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CHEMOSELECTIVE CYSTEINE
MODIFICATION OF PEPTIDES AND
PROTEINS USING 1H-ISOINDOLIUM-
BASED ELECTRON-DEFICIENT ALLENES
O WA YI
MPhil
The Hong Kong Polytechnic University

2021
The Hong Kong Polytechnic University
Department of Applied Biology and Chemical Technology
Chemoselective Cysteine Modification of Peptides

and Proteins using 1H-Isoindolium-based Electron-
Deficient Allenes
O Wa Yi
A thesis submitted in partial fulfilment of the requirements for

the degree of Master of Philosophy
September 2020
CERTIFICATE OF ORIGINALITY
I hereby declare that this thesis is my own work and that, to the best of my knowledge
and belief, it reproduces no material previously published or written, nor material that
has been accepted for the award of any other degree or diploma, except where due
acknowledgement has been made in the text.
_______________________
O WA YI
I
ABSTRACT
Site-selective modification of peptides and proteins has been recognized as a
powerful tool for biological studies. It is of ongoing interest to develop new
modification strategies with high selectivity and efficiency under mild reaction
conditions. Cysteine with low abundance and high nucleophilicity is an ideal residue
for selective modification. A novel approach for cysteine modification of peptides and
proteins using 1H-isoindolium-based electron-deficient allenes was developed.
A novel electrophile-mediated cascade cyclization/iodination of propargylamine-
based 1,6-diyne has been developed for the synthesis of a new class of spiro 1H-
isoindoliums 3. Upon treatment with base, the alkyne moieties of 1H-isoindoliums were
transformed to allene functionalities to give the corresponding allene products. The
application of this new class of 1H-isoindolium-based electron-deficient allenes to
cysteine modification of peptides and proteins was presented. By treatment of cysteine-
containing peptides with 5 equivalents of allene reagents in aqueous media at 25 °C for
4 h, cysteine-modified peptides were obtained in good conversions up to 96% as
confirmed by LC–MS/MS analysis. Allene reagents with different functional groups
were compatible with this modification and the modified products were stable towards
excessive thiol-containing reagents and reducing agents. Further application of this
modification was demonstrated in protein modification and labeling. Fluorescent tags
could be labeled on the cysteine-containing proteins by sequential modification using
the azide-alkyne click reaction.
II
Based on the success of this newly developed cysteine modification method, a one-
step procedure for preparation of electron deficient allene reagents for bioconjugation
was established to enhance the efficiency of the modification. By reaction of
propargylamine-based 1,6-diynes with iodine monochloride for the formation of in situ
generated 1H-isoindoliums, followed by the addition of 1 equivalent of reducing agent,
stock solutions of in situ generated electron deficient allene reagents with high stability
could be prepared for direct use in bioconjugation.
With 1 equivalent of the in situ generated allene reagents, the modification of
peptides could proceed with high efficiency up to 99% conversion in 4 h and the
performance was comparable with the isolated allene reagents. The in situ generated
allene reagents could be further applied to protein modification and labeling. The results
demonstrated that this approach could serve as an alternative method for preparation of
allene reagents and be utilized in the newly developed cysteine modification using
electron-deficient allenes.
III
ACKNOWLEDGEMENT
I would like to express my deepest gratitude to my supervisor, Dr. Man-Kin Wong
for his supervision, support and encouragement as well as invaluable advices for my
research study in these two years.
I would like to convey my greatest appreciation to my group members: Dr. Ka-
Yan Karen Kung, Dr. Jian-Fang Cui, Mr. Bin Yang, Mr. Jie-Ren Deng, Mr. Jia-Jun
Jiang, Mr. Wai-Ming Yip, and Miss Hoi-Yi Sit for their precious guidance and patience
in providing useful and constructive recommendations for my research. I would also
like to express my sincere thanks to colleagues in Shenzhen Research Institute: Dr.
Qiong Yu and Mr. Li Wu Huang for their efforts and assistance in synthesis of some
compounds for this project.
Besides, I would like to thank all the staffs and technicians of the Department of
Applied Biology and Chemical Technology in the Hong Kong Polytechnic University
for their genuine help and technical support, especially Dr. Pui-Kin So and Dr. Melody
Yee-Man Wong for training and assistance in mass spectrometry.
I sincerely thank Dr. Man-Kin Wong and his research group again. It is my honor
to work and discuss with them in these two years and I will never forget this valuable
and memorable experience.
IV
TABLE OF CONTENT
CERTIFICATE OF ORIGINALITY I
ABSTRACT II
ACKNOWLEDGEMENT IV
TABLE OF CONTENT V
LIST OF FIGURES X
LIST OF TABLES XVII
LIST OF ABBREVIATIONS XVIII
Chapter 1 Introduction 1
1.1 Allene Chemistry 1
1.1.1 Synthesis of Allenes 3
1.1.2 Reactions of Allenes 10
1.2 Chemical Modification of Peptides and Proteins 16
1.2.1 Lysine and N-Terminal Modification 17
1.2.2 Modification of Other Natural Amino Acids 23
1.2.3 Modification of Unnatural Amino Acids 27
1.3 Cysteine Modification 30
1.3.1 Classical Methods 32
1.3.2 Transition Metal-Free Modifications 35
1.3.3 Transition Metal-Mediated Modifications 38
V
Chapter 2 Cysteine Modification of Peptides and Proteins using isolated
Electron-Deficient Allenes 40
2.1 Introduction 40
2.2 Results and Discussion 42
2.2.1 Modification of Cysteine-Containing Peptides Using Isolated Electron-
Deficient Allenes 42
2.2.1.1 Synthesis of Electron-Deficient Allenes 42
2.2.1.2 Optimization of Modification Reaction Conditions 45
2.2.1.3 Model Reaction of Cysteine Modification by Electron-Deficient
Allenes 49
2.2.1.4 Investigation of Cysteine Selectivity of the Modification 50
2.2.1.5 Investigation of Scope of Allene reagents 53
2.2.1.6 Stability Study of Allene-modified Bioconjugates 58
2.2.2 Modification of Cysteine-Containing Proteins Using Isolated Electron-
Deficient Allenes 59
2.2.2.1 Modification of BSA Protein with Allene Reagents 59
2.2.2.2 Control Experiments with Non-Free Cysteine-Containing Proteins 63
2.2.2.3 Fluorescent Labeling of Proteins 64
2.3 Conclusion 66
2.4 Suggestions for Future Research 67
VI
Chapter 3 Cysteine Modification of Peptides and Proteins using in situ
Generated Electron-Deficient Allenes 69
3.1 Introduction 69
3.2 Results and Discussion 70
3.2.1 Modification of Cysteine-Containing Peptides Using in situ Generated
3.2.1.1 Optimization of Reaction Conditions 70
3.2.1.2 Time Course Experiments of Cysteine Modification of Peptides by in
situ Generated Allene Reagents 75
3.2.1.3 Investigation of Scope of in situ Generated Allene Reagents 77
3.2.2 Modification of Cysteine-Containing Proteins Using in situ Generated
3.3 Conclusion 86
Chapter 4 Experimental Section 87
4.1 General procedures 87
4.2 Literature Reference of Compounds 89
4.3 Calculation of Peptide Conversion 89
4.4 Calculation of Protein Conversion 90
4.5 Synthesis of Propargylamine-based 1,6-diynes 2 90
4.5.1 Synthesis of Coumarin-derived Propargylamine-based 1,6-diyne 2g 94
4.6 Synthesis of 1H-isoindoliums 3 97
4.7 Synthesis of Electron-Deficient Allenes 4 103
VII
4.8 Synthesis of Model Compound 7 108
4.9 Modification of Peptides Using Isolated Electron-Deficient Allene Reagents 4
110
4.10 Time Course Studies on the Modification of Peptide AYEMWCFHQR 1a
Using Isolated Electron-Deficient Allene Reagents 4 at Different pH Values 110
Using Isolated Electron-Deficient Allene Reagents 4 at Different Temperatures 110
4.12 Studies on the Stability of the Modified Peptide 5a 111
4.13 Modification of Proteins Using Isolated Electron-Deficient Allene Reagents 4
111
4.14 Sequential Modification of Proteins via Huisgen Azide-Alkyne Cycloaddition
111
4.15 SDS-PAGE Analysis of Native and Modified Proteins 111
4.16 Preparation of in situ Generated Electron-Deficient Allene Reagents 4 112
4.17 Modification of Peptides Using in situ Generated Electron-Deficient Allene
Reagents 4 112
Using in situ Generated Electron-Deficient Allene Reagents 4 at Different pH
Values 113
Using in situ Generated Electron-Deficient Allene Reagents 4 at Different
Temperatures 113
VIII
Using in situ Generated Electron-Deficient Allene Reagents 4a-d 114
4.21 Modification of Proteins Using in situ Generated Electron-Deficient Allene
Reagents 4 114
4.22 MS Spectra 115
4.23 NMR Spectra 172
References 204
IX
LIST OF FIGURES
Chapter 1 Introduction
Figure 1.1 (a) Structure of allene; (b) Description of π bonding of allene. 1
Figure 1.2 Examples of naturally occurring allenic compounds. 2
Figure 1.3 Examples of allenic pharmacologically active compounds. 2
Chapter 2 Cysteine Modification of Peptides and Proteins using isolated Electron-
Deficient Allenes
Figure 2.1 The synthesis of allene 4a. 43
Figure 2.2 MS/MS spectrum of cysteine-modified peptide 5a. 45
Figure 2.3 Time course experiments of the formation of cysteine modified peptide 5a
at different pH values. 47
at different temperatures. 48
Figure 2.8 Mass spectrum of (a) BSA-1; (b) BSA-2 and (c) BSA-3. 61
X
Figure 2.9 Molecular feature extraction spectrum of cysteine modified
KGLVLIAFSQYLQQCPF after chymotrypsin digestion of BSA-1. 62
SQYLQQCPFDEHVKLVNELTEF after chymotrypsin digestion of BSA-1. 62
Figure 2.11 Mass spectrum of RNaseA after reaction with 4a. 63
Figure 2.12 Mass spectrum of lysozyme after reaction with 4a. 63
Figure 2.13 Mass spectrum of rhodamine-labeled BSA-4. 65
Figure 2.14 SDS-PAGE analysis of BSA, allene-modified BSA-3 and rhodamine-
labelled BSA-4. 65
Chapter 3 Cysteine Modification of Peptides and Proteins using in situ Generated
Electron-Deficient Allenes
Figure 3.1 LC–MS analysis of in situ generated allene reagent (TIC chromatogram).
71
Figure 3.2 LC–MS analysis of side product 5a' (TIC chromatogram). 73
Figure 3.3 MS/MS analysis of side product 5a'. 74
at (a) different pH values; (b) different temperatures. 76
Figure 3.5 Time course experiment of formation of modified peptides 5a-d. 81
Figure 3.6 Mass spectrum of (a) BSA-6; (b) BSA-7; (c) BSA-8 and (d) BSA-9. 83
XI
Figure 3.7 Mass spectrum of fluorescent-labeled BSA-10. 84
Figure 3.8 SDS-PAGE analysis of native BSA and BSA-10. 85
Chapter 4 Experimental Section
Figure 4.1 Extracted ion chromatogram of native peptide AYEMWCFHQR 1a. 115
Figure 4.2 Mass spectrum of native peptide AYEMWCFHQR 1a. 115
Figure 4.3 MS/MS spectrum of native peptide AYEMWCFHQR 1a. 116
Figure 4.4 Extracted ion chromatogram of cysteine-modified peptide 5a. 116
Figure 4.5 Mass spectrum of cysteine-modified peptide 5a. 117
Figure 4.7 Extracted ion chromatogram of cysteine-modified peptide 5b. 118
Figure 4.8 Mass spectrum of cysteine-modified peptide 5b. 118
Figure 4.9 MS/MS spectrum of cysteine-modified peptide 5b. 119
Figure 4.10 Extracted ion chromatogram of cysteine-modified peptide 5c. 120
Figure 4.11 Mass spectrum of cysteine-modified peptide 5c. 120
Figure 4.12 MS/MS spectrum of cysteine-modified peptide 5c. 121
Figure 4.13 Extracted ion chromatogram of cysteine-modified peptide 5d. 122
Figure 4.14 Mass spectrum of cysteine-modified peptide 5d. 122
Figure 4.15 MS/MS spectrum of cysteine-modified peptide 5d. 123
XII
Figure 4.16 Extracted ion chromatogram of cysteine-modified peptide 5e. 124
Figure 4.17 Mass spectrum of cysteine-modified peptide 5e. 124
Figure 4.18 MS/MS spectrum of cysteine-modified peptide 5e. 125
Figure 4.19 Extracted ion chromatogram of cysteine-modified peptide 5f. 126
Figure 4.20 Mass spectrum of cysteine-modified peptide 5f. 126
Figure 4.21 MS/MS spectrum of cysteine-modified peptide 5f. 127
Figure 4.22 Extracted ion chromatogram of cysteine-modified peptide 5g. 128
Figure 4.23 Mass spectrum of cysteine-modified peptide 5g. 128
Figure 4.24 MS/MS spectrum of cysteine-modified peptide 5g. 129
Figure 4.25 Extracted ion chromatogram of cysteine-modified peptide 5h. 130
Figure 4.26 Mass spectrum of cysteine-modified peptide 5h. 130
Figure 4.27 MS/MS spectrum of cysteine-modified peptide 5h. 131
Figure 4.28 Extracted ion chromatogram of native peptide STSSSCNLSK 1b. 132
Figure 4.29 Mass spectrum of native peptide STSSSCNLSK 1b. 132
Figure 4.30 MS/MS spectrum of native peptide STSSSCNLSK 1b. 133
Figure 4.34 Extracted ion chromatogram of cysteine-modified peptide 6b. 135
XIII
Figure 4.35 Mass spectrum of cysteine-modified peptide 6b. 135
Figure 4.36 MS/MS spectrum of cysteine-modified peptide 6b. 136
Figure 4.37 Extracted ion chromatogram of cysteine-modified peptide 6c. 137
Figure 4.38 Mass spectrum of cysteine-modified peptide 6c. 137
Figure 4.39 MS/MS spectrum of cysteine-modified peptide 6c. 138
Figure 4.40 Extracted ion chromatogram of cysteine-modified peptide 6d. 139
Figure 4.41 Mass spectrum of cysteine-modified peptide 6d. 139
Figure 4.42 MS/MS spectrum of cysteine-modified peptide 6d. 140
Figure 4.43 Extracted ion chromatogram of cysteine-modified peptide 6e. 141
Figure 4.44 Mass spectrum of cysteine-modified peptide 6e. 141
Figure 4.45 MS/MS spectrum of cysteine-modified peptide 6e. 142
Figure 4.46 Extracted ion chromatogram of cysteine-modified peptide 6f. 143
Figure 4.47 Mass spectrum of cysteine-modified peptide 6f. 143
Figure 4.48 MS/MS spectrum of cysteine-modified peptide 6f. 144
Figure 4.49 Extracted ion chromatogram of cysteine-modified peptide 6g. 145
Figure 4.50 Mass spectrum of cysteine-modified peptide 6g. 145
Figure 4.51 MS/MS spectrum of cysteine-modified peptide 6g. 146
Figure 4.52 Extracted ion chromatogram of cysteine-modified peptide 6h. 147
Figure 4.53 Mass spectrum of cysteine-modified peptide 6h. 147
XIV
Figure 4.54 MS/MS spectrum of cysteine-modified peptide 6h. 148
Figure 4.55 Extracted ion chromatogram of native peptide KSTFC 1c. 149
Figure 4.56 Mass spectrum of native peptide KSTFC 1c. 149
Figure 4.57 MS/MS spectrum of native peptide KSTFC 1c. 150
Figure 4.61 Extracted ion chromatogram of native peptide CSKFR 1d. 152
Figure 4.62 Mass spectrum of native peptide CSKFR 1d. 152
Figure 4.63 MS/MS spectrum of native peptide CSKFR 1d. 153
Figure 4.67 Extracted ion chromatogram of native peptide STSSSANLSK 1e. 156
Figure 4.68 Mass spectrum of native peptide STSSSANLSK 1e. 156
Figure 4.69 MS/MS spectrum of native peptide STSSSANLSK 1e. 157
Figure 4.70 Extracted ion chromatogram of native peptide STSSSHNLSK 1f. 158
Figure 4.71 Mass spectrum of native peptide STSSSHNLSK 1f. 158
Figure 4.72 MS/MS spectrum of STSSSHNLSK 1f. 159
XV
Figure 4.73 Extracted ion chromatogram of native peptide AYEMWSFHQR 1g. 160
Figure 4.74 Mass spectrum of native peptide AYEMWSFHQR 1g. 160
Figure 4.75 MS/MS spectrum of native peptide AYEMWSFHQR 1g. 161
Figure 4.76 Extracted ion chromatogram of native peptide WSKFR 1h. 162
Figure 4.77 Mass spectrum of native peptide WSKFR 1h. 162
Figure 4.78 MS/MS spectrum of native peptide WSKFR 1h. 163
Figure 4.79 Extracted ion chromatogram of native peptide YSKFR 1i. 164
Figure 4.80 Mass spectrum of native peptide YSKFR 1i. 164
Figure 4.81 MS/MS spectrum of native peptide YSKFR 1i. 165
Figure 4.82 Extracted ion chromatogram of native peptide PSKFR 1j. 166
Figure 4.83 Mass spectrum of native peptide PSKFR 1j. 166
Figure 4.84 MS/MS spectrum of native peptide PSKFR 1j. 167
Figure 4.85 Extracted ion chromatogram of native peptide GSKFR 1k. 168
Figure 4.86 Mass spectrum of native peptide GSKFR 1k. 168
Figure 4.87 MS/MS spectrum of native peptide GSKFR 1k. 169
Figure 4.88 Extracted ion chromatogram of native peptide ISKFR 1l. 170
Figure 4.89 Mass spectrum of native peptide ISKFR 1l. 170
Figure 4.90 MS/MS spectrum of native peptide ISKFR 1l. 171
XVI
LIST OF TABLES
Chapter 2 Cysteine Modification of Peptides and Proteins using isolated Electron-
Deficient Allenes
Table 2.1 Optimization of modification reaction conditions. 46
Table 2.2 Screening of aqueous solutions of the modification. 48
Table 2.3 Investigation of the cysteine selectivity. 51
Table 2.4 Investigation of the scope of electron-deficient allenes. 54
Table 2.5 Modification of cysteine-containing peptide STSSSCNLSK 1b with
electron-deficient allenes. 56
Table 2.6 Modification of cysteine-containing protein BSA with electron-deficient
allenes. 60
Chapter 3 Cysteine Modification of Peptides and Proteins using in situ Generated
Table 3.1 Optimization of modification conditions. 72
Table 3.2 Modification of cysteine-containing peptide AYEMWCFHQR 1a with in
situ generated electron-deficient allenes. 77
Table 3.3 Modification of cysteine-containing peptide STSSSCNLSK 1b with in situ
generated electron-deficient allenes. 79
XVII
LIST OF ABBREVIATIONS
keto-ABNO 9-azabicyclo[3.3.1]nonane-3-one-N-oxy
AcOH Acetic acid
ADCs Antibody-drug conjugates
BBS Borate buffered saline
BSA Bovine serum albumin
°C Celsius
CID Collision-induced dissociation
CuAAC Copper-catalyzed azide alkyne cycloaddition
Cys Cysteine
Da Dalton
Dha dehydroalanine
DOTA 1,4,7,10-tetraazacyclodecane1,4,7,10-tetraacetic acid
DTT Dithiothreitol
equiv Equivalent(s)
ee Enantiomeric excess
2-EBA 2-ethynylbenzaldehydes
ETD Electron transfer dissociation
FBDP 4-formylbenzene diazonium hexafluorophosphate
FDA U.S. Food and Drug Administration
FRET Förster resonance energy transfer
h Hour(s)
XVIII
HMPA Hexamethylphosphoramide
His Histidine
HPLC High Performance Liquid Chromatography
IEDDA Inverse electron-demand Diels–Alder cycloadditions
iPr Isopropyl
LC–MS Liquid chromatography–mass spectrometry
Lys Lysine
min minute(s)
mM millimolar
μM micromolar
MBH Morita–Baylis–Hillman
MDA malondialdehyde
Met Methionine
MSH O-mesi-tylenesulfonylhydroxylamine
MS/MS Tandem mass spectrometry
NHS N-hydroxysuccinimide
OPA ortho-phthalaldehyde
OTf Trifluoromethanesulfonate
2-PCA 2-pyridinecarboxyaldehyde
PEG Polyethylene glycol
PLP pyridoxal-5'-phosphate
Pro Proline
PTMs Post-translational modifications
XIX
PBS Phosphate-buffered saline
Phe Phenylalanine
pSer phosphoserine
QTOF Quadrupole Time-Of-Flight
R Generic functional groups
ReACT Redox-activated chemical tagging
RGD Arginylglycylaspartic acid
RNase Ribonuclease
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Ser Serine
SPAAC Strain-promoted azide alkyne cycloaddition
TCEP Tris(2-carboxyethyl)phosphine
TMV Tobacco mosaic virus
TNF Tumor necrosis factor
Tris Tris(hydroxymethyl)aminomethane
Trp Tryptophan
Tyr Tyrosine
UAAs Unnatural amino acids
UCB Union Chimique Belge
UV Ultraviolet
XX
Chapter 1 Introduction
1.1 Allene Chemistry
Allenes are the simplest class of cumulenes with the general formula R2C=C=CR2,
in which two π bonds are orthogonal to each other with an ideal C–C–C angle of 180o.
For an unsubstituted allene, the central carbon is sp-hybridized while the two terminal
carbons are sp2 hybridized. The remaining two p orbitals on the central carbon overlap
with the p orbitals on the terminal carbons to produce two π bonds that are
perpendicular to each other (Figure 1.1). The overall configuration of allenes resembles
to an elongated tetrahedron.1 Therefore, allenes can be chiral when there are having two
different substituents on each of the terminal carbon atoms.
Figure 1.1 (a) Structure of allene; (b) Description of π bonding of allene.
The unique structure of allenes provides various advantages for organic synthesis.
Allenes have higher reactivity compared with their alkenyl and alkynyl analogs, which
allows controllable selectivity under mild reaction conditions. Besides, the unsaturation
of allenes provides excellent flexibility and opportunity to perform multistep reactions.
The intrinsic axial chirality also allows allenes to be implemented for synthesis of
optically active compounds.2
1
Moreover, many natural products contain allene moieties. Nowadays, there are
about 150 natural products with known allenic or cumulenic structures, which shows
that allenes represent important structural elements for different classes of compounds.3
The majority of naturally occurring allenes can be divided into three classes: linear
allenes, allenic carotinoids and terpenoids, and bromoallenes, in which most of them
are chiral compounds showing interesting biological activities (Figure 1.2). This unique
property leads to the inspiration of introducing allenic moieties to pharmacologically
active compounds, such as steroids, prostaglandins and nucleosides, which can function
as enzyme inhibitors, cytotoxic or antiviral agents (Figure 1.3).
Figure 1.2 Examples of naturally occurring allenic compounds.3
Figure 1.3 Examples of allenic pharmacologically active compounds.3
2
The unique features of allenes have proven themselves to be valuable structural
building blocks of complex molecules, revealing novel applications in organic
synthesis, natural product synthesis and pharmaceutical chemistry. Therefore, it is of
importance to explore new methodologies for the synthesis and transformations of
allenes.
1.1.1 Synthesis of Allenes
In the past decades, various methods for synthesis of allenes have been developed.
Allenes can be prepared by using alkenes, conjugated enyes or alkynes as starting
materials through classical reaction types, including addition, elimination, substitution
and transition-metal catalyzed reactions.
Using alkenes as starting materials, 1,2-elimination represents a common type of
reactions for preparation of allenes (Scheme 1.1). For example, upon treatment with n-
butyl lithium, 2-bromo-3,3-difluoroallylic acetates could be converted to 1,1-
difluoroallenes through lithium-bromide exchange followed by 1,2-elimination of
lithium acetate to afford mono- and disubstituted 1,1-difluoroallenes (Scheme 1.2).4
Scheme 1.1 Synthesis of allenes by 1,2-elimination.
3
Scheme 1.2 Synthesis of 1,1-difluoroallenes via 1,2-elimination of lithium acetate.
Besides, Satoh and co-workers reported the synthesis of functionalized allenes by
the reaction of 1-chlorovinyl sulfoxides with iPrMgCl, which gave magnesium
carbenoids through sulfoxide-magnesium exchange, followed by the treatment with 2-
lithio-5-methoxyfuran (Scheme 1.3a).5a When the magnesium carbenoids were trapped
by lithium enolates of α-chloroalkanoates, fully substituted 2,3-allenonates could be
obtained (Scheme 1.3b).5b Moreover, Ogasawara et al. developed a SN2’ substitution
of 2-bromo-1,3-dienes with soft nucleophiles in the presence of catalytic
palladium/bisphosphine complexes, which resulted in the formation of α-functionalized
allenes. With the use of chiral phosphine on the palladium center, (alkylidene-p-
allyl)palladium species served as a key intermediate for highly enantioselective
preparation of axial chiral allenic compounds (Scheme 1.4).
Scheme 1.3 Synthesis of allenes conjugated with α,β-unsaturated esters with
magnesium alkylidene carbenoids.
4
Scheme 1.4 Palladium-catalyzed SN2' substitution of 2-bromo-1,3-dienes.
Conjugated enynes have also been widely used for allene synthesis, in which 1,4-
addition represented an efficient approach for preparation of multi-substituted
functionalized allenes. For example, Zhang and co-workers reported a highly
functionalized 1,2-allenes could be produced base-catalyzed Michael addition to
electron-deficient 1,3-conjugated enynes (Scheme 1.5).6a The regionselectivity of the
reaction was controlled by the choice of nucleophiles. This approach was further
developed to Pd-catalyzed three-component tandem Michael addition/cross-coupling
reactions, in which the reaction between electron-deficient enynes, nucleophiles and
aryl halides resulted in the formation of tetrasubstituted allenes (Scheme 1.6).6b
Scheme 1.5 Synthesis of allenes by base-catalyzed Michael addition.
5
Scheme 1.6 Synthesis of allenes by Pd0-catalyzed three-component tandem Michael
addition/cross-coupling reaction.
Besides, Ma and co-workers developed a highly selective protocol for preparation
of 1,1-diaryl allenes or 1,3-diaryl allenes by a tunable 1,3-lithium shift of
propargylic/allenylic lithium formed by the conjugate addition of 4-aryl-3-butyn-1-
enes, followed by sequential transmetalation and Pd(0)-catalyzed cross coupling with
aryl halides.7 The regioselectivity of the reaction was controlled by the addition of
HMPA, which promoted complete 1,3-lithium shift of the formed organolithium
species (Scheme 1.7).
Scheme 1.7 Synthesis of highly selective allenes through tunable 1,3-lithium shift.
Alkynes, which are the isomers of allenes, are the most widely used starting
materials for synthesis of allenes. Isomerization of alkynes in the presence of base is
one of the earliest methods for synthesis of allenes (Scheme 1.8).8 Generally, treatment
6
of strong bases such as alkali metal amide with simple alkynes at high temperatures
could lead to the formation of allenes. For alkynes coordinated to a transition metal or
consisting of α-hydrogen activated by alkene, arene, carbonyl or oxygen / nitrogen /
sulfur / phosphorus-containing groups, weaker bases could be used for the
isomerization.
Scheme 1.8 Isomerization of alkynes to allenes in the presence of base.
SN2’-substitution reaction of propargylic compounds is one of the common
methods for the synthesis of allenes and various synthetic strategies of allenes from
propargylic alcohols have been developed (Scheme 1.9). For example, haloallenes
served as useful substrates for substitution reactions and transition metal-catalyzed
cross coupling reactions. Different methods were developed for the synthesis of
haloallenes, such as the reaction of aryl-substituted propargyl alcohols with N-
halosuccinimide and triphenylphosphine developed by Bao,9a or bromination of 1-
arylpropargylic alcohol through Appel-type reaction developed by Konakahara.9b Also,
Ready and co-workers reported an efficient and stereospecific approach for
synthesizing allenes from propargylic alcohols.9c Cp2Zr(H)Cl was applied to the anti-
SN2’-type reductive substitution of the in situ generated magnesium or zinc alkoxides
of propargylic alcohols, giving allenes with high optical purities.
7
Scheme 1.9 Examples of allene synthesis from propargylic alcohols.
The success of propargyl alcohols in allene synthesis prompted Che and Wong to
use propargylamines for the synthesis of allenes (Scheme 1.10). In 2008, Che and Wong
reported that chiral 1,3-diphenyl propargylamines could be efficiently transformed into
chiral allenes using KAuCl4 as catalysts in CH3CN at 40 °C.10a High product yields (up
to 93% yield) and excellent enantioselectivities (up to 97% ee) were resulted.
Scheme 1.10 Gold-catalyzed synthesis of axially chiral allenes.
Furthermore, a silver(I)-mediated enantioselective synthesis of axially chiral
allenes was developed in 2010 (Scheme 1.11).10b With the optimized conditions, a
variety of axially chiral allenes were synthesized in high product yields up to 95% and
high enantioselectivities of 98–99% ee. The use of microwave irradiation allowed the
8
completion of the reaction in a much shorter time than that required under conventional
thermal conditions.
Scheme 1.11 Silver(I)-mediated synthesis of axially chiral allenes.
Recently, our group discovered a novel electrophile-mediated cascade
cyclization/iodination of propargylamine-based 1,6-diynes to generate spiro 1H-
isoindoliums, which can be transformed to the corresponding 1H-isoindolium-based
electron-deficient allenes upon treatment with triethylamine. To the best of our
knowledge, the synthesis of these kinds of electron deficient allenes has not been
reported.
H PF6 H PF6
R2 R2 I R2 2 I R2 2
N R R
1) I2 (1.05 equiv.), CH3CN, r.t., 1 h N Et3N (1 equiv.) N
CH2Cl2, 1 h R1
R3 2) AgPF6 (1.1 equiv.), r.t., 5 mins R1
R1
H
R3 R3
Propargylamine-based
1H-isoindoliums 3 Electron-deficient allenes 4
1,6-diynes 2
Scheme 1.12 Electrophile-mediated cascade cyclization / iodination of
propargylamine-based 1,6-diynes and transformation into 1H-isoindolium-based
electron-deficient allenes.
9
1.1.2 Reactions of Allenes
The high degree of unsaturation and a readily accessible π-bond system allow
allenes to undergo various types of reactions, such as electrophilic addition,
nucleophilic addition, radical addition and transition metal-catalyzed reactions. Allenes
can act as nucleophiles or electrophiles, depending on the substituents at the terminal
carbons (Scheme 1.13).
Scheme 1.13 Property of allenes with different substituents.
For electrophilic addition, allenes undergo the usual electrophilic addition
reactions, such as halogen and addition of hydrogen halides, but the regioselectivity is
different from the generally accepted order of cation stability (Scheme 1.14).
The protonation of an unsubstituted allene can lead to either the 2-propenyl cation
or the allyl cation. The resulting product for the addition of HBr to an unsubstituted
allene is 3-bromopropene, suggesting that the formation of an intermediate vinyl
carbocation is more favorable, as opposed to the relatively higher stability of allylic
carbocation than vinylic or primary carbocation. This regiochemistry can be explained
by the fact that the two adjacent π-bonds in the allene are perpendicular to each other.
For formation of stabilized allyl cation in allene, a 90° rotation of the C–C bond joining
the carbocation and the double bond should take place, which requires higher activation
10
energy.11 As a result, the formation of 2-propenyl cation by protonation at a terminal
carbon is kinetically favored. However, the presence of stabilizing groups, such as
phenyl and dialkyl groups, at terminal carbon atom of the allene can change the
regiochemistry.
Scheme 1.14 Regiochemistry of electrophilic addition of allenes.
For nucleophilic addition, the presence of electron-withdrawing groups leads to
the electron deficiency of the inner C=C double bond, which induces nucleophilic
addition at the central carbon atom. Generally, the addition of nucleophiles, such as
alcohols, phenols and thiols, is carried out in the presence of base and results in the
formation of two types of products (Scheme 1.15).12
In early examples of nucleophilic addition to electron-deficient allenes, the
formation of the non-conjugated product C is kinetically-controlled, while the
conjugated product D is the result of a thermodynamically-controlled reaction.12 Firstly,
the intermediate A is formed after nucleophilic attack on the central carbon atom of
allene, which requires a torsion of 90° to merge into the allylic carbanion B.
Intermediate A can only lead to product C by proton transfer, while the protonation of
B can result in both C and D.
11
Scheme 1.15 General mechanism of nucleophilic addition to allenes12
For radical addition of allenes, addition of different types of radicals, such as
carbon-, nitrogen-, phosphorus-, sulfur-and selenium-centered, to allenes has been
experimentally and theoretically studied.13 The intermolecular addition of a radical to
an unsymmetrically substituted allene can occur at the two terminal carbon atoms or
the central carbon (Scheme 1.16).
Radical attack at C1 or C3 results in a pair of radical intermediates III and IV. The
vinyl radical intermediate II or IV will isomerize from a π-type configuration to the
more energetically favored σ-type radicals III and V. Radical attack on C2 results in a
π-type alkyl radical VI or VIII, which rotates around the C2–C3 bond of VI or the C1–
C2 bond of VIII for the formation of resonance-stabilized allylic radical VII. The
preferred site for radical addition to allene depends on three factors: (1) the degree of
substitution; (2) the nature of the attacking radical and (3) reaction parameters such as
temperature and initial reactant concentrations.12 In general, selective α-addition to
12
allene occurs for unsubstituted allene, regardless of the nature of the radical, while
substituted allenes favor β-addition of radicals, resulting in the formation of stabilized
allylic intermediates.
Y R3
R3
X
X R4
R4 R2 R1
trapping
R2 R1 reagent Y
R1 R3
1 2 3 R3 X
a b g R4 R2 R1 R4 Y R4
X R2 R4
I II X X
R3 R3
R2 R1 R2 R1
III
R1 R1 Y
X X
R2 R2
R1 R3 R4 trapping R3 R4
R1
1 2 3 R3 X reagent Y
R4 R4
R2 R2 R3 R2
X R2 Y
IV X
I R1 X
4 R1
R3 R R3 R
4
R3 R 3 R4
R1 R1 R4
R1 R1
1 2 3 R3 R3 Y
R4
R4 R2 X
R2 R2 X trapping R2 X
X reagent Y
I VI
1
R1 R2 R
R1 R1 R3
1 2 3 R3 R2 R3 Y
R3
R4 R2
R2 X R4 X R4 X R4
X
I VII
VIII
X = C, F, Cl, Br, N, O, S, Se, Sn, etc.; Y = H, I, Se, S, etc.
Scheme 1.16 Regio- and stereoselectivity in intermolecular radical addition
of allenes.13
Transition metal-catalyzed transformations of allenes have also been widely
developed due to their high π-coordination ability towards transition metals. In
principle, transition metal-catalyzed cross-couplings of allenes with halogen or metal
substituents at one of the terminal carbons or substitution reactions of α-functionalized
allenes give products with unchanged cumulene π-system (Scheme 1.17a).12 Transition
13
metal-catalyzed couplings at the central carbon are often accompanied by subsequent
reactions which rule out the cumulene system in the final products (Scheme 1.17b).
Scheme 1.17 Types of reaction modes of transition metal-catalyzed
coupling of allenes.12
Among the transition metal-catalyzed reactions of allenes, palladium-catalyzed
reactions have gained considerable attention over the last two decades because of their
wide applicability in organic synthesis. Allenes readily undergo carbopalladation in two
pathways depending on the regiochemistry of the insertion reaction. In Path I, the
reaction produces a sp2 C-Pd species, while Path II results in a π-allylpalladium species
(Scheme 1.18a).14
a) b) R
R1
Pd
R3
Path I
Pd(0) + RX b- H elimination
R R2 R4
R Pd
R HPdX
R
RPdX R1 CH2R3 base
Path II
Pd R
+ R2 Pd R4
Pd Pd(0)
R1 CH2R3 L L
R
R2 R4 R1
CH2R3
Nu R2
Nu R4
+
R
R1 CH2R3
R4
R2 Nu
Scheme 1.18 Carbopalladation of allenes.14
14
In most cases, Pd firstly connects to RX, followed by insertion reaction into allene
to form a π-allylpalladium species which can undergo β-hydride elimination or
nucleophilic substitution (Scheme 1.18b).14 Apart from intermolecular reactions,
allenes with a nucleophilic functionality can undergo Pd-catalyzed cyclization to form
cyclic skeletons, which provides possible pathways for synthesis of natural products
(Scheme 1.19).15
Scheme 1.19 Pd-catalyzed cyclization of allenes.15
15
1.2 Chemical Modification of Peptides and Proteins
Proteins are a major class of biopolymers that play important roles in different
biological events in living organisms. Therefore, study of proteins is one of the
important research areas in science. Post-translational protein modifications (PTMs),
which refers to the modification of proteins after transcription and translation, is often
responsible for the diverse structures of proteins found in nature. Naturally occurring
protein PTMs play important roles in tuning the properties of proteins and modulating
their biological functions.16
Similar to the natural approach of expanding the function of biomolecules through
PTMs, the development of novel bioconjugation approaches provides new
opportunities for chemical biologists to modulate structure and function of
biomolecules through synthetic chemistry. Therefore, chemical modification of
peptides and proteins has emerged as an invaluable tool for biological studies and
development of targeted therapeutics. For example, fluorescent labeling of proteins
allows in vitro and in vivo imaging analysis of the structure, function and localization
of proteins in biological environments.17 The addition of polyethylene glycol (PEG)
can increase the circulation half-life of a therapeutic protein.18 Antibody–drug
conjugates (ADCs) consisting of a drug linked to a tumor-selective antibody have also
recently shown considerable promise as anticancer agents.19
Despite a wide range of modification methods developed, the progress in
bioconjugation is still limited. In comparison with organic transformations which can
be conducted in organic solvent and under comparatively harsh conditions, chemical
16
reactions for protein modification must tolerate biological ambient conditions at or
below 37 °C with moderate pH (pH 6–8) in aqueous solvent. Unlike standard reaction
conditions of organic synthesis at tens to hundreds mM concentrations, protein
modifications are generally conducted at μM concentrations or less. Therefore,
chemoselective synthesis in conventional organic chemistry cannot be generally
applied to modification of biomolecules. Another challenge for protein modification is
to achieve modification with high efficiency and site selectivity. In order to avoid
purification steps, modification methods at a single site with high conversion and
minimal generation of side products are desired.20, 21 Therefore, it is of great importance
to develop complementary reactions for site-selective protein modification.
1.2.1 Lysine and N-Terminal Modification
The use of chemical reagents that react with primary amines is one of the most
versatile techniques for peptide and protein modification. There are two groups of
primary amino groups in protein: the α-amino group on the N-terminus of polypeptide
chains and ε-amino groups on lysine residue (Lys, K). The N-terminal amine has pKa
≈8 while that of a lysine ε-amine has pKa ≈10, so they are protonated at physiological
pH and predominantly exposed on the surface protein tertiary structures, resulting in
easy accessibility to the modification reagents. Deprotonated primary amines are highly
nucleophilic which favor the modification in peptides and proteins. However,
protonation can significantly reduce their reactivity. Therefore, it is important to control
the pH when conducting the N-terminal or lysine modifications.
17
Depending on the reaction conditions, selective modification of either N-termini
or lysine residues can be achieved by using different chemical reagents. Classical
approaches of modifying the amino groups in protein includes using activated esters,
isothiocyanates and through reductive amination of aldehydes (Scheme 1.20). Among
these methods, N-hydroxysuccinimide activated esters represent the most popular
amine-specific reagents for protein bioconjugation and labelling. NHS-mediated
modifications work within mild pH ranges from 7 to 8, which is lower than that for
isothiocyanates (pH 9–9.5), which is favorable for modifying alkaline-sensitive
proteins. Although the possible side reactions between NHS-activated esters with
tyrosine, histidine, serine and threonine were reported, the higher reaction rate of NHS
towards free amine groups overcomes the drawbacks of these side reactions.22
Scheme 1.20 Classical methods of lysine modification.
Apart from the classical methods, various novel approaches for lysine
modification have been developed in the past decades. For example, Tanaka and co-
18
workers developed a novel lysine-based labeling of biomolecules based on a rapid 6π-
azaelectrocyclization (Scheme 1.21).23 By reaction with unsaturated aldehyde probes,
1,4,7,10-tetraazacyclodecane1,4,7,10-tetraacetic acid (DOTA) as a metal chelating
agent and fluorescent groups could be efficiently introduced to lysine residues. The
reaction was selective to lysine residues at the protein surfaces, while it was less
sensitive to internal lysines and N-terminal amines.
Scheme 1.21 Lysine modification through 6π-azaelectrocyclization.
In 2016, Li and co-workers reported the chemoselective lysine modification
methods using ortho-phthalaldehyde (OPA) and its derivatives through the formation
of phthalimidines (Scheme 1.22).24 This approach could be applied to generation of
PEGylated L-asparaginase with resistance to proteolytic degradation by trypsin and
Glu-C and retained 70% and 40% of activities, respectively.
Scheme 1.22 Lysine modification using ortho-phthalaldehyde (OPA).
In 2018, Bernardes and co-workers demonstrated chemo- and regioselective
modification of lysine using a sulfonyl acrylate reagent, which was highly selective to
19
the ε-amino group of the most reactive lysine in the presence of other nucleophilic
amino acids such as cysteine (Scheme 1.23). The chemoselectivity of the modification
was attributed to the transient hydrogen bonding of the lysine amine with the sulfone
moiety of the reagents. This reaction was successfully employed to proteins with
diverse structures, including a full-length IgG antibody.25
Scheme 1.23 Lysine modification using sulfonyl acrylate reagents.
In 1960s, an N-terminal conjugation method through transamination was
developed by Dixon and co-workers.26 Through transamination of the terminal residue,
a carbonyl group could be introduced into proteins for further functionalization by
formation of hydrazone or oxime bonds.27 However, this method was not applicable
due to the harsh reaction conditions. Francis and co-workers re-examined this approach
and developed the pyridoxal-5'-phosphate (PLP)-mediated transamination for
modifying the N-terminal residues of peptides (Scheme 1.24).28a By introducing
reactive ketone or aldehyde groups, further functionalization could be proceeded
through oxime or hydrazone formation. The modification method was highly efficient
and selective to the N-terminus of peptides and proteins. However, it was found that
there were side reactions between some residues (His, Trp, Lys, and Pro) and PLP.
20
Scheme 1.24 N-terminal modification through PLP-mediated transamination
followed by oxime ligation.
Considering the lower pKa of N-terminal α-amino group (pKa ~ 8) than that of
lysine ε-amino groups (pKa ~ 10), pH-controlled N-terminal selective modification
methods were developed. In 2006, Che and Wong discovered an N-terminal α amino
group ligation of peptides with alkyne moieties using [Mn(2,6-Cl2TPPCl)] as catalyst
and Oxone as terminal oxidant (Scheme 1.25).29a Detailed studies revealed that the in
situ-generated ketenes were the key intermediates for the modification, which could be
isolated for direct selective N-terminal modification.29b
Scheme 1.25 N-terminal modification of peptides with ketenes.
In 2015, Francis and co-workers developed a promising approach for one-step N-
terminal selective modification of proteins using 2-pyridinecarboxyaldehyde (2-PCA)
derivatives (Scheme 1.26).28b The reaction between 2-PCA and the N-terminal residue
21
formed an N-terminal imine, followed by a nucleophilic attack of the adjacent amide
nitrogen on its electrophilic carbon, resulting in the formation of cyclic imidazolidinone
products.
In 2017, Chou and co-workers reported an N-terminal modification method of
peptides and proteins through reductive alkylation with aldehyde derivatives (Scheme
1.27).30 The modification was efficient with all N-terminal residues with an exception
of N-terminal cysteines, which resulted in thiazolidine derivatives. The modified
peptide and protein derivatives with bioorthogonal groups allowed further
functionalizations, such as fluorophores or biotins, for biological studies.
Scheme 1.26 N-terminal modification using 2-PCA derivatives.
Scheme 1.27 N-terminal modification using aldehyde derivatives.
Most recently, our group reported an N-terminal selective modification method of
peptides and proteins using 2-ethynylbenzaldehydes (2-EBA).31 By performing the
reaction in slightly acidic phosphate buffer using 2-ethynyl-4-hydroxy-5-
methoxybenzaldehyde, modification of a library of peptides XSKFR (X = either one of
22
20 natural amino acids) resulted in good-to-excellent N-terminal selectivity up to >99:1
(Scheme 1.28). N-terminal modification was also applied to protein modification,
including lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox
arginase mutant (BCArg mutant).
Scheme 1.28 N-terminal selective modification of peptides and proteins
using 2-EBA.
1.2.2 Modification of Other Natural Amino Acids
Tyrosine (Tyr, Y) is a choice of amino acid for site-specific modification of
proteins because of its lower abundance on the surface of proteins compared with Lys
(Scheme 1.29). In 2004, Francis and co-workers have demonstrated the chemoselective
azo coupling reaction using diazonium compounds to modify Tyr residues on the
exterior and interior surfaces of the tobacco mosaic virus (TMV) and bacteriophage
MS2 respectively.32a-b In 2006, they reported the use of π-allylpalladium complexes for
selective Tyr O- alkylation with a rhodamine dye and lipophilic moieties.32c The same
group also developed a three component Mannich-type reaction between Tyr residues,
aldehyde and aniline derivatives, which successfully modified solvent-exposed Tyr in
lysozyme, RNase A, and chymotrypsinogen A.32d-f
23
Apart from Francis’s group, Barbas and co-workers also developed two Tyr
modification strategies. In 2010, they reported an aqueous ene-type reaction for Tyr
modification using cyclic diazodicarboxamides, in which therapeutic antibody
herceptin was successfully labeled with an integrin binding cyclic RGD peptide under
optimized reaction conditions.33a In 2012, a novel 4-formylbenzene diazonium
hexafluorophosphate (FBDP) was developed for tyrosine-selective modification of
peptides and proteins, which was also applicable to labeling of the antibody
trastuzumab and cell surface labeling.33b
Scheme 1.29 Examples of tyrosine modification.
24
Tryptophan (Trp, W) is another for site-selective modification as it is a low
abundance amino acid with 1.1% frequency but exists in the primary sequence of
approximately 90% of native proteins (Scheme 1.30).34, 35 Some metal-mediated and
transition metal free modifications of Trp have been reported. For example, Francis and
co-workers reported rhodium carbenoids for Trp modification in 2004, yielding N-
alkylated and 2-alkylated products under acidic conditions (pH 1.5–3.5).36a The
protocol was improved to be performed at mild pH (pH 6–8) by replacing H2NOH
buffer with tert-BuNHOH.36b In 2007, Lindner and co-workers reported a condensation
reaction of malondialdehyde (MDA) with the indole nitrogen of tryptophan residues in
peptides under strongly acidic conditions.37 The resulting acroleinyl label at the indole
nitrogen could be further converted to hydrazone using hydrazide compounds, while
hydrazines led to cleavage of MDA adduct and releasing the free indole group.
In 2016, Kanai and co-workers reported a transition-metal free Trp-selective
bioconjugation method using an organic radical compound, 9-
azabicyclo[3.3.1]nonane-3-one-N-oxy (keto-ABNO) with NaNO2.38 Treatment of
peptides with the reagents in acidic aqueous solutions (0.1 – 0.5% AcOH) at room
temperature within 30 min gave keto-ABNO adducts which resulted from C–O bond
formation at the 3-position of Trp residues. This strategy was further applied to
modifications of lysozyme, myoglobin, concanavalin A, BSA and β2-microglobulin
antibody.
25
Scheme 1.30 Examples of tryptophan modification.
Methionine (Met, M) is the second rarest amino acid in proteins and has limited
surface-accessibility. Therefore, it is considered as a potential handle for highly
selective modification.39 There are limited numbers of Met modification methods
reported (Scheme 1.31). In 2012, Deming and co-workers developed a “click”-type
reaction for modification of methionine-containing peptides based on the
chemoselective alkylation of thioether groups in Met.40a This method allowed
introduction of a broad range of functional groups, such as carbohydrates, alkenes and
alkynes. The resulting sulfonium product could be reversibly dealkylated by addition
of nucleophiles, such as 2-mercaptoethanol, thiourea, 2-mercaptopyridine and
glutathione.40b
In 2017, Toste, Chang and co-workers developed a general and versatile Met-
selective bioconjugation method using redox-activated chemical tagging (ReACT).41
26
The modification was based on the oxidative sulfur imidation reaction between Met
and oxaziridine derivatives. The reagents enabled specific Met labeling from a single
protein to whole proteome level under mild biocompatible conditions. The broad utility
of the ReACT technology was demonstrated by synthesis of antibody–drug conjugates
and identification of hyper-reactive Met residues in whole proteomes.
Scheme 1.31 Examples of methionine modification.
1.2.3 Modification of Unnatural Amino Acids
The incorporation of unnatural amino acids (UAAs) to protein allows introduction
of chemical handles for subsequent bioorthogonal reactions. Different functional
groups, such as azide, alkyne, alkene and tetrazine can be chemically or genetically
incorporated in biomolecules, which can then further conjugate with specially designed
bioorthogonal probes.42 Various bioorthogonal reactions to target biomolecules have
been developed, including Staudinger ligation, copper-catalyzed azide alkyne
27
cycloaddition (CuAAC), strain-promoted azide alkyne cycloaddition (SPAAC), inverse
electron-demand Diels–Alder cycloadditions (IEDDA) and photoclick cycloaddition
(Scheme 1.32).
Scheme 1.32 Bioorthogonal reactions for protein labeling.
The Staudinger ligation between azides and triarylphosphines was demonstrated
by Bertozzi and co-workers.43a This technique was initially used for modification of
azido-glycoproteins on the surface of live cells, which was then further extended to the
modification of azide-incorporated proteins.43b-c Another example of azide
functionality was presented in CuAAC developed by Sharpless43d and Meldal, 43e which
was the Huisgen 1,3-dipolar-cycloaddition with alkynes in the presence of Cu(I)
catalysts. Because of the high specificity and fast reaction rate, CuAAC has been widely
28
used for protein modification. However, cytotoxicity caused by Cu(I) metal has
remained a limitation for modification of live cells, which led to the exploration of
alternative cycloaddition-type reactions. In 2004, Bertozzi’s group developed SPAAC,
in which highly strained cyclooctynes could react with azide-‘tagged’ glycoproteins at
room temperature without ligands or catalysts.44a After further studies for improving
the reaction rates, SPAAC has been broadly used in live mammalian cells and
animals.44b-d
Besides, Fox and co-workers developed the use of IEDDA reactions for
bioconjugation, which involved the cycloaddition between tetrazine dienophiles and
dienes.45a Because of the extremely fast reaction rate and high selectivity, IEDDA has
been widely used for modifications and fluorescent labeling of proteins in live
mammalian cells.45b-d
An alternative approach of cycloaddition-type modification of proteins has been
reported by Lin. Tetrazole-modified protein can undergo [3+2]-cycloadditions with
unactivated alkenes under irradiation with UV light.46a Attachment of alkenes to
proteins followed by treatment with tetrazoles proceeded with similar efficiency, which
has been used to for modification of alkenyl-UAAs such as homoallylglycine46b and
cyclopropenes.46c
Most recently, our group reported a novel approach for selective modification of
alkyne-linked peptides and proteins through alkynylation reaction of 6-membered ring
cyclometalated gold(III) complexes [Au(C^N)Cl2] with terminal alkynes (Scheme
1.33).47 After insertion of terminal alkyne to peptide, modification using 6-membered
29
ring cyclometalated gold (III) complexes afforded alkynylation product in > 99%
conversion. This approach could be further applied to fluorescent labeling of alkyne-
linked proteins using dansyl and BODIPY-linked gold(III) complexes.
Scheme 1.33 Chemoselective modification of alkyne-linked peptides and proteins by
cyclometalated gold(III) (C^N) complex-mediated alkynylation.
1.3 Cysteine Modification
Among the 20 natural amino acids, cysteine (Cys, C) residue has a unique
reactivity. Under physiological conditions, it has a high propensity to form the
nucleophilic thiolate ion (Scheme 1.34).48 The high nucleophilicity of the thiol moiety
offers advantages for developing modification approaches with high efficiency and
excellent site-selectivity.
Scheme 1.34 Formation of thiolate in cysteine residues.48
30
Conventional protein bioconjugation methods rely on reactions at nucleophilic
amino acids, particularly lysine or cysteine residues. However, the high abundance of
lysine residues on protein surfaces leads to the difficulties in the development of
selective labeling of a single lysine residue. In comparison, cysteine has a relatively
low natural abundance (~1.9%) and can be readily introduced by direct mutagenesis,
which allows the control of modification at a predetermined site.18 Therefore, cysteine
is considered as an ideal residue for selective modification and wide range of cysteine
modification strategies have been developed over the past decades.
Cysteine can be easily modified with suitable electrophiles, such as α-
halocarbonyls and Michael acceptors. However, some of the resulting bioconjugates
suffer the stability problems, in which they may undergo retro-Michael additions and
other thiol-promoted exchange reactions.17 These limitations prompt the development
of a number of metal-free and transition metal-mediated cysteine modifications.
Moreover, cysteine-selective functionalization of peptides and proteins occupies a
remarkable place in the field of bioconjugation as all of the recently FDA approved
ADCs are developed based on conjugation with cysteine residues in the polypeptide
chain.49 Therefore, it is of importance to explore new strategies of cysteine modification
for the generation of functional bioconjugates.
31
1.3.1 Classical Methods
Classical methods of cysteine modification include substitution with haloalkyl
substrates, Michael Addition with maleimides, disulfide formation and reaction of Dha
with thiols.
One of the earliest methods for modification of cysteine residues is by SN2
substitution with α-halocarbonyl and haloalkyl substrates (Scheme 1.35). Particularly,
reagents with iodide and bromide groups are commonly used. As early as 1935,
iodoacetamide was used to modify and study cysteine residues in proteins.50 A variety
of examples using such reagents for alkylating or arylating cysteine residues in proteins
have been reported over the past decades. For example, Davis and co-workers reported
that the p-iodobenzyl cysteine moiety on peptides and proteins could act as a coupling
partner or Suzuki–Miyaura coupling with phenylboronic acids.51 However, it was
reported that the haloalkyl substitution has a potential cross-reactivity with other amino
acids containing nucleophilic side chains, such as lysine and histidine.
Scheme 1.35 Modification of cysteine through nucleophilic substitution.
32
Another conventional approach of cysteine modification is the use of maleimides
(Scheme 1.36). Maleimides are Michael acceptors, which react with cysteine thiolates
to form thiosuccinimide bonds. The conjugation reaction between maleimide and the
cysteine residue in proteins was first reported by Moore and Ward in 1956, which
described the use of bis-maleimides as crosslinking agents of bovine plasma albumin
and wool keratin. To date, the use of maleimides still remains the preferred method of
cysteine modification of proteins for a number of reasons. Maleimides are high
selectivity towards thiol groups and the maleimides-thiols coupling reaction can be
performed in biocompatible conditions. Moreover, the maleimide moiety can be easily
functionalized with different conjugation partners such as fluorophores. This
conjugation reaction is also widely used in medicinal chemistry, in which antibody-
based therapeutics formed by maleimide-thiol coupling is a rapidly growing class of
biotherapeutic drugs. For example, Cimzia, a PEGylated anti-TNF construct developed
by UCB, has been approved by FDA as therapeutics drugs for Crohn’s disease and
arthritis.52 Despite its wide applications in research and industry fields, it still remains
problem of instability of thiosuccinimide adducts, which are known to undergo retro-
Michael additions or other thiol exchange reactions under physiological conditions.
Scheme 1.36 Modification of cysteine with maleimide reagents.
33
Disulfide bond formation is important for folding and maintaining tertiary
structural integrity in proteins. It has also been used as a common approach for chemical
modification of cysteine-containing proteins with other thiol-containing moieties. The
formation of disulfide bonds can be simply promoted by air oxidation, but have a
drawback of protein dimer formation. To prevent this, activated reagents RSX (X =
SO2R’, SeR’, I, Br, Cl) are usually used (Scheme 1.37). For example, Ellman reagent,
5,5’-dithiobis(2-nitrobenzoic acid), is a common reagent for activating cysteine thiols
on proteins,53 which have also been used for antibody–drug conjugates formation.54
Scheme 1.37 Common methods for disulfide formation.
Cysteine as a precursor to dehydroalanine (Dha) is of high importance for
chemical protein modification. Dha is a naturally occurring amino acid formed by
serine (Ser) dehydration or phosphoserine (pSer) elimination. There are various
methods for inserting dehydroalanine into peptides and proteins using serine as a
precursor. However, serine has a high natural abundance and often requires harsh
conditions for modifications, which causes difficulties in site-selectivity and their
applicability to a wide range of proteins.55 This leads to the development of method for
34
transforming cysteine to Dha.56 For example, in 2008, Davis and co-workers reported
an oxidative amidation / Cope-type elimination of cysteine to Dha using O-mesi-
tylenesulfonylhydroxylamine (MSH) (Scheme 1.38).57 Chemically, Dha is an α,β-
unsaturated carbonyl moiety that can undergo Michael-type addition reactions with
nucleophiles, which allow further functionalization of proteins.
Scheme 1.38 Transformation of cysteine to Dha in protein using MSH.
1.3.2 Transition Metal-Free Modifications
Apart from conventional approaches, different new methods for cysteine
modification were developed.
In 2009, Che and Wong first reported the use of electron-deficient alkynes,
including alkynoic amides, esters and alkynones, to perform modification of cysteine-
containing peptides and proteins through the formation of vinyl sulfide linkages
(Scheme 1.39).58a It was proposed that the modification proceeded through the
nucleophilic addition of thiols to the electron-deficient alkynes. The reaction was
facilitated in solutions at alkaline pH (pH 7.0–9.0), presumably due to the increased
concentration of thiolate ions in alkaline solution. The vinyl sulfide linkage was stable
towards TCEP while the alkynone-modified peptides could be cleaved to regenerate
the unmodified peptides by treatment with thiols.
35
Scheme 1.39 Modification of cysteine-containing peptides and proteins using
electron-deficient alkynes.
Based on this thiol-specific cleavage reaction, this cysteine modification was
further employed to develop a highly selective FRET-based fluorescent probe for
detection of cysteine and homocysteine.58b-c The probe was able to be applied to cellular
imaging. Besides, the introduction of luminescent iridium(III) complexes to the FRET-
based probes for the detection of thiols was also demonstrated.58d
In 2012, our group reported the amine-catalyzed Morita–Baylis–Hillman (MBH)
bioconjugation reaction (Scheme 1.40). With the formation of the β-hydroxyl-α-
methylene-carbonyl moiety, multifunctional modification of cysteine-containing
peptides and proteins with fluorescent probes/ biotin tags could be performed in slightly
basic aqueous media (pH 8.0).59
Scheme 1.40 Multifunctional modification of cysteine-containing peptides and
proteins by Morita–Baylis–Hillman reaction.
36
In 2016, Pentelute and co-workers reported a π-clamp-mediated site-selective
conjugation with perfluoroaromatic reagents (Scheme 1.41).60 A four-amino-acid
sequence (Phe-Cys-Pro-Phe) called the “π-clamp” tuned the reactivity of a cysteine
thiol to recognize the perfluoroaromatic reaction partner, which allowed selective
modification of one cysteine residue in proteins with multiple endogenous cysteine
residues, including antibodies and cysteine-containing enzymes.
Scheme 1.41 π-Clamp-mediated site-specific cysteine conjugation.
In 2019, Bernardes and co-workers developed an efficient and irreversible
cysteine-selective bioconjugation method through quaternization of vinyl- and alkynyl
pyridines (Scheme 1.42).61 Quaternization of the nitrogen of vinyl and alkynyl
pyridines transformed these molecules to reactive electrophiles towards thiols, allowing
selective bioconjugation of cysteine-containing protein. Also, the introduction of a +1
charge to the overall net charge of proteins by the quaternized vinyl pyridinium reagent
could modulate the electrophoretic mobility of proteins.
Scheme 1.42 Cysteine bioconjugation method through quaternization of vinyl- and
alkynyl pyridines.
37
1.3.3 Transition Metal-Mediated Modifications
Transition metal-mediated transformations are widespread in organic synthesis.
Due to the stringent requirements for bioconjugation and the existence of multiple
functional groups in biomolecules, only a few examples of transition-metal based
bioconjugation methods have been reported. Cysteine residue has metal binding ability,
in which the thiolate groups are capable of binding to metal ions such as iron, zinc and
cadmium.62 This unique property leads to the development of metal-mediated
modifications of cysteine.
Transition metals can serve as mediators for selective modifications at cysteine.
In 2014, our group first reported a gold-mediated cysteine modification method using
cyclometalated gold(III) complexes with bidentate C,N-donor ligands and ancillary
ligands (Scheme 1.43).63a The cyclometallated gold(III) complexes could selectively
modify the cysteine residues of peptides to form gold–cysteine adducts in > 90%
conversion in 2 h at room temperature. Increasing the temperature to 37 °C resulted in
reductive elimination of the gold–cysteine adducts to give alkylated cysteine.
Scheme 1.43 Cysteine modification using cyclometalated gold(III) complexes via
ligand controlled C–S bond formation.
38
In 2015, Buchward, Pentelute and co-workers reported a cysteine modification
method using organometallic palladium reagents (Scheme 1.44).64 Treatment of low
micromolar concentrations of palladium(II) complexes with cysteine-containing
peptides gave C–S reductive elimination products in excellent conversions within 5 min.
This strategy could be used for functionalization of unprotected peptides, such as
fluorescent tags and affinity labels. Further applications in peptide stapling and
construction of antibody–drug conjugates (ADCs) were also demonstrated.
Scheme 1.44 Cysteine modification using palladium(II) complexes.
Most recently, our group developed a novel isoxazolinium-mediated cysteine
modification of peptides and proteins (Scheme 1.45).63b The use of a stoichiometric
amount of isoxazolinium reagents generated in situ from a catalytic amount of silver
salts can efficiently modify cysteine-containing peptides and proteins. The modified
bioconjugate was stable towards thiol-containing reagents, reducing reagents and
oxidizing reagents. Under irradiation of UV-A light, the phenylacyl thioether linkage
bearing an alkyne moiety of can be rapidly cleaved to form a thioaldehyde moiety,
which could be converted back to cysteine by reduction.
Scheme 1.45 Isoxazolinium-mediated cysteine modification of peptides and proteins.

39
Chapter 2 Cysteine Modification of Peptides and Proteins using
isolated Electron-Deficient Allenes
2.1 Introduction
In 2013, our group first reported the gold-mediated selective cysteine modification
of peptides (Scheme 2.1).65 Gold compounds were effective in activating the π-system
of allenes for addition reactions with nucleophiles. In the presence of AuCl–AgOTf, a
diversity of allenes were successfully coupled with the thiol group of cysteine-
containing peptides to afford hydroxy vinyl thioethers instead of the commonly
observed 1,2 / 1,3-adducts. Another example of thiol-allene coupling was reported by
Loh and co-workers, which utilized the C-substituted terminal allenamide moieties for
cysteine modification of peptides (Scheme 2.2).66 The strategy was based on the 1,4-
Michael addition of the thiol group of cysteine residues to allenamides. This
modification method was further applied to fluorescent labeling of proteins. Despite
these examples, comprehensive study on the applicability of allenes on protein
modification remains largely elusive.
Scheme 2.1 Gold-mediated selective cysteine modification of peptides.
40
Scheme 2.2 Cysteine modification using allenamides.
Recently, our group disclosed a novel electrophile-mediated cascade cyclization /
iodination of propargylamine-based 1,6-diynes to generate spiro 1H-isoindolium,
which can be transformed to the corresponding 1H-isoindolium-based electron
deficient allenes upon treatment with triethylamine.
Given the electrophilic allenes and strong nucleophilicity of cysteine residues in
peptides and proteins, it is envisioned that the novel electron-deficient allenes would
be a useful handle for bioconjugation of cysteine-containing peptides and proteins. It is
hypothesized that the 1H-isoindolium-based electron deficient allenes would have
excellent selectivity and reactivity in metal-free cysteine modification. A detailed study
of cysteine modification of peptides and proteins using electron-deficient allenes was
conducted.
Reaction conditions were first optimized by screening with different amounts of
reagents, buffer media and temperature. Then, detailed studies of modification products
were performed by employing electron-deficient allene derivatives and various
cysteine-containing peptides. Moreover, the stability of modified peptides was
examined by treatment with excessive thiol-containing reagents and reducing agents.
41
The optimized modification protocol was then utilized for modification of proteins
with free cysteine residues, and the modification was confirmed by chymotrypsin
digestion followed by LC–MS analysis. This method would be applied for fluorescent
labeling of proteins.
2.2 Results and Discussion
2.2.1 Modification of Cysteine-Containing Peptides Using Isolated Electron-
Deficient Allenes
2.2.1.1 Synthesis of Electron-Deficient Allenes
The new class of electron deficient allenes was synthesized by electrophile-
mediated cascade cyclization / iodination of propargylamine-based 1,6-diynes 2, in
which propargylamine-based 1,6-diynes 2 could be synthesized by gold(III)-catalyzed
three-component coupling reaction of aldehydes, amines and alkynes.67
Scheme 2.3 General scheme for synthesis of propargylamine-based 1,6-diynes 2.
42
Treatment of propargylamine-based 1,6-diynes 2 with iodine generated the spiro
1H-isoindoliums 3, which were very sensitive to base. Upon treatment with
triethylamine, the alkyne moieties was easily isomerized to the corresponding 1H-
isoindolium-based electron-deficient allenes 4.
H PF6 H PF6
R2 R2 I R2 2 I R2 2
N R R
1) I2 (1.05 equiv.), CH3CN, r.t., 1 h N Et3N (1 equiv.) N
CH2Cl2, 1 h R1
R3 2) AgPF6 (1.1 equiv.), r.t., 5 mins R1
R1
H
R3 R3
1H-isoindoliums 3 Electron-deficient allenes 4
1,6-diynes 2
Scheme 2.4 General scheme for synthesis of electron-deficient allenes.
Besides, ICl also worked well as an electrophile affording the corresponding
products 2 and this method is practical and easily scalable. Electron-deficient allene 3a
could be also obtained from 2a with 87% yield under basic conditions in a one-pot
reaction.
Figure 2.1 The synthesis of allene 4a.
43
The reaction mechanism for this novel iodine-mediated cascade iodination /
cyclization was proposed. Electrophilic addition of iodine to the terminal alkyne of 2
gives iodonium salt A. Then, the nitrogen atom of A attacks the iodonium moiety to
afford quaternary ammonium salt B. Subsequent anion exchange with AgPF6 gives
alkyne 3. Under basic conditions, isomerization of alkyne B to allene C followed by
anion exchange with AgPF6 provides allene 4.
I
H I H
R2 PF6
R2 R2 I 2 I R2 R2
N N R 2 I 2
I2 N R AgPF6 N R
R3
R1 R1 R1 R1
H AgI
R3 R3 Et3N
2 A B R3 3
Et3NH
H PF6
H I H I
I R2 I R2 I R2
2
N R AgPF6 N R
2
N R
2
R1 R1 R1
H H Et3N
AgI H NEt3
R3 R3 R3
4 D C
Scheme 2.5 Proposed reaction mechanism.
Compounds 4b, e-f were prepared by our group members and used for the reaction
condition screening of the bioconjugation studies in the following section. Given the
reaction screening results, I have designed and synthesized functionalized electron-
deficient allenes 4g-h for cysteine-selective peptide and protein labeling.
44
2.2.1.2 Optimization of Modification Reaction Conditions
Cysteine-containing peptide AYEMWCFHQR 1a and allene reagent 4a were
employed as substrates for reaction condition screening. By treatment of peptide
AYEMWCFQHR 1a (0.1 mM) with allene reagent 4a (1 equivalent) in potassium
phosphate buffer pH 8.0 buffer/CH3CN (9:1) at 25 °C for 4 h, modified peptide 5a was
afforded in 92% conversion with all other amino acid residues remaining intact as
confirmed by LC–MS/MS analysis (Table 2.1, Entry 1). Increasing the loading of allene
from 1 to 5 equivalents led to an improvement of the conversion to 95% (Entries 5-8).
Figure 2.2 MS/MS spectrum of cysteine-modified peptide 5a.
45
Table 2.1 Optimization of modification reaction conditions.a
H PF6
I O Cl O
H
N I
PF6
N H
H
4a S
AYEMWCFHQR Cl
Potassium phosphate buffer/ CH3CN (9:1) AYEMW FHQR
1a N
25 ºC, 4 h H
O
5a
Entry 4a (equiv.) pH values Temperature (°C) Conversion (%)b
1 1 8.0 25 92
2 1 5.8 25 19
3 1 6.5 25 36
4 1 7.4 25 74
5 2 8.0 25 93
6 3 8.0 25 94
7 4 8.0 25 95
8 5 8.0 25 95
9 5 8.0 37 96
10 5 5.8 25 62
11 5 6.5 25 90
12 5 7.4 25 93
a
Conditions of the modifications: treatment of AYEMWCFHQR 1a (0.1 mM) with
allene reagent 4a in 50 mM potassium phosphate buffer/CH3CN (9:1) at different pH
values and temperature for 4 h. b Conversion of the modification was determined by
LC–MS analysis.
46
Time course analysis was performed by conducting the reactions at different pH
values. When potassium phosphate buffer with different pH values (5.8, 6.5, 7.4 and
8.0) were used for the bioconjugation, lower conversion was resulted under slightly
acidic conditions (Figure 2.3). The conversions could be improved by increasing the
pH values of buffer (Table 2.1, Entries 1-4). This observation could be explained by
the pKa value of the thiol group of cysteine (pKa = ~ 8.5). Basic conditions could
promote the deprotonation of the thiol group to the thiolate group, resulting in stronger
nucleophilicity that facilitated the modification.
Time course experiment of the formation of modified peptide 5a at different
temperatures was also performed (Figure 2.4). The reaction proceeded relatively slow
at 4 °C. When the reaction was performed at 37 °C, modified peptide 5a in > 70%
conversion was observed after 30 min. Comparable results were obtained when the
modification was conducted at room temperature or 37 °C for 4 h (Table 2.1, Entry 9),
suggesting that the reaction proceeded efficiently at room temperature or 37 °C.
100
pH 5.8 buffer/ CH3CN (9:1)
80 pH 6.5 buffer/ CH3CN (9:1)
Conversion (%)

40
20
0
0 1 2 3 4
Time (h)
at different pH values.
47
100
4 °C
80 25 °C
Conversion (%)
37 °C
60
40
20
0
0 1 2 3 4
Time (h)
at different temperatures.
With the optimized conditions (using 5 equivalents of allene reagent 4a), the
modification proceeded smoothly under potassium phosphate, Tris-HCl, ammonium
bicarbonate, borate buffered saline (BBS) or sodium borate buffer medium, giving
conversions in 92-96% (Table 2.2).
Table 2.2 Screening of aqueous solutions of the modification.a
H PF6
I O Cl O
H
N I
PF6
N H
H
4a S
AYEMWCFHQR Cl
aqueous solution/ CH3CN (9:1) AYEMW FHQR
1a N
25 ºC, 4 h H
O
5a
Entry Aqueous solutions Conversion (%)b

1 50 mM Potassium phosphate buffer (pH 8.0) 95
2 50 mM Tris-HCl (pH 8.0) 96
48
3 50 mM NH4HCO3 buffer (pH 8.0) 92
4 50 mM BBS (pH 8.0) 95
5 50 mM Sodium borate buffer (pH 8.0) 96
a
allene reagent 4a in aqueous solutions/CH3CN (9:1) at 25 °C for 4 h. b Conversion of
the modification was determined by LC–MS analysis.
2.2.1.3 Model Reaction of Cysteine Modification by Electron-Deficient Allenes
To investigate the structure of electron-deficient allene-modified peptide 5a, a
model study was conducted using benzyl mercaptan as substrate for the thiol-allene
reaction. Firstly, 1H-isoindoliums 3a was mixed with triethylamine for 1 h to generate
electron-deficient allene 4a (1.1 equiv.) under basic condition. After that, benzyl
mercaptan was added to the reaction mixture for further reaction of 3 h at room
temperature in H2O/CH3CN (1:3). After the reaction, the corresponding vinyl thioether
product 7 was isolated in 63% yield and the structure of product was confirmed by 1H
NMR analysis.
O
H
SH I PF6
N H
H PF6 H PF6
O I O Cl
I Et3N (1 equiv.) (1 equiv.)
N N
S Cl
CH3CN, r.t., 1 h CH3CN/H2O 3:1, r.t., 3 h
Cl H
3a 4a
7
Scheme 2.5 Model reaction of thiol with electron-deficient allenes.
49
2.2.1.4 Investigation of Cysteine Selectivity of the Modification
To examine the cysteine selectivity of the modification, another cysteine-
containing peptide STSSSCNLSK 1b, which consisted of different residues compared
with AYEMWCFHQR 1a, was treated with allene reagent 4a to afford modified
peptide 6a in 96% conversion (Table 2.3, Entry 2). From the MS/MS analysis, only the
cysteine residue on the peptides was modified with all other residues remaining intact
(Figure 2.5). The result indicated that this modification had high chemoselectivity
towards the thiol moiety of cysteine residue in the presence of other nucleophilic
residues, such as methionine, tryptophan, histidine etc.
Treatment of C-terminal cysteine-containing peptide KSTFC 1c and N-terminal
cysteine-containing peptide CSKFR 1d with allene reagent 4a gave modified peptide
8a and 9a in 93% and 94% conversion, respectively. LC–MS/MS analysis showed that
modification was on the cysteine residue with other residues remaining intact (Figures
2.6 and 2.7). Control experiments using peptides 1e-l without free cysteine residue gave
no modification (Table 2.3, Entries 5-13). This showed that the modification was highly
selective to cysteine residues.
50
Table 2.3 Investigation of the cysteine selectivity.a
H PF6 O
H
I O Cl I
N PF6
N H
H S
4a Cl
Peptides
pH 8.0 potassium N
1a-l H
phosphate buffer/ CH3CN (9:1), O
25 ºC, 4 h
5a, 6a, 8a and 9a
H O H O H O
I PF6 I PF6 I PF6
N H N H N H
S Cl S Cl S Cl
STSSS NLSK KSTF OH SKFR
N N H2N
H O H O O
6a 8a 9a
Entry Peptides Conversion (%)b

1 AYEMWCFHQR 1a 95
2 STSSSCNLSK 1b 96
3 KSTFC 1c 93
4 CSKFR 1d 94
5 STSSSANLSK 1e 0
6 STSSSHNLSK 1f 0
7 AYEMWSFHQR 1g 0
8 WSKFR 1h 0
9 YSKFR 1i 0
10 PSKFR 1j 0
12 GSKFR 1k 0
13 ISKFR 1l 0
a
Conditions of the modifications: treatment of peptides 1a-l (0.1 mM) with allene
reagent 4a in 50 mM pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C for 4
h. b Conversion of the modification was determined by LC–MS analysis.
51

52
2.2.1.5 Investigation of Scope of Allene reagents
The scope of allene reagents for the bioconjugation was investigated. Electron-
deficient allenes 5b-h were screened with cysteine containing peptide
AYEMWCFHQR 1a (Table 2.4). Allene reagents containing a wide range of para-
substituted phenyl groups at R1 position, such as methyl and halogen, gave modified
peptides in excellent conversion between 89% and 95% (Entries 1-6). Different amine
components at R2 were also compatible with the modification (Entries 6-7). The results
showed that the modification was well tolerated with allene reagents with different
substituents.
53
For functional labeling of the cysteine-containing peptides, coumarin-derived
allene 4g was employed for the modification and gave the corresponding modified
peptides 5g in 31% conversion (Entry 7). Modification with allene reagent 4h bearing
the alkyne moiety, which could be applied for sequential modification using click
reaction was also conducted, giving the formation of 5h in 94% conversion (Entry 8).
Bioconjugation of another cysteine-containing peptide STSSSCNLSK 1b with the
allene reagents was also performed. Modified peptides 6b-h were obtained in good to
excellent conversion between 59% and 96% (Table 2.5), indicating the high efficiency
of this modification.
Table 2.4 Investigation of the scope of electron-deficient allenes.a
H PF6
I R2 2 H
R I R2 PF6
N
R2
R1 N H
H R1
R3 R3
S
4
AYEMWCFHQR AYEMW FHQR
pH 8.0 potassium N
1a H
phosphate buffer/ CH3CN (9:1), O
25 ºC, 4 h
5a-h
Entry Allene Modified peptide Conversion (%)b
1 5a 95
54
2 5b 93
3 5c 91
4 5d 93
5 5e 89
6 5f 90
7 5g 31
55
8 5h 94
a
allene reagent 4 in 50 mM pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C
for 4 h. b Conversion of the modification was determined by LC–MS analysis.
Table 2.5 Modification of cysteine-containing peptide STSSSCNLSK 1b with
electron-deficient allenes.a
H PF6
I R2 2
R H
N I R2 PF6
R1 R2
N H
H
R1
R3
4 R3
S
STSSSCNLSK
pH 8.0 potassium STSSS NLSK
1b N
phosphate buffer/ CH3CN (9:1), H
O
25 ºC, 4 h
6a-h
Entry Allene Modified peptide Conversion (%)b
1 6a 96
2 6b 96
56
3 6c 93
4 6d 87
5 6e 84
6 6f 96
7 6g 59
8 6h 93
57
a
Conditions of the modifications: treatment of STSSSCNLSK 1b (0.1 mM) with
allene reagents 4 in 50 mM pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C
for 4 h. b Conversion of the modification was determined by LC–MS analysis.
2.2.1.6 Stability Study of Allene-modified Bioconjugates
Previous studies have shown that some cysteine-selective reagents could be
removed after bioconjugation. For example, thiosuccinimide adducts formed by the
modification of cysteine with maleimides are now known to undergo retro-Michael
additions or thiol exchange reactions which cause cleavage of bioconjugates.68 This
would result in formation of heterogenous conjugate mixtures, limiting the applications
in in vivo studies.69 Therefore, stability study of the modified bioconjugates was
conducted for the allene-modified bionconjugates.
Investigations were conducted by treatment of modified peptide 5a with excessive
thiol-containing reagents and reducing reagents. Treatment of modified peptide 5a with
500 equivalents of L-cysteine and DL-homocysteine, respectively, showed that the
modified peptide 5a still remained intact after 3 h as confirmed by LC–MS analysis.
Besides, treatment of the modified peptide 5a with 500 equivalents of common
reducing reagents, DTT and sodium ascorbate, also showed no interference with the
modified bioconjugates.
These results indicated that the modified bioconjugates were stable toward
environments with thiol-containing reagents and common reducing reagents. Therefore,
the modification was regarded as a stable and irreversible cysteine modification.
58
2.2.2 Modification of Cysteine-Containing Proteins Using Isolated Electron-
Deficient Allenes
2.2.2.1 Modification of BSA Protein with Allene Reagents
The electron-deficient allenes were further employed to protein modification.
Bovine serum albumin (BSA) with a single free cysteine residue were utilized for
bioconjugation. Treatment of BSA (0.1 mM) with allene reagents 4a, 4d and 4h (10
equivalents) in PBS pH 7.4 buffer/CH3CN (9:1) at 25 °C for 4 h afforded modified
proteins in 56-70% conversion by LC–MS analysis (Table 2.6).
To identify the site of modification in protein, chymotrypsin digestion was
performed on the allene- modified protein BSA-1. By LC–MS analysis, two incomplete
digested peptide fragments, Lys20-Phe36 ([M+3H]3+ m/z 806.7) and Ser28-Phe49
([M+3H]3+ m/z 1044.1), were obtained (Figures 2.9 and 2.10). The expected mass of
this two peptide fragments from the native protein should be 1954.0 Da and 2666.3 Da
respectively. From the molecular feature extraction spectrum of modified protein BSA-
1, the mass of the two peptide fragments was shifted to 2417.1 Da and 3132.3 Da,
respectively. The increase in the mass of this two cysteine-containing fragment peptides
after modification indicated the incorporation of one molecule of 4a (M.W. = 462) into
the BSA peptide sequence. However, control experiment of chymotrypsin digestion of
native BSA protein is needed to confirm the mass shift of the two peptide fragments
and LC–MS/MS analysis of the modified peptide fragments is required to show that the
59
modification was on free cysteine residue Cys34 of BSA with other residues remaining
intact.
Table 2.6 Modification of cysteine-containing protein BSA with electron-deficient
allenes.a
H PF6 H
I R2 2 PF6
I R2
R2 R
N N H
R1
R1
SH S
H R3
R3
4
pH 7.4 PBS buffer,

25 ºC, 4 h
protein modified protein
Entry Allene Modified protein Conversion (%)b
1 BSA-1 65
2 BSA-2 70
3 BSA-3 56
a
Conditions of the modifications: treatment of BSA protein (0.1 mM) with allene
reagent 4 in 50 mM pH 7.4 PBS buffer/CH3CN (9:1) at 25 °C for 4 h. b Conversion of
the modification was determined by LC–MS analysis.
60
(a)
(b)
(c)
Figure 2.8 Mass spectrum of (a) BSA-1; (b) BSA-2 and (c) BSA-3.
61
KGLVLIAFSQYLQQCPF after chymotrypsin digestion of BSA-1.
SQYLQQCPFDEHVKLVNELTEF after chymotrypsin digestion of BSA-1.
62
2.2.2.2 Control Experiments with Non-Free Cysteine-Containing Proteins
Control experiments were conducted by treatment of non-free cysteine-containing
proteins, RNaseA and lysozyme in PBS pH 7.4 buffer at room temperature for 4 h. The
result showed that no modification was found. This result suggested that allene reagents
were highly selective to the cysteine residues on proteins and could be applied to
cysteine modification of proteins with high efficiency (Figures 2.11 and 2.12).
Figure 2.11 Mass spectrum of RNaseA after reaction with 4a.
Figure 2.12 Mass spectrum of lysozyme after reaction with 4a.
63
2.2.2.3 Fluorescent Labeling of Proteins
For the fluorescent labeling of proteins, alkyne handles were first incorporated on
the free cysteine residues of BSA using allene reagent 4h. After washing out the
excessive reagents, the alkyne-functionalized proteins BSA-3 were labeled with red
fluorescent rhodamine dye 10 by azide-alkyne Huisgen cycloadditions in 40%
conversion (Scheme 2.6 and Figure 2.13). SDS-PAGE analysis revealed that,
rhodamine-labeled protein BSA-4 were observed using fluorescent analysis, while no
fluorescent signal was observed for native BSA or alkyne-functionalized protein BSA-
3 (Figure 2.14). Employing Coomassie blue staining on the same gel, deep blue color
signals of native, alkyne-functionalized as well as rhodamine-labeled proteins were
observed, indicating that the fluorescent tag has been labeled on the proteins using the
allene-mediated cysteine modification and a sequential azide-alkyne click reaction.
PF6
H H O
I O I
PF6
N N H
SH
H S
4h
pH 7.4 PBS buffer,

protein 25 ºC, 4 h
N O N
N O N
O
O
N
H O
N N
I O
N PF6
N3
N H
O
Rhodamine azide 10,
S
CuSO4, TBTA, TCEP N
N N
pH 7.4 PBS buffer,
37 ºC, 1 h
Scheme 2.6 Modification of protein with alkyne-functionalized allene 4h and
sequential copper(I)-catalyzed azide-alkyne cycloadditions with rhodamine azide.
64
Figure 2.13 Mass spectrum of rhodamine-labeled BSA-4.
Figure 2.14 SDS-PAGE analysis of BSA, allene-modified BSA-3 and rhodamine-
labelled BSA-4.
65
2.3 Conclusion
In summary, a metal-free chemoselective cysteine modification using 1H-
isoindolium-based electron-deficient allenes was developed. We have developed a
novel electrophile-mediated cascade cyclization / iodination of propargylamine-based
1,6-diyne 2 for synthesis of a new class of spiro 1H-isoindoliums 3. Upon treatment
with base, the alkyne moieties of 1H-isoindoliums 2 were transformed to allene
functionalities to give the corresponding allene products 3. This new class of electron
deficient allenes could be employed for cysteine modification of peptides and proteins
through the interaction between allene and the nucleophilic thiol moiety on cysteine
residue.
A comprehensive study on this newly developed bioconjugation reaction was
presented. The modification could proceed with high efficiency (up to 96% conversion
in 4 h) and high chemoselectivity toward cysteine residue. Allene reagents with
different functional groups were compatible with this modification. The modified
products were stable towards excessive thiol-containing reagents and reducing agents.
This modification was further applied to protein modification and labeling.
Fluorescent tags could be labeled on the cysteine-containing proteins by sequential
modification using the azide-alkyne click reaction. This newly developed modification
would become a complementary method towards the currently used cysteine
modification protocols.
66
2.4 Suggestions for Future Research
In the present work, a highly selective cysteine modification of peptides and
proteins using 1H-isoindolium-based electron-deficient allenes was developed. For
peptide modification, the two cysteine-containing peptides 1a and 1b used for the study
are linear peptides consisting of different amino acid residues. The results showed that
the modification could proceed smoothly in the presence of amino acids with bulky or
aromatic side chains, including alanine, tyrosine, and tryptophan. In the future, it is
envisioned that the modification can be further applied to the modification of cyclic
peptides for the potential development of therapeutic agents.70 Besides, the
modification was well tolerated with allene reagents with different substituents. Based
on this results, it is suggested that other functionalized allene reagents can be prepared,
such as PEG probes for improving the water solubility and biocompatibility, so as to
further expand their applications on selective labelling of cysteine-containing peptides.
For protein modification, upon chymotrypsin digestion of allene-modified BSA
protein, the cysteine-containing peptide fragments were found, but the analysis and
identification of modification site was not yet completed. Therefore, it is suggested that
LC–MS/MS analysis should be performed in the future so as to confirm the location of
modification. Apart from chymotrypsin, it is suggested that the choice of protease for
protein digestion can be further explored, such as trypsin, Glu-C, or a combination of
multiple proteases, so as to optimize the digestion protocol for LC–MS/MS analysis.
Moreover, as protein digestion produces relatively long peptide fragments which may
be more difficult to interpret, the conditions for MS/MS analysis of tryptic peptide
67
fragments can be further optimized. For example, apart from performing MS/MS with
collision-induced dissociation (CID), electron transfer dissociation (ETD) may be an
alternative and effective approach for analysis of peptide fragments while the fragile
modification remaining intact. By using different fragmentation methods for MS/MS
analysis, more information of peptide fragmentation can be obtained.
68
Chapter 3 Cysteine Modification of Peptides and Proteins using in situ
Generated Electron-Deficient Allenes
3.1 Introduction
A detailed study of cysteine modification of peptides and proteins using electron-
deficient allenes was presented in Chapter 2, in which the electron deficient allene
reagents were prepared by multi-step chemical synthesis. Based on the success of
development of the modification, we would like to enhance the efficiency of this
modification by developing a simple, one-step procedure for preparation of electron
deficient allenes.
The investigation was initiated by preparation of in situ generated electron
deficient allene reagents by the reaction between propargylamine-based 1,6-diynes and
iodine monochloride, which resulted in the formation of in situ generation of 1H-
isoindolium. With the addition of reducing agent for quenching of reaction, the reaction
mixture could become a stock solution of in situ generated electron deficient allene with
high stability for direct use in bioconjugation.
Reaction conditions were optimized by screening different amount of allene
reagents with reducing agents. Then, studies of the modification were performed by
employing in situ generated electron-deficient allene reagents prepared from
propargylamine-based 1,6-diynes with different substituents. Moreover, the
modification was further applied to protein modification.
69
3.2 Results and Discussion
3.2.1 Modification of Cysteine-Containing Peptides Using in situ Generated
3.2.1.1 Optimization of Reaction Conditions
To prepare the stock solution of in situ generated electron-deficient allene reagents,
a mixture of propargylamine-based 1,6-diyne 2 and iodine monochloride (ICl, 1.05
equivalents) was stirred in room temperature for 1 h to allow cyclization / iodination
for in situ generation of 1H-isoindolium 3 (Scheme 3.1). After one hour, the reaction
mixture was further diluted to 5 mM in CH3CN. Under basic reaction conditions, the
1H-isoindoliums 3 could isomerize to electron-deficient allenes 4 which react with the
thiol groups on cysteine residues.

H
R2 R2 I R2 2 Cl
N R
N
1) ICl (1.05 equiv.), CH3CN, r.t., 1 h
R3 2) reducing agent R1
R1
R3
2 3
Scheme 3.1 General procedure of preparation of in situ generated allene reagents.
As an initial attempt, propargylamine-based 1,6-diyne 2a was mixed with ICl for
the formation of in situ generated 1H-isoindolium 3a. After the reaction for 1 h,
reducing agent was added to the reaction mixture so as to quench the excess ICl. The
purpose of adding reducing agent was to prevent unwanted oxidation of cysteine
residues on peptides. The cysteine sulfhydryl group is nucleophilic and easily oxidized,
70
which can be readily converted to sulfenic acids and disulfides under oxidizing
conditions.48 Iodine monochloride is an oxidizing agent which potentially causes
oxidation of cysteine, which can block the target residue and hamper the modification
of peptides with allene reagents. Therefore, the addition of reducing agent in the
reaction mixtures could protect cysteine from oxidation.
The stock solution was directly used for bioconjugation with cysteine-containing
peptide AYEMWCFHQR 1a. Firstly, different common reducing agents, including
tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), sodium borohydride and
sodium ascorbate, were used for selection of reducing agent and optimization of
reaction conditions (Table 3.1, Entries 1-4). From the mass spectrum of the stock
solution, formation of 1H-isoindolium could be observed with m/z = 462.0129 (Figure
3.1). The stock solution showed excellent stability and could be stored at –20 °C for
repeated use.
Figure 3.1 LC–MS analysis of in situ generated allene reagent (TIC chromatogram).
With 1 equivalent of TCEP, treatment of cysteine-containing peptide
AYEMECFHQR 1a (0.1 mM) with in situ generated electron-deficient allene reagent
71
4a (1 equivalent) in pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C for 4 h
afforded modified peptides 5a in 98% conversion. Increasing the amount of in situ
generated allene reagent 4a from 1 to 5 equivalents gave comparable results of
modification. Increasing the amount of TCEP lowered the overall conversion of
modification (Table 3.1, Entry 5). Additionally, formation of side product 5a’ was
observed when increasing the TCEP loading to 2 equivalents (Figure 3.2).
Table 3.1 Optimization of modification conditions.a
O
H
O Cl
N I
N
2) reducing agent
Cl
Cl
3a
2a
H O
I
Cl
N H
AYEMWCFHQR 1a
S
pH 8.0 potassium phosphate Cl
buffer/ CH3CN (9:1) AYEMW FHQR
N
25 ºC, 4 h H
O
5a
Propargylamine- Amount of
Conversion
Entry based 1,6-diynes Reducing Agent Reducing
(%)b
(equiv.) Agent (equiv.)
1 1 TCEP 1 98
2 1 NaBH4 1 95
3 1 DTT 1 95
4 1 Sodium ascorbate 1 93
5 1 TCEP 2 89
6 2 TCEP 1 99
7 3 TCEP 1 99
72
8 4 TCEP 1 99
9 5 TCEP 1 99
a
different amounts of in situ generated allene reagent 4a prepared from
propargylamine-based 1,6-diyne 2a in 50 mM pH 8.0 potassium phosphate
b
buffer/CH3CN (9:1) at 25 °C for 4 h. Conversion of the modification was
determined by LC–MS analysis.
H Cl
I O O O
H
N I
Cl N
N H
Cl
+
S
Cl S
5 mM stock solution in CH3CN
with TCEP (2 equiv.) AYEMW FHQR
N AYEMW FHQR
AYEMWCFHQR 1a H N
O H Cl
O
5a (89% conversion) 5a’ (8% conversion)
Scheme 3.2 Formation of side product 5a'.
Figure 3.2 LC–MS analysis of side product 5a' (TIC chromatogram).
Control experiment was conducted by treatment of cysteine-containing peptide
AYEMWCFHQR 1a with 10 equivalents of propargylamine-based 1,6-diyne 2a, which
showed the formation of 5a’ as the single product in 30% conversion. The cysteine
73
residue on the peptides was modified while all other residues remained intact as shown
in LC–MS/MS analysis (Figure 3.3). This result revealed that product 5a’ was
suggested to form by cysteine modification through thiol-yne reaction between the thiol
group on cysteine and the alkyne group on the propargylamine-based 1,6-diyne 2a,
which could possibly be proceeded through radical processes or under base-catalyzed
conditions as reported in previous literature examples.58a, 71
Figure 3.3 MS/MS analysis of side product 5a'.
Although the modification of propargylamine-based 1,6-diynes on cysteine was
observed, the reactivity was far lower than that of electron-deficient allene reagents.
74
Besides, the formation of product 5a’ could be minimized (< 2% conversion) under
optimized reaction conditions. Therefore, this observation was assumed to have little
effect on the selectivity of the modification.
3.2.1.2 Time Course Experiments of Cysteine Modification of Peptides by in situ
Generated Allene Reagents
To compare the performance of bioconjugation using the in situ generated allene
reagents with that using the isolated electron-deficient allene reagents, time course
experiments of the formation of modified peptide 5a at different pH values and
temperatures were performed. At different pH values, bioconjugation of peptide with
the in situ generated allene reagents gave comparable results with that conducted using
the isolated reagents, in which better conversion could be obtained in buffer with higher
pH values. The reaction rates were lower in slightly acidic media (pH 5.8 and pH 6.5)
at the first one hour of the reaction, which could be attributed to the low conversion of
1H-isoindoliums 3 to electron-deficient allenes 4 at these pH values. The isomerization
of 1H-isoindoliums 3 to electron-deficient allenes 4 should be promoted under basic
reaction conditions. At slightly acidic reaction conditions, the reagents remained in
their alkyne forms, which was less reactive than allenes towards thiol group on cysteine.
As a result, lower reaction rates were observed when the bioconjugation was conducted
at pH 5.8 and 6.5.
When the reaction was performed at room temperature or 37 °C, modified peptide
5a in > 80% conversion was observed at the first hour of the reaction. The conjugation
75
could also proceed smoothly at 4 °C, giving approximately 80% conversion after 4 h.
The result revealed that the use of the in situ generated allene reagents generally gave
improved conversion of modified peptides compared with the isolated allene reagents.
This could be explained by the presence of TCEP in the in situ generated allene reagents,
which could prevent the cysteine-containing peptides from dimerization. Therefore, the
cysteine residues on peptides could remain active for reaction with the allene reagents,
resulting in higher conversion of modified peptides.
(a) 100

Conversion (%)

40
20
0
0 1 2 3 4
Time (h)
(b)
100
4 °C
80 25 °C
Conversion (%)
37 °C
60
40
20
0
0 1 2 3 4
Time (h)
Figure 3.4 Time course experiments of the formation of cysteine modified peptide
5a at (a) different pH values; (b) different temperatures.
76
3.2.1.3 Investigation of Scope of in situ Generated Allene Reagents
The optimized reaction conditions of employing 1 equivalent of in situ generated
allene reagent bearing 1 equivalent of TCEP and pH 8.0 potassium phosphate buffer at
25 °C for 4 h was utilized for expansion of the scope with other in situ generated allene
reagents prepared from different propargylamine-based 1,6-diynes 2. Two cysteine-
containing peptides, AYEMWCFHQR 1a and STSSSCNLSK 1b, were treated with in
situ generated allene reagents 4a-d, which differed in the substituents on the para-
substituted phenyl ring at the R1 position. Modified peptides 5a-d and 6a-d were
obtained in 96-98% and 98-99% conversion, respectively, suggesting that the
modification was compatible with in situ generated allene reagents with different
substituents. Fluorescent allene reagent 4g prepared from coumarin-derived
propargylamine-based 1,6-diyne 2g was also applied to the modification, giving
modified peptide 5g and 6g in 52% and 85% conversion, respectively.
Table 3.2 Modification of cysteine-containing peptide AYEMWCFHQR 1a with in
situ generated electron-deficient allenes.a
H
R2 R2 I R2 2 Cl
N R
N
R3 2) TCEP (1 equiv.) R1
R1
R3
2 3
H
I R2 Cl
R2
N H
AYEMWCFHQR 1a R1
R3 S
pH 8.0 potassium phosphate
buffer/ CH3CN (9:1) AYEMW FHQR
N
25 ºC, 4 h H
O
5a-d, g
77
Entry Modified peptide Conversion (%)b
1,6-diynes 2
1 5a 98
2 5b 96
3 5c 96
4 5d 98
5 5g 52
a
in situ generated allene reagents 4 prepared from different propargylamine-based 1,6-
diynes 2 in 50 mM pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C for 4 h.
b
Conversion of the modification was determined by LC–MS analysis.
78
Table 3.3 Modification of cysteine-containing peptide STSSSCNLSK 1b with in situ
generated electron-deficient allenes.a

H
R2 R2 I R2 2 Cl
N R
N
R1
R3
2 3
H
I R2 2 Cl
R
N H
STSSSCNLSK 1b R1
R3 S
pH 8.0 potassium phosphate
buffer/ CH3CN (9:1) STSSS NLSK
N
25 ºC, 4 h H
O
6a-d, g
Entry Modified peptide Conversion (%)b
1,6-diynes 2
1 6a 98
2 6b 98
3 6c 99
79
4 6d 99
5 6g 85
a
Conditions of the modifications: treatment of STSSSCNLSK 1b (0.1 mM) with in
situ generated allene reagents 4 prepared from different propargylamine-based 1,6-
diynes 2 in 50 mM pH 8.0 potassium phosphate buffer/CH3CN (9:1) at 25 °C for 4
h. b Conversion of the modification was determined by LC–MS analysis.
For in situ generated allene reagents 4a-d prepared from propargylamine-based
1,6-diynes 2a-d, the substituents on the phenyl ring showed increased electron
withdrawing property, in which 2d (-CN) > 2c (-CF3) > 2a (-Cl) > 2b (-H). Time course
experiment of the formation of their corresponding bioconjugates 5a-d was conducted
to examine the effect of electron withdrawing group on the efficiency of the
modification. All reagents gave excellent conversion to desired products after
modification for 4 h, but there was difference in reaction rate among the reagents at the
beginning of the modification. At the first half hour of the reaction, reagents with
stronger electron withdrawing substituents gave higher conversion to modified peptides,
in which 5d (-CN) > 5c (-CF3) > 5a (-Cl) > 5b (-H). This observation could be
potentially explained by the increased electrophilicity of allene reagents caused by the
80
presence of strong electron withdrawing groups, which facilitated the nucleophilic
addition of thiol group and resulting in higher reaction rate of modification.
Figure 3.5 Time course experiment of formation of modified peptides 5a-d.
3.2.2 Modification of Cysteine-Containing Proteins Using in situ Generated
The in situ generated electron-deficient allene reagents were further employed to
protein modification. Bovine serum albumin (BSA) with a single free cysteine residue
were used for bioconjugation with in situ generated allene reagents 4a-d. With 1
equivalent of allene reagent 4a, modified protein BSA-1 was obtained in 28%
conversion by LC–MS analysis. When the loading of allene reagent was increased from
1 to 5 equivalents, both single-site and multisite modifications of protein were observed
and the results were consistent with four allene reagents 4a-d.
81
Taking modified protein BSA-6 as the example (Figure 3.6a), two bioconjugates
corresponding to single-site modification were found. The peak with m/z 66765.96 and
m/z 66892.00 indicated that one molecule of 2a (M.W.= 335) and 4a (M.W.= 462) was
incorporated into the protein, respectively. For multisite modification, six
bioconjugates were found. Random incorporation of one molecule of 4a and one
molecule of 2a (m/z 67227.51), two molecules of 4a (m/z 67354.50), two molecules of
4a and one molecule of 2a (m/z 67690.64), three molecules of 4a (m/z 67817.08), three
molecules of 4a and one molecule of 2a (m/z 68153.05), or four molecules of 4a (m/z
68281.26) was observed from the mass spectrum, indicating that the modification
occurred on not only the free cysteine Cys34, but also the cysteine residues produced
from reduced disulfide bonds. This observation was possibly attributed to the presence
of TCEP in the in situ generated allene reagents, leading to disulfide bond reduction
and resulting in multiple free cysteine residues available for modification.
H
R2 R2 I R2 2 Cl
N R
N
R1
R3
2 3
H
SH I R2 2 Cl
R
N H
R1
R3 S
protein
pH 7.4 PBS buffer, 25 ºC, 4 h
modified protein
Scheme 3.3 Modification of cysteine-containing protein BSA with in situ generated
electron-deficient allenes.a
82
(a)
(b)
(c)
(d)
Figure 3.6 Mass spectrum of (a) BSA-6; (b) BSA-7; (c) BSA-8 and (d) BSA-9.
83
Bioconjugation of BSA with 10 equivalents of fluorescent allene reagent 4g was
performed to give modified protein BSA-10. Two single-site modifications were
observed, including incorporation of one molecule of 4g and 2g in 8% and 19%
conversion, respectively (Figure 3.7). The high conversion of 4g-modified protein than
2g-modified protein could be attributed to their difference in steric bulkiness.
Propargylamine-based 1,6-diyne 2g was an uncharged compound that could access to
the charged protein surface more effectively than the charged and bulkier electron
deficient allene 4g. As a result, the reaction of protein with 2g was more favorable.
According to the SDS-PAGE analysis, modified protein BSA-10 was observed by
fluorescent analysis while no fluorescent signal was observed for native BSA (Figure
3.8). Employing Coomassie blue staining on the same gel, deep blue color signals of
native protein and BSA-10 were observed, indicating that the fluorescent tag has been
labeled on the protein.
Figure 3.7 Mass spectrum of fluorescent-labeled BSA-10.
84
Figure 3.8 SDS-PAGE analysis of native BSA and BSA-10.
Disulfide bond formation is essential for the folding, activity and stability of
proteins. The modification of proteins with the in situ generated allene reagents resulted
in multisite modified bioconjugates, which indicated the cleavage of protein disulfide
bonds. In this regard, LC–MS/MS analysis is required to determine the sites of
modification and Circular Dichroism can be used to examine if the modifications alter
the secondary and tertiary structures of the proteins. Despite the formation of
heterogenous products, this modification method still showed high selectivity towards
cysteine residues in proteins.
85
3.3 Conclusion
In summary, a cysteine modification using in situ generated electron-deficient
allenes was developed. Based on the newly developed cysteine modification method
using isolated 1H-isoindolium-based electron deficient allene reagents presented in
Chapter 2, a one-step procedure for preparation of electron deficient allene reagents for
bioconjugation was established.
The in situ generated electron deficient allene reagents were prepared by reaction
of propargylamine-based 1,6-diynes 2 with iodine monochloride, resulting in the
formation of in situ generation of 1H-isoindoliums 3. With 1 equivalent of reducing
agent, the stable reaction mixtures could serve as stock solutions of in situ generated
electron deficient allene reagents for direct use in bioconjugation.
The modification of peptides using the in situ generated allene reagents could
proceed with high efficiency (up to 99% conversion in 4 h) and the results were
comparable with those obtained using the isolated allene reagents. These findings
showed that this approach could serve as an alternative method for preparation of allene
reagents and be utilized in the newly developed cysteine modification using electron-
deficient allenes.
For protein modification and labeling, single-site modification was resulted to give
cysteine-modified protein in 28% conversion using 1 equivalent of allene reagent, while
multisite modification was observed by increasing the amount of reagent.
86
Chapter 4 Experimental Section
4.1 General procedures
All reagents were commercially available and used without further purification. Milli-
Q® water used as reaction solvent in peptide and protein modification and LC–MS was
deionised using a Milli-Q® Gradient A10 system (Millipore, Billerica, USA). Flash
column chromatography was performed using silica gel 60 (230-400 mesh ASTM) with
ethyl acetate/n-hexane or methanol/dichloromethane as eluent. 1H and 13

C NMR
spectra were recorded on a Bruker DPX-400, Varian Unity Inova 400 NB spectrometers.
All chemical shifts are quoted on the scale in ppm using TMS or residual solvent as the
internal standard. Coupling constants (J) are reported in Hertz (Hz) with the following
splitting abbreviations: s = singlet, br s = broad singlet, d = doublet, dd = double doublet,
t = triplet and m = multiplet. All the mass spectra were obtained on an ESI source of
Agilent 6540 Ultra High Definition (UHD) Accurate-Mass Q-TOF LC/MS systems in
the positive ion mode.
LC–MS analyses for peptide identification were performed by using an Agilent 6540
UHD Accurate-Mass Q-TOF LC/MS system equipped with an ion spray source and an
Agilent 1290 Infinity LC, using an Agilent ZORBAX RRHD SB300-C18 (1.8 μm, 2.1
x 100 mm) column. 3 μL of sample was injected with a flow rate of the flow rate was
0.2 mL/min. Mobile phase A was made of Milli-Q® water containing 0.1% formic acid.
Mobile phase B was made of HPLC grade methanol containing 0.1% formic acid. The
initial conditions for separation were 5% B for 3 min, followed by a linear gradient to
87
95% B by 17 min. The composition was maintained for 10 min, followed by a linear
gradient to 5% B by 1 min. The composition was maintained for 4 min. Operating
conditions optimized for the detection of reaction mixture were the following: Gas
temperature: 300 °C, Drying gas: 8 L/min, Nebulizer: 35 psig, Sheath gas temperature:
270 °C, Sheath gas flow: 11 L/min, VCap: 3500 V, Nozzle voltage: 1000 V.
LC–MS analyses for protein identification were performed by using an Agilent 6540
UHD Accurate-Mass Q-TOF LC/MS system equipped with an ion spray source and an
Agilent 1290 Infinity LC, using an Agilent ZORBAX RRHD SB300-C3 (1.8 μm, 2.1
x 100 mm) column. 3 μL of sample was injected with a flow rate of 0.2 mL/min. Mobile
phase A was made of Milli-Q® water containing 0.1% formic acid. Mobile phase B was
made of HPLC grade methanol containing 0.1% formic acid. The initial conditions for
separation were 5% B for 3 min, followed by a linear gradient to 95% B by 17 min.
The composition was maintained for 10 min, followed by a linear gradient to 5% B by
1 min. The composition was maintained for 4 min. Operating conditions optimized for
the detection of reaction mixture were the followings: Gas temperature: 300 °C, Drying
gas: 8 L/min, Nebulizer: 35 psig, Sheath gas temperature: 350 °C, Sheath gas flow: 11
L/min, VCap: 3500 V, Nozzle voltage: 1000 V.
88
4.2 Literature Reference of Compounds
M. A. Cinellu, A. Zucca, S. Stoccoro, G.

Minghetti, M. Manassero and M.
Sansoni, J. Chem. Soc., Dalton Trans.,
1995, 2865–2872.
G. Charron, M. M. Zhang, J. S. Yount, J.

Wilson, A. S. Raghavan, E. Shamir and
H. C. Hang, J. Am. Chem. Soc., 2009,
131, 4967–4975.
M. L. Mason, R. F. Lalisse, T. J.
Finnegan, C. M. Hadad, D. A. Modarelli,
J. R. Parquette, Langmuir, 2019, 35,
12460−12468.
4.3 Calculation of Peptide Conversion
The crude reaction mixture of unmodified peptide (starting) and modified peptide
(product) was subjected to LC–MS analysis with elution time of 35 min. After data
processing by MassHunter, peptide conversion at different time intervals was
determined by measuring the relative peak intensities of starting and product in the
mass spectrum as follows:
𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (%) =
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝑒𝑎𝑘 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑆𝑡𝑎𝑟𝑡𝑖𝑛𝑔

(1 − ) × 100%
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝑒𝑎𝑘 𝐼𝑛𝑡𝑒𝑠𝑖𝑡𝑖𝑒𝑠 𝑜𝑓 𝑆𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑎𝑛𝑑 𝑃𝑟𝑜𝑑𝑢𝑐𝑡
89
4.4 Calculation of Protein Conversion
The crude reaction mixture of unmodified protein (starting) and modified protein
(product) was subjected to LC–MS analysis with elution time of 35 min. After data
processing by MassHunter, protein conversion at different time intervals was
determined by measuring the relative peak intensities of starting and product in the
mass spectrum as follows:
𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (%) =
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝑒𝑎𝑘 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑆𝑡𝑎𝑟𝑡𝑖𝑛𝑔

(1 − ) × 100%
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝑒𝑎𝑘 𝐼𝑛𝑡𝑒𝑠𝑖𝑡𝑖𝑒𝑠 𝑜𝑓 𝑆𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑎𝑛𝑑 𝑃𝑟𝑜𝑑𝑢𝑐𝑡
4.5 Synthesis of Propargylamine-based 1,6-diynes 2
To synthesize i, a mixture of aldehyde (10 mmol, 1 equiv.), amine (12 mmol, 1.2 equiv.),
alkyne (15 mmol, 1.5 equiv.) and gold catalyst (2 mol%) in ClCH2CH2Cl was stirred at
60 °C overnight. After the reaction, the resulting mixture was concentrated under
90
reduced pressure and purified by flash column chromatography using EtOAc/hexane
(1:3) as eluent to give product i.
To synthesize 2, a mixture of i (5 mmol, 1 equiv.) and K2CO3 (5 mmol, 1 equiv.) in 10
mL of CH3OH was stirred at room temperature for 30 min. After the reaction, the
reaction mixture was diluted with 20 mL of H2O. The mixture was then extracted with
CH2Cl2 (20 mL × 3). The solvent was removed by rotary evaporation and purified by
flash column chromatography using EtOAc/hexane (1:3) as eluent to give product 2.
Pale yellow solid, 62% yield.
1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.6 Hz, 1H), 7.57 – 7.52 (m, 1H), 7.44 –
7.34 (m, 3H), 7.28 (dd, J = 8.8, 7.3 Hz, 3H), 5.17 (s, 1H), 3.76 – 3.65 (m, 4H), 3.34 (s,
1H), 2.72 – 2.59 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 140.38, 134.44, 133.41, 133.15, 128.83, 128.77, 128.72,
127.92, 122.68, 121.53, 86.88, 86.74, 82.18, 81.85, 67.18, 59.96, 50.26.
HRMS (ESI): [M+H]+ Calcd. for [C21H19ClNO]+ 336.1150, found 336.1157.
91
7.46 (m, 2H), 7.41 – 7.35 (m, 1H), 7.34 – 7.25 (m, 4H), 5.19 (s, 1H), 3.76 – 3.66 (m,
4H), 3.34 (s, 1H), 2.76 – 2.61 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 140.64, 133.37, 131.94, 128.93, 128.70, 128.45, 128.42,
127.84, 123.09, 122.69, 88.05, 85.61, 82.09, 81.94, 67.23, 59.97, 50.24.
HRMS (ESI): [M+H]+ Calcd. for [C21H20NO]+ 302.1539, found 302.1546.
1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.7 Hz, 1H), 7.58 (s, 5H), 7.41 – 7.35 (m,
1H), 7.30 (t, J = 7.5 Hz, 1H), 5.21 (s, 1H), 3.75 – 3.66 (m, 4H), 3.35 (s, 1H), 2.67 (ddt,
J = 20.2, 10.7, 5.1 Hz, 4H).
13C NMR (100 MHz, CDCl3) δ 140.17, 133.49, 132.21, 130.39, 128.81, 128.77, 128.03,
126.87, 125.46, 125.42, 125.39, 125.35, 122.75, 122.72, 88.41, 86.73, 82.27, 82.25,
81.80, 67.19, 59.99, 50.30.
HRMS (ESI): [M+H]+ Calcd. for [C22H19F3NO]+ 370.1413, found 370.1418.
92
Yellow solid, 40% yield.
1H NMR (400 MHz, CDCl3) δ 7.69 – 7.63 (m, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.55 (d,
J = 7.8 Hz, 3H), 7.38 (td, J = 7.6, 1.3 Hz, 1H), 7.29 (td, J = 7.5, 1.2 Hz, 1H), 5.20 (s,
1H), 3.69 (pt, J = 5.5, 2.8 Hz, 4H), 3.35 (s, 1H), 2.73 – 2.58 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 133.50, 132.46, 132.16, 128.77, 128.70, 128.09, 82.34,
67.12, 60.00, 50.29.
HRMS (ESI): [M+H]+ Calcd. for [C22H19N2O]+ 327.1492, found 327.1497.
1H NMR (400 MHz, CDCl3) δ 7.70 (dd, J = 7.8, 1.3 Hz, 1H), 7.55 (dd, J = 7.6, 1.4 Hz,
1H), 7.43 (s, 4H), 7.38 (td, J = 7.6, 1.5 Hz, 1H), 7.31 – 7.26 (m, 1H), 5.19 (s, 1H), 3.70
(dt, J = 5.8, 3.8 Hz, 4H), 3.34 (s, 1H), 3.17 (s, 1H), 2.74 – 2.59 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 140.39, 133.43, 132.18, 131.84, 128.87, 128.75, 127.94,
123.55, 122.70, 122.12, 87.82, 87.49, 83.36, 82.18, 81.87, 67.21, 60.02, 50.28.
HRMS (ESI): [M+H]+ Calcd. for [C23H20NO]+ 326.1539, found 326.1539.
93
4.5.1 Synthesis of Coumarin-derived Propargylamine-based 1,6-diyne 2g
To synthesize ii, a mixture of 7-(diethylamino)coumarin-3-carboxylic acid (2 mmol, 1
equiv.), N-hydroxysuccinimide (2.6 mmol, 1.3 equiv.), 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDCI) (2.6 mmol, 1.3 equiv.) and 4-
dimethylaminopyridine (DMAP) (2 mmol, 1 equiv.) in CH2Cl2 (15 mL) was stirred in
room temperature for 8 h. After the reaction, the reaction mixture was diluted with 30
mL of CH2Cl2. The mixture was washed with H2O (30 mL × 2), and then dried over
anhydrous MgSO4. The solvent was removed by rotary evaporation to give residue
which was purified by flash column chromatography using CH2Cl2/EtOAc (10:1) as
eluent to give product ii.
To synthesize iii, a mixture of compound ii (1.57 mmol, 1 equiv.), 4-amino-1-Boc-
piperidine (1.57 mmol, 1 equiv.) and DMAP (0.31 mmol, 0.2 equiv.) in CH2Cl2 (15 mL)
94
was stirred at room temperature for 8 h. After the reaction, the solvent was removed by
rotary evaporation. The mixture was re-dissolved by TFA/CH2Cl2 (1:1, 5 mL:5 mL)
and was stirred in room temperature for 1 h. After the reaction, the reaction mixture
was diluted with 20 mL of H2O. The mixture was neutralized to pH 8.0 by 1 M NaOH
solution. The mixture was extracted with CH2Cl2 (30 mL × 3), and then dried over
anhydrous MgSO4. The solvent was removed by rotary evaporation to give residue
which was purified by flash column chromatography using CH2Cl2/CH3OH (10:1) as
eluent to give product iii.
Yellow Powder; 91% isolated yield.
1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 7.6 Hz, 1H), 8.69 (s, 1H), 7.43 (d, J = 9.0
Hz, 1H), 6.64 (dd, J = 9.0, 2.4 Hz, 1H), 6.50 (d, J = 2.3 Hz, 1H), 4.02 – 4.12 (m, 1H),
3.45 (q, J = 7.1 Hz, 4H), 3.10 (dd, J = 9.0, 3.7 Hz, 2H), 2.80 – 2.65 (m, 2H), 2.01 (d, J
= 9.6 Hz, 2H), 1.56 – 1.38 (m, 2H), 1.24 (t, J = 7.1 Hz, 6H).
3) δ 162.88, 162.58, 157.82, 152.73, 148.23, 131.26, 110.34,

13C NMR (100 MHz, CDCl
110.09, 108.55, 96.77, 46.13, 45.22, 44.51, 31.84, 12.57.
HRMS (ESI): [M+H]+ Calcd. for [C19H26N3O3]+ 344.1969, found 344.1996.
To synthesize iv, a mixture of 2-[(trimethylsilyl)ethynyl]benzaldehyde (10 mmol, 1
equiv.), iii (12 mmol, 1.2 equiv.), 1-chloro-4-ethynylbenzene (15 mmol, 1.5 equiv.) and
95
gold catalyst (2 mol%) in ClCH2CH2Cl was stirred at 60 °C overnight. After the
reaction, the resulting mixture was concentrated under reduced pressure and purified
by flash column chromatography using EtOAc/CH2Cl2 (1:4) as eluent to give product
iv.
To synthesize 2g, a mixture of iv (5 mmol, 1 equiv.) and K2CO3 (5 mmol, 1 equiv.) in
10 mL of CH3OH was stirred at room temperature for 30 min. After the reaction, the
reaction mixture was diluted with 20 mL of H2O. The mixture was then extracted with
CH2Cl2 (20 mL × 3). The solvent was removed by rotary evaporation and purified by
flash column chromatography using EtOAc/CH2Cl2 (1:4) as eluent to give product 2g.
1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 7.8 Hz, 1H), 8.68 (s, 1H), 7.70 (d, J = 7.6
Hz, 1H), 7.57 – 7.52 (m, 1H), 7.46 – 7.34 (m, 4H), 7.31 – 7.27 (m, 2H), 6.63 (dd, J =
9.0, 2.3 Hz, 1H), 6.49 (d, J = 2.1 Hz, 1H), 5.22 (s, 1H), 4.00 (td, J = 13.8, 6.8 Hz, 1H),
3.44 (q, J = 7.1 Hz, 4H), 3.35 (s, 1H), 2.92 (dd, J = 29.3, 11.5 Hz, 2H), 2.69 – 2.56 (m,
1H), 2.53 – 2.42 (m, 1H), 2.07 – 1.91 (m, 2H), 1.78 (s, 1H), 1.74 – 1.51 (m, 2H), 1.23
(t, J = 7.1 Hz, 6H).
96
13C NMR (100 MHz, CDCl3) δ 162.84, 162.46, 157.76, 152.62, 148.07, 141.18, 134.27,
133.23, 131.20, 128.75, 127.73, 122.69, 121.75, 110.67, 110.03, 108.56, 96.74, 87.01,
82.07, 59.84, 47.06, 46.66, 45.21, 32.11, 12.60.
HRMS (ESI): [M+H]+ Calcd. for [C36H35ClN3O3]+ 592.2361, found 592.2377.
4.6 Synthesis of 1H-isoindoliums 3
A mixture of 2 (1.0 mmol), electrophile (1.05 mmol, 1.05 equiv.) in 5 mL of CH3CN
was stirred at room temperature for 1 h, and then AgPF6 (1.1 mmol, 1.1 equiv.) was
added to the reaction mixture and stirred for 5 min. The resulting precipitate (AgI) was
separated by suction filtration and washed by CH2Cl2. The filtrate was concentrated
under vacuum and the residue was purified by flash column chromatography using
CH2Cl2/CH3OH (50:1) to give product 3.
Grey solid, 90% yield.
1H NMR (400 MHz, d6-DMSO) δ 8.60 (d, J = 7.7 Hz, 1H), 8.26 (s, 1H), 7.84 – 7.68
(m, 3H), 7.59 – 7.53 (m, 2H), 7.50 – 7.44 (m, 2H), 6.99 (s, 1H), 4.42 (td, J = 10.4, 2.8
97
Hz, 1H), 4.32 (td, J = 14.2, 2.4 Hz, 2H), 4.20 – 3.99 (m, 3H), 3.87 – 3.77 (m, 1H), 3.66
(d, J = 12.1 Hz, 1H).
13C NMR (100 MHz, d6-DMSO) δ 146.60, 136.25, 135.29, 133.80, 132.60, 130.21,
129.08, 129.00, 125.90, 124.82, 118.45, 92.86, 80.29, 79.03, 65.67, 62.94, 61.96, 61.72,
56.44.
HRMS (ESI): [M-PF6]+ Calcd. for [C21H18ClINO]+ 462.0116, found 462.0114.
Grey solid, 81% yield.
(m, 3H), 7.50 (dd, J = 25.8, 6.9 Hz, 3H), 7.42 (t, J = 7.5 Hz, 2H), 6.99 (s, 1H), 4.40 (t,
J = 11.6 Hz, 1H), 4.32 (t, J = 14.0 Hz, 2H), 4.19 – 4.11 (m, 1H), 4.07 (d, J = 12.9 Hz,
1H), 4.01 (d, J = 13.5 Hz, 1H), 3.81 (t, J = 11.5 Hz, 1H), 3.65 (d, J = 12.9 Hz, 1H).
13C NMR (150 MHz, d6-DMSO) δ 146.57, 136.40, 132.57, 132.01, 130.44, 130.17,
129.05, 128.84, 125.86, 124.76, 119.50, 94.01, 79.31, 79.02, 65.79, 62.83, 61.92, 61.69,
56.38, 30.68.
HRMS (ESI): [M-PF6]+ Calcd. for [C21H19INO]+ 428.0506, found 428.0505.
98
White solid, 40% yield.
(m, 7H), 7.03 (s, 1H), 4.48 – 4.38 (m, 1H), 4.36 – 4.25 (m, 2H), 4.14 (t, J = 12.2 Hz,
2H), 4.02 (d, J = 13.6 Hz, 1H), 3.86 – 3.73 (m, 1H), 3.67 (d, J = 12.7 Hz, 1H).
13C NMR (100 MHz, d6-DMSO) δ 146.55, 136.04, 132.86, 132.58, 130.27, 129.07,
125.84, 125.70, 125.66, 125.63, 125.59, 124.82, 123.85, 122.35, 92.25, 81.62, 79.15,
65.45, 63.07, 61.95, 61.68, 56.54.
19
F NMR (376 MHz, d6-DMSO) δ -62.55, -70.21, -72.10.
HRMS (ESI): [M-PF6]+ Calcd. for [C22H18F3INO]+ 496.0380, found 496.0388.
1H NMR (400 MHz, d6-DMSO) δ 8.60 (d, J = 8.7 Hz, 1H), 8.27 (s, 1H), 7.91 (d, J =
8.3 Hz, 2H), 7.75 (t, J = 10.1 Hz, 5H), 7.03 (s, 1H), 4.42 (t, J = 10.8 Hz, 1H), 4.35 –
4.25 (m, 2H), 4.13 (t, J = 10.1 Hz, 2H), 4.01 (d, J = 13.4 Hz, 1H), 3.80 (t, J = 11.7 Hz,
1H), 3.66 (d, J = 12.2 Hz, 1H).
99
13C NMR (100 MHz, d6-DMSO) δ 146.54 (s), 135.93 (s), 132.67 (d, J = 18.6 Hz),
130.27 (s), 129.07 (s), 125.83 (s), 124.82 (s), 124.33 (s), 118.06 (s), 112.55 (s), 92.16
(s), 82.81 (s), 79.13 (s), 65.42 (s), 63.10 (s), 61.95 (s), 61.67 (s), 56.55 (s).
HRMS (ESI): [M-PF6]+ Calcd. for [C22H18IN2O]+ 453.0458, found 453.0461.
Brown solid, 93% yield.
1H NMR (600 MHz, CD3CN) δ 8.64 (d, J = 7.8 Hz, 1H), 7.78 (s, 1H), 7.75 (t, J = 7.5
Hz, 1H), 7.72 – 7.67 (m, 2H), 7.55 – 7.49 (m, 2H), 7.15 (t, J = 8.8 Hz, 2H), 6.43 (s,
1H), 4.26 (d, J = 13.8 Hz, 1H), 4.24 – 4.15 (m, 2H), 4.14 – 4.02 (m, 3H), 3.75 (ddd, J
= 14.3, 10.7, 3.9 Hz, 1H), 3.58 (dd, J = 13.3, 2.1 Hz, 1H).
13C NMR (150 MHz, CD3CN) δ 164.24 (d, J = 250.5 Hz), 147.64, 136.67, 135.26 (d,
J = 8.8 Hz), 133.45, 130.93, 129.83, 127.33, 125.42, 116.82 (d, J = 3.5 Hz), 116.68 (d,
J = 22.7 Hz), 94.90, 78.31, 75.85, 67.58, 63.24, 62.72, 62.60, 57.15.
19F NMR (565 MHz, CD3CN) δ -72.12, -73.37, -109.10.
HRMS (ESI): [M-PF6]+ Calcd. for [C21H18FINO]+ 446.0412, found 446.0410.
100
(m, 3H), 7.37 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 6.76 (s, 1H), 4.20 (t, J =
13.3 Hz, 1H), 4.00 (d, J = 11.9 Hz, 1H), 3.63 (t, J = 12.0 Hz, 1H), 3.55 (d, J = 12.0 Hz,
1H), 2.35 – 2.20 (m, 4H), 2.13 (s, 2H), 1.83 (d, J = 12.7 Hz, 2H), 1.68-1.57 (m, 1H).
13C NMR (150 MHz, d6-DMSO) δ 147.57, 140.38, 136.93, 132.44, 131.80, 129.98,
129.46, 129.30, 125.73, 124.71, 116.64, 93.03, 79.40, 77.80, 64.88, 64.54, 57.56, 21.56,
21.34, 21.07, 20.18.
HRMS (ESI): [M-PF6]+ Calcd. for [C23H23IN]+440.0870, found 440.0837.
7.70 (m, 2H), 7.66 – 7.61 (m, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H),
7.38 – 7.34 (m, 2H), 7.28 (s, 2H), 6.65 – 6.61 (m, 1H), 6.45 – 6.39 (m, 2H), 4.13 (d, J
= 13.4 Hz, 1H), 4.06 – 3.95 (m, 2H), 3.92 – 3.81 (m, 1H), 3.43 (d, J = 6.9 Hz, 4H), 2.74
101
(td, J = 10.6, 4.5 Hz, 1H), 2.60 (t, J = 10.6 Hz, 1H), 2.49 – 2.42 (m, 1H), 2.37 – 2.26
(m, 1H), 1.22 – 1.20 (m, 6H).
13C NMR (100 MHz, CDCl3) δ 163.56, 163.36, 157.85, 153.16, 148.45, 147.63, 136.79,
136.07, 133.95, 133.68, 133.28, 131.62, 130.35, 129.34, 129.11, 126.74, 125.35,
118.38, 110.55, 109.29, 108.49, 96.57, 94.93, 78.49, 73.80, 69.12, 60.49, 54.40, 45.31,
41.93, 26.90, 26.41, 12.60.
HRMS (ESI): [M-PF6]+ Calcd. for [C36H34ClIN3O3]+ 718.1328, found 718.1346.
(m, 2H), 7.57 – 7.49 (m, 4H), 6.99 (s, 1H), 4.40 (d, J = 17.0 Hz, 2H), 4.35 – 4.25 (m,
2H), 4.08 (td, J = 30.1, 27.2, 12.7 Hz, 4H), 3.86 – 3.74 (m, 1H), 3.65 (d, J = 12.6 Hz,
1H).
13C NMR (100 MHz, d6-DMSO) δ 146.52, 136.20, 132.53, 132.22, 131.97, 130.17,
129.03, 125.81, 124.75, 123.50, 119.87, 93.19, 83.66, 82.59, 81.06, 79.04, 65.68, 62.90,
61.91, 61.66, 56.43.
102
4.7 Synthesis of Electron-Deficient Allenes 4
A mixture of 3 (0.5 mmol) and Et3N (0.5 mmol, 1 equiv) in 5 mL of CH2Cl2 (5 mL)
was stirred at room temperature for 1 h. The product was separated by suction filtration
and washed by CH2Cl2 to give the product 4.
1H NMR (400 MHz, d6-DMSO) δ 8.79 (dd, J = 6.8, 1.9 Hz, 1H), 8.44 (s, 1H), 8.03 (s,
1H), 7.78 – 7.71 (m, 2H), 7.71 – 7.65 (m, 2H), 7.56 (s, 1H), 7.54 (s, 1H), 7.48 – 7.42
(m, 1H), 4.49 (t, J = 11.6 Hz, 1H), 4.35 (d, J = 12.5 Hz, 1H), 4.28 – 4.01 (m, 7H).
13C NMR (100 MHz, d6-DMSO) δ 195.44, 148.62, 135.10, 132.96, 130.97, 130.76,
130.63, 129.52, 129.20, 129.01, 124.82, 122.30, 121.35, 112.79, 78.09, 64.18, 63.20,
60.86, 60.76.
HRMS (ESI): [M-PF6]+ Calcd. For [C21H18ClINO]+ 462.0116, found 462.0153.
103
1H NMR (600 MHz, d6-DMSO) δ 8.88 – 8.65 (m, 1H), 8.45 (s, 1H), 8.02 (s, 1H), 7.77
– 7.70 (m, 2H), 7.66 (dd, J = 4.3, 3.1 Hz, 2H), 7.53 – 7.42 (m, 4H), 4.53 (t, J = 12.4 Hz,
1H), 4.36 (d, J = 12.1 Hz, 1H), 4.30 – 4.04 (m, 6H).
13C NMR (150 MHz, d6-DMSO) δ 195.22, 148.66, 132.95, 130.87, 130.75, 130.49,
130.17, 129.51, 129.07, 128.95, 124.83, 122.23, 121.23, 113.80, 78.01, 64.11, 63.32,
60.87, 60.73.
1H NMR (400 MHz, d6-DMSO) δ 8.79 (d, J = 8.4 Hz, 1H), 8.45 (s, 1H), 8.13 (s, 1H),
7.91 – 7.80 (m, 4H), 7.74 (tt, J = 8.3, 3.9 Hz, 2H), 7.51 – 7.44 (m, 1H), 4.47 (t, J = 11.1
Hz, 1H), 4.37 (d, J = 12.5 Hz, 1H), 4.30 – 4.07 (m, 6H).
104
13C NMR (100 MHz, d6-DMSO) δ 196.30, 148.57, 134.55, 132.93, 131.05, 130.44,
129.71, 129.04, 126.18, 124.81, 122.32, 121.46, 112.64, 78.20, 64.28, 63.17, 60.79,
54.89.
19
F NMR (376 MHz, d6-DMSO) δ -62.26, -70.21, -72.10.
HRMS (ESI): [M-PF6]+ Calcd. for [C22H18F3INO]+ 496.0380, found 496.0389.
1H NMR (400 MHz, d6-DMSO) δ 8.79 (d, J = 7.9 Hz, 1H), 8.45 (s, 1H), 8.11 (s, 1H),
7.95 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.74 (p, J = 7.3 Hz, 2H), 7.46 (d, J =
7.9 Hz, 1H), 4.49 – 4.33 (m, 2H), 4.30 – 4.06 (m, 6H).
13C NMR (100 MHz, d6-DMSO) δ 196.69 (s), 148.55 (s), 135.07 (s), 133.19 (s), 132.92
(s), 131.08 (s), 130.36 (s), 129.69 (s), 129.05 (s), 124.81 (s), 122.34 (s), 121.56 (s),
118.41 (s), 112.70 (s), 112.40 (s), 78.16 (s), 64.30 (s), 63.09 (s), 60.83 (d, J = 6.6 Hz).
HRMS (ESI): [M-PF6]+ Calcd. for [C22H18IN2O]+ 453.0458, found 453.0462.
105
1H NMR (600 MHz, d6-DMSO) δ 8.80 (s, 1H), 8.44 (s, 1H), 8.03 (s, 1H), 7.73 (s, 4H),
7.45 (s, 1H), 7.33 (s, 2H), 4.50 (t, J = 12.2 Hz, 1H), 4.35 (d, J = 12.4 Hz, 1H), 4.29-
4.02 (m, 6H).
13C NMR (150 MHz, d6-DMSO) δ 194.92, 163.24 (d, J = 248.5 Hz), 148.60, 132.91,
131.39, 131.36 (d, J = 8.5 Hz), 130.79 (d, J = 23.1 Hz), 131.33, 130.86, 130.71, 128.96,
124.79, 122.24, 121.26, 116.54 (d, J = 21.9 Hz), 112.78, 77.94, 64.12, 63.15, 60.83,
60.73.
19F NMR (565 MHz, d6-DMSO) δ -69.46, -70.42, -108.79.
HRMS (ESI): [M-PF6]+ Calcd. For [C21H18FINO]+ 446.0412, found 446.0370.
– 7.66 (m, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.44 – 7.36 (m, 1H), 7.29 (d, J = 8.0 Hz, 2H),
106
4.25 (d, J = 11.4 Hz, 1H), 4.10 – 3.89 (m, 3H), 2.46 (dd, J = 13.2, 8.1 Hz, 1H), 2.11 (d,
J = 11.2 Hz, 1H), 1.88 (d, J = 12.0 Hz, 2H), 1.81 – 1.62 (m, 2H).
13C NMR (100 MHz, d6-DMSO) δ 195.64, 149.45, 140.31, 132.91, 131.02, 130.69,
130.08, 128.92, 128.83, 127.45, 124.67, 122.08, 120.68, 112.42, 77.17, 66.27, 65.06,
21.01, 20.46, 20.26, 19.03.
HRMS (ESI): [M-PF6]+ Calcd. for [C23H23IN]+ 440.0870, found 440.0862.
1H NMR (400 MHz, CDCl3) δ 9.25 (d, J = 6.0 Hz, 1H), 8.78 (d, J = 7.3 Hz, 1H), 8.53
(s, 1H), 8.02 (s, 1H), 7.80 (s, 1H), 7.59 (q, J = 7.5 Hz, 3H), 7.43 (s, 2H), 7.41 (s, 2H),
7.39 (s, 2H), 6.68 – 6.63 (m, 1H), 6.44 (s, 1H), 4.33 – 4.28 (m, 1H), 4.21 (d, J = 6.7 Hz,
1H), 4.10 (s, 1H), 3.99 (d, J = 8.6 Hz, 1H), 3.43 (d, J = 6.9 Hz, 5H), 2.54 – 2.32 (m,
4H), 1.23 (d, J = 5.8 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 194.48, 163.65, 157.88, 153.22, 149.21, 148.41, 137.06,
133.69, 131.66, 131.33, 130.47, 130.35, 130.27, 130.13, 128.67, 128.17, 126.04,
124.55, 122.92, 115.35, 110.62, 109.39, 108.53, 96.67, 73.05, 63.41, 62.50, 45.36,
41.01, 25.60, 25.49, 12.63.
HRMS (ESI): [M-PF6]+ Calcd. for [C36H34ClIN3O3]+ 718.1328, found 718.1345.
107
– 7.69 (m, 2H), 7.66 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.47 – 7.41 (m, 1H),
4.47 (t, J = 11.5 Hz, 1H), 4.35 (d, J = 19.5 Hz, 2H), 4.17 (ddt, J = 45.9, 16.3, 9.0 Hz,
6H).
13C NMR (100 MHz, d6-DMSO) δ 195.77, 148.53, 132.86, 132.56, 130.87, 130.63,
130.54, 129.17, 124.73, 122.22, 121.28, 113.10, 82.94, 77.98, 64.11, 63.13, 60.79,
60.68.
4.8 Synthesis of Model Compound 7
H PF6 H PF6
O I O Cl
I Et3N (1 equiv.)
N N
CH3CN, r.t., 1 h
Cl H
3a 4a
O
H
SH I PF6
N H
(1 equiv.)
S Cl
CH3CN/H2O 3:1, r.t., 3 h
108
A mixture of 1H-isoindolium 3a (0.2 mmol) and Et3N (0.2 mmol, 1 equiv) in 3 mL of
CH3CN was stirred at room temperature for 1 h. Then, benzyl mercaptan (0.2 mmol, 1
equiv) and 1 mL of H2O was added to the mixture and stirred for 3 h. After the reaction,
the reaction mixture was diluted with 3 mL of CH2Cl2. The mixture was then extracted
with CH2Cl2 (3 mL × 2). The solvent was removed by rotary evaporation and purified
by flash column chromatography using CH2Cl2/CH3OH (50:1) as the eluent to give the
product in 63% isolated yield.
1H NMR (400 MHz, CD3CN) δ 8.68 (d, J = 6.9 Hz, 1H), 7.77 – 7.64 (m, 3H), 7.62 –
7.55 (m, 3H), 7.48 (d, J = 8.4 Hz, 2H), 7.34 – 7.25 (m, 3H), 7.20 (d, J = 6.6 Hz, 2H),
7.15 (s, 1H), 6.44 (s, 1H), 3.94 – 3.84 (m, 2H), 3.81 (d, J = 12.8 Hz, 1H), 3.77 – 3.64
(m, 2H), 3.48 – 3.36 (m, 2H), 3.32 (d, J = 13.1 Hz, 1H), 3.12 (t, J = 12.3 Hz, 2H).
13C NMR (100 MHz, CD3CN) δ 149.72, 139.23, 137.64, 136.97, 136.16, 134.16,
133.78, 132.41, 131.93, 131.70, 131.19, 131.00, 130.59, 130.10, 129.22, 127.75,
126.51, 74.18, 73.48, 66.24, 63.73, 63.38, 55.55, 38.65.
HRMS (ESI): [M-PF6]+ Calcd. for [C28H26ClINOS]+ 586.0463, found 586.0461.
109
4.9 Modification of Peptides Using Isolated Electron-Deficient Allene Reagents 4
A mixture of 10 µL of peptides 1 (1 mM in H2O), 5 µL of allene reagents 4 (10 mM in
CH3CN), 5 µL of CH3CN and 80 µL of 50 mM pH 8.0 potassium phosphate buffer was
treated in a 1.5 mL Eppendorf tube at 25 ºC for 4 h. The modified product was
characterized by LC–MS and LC–MS/MS analysis.
Using Isolated Electron-Deficient Allene Reagents 4 at Different pH Values
A mixture of 20 μL of peptides 1a (1 mM in H2O), 2 μL of allene reagents 4a (10 mM
in CH3CN), 18 μL of CH3CN and 160 μL of 50 mM potassium phosphate buffer with
different pH values was treated in a 1.5 mL Eppendorf tube at 25 ºC for 0–4 h. At each
time point, 25 μL of the resulting mixture was collected and diluted with 25 µL of Milli-
Q® water. The resulting mixture was characterized by LC–MS and LC–MS/MS
analysis to determine the conversion.
Using Isolated Electron-Deficient Allene Reagents 4 at Different Temperatures
A mixture of 20 μL of peptides 1a (1 mM in H2O), 2 μL of allene reagents 4a (10 mM
in CH3CN), 18 μL of CH3CN and 160 μL of 50 mM pH 8.0 potassium phosphate buffer
was treated in a 1.5 mL Eppendorf tube at different temperatures for 0–4 h. At each
time point, 25 μL of the resulting mixture was collected and diluted with 25 µL of Milli-
Q® water. The resulting mixture was characterized by LC–MS and LC–MS/MS
analysis to determine the conversion.
110
4.12 Studies on the Stability of the Modified Peptide 5a
A mixture of 25 μL of modified peptide 5a (0.1 mM in 50 mM pH 8.0 PBS buffer/
CH3CN (9:1)) and 25 μL of thiol-containing reagents (L-cysteine, DL-homocysteine,
and dithiothreitol (DTT)), reducing reagent (sodium ascorbate) (50 mM in H2O) was
treated in a 1.5 mL Eppendorf tube at 25 ºC for 3 h. The resulting mixture was
characterized by LC–MS and LC–MS/MS analysis to determine the conversion.
4.13 Modification of Proteins Using Isolated Electron-Deficient Allene Reagents 4
A mixture of 10 μL of proteins (BSA, insulin, RNaseA or lysozyme) (1 mM in 50 mM
pH 7.4 PBS buffer), 10 μL of allene reagents (10 mM in CH3CN), 80 μL of 50 mM pH
7.4 PBS buffer was treated in a 1.5 mL Eppendorf tube at 25 ºC for 4 h. The modified
product was purified by Bio-Rad Bio-Spin® 6 column prior to LC–MS analysis.
4.14 Sequential Modification of Proteins via Huisgen Azide-Alkyne Cycloaddition
A mixture of 50 μL of alkyne-functionalized proteins BSA-3 (0.1 mM in 50 mM PBS
buffer), 5 μL of rhodamine azide 10 (50 mM in DMSO), 5 μL of TBTA (5 mM in
DMSO), 5 μL of TCEP (5 mM in H2O), 5 μL of CuSO4 solution (5 mM in H2O) and
30 μL of 50 mM PBS buffer was treated in a 1.5 mL Eppendorf tube at 37 ºC for 1 h.
The modified product was characterized by LC–MS analysis.
4.15 SDS-PAGE Analysis of Native and Modified Proteins
The native BSA and modified proteins (BSA-3, BSA-4, BSA-10) (10 μL) was mixed
with 2X loading buffer (10 μL) in a 0.5 mL Eppendorf tube and then boiled for 5 min.
111
Samples were analysed by SDS-PAGE by loading a sample of the boiled solution (10
μL) in each lane of a 12% SDS-PAGE gel and running in a SE 250 Mini-Vertical Unit
(Amersham, USA) at 125 V at room temperature for 110 min. After SDS-PAGE
separation, the fluorescence of gel was scanned by Azure C600 gel-imaging system.
The gel was stained with Coomassie Blue G250 for 10 min, and destained in the
methanol/acetic acid solution (45% methanol, 10% acetic acid) for 10 min. The
destained gel was scanned by Azure C600 gel-imaging system.
4.16 Preparation of in situ Generated Electron-Deficient Allene Reagents 4
A mixture of propargylamine-based 1,6-diyne 2 (5 μmol) and iodine monochloride
(5.25 μmol, 0.26 μL) in 500 μL of CH3CN was stirred at room temperature for 1 h.
After that, the reaction mixture was diluted with 450 μL of CH3CN and 50 μL of TCEP
solution (100 mM in H2O) was added to the mixture. The reagent was stored at –20 °C
for repeated use.
4.17 Modification of Peptides Using in situ Generated Electron-Deficient Allene
Reagents 4
A mixture of 10 µL of peptides 1 (1 mM in H2O), 2 µL of in situ generated allene
reagents 4 (5 mM in CH3CN), 8 µL of CH3CN and 80 µL of 50 mM pH 8.0 potassium
phosphate buffer was treated in a 1.5 mL Eppendorf tube at 25 ºC for 4 h. The modified
product was characterized by LC–MS and LC–MS/MS analysis.
112
Using in situ Generated Electron-Deficient Allene Reagents 4 at Different pH
Values
A mixture of 20 μL of peptides 1a (1 mM in H2O), 4 μL of in situ generated allene
reagents 4a (5 mM in CH3CN), 16 μL of CH3CN and 160 μL of 50 mM potassium
phosphate buffer with different pH values was treated in a 1.5 mL Eppendorf tube at
25 ºC for 0–4 h. At each time point, 25 μL of the resulting mixture was collected and
diluted with 25 µL of Milli-Q® water. The resulting mixture was characterized by LC–
MS and LC–MS/MS analysis to determine the conversion.
Using in situ Generated Electron-Deficient Allene Reagents 4 at Different
Temperatures
A mixture of 20 μL of peptides 1a (1 mM in H2O), 4 μL of in situ generated allene
reagents 4a (5 mM in CH3CN), 16 μL of CH3CN and 160 μL of 50 mM pH 8.0
potassium phosphate buffer was treated in a 1.5 mL Eppendorf tube at different
temperatures for 0–4 h. At each time point, 25 μL of the resulting mixture was collected
and diluted with 25 µL of Milli-Q® water. The resulting mixture was characterized by
LC–MS and LC–MS/MS analysis to determine the conversion.
113
Using in situ Generated Electron-Deficient Allene Reagents 4a-d
A mixture of 20 μL of peptides 1a (1 mM in H2O), 4 μL of the in situ generated allene
reagents 4a-d (5 mM in CH3CN), 16 μL of CH3CN and 160 μL of 50 mM pH 8.0
potassium phosphate buffer was treated in a 1.5 mL Eppendorf tube at 25 ºC for 0–4 h.
At each time point, 25 μL of the resulting mixture was collected and diluted with 25 µL
of Milli-Q® water. The resulting mixture was characterized by LC–MS and LC–
MS/MS analysis to determine the conversion.
4.21 Modification of Proteins Using in situ Generated Electron-Deficient Allene
Reagents 4
A mixture of 5 μL of BSA protein (1 mM in 50 mM pH 7.4 PBS buffer), 10 μL of
allene reagents (5 mM in CH3CN), 85 μL of 50 mM pH 7.4 PBS buffer was treated in
a 1.5 mL Eppendorf tube at 25 ºC for 4 h. The modified product was purified by Bio-
Rad Bio-Spin® 6 column prior to LC–MS analysis.
114
4.22 MS Spectra
Figure 4.1 Extracted ion chromatogram of native peptide AYEMWCFHQR 1a.
Figure 4.2 Mass spectrum of native peptide AYEMWCFHQR 1a.
115
b-ions 72 235 364 495 681 784 931 1068 1196
H 2N A Y E M W C F H Q R COOH
1299 1136 1007 876 690 587 440 303 175 y-ions
Figure 4.3 MS/MS spectrum of native peptide AYEMWCFHQR 1a.
Figure 4.4 Extracted ion chromatogram of cysteine-modified peptide 5a.
116
Figure 4.5 Mass spectrum of cysteine-modified peptide 5a.
117
Figure 4.7 Extracted ion chromatogram of cysteine-modified peptide 5b.
Figure 4.8 Mass spectrum of cysteine-modified peptide 5b.
118
Figure 4.9 MS/MS spectrum of cysteine-modified peptide 5b.
119
Figure 4.10 Extracted ion chromatogram of cysteine-modified peptide 5c.
Figure 4.11 Mass spectrum of cysteine-modified peptide 5c.
120
Figure 4.12 MS/MS spectrum of cysteine-modified peptide 5c.
121
Figure 4.13 Extracted ion chromatogram of cysteine-modified peptide 5d.
Figure 4.14 Mass spectrum of cysteine-modified peptide 5d.
122
Figure 4.15 MS/MS spectrum of cysteine-modified peptide 5d.
123
Figure 4.16 Extracted ion chromatogram of cysteine-modified peptide 5e.
Figure 4.17 Mass spectrum of cysteine-modified peptide 5e.
124
Figure 4.18 MS/MS spectrum of cysteine-modified peptide 5e.
125
Figure 4.19 Extracted ion chromatogram of cysteine-modified peptide 5f.
Figure 4.20 Mass spectrum of cysteine-modified peptide 5f.
126
Figure 4.21 MS/MS spectrum of cysteine-modified peptide 5f.
127
Figure 4.22 Extracted ion chromatogram of cysteine-modified peptide 5g.
Figure 4.23 Mass spectrum of cysteine-modified peptide 5g.
128
Figure 4.24 MS/MS spectrum of cysteine-modified peptide 5g.
129
Figure 4.25 Extracted ion chromatogram of cysteine-modified peptide 5h.
Figure 4.26 Mass spectrum of cysteine-modified peptide 5h.
130
Figure 4.27 MS/MS spectrum of cysteine-modified peptide 5h.
131
NH 2
H 3C OH OH SH
O H O H O H O H O
H 2N N N N N O
N N N N N
H O H O H O H O H OH
OH OH OH NH 2 OH
O
STSSSCNLSK
Figure 4.28 Extracted ion chromatogram of native peptide STSSSCNLSK 1b.
Figure 4.29 Mass spectrum of native peptide STSSSCNLSK 1b.
132
b-ions 88 189 276 363 450 667 780 867 1241
H 2N S T S S S C N L S K COOH
926 825 738 651 564 461 347 234 147 y-ions
Figure 4.30 MS/MS spectrum of native peptide STSSSCNLSK 1b.
133
134
Figure 4.34 Extracted ion chromatogram of cysteine-modified peptide 6b.
Figure 4.35 Mass spectrum of cysteine-modified peptide 6b.
135
Figure 4.36 MS/MS spectrum of cysteine-modified peptide 6b.
136
Figure 4.37 Extracted ion chromatogram of cysteine-modified peptide 6c.
Figure 4.38 Mass spectrum of cysteine-modified peptide 6c.
137
Figure 4.39 MS/MS spectrum of cysteine-modified peptide 6c.
138
Figure 4.40 Extracted ion chromatogram of cysteine-modified peptide 6d.
Figure 4.41 Mass spectrum of cysteine-modified peptide 6d.
139
Figure 4.42 MS/MS spectrum of cysteine-modified peptide 6d.
140
Figure 4.43 Extracted ion chromatogram of cysteine-modified peptide 6e.
Figure 4.44 Mass spectrum of cysteine-modified peptide 6e.
141
Figure 4.45 MS/MS spectrum of cysteine-modified peptide 6e.
142
Figure 4.46 Extracted ion chromatogram of cysteine-modified peptide 6f.
Figure 4.47 Mass spectrum of cysteine-modified peptide 6f.
143
Figure 4.48 MS/MS spectrum of cysteine-modified peptide 6f.
144
Figure 4.49 Extracted ion chromatogram of cysteine-modified peptide 6g.
Figure 4.50 Mass spectrum of cysteine-modified peptide 6g.
145
Figure 4.51 MS/MS spectrum of cysteine-modified peptide 6g.
146
Figure 4.52 Extracted ion chromatogram of cysteine-modified peptide 6h.
Figure 4.53 Mass spectrum of cysteine-modified peptide 6h.
147
Figure 4.54 MS/MS spectrum of cysteine-modified peptide 6h.
148
Figure 4.55 Extracted ion chromatogram of native peptide KSTFC 1c.
Figure 4.56 Mass spectrum of native peptide KSTFC 1c.
149
Figure 4.57 MS/MS spectrum of native peptide KSTFC 1c.
150
151
Figure 4.61 Extracted ion chromatogram of native peptide CSKFR 1d.
Figure 4.62 Mass spectrum of native peptide CSKFR 1d.
152
Figure 4.63 MS/MS spectrum of native peptide CSKFR 1d.
153
154
155
Figure 4.67 Extracted ion chromatogram of native peptide STSSSANLSK 1e.
Figure 4.68 Mass spectrum of native peptide STSSSANLSK 1e.
156
b-ions 88 198 276 363 450 521 635 748 835
H 2N S T S S S A N L S K COOH
981 894 793 706 619 532 347 234 147 y-ions
Figure 4.69 MS/MS spectrum of native peptide STSSSANLSK 1e.
157
Figure 4.70 Extracted ion chromatogram of native peptide STSSSHNLSK 1f.
Figure 4.71 Mass spectrum of native peptide STSSSHNLSK 1f.
158
b-ions 88 189 276 363 450 587 673 786 873
H 2N S T S S S H N L S K COOH
960 859 772 685 598 461 347 234 147 y-ions
Figure 4.72 MS/MS spectrum of STSSSHNLSK 1f.
159
Figure 4.73 Extracted ion chromatogram of native peptide AYEMWSFHQR 1g.
Figure 4.74 Mass spectrum of native peptide AYEMWSFHQR 1g.
160
b-ions 72 235 364 495 681 768 915 1052 1180
H 2N A Y E M W S F H Q R COOH
1283 1120 991 860 674 587 440 303 175 y-ions
Figure 4.75 MS/MS spectrum of native peptide AYEMWSFHQR 1g.
161
Figure 4.76 Extracted ion chromatogram of native peptide WSKFR 1h.
Figure 4.77 Mass spectrum of native peptide WSKFR 1h.
162
Figure 4.78 MS/MS spectrum of native peptide WSKFR 1h.
163
Figure 4.79 Extracted ion chromatogram of native peptide YSKFR 1i.
Figure 4.80 Mass spectrum of native peptide YSKFR 1i.
164
Figure 4.81 MS/MS spectrum of native peptide YSKFR 1i.
165
Figure 4.82 Extracted ion chromatogram of native peptide PSKFR 1j.
Figure 4.83 Mass spectrum of native peptide PSKFR 1j.
166
Figure 4.84 MS/MS spectrum of native peptide PSKFR 1j.
167
Figure 4.85 Extracted ion chromatogram of native peptide GSKFR 1k.
Figure 4.86 Mass spectrum of native peptide GSKFR 1k.
168
Figure 4.87 MS/MS spectrum of native peptide GSKFR 1k.
169
Figure 4.88 Extracted ion chromatogram of native peptide ISKFR 1l.
Figure 4.89 Mass spectrum of native peptide ISKFR 1l.
170
Figure 4.90 MS/MS spectrum of native peptide ISKFR 1l.
171
4.23 NMR Spectra
1H NMR
13C NMR
172
1H NMR
13C NMR
173
1H NMR
13C NMR
174
1H NMR
13C NMR
175
19F NMR
176
1H NMR
13C NMR
177
1H NMR
13C NMR
178
19F NMR
179
1H NMR
13C NMR
180
1H NMR
13C NMR
181
1H NMR
13C NMR
182
1H NMR
13C NMR
183
1H NMR
13C NMR
184
1H NMR
13C NMR
185
19F NMR
186
1H NMR
13C NMR
187
1H NMR
13C NMR
188
19F NMR
189
1H NMR
13C NMR
190
1H NMR
13C NMR
191
1H NMR
13C NMR
192
1H NMR
13C NMR
193
1H NMR
13C NMR
194
1H NMR
13C NMR
195
19F NMR
196
1H NMR
13C NMR
197
1H NMR
13C NMR
198
19F NMR
199
1H NMR
13C NMR
200
1H NMR
13C NMR
201
1H NMR
13C NMR
202
1H NMR
13C NMR
203
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BASED ELECTRON-DEFICIENT ALLENES

The Hong Kong Polytechnic University

Chemoselective Cysteine Modification of Peptides

A thesis submitted in partial fulfilment of the requirements for

acknowledgement has been made in the text.

Site-selective modification of peptides and proteins has been recognized as a

powerful tool for biological studies. It is of ongoing interest to develop new

proteins using 1H-isoindolium-based electron-deficient allenes was developed.

A novel electrophile-mediated cascade cyclization/iodination of propargylamine-

transformed to allene functionalities to give the corresponding allene products. The

application of this new class of 1H-isoindolium-based electron-deficient allenes to

cysteine modification of peptides and proteins was presented. By treatment of cysteine-

containing peptides with 5 equivalents of allene reagents in aqueous media at 25 °C for

4 h, cysteine-modified peptides were obtained in good conversions up to 96% as

confirmed by LC–MS/MS analysis. Allene reagents with different functional groups

excessive thiol-containing reagents and reducing agents. Further application of this

modification was demonstrated in protein modification and labeling. Fluorescent tags

could be labeled on the cysteine-containing proteins by sequential modification using

the azide-alkyne click reaction.

was established to enhance the efficiency of the modification. By reaction of

propargylamine-based 1,6-diynes with iodine monochloride for the formation of in situ

generated 1H-isoindoliums, followed by the addition of 1 equivalent of reducing agent,

could be prepared for direct use in bioconjugation.

With 1 equivalent of the in situ generated allene reagents, the modification of

I would like to express my deepest gratitude to my supervisor, Dr. Man-Kin Wong

research study in these two years.

I would like to convey my greatest appreciation to my group members: Dr. Ka-

in providing useful and constructive recommendations for my research. I would also

like to express my sincere thanks to colleagues in Shenzhen Research Institute: Dr.

compounds for this project.

Yee-Man Wong for training and assistance in mass spectrometry.

and memorable experience.

LIST OF TABLES XVII

LIST OF ABBREVIATIONS XVIII

1.1 Allene Chemistry 1

1.1.1 Synthesis of Allenes 3

1.1.2 Reactions of Allenes 10

1.2 Chemical Modification of Peptides and Proteins 16

1.2.1 Lysine and N-Terminal Modification 17

1.2.2 Modification of Other Natural Amino Acids 23

1.2.3 Modification of Unnatural Amino Acids 27

1.3 Cysteine Modification 30

1.3.1 Classical Methods 32

1.3.2 Transition Metal-Free Modifications 35

1.3.3 Transition Metal-Mediated Modifications 38

2.2 Results and Discussion 42

2.2.1 Modification of Cysteine-Containing Peptides Using Isolated Electron-

2.2.1.1 Synthesis of Electron-Deficient Allenes 42

2.2.1.2 Optimization of Modification Reaction Conditions 45

2.2.1.3 Model Reaction of Cysteine Modification by Electron-Deficient

2.2.1.4 Investigation of Cysteine Selectivity of the Modification 50

2.2.1.5 Investigation of Scope of Allene reagents 53

2.2.1.6 Stability Study of Allene-modified Bioconjugates 58

2.2.2 Modification of Cysteine-Containing Proteins Using Isolated Electron-

2.2.2.1 Modification of BSA Protein with Allene Reagents 59

2.2.2.2 Control Experiments with Non-Free Cysteine-Containing Proteins 63

2.2.2.3 Fluorescent Labeling of Proteins 64

2.4 Suggestions for Future Research 67

Generated Electron-Deficient Allenes 69

3.2 Results and Discussion 70

3.2.1 Modification of Cysteine-Containing Peptides Using in situ Generated