consequences on substrate proteins
and demonstrate that it is indeed enzymatically stable and, therefore, an excellent surrogate
for future experiments with synthetic proteins and peptides, including α-synuclein.

97

Results
Synthesis of S-GlcNAcylated amino acids for peptide and protein synthesis.
Synthetic routes to generate S-GlcNAc involve the conjugation of β-anomeric thiolates of
glucosamine with alanine derivatives typically containing a good leaving group at the β-
carbon of the amino acid (Figure 4-2A) (Cohen and Halcomb, 2002; Ohnishi et al., 2000).
For example, β-iodo-alanine can be employed as the electrophile, but elimination can occur
to generate dehydroalanine derivatives, which upon nucleophilic attack by the thiolates
lead to an undesirable mixture of diastereomers at the α-carbon (Ohnishi et al., 2000).
Although this side-reaction can be overcome through the use of a modified Mitsunobu
reaction or a cyclic sulfamidate leaving group, they suffer from moderate yields or
extensive protecting group chemistry. Moreover, the final glycosylated amino acid was
often protected in ways that were incompatible with Fmoc-based solid-phase peptide
synthesis. Recently, Polt and co-workers reported the use of minimally competent Lewis
acids, in particular In(III)Br
3
, to promote the conjugation of β-D-glucopyranose peracetates
with Fmoc-L-cysteine to afford the corresponding thioglycosides in good yields (Szabó et
al., 2016). Notably, the weak lewis acid was used in catalytic amounts and the reaction
generated exclusively the β-anomer. We envisioned that this same synthetic route could
potentially be adapted to glucosamine per-O-acetylated monosaccharides with a carbamate
protecting group at the 2-N-position.

98

Figure 4-2. Synthetic routes to S-GlcNAcylated amino acids for solid phase peptide synthesis.
(A) Previous routes involved nucleophilic displacement of leaving groups (LG) on alanine
derivatives. (B) The new synthetic route to S-GlcNAcylated cysteine developed here. Reagents: (a)
20 mol % InBr 3, Fmoc-Cys-OH, CH 2Cl 2, reflux, 1 - 16 h; (b) 20 mol % InBr 3, Fmoc-Cys-OH,
dichloroethane, reflux, 16 h, 90%; (c) Zn dust, AcOH, Ac 2O, 16 h; (d) pentafluorophenyl
trifluoroacetate, pyridine, 3 h, 91%; (e) Zn dust, AcOH, Ac 2O, 16 h, 70%.

In our initial attempt to use this method to prepare S-GlcNAcylated cysteine (Figure 4-2B),
we chose to use the 2,2,2-tricholorethylcarbamate (Troc) protecting group, as the
corresponding per-O-acetyl-glucosamine sugar donor (Ac
4
GlcNTroc) can be readily
prepared on multigram scales in high yield with no requirement for column
chromatography (Marotta et al., 2012; Mitchell et al., 2001). Incubation of Ac
4
GlcNTroc
with N-Fmoc-L-cysteine under the conditions similar to those for glucose monosaccharides
99

(20 mol% InBr
3
, refluxed in CHCl
3
, 1 h) resulted in a mixture of products that could be
detected by crude ESI-MS. We clearly observed the formation of a small amount of the
desired product (4.1), but also a mixture of the glycosyl bromide of undetermined
stereochemistry and the 2-Troc Oxazoline generated from the anchimeric assistance of the
2-N-Troc group. Interestingly, none of the starting material (Ac
4
GlcNTroc) could be
detected; however, continued refluxing for 16 h did not change the product/intermediate
distribution. These initial results suggested that the amount of lewis acid (20 mol%) used
was sufficient to activate all of the donor sugar and that higher temperatures might be
needed to drive the reaction to completion. We therefore exchanged chloroform for 1,2-
dichloroethane as the solvent and repeated the reaction while refluxing at 84 °C. Although
we were concerned that the higher temperature could potentially decrease the anomeric
stereoselectivity of the reaction, we were pleased to see that the increased temperature
resulted in the formation of the glycosylated cysteine (4.1) in 90% yield with complete β-
selectivity as determined by the coupling constant (J= 12.1 Hz) in the
1
H NMR. Notably,
this excellent yield represents a significant improvement over the previous routes described
above, whose yields were modest (~50%) in the key coupling step or required significant
protecting group manipulations before solid phase peptide synthesis. Following the
successful glycosylation to form (4.1), we attempted to first remove the Troc-protecting
group and acetylate the resulting free amine in one pot using activated zinc dust in the
presence of acetic anhydride. Unfortunately, the major product of the reaction was the
cyclic lactam formed by the attack of the free amine on the C-terminal mixed anhydride
generated in situ. This type of side reaction had been observed in the past and was
100

overcome by the protection of the carboxylate as a pentafluorophenyl (Pfp) ester,
(Meinjohanns et al., 1995) which conveniently activates the amino acid for peptide
synthesis. Therefore, we also first protected the C-terminal acid of (4.1) as a
pentafluorophenyl (Pfp) ester (4.2) in 91% yield. With the C-terminal acid protected, the
one pot deprotection/acetylation of (4.2) proceeded smoothly to afford compound 4.3,
ready for solid-phase peptide synthesis, in 70% yield and an overall yield of 57%.

S-GlcNAc is a good structural mimic of O-GlcNAc in the context of a model peptide.
After synthesizing the thioglycoside monomer, we next explored whether S-GlcNAc is a
good structural mimic for O-GlcNAc. While it is well documented in the literature that O-
GlcNAcylation can change the activity or biophysical properties of proteins, the structural
details of the O-GlcNAc modifications are mostly unknown. One notable exception to this
trend is the analysis of O-GlcNAcylation on a peptide derived from the N-terminus of the
murine estrogen receptor (residues 7-23) that is endogenously modified at serine 16 (Chen
et al., 2006). More specifically, a combination of NMR spectroscopy and molecular
dynamics simulations showed that O-GlcNAcylation promotes a turn in the peptide directly
around the glycosylated residue. Following this blueprint, we synthesized the same O-
GlcNAcylated peptide (Ac-AVMNYSVPSgSTGNLEGG-NH
2
) and the corresponding S-
GlcNAcylated analog (Ac-AVMNYSVPSgCTGNLEGG-NH
2
), which were purified by
RP-HPLC and characterized by mass spectrometry (Figure 4-3). Importantly, we did not
observe any major impurities arising from either peptide synthesis, indicating that the S-
GlcNAc building-block is stable for solid phase synthesis. With these peptides in hand, we
101

Figure 4-3. Characterization of O-GlcNAcylated and S-GlcNAcylated model peptides. The O-
GlcNAcylated (blue) and S-GlcNAcylated (orange) peptides corresponding to a fragment of the β-
estrogen receptor were prepared by solid phase peptide synthesis, purified by RP-HPLC and
characterized by ESI-MS. RP-HPLC conditions were 0-70% buffer B over 60 min; buffer A: 0.1%
TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

first compared the peptides using CD spectroscopy (Figure 4-4). The two peptides show
very similar CD spectra, with a minimum around 200 nm that is indicative of a largely
unfolded conformation and a small negative shoulder at around 230 nm that could result
from some secondary structure. These results are very consistent with previously published
data on the O-GlcNAcylated peptide (Chen et al., 2006). The spectra from the S-
GlcNAcylated peptide does show a slightly more pronounced shoulder at 230 nm,
indicating that this modification might induce the peptide turn to a greater extent. We then
used a combination of NMR spectroscopy and computational modeling to elucidate the
102

Figure 4-4. O-GlcNAcylation and S-GlcNAcylation have similar small effects on peptide
structure by CD. CD spectra were collected for freshly dissolved peptides at 50 μM concentration
in 10 mM phosphate buffer at a pH of 7.4.

impact of the single-atom substitution. More specifically, we first performed temperature
controlled 2D TOCSY and NOESY experiments to study the effect of S-GlcNAc and O-
GlcNAc on the secondary structure of the peptides. Using the sequential assignment
strategy, the NMR resonances were then assigned to each amino acid on both peptides. To
determine if the glycosylated amino acids induce similar secondary structure, the Hα and
HN chemical shifts from both peptides were compared to each other as well as to their
deviations from 'random coil' Hα and HN chemical shift values (Figure 4-5) (De Simone
et al., 2009). It is well established that deviations from random coil shifts are indicators of
secondary structure. As evident from Figure 4-5, not only do both glycosylated peptides
show deviations from random coil, but the pattern of deviations look very similar. These
103

results indicate the possibility that the two motifs are inducing the same effect on secondary
structure, consistent with previously published data (Chen et al., 2006).

Figure 4-5. S-GlcNAc is a good structural mimic of O-GlcNAc. Comparison of ab initio folding,
QM modeling, and backbone HN and Hα chemical shift deviations (CSDs) for model O/S-GlcNAc
modified peptides. Ab initio folding was conducted within the Rosetta Molecular Design package
(see SI for 'folding funnels' and O/S GlcNAc residue parameterizations). The lowest energy
structure from the ab initio folding is presented. The β-hairpin conformation observed in the lowest
energy ab initio modeling was extracted and geometrically optimized at the B3LYP/6-311+G(2d,p)
level of theory.

104

To computationally model the effects of O- and S-GlcNAcylation on peptide structure,
these PTMs were parameterized to facilitate accurate side-chain rotamer repacking within
the Rosetta Molecular Design package. This allowed for ab initio modeling of these
peptides, which entailed initiating ~25,000 ‘decoy' structures and minimizing via a Monte-
Carlo search of the conformation space of the peptides. This procedure resulted in
converged lowest energy structures for both the O- and S-GlcNAcylated peptides
prominently featuring a hydrogen-bond between the side-chain amide of GlcNAc and the
side-chain hydroxyl group of the i-1 Thr residue, stabilizing a β-hairpin conformation
(Figure 4-5, see Figure 4-6 for ab initio 'folding funnels’). To gain additional insights into
the relative folding energetics of these modifications we isolated the β-hairpin structures
observed in the lowest energy conformations of the ab initio modeling followed by high-
level quantum mechanic-based optimizations and single point energy calculations at the
B3LYP and M06 levels of theory (Figure 4-5 and Figure 4-7). A comparison of the
differences in energies between a β-hairpin and an extended peptide conformation for each
PTM reveal very similar energetics for these two peptide model systems (in kcal/mol, -
3.531/-2.781 for SgCTG and -3.725/-2.941 for SgSTG at the B3LYP/M06 levels of
theory). These findings, as well as the extremely minimal differences between the Hα and
HN chemical shift deviations from the 2D NMR, strongly support the conclusion the
single-atom substitution has a minimal effect on structure and peptide folding energetics.
However, it is important to point out that S-linked oligosaccharides are known to occupy
different conformational populations when compared to their O-linked counterparts,
105

(Driguez, 2001) raising the possibility that S-GlcNAc will occupy different conformations
in certain contexts.

Figure 4-6. ab initio folding of O vs. S GlcNAcylated model peptides. RMSD (root mean square
deviation) is relative to the lowest energy conformation in each funnel. Energy is in Rosetta Energy
Units (R.E.U.) utilizing the beta score function.

106

Figure 4-7. QM optimized models of extended and β-hairpin conformations of O- and S-
GlcNAcylated tetra-peptides. Models were geometrically optimized at the B3LYP/6-
311+G(2d,p) level of theory utilizing polarizable continuum model (PCM) solvation. Single point
energies are also presented for conformations at the M06-2X/6-311+G(2d,p) level of theory.
Energies for the β-hairpin models are presented relative to that of the extended conformation.
Lowest energy glycosylated side-chain dihedral angle rotamers as well as the hydrogen bond length
between the GlcNAc HN and i-1 Thr hydroxyl are presented.

S-GlcNAc is enzymatically stable against hOGA in peptides and proteins.
As noted above, there have been contradictory reports concerning the ability of different
OGA enzymes to hydrolyze thioglycoside bonds to GlcNAc (Macauley et al., 2005;
Maynard et al., 2016). To evaluate whether hOGA is capable of removing S-GlcNAc on a
peptide substrate, purified recombinant hOGA (1 μM) was incubated with either the O-
GlcNAc or S-GlcNAc peptides described above (50 μM) in PBS buffer (pH = 7.4) at 37
°C for up to 72 h. Following incubation, hOGA was denatured by heating the samples to
100 °C for 5 min. Immediately after, the reactions were cooled and analyzed by RP-HPLC
and ESI-MS. As expected, hOGA readily removed the O-GlcNAc modification on the
107

peptide (Figure 4-8 and Figure 4-9). In contrast, we observed no deprotection with the S-
GlcNAc modified peptide up to 72 hours (Figure 4-8 and Figure 4-9), indicating that it is
indeed an enzymatically stable surrogate for O-GlcNAcylation at the peptide level.

Figure 4-8. In the context of a peptide, S-GlcNAc is completely stable against human OGA
enzymatic deglycosylation. O-GlcNAcylated (blue) or S-GlcNAcylated (orange) peptides (50
μM, in PBS at pH 7.4) were incubated with human OGA (1 μM) at 37 °C for up to 72 hours. The
S-GlcNAcylated peptide HPLC-trace is offset in y-direction for clarity. mAU indicates milli
absorbance units. RP-HPLC conditions were 0-70% buffer B over 60 min; buffer A: 0.1% TFA in
H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

108

Figure 4-9. S-GlcNAcylation in a peptide is resistant to O-GlcNAcase (OGA) mediated
deglycosylation. The O-GlcNAcylated (blue) and S-GlcNAcylated (orange) peptides were
incubated with recombinant human OGA. After the indicated lengths of time, aliquots were
removed and the reaction mixture was analyzed by RP-HPLC and any the identity of the peptides
was determined by ESI-MS. RP-HPLC conditions were 0-70% buffer B over 60 min; buffer A:
0.1% TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

109

Following these results, we wanted to assess the enzymatic stability of S-GlcNAc in a
therapeutically relevant protein substrate. Given our previous work on examining the site-
specific consequences of O-GlcNAcylation on α-synuclein (Lewis et al., 2017; Marotta et
al., 2015), the aggregation-prone protein in Parkinson’s disease and other
synucleinopathies, we decided to use α-synuclein as our model OGA substrate. More
specifically, we chose to examine O- and S-GlcNAcylation at residue 87 (normally serine
in α-synuclein), as we have demonstrated that O-GlcNAcylation at this site has interesting
effects on α-synuclein (Lewis et al., 2017), particularly in comparison to phosphorylation
at the same site (Paleologou et al., 2010). To synthesize α-synuclein S-GlcNAcylated at
residue 87, termed α-synuclein(gC87), we used an EPL strategy in combination with one
synthetic S-GlcNAcylated peptide and two recombinant α-synuclein fragments (Figure 4-
10A). Accordingly, α-synuclein(gC87) was retrosynthetically deconstructed (Figure 4-
10A) into a recombinant protein thioester (8, residues 1–75), a synthetic glycopeptide (9,
residues 76–90), and a recombinant protein with an N-terminal cysteine residue (10,
residues 91–140). Protein fragment 10 was heterogeneously expressed in Escherichia coli.
Notably, we found that the initiating methionine residue was conveniently removed during
expression by an endogenous methionine aminopeptidase (Lewis et al., 2017).
Glycopeptide 9 was prepared using standard Fmoc-based solid-phase peptide synthesis on
the Dawson aminobenzoyl resin that enables the generation of C-terminal peptide
thioesters upon linker activation. On-resin deprotection of the acetyl groups on the S-
GlcNAc moiety was accomplished using hydrazine before cleavage and purification of the
glycopeptide by RP-HPLC (Figure 4-11). Importantly, the N-terminal cysteine residue
110

Figure 4-10. Semisynthesis of S-GlcNAcylated α-synuclein. A) α-Synuclein was
retrosynthetically deconstructed into a recombinant protein thioester (8) obtained using intein
chemistry, a synthetic thioester-peptide (9) and a recombinant protein (10). B) These fragments
were then combined through iterative ligation reactions. C) Characterization of synthetic S-
GlcNAcylated α-synuclein using RP-HPLC and electrospray ionization mass spectrometry (ESI-
MS). Analysis by RP-HPLC showed that synthetic α-synuclein(gC87) was pure, as evidenced by
the appearance of only one, sharp peak. Characterization by ESI-MS gave a range of charge states
that could be deconvoluted to a molecular mass (14,682.7 ± 0.6 Da) in excellent agreement with
the predicted weight of 14,679 Da. RP-HPLC conditions were 0-70% buffer B over 60 min; buffer
A: 0.1% TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

remained protected as a thioproline to prevent auto-ligation. Incubation of protein 10 and
peptide 9 resulted in formation of the ligation product in high yield. The new N-terminal
cysteine residue was then deprotected in the same pot by treatment with methoxylamine to
give the corresponding protein ready for the next ligation reaction (Figure 4-12). To
prepare protein thioester 8, the appropriate α-synuclein fragment (residues 1–75) was
recombinantly expressed as an N-terminal fusion with an engineered DnaE intein from
Anabaena variabilis. Incubation of this thioester with the S-GlcNAcylated protein
fragment gave the full-length α-synuclein product (Figure 4-13). Finally, radical-based
111

desulfurization was used to convert the two cysteine residues required for the ligations back
to the native alanine residues, yielding α-synuclein(gC87) (Figure 4-10B and Figure 4-14).

Figure 4-11. Characterization of S-GlcNAcylated thioester peptide 9. The thioester peptide
corresponding to residues 76-90 of α-synuclein (10) was prepared using solid phase peptide
synthesis on the Dawson linker. The peptide was cleaved from the resin and deprotected, before
thiolysis, purification by RP-HPLC, and characterization by ESI-MS. RP-HPLC conditions were
0-70% buffer B over 60 min; buffer A: 0.1% TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

112

Figure 4-12. Ligation of peptide 9 and protein 10 and the subsequent deprotection to give the
corresponding α-synuclein fragment. The ligation reaction was followed by RP-HPLC and the
identity of intermediate product C was confirmed by ESI-MS. This was followed by deprotection
in the same pot to give the product D, which again was confirmed by ESI-MS. MPAA = 4-
mercaptophenylacetic acid. RP-HPLC conditions were 0-70% buffer B over 60 min; buffer A: 0.1%
TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

113

Figure 4-13. Ligation with protein thioester 8 to yield full-length S-GlcNAcylated α-synuclein.
The ligation reaction was followed by RP-HPLC and the identities of the product C was confirmed
by ESI-MS. MPAA = 4-mercaptophenylacetic acid. RP-HPLC conditions were 0-70% buffer B
over 60 min; buffer A: 0.1% TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

Figure 4-14. Desulfurization of S-GlcNAcylated α-synuclein. The desulfurization reaction was
followed by RP-HPLC and the identities of the product B was confirmed by ESI-MS. RP-HPLC
conditions were 0-70% buffer B over 60 min; buffer A: 0.1% TFA in H 2O, buffer B: 0.1% TFA,
90% ACN in H 2O.

114

To test directly whether hOGA is capable of cleaving S-GlcNAc from α-synuclein(gC87),
we again used the same HPLC assay with purified recombinant OGA that was employed
for the peptides above. As a positive control, the O-GlcNAcylated α-synuclein, α-
synuclein(gS87), was prepared using EPL as previously described (Lewis et al., 2017).
Both α-synuclein(gC87) and α-synuclein(gS87) were separately diluted to a concentration
of 25 μM in PBS buffer (pH = 7.4) and incubated with hOGA (1 μM) at 37 °C for up to 72
h. Following incubation, hOGA was denatured and the reactions were centrifuged to
remove any possible aggregates in the reaction. Following centrifugation, the samples were
directly injected onto the HPLC and monitored at 214 nm. As expected, hOGA readily
hydrolyzed O-GlcNAc modified α-synuclein, which was confirmed by HPLC and ESI-MS
(Figure 4-15). In contrast, we observed no cleavage of the S-GlcNAc modified α-synuclein
(Figure 4-15). These results further validate that S-GlcNAc can be used as an enzymatically
stable surrogate for O-GlcNAcylation for in vivo and cellular studies.

S-GlcNAc has the same biophysical effects on α-synuclein as O-GlcNAcylation.
To compare the impact of O-GlcNAcylation and S-GlcNAcylation on the gross secondary
structure of α-synuclein, we used circular dichroism (CD) spectroscopy using either
unmodified α-synuclein, α-synuclein(gC87), or α-synuclein(gS87). Unmodified α-
synuclein was recombinantly expressed and purified by RP-HPLC. Unmodified α-
synuclein exists as an unstructured random coil in solution, and consistent with our
previous results (Lewis et al., 2017), O-GlcNAcylation at serine 87 did not induce any
secondary structure in the protein. Not surprisingly based on our structural data above, the
115

Figure 4-15. S-GlcNAcylation of α-synuclein is enzymatically stable. α-Synuclein(gS87) or α-
synuclein(gC87) (25 μM, in PBS at pH 7.4) were incubated separately in triplicate with OGA (1
μM) at 37 °C for 72 h. S-GlcNAc is offset in y-direction for clarity. mAU indicates milli absorbance
units. Hydrolysis of GlcNAcylated α-synuclein was analyzed by HPLC at 72 hour time point.
Deglycosylation was quantitated by area percent using high-performance liquid chromatography
(HPLC) at 214 nm and the deglycosylated product was characterized ESI-MS. Results are the mean
± s.e.m. of three separate biological experiments. RP-HPLC conditions were 35-60% buffer B over
60 min; buffer A: 0.1% TFA in H 2O, buffer B: 0.1% TFA, 90% ACN in H 2O.

spectra of α-synuclein(gC87) looked identical to both the unmodified and O-GlcNAcylated
protein controls, demonstrating that this single-atom substitution has essentially no impact
on secondary structure of α-synuclein in solution (Figure 4-16). Next, we explored the
effect of S-GlcNAcylation at residue 87 on the endogenous function of α-synuclein. α-
Synuclein is known to interact with negatively charge membranes and vesicles, which
induce the protein to become α-helical in structure. This interaction has been shown to be
116

important for both the direct remodeling of membranes (Jao et al., 2008; Middleton and
Rhoades, 2010; Mizuno et al., 2012; Varkey et al., 2010) and interactions with other vesicle
trafficking proteins (Burré et al., 2010; Burré et al., 2014). Notably, this protein-membrane
interaction can readily be measured using CD spectroscopy; therefore, we incubated either
unmodified α-synuclein, α-synuclein(gS87), or α-synuclein(gC87) with vesicles formed
from 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)] (POPG) in a 1:100
protein to lipid ratio. After 20 min, we measured the secondary structure of the α-synuclein
proteins by CD and found that neither O-GlcNAcylation or S-GlcNAcylation at residue 87
had any effect on the membrane-binding properties of α-synuclein (Figure 4-17A). These
results are consistent with our previous data on α-synuclein(gS87) and show that S-
GlcNAcylation is again a grossly equivalent modification in this context. Next, we
examined the effect of S-GlcNAcylation at residue 87 on α-synuclein aggregation using
thioflavin T (ThT) fluorescence, which is a dye that intercalates into the fibers that are
formed by amyloid proteins including α-synuclein. Unmodified α-synuclein, α-
synuclein(gS87), or α-synuclein(gC87) were subjected to aggregation by incubation at 37
°C with constant agitation (1,000 r.p.m.) at a protein concentration of 25 μM. After 0, 72,
120, and 168 hours aliquots of the reaction were removed and analyzed (Figure 4-17B).
Consistent with our previous results, O-GlcNAcylation at residue 87 did not completely
block protein aggregation but did significantly inhibit it. Notably, S-GlcNAcylation at the
same position had essentially the same effect, further supporting it as functionally
equivalent to the native modification. Taken together, these data demonstrate that the
substitution of oxygen for sulfur to generate S-GlcNAcylation does not dramatically
117

change the biophysical properties of α-synuclein, and we believe that the conservative
nature of this modification will also be well tolerated in the context of other proteins.

Figure 4-16. Neither O-GlcNAcylation or S-GlcNAcylation induces secondary structure in α-
synuclein. CD spectra were collected for freshly dissolved proteins at 7.5 μM concentration.

118

Figure 4-17. S-GlcNAcylation has identical effects as O-GlcNAcylation on the membrane
binding and aggregation of α-synuclein. A) Neither O-GlcNAcylation or S-GlcNAcylation at
residue 87 effects α-synuclein membrane binding. Recombinant α-synuclein, α-synuclein(gS87),
or α-synuclein(gC87) were incubated with an 100-fold excess of POPG preformed vesicles and
analyzed using circular dichroism (CD). All of the proteins gave essentially indistinguishable CD
spectra consistent with the formation of an extended α-helix. POPG = 1-palmitoyl-2-oleoyl-sn-
glycero-3-[phospho-RAC-(1-glycerol)]. B) O-GlcNAcylation and S-GlcNAcylation are equally
inhibitory towards α-synuclein aggregation. Recombinant α-synuclein, α-synuclein(gS87), or α-
synuclein(gC87) were subjected to aggregation conditions (25 μM concentration and agitation at
37 °C) for the indicated lengths of time before analysis of aliquots by ThT fluorescence (λ ex = 450
nm, λ em = 482 nm). The y-axis shows the fold-increase of fluorescence compared with the
corresponding protein at t = 0. Error bars represent ±s.e.m from the mean of three biological
replicates, and statistical significance was calculated using a two-tailed Student’s t-test.

119

Discussion
Here, we have reported the facile synthesis and subsequent characterization of S-
GlcNAcylated analogs of O-GlcNAcylation and show that this modification is structurally
similar and enzymatically stable in a model peptide and protein. First, we developed an
improved synthetic route to access a GlcNAc-modified cysteine amino acid by harnessing
In(III)Br
3
as a catalyst. This reaction is stereospecific, exclusively generating the β-anomer,
and does not require the strictly anhydrous conditions of typical O-glycosylation
conditions. Furthermore, in the case of S-GlcNAcylated cysteine the reaction scheme
requires only modest protecting group manipulations and provides the monomer protected
and activated for solid phase peptide synthesis. We then experimentally validate, for the
first time, that S-GlcNAc is a suitable structural mimic for O-GlcNAc in the context of a
model peptide sequence-derived from the β-estrogen receptor. As noted above, this peptide
was previously characterized using a combination of NMR spectroscopy and molecular
modeling, and the authors found that O-GlcNAcylation at serine 16 stabilizes a β-turn in
residues 15-18 (Chen et al., 2006). Consistent with these results, we also observe a β-turn
in our O-GlcNAcylated peptide and that the corresponding S-GlcNAcylated peptide has an
almost identical structure, as comparison of the chemical shift deviations of the Hα and
HN protons revealed a minimal perturbation to the overall fold of the peptide, and we
believe the chemical deviation of the threonine residue located just C-terminal to the
modified residue could result from the electronic differences between sulfur and oxygen.
Importantly, we used the newest version of the Rosetta score function that has been used
to model and design glycopeptides (Alford et al., 2017; Labonte et al., 2017). Substitution
120

of O-GlcNAc with S-GlcNAc in additional synthetic peptides and proteins may present
divergent structural features. For example, the linkages will have different bond-lengths
and may occupy different conformational space due to changes in the exo-anomeric effect.
However, we expect that this single atom substitution will be a reasonable structural mimic
in the vast majority of scenarios. Unfortunately, there is a dearth of detailed data on the
structural effects of O-GlcNAcylation in the context of peptides, so other model systems
where the natural modification has a defined effect are lacking. Therefore, the exact
differences between the O- and S-linkages will need to be further evaluated in different
biological and biochemical contexts.

Encouraged by our peptide data, we also used an EPL synthetic strategy to prepare the
protein α-synuclein bearing S-GlcNAc at residue 87, α-synuclein(gC87), and compared
this protein with the semisynthetic O-GlcNAcylated protein that we previously
characterized (Lewis et al., 2017). Separate incubation of these proteins, as well as the O-
GlcNAcylated or S-GlcNAcylated β-estrogen receptor peptides, with hOGA showed that
while O-GlcNAc can be readily removed the S-GlcNAc modification was completely
stable. These data are consistent with previous results obtained by using a bacterial
homolog of OGA (Maynard et al., 2016) and show that the hydrolysis of GlcNAc
thioglycosides by hOGA observed by Vocadlo and co-workers is most likely confined to
more activated leaving groups (Macauley et al., 2005). Notably, the activity of OGA for
processing several glycosylated proteins has been shown to be independent of the
underlying primary sequence, suggesting that the S-GlcNAc analog should be stable in the
121

context of essentially any protein. Because S-GlcNAc is stable, but presumably binds to
OGA active site, it could also function as a substrate-based inhibitor of the enzyme, and
this possibility should be considered in future cellular or in vivo experiments. In the case
of α-synuclein, we also find that O-GlcNAc and S-GlcNAc similarly have no effect on the
secondary structure of α-synuclein in solution or its ability to bind and form an α-helix in
the presence of negatively charged membranes. Furthermore, both modifications inhibit
the aggregation of α-synuclein, which is a causative factor in the development and
progression of Parkinson’s disease. These results further support a protective role for O-
GlcNAcylation in neurodegenerative diseases. Interestingly, proteomic analysis of
mammalian samples recently identified endogenous S-GlcNAcylation of cysteine residues
in proteins, and the same authors demonstrated that OGT can indeed modify cysteines of
peptides in vitro. This establishes a physiological relevance to studying and synthesizing
these modified peptides and proteins for the biological investigation of endogenous site-
specific S-GlcNAcylation. In summary, we demonstrate that S-GlcNAc analogs can be
used to extend the use of semisynthetic proteins to experiments involving living cells or
animals without the complication of O-GlcNAc removal by OGA in the course of the
analysis. We anticipate future studies will utilize our general synthetic strategy to both
exploit S-GlcNAcylation as a stable O-GlcNAc analog and install native cysteine
modifications to explore the consequences of both of these important posttranslational
modifications in vitro and in vivo.

122

Materials and Methods
All solvents and reagents were purchased from commercial sources (Sigma-Aldrich, Fluka,
EMD, Novagen, etc.) and used without any further purification. All aqueous solutions were
prepared using ultrapure laboratory grade water (deionized, filtered, sterilized) obtained
from an in-house ELGA water purification system and filter sterilized with 0.45 μm syringe
filters (VWR) before use. Growth media (LB broth, Miller, Novagen and TB broth, Sigma)
were prepared, sterilized, stored, and used according to the manufacturer. Antibiotics were
prepared as stock solutions at a working concentration of 1000x (ampicillin sodium salt,
EMD 100 mg mL-1, kanamycin sulfate, EMD, 50 mg mL-1) and stored at -20 °C. All
bacterial growth media and cultures were handled using sterile conditions under open
flame. All silica gel column chromatography was performed using 60 Å silica gel (EMD)
and all thin-layer chromatography performed using 60 Å, F254 silica gel plates (EMD)
with detection by ceric ammonium molybdate (CAM) and/or UV light. Reverse phase high
performance liquid chromatography (RP-HPLC) was performed using an Agilent
Technologies 1200 Series HPLC with Diode Array Detector. Unless otherwise stated the
HPLC buffers used were buffer A: 0.1% TFA in H
2
O, buffer B: 0.1% TFA, 90% ACN in
H
2
O. Mass spectra were acquired on an API 3000 LC/MS-MS System (Applied
Biosystems/MDS SCIEX).
1
H NMR spectra were acquired on either a Varian Mercury 400
MHz or Varian VNMRS 500 MHz magnetic resonance spectrometer. TOCSY and NOESY
spectra were acquired on a Varian VNMRS-600 3-channel NMR spectrometer equipped
with a CryoProbe.

123

Chemical Synthesis.
Compound 4.1 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine.
N-Troc-Peracetyl-β-D-glucosamine (1.45 mmol, 2.5 eq), InBr
3

(41.3 mg, 0.116 mmol, 0.2 eq), and N-Fmoc-L-Cys-OH (200 mg, 0.582 mmol, 1 eq) were
suspended in 1,2-dichloroethane

(5.8 mL). The reaction mixture was heated to reflux.
Reaction progress was monitored by mass spectroscopy and TLC (EtOAc/MeOH/H2O
7:2:1). The reaction mixture was concentrated in vacuo and azeotroped with toluene
multiple times to remove the acetic acid generated in situ. The residue was purified by flash
chromatography (MeOH/CH
2
Cl
2
/AcOH 4:96:0.1) and concentrated in vacuo to afford an
off-white solid (422 mg, 90%).
1
H NMR (600 MHz, Chloroform-d) δ 7.75 (d, J = 7.6 Hz,
2H), 7.61 – 7.57 (m, 2H), 7.41 – 7.37 (m, 2H), 7.33 – 7.29 (m, 2H), 5.99 (d, J = 7.6 Hz,
1H), 5.35 (d, J = 9.3 Hz, 1H), 5.17 (t, J = 9.8 Hz, 1H), 5.05 (t, J = 9.7 Hz, 1H), 4.79 (d, J =
12.1 Hz, 1H), 4.72 – 4.62 (m, 2H), 4.52 – 4.35 (m, 3H), 4.21 (d, J = 16.0 Hz, 3H), 3.74 (q,
J = 10.0 Hz, 1H), 3.63 (s, 1H), 3.32 (d, J = 14.5 Hz, 1H), 3.09 (d, J = 14.1 Hz, 1H), 2.05
(s, 3H), 2.03 (s, 3H), 2.01 (s, 3H).
13
C NMR (151 MHz, Chloroform-d) δ 168.19, 166.85,
140.89, 138.68, 125.22, 124.56, 122.43, 117.46, 82.08, 74.63, 74.42, 74.21, 73.16, 71.81,
70.72, 66.10, 64.89, 59.88, 52.48, 50.81, 44.45, 30.40, 18.15, 18.03. HRMS: calculated for
(M+H)
+
, 805.1004; found, 805.1012. IR (KBr pellet) cm
-1
3344.6, 3067.2, 2955.4, 1754.3,
1535.5, 1478.7, 1450.5, 1370.5, 1222.4, 1048.4, 948.5, 917.5, 819.4.

O
AcO
AcO
TrocHN
OAc
S
FmocHN
OH
O
124

Compound 4.2 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
Pfp Ester. Anhydrous pyridine (2.23 mmol, 4.5 eq) was added to
a stirring solution of compound 4.1 (400mg, 0.496 mmol, 1 eq) in anhydrous DMF (5.7
mL) under N
2
. To this solution, Pentafluorophenyl trifluoroacetate (1.48 mmol, 3 eq), was
added dropwise under N
2
. The reaction was allowed to stir at room temperature overnight.
Reaction progress was monitored by TLC (35% EtOAc/Hexane). Once complete, the
reaction was concentrated in vacuo and azeotroped with toluene multiple times to remove
the TFA generated in situ. The residue was purified by flash chromatography
(Acetone/Hexane 40:60) and concentrated in vacuo to afford an off-white solid (440 mg,
91%).
1
H NMR (500 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.63 (dd, J = 13.8,
7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.36 – 7.31 (m, 2H), 6.21 (d, J = 7.7 Hz, 1H), 5.35
(d, J = 9.3 Hz, 1H), 5.22 (t, J = 9.9 Hz, 1H), 5.07 (t, J = 9.7 Hz, 1H), 4.89 (td, J = 7.8, 3.7
Hz, 1H), 4.78 (d, J = 11.9 Hz, 1H), 4.69 (d, J = 10.3 Hz, 1H), 4.61 – 4.50 (m, 2H), 4.43 (t,
J = 8.9 Hz, 1H), 4.28 (t, J = 6.9 Hz, 1H), 4.16 – 4.05 (m, 2H), 3.83 (q, J = 10.0 Hz, 1H),
3.72 – 3.67 (m, 1H), 3.50 (dd, J = 14.7, 4.0 Hz, 1H), 3.08 (dd, J = 14.8, 8.3 Hz, 1H), 2.06
(s, 3H), 2.04 (s, 3H), 2.00 (s, 3H).
13
C NMR (126 MHz, Chloroform-d) δ 170.67, 169.29,
166.95, 156.02, 154.30, 143.74, 143.49, 141.32, 127.80, 127.14, 125.08, 124.94, 120.07,
83.71, 76.29, 74.49, 73.02, 68.27, 67.31, 62.10, 54.88, 53.70, 47.11, 31.58, 20.59, 20.50.
HRMS: calculated for (M+H)
+
, 971.0846; found, 971.0833. IR (KBr pellet) cm
-1
3339.8,
3068.4, 2955.5, 2670.0, 2461.8, 1753.4, 1519.4, 1450.9, 1374.5, 1223.3, 994.8, 916.0,
878.0, 817.5.
O
AcO
AcO
TrocHN
OAc
S
FmocHN
OPfp
O
125

Compound 4.3 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
Pfp Ester (S-GlcNAc). Compound 4.2 (810 mg, 0.833 mmol, 1 eq)
was dissolved with 12 mL of 3:2:1 THF:Ac
2
O:AcOH under N
2
. Zinc dust (1.08 g, 16.52
mmol, 36.5 eq) was added to the reaction flask. Reaction was allowed to stir at room
temperature overnight. Upon disappearance of the starting material by TLC, the reaction
was filtered through celite and filtrant concentrated in vacuo. Residue was azeotroped
multiple times with toluene to afford an off-white powder. Product was recrystallized from
EtOAc using Hexanes (488 mg, 70%).
1
H NMR (600 MHz, Chloroform-d) δ 7.75 (d, J =
7.5 Hz, 2H), 7.62 (t, J = 8.2 Hz, 2H), 7.38 (td, J = 7.5, 2.7 Hz, 2H), 7.30 (td, J = 7.5, 2.6
Hz, 2H), 6.35 (d, J = 7.8 Hz, 1H), 5.73 (d, J = 9.2 Hz, 1H), 5.17 (t, J = 9.8 Hz, 1H), 5.05
(t, J = 9.7 Hz, 1H), 4.84 (td, J = 8.1, 3.9 Hz, 1H), 4.63 (d, J = 10.3 Hz, 1H), 4.51 (dd, J =
10.6, 6.9 Hz, 1H), 4.41 (dd, J = 10.8, 7.0 Hz, 1H), 4.25 (t, J = 6.9 Hz, 1H), 4.11 (td, J = 7.7,
6.2, 3.3 Hz, 2H), 4.05 (dd, J = 12.4, 5.6 Hz, 1H), 3.70 – 3.66 (m, 1H), 3.46 (dd, J = 14.4,
3.9 Hz, 1H), 3.09 (dd, J = 14.4, 8.4 Hz, 1H), 2.03 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.92
(s, 3H).
13
C NMR (151 MHz, Chloroform-d) δ 171.02, 170.58, 170.53, 169.17, 167.02,
156.03, 143.68, 143.59, 141.74, 141.28, 141.25, 140.07, 138.69, 137.01, 127.74, 127.71,
127.11, 127.08, 125.07, 124.97, 120.00, 119.98, 83.81, 76.32, 73.33, 68.21, 67.19, 62.13,
53.77, 52.89, 47.10, 31.21, 23.15, 20.62, 20.61, 20.54, 20.47. HRMS: calculated for
(M+H)
+
, 839.1909; found, 839.1918. IR (KBr pellet) cm
-1
3334.7, 3068.7, 2926.23, 2854.6,
2669.3, 2461.4, 1752.9, 1518.2, 1450.7, 1373.4, 1218.5, 994.5, 915.9, 874.0, 815.7.

O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
126

Peptide Synthesis. All peptides were synthesized using standard Fmoc solid-phase
chemistry on Rink amide ChemMatrix® (PCAS BioMatrix, 0.45 mmol g-1) or Dawson
Dbz AM® (Novabiochem, 0.49 mmol g-1) resin using HBTU (5 eq, Novabiochem) and
DIEA (10 eq, Sigma) in DMF for 1 hr. For activated glycosylated amino acids, 2 eq of
monomer in 3 mL DMF was coupled overnight. N-terminal acetylation was conducted
using Ac
2
O (3 eq) and pyridine (2 eq) in DMF for 1 hr. When peptides were completed,
acetyl groups were deprotected with hydrazine monohydrate (80% v/v in MeOH) twice for
30 min with mixing. Following deprotection, Dawson linker peptides were activated with
p-nitrophenyl-chloroformate (5 eq in DCM, 1.5 hr, mixing) followed by treatment with
excess DIEA (0.5 M in DMF) for 30 min to cyclize the Dbz linker. All peptides were then
cleaved (95:2.5:2.5 TFA/H
2
O/Triisopropylsilane) for 3.5 h at room temperature,
precipitated out of cold ether, and purified by reverse-phase HPLC using preparative
chromatography. To generate thioester peptides, crude peptide were resuspended in
thiolysis buffer (150 mM NaH
2
PO
4
, 150 mM MESNa, pH 7.0) and incubated at room
temperature for 2 h prior to purification. All peptides were characterized by mass analysis
using ESI-MS and sequence purity was assessed by analytical HPLC.

Protein Expression and Purification. General expression and purification methods were
similar to those previously reported. Briefly, BL21(DE3) chemically competent E. coli
(VWR) were transformed with plasmid DNA by heat shock and plated on selective LB
agar plates containing 50 μg mL-1 kanamycin (C-terminus) or 100 μg mL-1 ampicillin (N-
terminus). Single colonies were then inoculated, grown to OD600 of 0.6 at 37 °C shaking
Type to enter text
127

at 250 rpm, and then expression was induced with IPTG (final concentration: 0.5 mM) at
25 °C shaking at 250 rpm for 18 h. Pellets were harvested by centrifugation (8,000 x g, 30
min, 4 °C) and lysed. C-terminus was acidified (pH 3.5 with HCl), centrifuged, dialyzed
against 3 x 1 L of 1% acetic acid in water (degassed with N
2
, 1 hr per L), and purified by
HPLC. N-terminus was loaded onto a Ni-NTA purification column (HisTrap FF Crude,
GE Healthcare) and eluted fractions were dialyzed against 3 x 1 L (100 mM NaH
2
PO
4
, 150
mM NaCl, 1 mM EDTA, 1mM TCEP HCl, pH 7.2) and concentrated. Sodium
mercaptoethane sulfonate (MESNa) was added to a final concentration of 200 mM along
with fresh TCEP (2 mM final concentration) overnight to generate the protein thioester,
and then purified by HPLC. Pure protein fragments were characterized by analytical RP-
HPLC and ESI-MS.

Expressed Protein Ligation. General ligation and purification methods were similar to
those previously reported. Briefly, C-terminal fragment (2 mM) and peptide fragment (4
mM) were dissolved in ligation buffer (300 mM NaH
2
PO
4
, 6 M guanidine HCl, 100 mM
MESNa, 1 mM TCEP, pH 7.8) and allowed to react at room temperature. Following
completion, as monitored by HPLC, the pH was adjusted to 4 with HCl and methoxylamine
was added (final concentration 100 mM) to deprotect the N-terminal thiazolidine group.
Following HPLC purification, N-terminal fragment (8 mM) and ligation product fragment
(2 mM) were dissolved in ligation buffer, and following completion was purified by HPLC.
Finally, radical desulfurization was carried out using desulfurization buffer (200 mM
NaH
2
PO
4
, 6 M guanidine HCl, 300 mM TCEP, pH 7.0) containing 2% v/v ethanethiol,
128

10% v/v tertbutyl-thiol, and the radical initiator VA-061 (as a 0.2 M stock in MeOH). The
reaction was heated to 37 °C for 15 h and then purified by RP-HPLC to yield synthetic α-
synuclein.

Circular Dichroism (CD). Circular dichroism spectra were collected on a Jasco J-815 CD
Spectrometer. Sample aliquots were diluted to 50 μM for peptides or 7.5 μM for proteins
in 10 mM phosphate buffer at a pH of 7.4 for peptides or 7.5 for proteins. Spectra were
collected from 250-190 nm with a 0.1 nm data pitch, 50 nm min
-1
scanning speed, data
integration time of 4 sec, 1 nm bandwidth, 1 mm path length with 3 accumulations, at 25
°C.

Peptide NMR Spectroscopy. NMR spectra were collected using a Varian (Palo Alto, CA)
VNMRS-600 3-channel NMR spectrometer equipped with a CryoProbe. All samples were
prepared in H
2
O and D
2
O that had been purged of dissolved O
2
gas. 1D and 2D
1
H NMR
were collected at protein concentrations of 1.0 mM in 90% H
2
O:10% D
2
O solutions
containing 10mM phosphate at pH 3.5 (uncorrected). Total correlated spectroscopy
(TOCSY) spectra were collected utilizing a mixing time of 120 ms. 2D
1
H −
1
H Nuclear
Overhauser Effect SpectroscopY (NOESY) spectra were collected utilizing a 250 ms
mixing time. All spectra were acquired at 5 °C with 512 points in f1 and 2024 points in f2.
Chemical shift deviations from ‘random coil’ values shown in Figure 3. were reported
relative to a DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) standard.

129

Ab initio folding and protein modeling. Peptides containing both serine O-GlcNAc and
cysteine S-GlcNAc modifications were modeled in the Rosetta Molecular Modeling
program (Drew et al., 2013; Leaver-Fay et al., 2011). In silico parameters utilized for
modeling of the serine and cysteine GlcNAc modifications can be found in the supporting
information. For ab initio peptide modeling, 25,000 decoy structures were initialed with
randomized φ and ψ backbone dihedral angles. Side-chains, including GlcNAc modified
residues, were repacked and sequences minimized with respect to total molecular energy
utilizing a Monte Carlo algorithm. The lowest energy structure for both serine O-GlcNAc
and cysteine S-GlcNAc containing model peptides can be seen in Figure 3. The resulting
plots of Rosetta calculated total molecular energy vs. RMSD (root mean square deviation)
to the lowest energy structure (typically referred to as the ‘folding funnel’) can be seen in
Figure S2 (SI).

Human O-GlcNAcase (hOGA) Assay. Peptides (50 uM, in PBS at pH 7.4) were incubated
with hOGA (1 uM) in 200 uL reactions and allowed to incubated at 37 °C for up to 72
hours. Proteins (25 uM, in PBS at pH 7.4) were incubated with hOGA (1 uM) in 125 uL
reactions and allowed to incubate at 37 °C for up to 48 hours. Following incubation, hOGA
was denatured by heating at 100 °C for 5 min. Immediately after, reactions were cooled
and analyzed by high-performance liquid chromatography (HPLC).

Circular dichroism (CD) of α-synuclein with lipids. All circular dichroism (CD) spectra
were collected with Jasco-J-815 spectrometer at room temperature. Samples were prepared
130

by mixing 1:100 ratio of a protein with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-
(1-glycerol)] (POPG) and incubated at room temperature for 20 min. Dried lipid films were
solubilized in 10 mM phosphate buffer at pH 7.4 by vortexing. All spectra (190-250 nm)
were collected with scan rate of 50 nm min
-1
, band width of 1 nm, data integration time of
8 sec, and a 0.1 nm step resolution. Appropriate buffer spectra were subtracted from the
final spectra.

Aggregation reaction. Synthetic or recombinant protein was dissolved with bath
sonication in a reaction buffer (10 mM phosphate, 0.05% sodium azide, pH 7.4) to make
its final concentration 25 µM. The solution was centrifuged at 15,000 rpm for 15 min at 4
℃ to remove any debris, and the supernatant was aliquoted into triplicate reactions. The
samples were incubated at 37 ℃ with constant agitation (1000 rpm) in a Thermomixer
F1.5 (Eppendorf) for 7 d. At each time point, solution was aliquoted for ThT analysis.

Thioflavin T Fluorescence. Samples from aggregation assay reaction were diluted in 96-
well plate to a concentration of 1.25 μM with a reaction buffer (10 mM phosphate, pH 7.4,
0.05% NaN
3
) containing 10 μM Thioflavin T. The plate was read by Synergy H4 hybrid
reader (BioTek). The plate was shaken at 300 rpm for 3 min, followed by data collection
(λ
ex
= 450 nm, 20 nm band path, λ
em
= 482 nm, 9.5 nm band path, reading from the bottom
of a plate, gain = 100, read height was 5.00 mm).

131

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137

Chapter 5. O-GlcNAc modification inhibits the calpain-mediated
cleavage of α-synuclein
§

Introduction
O-GlcNAc modification (O-GlcNAcylation) refers to the modification of serine or
threonine side chains by the monosaccharide N-acetyl-glucosamine (Figure 5-1A) (Bond
and Hanover, 2015; Zachara and Hart, 2002). This posttranslational modification occurs
on intracellular proteins and has been found in the nucleus, cytosol, and mitochondria. This
modification is added by the enzyme O-GlcNAc transferase (OGT and is removed by the
enzyme O-GlcNAcase (OGA) (Vocadlo, 2012), neither which display a strong preference
for any particular amino acid sequence. Many different classes of proteins are known to be
modified by O-GlcNAc, including several of the proteins that are known to form toxic
aggregates in neurodegenerative diseases (Yuzwa and Vocadlo, 2014), suggesting that O-
GlcNAc may affect the normal and/or pathological functions of these proteins. Notably,
Vocadlo and co-workers demonstrated that enzymatic O-GlcNAcylation of the
microtubule stabilizing protein tau inhibited its aggregation in vitro (Yuzwa et al., 2012).
Furthermore, they developed a potent small-molecule inhibitor of OGA, Thiamet-G, that
can raise O-GlcNAcylation levels in both cells and in vivo. Treatment of an Alzheimer's
disease mouse model with this inhibitor resulted in lower levels of tau
hyperphosphorylation, a hallmark of tau aggregation, and fewer protein aggregates in the

§
Paul M. Levine, Ana Galesic, Aaron Balana, Nicholas P. Marotta, and Yuka E. Lewis (University of
Southern California) contributed to the work presented in this chapter.
138

mouse brains (Yuzwa et al., 2008; Yuzwa et al., 2012). Together these results support a
potential therapeutic strategy where increasing O-GlcNAcylation levels will slow protein
aggregation and the progression of disease.

We have focused on the roles of posttranslational modifications, including O-
GlcNAcylation, on the aggregation and degradation of α-synuclein, the major aggregating
protein in Parkinson’s disease (PD) and related Lewy body dementias (Lashuel et al.,
2013). α-Synuclein is 140 amino acids in length and is enriched at pre-synaptic termini,
where it appears to play a role in vesicle trafficking and membrane remodeling (Emanuele
and Chieregatti, 2015). α-Synuclein exists as a natively unstructured protein in the cytosol
but will form an extended α-helix when it comes into contract with cellular membranes
(Jao et al., 2008; Mizuno et al., 2012; Varkey et al., 2010), and this equilibrium is likely
important for its normal functions. During the progression of neurodegeneration, however,
α-synuclein forms β-sheet rich aggregates that contain amyloid fibers (Fink, 2006). Several
point mutants of α-synuclein that increase its aggregation have been identified that cause
early-onset PD, demonstrating a causative role for this process in human disease (Chartier-
Harlin et al., 2004; Krüger et al., 1998; Polymeropoulos et al., 1997; Singleton et al., 2003;
Zarranz et al., 2004). Notably, a growing set of data shows that these fibers can spread
along neuronal connections where they can seed the aggregation of additional protein
(Brettschneider et al., 2015; Recasens and Dehay, 2014). α-Synuclein is subjected to a
range of posttranslational modifications that can play important roles in PD (Oueslati et
al., 2010). While enzymatic modification of α-synuclein has enabled the analysis of certain
139

Figure 5-1. O-GlcNAc modification and calpain cleavage of α-synuclein. (A) O-GlcNAc
modification is the dynamic addition of N-acetyl-glucosamine to serine and threonine residues of
intracellular proteins. (B) α-Synuclein, the major aggregating protein in Parkinson’s disease, is O-
GlcNAcylated at several residues in vivo. (C) Monomeric α-synuclein has been shown to be
proteolytically cleaved by the enzyme calpain after several residues.

modifications, their site-specific installation using synthetic protein chemistry has played
an instrumental role in the evaluation of modified α-synuclein biochemistry (Pratt et al.,
2015). For example, Brik and Lashuel used chemistry to demonstrate that ubiquitination of
α-synuclein can inhibit its aggregation and promote its degradation by the proteasome
(Abeywardana et al., 2013; Haj-Yahya et al., 2013; Hejjaoui et al., 2010; Meier et al.,
2012). We also used a similar chemical strategy to show that these effects are dependent
on the site of modification and that the ubiquitin-like modifier SUMO will also inhibit α-
synuclein aggregation (Abeywardana and Pratt, 2015). In the case of O-GlcNAcylation, a
variety of in vivo proteomics experiments have identified many different O-GlcNAcylation
sites on α-synuclein (Figure 5-1B) (Alfaro et al., 2012; Morris et al., 2015; Wang et al.,
2009; Wang et al., 2010). We have previously used synthetic protein chemistry to prepare
α-synuclein with O-GlcNAcylation at two of these sites, threonine 72 (T72) and serine 87
140

(S87) (Lewis et al., 2017; Marotta et al., 2015). Using a variety of biochemical
experiments, we found that neither of these modifications inhibits the interaction of α-
synuclein with membranes; however, both modifications inhibit protein aggregation, with
O-GlcNAcylation at T72 having a stronger inhibitory effect.

Here, we use synthetic protein chemistry to explore the potential cross-talk between these
modifications and another critical posttranslational modification of α-synuclein,
proteolytic cleavage. While the majority of aggregated α-synuclein in the brains of PD
patients is full-length, several truncations have been observed (Li et al., 2005; Liu et al.,
2005). Many of these cleavages remove the C-terminal region of α-synuclein and increase
protein aggregation in vitro and in over expression experiments (Anderson et al., 2006).
Although it is not exactly clear which proteases are responsible for the cleavage of α-
synuclein in vivo, several different enzymes have been implicated. One of those enzymes
is the calcium-dependent protease calpain. Calpain has been shown to cleave α-synuclein
to generate several different fragments including C-terminal truncated forms, 1-57, 1-73,
1-75, and 1-83 (Figure 5-1C) (Mishizen-Eberz et al., 2003). Notably, these cleavage sites
lie within the region of α-synuclein that is necessary for aggregation and therefore inhibit
aggregation in vitro (Mishizen-Eberz et al., 2005). Additionally, α-synuclein fibers can be
cleaved near the C-terminus after residues 114 and 112, which promotes the seeded
aggregation of additional soluble protein (Mishizen-Eberz et al., 2005). The same α-
synuclein fragments are found in PD brains where they co-localize with calpain (Dufty et
al., 2007), and the levels of calpain activity are correlated with disease progression in PD
141

mouse models (Crocker et al., 2003; Diepenbroek et al., 2014). Given, the close proximity
of the calpain cleavage sites to O-GlcNAc modification at T72 and S87, we chose to
directly test if these O-GlcNAcylation events would affect α-synuclein proteolysis. To
accomplish this goal, we first used an expressed protein ligation strategy to prepare site-
specifically O-GlcNAcylated α-synuclein, which we then incubated with calpain. As
expected, we found that this protease can rapidly cleave unmodified α-synuclein to
generate the previously identified protein fragments. In contrast, both sites of O-
GlcNAcylation strongly inhibited proteolysis, and the minor cleavage products that were
formed differed between the two modified α-synuclein proteins. These results suggest that
in addition to its direct inhibitory effect on protein aggregation, α-synuclein O-
GlcNAcylation has the potential to inhibit α-synuclein proteolysis.

Results
In order to prepare α-synuclein with O-GlcNAcylation at either T72 or S87, we followed
our previously published synthetic routes. More specifically α-synuclein O-GlcNAcylated
at T72, α-synuclein(gT72), was retrosynthetically deconstructed (Figure 5-2A) into a
recombinant protein thioester 1 consisting of residues 1-68, a synthetic O-GlcNAcylated
peptide 2 (residues 69-75), and a recombinant protein 3 (residues 76-140) with an N-
terminal cysteine residue. Likewise, α-synuclein(gS87) can be constructed (Figure 5-2A)
from a recombinant protein thioester 4 (residues 1-75), a glycopeptide thioester 5 (residues
76-90), and a recombinant protein 6 corresponding to residues 91-140. For both proteins,
the recombinant thioesters were prepared using intein-fusions. More specifically, the
142

synuclein fragments were cloned as in-frame genetic fusions to an engineered DnaE intein
from Anabaena variabilis and His tag for purification developed by the Muir lab (Shah et
al., 2012). These proteins were heterologously expressed in E. coli, followed by
purification by cobalt chromatography, and thiolysis of the intein. The corresponding
protein thioesters (1 and 4) where then isolated by RP-HPLC and concentrated by
lyophilization. The glycopeptides 2 and 5 were prepared by solid phase peptide synthesis
on commercially available Dawson Dbz resin, which enables the generation of peptide
thioesters. Notably, these peptides contained a N-terminal thioproline residue to prevent
auto-ligation. The O-GlcNAcylated serine and threonine building blocks were prepared
from the corresponding Fmoc-protected amino acids and glucosamine using a
thioglycoside approach (Marotta et al., 2012). Recombinant proteins 3 and 6 were
expressed in E. coli followed by RP-HPLC purification, and conveniently the initiator

Figure 5-2. Synthesis of α-synuclein bearing site-specific O-GlcNAcylation. (A) α-Synuclein
bearing an O-GlcNAc modification at either threonine 72 (T72) or serine 82 (S87) was
retrosynthetically deconstructed into two recombinant proteins and a synthetic peptide that were
assembled by native chemical ligation. (B) Both protein products were characterized by RP-HPLC
and ESI-MS.

143

methionine residues were removed during expression. With these proteins in hand, iterative
ligation reactions were carried out by first reacting fragments 2 and 3 or 5 and 6, followed
by removal of the N-terminal protecting group. After purification by RP-HPLC these
proteins were then incubated with the appropriate protein thioester (either 1 or 3) to give
full-length α-synuclein. Finally, radical-based desulfurization was used to transform the
cysteine residues required for the ligation reactions to the native alanine residues, resulting
in α-synuclein(gT72) or α-synuclein(gS87) with no primary sequence mutations. These
synthetic proteins were purified by RP-HPLC and the identities were confirmed by ESI-
MS as previously described (Figure 5-2B).

To determine if O-GlcNAcylation at T72 or S87 affected α-synuclein cleavage by calpain,
we used both SDS-PAGE and RP-HPLC. More specifically, recombinant calpain (2 units
mL
-1
) was incubated with 50 μM concentrations of either unmodified α-synuclein (from
recombinant expression), α-synuclein(gT72), or α-synuclein(gS87) in HEPES buffer
containing 5 mM DTT (pH = 7.2) at 37 °C for up to 30 min. After different lengths of time
(0, 5, 10, 15, and 30 min), the enzymatic reactions were quenched by addition of SDS-
containing loading buffer and heating to 100 °C for 10 min. The proteins were then
separated by SDS-PAGE and the proteins were detected by colloidal silver staining and
imaged on a ChemiDoc XRS (Bio-Rad) (Figure 5-3). As expected, wild-type α-synuclein
was rapidly degraded while, α-synuclein(gT72) and α-synuclein(gS87) were very resistant
to cleavage. Interestingly, the pattern of cleavage bands that appear at lower molecular
144

Figure 5-3. O-GlcNAcylation blocks the cleavage of α-synuclein by calpain. Unmodified α-
synuclein or O-GlcNAcylated protein, α-synuclein(gT72) or α-synuclein(gS87), were incubated
with calpain for the indicated lengths of time before termination of the enzymatic reaction by
addition of SDS-loading buffer. The proteins were then separated by SDS-PAGE and visualized
by colloidal silver staining.

weights are different for the two O-GlcNAcylated proteins, indicating that the site of the
modification can play a role in the inhibition. In an effort to identify the calpain-dependent
cleavage products that were formed, we also analyzed the enzymatic reaction by RP-HPLC
and ESI-MS. The same reactions were initiated and allowed to proceed for 30 min. At this
time, the reactions were quenched by heating at 100 °C for 10 min. Upon cooling, 100 uL
of reaction was directly injected into the HPLC and fractions were collected for mass
analysis (Figure 5-4). Consistent with SDS-PAGE, no full-length, unmodified α-synuclein
was detected after 30 min of incubation with calpain. The major peaks detected in the
unmodified cleavage reaction were analyzed by ESI-MS and corresponded to known
cleavage sites of calpain after residues 57, 73, 75, and 83. Consistent with our SDS-PAGE
analysis, full-length α-synuclein(gT72) and α-synuclein(gS87) were identified as the main
peak in their respective chromatograms. The majority of the small cleavage products that
were analyzed by ESI-MS did not produce strong signals due to their low abundance.
However, we were able to identify cleavage of α-synuclein(gT72) after residue 33 and α-
145

synuclein(gS87) after residue 57. Cleavage after residue 33 is notable, as it has not been
observed from the proteolysis of unmodified α-synuclein.

Discussion
Here, we have described the further application of synthetic protein chemistry to
investigate the roles of posttranslational modifications on proteins. Specifically, we
demonstrate that α-synuclein that has been site-specifically O-GlcNAcylated is notably
resistant to cleavage by the enzyme calpain, which has been shown to proteolyze α-
synuclein in vitro and potentially during PD. The exact consequences of calpain proteolysis
are unknown in PD. α-Synuclein fragments that can result from calpain cleavage have been
found in aggregates from diseased brains (Dufty et al., 2007), but cleavage of α-synuclein
at many of the sites in the middle of the protein are known to generate fragments that do
not aggregate in vitro (Mishizen-Eberz et al., 2005). In vivo models of PD show that calpain
activity correlates with severity of the disease phenotype, but this could be due to reasons
other than α-synuclein cleavage (Crocker et al., 2003; Diepenbroek et al., 2014). Therefore,
the exact physiological consequences of O-GlcNAc blockage of α-synuclein proteolysis is
not clear. However, since O-GlcNAc directly inhibits protein aggregation, we believe that
it should still be viewed as an overall protective modification. Additionally, we predict that
O-GlcNAc could similarly inhibit the cleavage of α-synuclein by the as of yet unidentified
proteases that generate aggregation-prone protein fragments of α-synuclein in PD.
Notably, O-GlcNAcylation is able to inhibit α-synuclein cleavage at sites that are fairly
distant in the primary protein sequence. For example, while α-synuclein(gS87) is only
146

cleaved at low levels after residue 57. This suggests that O-GlcNAc is not simply acting as
steric bulk in the active site of calpain. We believe that this is due to the natively unfolded
nature of α-synuclein, which we predict can cause non-specific interactions with a variety
of proteins. These interactions that mediate some of the binding to calpain could be distant
from the protease active site, enabling other areas of α-synuclein to be cleaved. Our
previous experiments with O-GlcNAcylation and protein aggregation indicate that this
modification promotes the solubility of α-synuclein, which could explain our results here
by preventing non-specific interactions with calpain. In summary, we have used synthetic
protein chemistry to demonstrate that O-GlcNAcylation of α-synuclein can have site-
specific inhibitory effects on its cleavage, with potentially important implications for the
generation of aggregation-prone fragments in PD.

147

Figure 5-4. Identification of the α-synuclein-derived fragments after calpain cleavage. (A)
Unmodified α-synuclein was incubated with calpain for 30 min and the enzymatic reaction was
then quenched by heating to 100 °C. Protein fragments were then separated by RP-HPLC and
identified using ESI-MS. (B and C) α-Synuclein(gT72) or α-synuclein(gS87) were subjected to
calpain cleavage and analysis as in A.

148

Materials and Methods
Peptide Synthesis. All peptides were synthesized using standard Fmoc solid-phase
chemistry on Dawson Dbz AM® (Novabiochem, 0.49 mmol g-1) resin using HBTU (5 eq,
Novabiochem) and DIEA (10 eq, Sigma) in DMF for 1 hr. For activated glycosylated
amino acids, 2 eq of monomer in 3 mL DMF was coupled overnight. When peptides were
completed, acetyl groups were deprotected with hydrazine monohydrate (80% v/v in
MeOH) twice for 30 min with mixing. Following deprotection, Dawson linker peptides
were activated with p-nitrophenyl-chloroformate (5 eq in DCM, 1.5 hr, mixing) followed
by treatment with excess DIEA (0.5 M in DMF) for 30 min to cyclize the Dbz linker. All
peptides were then cleaved (95:2.5:2.5 TFA/H
2
O/Triisopropylsilane) for 3.5 h at room
temperature, precipitated out of cold ether, lyophilzed. Following lyophilization, crude
peptides were resuspended in thiolysis buffer (150 mM NaH
2
PO
4
, 150 mM MESNa, pH
7.0) and incubated at room temperature for 2 h prior to purification. All peptides were
purified by reverse-phase HPLC using preparative chromatography, characterized by mass
analysis using ESI-MS, and sequence purity was assessed by analytical HPLC. ESI-MS:
calculated for peptide 2 (M+H)
+
, 1017.1; found, 1018.2. ESI-MS: calculated for peptide 5
(M+H)
+
, 1017.1; found, 1018.2.

Protein Expression and Purification. General expression and purification methods were
similar to those previously reported. Briefly, BL21(DE3) chemically competent E. coli
(VWR) were transformed with plasmid DNA by heat shock and plated on selective LB
agar plates containing 50 μg mL-1 kanamycin (C-terminus) or 100 μg mL-1 ampicillin (N-
149

terminus). Single colonies were then inoculated, grown to OD600 of 0.6 at 37 °C shaking
at 250 rpm, and then expression was induced with IPTG (final concentration: 0.5 mM) at
25 °C shaking at 250 rpm for 18 h. Pellets were harvested by centrifugation (8,000 x g, 30
min, 4 °C) and lysed. C-terminus was acidified (pH 3.5 with HCl), centrifuged, dialyzed
against 3 x 1 L of 1% acetic acid in water (degassed with N
2
, 1 hr per L), and purified by
HPLC. N-terminus was loaded onto a Ni-NTA purification column (HisTrap FF Crude,
GE Healthcare) and eluted fractions were dialyzed against 3 x 1 L (100 mM NaH
2
PO
4
, 150
mM NaCl, 1 mM EDTA, 1mM TCEP HCl, pH 7.2) and concentrated. Sodium
mercaptoethane sulfonate (MESNa) was added to a final concentration of 200 mM along
with fresh TCEP (2 mM final concentration) overnight to generate the protein thioester,
and then purified by HPLC. Pure protein fragments were characterized by analytical RP-
HPLC and ESI-MS.

Expressed Protein Ligation. General ligation and purification methods were similar to
those previously reported. Briefly, C-terminal fragment (2 mM) and peptide fragment (4
mM) were dissolved in ligation buffer (300 mM NaH
2
PO
4
, 6 M guanidine HCl, 100 mM
MESNa, 1 mM TCEP, pH 7.8) and allowed to react at room temperature. Following
completion, as monitored by HPLC, the pH was adjusted to 4 with HCl and methoxylamine
was added (final concentration 100 mM) to deprotect the N-terminal thiazolidine group.
Following HPLC purification, N-terminal fragment (8 mM) and ligation product fragment
(2 mM) were dissolved in ligation buffer, and following completion was purified by HPLC.
Finally, radical desulfurization was carried out using desulfurization buffer (200 mM
150

NaH
2
PO
4
, 6 M guanidine HCl, 300 mM TCEP, pH 7.0) containing 2% v/v ethanethiol,
10% v/v tertbutyl-thiol, and the radical initiator VA-061 (as a 0.2 M stock in MeOH). The
reaction was heated to 37 °C for 15 h and then purified by RP-HPLC to yield synthetic α-
synuclein.

Calpain Cleavage of α-Synuclein Proteins. Cleavage of purified α-synuclein proteins (50
μM, 150 μL reaction volume) was carried out using calpain (EMD Millpore, 2 units mL
-1
)
in buffer containing 40 mM HEPES, pH 7.5, and 5 mM dithiothreitol at 37 °C. Calpain
cleavage was initiated by the addition of calcium (1 mM final concentration). Aliquots
were removed from the reaction mixture and quenched with SDS-containing loading buffer
at various time points, heated at 100 °C for 10 min, and stored at –20 °C until SDS-PAGE
was performed.

Silver Staining. SDS-PAGE was performed using 16.5% Criterion Tris-Tricine
polyacrylamide precast gels. Silver staining of the gels was performed in accordance with
the instructions provided in the Pierce Silver Stain Plus. Briefly, the resolved proteins were
fixed by incubating the gel in 200 ml of fixative enhancer solution (30% ethanol, 10%
acetic acid, 60% deionized distilled water) for 30 min at 25 C. Following that, the gel was
rinsed twice with 10% ethanol for 10 min and twice with 10% deionized distilled water for
10 min. Gel was then subjected to sensitizer solution (100uL sensitizer in 50 mL deionized
distilled water) for 1 minute followed by washing with deionized distilled water for 2 min.
Next, gel was stained (1 mL enhancer in 50 mL stain solution) for 30 min followed by
151

washing with deionized distilled water for 1 min. Lastly, gel was developed (1 mL
enhancer in 50 mL developer) for 2 min followed by a 5% acetic acid wash for 10 min.
Stained gel was then imaged using ChemiDoc XRS (Bio-Rad).

HPLC/ESI-MS Analysis. Reverse phase high performance liquid chromatography (RP-
HPLC) analysis was performed on digested proteins following a 30 minute incubation with
calpain. Briefly, the reactions were quenched by heating to 100 C for 30 min followed by
direct injection onto the HPLC (0-70% buffer B over 60 min). The chromatogram was
monitored at 210 nm and fractions were collected for ESI-MS. Each individual fraction
was then acquired on an API 3000 LC/MS-MS system to identify the corresponding
cleaved peptide products. buffer A: 0.1% TFA in H
2
O, buffer B: 0.1% TFA, 90% ACN in
H
2
O.

152

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178

Appendix A: NMR Spectra
179

Compound 2.1 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-serine (β-Ac 3GlcNAc Serine)
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 5 6
3 . 0 3
3 . 1 7
2 . 9 7
1 . 0 9
1 . 0 4
0 . 8 9
1 . 3 1
0 . 8 4
1 . 2 7
1 . 1 4
2 . 1 1
1 . 1 5
0 . 8 1
1 . 1 0
1 . 0 6
2 . 2 4
2 . 2 9
1 . 8 8
2 . 2 1
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
180

Compound 2.1 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-serine (β-Ac3GlcNAc Serine)
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
1 9 . 1 2
1 9 . 1 6
1 9 . 2 1
2 1 . 4 7
4 6 . 9 4
4 8 . 4 2
5 3 . 9 5
6 1 . 7 8
6 6 . 5 9
6 8 . 7 0
6 9 . 0 9
7 1 . 5 5
7 2 . 5 4
1 0 0 . 4 7
1 1 9 . 5 1
1 2 4 . 8 1
1 2 6 . 7 9
1 2 7 . 3 8
1 4 1 . 1 4
1 4 3 . 8 6
1 5 6 . 8 7
1 6 9 . 8 3
1 7 0 . 3 7
1 7 0 . 9 1
1 7 2 . 3 1
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
181

Compound 2.2 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Serine Pfp Ester (β-Ac3GlcNAc Ser-OPfp)
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 5 8
3 . 0 5
5 . 4 6
1 . 0 5
1 . 1 8
1 . 0 5
1 . 3 4
1 . 1 9
1 . 1 5
2 . 0 1
1 . 2 4
0 . 8 7
0 . 8 4
1 . 1 7
1 . 0 2
0 . 9 7
0 . 9 4
2 . 0 1
2 . 0 6
1 . 8 0
2 . 0 3
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
182

Compound 2.2 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Serine Pfp Ester (β-Ac 3GlcNAc Ser-OPfp)
0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 11 0 12 0 1 3 0 1 4 0 15 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
2 0 . 5 8
2 3 . 2 1
4 7 . 1 2
5 4 . 3 0
5 4 . 9 4
6 1 . 9 1
6 7 . 2 1
6 8 . 2 6
6 8 . 3 6
7 1 . 9 0
7 2 . 1 3
1 0 0 . 5 8
1 1 9 . 9 5
1 2 5 . 1 3
1 2 7 . 1 0
1 2 7 . 7 5
1 4 1 . 2 5
1 4 3 . 6 0
1 4 3 . 7 0
1 5 6 . 0 2
1 6 6 . 2 5
1 6 9 . 3 2
1 7 0 . 5 9
1 7 0 . 8 6
1 7 0 . 9 1
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
183

Compound 2.3 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-threonine (β-Ac3GlcNAc Threonine)
0 . 5 1. 0 1. 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
3 . 0 0
2 . 5 4
2 . 8 4
2 . 7 1
2 . 8 9
0 . 9 7
0 . 8 6
0 . 9 4
1 . 0 3
1 . 0 9
1 . 1 6
1 . 9 5
1 . 1 1
0 . 8 6
0 . 9 2
0 . 9 6
2 . 3 3
2 . 4 6
1 . 9 8
2 . 3 3
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
184

Compound 2.3 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-threonine (β-Ac3GlcNAc Threonine)
0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
1 6 . 6 0
1 9 . 1 1
1 9 . 1 5
1 9 . 2 5
2 1 . 4 1
4 8 . 4 2
5 4 . 1 0
5 8 . 4 5
6 1 . 7 7
6 6 . 7 8
6 8 . 7 2
7 1 . 3 6
7 2 . 4 0
7 5 . 3 1
9 9 . 4 9
1 1 9 . 4 9
1 2 4 . 8 6
1 2 6 . 7 4
1 2 7 . 3 6
1 4 1 . 1 3
1 4 3 . 8 6
1 5 7 . 5 6
1 6 9 . 8 1
1 7 0 . 3 7
1 7 1 . 0 2
1 7 2 . 2 8
O
AcO
AcO
AcHN
OAc
O
FmocHN
OH
O
185

Compound 2.4 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Threonine Pfp Ester (β-Ac 3GlcNAc Thr-OPfp)
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
3 . 0 0
3 . 2 3
3 . 1 2
3 . 3 7
2 . 8 2
1 . 2 3
1 . 0 5
1 . 1 7
1 . 1 9
1 . 2 3
2 . 6 0
1 . 1 3
1 . 0 1
1 . 0 2
1 . 1 5
1 . 0 5
1 . 0 0
0 . 9 8
2 . 2 9
2 . 4 3
2 . 0 1
2 . 2 9
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
186

Compound 2.4 O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Threonine Pfp Ester (β-Ac3GlcNAc Thr-OPfp)
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
1 6 . 4 9
2 0 . 4 1
2 0 . 5 6
2 0 . 6 5
2 3 . 3 4
4 7 . 1 1
5 5 . 0 9
5 8 . 6 9
6 1 . 8 6
6 7 . 3 5
6 8 . 4 0
7 1 . 7 2
7 1 . 8 7
7 3 . 2 0
9 8 . 1 8
1 1 9 . 9 2
1 2 5 . 1 6
1 2 7 . 0 3
1 2 7 . 7 2
1 4 1 . 2 5
1 4 3 . 5 7
1 4 3 . 8 2
1 5 6 . 6 2
1 6 6 . 4 6
1 6 9 . 2 7
1 7 0 . 4 6
1 7 0 . 6 0
1 7 1 . 0 9
O
AcO
AcO
AcHN
OAc
O
FmocHN
OPfp
O
187

Compound 2.5 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine (β-Ac3GlcNAc Cysteine)
0 . 5 1 . 0 1. 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 8 1
3 . 1 3
3 . 0 2
2 . 8 5
0 . 9 3
0 . 9 5
1 . 0 0
0 . 9 9
1 . 4 0
1 . 0 4
1 . 1 1
2 . 1 2
0 . 9 9
0 . 9 2
1 . 0 3
1 . 0 3
2 . 0 9
1 . 8 9
1 . 7 9
1 . 9 1
O
AcO
AcO
AcHN
OAc
S
FmocHN
OH
O
188

Compound 2.5 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine (β-Ac 3GlcNAc Cysteine)
0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 10 12 0 1 3 0 14 0 1 5 0 1 6 0 17 0 1 8 0
f 1 ( p p m )
1 9 . 1 0
1 9 . 1 3
1 9 . 2 1
2 1 . 2 6
3 1 . 1 9
4 6 . 9 1
4 7 . 1 4
4 7 . 2 8
4 7 . 4 2
4 7 . 5 6
4 7 . 7 0
4 7 . 8 5
4 7 . 9 9
5 2 . 8 0
6 2 . 0 6
6 6 . 7 4
6 8 . 6 8
7 3 . 6 7
7 5 . 5 8
8 3 . 1 4
1 1 9 . 5 1
1 2 4 . 8 5
1 2 6 . 7 9
1 2 6 . 8 1
1 2 7 . 3 9
1 4 1 . 1 3
1 4 3 . 7 6
1 4 3 . 8 0
1 5 6 . 9 9
1 6 9 . 7 7
1 7 0 . 3 6
1 7 1 . 0 0
1 7 1 . 9 0
O
AcO
AcO
AcHN
OAc
S
FmocHN
OH
O
189

Compound 2.6 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester (β-Ac3GlcNAc Cys-OPfp)
0 . 5 1 . 0 1. 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 5 3
2 . 6 8
3 . 4 0
2 . 8 5
0 . 9 0
0 . 8 7
0 . 9 6
0 . 9 4
1 . 8 2
0 . 9 1
0 . 8 0
1 . 0 0
0 . 8 5
0 . 8 4
0 . 9 3
0 . 9 2
0 . 9 5
0 . 8 3
2 . 0 0
2 . 0 1
1 . 7 4
1 . 9 6
O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
190

Compound 2.6 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester (β-Ac 3GlcNAc Cys-OPfp)
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
2 0 . 4 8
2 0 . 5 4
2 0 . 6 1
2 3 . 1 2
2 9 . 6 7
3 1 . 2 8
4 7 . 1 0
5 2 . 9 3
5 3 . 7 3
6 2 . 1 1
6 7 . 2 0
6 8 . 1 2
7 3 . 3 4
7 6 . 3 6
8 3 . 8 2
1 1 9 . 9 8
1 2 0 . 0 0
1 2 4 . 9 5
1 2 5 . 0 5
1 2 7 . 0 8
1 2 7 . 1 1
1 2 7 . 7 1
1 2 7 . 7 4
1 4 1 . 2 6
1 4 1 . 2 8
1 4 3 . 5 6
1 4 3 . 6 6
1 5 6 . 0 4
1 6 6 . 9 8
1 6 9 . 1 8
1 7 0 . 5 7
1 7 0 . 7 3
1 7 1 . 0 9
O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
191

1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 8 9
2 . 9 7
3 . 0 2
2 . 9 6
1 . 0 5
1 . 1 5
0 . 9 6
0 . 9 4
1 . 0 2
1 . 0 0
1 . 0 4
Compound 2.7 2-Methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline (2-Methyl Oxazoline)
O
AcO
AcO
OAc
N
O
192

1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
3 . 0 3
3 . 1 7
3 . 0 9
0 . 8 9
0 . 9 5
0 . 5 5
0 . 8 2
3 . 3 9
2 . 6 4
1 . 9 0
0 . 9 0
1 . 0 1
1 . 0 4
0 . 9 1
0 . 8 9
2 . 3 3
2 . 3 7
2 . 0 5
2 . 0 7
Compound 4.1 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
O
AcO
AcO
TrocHN
OAc
S
FmocHN
OH
O
193

O
AcO
AcO
TrocHN
OAc
S
FmocHN
OH
O
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
1 8 . 0 3
1 8 . 1 4
2 7 . 1 0
3 0 . 3 9
4 4 . 4 5
5 0 . 8 1
5 2 . 4 7
5 9 . 8 9
6 4 . 8 9
6 6 . 1 0
7 0 . 7 2
7 1 . 8 1
7 3 . 1 6
8 2 . 0 7
9 2 . 7 5
1 1 7 . 4 6
1 2 2 . 4 3
1 2 2 . 5 2
1 2 4 . 5 6
1 2 5 . 2 2
1 3 8 . 6 8
1 4 0 . 8 9
1 4 1 . 2 1
1 5 1 . 7 2
1 5 3 . 8 6
1 6 6 . 8 5
1 6 8 . 1 9
1 6 8 . 6 3
Compound 4.1 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine
194

1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 5 9
2 . 7 6
3 . 2 6
0 . 8 4
0 . 8 7
0 . 9 8
1 . 0 8
2 . 0 1
1 . 1 2
0 . 8 7
2 . 2 0
0 . 9 7
0 . 9 5
0 . 9 8
0 . 9 6
0 . 8 9
0 . 7 8
0 . 7 3
2 . 1 3
2 . 0 8
1 . 9 5
2 . 0 3
O
AcO
AcO
TrocHN
OAc
S
FmocHN
OPfp
O
Compound 4.2 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester
195

Compound 4.2 S-(2-N-Troc-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester
O
AcO
AcO
TrocHN
OAc
S
FmocHN
OPfp
O
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
2 0 . 5 0
2 0 . 5 9
3 1 . 5 8
4 7 . 1 1
5 3 . 6 9
5 4 . 8 8
6 7 . 3 1
6 8 . 2 7
7 3 . 0 2
7 4 . 4 9
7 6 . 2 9
8 3 . 7 1
9 5 . 2 2
1 2 0 . 0 6
1 2 4 . 9 4
1 2 5 . 0 8
1 2 7 . 1 4
1 2 7 . 8 1
1 4 1 . 3 2
1 4 3 . 4 9
1 4 3 . 7 4
1 5 4 . 3 0
1 5 6 . 0 2
1 6 6 . 9 5
1 6 9 . 2 8
1 7 0 . 5 4
1 7 0 . 6 7
196

1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0
f 1 ( p p m )
2 . 6 5
2 . 6 1
2 . 7 4
3 . 1 0
0 . 9 3
0 . 9 0
0 . 9 7
1 . 0 9
1 . 8 0
1 . 0 4
0 . 9 9
1 . 1 0
0 . 9 8
0 . 9 2
0 . 9 7
0 . 9 6
0 . 9 9
0 . 8 6
1 . 9 2
2 . 0 2
1 . 6 8
1 . 9 2
Compound 4.3 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester
O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
197

O
AcO
AcO
AcHN
OAc
S
FmocHN
OPfp
O
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0
f 1 ( p p m )
2 0 . 4 7
2 0 . 5 4
2 0 . 6 2
2 3 . 1 5
3 1 . 2 1
4 7 . 1 0
5 2 . 8 9
5 3 . 7 7
6 2 . 1 3
6 7 . 1 9
6 8 . 2 1
7 3 . 3 3
7 6 . 3 2
8 3 . 8 1
1 1 9 . 9 8
1 2 0 . 0 0
1 2 4 . 6 0
1 2 4 . 9 7
1 2 5 . 0 5
1 2 5 . 0 7
1 2 7 . 0 8
1 2 7 . 1 1
1 2 7 . 7 1
1 2 7 . 7 4
1 3 7 . 0 1
1 3 8 . 7 9
1 3 8 . 8 9
1 4 0 . 5 8
1 4 1 . 2 5
1 4 1 . 2 8
1 4 3 . 5 9
1 4 3 . 6 8
1 5 6 . 0 3
1 6 7 . 0 2
1 6 9 . 1 7
1 7 0 . 5 3
1 7 0 . 5 8
1 7 1 . 0 2
Compound 4.3 S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-Cysteine Pfp Ester

Abstract (if available)

Abstract O-GlcNAcylation is a post-translational modification that involves the β-linkage of a single unit of N-acetyl-D-glucosamine (GlcNAc) to the alcoholic residues (serine/threonine) of nuclear, cytosolic, and mitochondrial proteins. Despite the identification of thousands of O-GlcNAcylated proteins involved in several important cellular processes and diseases, the functional outcome of this site-specific modification on the protein’s biophysical and biochemical properties remains difficult to evaluate. This is in part due to the difficulty in isolating homogenous samples of O-GlcNAcylated proteins from natural sources and the limiting number of mutational and chemical strategies available to study this modification. The advent of solid-phase peptide synthesis (SPPS) has revolutionized the field of O-GlcNAcylation by enabling the chemical synthesis of peptides bearing site-specific O-GlcNAc units. These well-defined glycopeptides have in-turn facilitated the preparation of homogeneously O-GlcNAcylated proteins suitable for in vitro biological studies. However, the chemical synthesis of the glycosylated amino acids required for SPPS still remains a challenge. Described here is the facile synthesis of GlcNAc β-glycosides and the application of these building blocks for the construction of homogenous site-specifically modified proteins for in vitro functional studies. Particularly, we focus on the semi-synthetic preparation of the site-specifically O-GlcNAcylated neuronal protein, α-synuclein. This protein has been demonstrated to play a causative role in synucleinopathies such as Parkinson’s disease, where it aggregates to form toxic protein deposits known as Lewy bodies. Using a variety of in vitro assays, we demonstrate that site-specific O-GlcNAcylation of α-synuclein inhibits its propensity to aggregate.

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Investigating the role of O-GlcNAcylation in α-synuclein aggregation

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Understanding the roles of posttranslational modifications in aggregation using synthetic proteins

Asset Metadata

Creator Deleon, Cesar Augusto (author)

Core Title Novel synthesis of β-glycosides for SPPS of GLCNAC glycoproteins and study of their site-specific biochemical and biophysical consequences

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/26/2018

Defense Date 06/08/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag glycosylation,Indium bromide,OAI-PMH Harvest,OGA,O-GlcNAc,post-translational modifications,protein aggregation,S-GlcNAc,site-specific function,solid-phase peptide synthesis,synuclein,β-glycosides

Format application/pdf (imt)

Language English

Advisor Pratt, Matthew Robert (committee chair), Langen, Ralf (committee member), Qin, Peter (committee member)

Creator Email cadeleon@usc.edu,longuda_08@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-26414

Unique identifier UC11670332

Identifier etd-DeleonCesa-6490.pdf (filename),usctheses-c89-26414 (legacy record id)

Legacy Identifier etd-DeleonCesa-6490.pdf

Dmrecord 26414

Document Type Dissertation

Format application/pdf (imt)

Rights Deleon, Cesar Augusto

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA