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Peering into the cell: click chemistry and novel dyes towards enhanced biomedical imaging

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Peering into the cell: click chemistry and novel dyes towards enhanced biomedical imaging

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Content Copyright 2022 Jose Ricardo Moreno

Peering Into The Cell:
Click Chemistry and Novel Dyes Towards Enhanced Biomedical Imaging
by
Jose Ricardo Moreno

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
December 2022
ii
Dedication
Para mi familia

iii
Acknowledgements
My journey to completing this PhD has been challenging, rewarding, and humbling. I would
not be here without the encouragement, support, love of the community I have found at USC and
in Los Angeles. First, I would like to thank my advisor, Prof. Valery V. Fokin, for providing me a
challenging and empowering environment to explore chemistry these last few years. It has all
worked out the way it should have, and I’m honored to have spent my time in graduate school in
your lab. For your tutelage, mentorship, and friendship, I’m eternally grateful. To my parents, thank
you for your unconditional encouragement, support, and love; you have inspired me to keep going
after my dreams despite the odds. I can’t wait to see what the future will hold! To Micky and Jeam,
thank you for your love and care you both have showered me with the last few years. To Prof.
Cristina Zavaleta, I’ve deeply enjoyed working with you and your lab in my time at USC. The
synthetic dyes project pushed me harder as a synthetic chemist and nearly broke me several times –
thankfully it only made me stronger. Moreover, I will cherish our conversations about science,
heritage, culture, pride, duty to community, and the simple joys of RuPaul’s Drag Race. To Prof.
Travis Williams, thank you for believing in me when I came to you for help in the middle of my
PhD – I would not be here without your guidance and support. To Prof. Myrna Jacboson Myers, I
have become a better person with each conversation that we’ve had about science and life. Your
wisdom and guidance got me where I needed to be. Thank you for believing in me when I doubted
myself. To Prof. Paul Nash, thank you for your constant encouragement and friendship throughout
my time at USC. They don’t make them like you anymore! To Guadalupe, it was deeply refreshing
and grounding to have you as my soundboard throughout my time in graduate school; I’m honored
to have you and Sean in my life. I’ll see you on the East coast! To Lisa, thank you for being my best
friend throughout graduate school. You are one of the bravest and smartest people I know and one
of the few that will snap me back to reality. You’re one of the good ones :]
iv
To Shubangi, thank you for always being there to bounce off ideas and your support in the
Fokin group. Seeing and working with you in lab was always a joy and deeply inspiring. To Shelby,
you have been my lucky charm with it came to the dye project – everything seemed to work when
you are around! I enjoyed the last few years watching you grow as a scientist and I’m excited to see
where life will take you. To Sydney, you’re brilliant, don’t let anyone tell you otherwise. To Rudra,
you’ve been something of kid brother to me in the lab. Keep the lab in good spirits :] To Will, thank
you for your kindness throughout our time in lab. I could always count on you for support and
warmth. To the rest members of the Fokin group, thank you.
To the girls, and by that, I mean Jimmy, Drystan, Xichen, David, and Matt. Thank you for
always being there for me in moments of joy and moments of resiliency. To Ella, our times seeing
Shakespeare and live theatre have been some of my favorite moments during graduate school – they
kept me human. To Robert, thank you for stepping into my life at the moment that you did. I can’t
wait to see what life will bring. To Sean and Rich, the dinners we had and the wild guests who came
through are some of the highlights of grad school. But more than that, your friendship was the
greatest of gifts.
To my high school chemistry teacher, Ms. Debroah Maner, thank you for your guidance and
instilling a love of chemistry
To my grandma and grandpa, valio la pena.

v
Table of Contents
Dedication ........................................................................................................................................................... ii
Acknowledgements ........................................................................................................................................... iii
List of Figures ................................................................................................................................................... vii
Table of Schemes ............................................................................................................................................... x
Abbreviations ..................................................................................................................................................... xi
Abstract ................................................................................................................................................................ ii
Chapter 1 - Synthesis of Peptide-Dye conjugates for Isolation of Beta-Pancreatic Cells ...................... 1
Introduction ................................................................................................................................................... 1
Results and Discussion: ................................................................................................................................ 3
Conclusions: ................................................................................................................................................... 5
General Procedures: ...................................................................................................................................... 6
Experimentals ................................................................................................................................................. 7
Representative Spectra ................................................................................................................................ 16
References: .................................................................................................................................................... 31
Chapter 2 - ELISA development and detection of SARS-CoVID-19 Antibodies ................................ 33
Introduction ................................................................................................................................................. 33
Results and Discussion: .............................................................................................................................. 35
Conclusion: ................................................................................................................................................... 53
Experimentals: .............................................................................................................................................. 54
References: .................................................................................................................................................... 67
Chapter 3 - Dyes and Pigment Handles for Nanoparticles in Biomedical Imaging .............................. 69
Introduction: ................................................................................................................................................ 69
Results and Discussion: .............................................................................................................................. 70
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonic acid ..................... 70
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonamide ..................... 75
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonyl trifluoride .......... 76
Conclusions: ................................................................................................................................................. 76
Experimental ................................................................................................................................................ 79
Reaction Optimization of Sulfonic Acid Reaction Procedure ......................................................... 85
Representative Spectra ................................................................................................................................ 86
References: .................................................................................................................................................. 102
vi
Chapter 4 - Design, Synthesis, and Applications of Metabolic Chemical Reporters as Tools for
Probing Glycosylation Pathways ................................................................................................................. 103
Introduction: .............................................................................................................................................. 103
Results and Discussion ............................................................................................................................. 106
Synthesis of 5-Alkyne GlcNAc ........................................................................................................... 106
Labelling of 5-AlkyneGlcNAc ............................................................................................................ 108
Synthesis of 5-Alkyne ManNAc ......................................................................................................... 108
Conclusions: ............................................................................................................................................... 109
Experimental: ............................................................................................................................................. 112
Representative Spectra .............................................................................................................................. 122
References: .................................................................................................................................................. 135
Bibliography .................................................................................................................................................... 139

vii
List of Figures
Figure 1.1 Beta Pancreatic Cell Project Overview ............................................................................. 2
Figure 1.2 Copper Catalyzed Alkyne-Azide Cycloaddition of Peptide with Dye ............................... 3
Figure 1.3
1
H NMR Spectrum of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) ........................................................................................................ 16
Figure 1.4
13
C NMR Spectrum of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) ........................................................................................................ 17
Figure 1.5
1
H NMR Spectrum of 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane ................. 18
Figure 1.6
13
C NMR Spectrum of 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane ................ 19
Figure 1.7
1
H NMR Spectrum of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine ................. 20
Figure 1.8
13
C NMR Spectrum of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine ................. 21
Figure 1.9
1
H NMR Spectrum of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) ........................................................................................................ 22
Figure 1.10
13
C NMR Spectrum of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) ........................................................................................................ 23
Figure 1.11
1
H NMR Spectrum of 1,2-bis(2-azidoethoxy)ethane .................................................... 24
Figure 1.12
13
C NMR Spectrum of 1,2-bis(2-azidoethoxy)ethane .................................................... 25
Figure 1.13
1
H NMR Spectrum of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine ............................... 26
Figure 1.14
13
C NMR Spectrum of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine ............................... 27
Figure 1.15
1
H NMR Spectrum of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-difluoro-7-(4-
methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-
yl)propanamido)hexanamide .................................................................................................. 28
Figure 1.16 g-COSY NMR Spectrum of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-difluoro-7-
(4-methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-
yl)propanamido)hexanamide .................................................................................................. 29
Figure 1.17 NMR assignments table of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-difluoro-7-(4-
methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-
yl)propanamido)hexanamide .................................................................................................. 30
Figure 2.1 RBD Fragment Concentration Series in PBSA Buffer ................................................... 35
Figure 2.2 RBD Fragment Concentration Series in Bicarbonate Buffer .......................................... 35
Figure 2.3 IgG Antibody Dilution Series for RBD at 0.3 µg /mL ................................................... 36
Figure 2.4 IgG Antibody Dilution Series for RBD at 0.6 µg /mL ................................................... 36
Figure 2.5 Sample Dilution Series for RBD 0.3 µg /mL ................................................................. 37
Figure 2.6 Sample Dilution Series for RBD 0.6 µg /mL ................................................................. 37
Figure 2.7 Serum Sample Dilution Series ........................................................................................ 38
Figure 2.8 Sample Dilution Series at 0.3 µg /mL RBD and IgG Concentration .............................. 38
Figure 2.9 Sample Dilution Series at 0.6 µg /mL and IgG Concentration ....................................... 39
Figure 2.10 Antibody Dilution Series at RBD 0.3 µg /mL and 1:25 Serum Dilution ....................... 39
Figure 2.11 Antibody Dilution Series at RBD 0.6 µg /mL and 1:25 Serum Dilution ....................... 40
Figure 2.12 Paired Serum and Oral Samples on RBD 0.3 µg /mL and 1:2000 IgG ......................... 41
Figure 2.13 Serum Sample Diluted in Negative Saliva Series on RBD 0.3 µg/mL and 1:2000 IgG . 41
Figure 2.14 Serum IgG detection across 90 random clinical samples .............................................. 43
Figure 2.15 Serum Panel Samples by PCR Status ............................................................................ 43
Figure 2.16 RBC Fragment Concentration Series at 1:500 IgG Concentration ................................ 46
viii
Figure 2.17 RBC Fragment Concentration Series at 1:2000 IgG Concentration .............................. 46
Figure 2.18 IgG Antibody Dilution Series at 1.2 μg/mL of RBD and 1:5 dilution of mouthwash
samples .................................................................................................................................. 47
Figure 2.19 Mouthwash sample dilution series on 1.2 µg/mL of RBD and 1:100 IgG .................... 48
Figure 2.20 Expanded Mouthwash Panel at 1.2 µg /mL RBD, 1:5 MW dilution, and 1:100 IgG
dilution ................................................................................................................................... 49
Figure 2.21 Mouthwash Panel Samples Split by PCR Status ........................................................... 49
Figure 2.22 Comparison of Diluent Buffers with Mouthwash Samples ........................................... 51
Figure 2.23 Comparison of Diluent Buffer for Mouthwash Samples .............................................. 52
Figure 2.24 Spin Column Use Increased Separation Between Negative and Positive Populations of
Patient Samples ...................................................................................................................... 53
Figure 3.1 Overlap of Trisulfonic Acid and Disulfonic Acid on C18 HPLC ................................... 71
Figure 3.2 Reaction Optimization of 8-Aminopyrene-1,3,6-trisufonic acid ................................ 74
Figure 3.3
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride ................................. 86
Figure 3.4
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride ................................ 87
Figure 3.5 LC/MS Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride in Negative Mode ...... 88
Figure 3.6 IR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride ........................................... 88
Figure 3.7
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride ................................. 89
Figure 3.8
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride ................................. 90
Figure 3.9
19
F NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride ................................. 91
Figure 3.10 LC/MS Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride in Positive Mode ...... 92
Figure 3.11 IR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride .......................................... 92
Figure 3.12 MS/MS MRM Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride ....................... 93
Figure 3.13
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide ......................................... 94
Figure 3.14
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide ......................................... 95
Figure 3.15 HSQC NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide ................................... 96
Figure 3.16 UV/Vis Spectrum of 8-aminopyrene-1,3,6-trisulfonamide ........................................... 96
Figure 3.17
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonic acid ......................................... 97
Figure 3.18
1
H NMR Spectrum of triethoxy(3-isothiocyanatopropyl)silane ..................................... 98
Figure 3.19
13
C NMR Spectrum of triethoxy(3-isothiocyanatopropyl)silane .................................... 99
Figure 3.20
1
H NMR Spectrum of 8-(3-(3-(diethoxy(methoxy)silyl)propyl)thioureido)pyrene-1,3,6-
trisulfonamide ...................................................................................................................... 100
Figure 3.21
13
C NMR Spectrum of 8-(3-(3-(diethoxy(methoxy)silyl)propyl)thioureido)pyrene-1,3,6-
trisulfonamide ...................................................................................................................... 101
Figure 4.1 Metabolic Engineering using modified monosaccharides ............................................. 103
Figure 4.2 The Hexosamine Salvage Pathway ............................................................................... 104
Figure 4.3 5-Alkyne GlcNAc and 5-Alkyne ManNAc ................................................................... 105
Figure 4.4 Potential instability of 5Az derivative ........................................................................... 105
Figure 4.5 Protein incorporation of 5-AlkyneGlcNAc .................................................................. 108
Figure 4.6
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-glucoside ............................. 122
Figure 4.7
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-glucoside ............................. 123
Figure 4.8
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-α-D-glucoside ............................................................................................................... 124
Figure 4.9
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-α-D-glucoside ............................................................................................................... 125
ix
Figure 4.10
1
H NMR Spectrum of Methyl 2-acetamido-6-aldehydo-2-deoxy-3,4-O-(2,3-
dimethoxybutane-2,3-diyl)-α-D-glucoside ............................................................................. 126
Figure 4.11
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-5-ethynyl-α-D-glucoside ............................................................................................... 127
Figure 4.12
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-5-ethynyl-α-D-glucoside ............................................................................................... 128
Figure 4.13
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-mannoside ......................... 129
Figure 4.14
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-mannoside ........................ 130
Figure 4.15
1
H NMR Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-
mannoside ............................................................................................................................ 131
Figure 4.16
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-α-D-mannoside ............................................................................................................ 132
Figure 4.17
1
H NMR Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-5-ethynyl-
α-D-mannoside .................................................................................................................... 133
Figure 4.18
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-
diyl)-5-ethynyl-α-D-mannoside ............................................................................................. 134

x

Table of Schemes
Scheme 1.1 Synthetic Scheme for Azide Linkers .............................................................................. 3
Scheme 1.2 Synthesis of BODIPY-Linker Conjugate ....................................................................... 4
Scheme 3.1 Retrosynthesis of G8-Silane Monomer ........................................................................ 70
Scheme 3.2 Bucherer Mechanism of Naphthalene .......................................................................... 70
Scheme 3.3 Sulfonation of Aminopyrene with Fuming Sulfuric Acid Leads to Mixture of the Tri-
and Di-sulfonic Acid Products ............................................................................................... 71
Scheme 3.4 Revised Synthetic Scheme for Sulfonic Acids .............................................................. 72
Scheme 3.5 Reaction Progression for Sulfonyl Chlorides ................................................................ 72
Scheme 3.6 Revised Synthetic Scheme for G8-Sulfonic Acid Silane Monomer ............................... 75
Scheme 3.7 Synthetic Scheme for G8-Sulfonamide Silane Monomer .............................................. 75
Scheme 3.8 Synthetic Scheme for G8-Sulfonyl fluoride silane monomer ........................................ 76
Scheme 4.1 Retrosynthesis of 5-Alkyne GlcNAc .......................................................................... 106
Scheme 4.2 Electrophilic Aldehyde at C5 position undergoes beta elimination ............................. 106
Scheme 4.3 Synthesis of 5AlkGlcNAc .......................................................................................... 107
Scheme 4.4 Synthesis of 5-ManNAcAlkyne .................................................................................. 109

xi
Abbreviations
o
C degrees Celsius
Å angstrom
Ac acetyl
Ace-2 angiotensin converting enzyme 2
AIDS acquired immunodeficiency syndrome
Approx. approximately
Bicarb bicarbonate buffer
BOC tertI-butyloxycarbonyl
bs broad singlet
BSA bovine serum albumin
BSL2 Biosafety level 2
bd broad doublet
CuAAC copper-catalyzed azide-alkyne cycloadditions
δ chemical shift
d deuterated
d doublet
dd doublet of doublets
DCM dichloromethane
DIEA diisopropylethylamine
DMAP N,N-dimethylaminopyridine
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
ELISA enzyme-linked immunosorbent assay
Eq equivalents
Et ethyl
EtOH ethanol
EtOAc ethyl acetate
g gram
Glc glucose
GlcNAc N-acetyl glucosamine
GLPR-1 glucagon-like peptide 1 receptor
GSD Gold Standard Diagnostics
h hour
hept heptete
Hex hexane
HIV human immunodeficiency virus
HMBC heteronuclear multiple bond correlation
HMPA hexamethylphosphoric triamide (hexamethylphosphoramide)
HRP horseradish peroxidase
HPLC high-performance liquid chromatography
HSQC heteronuclear single quantum correlation
Hz hertz
IgA immunoglobulin A
IgG immunoglobulin G
IgM immunoglobulin M
xii
IR infrared
J coupling constant
Ka association constant
K kelvin
k reaction rate
L liter
λ wavelength
m multiplet
M molar
M
+
molecular ion
Man mannose
ManNAc N-acetyl mannosamine
Me methyl
MeOH methanol
mg milligram
MHz megahertz
min minute
mL milliliter
mM millimolar
mmol millimole
Ms methylsulfonyl (mesyl)
MW mouthwash
m/z mass-to-charge ratio
μl microliter
μM micromolar
μmol micromole
nM nanomolar
nm nanometer
NMR nuclear magnetic resonance
N protein nucleocapsid protein of the SARS-CoVID-19 virus
OD optical density
PBS phosphate sodium chloride buffer
PBSA phosphate sodium chloride buffer with bovine serum albumin
PCR polymerase chain reaction
pH negative log of hydrogen ion concentration
Ph phenyl
ppb parts per billion
ppm parts per million
q quartet
quint. quintet
RBD receptor binding domain of the SARS-CoVID-19 virus
Rf retention factor
RNA ribonucleic acid
rt room temperature
rt-qPCR reverse transcription-quantitative polymerase chain reaction
s singlet
S protein spike protein of the SARS-CoVID-19 virus
TEA triethylamine
xiii
TLC thin-layer chromatography
THF tetrahydrofuran
THPTA Tris(3-hydroxypropyltriazolylmethyl)amine
TMB 3,3',5,5'-tetramethylbenzidine
TTTA tris((1-tert-butyl-1H-1,2,3-triazolyl)methyl)amine
ii
Abstract
The ability to utilize the skills and insights of organic chemistry towards understanding the
fundamental mechanisms of the cell has been a central theme in this dissertation. Each of these
projects has been directed to make new and useful tools for exploration of the cell and
understanding it’s underlying mechanisms.
Chapter 1 focused on the synthesis of exendin-4 dye conjugates for the isolation of beta
pancreatic cells in heterogenous populations of cells. These peptide-dye conjugates were coupled
through the copper-catalyzed azide-alkyne cycloaddition and bind selectively to the GLPR-1
receptor of pancreatic β-cells. Using these tools, we have developed a framework for isolating beta
pancreatic cells via their unique cellular receptors.
Chapter 2 focused on the development of ELISA assays for the detection of antibodies in
response to the SARS-CoVID-19 pandemic. In the process of these studies, we first developed an
IgG antibody ELISA in serum and then extend our assay to work in a variety of saliva samples. Our
studies revealed that the difficulty in detecting antibodies in saliva was not just the low
concentrations of IgG present in the mouth but also the confounding interactions of enzymes,
proteins, and food material present in the mouth. Despite these difficulties, these assays can be
quickly utilized the field and the development framework can be used to develop new ELISA assays
for future pandemics.
Chapter 3 focused on the development of fluorescent pyrene-based silica nanoparticles that
were inspired by 8-hydroxypyrene-1,3,6-sulfonic acid. In the process, we developed a synthetic
strategy to smoothly access 8-aminoypyrene-1,3,6-sulfonic acid, 8-aminoypyrene-1,3,6-sulfonamides,
and 8-aminoypyrene-1,3,6-sulfonyl fluorides dyes. We have demonstrated that 8-aminoypyrene-
1,3,6-sulfonamides can be covalently incorporated into the silica framework of silica nanoparticles.
iii
Chapter 4 focused on development of non-natural alkyne modified N-acetylglucosamine and
N-acetylmannosamine derivatives to probe the metabolic promiscuity. After developing a synthetic
pathway towards the 5-Alkyne GlcNAc, we incubated 3T3 cells with the compound and determined
that this non-natural sugar was incorporated into proteins despite not having an entry into cellular
metabolism via the hexosamine salvage pathway.

1
Chapter 1 - Synthesis of Peptide-Dye conjugates for Isolation of
Beta-Pancreatic Cells
Introduction
Beta pancreatic cells are located in the pancreas and, in conjugation with alpha pancreatic
cells, are responsible for tightly regulating glucose levels in the blood via production of insulin and
glucagon, respectively.
1-7
Dysregulation of alpha and beta pancreatic cells, or lack thereof, can result
in the body’s inability to absorb and metabolize glucose – leading to diabetes. In Type 1 diabetes, the
pancreas produces too little insulin or is unable to make insulin. In Type 2 diabetes, the pancreas is
unable to produce enough insulin or the pancreatic cells are unable to respond to insulin.
3
Currently,
26.9 million people in the United States live with diabetes. Management of diabetes for individual
patients requires regimented check of their own blood glucose levels using fingerpicks of blood and
a blood glucose meter. If the blood glucose levels are too high, the patient self-administers a dose of
insulin to prevent hyperglycemia. If the blood glucose levels are too low, the patient can experience
shakiness, lack of coordination, and must eat simple carbohydrates in order to prevent
hypoglycemia. In comparison to other chronic conditions like HIV/AIDS (that require the daily
intake of nucleoside reverse transcriptase inhibitors), diabetes, in the best-case scenario, demands
constant monitoring of bodily glucose level, from food intake to insulin injection. We need better
treatments for diabetes, and that begins with getting a better understanding of the underlying cell
biology of the beta pancreatic cell.
2

Figure 1.1 Beta Pancreatic Cell Project Overview

The beta pancreatic cell is unique in being the only cell in the pancreases that expresses the
glucagon-like peptide-1 receptor (GLPR1) on its cell membrane.
1-2, 4
GLPR1 is a G protein-coupled
receptor and is encoded by the gene GLP1R. This receptor can bind to glucagon and begin the
cellular cascade for production of insulin with the cell. It is also known to bind to exendin-4, a 39-
amino acid peptide that was first discovered in the saliva of the Gila monster. In sequent years,
exendin-4 has been transformed into a drug used to treat diabetes by binding to the GLPR1 receptor
and encouraging the beta cell to produce insulin.
5, 8-9
We proposed to take advantage of this feature
of the beta pancreatic cell and design a peptide-dye conjugate that would be used with flow
cytometry to isolate beta pancreatic cells from a heterogenous cell population.
Currently beta pancreatic cells are isolated using fluorescent antibodies that bind to GLP1R
and then sorted using flow cytometry. However, after this process, the antibodies remain bound to
the beta pancreatic cells at the GLP1Rs. The GLP1R is used to bind to glucagon and turn on the
release of calcium in the beta cell and begin the production of insulin. If the beta pancreatic cell is
isolated using an antibody, then the antibody is bound tightly to the cell’s receptor. Because the
N
N
N
Exendin-4
TAG
GL1PR
Flow Cytometry
Sorting
Hetetrogenous Cell Population
Tagged Pancreatic β-cell Population
3
receptor is bound to the antibody, the downstream pathways of the beta pancreatic cells are turned
on – any subsequent studies from this cell population would be biased in this pathway. Our
approach uses a small peptide-dye conjugate that is designed to bind to the receptor and wash away
– leaving a population of pancreatic beta cells that are not biased towards a specific pathway from
activation/blockage of the GLPR1 receptor.

Figure 1.2 Copper Catalyzed Alkyne-Azide Cycloaddition of Peptide with Dye
Previous work from Clardy et. al had demonstrated that these peptide-dye linkers could be
used to separate cells that artificially expressed the GLPR1 receptor using flow cytometry but were
not tested in primary tissues.
10
We proposed to take advantage of our work in click chemistry in
order to prepare sufficient peptide-dye conjugates using an enhanced method and tests our
conjugates in primary samples of beta pancreatic cells. Our peptide-dye conjugate will serve as the
azide partner and the exendin-4 functionalized with an alkyne will serve as our alkyne partner.
Results and Discussion:

Scheme 1.1 Synthetic Scheme for Azide Linkers
N
3 TAG
N
N
N
Exendin-4
TAG
Exendin-4
Copper(I) catalyzed azide-
alkyne cycloaddition
(CuAAC)
TAG
N O N
S
S
O
O
O
O
O NH
O
R
O
N
B
N
F F
O
HN
O
R
HO
O
OH
n = 2,3
Tosyl Chloride, Triethylamine
DCM, 0C to 20C
TsO
O
O
n = 2,3
S
O
O
Sodium Azide
DMF, 90C
N
3
O
N
3
n = 2,3
1. PPh
3
, THF, 20C
2. H
2
O, 20C
N
3
O
NH
2
n = 2,3
4
We initially proposed starting with tetraethylene glycol and triethylene glycol and tosylating
both sides of the molecule using tosyl chloride and triethylamine as the base. These reactions were
complete in yields of the 90% or above and we progressed to the next step of substituting the tosyl
groups with azides. We subjected the ditosylated materials to direct displacement with sodium azide
in DMF at 90
o
C to get our diazide materials in 90% yield or higher. In the next step, the selective
reduction of only one of the azides proved to be an issue of isolation. While we could reduce one of
the azides, the separation of the reduced amine using traditional flash chromatography proved to be
difficult. We were able to separate the spent triphenylphosphine oxide from the product by flushing
the column with ethyl acetate; however, the amine product would not move from the baseline of the
column. By changing the solvent system from ethyl acetate in hexanes to 50% acetone in methanol,
we were able to separate the product from the baseline and isolate it – however the isolated material
was not the free base amine, but an imine formed on the column between the condensation of the
amine and acetone in the elutant. Despite this unexpected chemistry in the purification, we were able
to hydrolyze the imine using distilled water on a rotary evaporator to recover the free amine.

Scheme 1.2 Synthesis of BODIPY-Linker Conjugate

H
2
N
O
O
N
3
O
N
B
N
F F
O
HN
O
NH
O
O
N
3
O
N B
N
F
F
O
N
H
O O
N
O
O
NEt
3
,
MeOH, r.t., 3 hrs, 43.7%
5

Attachment of linker to dye
Once we had access to the linkers, we began to attach the dyes to the linkers. The dyes had
NHS esters that in the presence of an amine moiety would quickly form the amide bond. We
performed our reactions using Texas Red-X, succinimidyl ester (ThermoFisher, T6134) and
BODIPY TMR-X NHS ester (succinimidyl ester, ThermoFisher, D6117) with both of our linkers.
Our first attempts to purify the dye-linker conjugates required the use of the Prep HPLC and in the
process significantly reduced our yield of the dye-linker conjugates. In the end we turned to normal
phase column chromatography using 10% methanol in dichloromethane to purify our dye-linkers to
acceptable yields.

Conclusions:
We have synthesized BODIPY and Texas-Red dye-linkers for use to couple with alkyne
functionalized Exendin-4. After performing the copper-catalyzed alkyne azide cycloaddition, we
have been able to make peptide that with the required dye conjugates. Our next steps are to purify
the materials on the prep HPLC and characterize them. Once complete they will be available for
testing and isolating beta pancreatic cells in heterogenous cell populations.

6
General Procedures:
All reactions were carried out under a nitrogen atmosphere with dry solvents under
anhydrous conditions, unless otherwise noted. Tetrahydrofuran (THF) was purchased from Fisher
and dried using a lab installed benzophenone-sodium still before use. ACS reagent grade toluene was
purchased from Fisher, ACS reagent grade diethyl ether (Et 2O) was purchased from Fisher, DriSolv
dimethylformamide (DMF) was purchased from Fisher, DriSolv methanol (MeOH) was purchased
from Fisher, and DriSolv dichloromethane (DCM) was purchased from Fisher and used without
further purification. Triethylene glycol and tetraethylene glycol were purchased from Sigma Aldrich
and used without any further purification. 4-toluenesulfonyl chloride was purchased from Sigma
Aldrich and was recrystallized from water before use. Triphenylphosphine was purchased from
Sigma Aldrich and used without further purification. Crystalline sodium azide was purchased from
Fisher (S2271-100) and used without any further purification. PBS buffer solution (Gibco 10010072)
was purchased from Fisher Scientific and used without any further purification. Texas Red-X,
succinimidyl ester (ThermoFisher, T6134) and BODIPY TMR-X NHS ester (succinimidyl ester,
ThermoFisher, D6117) were purchased from ThermoFisher and used without any further
purification. Yields refer to chromatographically and spectroscopically (
1
H NMR) homogenous
materials, unless otherwise stated. Reactions were monitored both by thin-layer chromatography
(TLC) carried out on 0.2 mm E. Merck silica gel plates (60F-254) using UV light as visualizing agent
and by Agilent LC/MSD. E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash
column chromatography. NMR spectra were recorded either on Varian Mercury 400, Varian
400MR, Varian VNMRS 500, or Varian VNMRS 600 and calibrated using residual undeutrated
solvent (CDCl 3: 𝛿 H = 7.26 ppm, 𝛿 C = 77.16 ppm; acetone-d6: 𝛿 H = 2.05 ppm, 𝛿 C = 29.92 ppm) as an
internal reference. The following abbreviations were used to designate the multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, br = broad.
7
Experimentals
((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate)

The ditosylate was obtained according to a literature procedure with slight modifications.
11

In a reaction flask with a stir bar, tetraethylene glycol (1.0 g, 5.15 mmol, 1.0 eq) was added under
nitrogen. After addition of tetraethylene glycol, dichloromethane (10.4 mL) was added under
nitrogen and allowed to stir for 10 minutes. After 10 minutes, the reaction flask was then cooled to -
10
o
C in a sodium chloride-ice/water bath for 10 minutes. After 10 minutes, 4-toluenesulfonyl
chloride (2.16 g, 11.33 mmol, 2.2 eq) was added in one portion and allowed to stir for 5 minutes.
After 5 minutes, triethylamine (1.58 mL, 11.33 mmol, 2.2 eq) was added dropwise over 2 minutes to
the reaction flask. Once addition was complete, the reaction was allowed to stir for 16 hours at room
temperature. After 16 hours, the reaction was quenched with addition of saturated ammonium
chloride aqueous solution (10 mL) and was allowed to stir for 20 minutes at room temperature.
After 20 minutes, the mixture was diluted with dichloromethane (20 mL) and transferred to a
separatory funnel. The organic layer was separated, and the aqueous layer was extracted with
dichloromethane (10 mL) three times in a separatory funnel. The organic layers were combined in a
separatory funnel and then was washed with saturated ammonium chloride aqueous solution (10
mL) three times, then washed with saturated sodium bicarbonate aqueous solution three times (10
mL), and then washed once with saturated sodium chloride aqueous solution (10 mL). The
combined organic layers were then dried with magnesium sulfate and allowed to stand for 20
minutes. The mixture was filtered through filter paper on a Büchner funnel, and the filtrate was
collected into a round bottom flask. The round bottom flask was put on a rotary evaporator and the
dichloromethane was evaporated under reduced pressure. The crude product was then purified on a
O
O
O
O O
S
O
O
S
O
O
8
silica gel column using 40% ethyl acetate in hexanes to get the titled product (2.59g, 100%).
1
H NMR
(400 MHz, Chloroform-d) δ 7.79 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.18 – 4.13 (m, 2H),
3.71 – 3.65 (m, 2H), 3.56 (s, 4H), 2.44 (s, 3H).
13
C NMR (101 MHz, cdcl 3) δ 144.93, 133.10, 129.94,
128.06, 70.83, 70.65, 69.37, 68.78, 21.73.
1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane

`
The diazide was obtained according to a literature procedure with slight modifications.
11
In a
reaction flask with a stir bar, ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) (1.0 g, 1.99 mmol, 1.0 eq) was added under nitrogen. After addition, DMF
(10 mL) was added under nitrogen and allowed to stir for 10 minutes. After 10 minutes, sodium
azide (259 mg, 3.98 mmol, 2.0 eq) was dissolved in distilled water (1 mL) and then added dropwise
over 2 minutes to the reaction flask. After addition, the reaction was allowed to stir at 90
o
C for 3
hours. After 3 hours, the reaction flask was allowed to cool to room temperature for 20 minutes.
Once the reaction flask was cooled, distilled water (50 mL), saturated sodium bicarbonate aqueous
solution (25 mL), and saturated sodium chloride aqueous solution (25 mL) was added to the reaction
flask and allowed to stir for 10 minutes. After 10 minutes, the mixture was transferred to a
separatory funnel and extracted with diethyl ether (25 mL) three times. After extraction, the organic
layers were combined in a separatory funnel and then washed with saturated sodium chloride
aqueous solution (10 mL). The combined organic layers were then dried with magnesium sulfate and
allowed to stand for 20 minutes. The mixture was filtered through filter paper on a Büchner funnel,
and the filtrate was collected into a round bottom flask. The round bottom flask was put on a rotary
evaporator and the diethyl ether was evaporated under reduced pressure. The crude product was
then purified on a silica gel column using 45% ethyl acetate in hexanes to give titled product (458.7
O
O
O
N
3
N
3
9
mg, 94.4%).
1
H NMR (400 MHz, chloroform-d) δ 3.72 – 3.63 (m, 12H), 3.39 (t, J = 5.1 Hz, 4H).
13
C
NMR (101 MHz, cdcl 3) δ 70.86, 70.85, 70.18, 50.83.
2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine

The product was obtained according to a literature procedure with slight modifications.
11
In
a reaction flask with a magnetic stir bar, 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane (2.23
g, 9.12 mmol, 1.0 eq) was added under nitrogen. After addition, THF (90 mL, 0.1 M) from a
benzophenone-sodium still was added under nitrogen and allowed to stir for 10 minutes. The
reaction flask was then cooled to 0
o
C in an ice/water bath for 20 minutes. After 20 minutes,
triphenylphosphine (2.28 g, 8.21 mmol, 0.9 eq) was dissolved in THF (5 mL) and added dropwise
over 5 minutes to the reaction flask. After complete addition, the reaction flask was allowed to stir
for 5 hours at 50
o
C. After 5 hours, distilled water (1 mL) was added dropwise over 5 minutes to the
reaction flask. After complete addition, the reaction flask was allowed to stir at 50
o
C for 12 hours.
After 12 hours, the reaction mixture was cooled to room temperature for 20 minutes. After 20
minutes, the reaction flask was transferred to a rotary evaporator and the THF was evaporated
under reduced pressure. The crude product was then transferred to a silica gel column and the
column was flushed with 100% ethyl acetate to remove triphenylphosphine oxide -- Rf (100% ethyl
acetate): 0.35. Once all triphenylphosphine oxide has been removed, the solvent system was
switched to 1:1 acetone/methanol and the product was collected off the column as an imine
condensation product of acetone and the desired amine. All column fractions with imine product
were pooled into a round bottom flask, then transferred to a rotary evaporator, and the methanol
and acetone was evaporated under reduced pressure. After evaporation of organic solvents, distilled
O
O
O
N
3
NH
2
10
water (10 mL) was added to the flask and the flask was transferred to a rotary evaporator, and the
water was removed under reduced pressure. After evaporation of water, distilled water (10 mL) was
added to the flask and the evaporation process was repeated three times on the rotary evaporator to
give the titled product as a clear oil (1.84 g, 92%).
1
H NMR (400 MHz, cdcl 3) δ 3.63 (s, 8H), 3.47 (s,
2H), 3.35 (s, 1H), 2.82 (s, 2H), 1.53 (s, 2H).
13
C NMR (101 MHz, cdcl 3) δ 70.81, 70.76, 70.73, 70.37,
70.14, 50.80.
(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate)

The product was obtained according to literature procedure with slight modifications.
12
In a
reaction flask with a magnetic stir bar, triethylene glycol (2.0 g, 13.3 mmol, 1.0 eq) was added under
nitrogen. After addition, dichloromethane (133 mL, 0.1 M) was added under nitrogen and allowed to
stir for 5 minutes. After 5 minutes, the reaction flask was then cooled to -10
o
C in a sodium
chloride-ice/water bath for 20 minutes. After 20 minutes, 4-toluenesulfonyl chloride (5.59 g, 29.3
mmol, 2.2 eq) was added in one portion to the reaction flask and allowed to stir for 10 minutes.
After 10 minutes, triethylamine (4.09 mL, 29.3 mmol, 2.2 eq) was added dropwise over 2 minutes to
the reaction flask. Once addition was complete, the reaction was allowed to stir for 4 hours at room
temperature. After 4 hours, the reaction was quenched with addition of saturated ammonium
chloride aqueous solution (10 mL) and was allowed to stir for 20 minutes. After 20 minutes, the
mixture was diluted with dichloromethane (20 mL) and transferred to a separatory funnel. The
organic layer was separated, and the aqueous layer was extracted with dichloromethane (10 mL)
three times in a separatory funnel. The organic layers were combined in a separatory funnel and then
O
O
O
O
S
S
O
O
O O
11
was washed with saturated ammonium chloride aqueous solution (10 mL) three times, then washed
with saturated sodium bicarbonate aqueous solution three times (10 mL), and then washed once
with saturated sodium chloride aqueous solution (10 mL). The combined organic layers were then
dried with magnesium sulfate and allowed to stand for 20 minutes. After 20 minutes, the mixture
was filtered through filter paper on a Büchner funnel, and the filtrate was collected into a round
bottom flask. The round bottom flask was put on a rotary evaporator and the dichloromethane was
evaporated under reduced pressure. The crude product was then purified on a silica gel column
using 40% ethyl acetate in hexanes give the titled product as a white solid (4.22 g, 68.7%).
1
H NMR
(400 MHz, cdcl 3) δ 7.78 (d, J = 8.3 Hz, 4H), 7.33 (d, J = 8.0 Hz, 4H), 4.13 (s, 4H), 3.66 (s, 4H), 3.52
(s, 4H), 2.44 (s, 6H).
13
C NMR (101 MHz, cdcl 3) δ 144.99, 133.09, 130.36, 129.97, 128.07, 127.17,
70.80, 69.33, 68.85, 21.75.
1,2-bis(2-azidoethoxy)ethane

The product was obtained according to a literature procedure with slight modifications.
12
In
a reaction flask equipped with a magnetic stir bar, (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) (4.22 g, 9.20 mmol, 1.0 eq) was added under nitrogen. After addition,
DMF (92 mL, 0.1 M) was added under nitrogen and allowed to stir for 10 minutes. After 10
minutes, sodium azide (259 mg, 3.98 mmol, 2.0 eq) was dissolved in distilled water (1 mL) and then
added dropwise over 2 minutes to the reaction flask. After addition, the reaction was allowed to at
90
o
C for 24 hours. After 24 hours, the reaction flask was allowed to cool to room temperature for
20 minutes. Once the reaction flask was cooled, distilled water (50 mL), saturated sodium
bicarbonate aqueous solution (25 mL), and saturated sodium chloride aqueous solution (25 mL) was
added to the reaction flask and allowed to stir for 10 minutes. After 10 minutes, the mixture was
transferred to a separatory funnel and extracted with diethyl ether (25 mL) three times. After
N
3
O
O
N
3
12
extraction, the organic layers were combined in a separatory funnel and then washed with saturated
sodium chloride aqueous solution (10 mL). The combined organic layers were then dried with
magnesium sulfate and allowed to stand for 20 minutes. The mixture was filtered through filter
paper on a Büchner funnel, and the filtrate was collected into a round bottom flask. The round
bottom flask was put on a rotary evaporator and the diethyl ether was evaporated under reduced
pressure. The crude product was then purified a silica gel column using 40% ethyl acetate in hexanes
to give the titled product as a clear oil (1.32 g, 72%).
1
H NMR (400 MHz, cdcl 3) δ 3.71 – 3.65 (m,
8H), 3.43 – 3.35 (m, 4H).
13
C NMR (101 MHz, cdcl 3) δ 77.49, 77.16, 76.84, 70.86, 70.26, 50.81.
2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine

The product was obtained according to a literature procedure with slight modifications.
12
In
a reaction flask equipped with a magnetic stir bar, 1,2-bis(2-azidoethoxy)ethane (787 mg, 3.93 mmol,
1.0 eq) was added under nitrogen. After addition, THF (39 mL, 0.1 M) from a benzophenone-
sodium still was added under nitrogen and allowed to stir for 10 minutes. The reaction flask was
then cooled to 0
o
C in an ice bath for 20 minutes. After cooling, triphenylphosphine (228 mg, 3.54
mmol, 0.9 eq) was dissolved in THF (5 mL) and added dropwise over 5 minutes to the reaction
flask. After complete addition, the reaction flask was allowed to stir at 50
o
C for 5 hours. After 5
hours, distilled water (1 mL) was added to the reaction flask and allowed to stir at 50
o
C for 12
hours. After 12 hours, the reaction mixture was cooled to room temperature for 20 minutes. After
20 minutes, the reaction flask was transferred to a rotary evaporator and the THF was evaporated
under reduced pressure. The crude product was then transferred to a silica gel column and the
column was flushed with 100% ethyl acetate to remove triphenylphosphine oxide -- Rf (100% ethyl
acetate): 0.35. Once all triphenylphosphine oxide has been removed, the solvent system was
switched to 1:1 acetone/methanol and the product was collected off the column as an imine
O
N
3
O
NH
2
13
condensation product of acetone and the desired amine. All column fractions with imine product
were pooled into a round bottom flask, then transferred to a rotary evaporator, and the methanol
and acetone was evaporated under reduced pressure. After evaporation of organic solvents, distilled
water (10 mL) was added to the flask and the flask was transferred to a rotary evaporator, and the
water was removed under reduced pressure. After evaporation of water, distilled water (10 mL) was
added to the flask and this process was repeated three times on the rotary evaporator to give the
titled product as a clear oil (479.9 mg, 70%).
1
H NMR (400 MHz, cdcl 3) δ 3.71 – 3.57 (m, 6H), 3.50
(t, J = 5.4 Hz, 2H), 3.41 – 3.34 (m, 2H), 2.85 (t, J = 5.2 Hz, 2H), 1.56 (s, 3H).
13
C NMR (101 MHz,
cdcl 3) δ 73.65, 70.77, 70.42, 70.16, 50.78, 41.88.
N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-
5H-4λ
4
,5λ
4
-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-yl)propanamido)hexanamide

The product was obtained according to a literature procedure with slight modifications.
10
In
a reaction flask equipped with a magnetic stir bar, 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine (14.32
mg, 0.082 mmol, 10 eq) was added under nitrogen. After addition, methanol (2 mL, 0.04 M) was
added under nitrogen and allowed to stir for 5 minutes. After 5 minutes, triethylamine (5.7 μL, 0.04
mmol, 5 eq) is added in one portion under nitrogen and stirred for 1 minute. After addition,
O
N
B
N
F F
O
HN
O
NH
O
O
N
3
14
BODIPY-TMR-X NHS ester (5 mg, 0.008 mmol, 1 eq, Invitrogen: D6117) is added in one portion
under nitrogen. After addition, the reaction flask was covered in aluminum foil and was allowed to
stir for 3 hours at room temperature. After 3 hours, the reaction flask was transferred to a rotary
evaporator and the methanol was evaporated under reduced pressure. The crude product was then
purified on a silica gel column using 10% MeOH in DCM to give the titled product as a purple film
(2.4 mg, 43.7%).
1
H NMR (400 MHz, cdcl 3) δ 7.87 (d, J = 9.1 Hz, 2H), 7.09 (s, 1H), 7.00 – 6.94 (m,
3H), 6.54 (d, J = 4.2 Hz, 1H), 3.86 (s, 3H), 3.63 (s, 2H), 3.56 (s, 3H), 3.51 (s, 2H), 3.44 (s, 3H), 3.34
(s, 2H), 2.76 (t, J = 7.6 Hz, 2H), 2.53 (s, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.21 (s, 2H), 2.17 (s, 1H), 2.09
(s, 1H), 1.25 (s, 9H), 0.88 (s, 2H).
Copper Click Reaction for Dye to Peptide:
Stock Solution Preparation:
In a 2 mL Eppendorf tube labeled “stock ligand mixture”, TTTA (12 mg, 0.1 mmol) and
THPTA (12 mg, 0.1 mmol) was added and dissolved in distilled water (1 mL). In a 2 mL Eppendorf
tube labeled “stock copper sulfate”, copper sulfate pentahydrate (16 mg, 0.1 mmol) was added and
dissolved in distilled water (1 mL). In a 2 mL Eppendorf tube labelled “stock aminoguanidine”,
aminoguanidine (11 mg, 0.1 mmol) was added and dissolved in distilled water (1 mL). In a 2 mL
Eppendorf tube, sodium ascorbate (20 mg, 0.1 mmol) was added and dissolved in distilled water (1
mL).
Premixing of Copper Catalyst:
In a 200 μL PCR tube, 25 μL from the “stock copper sulfate” solution and 125 μL from the
“stock ligand mixture” solution was added. After addition, the PCR tube was vortexed for 20
seconds and the solution turned a deep blue color. Once the deep blue color has persisted, the
catalyst was ready for use.

15
Cu(I)-catalyzed azide-alkyne cycloaddition Protocol:
In a 2 mL Eppendorf tube, reagents were added in the following order:
1. 25 μL of azide dye label (1.2 eq, 240 μM) in DMSO
2. 400 μL of PBS buffer solution
3. 10 μL of exendin-4 alkyne peptide (1.0 eq, 200 μM) in distilled water
4. 25 μL of aminoguanidine stock
5. 25 μL of sodium ascorbate stock
6. 15 μL of premixed copper sulfate and ligand stock
Once all the solutions were added, the solution was mixed using a 1 mL micropipette to pipette the
solution up and down the pipette tip for 10 seconds. Once mixed, the Eppendorf tube was covered
with foil and allowed to react at room temperature for 1 hour. After 1 hour, methanol (705 μL),
chloroform (617 μL), and distilled water (176 μL) was added to the Eppendorf tube. After addition,
the Eppendorf tube was centrifuged at 5000 rpm for 15 minutes at 4
o
C. After centrifugation, a
protein disc was situated between an aqueous top layer and an organic bottom layer. The aqueous
layer and organic layer were removed using a micropipette. After removal, methanol (500 μL),
chloroform (150 μL), and distilled water (600 μL) was added to the Eppendorf tube and vortexed
for 5 minutes. After 5 minutes, the tube was centrifuged at 14000 rpm for 10 minutes at 4
o
C. After 5
minutes, a protein disc was situated between an aqueous top layer and an organic bottom layer. The
aqueous layer and organic layer were removed using a micropipette. The Eppendorf tube with the
protein disc was turned upside down on a paper towel and allowed to dry for 1 hour at room
temperature. After 1 hour, the Eppendorf tube was closed and transferred to a -20
o
C freezer before
purification on an HPLC.
16
Representative Spectra

Figure 1.3
1
H NMR Spectrum of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)
bis(4-methylbenzenesulfonate)
17

Figure 1.4
13
C NMR Spectrum of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)
bis(4-methylbenzenesulfonate)
18

Figure 1.5
1
H NMR Spectrum of 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane
19

Figure 1.6
13
C NMR Spectrum of 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane
20

Figure 1.7
1
H NMR Spectrum of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine
21

Figure 1.8
13
C NMR Spectrum of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine
22

Figure 1.9
1
H NMR Spectrum of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate)
23

Figure 1.10
13
C NMR Spectrum of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate)
24

Figure 1.11
1
H NMR Spectrum of 1,2-bis(2-azidoethoxy)ethane
25

Figure 1.12
13
C NMR Spectrum of 1,2-bis(2-azidoethoxy)ethane
26

Figure 1.13
1
H NMR Spectrum of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine
27

Figure 1.14
13
C NMR Spectrum of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine
28

Figure 1.15
1
H NMR Spectrum of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-difluoro-7-
(4-methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-
yl)propanamido)hexanamide

29

Figure 1.16 g-COSY NMR Spectrum of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-
difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-
f][1,3,2]diazaborinin-2-yl)propanamido)hexanamide

30

Figure 1.17 NMR assignments table of N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-6-(3-(5,5-
difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-
f][1,3,2]diazaborinin-2-yl)propanamido)hexanamide

31
References:

1. Neidigh, J. W.; Fesinmeyer, R. M.; Prickett, K. S.; Andersen, N. H., Exendin-4 and
Glucagon-like-peptide-1: NMR Structural Comparisons in the Solution and Micelle-Associated
States. Biochemistry 2001, 40 (44), 13188-13200.

2. Underwood, C. R.; Garibay, P.; Knudsen, L. B.; Hastrup, S.; Peters, G. H.; Rudolph, R.;
Reedtz-Runge, S., Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular
Domain of the Glucagon-like Peptide-1 Receptor*. Journal of Biological Chemistry 2010, 285 (1), 723-
730.

3. Lee, Y.; Berglund, E. D.; Yu, X.; Wang, M.-Y.; Evans, M. R.; Scherer, P. E.; Holland, W. L.;
Charron, M. J.; Roth, M. G.; Unger, R. H., Hyperglycemia in rodent models of type 2 diabetes
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13217-13222.

4. Graaf, C. d.; Donnelly, D.; Wootten, D.; Lau, J.; Sexton, P. M.; Miller, L. J.; Ahn, J.-M.; Liao,
J.; Fletcher, M. M.; Yang, D.; Brown, A. J. H.; Zhou, C.; Deng, J.; Wang, M.-W., Glucagon-Like
Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes.
Pharmacological Reviews 2016, 68 (4), 954-1013.

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development of the glucagon-like peptides. The Journal of Clinical Investigation 2017, 127 (12), 4217-
4227.

6. Wang, M.-y.; Yu, X.; Lee, Y.; McCorkle, S. K.; Chen, S.; Li, J.; Wang, Z. V.; Davidson, J. A.;
Scherer, P. E.; Holland, W. L.; Unger, R. H.; Roth, M. G., Dapagliflozin suppresses glucagon
signaling in rodent models of diabetes. Proceedings of the National Academy of Sciences 2017, 114 (25),
6611-6616.

7. Evers, A.; Haack, T.; Lorenz, M.; Bossart, M.; Elvert, R.; Henkel, B.; Stengelin, S.; Kurz, M.;
Glien, M.; Dudda, A.; Lorenz, K.; Kadereit, D.; Wagner, M., Design of Novel Exendin-Based Dual
Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists. Journal of Medicinal Chemistry 2017, 60
(10), 4293-4303.

8. Andersen, A.; Lund, A.; Knop, F. K.; Vilsbøll, T., Glucagon-like peptide 1 in health and
disease. Nature Reviews Endocrinology 2018, 14 (7), 390-403.

32
9. Drucker, D. J., Advances in oral peptide therapeutics. Nature Reviews Drug Discovery 2020, 19
(4), 277-289.

10. Clardy, S. M.; Keliher, E. J.; Mohan, J. F.; Sebas, M.; Benoist, C.; Mathis, D.; Weissleder, R.,
Fluorescent Exendin-4 Derivatives for Pancreatic β-Cell Analysis. Bioconjugate Chemistry 2014, 25 (1),
171-177.

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Pattern Gold Substrates. Angewandte Chemie International Edition 2012, 51 (9), 2151-2154.

12. Budin , G.; Dimala , M. M.; Lamour, V.; Oudet, P.; Mioskowski, C.; Meunier, S.; Brino, L.;
Wagner, A., A Chemical Labeling Strategy for Proteomics under Nondenaturing Conditions.
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13. Neidigh, J. W.; Fesinmeyer, R. M.; Prickett, K. S.; Andersen, N. H., Exendin-4 and
Glucagon-like-peptide-1: NMR Structural Comparisons in the Solution and Micelle-Associated
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33
Chapter 2 - ELISA development and detection of SARS-CoVID-19
Antibodies
Introduction
“The pandemic which has just swept round the earth has been without precedent. There
have been more deadly epidemics, but they have been more circumscribed; there have been
epidemics almost as widespread, but they have been less deadly. Floods, famines, earthquakes and
volcanic eruptions have all written their stories in terms of human destruction almost too terrible for
comprehension, yet never before has there been a catastrophe at once so sudden, so devastating and
so universal.
The most astonishing thing about the pandemic was the complete mystery which
surrounded it. Nobody seemed to know what the disease was, where it came from or how to stop it.
Anxious minds are inquiring today whether another wave of it will come again.”
13

That quote by Major George A. Soper is not in reference to the SARS-CoVID-19 pandemic
but rather to the Spanish Flu of 1918. In the span of 100 years, the tools of science and medicine
have drastically advanced and have proved new ways of understanding the underlying mechanisms
of new diseases. Few events in human history have drastically and fundamentally changed the way
that human society functions. The SARS-CoVID-19 pandemic is one of them.
In late 2019, the SARS-CoVID-19 virus originated in Wuhan, China
14
and it subsequently
spread across the world resulting in shutting down of international borders, the rise of stay at home
orders, and a race by the scientific community to understand the progression of disease and its
treatments.
15-16
To this end, we wanted to contribute to the understanding of the coronavirus by
designing an ELISA diagnostic tool that would be able to detect antibodies in serum and saliva. To
achieve this goal, we looked to the previous diagnostic work based on detection of the HIV virus
17-18
,
dengue virus
19
, giardia duodenalis
20
, pneumococcal infection
21
, trypanosoma cruzi
22
, porcine delta
coronavirus
23
, and previous iterations of the SARS-CoVID virus
24
from 2003. In addition, detection
of IgA and IgG antibodies in tears and saliva has been established since the 1960s – albeit at much
much lower concentrations than in serum (0.4 – 16 mg/mL).
25-27

34
Building upon the work with other ELISA kits
28
and the developing literature on antibodies
for SARS-CoVID-19
16, 29
, we focused on developing an IgG antibody test utilizing the RBD
fragment as the basis of our ELISA assay. The RBD fragment was chosen over the N and S protein
of the SARS-CoVID-19 virus because we hypothesized that the RBD segment was central to the
ability of the virus to bind to the ACE-2 receptor and gain entry to the cell.
30
We parameterized the
following: coating buffer, fragment concentration, sample type, sample dilution, antibody dilution,
TMB substrate, and diluent buffer using a design of experiments approach. The ELISA was tested
against a consistent panel of patient samples: 6 positives (positive by PCR and by other commercial
antibody tests), 3 negatives (negative by PCR and by other commercial antibody tests), and two
samples purchased before the advent of SARS-CoVID-19. The following work demonstrates our
progress towards the detection of IgG, IgA, IgM SARS-CoVID-19 antibodies in serum and salvia.

35

Results and Discussion:

Figure 2.1 RBD Fragment Concentration Series in PBSA Buffer

Figure 2.2 RBD Fragment Concentration Series in Bicarbonate Buffer

Optimization of ELISA with RBD in Serum
Our first goal was to determine the concentration of RBD and buffer used to for our assay.
We prepared two plates with varying concentrations of RBD (0.3 µg /mL; 0.6 µg /mL; 0.9 µg /mL;
1.2 µg /mL; 1.5 µg /mL; 1.8 µg /mL; 2.1 µg /mL) in two different buffers (PBSA at pH 7.2 and
Bicarbonate at pH 9.6). The following parameters were held constant in the experiment: sample
dilution (1:50), antibody (IgG), antibody dilution (1:500), and TMB substrate. The positive samples
were A0006, A0035, A0040, A0044, A0054, and A0055; these are positive by PCR and by antibody
from other kits. The negative samples were A0088, A0091, A0096; these are negative by PCR and by
antibody from other kits. The other two samples were CU170904-108-1722017B-N-21 and
36
CU170904-108-1722017B-N-18 -- these negative serum samples that were purchased and collected
before the advent of SARS-CoVID-19. From the data, there was a good cutoff for the fragment
concentration around 0.3 µg /mL and 0.6 µg/mL. In addition, the data also showed that RBD in
PBSA buffer (Figure 2.1) was more consistent across RBD fragment concentration in comparison to
RBD in the bicarbonate buffer (Figure 2.2). There was a concern with the non-specific reactivity of
sample CU170904-108-1722017B-N-18 in the ELISA and we varied the serum concentration and
the secondary antibody concentration to address the non-specific reactivity.

Figure 2.3 IgG Antibody Dilution Series for RBD at 0.3 µg /mL

Figure 2.4 IgG Antibody Dilution Series for RBD at 0.6 µg /mL
RBD 0.6 µg/mL
RBD 0.3 µg/mL
37

Figure 2.5 Sample Dilution Series for RBD 0.3 µg /mL

Figure 2.6 Sample Dilution Series for RBD 0.6 µg /mL
Our next goal was to reduce the non-specific signal seen in our ELISA that originated from
the negative serum sample CU170904-108-1722017B-N-18. We prepared two plates with varying
concentrations of RBD (0.3 µg /mL; 0.6 µg /mL) in PBSA at pH 7.2. The following parameters were
held constant in the experiment for each plate: fragment concentration (0.3 µg /mL or 0.6 µg /mL),
antibody (IgG), and the testing samples from the previous experiment. The same samples were used
for this experiment as before. From the data, increasing serum sample dilution from 1:25 to 1:100
decreases non-specific signal in negative serum. In addition, increasing antibody dilution from 1:500
RBD 0.6 µg/mL
RBD 0.3 µg/mL
38
to 1:2000 decreases non-specific signal in negative serum. With this insight, we moved to test a
combination of 1:100 sample dilution with 1:2000 IgG dilution into our protocol.

Figure 2.7 Serum Sample Dilution Series

Figure 2.8 Sample Dilution Series at 0.3 µg /mL RBD and IgG Concentration
RBD 0.3 µg/mL | 1:2000 IgG

1:25 (-) | 0.3 µg/mL
1:25 (-) | 0.6 µg/mL

1:25 (+) | 0.3 µg/mL
1:25 (+) | 0.6 µg/mL
1:50 (-) | 0.3 µg/mL
1:50 (-) | 0.6 µg/mL
1:50 (+) | 0.3 µg/mL
1:50 (+) | 0.6 µg/mL
1:100 (-) | 0.3 µg/mL
1:100(-) | 0.6 µg/mL
1:100(+) | 0.3 µg/mL
1:100(+) | 0.6 µg/mL
39

Figure 2.9 Sample Dilution Series at 0.6 µg /mL and IgG Concentration

Figure 2.10 Antibody Dilution Series at RBD 0.3 µg /mL and 1:25 Serum Dilution
RBD 0.3 µg/mL | 1:25 Serum Dilution
RBD 0.6 µg/mL | 1:2000 IgG
40

Figure 2.11 Antibody Dilution Series at RBD 0.6 µg /mL and 1:25 Serum Dilution

Our next goal was to test the combination of 1:100 sample dilution and 1:2000 IgG dilution
for our protocol. To eliminate the possibility that decreasing the non-specific signal was with IgG
dilution or sample dilution, we performed an IgG dilution of 1:2000 to 1:25000 and a sample
dilution of 1:25 to 1:100. We prepared two plates with varying concentrations of RBD (0.3 µg /mL;
0.6 µg /mL) in PBSA at pH 7.2. The following parameters were held constant in the experiment for
each plate: fragment concentration (0.3 µg /mL or 0.6 µg /mL), antibody (IgG), TMB, and the
testing set from the previous experiment. All the experiments were run using human serum. In
Figure 2.10, the dilution of the IgG antibody from 1:2000 to 1:25000 showed a decrease in the non-
specific signal from CU170904-108-1722017B-N-18; however, it also demonstrated a decrease in the
overall signals of the positives. In Figure 2.8 and Figure 2.9, dilution of the sample from 1:25 to
1:100 showed a decrease in the non-specific signal from sample CU170904-108-1722017B-N-18. In
Figure 2.7, when combining the dilution and antibody conditions, we saw that the non-specific
negative signal was eliminated at antibody dilution 1:2000 and sample dilution of 1:100
RBD 0.6 µg/mL | 1:25 Serum Dilution
41

Figure 2.12 Paired Serum and Oral Samples on RBD 0.3 µg /mL and 1:2000 IgG

Figure 2.13 Serum Sample Diluted in Negative Saliva Series on RBD 0.3 µg/mL and 1:2000
IgG
RBD 0.3 µg/mL | 1:2000 IgG
RBD 0.3 µg/mL | 1:2000 IgG
42
After we eliminated the non-specific signals in the negative serum samples, we turned our
attention to expanding our sample panel and testing oral fluid samples. To that end, we prepared
two plates of RBD at 0.3 µg /mL in PBSA at pH 7.2. The panel was expanded to include 60 samples
(29 positive samples by PCR and antibody & 31 negative samples by PCR and antibody). These 60
samples composed of one undiluted saliva sample collected by the OraSure oral fluid collection
device and one serum sample diluted to 1:100 in diluent buffer A. After we performed our ELISA
using these samples, the data in Figure 2.12 appeared inconsistent -- namely that positive PCR
samples were not resulting in positive antibody detection in oral samples. In addition, positive PCR
samples were also not resulting in positive antibody detection in serum samples. It was possible that
the assay was detecting false positive and false negatives, at which point we would need to retool our
protocol, or that the PCR status of a sample was assigned incorrectly from the beginning, or the
patients exposed to the SARS-CoVID-19 failed to seroconvert. Regarding the assay not working, the
results from the training set does not support this conclusion – our data showed well separated
positive and negative populations. The assay data could result from detecting IgG non-specifically
however this would have been seen in the training population and was not the case. We could not
discount the possibility that individuals exposed to SARS-CoVID-19 did not seroconvert and
therefore their antibody levels are undetectable. Further, we could not discount the possibility that
some of the patient samples were not assigned the correct PCR status. Regarding the oral samples,
this assay was not optimized for oral samples and must be developed on its own. (Figure 2.12)
In addition, a spike-in experiment was performed using the 11 samples previously chosen as
a testing set. All the samples in the testing panel were prepared to their desired dilution (1:100,
1:1250, 1:500, 1:1000, 1:2000) using negative saliva collected using the OraSure oral fluid collection
device -- i.e., to make up a 1:100 would be 1μL of serum into 99 μL of negative saliva. The dilutions
were then added directly into the wells and the protocol was followed without any changes. In
43
Figure 2.13, we were able to detect antibodies in the spike-in experiment at dilutions of 1:100, 1:250,
and 1:500 positive antibody serum in negative saliva. This experiment suggested that SARS-CoVID-
19 antibodies could be detected in saliva samples.

Figure 2.14 Serum IgG detection across 90 random clinical samples

Figure 2.15 Serum Panel Samples by PCR Status

RBD 0.3 µg/mL | 1:100 Serum | 1:2000 IgG
RBD 0.3 µg/mL | 1:100 Serum | 1:2000 IgG
44
To address the concerns with the assay, we expanded our clinical sample and performed the
assay on samples with multiple timepoints. To eliminate any origin of human error, we programmed
a GSD’s thunderbolt analyzer machine in order to automate the ELISA assay using serum. In Figure
2.14, all the serum samples were from the first time point -- the panel has 52 positive samples by
PCR and 38 negative samples by PCR. From start to finish, the assay took 2 hours. The data from
this sample set uncovered that several false positives (A0059, A0148, A0022, A0026, A0094, A0010,
A0013, A0045, A0034, A0087). For A0059, it was tested on the Euroimmun kit with IgG and
showed that was antibody positive but PCR negative. For samples A0148, A0022, A0026, A0094,
and A0034, they were tested using the Euroimmun kit against IgG and were determined to be
SARS-CoVID-19 antibody positive but PCR negative. Samples A0010, A0013, A0045, and A0087
were determined to be negative by either the GSD or the Euroimmun kits. Further, A0010 had the
highest detectable levels of antibodies. In the positive sample set, we have two false negatives
(A0049, A0018). For A0049, it was determined to be negative by the Euroimmun kit with IgG. For
A0018, it was determined to be positive by the GSD antibody kit. Our assay points that there was
disagreement about IgG antibodies for samples for Euroimmun and GSD commercial ELISA kits –
this may be explained by the difference in antigen. The Euroimmun ELISA kits use the SARS-
CoVID-19 S protein as their antigen while the GSD ELISA kits use the SARS-CoVID-19 N protein
as their antigen. In serum samples that were separated by PCR status (Figure 2.15), the data that the
assay can distinguish between positive and negative samples. These data showed that if samples have
always tested negative via PCR then they will also test negative for antibodies in the assay. If samples
have always tested positive via PCR, they will have detectable SARS-CoVID-19 antibody levels. If
samples have changed PCR status from positive to negative during the study, then their antibodies
can be detected however the levels within that subset of samples was not consistent – these data
45
suggested that antibody levels decline over time. While this assay and its procedure were able to
detect antibodies in serum, it must be optimized for detecting antibodies in saliva.

46

Optimization of ELISA for Saliva:

Figure 2.16 RBC Fragment Concentration Series at 1:500 IgG Concentration

Figure 2.17 RBC Fragment Concentration Series at 1:2000 IgG Concentration

After developing the RBD ELISA assay for serum samples, we turned our attention to
developing our assay to detect antibodies in saliva. To determine the concentration of RBD
fragment for oral samples, two plates of varying concentrations of RBD (0 µg /mL - 2.1 µg /mL) in
RBD Fragment Concentration (µg/mL)
RBD Fragment Concentration (µg/mL)
47
PBSA at pH 7.2 were made. The following parameters were held constant in the experiment for
each plate: antibody (IgG), and a new training set was selected. The following parameters were held
constant in the experiment for each plate: antibody (IgG) and mouthwash sample dilution (1:5). The
positive samples were A0035, A0044, A0055, A0119, A0121, and A0127; these were positive by
PCR. From these samples, we determined the following: In Figure 2.16, there were positive signals
around 1.2 µg /mL, meanwhile the negative samples were at undetectable levels. When comparing
Figure 2.16 and Figure 2.17, the data demonstrated that we needed to maintain at least a
concentration of 1:500 in IgG secondary antibody to detect antibodies in saliva.

Figure 2.18 IgG Antibody Dilution Series at 1.2 μg/mL of RBD and 1:5 dilution of
mouthwash samples
Our next goal was to determine the contribution that the IgG secondary antibody has on the
detection of antibodies in the oral samples. Figure 2.18 showed the series of dilutions for the
RBD 1.2 µg/mL | MW 1:5 Dilution
48
secondary antibody and we demonstrated that the best distinction between negatives and positives
samples was at secondary IgG concentrations of 1:100. Any concentration less would not be able to
distinguish between negative samples and positive samples.

Figure 2.19 Mouthwash sample dilution series on 1.2 µg/mL of RBD and 1:100 IgG
Our next experiment focused on how the dilution of the mouthwash samples affected the
detection of the antibodies. Figure 2.19 showed the series of dilutions for the oral samples and, from
the data, the best distinction between negatives and positives samples were at sample dilution 1:2 in
diluent buffer A.
RBD 1.2 µg/mL | 1:100 IgG
49

Figure 2.20 Expanded Mouthwash Panel at 1.2 µg /mL RBD, 1:5 MW dilution, and 1:100
IgG dilution

Figure 2.21 Mouthwash Panel Samples Split by PCR Status

RBD 1.2 µg/mL | 1:5 MW | 1:100 IgG
RBD 1.2 µg/mL | 1:2 MW | 1:100 IgG
50
After the assay showed a separation between the negative and positive mouthwash samples,
we were eager to expand our sampling from our test panel to a larger collection of oral samples.
When we expanded the panel to include more patient samples, we saw unexpected behavior in the
presentation of antibodies in saliva – specifically in Figure 2.20 that antibodies were not consistent
for samples subset by PCR status. For sample A0021, which has remained PCR negative, there were
two time points (2 and 4) that were separated by 23 days, the OD signal at the 2nd time point was
0.314 and the OD signal at the 4th time point was 0.11 – these data showed that antibodies levels
can drop over time. For sample A0111, the PCR status changed from positive to negative during the
interval of time points 2 and 3 (separated by 7 days); the OD signal for the 2nd time point is 3.86
and the OD signal for the 3rd time point was 3.85 – these data suggested that antibody levels can
remain constant after 7 days after a change of PCR status from positive to negative. In Figure 2.20,
the outliers in the always PCR (-) column was sample A0008. A0008 was odd because it tested highly
for IgG antibodies using oral samples however, when the A0008 sample was tested using serum –
the antibody levels in serum were at levels similar to negative samples. These data suggested that
there was a difference between SARS-CoVID-19 antibodies levels detected in serum and saliva.
Also, in Figure 2.20, oral samples of A0013 (OD: 1.2) and A0027 (OD: 1.5) tested high though they
have remained PCR negative the whole time – these data would suggest that these two samples were
exposed to SARS-CoVID-19 before the study and have maintained detectable levels of RBD
antibodies. We noted that as some samples have transitioned from PCR positive to PCR negative,
the signal for detected SARS-CoVID-19 antibodies dropped to levels on antibodies found in
samples never exposed to SARS-CoVID-19. These data would suggest a differentiation happening
in the clinical samples, namely that there exists a clinical population that maintains detectable levels
of RBD IgG antibodies and another population that after exposure would not maintain detectable
levels of RBD IgG antibodies in their serum or saliva. We hypothesized that detection of antibodies
51
in saliva decreases over time and thus antibodies level can return quickly to pre-exposure levels
because the patient does not seroconvert after exposure to SARS-CoVID-19. The possibility of the
patients not seroconverting was supported also by our earlier results with serum in Figure 2.15. In
Figure 2.15 and Figure 2.21, the serum and mouthwash results have the same pattern for PCR
status.

Figure 2.22 Comparison of Diluent Buffers with Mouthwash Samples
RBD 1.2 µg/mL | 1:5 MW | 1:100 IgG
52

Figure 2.23 Comparison of Diluent Buffer for Mouthwash Samples
We further wanted to optimize our assay by adjusting the composition of the diluent buffer
used in the assay. We hypothesized that the proteins and enzymes found in oral samples could be
interfering with the detection of IgG antibodies. We decided to change the components of the
diluent buffers for the samples in order to disrupt any unwanted binding to the ELISA plate. In the
course of our studies, we determined that our original buffer composition was the best for our
purposes. (Figure 2.22 and Figure 2.23). After buffer optimization, we decided to look into using
spin column to concentrate oral samples and use those samples in our assays. From that experiment,
RBD 1.2 µg/mL MW Diluent Buffer Comparison
53
the data showed that using an Amicon Ultra-0.5 mL centrifugal filters (100 kDa) increased
separation between negative and positive populations in clinical mouthwash samples. (Figure 2.24 ).
With this data in hand, we settled on a protocol for oral samples that required the following 1.2
µg/mL of RBD fragment, 1:100 dilution of IgG secondary antibody, and use of the Amicon spin
columns to clean up samples for the ELISA assay.

Figure 2.24 Spin Column Use Increased Separation Between Negative and Positive
Populations of Patient Samples
Conclusion:
In response to the SARS-CoVID-19 virus, we developed an ELISA assay that could detect
IgG antibodies in serum and saliva. In the process, we were able to determine that success of
antibody detection relied upon having the correct amount of virus antigen fragment for binding, the
correct dilution of sample, and use of a spin column to remove small proteins and peptides that
would interfere with antibody detection. Transforming a working serum antibody ELISA assay to a
working saliva antibody ELISA assay required an increase in antigen concentration and
concentration of saliva samples in order to reliably separate negative and positive antibody
populations. This protocol and approach not only provided a workflow for detecting IgG antibodies
but also provided a framework to quickly develop new diagnostic tools for the next emerging
pathogen. We hope that this will give others a head start.
RBD 1.2 µg/mL | MW | 1:100 IgG
54

Experimentals:
All reagents that are not listed were purchased Fisher Scientific and used without further
purification. Goat antihuman IgG-HRP (109-035-088), goat anti-human IgM-HRP (109-035-043),
and goat antihuman IgA-HRP (109-035-011) were purchased from Jackson ImmunoResearch labs.
TMB (TMBE-1000) was purchased from Moss bio. Immulon 2HB flat bottom microtiter plates
(62402-972) were purchased from VWR. Bovine Serum Albumin Fraction V (10735078001) was
purchased from Sigma Aldrich. SARS-CoV-2 Spike S1 RBD Protein (His-Tag), rec. (9552) was
purchased from Meridian Bio Science. Heat inactivated sterile goat serum (D104-10-0500) was
purchased from Rockland. Sodium chloride, citric acid, sodium benzoate, and potassium sorbate
were purchased from Sigma Aldrich and used without further purification. When working with
clinical samples, perform all assay steps in a BSL2 hood. All plates were read using a Promega
GloMax Plate reader in UV-Vis mode. A GSD thunderbolt analyzer was purchased from Gold
Standard Diagnostics for automating the ELISA assay. The GSD thunderbolt analyzer can prepare
samples and plates, wash assay plates, incubate at different temperatures, and perform readings of
the ELISA plates using an internal spectrophotometer for UV/Vis readings. Optimization programs
(“Optimization 4” and “Optimization 2”) were custom programs made by GSD’s software
engineers for running our assay on the GSD thunderbolt analyzer.
Buffer Prep:
PBA coating buffer:
In a volumetric flask, sodium phosphate (689 mg, 10 mM), sodium chloride (4.383 g, 150
mM), and sodium azide (500 mg, 0.10%) were added. After addition, enough distilled water was
added to make up 500 mL of buffer. After addition, the solution was stirred for 15 minutes and then
pH adjusted to pH = 7.2 using 0.1 M of NaOH.
55
Carb-bicarbonate coating buffer:
In a volumetric flask, sodium carbonate (1.6 g), sodium bicarbonate (3.7 g), and sodium
azide (0.2 g) were added. After addition, enough distilled water was added to make up 1 L of buffer.
After addition, the solution was stirred for 15 minutes and then pH adjusted pH = 9.6 using 0.1 M
of NaOH.
Wash buffer:
In a volumetric flask, sodium phosphate (1.379 g, 10 mM), sodium chloride (8.766 g, 150
mM), 200 mg of Thimerosal (200 mg, 0.01%), Triton X-100 (0.5 mL, 0.05%), and Tween 20 (0.5
mL) was added. After addition, enough distilled water to make up 1 L was added to the flask. After
addition, the solution was stirred for 15 minutes and then pH adjusted to pH = 7.2 using 0.1 M of
NaOH.
Blocking buffer:
In a volumetric flask, sodium phosphate (689 mg, 10 mM), sodium chloride (4.383 g, 150
mM), sodium azide (0.5 g, 0.10%), and Fraction V BSA (5 g, 1.00%) was added. After addition,
enough distilled water to make up 500 mL was added to the flask. After addition, the solution was
stirred for 15 minutes and then pH adjusted to pH = 7.2 using 0.1 M of NaOH.
TMB Stop solution:
In a flask, distilled water (463 mL) was added. After addition, phosphoric acid (37 mL) was
added dropwise over 15 minutes to the flask to make 1 L of 1 M solution of phosphoric acid.
Diluent buffer A:
In a volumetric flask, sodium phosphate (689 mg, 10 mM), sodium chloride (4.383 g, 150
mM), thimerosal (50 mg, 0.01%), Fraction V BSA (500 mg, 0.10%), and Tween 20 (0.25 mL, 0.05%)
were added. After addition, enough distilled water to make up 500 mL was added to the flask. After
56
addition, the solution was stirred for 15 minutes and then pH adjusted pH = 7.2 using 0.1 M of
NaOH.
Diluent buffer B:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.314 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (750 mg, 0.50%), and Tween 20 (75 µL, 0.05%)
were added. After addition, enough distilled water to make up 150 mL was added to the flask. After
addition, the solution was stirred for 15 minutes and then pH adjusted pH = 7.2 using 0.1 M of
NaOH.
Diluent buffer C:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.314 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), Chaps (750 mg, 0.50%) and
Tween 20 (75 µL, 0.05%) were added. After addition, enough distilled water to make up 150 mL was
added. After addition, the solution was stirred for 15 minutes and then pH adjusted pH = 7.2 using
0.1 M of NaOH.
Diluent buffer D:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.314 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), heat inactivated normal goat
serum (15 mL, 10%), and Tween 20 (75 µL, 0.05%) were added. After addition, enough distilled
water to make up 150 mL was added. After addition, the solution was stirred for 15 minutes and
then pH adjusted to pH = 7.2 using 0.1 M of NaOH.
Diluent buffer E:
In a volumetric flask, TRIS (1.817 g, 100 mM), sodium chloride (1.314 g, 150 mM),
thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), and Tween 20 (75 µL, 0.05%) were
57
added. After addition, enough distilled water to make up 150 mL was added. After addition, the
solution was stirred for 15 mintues and then pH adjusted to pH = 7.8 using 0.1 M of NaOH.
Diluent buffer F:
In a volumetric flask, TRIS (1.817 g, 100 mM), sodium chloride (1.3149 g, 150 mM),
thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), and Tween 20 (75 µL, 0.05%) were
added. After addition, enough distilled water to make up 150 mL was added. After addition, the
solution was allowed to stir for 15 minutes and then pH adjusted to pH = 9.0 using 0.1 M of
NaOH.
Diluent buffer G:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.315 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), EDTA (438 mg, 10 mM), and
Tween 20 (75 µL, 0.05%) were added. After addition, enough distilled water to make up 150 mL was
added. After addition, the solution was allowed to stir for 15 minutes and then pH adjusted to pH =
7.2 using 0.1 M of NaOH.
Diluent buffer H:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.315 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), heat inactivated normal mouse
serum (15 mL, 10%), and Tween 20 (75 µL, 0.05%) were added. After addition, enough distilled
water to make up 150 mL was added. After addition, the solution was allowed to stir for 15 minutes
and then pH adjusted to pH = 7.2 using 0.1 M of NaOH.
Diluent buffer I:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.315 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), heat inactivated normal human
serum (15 mL, 10%), and Tween 20 (75 µL, 0.05%) were added. After addition, enough distilled
58
water to make up 150 mL was added. After addition, the solution was stirred for 15 minutes and
then pH to pH = 7.2 using 0.1 M of NaOH.
Diluent buffer J:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (4.383 g, 500
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), and Tween 20 (75 µL, 0.05%)
were added. After addition, enough distilled water to make up 150 mL was added. After addition,
the solution was stirred for 15 minutes and then pH adjusted to pH 7.2 using 0.1 M of NaOH.
Diluent buffer K:
In a volumetric flask, sodium phosphate (207 mg, 10 mM), sodium chloride (1.315 g, 150
mM), thimerosal (15 mg, 0.01%), Fraction V BSA (150 mg, 0.10%), and Tween 20 (0.3 mL, 0.2%)
were added. After addition, enough distilled water to make up 150 mL was added. After addition,
the solution was stirred for 15 minutes and then pH adjusted to pH = 7.2 using 0.1 M of NaOH.
Pre-COVID-19 serum samples:
These samples were purchased from Cureline (Brisbane, CA), and were collected before
September 2019 from healthy adults in the USA.
Post-COVID-19 serum and saliva samples:
Clinical samples were collected under UCLA Institutional Review Board (IRB) approved
study protocol IRB#20-000703. The UCLA IRB determined the protocol was minimal risk and
verbal informed consent was sufficient for the research under 45 CFR 46.117(c)(2). The study team
complied with all UCLA policies and procedures, as well as with all applicable Federal, State, and
local laws regarding the protection of human subjects in research as stated in the approved IRB. For
this study, we worked with four specimens obtained: oral fluid for viral RT-qPCR testing, blood via
venipuncture, and two saliva specimens.
Oral fluid swab for viral RNA:
59
This was obtained according to the reported guidelines for Curative’s oral fluid COVID-19
test. Briefly, participants coughed hard three times while shielding their cough via mask and/or
coughing into the crook of their elbow. They then swabbed the inside of their cheeks, along the top
and bottom gums, under the tongue, and finally on the tongue, to gather a sufficient amount of
saliva. Swabs were placed in a tube containing RNA Shield (Zymo Research) and transported at
room temperature before laboratory processing as described. RT-qPCR was performed to determine
positive and negative samples. Positive for viral RNA was determined as below 35 cycle threshold
(CT).

Blood sampling:
Participants underwent a standard venipuncture procedure. Briefly, licensed phlebotomists
collected a maximum of 15 mL whole blood into 3 red-top SST tubes (Becton–Dickinson, cat.
number 367988). Once collected, the sample was left at ambient temperature for 30–60 minutes to
coagulate, then was centrifuged at 2200–2500 rpm for 15 minutes at room temperature. Samples
were then placed on ice until delivered to the laboratory site where the serum was aliquoted to
appropriate volumes for storage at – 80
o
C until use.

Orasure saliva sample collection:
Orasure oral specimen collection devices (catalog number 3001-2870, Orasure, USA) were
used as instructed. The pad was brushed briefly on the lower gums and then held between the gum
and the cheek for 2–5 minutes. The pad was then placed into the storage tube, with the provided
storage solution. Samples were kept on ice until they reached the lab. The samples were processed as
recommended by the manufacturer

before being aliquoted and stored at − 80 °C until use.

60
Mouthwash saliva samples:
To a flask, sodium chloride (15 g, 3%), citric acid (1 g, 0.2%), sodium benzoate (375 mg,
0.0075%) and potassium sorbate (375 mg, 0.075%) were added. After addition, 500 mL of doubly
distilled water was added. After addition, the solution was then autoclaved at 121
o
C for 20 minutes.
After 20 minutes, the solution was pH adjusted to pH = 6.5 with 0.1 M of NaOH. Then, 4 mL
aliquots were then used to vigorously rinse the mouth for 1–2 min before being collected. Samples
were kept on ice until they reached the laboratory, where they were aliquoted (avoiding any large
particulates in the liquid) and stored at − 80 °C until use.

61
RBD Fragment ELISA protocol:
Preparation of plates (2 hours)
1. Calculate the # of wells needed.
2. Prepare fragment dilution in PBSA (pH 7.2) buffer for the wells.
3. Add 100 μL/well of RBD fragment in PBSA (pH 7.2).
4. Cover wells with plate sealer. Incubate plate for 60 minutes at 37
o
C in incubator.
5. Wash plates 3x with 200 μL of wash buffer (squirt bottle, aim at sides of wells). When
discarding the wash buffer, firmly grasp the plate in one hand and do a forceful throwing
motion into a waste container. Do 2 or 3 more rapid flicks keeping the plate inverted and
finally pat dry on paper towels.
6. Add 300 μL of blocking buffer to all wells.
7. Incubate 60 minutes (or up to 24 hours) at ambient temperature. For better results, block for
the full 24 hours.
8. Wash plates 3x with 200-300 μL wash buffer (squirt bottle, aim at sides of wells). When
discarding the wash buffer, firmly grasp the plate in one hand and do a forceful throwing
motion into a waste container. Do 2 or 3 more rapid flicks keeping the plate inverted and
finally pat dry on paper towels.
9. Plate is ready for samples.

62
Manual ELISA protocol for serum samples (1.5 hours)
10. In a BSL2 hood, prepare samples for each of the well in diluent buffer A.
11. In a BSL2 hood, add 100 μL/well from diluted samples
12. In a BSL2 hood, add 100 μL diluent buffer to no sample control wells.
13. In a BSL2 hood, add 100 μL of positive control and negative control to control wells and
then cover wells with plate sealer.
14. Incubate plate for 30 minutes at 37
o
C in incubator.
15. Prepare enzyme conjugate to the desired dilution in diluent buffer A.
16. In a BSL2 hood, wash plates 4x with 300 μL wash buffer (squirt bottle, aim at sides of wells).
When discarding the wash buffer, firmly grasp the plate in one hand and do a forceful
throwing motion into a waste container. Do 2 or 3 more rapid flicks keeping the plate
inverted and finally pat dry on paper towels.
17. Add 100 μL/well enzyme conjugate (IgG) diluted in diluent buffer. (Keep covered with foil
from this step forward).
18. Incubate for 30 minutes at 37
o
C in incubator
19. Wash plates 4x using 300 μL with wash buffer (squirt bottle, aim at sides of wells). When
discarding the wash buffer, firmly grasp the plate in one hand and do a forceful throwing
motion into a waste container. Do 2 or 3 more rapid flicks keeping the plate inverted and
finally pat dry on paper towels.
20. Add 100 μL TMB/well incubate for 10 minutes at 25
o
C
21. Add 100 μL stop solution/well
22. Read plates at 450 nm and 600 nm using the Promega GloMax Platereader in UV-Vis mode
within 15 minutes.
63
23. To get the OD of the sample, subtract the reading at 450 nm for the blank well from the 450
nm for the sample well.

Manual ELISA for mouthwash samples (1.5 hours)
10. In a BSL2 hood, take 200 μL of the supernatant from the and concentrate using a Amicon
Ultra-0.5 mL centrifugal filters (100 kDa) and spin down at 15,000 rpm for 10 minutes.
11. In a BSL2 hood, take the 50 μL from the centrifugal filters and reconstitute in 60 μL of
diluent buffer G.
12. In a BSL2 hood, add 100 μL/well of supernatant from diluted samples.
13. In a BSL2 hood, add 100 μL Diluent buffer A to no sample control wells.
14. Cover wells with plate sealer. Incubate the plate for 30 minutes at 37
o
C in an incubator.
15. Prepare enzyme conjugate to the desired dilution in diluent buffer A.
16. In a BSL2 hood, wash plates 4x with 300 μL wash buffer (squirt bottle, aim at sides of wells).
When discarding the wash buffer, firmly grasp the plate in one hand and do a forceful
throwing motion into a waste container. Do 2 or 3 more rapid flicks keeping the plate
inverted and finally pat dry on paper towels.
17. Add 100 μL/well of enzyme conjugate (IgG) diluted in diluent buffer A (Keep covered with
foil from this step forward).
18. Incubate the plate for 30 minutes at 37
o
C in incubator.
19. Wash plates 4x using 300 μL with wash buffer (squirt bottle, aim at sides of wells). Flick and
blot technique. When discarding the wash buffer, firmly grasp the plate in one hand and do a
forceful throwing motion into a waste container. Do 2 or 3 more rapid flicks keeping the
plate inverted and finally pat dry on paper towels.
20. Add 100 μL TMB/well and incubate for 10 minutes at 25
o
C .
64
21. Add 100 μL stop solution/well
22. Read plates at 450 nm and 600 nm using the Promega GloMax Platereader in UV-Vis mode
within 15 minutes.
23. To get the OD of the sample, subtract the reading at 450 nm for the blank well from the 450
nm for the sample well.
Automated Serum ELISA using GSD thunderbolt analyzer (2 hours)
10. Check GSD thunderbolt analyzer for weekly cleaning with alconox, prime with wash buffer,
and other maintenance.
11. Add 65 μL of serum sample to microcentrifuge tubes.
12. Add microcentrifuge tubes to racks in GSD thunderbolt analyzer.
13. Add the respective plate for serum sample and desired antibody of study (IgG = 0.3 μg/mL
of RBD; IgA = 1.2 μg/mL of RBD)
14. Add diluent buffer A into the diluent buffer bottle on the GSD thunderbolt analyzer.
15. Add correct conjugate in diluent buffer A to conjugate bottle on the GSD thunderbolt
analyzer (IgG =1:2000 in diluent buffer A; IgA = 1:500 in diluent buffer A)
16. Add TMB substrate into the substrate bottle on the GSD thunderbolt analyzer.
17. Add 1 M H 3PO 4 into the stop bottle on the GSD thunderbolt analyzer.
18. On the GSD thunderbolt analyzer, in the program drop down menu find the programs
optimized for the assay. They will be called “Optimization 4” for running serum samples and
“Optimization 2” for running oral samples.
19. The GSD thunderbolt analyzer will run the assay automatically based upon the protocol for
the manual ELISA assays for serum or saliva. After the GSD thunderbolt analyzer
completed, collect the printed results from the printer connected to the GSD thunderbolt
analyzer.
65
20. To get the OD of the sample, subtract the reading at 450 nm for the blank well from the 450
nm for the sample well.
21. Turn off the GSD thunderbolt analyzer.
Automated mouthwash sample ELISA using GSD thunderbolt analyzer (2 hours)
10. Check GSD thunderbolt analyzer for weekly cleaning with alconox, prime with wash buffer,
and other maintenance.
11. In a BSL2 hood, take 200 μL of the supernatant for the mouthwash sample and concentrate
using a Amicon Ultra-0.5 mL centrifugal filters (100 kDa) and spin down at 15,000 rpm for
10 mins
12. In a BSL2 hood, transfer the 50 μL from the centrifugal filters into a microcentrifuge tube
and reconstitute in 30 μL of diluent buffer G.
13. Add microcentrifuge tubes to racks in GSD thunderbolt analyzer.
14. Add the respective RBD coated plate for mouthwash samples and desired antibody of study
(IgG = 1.2 μg/mL of RBD)
15. Add diluent buffer D into the diluent buffer bottle on GSD thunderbolt analyzer.
16. Add correct conjugate in diluent buffer A to conjugate bottle on GSD thunderbolt analyzer.
(IgG =1:100 in diluent buffer A)
17. Add TMB into the substrate bottle on GSD thunderbolt analyzer.
18. Add 1 M H 3PO 4 into the stop bottle on GSD thunderbolt analyzer.
19. On the GSD thunderbolt analyzer, in the program drop down menu find the programs
optimized for the assay. It will be called “Optimization 3” for running mouthwash samples
20. The GSD thunderbolt analyzer will run the assay automatically based upon the protocol for
the manual ELISA assays for serum or saliva. After the GSD thunderbolt analyzer has
66
completed, collect the printed results from the printer connected to the GSD thunderbolt
analyzer.
21. To get the OD of the sample, subtract the reading at 450 nm for the blank well from the 450
nm for the sample well.
22. Turn off the GSD thunderbolt analyzer.

67
References:

1. Soper, G. A., The Lessons of the Pandemic. Science 1919, 49 (1274), 501.

2. Worobey, M.; Levy, J. I.; Serrano, L. M.; Crits-Christoph, A.; Pekar, J. E.; Goldstein, S. A.;
Rasmussen, A. L.; Kraemer, M. U. G.; Newman, C.; Koopmans, M. P. G.; Suchard, M. A.;
Wertheim, J. O.; Lemey, P.; Robertson, D. L.; Garry, R. F.; Holmes, E. C.; Rambaut, A.; Andersen,
K. G., The Huanan Seafood Wholesale Market in Wuhan was the early epicenter of the COVID-19
pandemic. Science 0 (0), abp8715.

3. Holshue, M. L.; DeBolt, C.; Lindquist, S.; Lofy, K. H.; Wiesman, J.; Bruce, H.; Spitters, C.;
Ericson, K.; Wilkerson, S.; Tural, A.; Diaz, G.; Cohn, A.; Fox, L.; Patel, A.; Gerber, S. I.; Kim, L.;
Tong, S.; Lu, X.; Lindstrom, S.; Pallansch, M. A.; Weldon, W. C.; Biggs, H. M.; Uyeki, T. M.; Pillai,
S. K., First Case of 2019 Novel Coronavirus in the United States. New England Journal of Medicine
2020, 382 (10), 929-936.

4. Liu, C.; Zhou, Q.; Li, Y.; Garner, L. V.; Watkins, S. P.; Carter, L. J.; Smoot, J.; Gregg, A. C.;
Daniels, A. D.; Jervey, S.; Albaiu, D., Research and Development on Therapeutic Agents and
Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Central Science 2020, 6 (3),
315-331.

5. Schramm, W.; Angulo Gustavo, B.; Torres Patricia, C.; Burgess-Cassler, A., A Simple Saliva-
Based Test for Detecting Antibodies to Human Immunodeficiency Virus. Clinical Diagnostic
Laboratory Immunology 1999, 6 (4), 577-580.

6. Hodinka, R. L.; Nagashunmugam, T.; Malamud, D., Detection of Human Immunodeficiency
Virus Antibodies in Oral Fluids. Clinical Diagnostic Laboratory Immunology 1998, 5 (4), 419-426.
7. Groen, J.; Koraka, P.; Velzing, J.; Copra, C.; Osterhaus, A. D. M. E., Evaluation of Six
Immunoassays for Detection of Dengue Virus-Specific Immunoglobulin M and G Antibodies.
Clinical Diagnostic Laboratory Immunology 2000, 7 (6), 867-871.

8. Pacheco, F. T. F.; Carvalho, S. S. d.; Santos, S. A.; Chagas, G. M. T. d.; Santos, M. C.; Santos,
J. G. S.; Júnior, H. d. C.-R.; Ribeiro, T. C. M.; Mattos, Â. P. d.; Gomes, M. A.; Soares, N. M.;
Teixeira, M. C. A., Specific IgG and IgA Antibody Reactivities in Sera of Children by Enzyme-
Linked Immunoassay and Comparison With Giardia duodenalis Diagnosis in Feces. Ann Lab Med
2020, 40 (5), 382-389.

9. Heaney, J. L. J.; Phillips, A. C.; Carroll, D.; Drayson, M. T., The utility of saliva for the
assessment of anti-pneumococcal antibodies: investigation of saliva as a marker of antibody status in
serum. Biomarkers 2018, 23 (2), 115-122.
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10. de Oliveira, L. C.; Pereira, N. B.; Moreira, C. H. V.; Bierrenbach, A. L.; Salles, F. C.; de
Souza-Basqueira, M.; Manuli, E. R.; Ferreira, A. M.; Oliveira, C. D. L.; Cardoso, C. S.; Ribeiro, A. L.
P.; Sabino, E. C., ELISA Saliva for Trypanosoma cruzi Antibody Detection: An Alternative for
Serological Surveys in Endemic Regions. The American Journal of Tropical Medicine and Hygiene 2020,
102 (4), 800-803.

11. Lu, M.; Liu, Q.; Wang, X.; Zhang, J.; Zhang, X.; Shi, D.; Liu, J.; Shi, H.; Chen, J.; Feng, L.,
Development of an indirect ELISA for detecting porcine deltacoronavirus IgA antibodies. Archives of
Virology 2020, 165 (4), 845-851.

12. Bisht, H.; Roberts, A.; Vogel, L.; Subbarao, K.; Moss, B., Neutralizing antibody and
protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant
polypeptide containing an N-terminal segment of the spike glycoprotein. Virology 2005, 334 (2), 160-
165.

13. Heikenfeld, J.; Jajack, A.; Feldman, B.; Granger, S. W.; Gaitonde, S.; Begtrup, G.; Katchman,
B. A., Accessing analytes in biofluids for peripheral biochemical monitoring. Nature Biotechnology
2019, 37 (4), 407-419.

14. Burns, C. A.; Ebersole, J. L.; Allansmith, M. R., Immunoglobulin A antibody levels in human
tears, saliva, and serum. Infection and Immunity 1982, 36 (3), 1019-1022.

15. Ellison, S. A.; Mashimo, P. A.; Mandel, I. D., Immunochemical Studies of Human Saliva. I.
The Demonstration of Serum Proteins in Whole and Parotid Saliva. Journal of Dental Research 1960, 39
(5), 892-898.

16. MacMullan, M. A.; Ibrayeva, A.; Trettner, K.; Deming, L.; Das, S.; Tran, F.; Moreno, J. R.;
Casian, J. G.; Chellamuthu, P.; Kraft, J.; Kozak, K.; Turner, F. E.; Slepnev, V. I.; Le Page, L. M.,
ELISA detection of SARS-CoV-2 antibodies in saliva. Scientific Reports 2020, 10 (1), 20818.

17. Ju, B.; Zhang, Q.; Ge, J.; Wang, R.; Sun, J.; Ge, X.; Yu, J.; Shan, S.; Zhou, B.; Song, S.; Tang,
X.; Yu, J.; Lan, J.; Yuan, J.; Wang, H.; Zhao, J.; Zhang, S.; Wang, Y.; Shi, X.; Liu, L.; Zhao, J.; Wang,
X.; Zhang, Z.; Zhang, L., Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature
2020, 584 (7819), 115-119.

18. Samavati, L.; Uhal, B. D., ACE2, Much More Than Just a Receptor for SARS-COV-2.
Frontiers in Cellular and Infection Microbiology 2020, 10.

69
Chapter 3 - Dyes and Pigment Handles for Nanoparticles in
Biomedical Imaging
Introduction:
In collaboration with the Zaveleta Lab, our lab has been working towards
developing nanoparticles for multiplex biomedical imaging that target cancer cells. Their lab has
developed methods to make liposomes that could encapsulate dyes, pigments, and other cargo and
image the nanoparticles in cell and mouse models. However, as with many liposomes, these lipid
base nanoparticles tend to leak their cargo as they reach their intend destination.
31
In this project, we
hypothesized that we could prevent leaking of the dyes by forming a covalent bond between the dye
and the silane nanoparticle monomer. We chose D&C Green #8, a pyrene based green dye
commonly used in food and cosmetics, for our dye. Substituted derivatives of 8-hydroxypyrene-
1,3,6-sulfonic acid has found use as blue emitting fluorescent dyes.
32
The spectral properties of the
fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes
with minimum spectral interference. Interest in the chemistry of pyrene systems dates back to the
early 1900s when pyrene was first isolated from fossil fuels.
33
Pyrene dyes were first explored as
starting materials for the production of azo dyes – aminopyrene was sulfonated and unsuccessfully
subjected to diazonium coupling conditions.
70
Results and Discussion:
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonic acid

Scheme 3.1 Retrosynthesis of G8-Silane Monomer

Scheme 3.2 Bucherer Mechanism of Naphthalene
In order to construct the fluorescent silica nanoparticles, we focused our attention on
creating access to the G8-sulfonic acid material. Our first retrosynthesis of these materials focused
upon using the Bucherer reaction to interconvert the hydroxy to the amino group on the pyrene
ring. There was precedent for this reaction in interconverting the hydroxy and amino groups on
naphthalene rings and we believed that we could also extend it to pyrene systems.
34
However any
attempts to pursue this avenue proved unfruitful. Mechanistically, the Bucherer reaction depends
upon the addition of a sulfoxide on an aromatic ring system to break aromaticity and allow
interconversion of hydroxy to amine.
35
However, in the case of G8, the ring unable to be sulfonated
again and thus the reaction does not progress forward. We confirmed that this avenue was also
OH
S S
S
O
O O
O
OH HO
HO
O
O
NH
2
S S
S
O
O O
O
OH HO
HO
O
O
Bucherer Rxn
NH
2
NH
2
S S
S
O
O O
O
OH HO
HO
O
O
Sulfonylation
N
H
S
S
S
O
O O
O
OH
HO
HO
O
O
Si
O
O
O
N
H
S
OH O
NaHSO
3
-NaHSO
3
HO SO
3
Na
NaHSO
3
-NaHSO
3
NH
3
H
2
O
H
2
N SO
3
Na
NH
NH
2
71
pursued in the 1930s by Tietze and Bayer to create aminopyrene derivatives to form azo dyes but
proved to be unsuccessful.
36

Scheme 3.3 Sulfonation of Aminopyrene with Fuming Sulfuric Acid Leads to Mixture of the
Tri- and Di-sulfonic Acid Products

Figure 3.1 Overlap of Trisulfonic Acid and Disulfonic Acid on C18 HPLC
We changed tactics and pursued a second strategy starting with sulfonylation of
aminopyrene. Pervious work towards these sulfonic acid derivatives had been achieved by several
different groups using fuming sulfuric acid to sulfonate the pyrene ring.
37-39
However, in our hands,
these conditions led to incomplete sulfonation of the pyrene ring and formation of a mixture of 8-
aminopyrene-1,3,6-trisulfonic acid and 6-aminopyrene-1,3-disulfonic acid. (Scheme 3.3) Attempts to
separate the two products required use of HPLC to separate and purify – for a reaction that was
conducted on a 400 mg scale, only 10 mg was isolated through these efforts. In response to this, we
changed course in our synthetic scheme.
NH
2 NH
2
S S
S
O
O O
O
OH HO
HO
O
O
Fuming Sulfuric Acid
60C, 24 hrs
NH
2
S S
O
O O
O
OH HO
m/z: 456.96 m/z: 377.00
72

Scheme 3.4 Revised Synthetic Scheme for Sulfonic Acids
We reconsidered our approach and decided to add the sulfonic acids by first forming
sulfonyl chlorides and then replacing the chlorides with hydroxyls. We were able to add sulfonyl
chlorides to the pyrene ring using chlorosulfonic acid to achieve yields up to 92%. It should be
noted that kinetically this reaction progresses in two steps: the first step was the quick addition of
two sulfonyl groups on the pyrene ring followed by the slow addition of the final sulfonyl chloride.
While the first step can be complete in a few minutes, the last addition can take up to 32 hours to
complete in neat chlorosulfonic acid at room temperature. The reaction progression is temperature
dependent and can be speed up to completion by increasing the temperature. It can also be stopped
at the first step by maintaining the reaction temperature at 0
o
C. This strategy allowed access to both
the di- and trisulfonyl chlorides. In addition to this, also allowed access to further derivatives by
converting the sulfonyl chlorides to sulfonyl fluorides and sulfonamides.

Scheme 3.5 Reaction Progression for Sulfonyl Chlorides
Our next step was to establish a subsequent path towards the sulfonic acid residues. Our
first attempts were to follow literature protocols that called for mixing of the sulfonyl chloride in
NH
2 NH
2
S S
S
O
O O
O
Cl Cl
Cl
O
O
NH
2
S S
S
O
O O
O
O O
O
O
O
[O]
[HO
-
]
NH
2 NH
2
S S
S
O
O O
O
Cl Cl
Cl
O
O
NH
2
S S
O
O O
O
Cl Cl
FAST SLOW
73
THF and basifying to pH 8 or 9. While this process would lead to the production of the sulfonic
acid, it also led to the production of a significant amount of residual salt (NaCl for NaOH or
NaHCO 3, KCl for KOH or KHCO 3) that proved difficult to isolate from the sulfonic acids.
Attempts to purify using traditional flash column chromatography, reverse phase chromatography,
cation exchange chromatography, anion exchange chromatography, centrifugation, precipitation,
crystallization, and salting out were unable to isolate product. Taking a step back, we looked how the
reactions conditions could be manipulated in order to push the reaction to completion and to
control the production of residual salt after the completion of the reaction. To this end, we explored
ways to optimize the reaction and subsequent isolation of the desired product.
Reaction and Isolation Optimization
In our reaction optimization, we looked at 8 different reaction conditions, compared the
UV/Vis curves integration of the sulfonic acid at three different wavelengths (254 nm, 365 nm, and
502 nm), and used that data to determine which reaction conditions produced the most sulfonic
acid. The reactions were done on the 5 mg scale at 10 mM concentrations and allowed to stir for 5
mins before quenching; the conditions and results were the following:

74

NaOH
(eqv)
A232
(μL)
Total H20
(μL)
Total THF
(μL)
Total
Volume (μL)
Total %
H20
Total %
THF
Peak
254
Peak
365
Peak
502
A1 6 250 233 767 1000 23 77 541 557 -744
B 6 250 46.6 953 999.6 5 95 31.5 24 -30
C 6 250 466 534 1000 47 53 364 362 -482
D 6 250 750 250 1000 75 25 165 136 -163
E 3 250 749.5 250 999.5 75 25 255 250 -330
F 0 250 375 625 1000 38 63 35 31 -38
G 1 250 393.8 605 998.8 39 61 100 96 -131
H 0 250 750 250 1000 75 25 252 255 -339
Table 1 Reaction Conditions for Sulfonic Acid Synthesis Optimization
Using this approach, we compared which factors in our reaction conditions would lead to
production of the sulfonic acid without leading to residual salt production. We compiled our
observations in Table 1. For conditions H vs F, they had 0 eq of NaOH present in the reaction and
they differed in the amount of water -- this comparison showed that an increase in water lead to
more sulfonic acid production. This observation was reinforced when comparing conditions A and
B – more water corresponded in more product at the end of the reaction. For conditions D vs. E vs.
H, they had the same conditions but differed in the equivalents of NaOH -- this comparison showed
Figure 3.2 Reaction Optimization of 8-Aminopyrene-1,3,6-trisufonic acid
75
that an increase of NaOH in the reaction resulted in more production of sulfonic acid and increased
the amount of residual salt found. For B vs C vs D, they had the same conditions with 6 eq of
NaOH but with differing amounts of water -- this comparison showed that best conditions for the
reaction was when ratio of THF to water was 1:1 in solution.
Taking these observations together, we settled on our reaction conditions (4 eq NaOH, 1:1
THF: Water volume) and can access the 8-aminopyrene-1,3,6-sulfonic acid with 100% yield – no
further purification of the material was necessary after quenching reaction.

Scheme 3.6 Revised Synthetic Scheme for G8-Sulfonic Acid Silane Monomer
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonamide

Scheme 3.7 Synthetic Scheme for G8-Sulfonamide Silane Monomer
Following the adapted strategy from the sulfonic acids, we started with aminopyrene and
converted it to the 8-aminopyrene-1,3,6-trisulfonyl trichloride using chlorosulfonic acid with a 92%
yield. After isolation of the sulfonyl trichloride intermediate, we can replace the chloride with amines
in THF under basic conditions. We initially used ammonium hydroxide with the hypothesis that the
reaction would result in the sulfonic acid, instead we had the 8-aminopyrene-1,3,6-trisulfonamide in
100 % yield. We then coupled 8-aminopyrene-1,3,6-trisulfonamide and triethoxy(3-
isothiocyanatopropyl)silane in refluxing toluene in order to result in our G8-sufonamide silane
NH
2 NH
2
S S
S
O
O O
O
Cl Cl
Cl
O
O
Chlorosulfonic acid
80C , 24 hrs, 92%
NH
2
S S
S
O
O O
O
O O
O
O
O
4.0 eq. NaOH
THF/Water, r.t., 5 min, 100%
N
H
S
S
S
O
O O
O
OH
HO
HO
O
O
Si
O
O
O
N
H
S
Si
O
O
O N
C
S
NEt
3
,DMSO, RT, 24 hrs
3Na
+
NH
2 NH
2
S S
S
O
O O
O
Cl Cl
Cl
O
O
Chlorosulfonic acid
r.t. , 24 hrs, 92%
5.0 eq. NH
4
OH
THF, r.t., 10 mins, 100%
N
H
S
S
S
O
O O
O
NH
2 H
2
N
H
2
N
O
O
Si
O
O
O
N
H
S
Si
O
O
O N
C
S
NEt
3
,Toluene, 110
o
C, 24 hrs, 77%
NH
2
S S
S
O
O O
O
NH
2 H
2
N
H
2
N
O
O
76
monomer in 77% yield. It should be noted that the final product has two ethoxy groups and one
methoxy group rather than three ethoxy groups in the final structure – in the course of transferring
the product to another vial, methanol was used to dissolve the final product. When observing the
NMRs of the final product, we saw that one carbon was missing in the
13
C NMR. What we
determined was that one ethoxy was exchanged with a methoxy – we believe this was coming from
the methanol used to transfer the product. Our material was then transferred to our collaborators
where it has been incorporated into silica nanoparticles.
Synthesis of 8-(3-(3-(triethoxysilyl)propyl)thioureido)pyrene-1,3,6-trisulfonyl trifluoride

Scheme 3.8 Synthetic Scheme for G8-Sulfonyl fluoride silane monomer
Following the adapted strategy from the sulfonic acids and sulfonamides, we started with
aminopyrene and converted it to the 8-aminopyrene-1,3,6-trisulfonyl trichloride using chlorosulfonic
acid with a 92% yield. After isolation of the sulfonyl trichloride intermediate, we can substitute the
chlorides with fluorides using KH 2F in THF/water (1:1) with a yield of 79%. We are currently
determining conditions for producing the G8-sulfonyl fluoride silane monomer.
Conclusions:
Our ultimate desire was to create fluorescent silica nanoparticles that could be used towards
the detection of cancer cells. Our first step was to establish a synthetic pathway for adapting D&C
Green #8 for covalent incorporation into the framework of silica nanoparticles. Towards that end,
we focused on using the amino group of aminopyrene ring systems as a handle to form thiourea
linkages to the silane. The first challenge was synthesizing reasonable amounts of 8-aminopyrene-
1,3,6-sulfonic acid that could be used for nanoparticle fabrication and optimization. We developed
NH
2 NH
2
S S
S
O
O O
O
Cl Cl
Cl
O
O
Chlorosulfonic acid
r.t. , 24 hrs, 92%
NH
2
S S
S
O
O O
O
F F
F
O
O
18 eq. KH
2
F
THF/Water, r.t., 5 min, 79%
N
H
S
S
S
O
O O
O
F
F
F
O
O
Si
O
O
O
N
H
S
Si
O
O
O N
C
S
NEt
3
, Toluene, RT, 24 hrs
77
and refined a sulfonation process for smoothly adding and controlling the amount of sulfonyl
chlorides to the 8-aminopyrene ring. After solving the sulfonation, we optimized the reaction
conditions to access the G8-sulfonic acid without the need of specialized isolation equipment. We
are currently moving towards forming the G8-sulfonic acid silane monomers and G8-sulfonyl
fluoride silane monomers for development into silica nanoparticles. Our synthesized G8-
sulfonamide silane monomers have already been incorporated into silica nanoparticles and will be
tested for stability and further functionalization of the silica core. Access to multiple G8-modified
silane monomers will enable the production of silica nanoparticles of various colors that can be
further modified for targeting cancer cells.

78
General Procedures:
All reactions were carried out under a nitrogen atmosphere with dry solvents under
anhydrous conditions, unless otherwise noted. Tetrahydrofuran (THF) was obtained from a lab
installed sodium still, ACS reagent grade toluene was purchased from Fisher, ACS reagent grade
diethyl ether (Et 2O) was purchased from Fisher, DriSolv dimethylformamide (DMF) was purchased
from Fisher, DriSolv methanol (MeOH) was purchased from Fisher, and DriSolv methylene
chloride (CH 2Cl 2) was purchased from Fisher. Yields refer to chromatographically and
spectroscopically (
1
H NMR) homogenous materials, unless otherwise stated. Aminopyrene,
chlorosulfonic acid, and fuming sulfuric acid were purchased from Sigma Aldrich and used without
further purification. Reactions were monitored both by thin-layer chromatography (TLC) carried out
on 0.2 mm E. Merck silica gel plates (60F-254) using UV light as visualizing agent and by Agilent
LC/MSD. E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash column
chromatography. NMR spectra were recorded either on Varian Mercury 400, Varian 400MR, Varian
VNMRS 500, or Varian VNMRS 600 and calibrated using residual undeutrated solvent (CDCl 3 : 𝛿 H
= 7.26 ppm, 𝛿 C = 77.16 ppm; Acetone-d6 : 𝛿 H = 2.05 ppm, 𝛿 C = 29.92 ppm; DMSO-d6 : 𝛿 H = 2.50
ppm, 𝛿 C = 39.52 ppm) as an internal reference. The following abbreviations were used to designate
the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, br
= broad. Infrared (IR) spectra were recorded on Agilent Cary 630 FTIR. UV-Vis spectra were
recorded on Agilent Cary 60 UV-Vis. Low-resolution mass spectra were recorded on Agilent
LC/MSD (ESI).

79
Experimental
8-aminopyrene-1,3,6-trisulfonyl trichloride

In a reaction flask equipped with a magnetic stir bar, aminopyrene (50 mg, 0.23 mmol) was
added under nitrogen. After addition, dichloromethane (2.3 mL, 0.1 M) was added under nitrogen
and allowed to stir for 10 minutes at room temperature. After 10 minutes, chlorosulfonic acid (0.566
mL, 8.51 mmol) was added dropwise via a glass syringe to the reaction flask – there was an evolution
of HCl gas. After complete addition, the reaction was allowed to stir at 80
o
C for 1 hour and checked
for completion using TLC -- Rf (100% ethyl acetate): 0.9. After 1 hour, dichloromethane (10 mL)
was added to the reaction flask. After addition, the reaction mixture was poured into an ice/water
bath and allowed to stir for 20 minutes at 0
o
C. After 20 minutes, the mixture was filtered through
filter paper on a Büchner funnel to collect the precipitate. The precipitate was dried under high
vacuum to give a brick-colored material. (108 mg, 92%).
1
H NMR (400 MHz, acetone) δ 9.34 (d, J =
9.5 Hz, 1H), 9.27 (s, 1H), 9.01 (d, J = 9.5 Hz, 1H), 8.90 (dd, J = 9.6, 5.8 Hz, 2H), 8.46 (s, 1H).
13
C
NMR (151 MHz, acetone) δ 150.32, 143.08, 135.19, 133.52, 132.86, 132.31, 130.78, 129.99, 129.63,
127.14, 126.25, 122.15, 119.79, 117.64, 117.04, 115.58. IR (cm
-1
): 3643.5, 3623.9, 3543.8, 3164.5,
3003.3, 2944.6, 2627.8, 2409.7, 2293.2, 2253.2, 1444.3, 1375.4, 1039.9, 918.8, 749.2. LRMS (ESI)
m/z: [M]
-
calculated for C 16H 8Cl 3NO 6S 3 512.77; found 512.00
.

NH
2
S S
S
O
O
O
O
Cl
O
O
Cl
Cl
80
8-aminopyrene-1,3,6-trisulfonyl trifluoride

In a reaction flask equipped with a magnetic stir bar, 8-aminopyrene-1,3,6-trisulfonyl
trichloride (40 mg, 0.077mmol) was added under nitrogen. After addition, THF (3.885 mL) from a
benzophenone-sodium still was added. After addition, the solution was allowed to stir for 3 minutes
at room temperature. After 3 minutes, KHF 2 (84.07 mg, 1.398 mmol) in distilled water (3.885 mL)
was added dropwise via a syringe over 2 minutes. After addition, the reaction was allowed to stir for
1 hour at room temperature. After 1 hour, diethyl ether (20 mL) was added to the solution and then
the solution was transferred to a separatory funnel. After transferring, the solution was extracted
with diethyl ether (25 mL) three times. After extraction, the organic layers were combined in a
separatory funnel and then washed with saturated sodium chloride aqueous solution (10 mL). The
combined organic layers were then dried with sodium sulfate and allowed to stand for 20 minutes.
After 20 minutes, the mixture was filtered through filter paper on a Büchner funnel, and the filtrate
was collected into a round bottom flask. The round bottom flask was put on a rotary evaporator and
the diethyl ether was evaporated under reduced pressure. The crude product was then purified a
silica gel column using 20% ethyl acetate in hexanes as eluent to give pure product (28.6 mg, 79%).
19
F NMR (376 MHz, acetone) δ 66.16, 62.80, 62.05.
1
H NMR (400 MHz, acetone) δ 9.19 (s, 1H),
8.95 (dd, J = 9.5, 2.8 Hz, 1H), 8.91 (d, J = 9.5 Hz, 1H), 8.58 (ddd, J = 9.6, 7.2, 2.7 Hz, 2H), 8.41 (s,
1H), 7.80 (s, 1H).
13
C NMR (100 MHz, acetone) δ 136.47, 134.97, 132.57, 131.16, 130.29, 126.76,
126.11, 122.45, 120.12, 119.25, 116.76. IR (cm
-1
): 3623.9, 3544.7, 3165.4, 3062.0, 3002.4, 2944.6,
NH
2
S S
S
O
O
O
O
F
O
O
F
F
81
2627.8, 2409.7, 2293.2, 2253.2, 1703.4, 1630.7, 1442.5, 1375.4, 1272.0, 1250.5, 1039.0, 918.8, 741.4,
704.5. LRMS (ESI) m/z: [M+H]
+
calculated for C 16H 8F 3NO 6S 3 462.9; found 464.1. The product was
also detected by collision-induced MS/MS employing MRM and mass transition ion-pair (precursor-
product ion transitions) for the titled product m/z 464.0 → 380.0.
8-aminopyrene-1,3,6-trisulfonamide

In a reaction flask equipped with a magnetic stir bar, 8-aminopyrene-1,3,6-trisulfonyl
trichloride (60.6 mg, 0.118 mmol) was added under nitrogen. After addition, THF (10 mL) from a
benzophenone-sodium still was added under nitrogen. After addition, the solution was allowed to
stir for 3 minutes at room temperature. After 3 minutes, ammonium hydroxide (0.5 mL) was added
dropwise via a syringe over 5 minutes. After addition, the reaction was allowed to stir for 1 hour at
room temperature. After 1 hour, diethyl ether (20 mL) was added to the solution and then the
solution was transferred to a separatory funnel. After transferring, the solution was extracted with
diethyl ether (25 mL) three times. After extraction, the organic layers were combined in a separatory
funnel and then washed with saturated sodium chloride aqueous solution (10 mL). The combined
organic layers were then dried with sodium sulfate and allowed to stand for 20 minutes. After 20
minutes, the mixture was filtered through filter paper on a Büchner funnel, and the filtrate was
collected into a round bottom flask. The round bottom flask was put on a rotary evaporator and the
diethyl ether was evaporated under reduced pressure. The crude material was purified on a column
of 100% ethyl acetate to give pure product. (56 mg, 100%).
1
H NMR (400 MHz, dmso-d6) δ 9.12 (s,
1H), 9.01 (d, J = 9.7 Hz, 1H), 8.79 (d, J = 9.6 Hz, 1H), 8.72 (d, J = 9.7 Hz, 1H), 8.66 (d, J = 9.7 Hz,
NH
2
S S
S
O
O
O
O
H
2
N
O
O
H
2
N
H
2
N
82
1H), 8.18 (s, 1H), 7.85 (d, J = 9.4 Hz, 6H), 7.46 (s, 2H).
13
C NMR (151 MHz, dmso-d6) δ 147.15,
141.09, 132.91, 132.41, 131.04, 130.07, 127.98, 126.25, 126.02, 125.81, 125.79, 121.69, 118.88,
115.05, 114.99, 114.09. UV/Vis (nm): 238.0, 286.5, 376.0, 429.5
8-aminopyrene-1,3,6-trisulfonic acid

The product was obtained according to an already known procedure with slight
modifications.
36-37
In a reaction flask equipped with a magnetic stir bar, sodium hydroxide (15.54 mg,
0.388 mmol) was added under nitrogen. After addition, distilled water (4.85 mL) and THF (2.425
mL) was added to the reaction flask and allowed to stir at room temperature for 2 minutes. After 2
minutes, 8-aminopyrene-1,3,6-trisulfonyl trichloride (50 mg, 0.097 mmol) in THF (2.425 mL) was
added dropwise via syringe to the reaction flask over 10 minutes. After 10 minutes, the reaction was
allowed to stir for 15 minutes at room temperature. The solution turned from a deep red color to a
deep green color over the course of the reaction. The reaction was monitored using TLC of the
sulfonic chloride starting material Rf (100% ethyl acetate): 0.9. The reaction was then transferred to
a round bottom flask. The round bottom flask was put on a rotary evaporator and the THF was
evaporated under reduced pressure to give titled product as a green powder (56.8 mg, 100%).
1
H
NMR (400 MHz, dmso-d6) δ 8.91 – 8.83 (m, 3H), 8.68 (d, J = 9.7 Hz, 1H), 8.23 (d, J = 9.8 Hz, 1H),
7.94 (s, 1H), 6.37 (s, 2H).

NH
2
S S
S
O
O
O
O
HO
O
O
HO
HO
83
triethoxy(3-isothiocyanatopropyl)silane

The isothiocyanate was obtained according to an already known procedure with slight
modifications.
40
In a reaction flask, carbon disulfide (0.816 mL, 13.55 mmol) was added under
nitrogen. After addition, the reaction flask was cooled to 0
o
C using an ice/water bath for 20
minutes. After 20 minutes, (3-aminopropyl)triethoxysilane (2.0 g, 2.13 mL) was dissolved in THF
(6.02 mL) from a benzophenone-sodium still and then added dropwise via a syringe to the solution
of CS 2 at 0
o
C over 15 minutes. After 15 minutes, the reaction was allowed to stir at 0
o
C for 3 hours.
After 3 hours, dicyandiamide (1.14 g, 13.55 mmol), triethylamine (41.56 μL, 0.298 mmol), and THF
(6.02 mL) from a benzophenone-sodium still were added at room temperature to the solution
containing the dithiocarbamic acid. After addition, the reaction was allowed to stir at 40
o
C for 30
hours. After 30 hours, the reaction was cooled to room temperature for 20 minutes. After 20
minutes, the reaction flask was transferred to a rotary evaporator and the THF as evaporated under
reduced pressure. After THF evaporation, the crude product was transferred to a separatory funnel.
After transferring, the solution was extracted with diethyl ether (25 mL) three times. After
extraction, the organic layers were combined in a separatory funnel and then washed with saturated
sodium chloride aqueous solution (10 mL). The combined organic layers were then dried with
sodium sulfate and allowed to stand for 20 minutes. After 20 minutes, the mixture was filtered
through filter paper on a Büchner funnel, and the filtrate was collected into a round bottom flask.
The round bottom flask was put on a rotary evaporator and the diethyl ether was evaporated under
reduced pressure. The crude material was purified via distillation at 100
o
C under high vacuum to
give titled product as a clear yellow oil (2.3 g, 96%).
1
H NMR (500 MHz, cdcl 3) δ 3.82 (qd, J = 7.0,
Si
O O
O
N
C
S
84
1.4 Hz, 6H), 3.50 (t, J = 6.7 Hz, 2H), 1.85 – 1.77 (m, 2H), 1.22 (td, J = 7.0, 1.4 Hz, 9H), 0.71 – 0.67
(m, 2H).
13
C NMR (151 MHz, cdcl 3) δ 58.69, 47.52, 24.25, 18.44, 14.35, 7.84.

8-(3-(3-(diethoxy(methoxy)silyl)propyl)thioureido)pyrene-1,3,6-trisulfonamide

In a reaction flask equipped with a magnetic stir bar, 8-aminopyrene-1,3,6-trisulfonamide (56
mg, 0.257 mmol) was added under nitrogen. After addition, ethyl acetate (12.5 mL, 0.1 M) and
toluene (12.5 mL, 0.1 M) were added under nitrogen. After addition, triethylamine (0.359 mL, 10 eq)
was added and the solution was allowed allow to stir for 10 minutes at room temperature. After 10
minutes, triethoxy(3-isothiocyanatopropyl)silane (74 μL, 0.283 mmol, 1.1 eq) was added dropwise
via a syringe over 5 minutes. After addition, the reaction was brought refluxed at 110
o
C and allowed
to stir for 4 hours – TLC showed consumption of isothiocyanate Rf (100% ethyl acetate): 0.85. After
4 hours, the solution was allowed to cool to room temperature for 20 minutes. After 20 minutes, the
solution was transferred to a rotary evaporator and ethyl acetate and toluene were evaporated under
reduced pressure. After evaporation, acetone (10 mL) was added to the reaction flask and the
solution was filtered through a pad of celite. The pad of celite was washed with acetone (30 mL) and
the filtrate was collected. After collection, the filtrate was transferred to a round bottom flask and
put on a rotary evaporator. After transferring, the acetone was evaporated under reduced pressure.
After evaporation, ethanol (3 mL) was added to crude product and then transferred to a rotary
evaporator to evaporate the ethanol under reduced pressure – this was performed three times. After
N
H
S
S
S
O
O
O
O
NH
2
H
2
N
H
2
N
O
O
Si
O
O
O
N
H
S
85
evaporation of ethanol, the crude product was then lyophilized to give titled product (140 mg, 77%).
1
H NMR (500 MHz, dmso-d6) δ 9.13 (s, 1H), 9.02 (d, J = 9.6 Hz, 1H), 8.80 (d, J = 9.6 Hz, 1H), 8.73
(d, J = 9.6 Hz, 1H), 8.67 (d, J = 9.7 Hz, 1H), 8.19 (s, 1H), 7.86 (d, J = 13.4 Hz, 6H), 7.48 (s, 2H),
3.75 (d, J = 7.2 Hz, 1H), 3.66 (s, 2H), 3.07 (s, 4H), 1.76 (s, 2H), 1.22 (s, 3H), 1.17 (t, J = 7.3 Hz, 6H),
0.84 (t, J = 6.8 Hz, 1H).
13
C NMR (126 MHz, dmso-d6) δ 147.13, 141.11, 132.96, 132.45, 131.06,
130.09, 128.01, 126.28, 126.04, 125.83, 125.71, 121.73, 118.93, 115.10, 115.01, 114.11, 61.23, 45.70,
29.03, 15.14, 8.59.
Reaction Optimization of Sulfonic Acid Reaction Procedure
To a 2 mL reaction flask, add NaOH (stock of 10 mg/mL in distilled water) -- for 6 equivalents of
NaOH 233 μL was added from the stock, for 3 equivalents of NaOH 116.5 μL was added from the
stock, and for 1 equivalent of NaOH 38.8 μL was added-- THF, and water to the reaction flask to
make a total volume of 750 μL. The reaction was then sonicated in the Vevor Ultrasonic cleaner for
10 seconds. After sonication, 8-aminopyrene-1,3,6-trisulfonylchloride (5 mg in 250 μL of THF) was
added to reaction flask and allowed to stir for 5 mins. After 5 minutes, a 1 μL aliquot of the reaction
solution was taken and injected into the Agilent LC/MSD. After injection, the sample was integrated
under the curve for 254 nm, 365 nm, and 502 nm to determine conversion of sulfonyl chloride to
sulfonic acid.
86

Representative Spectra

Figure 3.3
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride
87

Figure 3.4
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride
88

Figure 3.5 LC/MS Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride in Negative
Mode

Figure 3.6 IR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trichloride
89

Figure 3.7
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride
90

Figure 3.8
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride
91

Figure 3.9
19
F NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride

92

Figure 3.10 LC/MS Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride in Positive Mode

Figure 3.11 IR Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride
93

Figure 3.12 MS/MS MRM Spectrum of 8-aminopyrene-1,3,6-trisulfonyl trifluoride

NH
2
S S
S
O
O O
O
F F
F
O
O
Chemical Formula: C
16
H
8
F
2
NO
4
S
2
•
Exact Mass: 379.9863
94

Figure 3.13
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide
95

Figure 3.14
13
C NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide
96

Figure 3.15 HSQC NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonamide

Figure 3.16 UV/Vis Spectrum of 8-aminopyrene-1,3,6-trisulfonamide
Page 1 of 4
8/5/2019 4:36:15 AM
200 400 600 800
0.0
0.2
0.4
0.6
0.8
1.0
Wavelength (nm)
Abs
429.5
376.0
286.5
238.0
Zer
o
Rep
ort
Read Abs (800.0 nm)
Zero 0.0724
Scan Analysis Report
Report Time : Mon 05 Aug 03:49:34 AM 2019
Method
Batch:
Software version: 5.1.0.1016
Operator:
Instrument Parameters
Instrument Cary 60
Instrument Version 2.00
Start (nm) 800.0
Stop (nm) 200.0
X Mode Nanometers
Y Mode Abs
UV-Vis Scan Rate (nm/min) 300.00
UV-Vis Data Interval (nm) 0.50
UV-Vis Ave. Time (sec) 0.1000
Beam Mode Dual Beam
Baseline Correction On
Baseline Type Baseline correction
Baseline File Name
Baseline Std Ref File Name
Cycle Mode Off
Comments
Zero Report
Read Abs (800.0 nm)
97

Figure 3.17
1
H NMR Spectrum of 8-aminopyrene-1,3,6-trisulfonic acid
98

Figure 3.18
1
H NMR Spectrum of triethoxy(3-isothiocyanatopropyl)silane
99

Figure 3.19
13
C NMR Spectrum of triethoxy(3-isothiocyanatopropyl)silane
100

Figure 3.20
1
H NMR Spectrum of 8-(3-(3-(diethoxy(methoxy)silyl)propyl)thioureido)pyrene-
1,3,6-trisulfonamide
101

Figure 3.21
13
C NMR Spectrum of 8-(3-(3-(diethoxy(methoxy)silyl)propyl)thioureido)pyrene-
1,3,6-trisulfonamide
102

References:
1. Hou, X.; Zaks, T.; Langer, R.; Dong, Y., Lipid nanoparticles for mRNA delivery. Nature
Reviews Materials 2021, 6 (12), 1078-1094.

2. Whitaker, J. E.; Haugland, R. P.; Moore, P. L.; Hewitt, P. C.; Reese, M.; Haugland, R. P.,
Cascade Blue derivatives: Water soluble, reactive, Blue emission dyes evaluated as fluorescent labels
and tracers. Analytical Biochemistry 1991, 198 (1), 119-130.

3. Vollmann, H.; Becker, H.; Corell, M.; Streeck, H., Beiträge zur Kenntnis des Pyrens und
seiner Derivate. Justus Liebigs Annalen der Chemie 1937, 531 (1), 1-159.

4. Abdelkhalik, A. M.; Paul, N. K.; Jha, A., Concise synthesis of 12a-methyl-11-aryl-1,2-
dihydrobenzo[f]pyrrolo[1,2-a]quinolin-3(12aH)-ones as racemic 14-azaestrogen analogs. Steroids
2015, 98, 107-113.

5. Seeboth, H., The Bucherer Reaction and the Preparative Use of its Intermediate Products.
Angewandte Chemie International Edition in English 1967, 6 (4), 307-317.

6. Tietze, E.; Bayer, O., Die Sulfosäuren des Pyrens und ihre Abkömmlinge. Justus Liebigs
Annalen der Chemie 1939, 540 (1), 189-210.

7. Sharrett, Z.; Gamsey, S.; Hirayama, L.; Vilozny, B.; Suri, J. T.; Wessling, R. A.; Singaram, B.,
Exploring the use of APTS as a fluorescent reporter dye for continuous glucose sensing. Organic &
Biomolecular Chemistry 2009, 7 (7), 1461-1470.

8. Katritzky, A. R.; Kim, M. S.; Fedoseyenko, D.; Widyan, K.; Siskin, M.; Francisco, M., The
sulfonation of aromatic and heteroaromatic polycyclic compounds. Tetrahedron 2009, 65 (6), 1111-
1114.

9. Koeberg-Telder, A.; Cerfontain, H., Aromatic sulphonation. Part XL. Rates of sulphonation
ortho, meta, and para to a sulphonic acid group in methyl substituted benzenesulphonic acids.
Journal of the Chemical Society, Perkin Transactions 2 1973, (5), 633-637.

10. Yamamoto, T.; Terada, A.; Muramatsu, T.; Ikeda, K., Synthesis of Alkyl Isothiocyanates
From Primary Alkyl Amines Using Dicyandiamide as a Dehydrosulfurizing Agent. Organic
Preparations and Procedures International 1994, 26 (5), 555-557.

103

Chapter 4 - Design, Synthesis, and Applications of Metabolic
Chemical Reporters as Tools for Probing Glycosylation Pathways

Figure 4.1 Metabolic Engineering using modified monosaccharides
Introduction:
Nature functions on proteins. Proteins are formed through the combination of amino acid
monomers under the guide of genetic instructions encoded by nucleic acids like DNA and RNA.
DNA is transcribed into RNA and then translated into proteins. After translation, proteins can be
further developed and transformed to take on vastly different roles and responsibilities after
undergoing post-translational modifications: alkylation, glycosylation, oxidation, palmitoylation,
phosphorylation, ubiquitination – to name a few.
41
These post-translational modifications are in
response to the changes in the cellular environment that affect cellular homeostasis. The job of
biological systems is to maintain cellular homeostasis in order for the organism at large to thrive.
The organism can be unicellular or a multicellular system but with the single goal of survival. In
order to achieve that survival, the organism responds and adapts to their environment in the best
ways that it can or perishes. In order to better understand the relationship between post-translational
modifications and machinery of cellular homeostasis, we focused our attention on protein
glycosylation – in particular the incorporation of non-natural monosaccharides to proteins.
Carbohydrates are found throughout the cell and they decorate the cell surface of eukaryotic
cells where they play a central role in cell surface recognition between pathogens and the host cell –
for example, the influenza virus and sialic acid residues on erythrocytes.
42-45
In addition, selective
addition of glycans can direct transport of modified proteins to specific locations within the cell, in
O
OAc
O
OAc N
3
O
O
Copper(I) catalyzed azide-
alkyne cycloaddition
(CuAAC)
N
3
O
O N
N N
Protein
TAG
TAG
Protein
Cell Labelled Cell Lysate Tagged Cell Lysate
104

particular, mannose 6-phosphate addition to proteins directs transport to lysosomes.
46-47

Furthermore, glycan has made several appearances in therapeutics like lovenox, fragmin, Aranesp,
and arixtra.
48
While glycosylation has been understood to play a central role in these mechanisms,
progress towards a molecular understanding has been slow.
49-50

Figure 4.2 The Hexosamine Salvage Pathway
One way to investigate carbohydrate modifications of proteins is to feed cells modified
monosaccharides with either an alkyne or azide moiety, also known as metabolic chemical
reporters.
51-52
After lysis of the cell, we can incorporate tags upon modified proteins using copper(I)
catalyzed azide-alkyne cycloaddition for isolation and characterization. (Figure 4.1) This work was
first pioneered by the Bertozzi et al. in order to explore the promiscuity of sialyltransferases; this was
achieved by either adding an alkyne and azide moiety to the acetyl chains of N-acetylglucosamine
and N-acetylmannosamine amine.
53-56
From the current understanding, these monosaccharides are
able to enter into the hexosamine salvage pathway and incorporated into proteins.(Figure 4.2)
57-61
It
is presumed that the metabolism of certain unnatural sugars should not be easily incorporated into
105

the proteome of the cell; however, this is not the case. Despite not having a hydroxyl on the 6
position of modified N-acetylglucosamine and glucose derivatives, the Pratt group was able to
demonstrate that their monosaccharides with alkyne or azide moiety on the C-6 position are
incorporated into proteins – presumably these monosaccharides should not be incorporated because
they are unable to enter the hexosamine biosynthetic pathway and suggests an overlooked
promiscuity in this salvage pathway.
62-63

Figure 4.3 5-Alkyne GlcNAc and 5-Alkyne ManNAc
We wanted to further probe the promiscuity of this salvage pathway by synthesizing
monosaccharides that completely lack functional groups at C-6 position in order to challenge the
current understanding of what kind of monosaccharides the hexosamine salvage pathway will
tolerate. To this end, we chose 5-Alkyne GlcNAc and 5-Alkyne ManNAc as viable synthetic
candidates (Figure 4.3). The 5-Azido versions of the GlcNAc and ManNAc were not selected
because of potential instability by the azide at the C-5 position – the azide could leave (Figure 4.4).

Figure 4.4 Potential instability of 5Az derivative

O
OAc
AcHN
AcO
AcO
5-Alkyne GlcNAc
O
OAc
NHAc
AcO
AcO
5-Alkyne ManNAc
O
O
N
3
HO
HO
HAcN
H
H
O
O
N
3
HO
HO
HAcN
O
O
N
3
HO
HO
NHAc
H
H
O
O
N
3
HO
HO
NHAc
106

Results and Discussion
Synthesis of 5-Alkyne GlcNAc

Scheme 4.1 Retrosynthesis of 5-Alkyne GlcNAc

Scheme 4.2 Electrophilic Aldehyde at C5 position undergoes beta elimination
The synthesis of 5-AlkyneGlcNAc required the production of an alkyne at the C5 position.
To install the alkyne, we envisioned installing an aldehyde at the C5 position that we would convert
to an alkyne under Bestmann-Ohira reaction conditions. However, due to the inherent instability of
the aldehyde
64-66
, its propensity to undergo beta elimination
66-68
, and facile formation of hydrate
69-70
;
conventional synthetic strategies that required the use of acidic conditions
71-78
like Swern oxidation,
IPX, Dess Martin, PDC, and PCC to convert the primary alcohol at the C5 position to an aldehyde
were severely limited. Instead, we chose neutral conditions utilizing the Collins reagent.
79-81

Furthermore, selection of hydroxyl protecting groups was limited to protecting groups that were
compatible with alkynes. Thus, the synthetic strategy of utilizing butane 2,3-bisacetal protection on
the C3 and C4 hydroxyls was incorporated into our retrosynthesis. (Scheme 4.1)
O
OAc
AcHN
AcO
AcO
5-GlcNAcAlkyne
Deprotection
O
OMe
AcHN
O
O
OMe
OMe
Bestmann Reaction
O
OMe
AcHN
O
O
OMe
OMe
Oxidation
HO
O
OMe
AcHN
O
O
OMe
OMe
O H
O
OMe
AcHN
HO
HO
HO
Selective Protection
Anomeric Protection
O
OH
AcHN
HO
HO
HO
O
OAc
AcHN
AcO
O
O
O
OAc
NHAc
AcO
AcO
H
H
Beta Elimination
107

Scheme 4.3 Synthesis of 5AlkGlcNAc
Our sugar synthesis begins using N-acetylglucosamine (GlcNAc), we locked the pyranose
ring and protected the C1 hydroxyl by forming a cyclic acetal with the methoxy selectively in the
alpha position. We then protected the C3 and C4 hydroxyl by forming a 6,6-trans-fused bicycle
upon protection of the 1,2 diol with butane dione. After completing our protection strategy, the C6
hydroxyl was available for oxidation to the aldehyde. Attempts to oxidize the C6 hydroxyl to the
aldehyde were non-trivial – any presence of acid in the reaction resulted in decomposition of the
subsequent aldehyde. We were able to smoothly oxidize the C6 hydroxyl using Collins’ reagent and
telescoping the resultant product directly into the Bestmann-Ohira reaction to give the protected
product. Once the alkyne was installed, deprotection of the ring and acylation followed to give our
desired product.
O
HO
OH
NHAc
HO
HO
DOWEX 50
MeOH, Reflux, 24 hr
71%
O
HO
OMe
AcHN
HO
HO
CSA, Butane Dione, HC(OMe)
3
MeOH, Reflux, 24 hrs
83%
O
HO
OMe
AcHN
O
O
OMe
OMe
CrO
3
·2 Pyridine
DCM, 0C, 3 hrs
O
OMe
AcHN
O
O
OMe
OMe
K
2
CO
3
MeOH,
r.t.,16 hrs, 24%
(Over two steps)
MeO
P
N
2
MeO
O O
O
OMe
AcHN
O
O
OMe
OMe
i) 1M HCl, Reflux, 24 hrs
ii) Ac
2
O, Pyridine, r.t., 24hrs
(95% Over two steps)
O
OAc
NHAc
AcO
AcO
Ac
3
5AlkGlcNAc
O H
108

Figure 4.5 Protein incorporation of 5-AlkyneGlcNAc
Labelling of 5-AlkyneGlcNAc
Once synthesized, 3T3 cells were incubated with 5AlkGlcNAc and GlcNAlk for 16 hours at
200 uM. Upon lysis and tagging with Azide-Rodamine, the samples were imaged. The fluorescence
demonstrated that 5AlkGlcNAc was incorporated into 3T3 cellular proteins and has a distinctly
different banding pattern than GlcNAlk. (Figure 4.5)

Synthesis of 5-Alkyne ManNAc
After completing the synthesis of 5-Alkyne GlcNAc, we turned our attention to using the
same retrosynthesis scheme in order to make the 5-Alkyne ManNAc.

109

Scheme 4.4 Synthesis of 5-ManNAcAlkyne
Our sugar synthesis begins using N-acetylmannosamine (ManNAc), we locked the pyranose
ring and protected the C1 hydroxyl by forming a cyclic acetal with the methoxy selectively in the
alpha position. We then protected the C3 and C4 hydroxyl by forming a 6,6-trans-fused bicycle
upon protection of the 1,2 diol with butane dione. After completing our protection strategy, the C6
hydroxyl was available for oxidation to the aldehyde. Attempts to oxidize the C6 hydroxyl to the
aldehyde were non-trivial – any presence of acid in the reaction resulted in decomposition of the
subsequent aldehyde. We were able to smoothly oxidize the C6 hydroxyl using Collins’ reagent and
telescoping the resultant product directly into the Bestmann-Ohira reaction to give the protected
product.

Conclusions:
In summary, this synthetic strategy towards the production of 5-alkyne glucose and mannose
structures was concise and highly stereocontrolled for forming these class of glycan structures. The
synthetic availability of this compound and subsequent biological investigations will support further
identification of glycosyl transferases that are performing, what seems to be, unconventional
incorporation of exotic sugar moieties to the backbone of proteins. Further understanding this
mechanism would shed insight in this under explored pathway for discovery of new glycosylation
O
HO
OH
NHAc
HO
HO
DOWEX50
MeOH, Reflux, 24hrs
73%
O
HO
OMe
NHAc
HO
HO
CSA, Butadione, HC(OMe)
3
MeOH, Reflux, 24hrs
81%
O
HO
OMe
NHAc
O
O
OMe
OMe
O
OMe
NHAc
O
O
OMe
OMe
1) CrO
3
2Pyr, DCM, 3hrs
2)K
2
CO
3
, MeOH, r.t., 3hrs
10% (Over two steps)
MeO
P
MeO
O
O
1) 1M HCl, Reflux, 24hr
2) Ac
2
O, Pyridine, r.t., 24hr
O
OAc
NHAc
AcO
AcO
5-ManNAcAlkyne
N
2
110

pathways and their purposes in biology. Overall, these data offer new insight and pushing our
understanding of sugar incorporation in biological systems.

111

General Procedures:
All reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous
conditions, unless otherwise noted. Dry tetrahydrofuran (THF), toluene, diethyl ether (Et 2O), and
dichloromethane (DCM) were obtained. Yields refer to chromatographically and spectroscopically
(
1
H NMR) homogenous materials, unless otherwise stated. Reagents were purchased at the highest
commercial quality and used without further purification, unless otherwise stated. Reactions were
monitored both by thin-layer chromatography (TLC) carried out on 0.2 mm E. Merck silica gel
plates (60F-254) using UV light as visualizing agent. E. Merck silica gel (60, particle size 0.040-0.063
mm) was used for flash column chromatography. NMR spectra were recorded either on Varian
Mercury 400, Varian 400MR, Varian VNMRS 500, or Varian VNMRS 600 and calibrated using
residual undeutrated solvent (DMSO: 𝛿 H = 2.50 ppm, 𝛿 C = 39.52 ppm, CDCl 3: 𝛿 H = 7.26 ppm, 𝛿 C =
77.16 ppm; Acetone-d6: 𝛿 H = 2.05 ppm, 𝛿 C = 29.92 ppm) as an internal reference. The following
abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q =
quartet, quin = quintet, m = multiplet, br = broad.

112

Experimental:
Methyl 2-acetamido-2-deoxy-α-D-glucoside

In a round bottom flask with a stir bar, N-acetylglucosamine (1.0 g, 4.52 mmol) was added under
nitrogen. After addition, methanol (40 mL) was added under nitrogen and stirred for 10 minutes at
room temperature. After 10 minutes, dry dowex-50 (H
+
) (2.0 g) was added and the reaction mixture
stirred under reflux for 13 hours. After 13 hours, the dowex-50 (H
+
) resin was filtered off and rinsed
with MeOH (30 mL). The filtrate was transferred to a round bottom flask and put on a rotary
evaporator. The methanol was evaporated under reduced pressure. After evaporation, toluene (10
mL) was added 3x and evaporated under reduced pressure to give the desired product methyl 2-
acetamido-2-deoxy-α-D-glucoside (756.3 mg, 71%).
1
H NMR (500 MHz, dmso-d6) δ 7.73 (d, J = 8.3
Hz, 1H), 4.52 (d, J = 3.5 Hz, 1H), 3.68 – 3.62 (m, 4H), 3.48 – 3.45 (m, 1H), 3.45 – 3.41 (m, 1H),
3.34 – 3.29 (m, 2H), 3.23 (s, 3H), 3.15 – 3.09 (m, 1H), 1.82 (s, 3H).
13
C NMR (126 MHz, dmso-d6) δ
169.54, 97.97, 72.74, 70.89, 70.80, 60.90, 54.30, 53.79, 22.67.
Methyl 2-acetamido-2-deoxy-α-D-mannoside

In a round bottom flask with a stir bar, N-acetylmannosamine (1.963 g, 8.87 mmol) was added
under nitrogen. After addition, methanol (89 mL, 0.1 M) was added under nitrogen and allowed to
stir for 10 minutes at room temperature. under a nitrogen atmosphere. After 10 minutes, dry dowex-
50 (H
+
) (4.05 g) was added and the reaction mixture stirred under reflux for 13 hours. After 13
hours, the dowex-50 (H
+
) resin was filtered off and rinsed with MeOH (30 mL). The filtrate was
O
OMe
OH
HO
HO
AcHN
O
OMe
OH
O
O
NHAc
OMe
OMe
113

transferred to a round bottom flask and put on a rotary evaporator. The methanol was evaporated
under reduced pressure. After evaporation, toluene (10 mL) was added 3x and evaporated under
reduced pressure to give the desired product methyl 2-acetamido-2-deoxy-α-D-mannoside (730.4
mg, 35%).
1
H NMR (400 MHz, dmso-d6) δ 7.73 (d, J = 8.2 Hz, 1H), 4.99 (d, J = 4.3 Hz, 1H), 4.74
(d, J = 4.0 Hz, 1H), 4.52 (d, J = 3.5 Hz, 1H), 3.69 – 3.60 (m, 2H), 3.42 (d, J = 10.6 Hz, 3H), 3.34 –
3.28 (m, 1H), 3.23 (s, 3H), 3.17 – 3.08 (m, 1H), 1.82 (s, 3H).
13
C NMR (101 MHz, dmso-d6) δ 169.68,
98.01, 72.76, 70.92, 70.84, 60.95, 54.36, 53.85, 22.71.
Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-glucoside

In a round bottom flask with a stir bar, methyl 2-acetamido-2-deoxy-α-D-glucoside (756.3 mg, 3.21
mmol) was added under nitrogen. After addition, methanol (30 mL) was added under nitrogen and
allowed to stir for 20 minutes at room temperature. After 20 minutes, camphor sulfonic acid (74.6
mg, 0.321 mmol) was added in one portion to the stirred solution followed by trimethyl
orthoformate (1.853 g, 17.5 mmol) was added in one portion and then butane-2,3-dione (326.10
mgs, 3.78 mmol) was added in one portion and allowed to stir for 10 minutes at room temperature.
After 10 minutes, the reaction mixture was allowed to reflux for 16 hours. After 16 hours, the
mixture was pH adjusted to pH = 8 by addition of triethylamine and then transferred to a rotary
evaporator. After the transfer, the methanol is evaporated under reduced pressure. The crude
product was then purified by silica gel column chromatography (10% methanol in dichloromethane)
to give the titled product (914 mg, 81%).
1
H NMR (600 MHz, cdcl 3) δ 5.58 (d, J = 8.8 Hz, 1H), 4.73
(d, J = 3.6 Hz, 1H), 4.20 – 4.15 (m, 1H), 3.83 (dd, J = 11.1, 8.9 Hz, 1H), 3.78 (dd, J = 12.2, 2.0 Hz,
1H), 3.74 – 3.68 (m, 3H), 3.31 (s, 3H), 3.20 (d, J = 0.7 Hz, 6H), 1.97 (s, 3H), 1.23 (d, J = 0.7 Hz,
6H).
13
C NMR (151 MHz, cdcl 3) δ 170.12, 99.95, 99.61, 98.64, 69.96, 67.69, 66.71, 61.12, 55.07,
O
OMe
OH
O
O
AcHN
OMe
OMe
114

51.05, 47.89, 47.86, 23.41, 17.78, 17.68. Rf (10% MeOH in DCM): 0.425 Visualize with p-
anisaldehyde.
Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-mannoside

In a round bottom flask with a stir bar, methyl 2-acetamido-2-deoxy-α-D-mannoside (730 mg, 3.1
mmol) was added under nitrogen. After addition, anhydrous methanol (31 mL) was added under
nitrogen and allowed to stir for 10 minutes. After 10 minutes, camphor sulfonic acid (72 mg, 0.31
mmol) was added to the stirred solution followed by trimethyl orthoformate (1.791 g, 16.88 mmol)
and then butane-2,3-dione (315 mg, 3.66 mmol). After the addition, the reaction mixture was
allowed to stir under reflux conditions for 18 hours. After 18 hours, the mixture was pH adjusted to
pH = 8 by addition of triethylamine. After pH adjustment, the solution was transferred to a rotatory
evaporator and methanol was evaporated under reduced pressure. The crude product was then
purified by silica gel column chromatography (5% methanol in dichloromethane) to give the titled
product (482 mg, 44%).
1
H NMR (400 MHz, cdcl 3) δ 6.21 (d, J = 5.5 Hz, 1H), 4.87 (s, 1H), 4.26 –
4.19 (m, 1H), 4.12 (d, J = 5.7 Hz, 1H), 3.79 (d, J = 8.9 Hz, 2H), 3.72 (t, J = 9.3 Hz, 1H), 3.33 (d, J =
1.8 Hz, 3H), 3.27 (d, J = 1.8 Hz, 3H), 3.24 (d, J = 1.8 Hz, 3H), 2.03 (d, J = 1.8 Hz, 3H), 1.27 (dd, J =
4.5, 1.8 Hz, 6H).
13
C NMR (101 MHz, cdcl 3) δ 171.38, 100.42, 100.14, 99.95, 69.63, 65.18, 63.54,
61.10, 55.12, 52.04, 48.28, 48.12, 23.53, 17.84, 17.79.

O
OMe
OH
O
O
NHAc
OMe
OMe
115

Methyl 2-acetamido-6-aldehydo-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-
glucoside

In a reaction flask equipped with a magnetic stir bar, dry pyridine (0.8 mL, 9.96 mmol) was added to
anhydrous dichloromethane (8 mL) and allowed to stir at 25
o
C for 10 minutes – maintain the
temperature below 30
o
C. After 10 minutes, chromium trioxide (498 mg, 4.98 mmol) was added in
one portion to the reaction flask and allowed to stir for 1 hour. After one hour, the solution has
turned a deep burgundy color – signifying the formation of the Collins reagent. After Collins reagent
formation, a solution of methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-
glucoside (290 mg, 0.83 mmol) dissolved in anhydrous dichloromethane (5 mL) was added dropwise
over 10 minutes to the reaction flask and allowed to stir for 1 hour at room temperature. After 1
hour, the reaction mixture was diluted in dichloromethane (20 mL) and transferred to a separatory
funnel. The organic layers were washed 3x with saturated sodium bicarbonate aqueous solution (10
mL). After washing, the solution was dried over sodium sulfate for 20 minutes. After 20 minutes,
the solution was filtered and the filtrate was collected. The filtrate was transferred to a round bottom
flask and put on a rotary evaporator. After transferring, the dichloromethane was evaporated under
reduced pressure. A sample of the material was taken to the NMR to confirm the presence of
aldehyde. The material was then pushed into the next reaction – the product was very acid sensitive
and decomposition of the product did occur if the desired product was left to stand regards if under
nitrogen or placed in a -20
o
C freezer.
1
H NMR (400 MHz, cd 3cn) δ 9.63 (s, 1H), 6.34 (s, 1H), 4.71
(d, J = 3.4 Hz, 1H), 4.11 – 4.05 (m, 2H), 3.88 – 3.82 (m, 1H), 3.79 (d, J = 10.2 Hz, 1H), 3.37 (s, 3H),
3.22 (s, 3H), 3.16 (s, 3H), 1.89 (s, 3H), 1.22 (d, J = 5.6 Hz, 6H). Rf (10% MeOH in DCM): 0.5
Visualize with p-anisaldehyde.
O
OMe
O
O
O
AcHN
OMe
OMe
H
116

Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-5-ethynyl-α-D-glucoside

In a reaction flask equipped with a magnetic stir bar, methyl 2-acetamido-6-aldehydo-2-deoxy-3,4-O-
(2,3-dimethoxybutane-2,3-diyl)-α-D-glucoside (0.83 mmol), from the previous reaction, was
dissolved in anhydrous methanol (8.3 mL, 0.1M) under nitrogen. After addition, the solution was
allowed to stir for 20 minutes. After 20 minutes, freshly prepared dimethyl-(1-diazo-2-oxopropyl)-
phosphonate (3.32 mL, 0.996 mmol, 1.2 eq) in methanol was added dropwise over 5 minutes and
followed by addition of potassium carbonate (230 mg, 1.66 mmol, 2.0 eq). After complete addition,
the reaction mixture was diluted with dichloromethane (30 mL) and transferred to a separatory
funnel. After transfer, the organic layer was washed with saturated sodium bicarbonate solution.
After washing, the aqueous layer was extracted three times with dichloromethane (25 mL). After
extraction, the organic layers were combined and washed with saturated sodium chloride aqueous
solution (10 mL). After washing, the solution was dried over sodium sulfate for 20 minutes. After 20
minutes, the solution was filtered and the filtrate was collected. The filtrate was transferred to a
round bottom flask and put on a rotary evaporator. After the transfer, the dichloromethane was
evaporated under reduced pressure. The crude mixture was purified on silica gel using 10%
methanol in dichloromethane to give the titled product (69 mg, 24%).
1
H NMR (500 MHz, cdcl 3) δ
5.46 (d, J = 8.8 Hz, 1H), 4.76 (d, J = 3.6 Hz, 1H), 4.36 (dd, J = 10.0, 2.1 Hz, 1H), 4.24 (ddd, J =
10.6, 8.8, 3.6 Hz, 1H), 3.80 – 3.75 (m, 1H), 3.70 (t, J = 9.8 Hz, 1H), 3.39 (s, 3H), 3.29 (s, 3H), 3.22 (s,
3H), 2.47 (d, J = 1.5 Hz, 1H), 2.00 (s, 3H), 1.29 (s, 3H), 1.27 (s, 3H).
13
C NMR (126 MHz, cdcl 3) δ
170.01, 100.14, 100.11, 98.98, 79.60, 77.16, 74.15, 70.47, 67.68, 60.83, 55.71, 50.70, 47.95, 47.86,
23.51, 17.76, 17.69.

O
OMe
O
O
AcHN
OMe
OMe
117

Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-5-ethynyl-α-D-
mannoside

Aldehyde formation:
In a reaction flask equipped with a magnetic stir bar, dry pyridine (0.98 mL, 12.15 mmol) was added
under nitrogen. After addition, anhydrous dichloromethane (10.1 mL) was added and allowed to stir
25
o
C for 10 minutes. After 10 minutes, chromium trioxide (607 mg, 6.07 mmol) was added in one
portion to the reaction flask and allowed to stir for 1 hour at 25
o
C. After 1 hour, a solution of
methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-D-mannoside (354 mg, 1.01
mmol) dissolved in anhydrous dichloromethane (10.1 mL) and added dropwise via syringe over 10
minutes to the reaction flask and allowed to stir for 1 hour at room temperature. After 1 hour, the
reaction mixture was diluted in dichloromethane (20 mL) and transferred to a separatory funnel. The
organic layers were washed 3x with saturated sodium bicarbonate aqueous solution (10 mL). After
washing, the solution was dried over sodium sulfate for 20 minutes. After 20 minutes, the solution
was filtered and the filtrate was collected. The filtrate was transferred to a round bottom flask and
put on a rotary evaporator. After transferring, the dichloromethane was evaporated under reduced
pressure. A sample of the material was taken to the NMR to confirm the presence of aldehyde. The
material was then pushed into the next reaction.

O
OMe
O
O
O
NHAc
OMe
OMe
H
118

Alkyne Formation:

In a reaction flask equipped with a magnetic stirbar, methyl 2-acetamido-6-aldehydo-2-deoxy-3,4-O-
(2,3-dimethoxybutane-2,3-diyl)-α-D-mannoside (1.01 mmol), from the previous reaction, was
dissolved in anhydrous methanol (10.1 mL, 0.1 M). After addition, freshly prepared dimethyl-(1-
diazo-2-oxopropyl)-phosphonate (2.42 mL, 1.21 mmol, 1.2 eq) in methanol was added dropwise
over 10 minutes. After addition, potassium carbonate (279 mg, 2.02 mmol, 2.0 eq) was added in one
portion the flask. After addition, the reaction was allowed to stir for 3 hours at room temperature.
After 3 hours, the reaction mixture was diluted with dichloromethane (30 mL) and transferred to a
separatory funnel. After transfer, the organic layer was washed with saturated sodium bicarbonate
solution (10 mL). After washing, the aqueous layer was extracted three times with dichloromethane
(25 mL). After extraction, the organic layers were combined and washed with saturated sodium
chloride aqueous solution. After washing, the solution was dried over sodium sulfate for 20 minutes.
After 20 minutes, the solution was filtered and the filtrate was collected. The filtrate was transferred
to a round bottom flask and put on a rotary evaporator. After the transfer, the dichloromethane was
evaporated under reduced pressure. The crude mixture was purified on silica gel using 10%
methanol in dichloromethane to give the titled product (35.7 mg, 10% over two steps).
1
H NMR
(500 MHz, cdcl 3) δ 5.97 (d, J = 5.6 Hz, 1H), 4.87 (s, 1H), 4.22 (dd, J = 10.4, 5.0 Hz, 1H), 4.14 – 4.10
(m, 1H), 4.10 – 4.03 (m, 1H), 3.80 (d, J = 3.1 Hz, 1H), 3.33 (s, 3H), 3.26 (s, 3H), 3.24 (s, 3H), 2.31 (s,
1H), 2.03 (s, 3H), 1.27 (d, J = 4.7 Hz, 6H).
13
C NMR (126 MHz, cdcl 3) δ 171.23, 100.45, 100.07,
99.95, 67.62, 65.14, 63.58, 55.11, 52.03, 48.26, 48.10, 31.04, 23.62, 17.82, 17.77. Rf (100% EtOAc):
0.354 Stain with KMnO 4.

O
OMe
O
O
NHAc
OMe
OMe
119

(2R,3R,4R,5R,6R)-3-acetamido-6-ethynyltetrahydro-2H-pyran-2,4,5-triyl triacetate

In a reaction flask equipped with a magnetic stir bar, methyl 2-acetamido-2-deoxy-3,4-O-(2,3-
dimethoxybutane-2,3-diyl)-5-ethynyl-α-D-glucoside (69 mg, 0.2 mmol) was added under nitrogen.
After addition, 1 M HCl (2 mL, 0.1 M) was added under nitrogen and allowed to stir at 50
o
C for 12
hours. After 12 hours, toluene (15 mL) was added to the reaction mixture and then the reaction
mixture as transferred to a rotary evaporator. After transferring, the water and toluene was
evaporated under reduced pressure. After removing the toluene, crude product was dried under high
vacuum for 3 hours to remove any residual water and acid. After 3 hours, the crude product was
transferred to a reaction flask equipped with a magnetic stir bar, filled with a nitrogen atmosphere,
and then dry pyridine (4 mL) was added. To the stirring mixture, 0.1 mL of acetic anhydride (5 eq,
1.0 mmol) was added dropwise and allowed to stir for 16 hours. After 16 hours, toluene (20 mL)
was added to the reaction flask and the reaction flask was transferred to a rotary evaporator. After
transferring, the toluene and pyridine was evaporated under reduced temperature to give pure
product (64.6 mg, 95% over two step).
1
H NMR (500 MHz, cdcl 3) δ 6.19 (d, J = 3.6 Hz, 1H), 5.55
(d, J = 8.9 Hz, 1H), 5.27 (t, J = 9.7 Hz, 1H), 4.52 (dd, J = 10.0, 2.2 Hz, 1H), 4.50 – 4.43 (m, 2H),
2.47 (d, J = 2.1 Hz, 1H), 2.19 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.92 (s, 3H).
13
C NMR (126 MHz,
cdcl 3) δ 171.78, 169.99, 169.01, 168.48, 90.34, 78.42, 74.87, 70.21, 62.86, 60.65, 51.12, 29.83, 23.18,
21.05, 20.86.
Cell Culture:
T3T were grown in Dulbecco's Modified Eagle media (Corning) supplemented with 10% Fetal
Bovine Serum (Altanta Biologicals) and were grown at 37 °C and 5.0% CO
2
.

O
OAc
AcO
AcO
AcHN
120

Metabolic Labeling:
To cells at 80−85% confluency, media was exchanged for fresh media containing Ac
3
5AlkGlcNAc
or GlcNAlk for 16 hours at 200 µM (in DMSO), or DMSO vehicle was added as indicated. Upon
lysis and tagging with Azide-Rodamine, the samples were imaged.
Analysis by In-Gel Fluorescence:
Cells were collected by trypsinization and pelleted by centrifugation for 4 min at 2000 g, followed
by washing 2× with PBS (1 mL). Cell pellets were then resuspended in 100 μL of 1% NP-40 lysis
buffer [1% NP-40, 150 mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete, Mini,
EDTA-free Protease Inhibitor Cocktail Tablets (Roche) for 20 min and then centrifuged for 10 min
at 10,000 g at 4 °C. The supernatant (soluble cell lysate) was collected and the protein concentration
was determined by BCA assay (Pierce, ThermoScientific). Protein concentration was normalized to 1
μg μL
−1
, and to 200 μg of protein, newly made click chemistry cocktail (12 μL) was added [Alkyne-
TAMRA tag (Click Chemistry tools, 100 μM, 10 mM stock solution in DMSO); tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50 mM freshly prepared stock solution in
water); tris[(1-benzyl-1-H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (100 μM, 10 mM stock solution in
DMSO); CuSO
4
·5H
2
O (1 mM, 50 mM freshly prepared stock solution in water)]. The reaction was
gently vortexed and allowed to sit at room temperature for 1 hour. Upon completion, 1 mL of ice
cold methanol was added to the reaction, and it was placed at −20 °C for 2 hours to precipitate
proteins. The reactions were then centrifuged at 10,000 g for 10 minutes at 4 °C. The supernatant
was removed, the pellet was allowed to air-dry for 15 min, and then 50 μL 4% SDS buffer (4% SDS,
150 mM NaCl, 50 mM TEA, pH 7.4) was added to each sample. The mixture was sonicated in a
water bath sonicator to ensure complete dissolution, and 50 μL of 2× SDS-free loading buffer (20%
glycerol, 0.2% bromophenol blue, 1.4% β-mercaptoethanol, pH 6.8) was then added. The samples
121

were boiled for 5 minutes at 97 °C, and 40 μg of protein was then loaded per lane for SDS-PAGE
separation. Following SDS-PAGE separation, gels were scanned on a Typhoon 9400 Variable Mode
Imager (GE Healthcare) using a 532 nm for excitation and 30 nm bandpass filter centered at 610 nm
for detection.

122

Representative Spectra

Figure 4.6
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-glucoside
123

Figure 4.7
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-glucoside
124

Figure 4.8
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-
2,3-diyl)-α-D-glucoside
125

Figure 4.9
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-
2,3-diyl)-α-D-glucoside
126

Figure 4.10
1
H NMR Spectrum of Methyl 2-acetamido-6-aldehydo-2-deoxy-3,4-O-(2,3-
dimethoxybutane-2,3-diyl)-α-D-glucoside
127

Figure 4.11
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-
2,3-diyl)-5-ethynyl-α-D-glucoside
128

Figure 4.12
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-
2,3-diyl)-5-ethynyl-α-D-glucoside
129

Figure 4.13
1
H NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-mannoside
130

Figure 4.14
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-α-D-mannoside
131

Figure 4.15
1
H NMR Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-α-
D-mannoside
132

Figure 4.16
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-
dimethoxybutane-2,3-diyl)-α-D-mannoside
133

Figure 4.17
1
H NMR Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-dimethoxybutane-2,3-diyl)-5-
ethynyl-α-D-mannoside
134

Figure 4.18
13
C NMR Spectrum of Methyl 2-acetamido-2-deoxy-3,4-O-(2,3-
dimethoxybutane-2,3-diyl)-5-ethynyl-α-D-mannoside

135

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Abstract (if available)

Abstract The ability to utilize the skills and insights of organic chemistry towards understanding the fundamental mechanisms of the cell has been a central theme in this dissertation. Each of these projects has been directed to make new and useful tools for exploration of the cell and understanding it’s underlying mechanisms.
Chapter 1 focused on the synthesis of exendin-4 dye conjugates for the isolation of beta pancreatic cells in heterogenous populations of cells. These peptide-dye conjugates were coupled through the copper-catalyzed azide-alkyne cycloaddition and bind selectively to the GLPR-1 receptor of pancreatic β-cells. Using these tools, we have developed a framework for isolating beta pancreatic cells via their unique cellular receptors.
Chapter 2 focused on the development of ELISA assays for the detection of antibodies in response to the SARS-CoVID-19 pandemic. In the process of these studies, we first developed an IgG antibody ELISA in serum and then extend our assay to work in a variety of saliva samples. Our studies revealed that the difficulty in detecting antibodies in saliva was not just the low concentrations of IgG present in the mouth but also the confounding interactions of enzymes, proteins, and food material present in the mouth. Despite these difficulties, these assays can be quickly utilized in the field and the development framework can be used to develop new ELISA assays for future pandemics.
Chapter 3 focused on the development of fluorescent pyrene-based silica nanoparticles that were inspired by 8-hydroxypyrene-1,3,6-sulfonic acid. In the process, we developed a synthetic strategy to smoothly access 8-aminoypyrene-1,3,6-sulfonic acid, 8-aminoypyrene-1,3,6-sulfonamides, and 8-aminoypyrene-1,3,6-sulfonyl fluorides dyes. We have demonstrated that 8-aminoypyrene-1,3,6-sulfonamides can be covalently incorporated into the silica framework of silica nanoparticles.
Chapter 4 focused on development of non-natural alkyne modified N-acetylglucosamine and N-acetylmannosamine derivatives to probe the metabolic promiscuity. After developing a synthetic pathway towards the 5-Alkyne GlcNAc, we incubated 3T3 cells with the compound and determined that this non-natural sugar was incorporated into proteins despite not having an entry into cellular metabolism via the hexosamine salvage pathway.

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Asset Metadata

Creator Moreno, Jose Ricardo (author)

Core Title Peering into the cell: click chemistry and novel dyes towards enhanced biomedical imaging

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2022-12

Publication Date 08/31/2022

Defense Date 08/31/2022

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

Tag click chemistry,dyes,nanoparticles,OAI-PMH Harvest,pigments

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Fokin, Valery (committee chair), Williams, Travis (committee member), Zavaleta, Cristina (committee member)

Creator Email josericm@usc.edu,jrmoreno1309@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111384551

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Source 20220901-usctheses-batch-976 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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