design & synthesis of peptidomimetics adopting secondary
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University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
4-2-2014
Design & Synthesis of Peptidomimetics AdoptingSecondary Structures for Inhibition of p53/MDM2 Protein-protein Interaction and MultipleMyeloma Cell AdhesionHyun Joo KilUniversity of South Florida, [email protected]
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Scholar Commons CitationKil, Hyun Joo, "Design & Synthesis of Peptidomimetics Adopting Secondary Structures for Inhibition of p53/MDM2 Protein-proteinInteraction and Multiple Myeloma Cell Adhesion" (2014). Graduate Theses and Dissertations.https://scholarcommons.usf.edu/etd/5051
Design & Synthesis of Peptidomimetics Adopting Secondary Structures for Inhibition of
P53/MDM2 Protein-protein Interaction and Multiple Myeloma Cell Adhesion
by
Hyun Joo Kil
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida
Major Professor: Mark McLaughlin, Ph.D.
Wayne Guida, Ph.D.
Jianfeng Cai, Ph.D.
Juan Del Valle, Ph.D.
Date of Approval
April 2, 2014
Key Words: solution phase synthesis,-helix, -hairpin, peptoid-peptide hybrids
Copyright © 2014, Hyun Joo Kil
ACKNOWLEDGMENTS
I would like to thank you to my parents and my sister. They always have supported
and encouraged my dream. Even when I was in slump, they tried to guide me to the
right path. It is not too much to say that my Ph D. and my dissertation are the work of
my whole family.
Also, my advisor, Dr. Mark McLaughlin, has helped me a lot during my Ph. D
program. First, he gave me an opportunity to be involved in medicinal chemistry. When
I was a master´s student whose field was physical organic chemistry, I started to be
interested in medicinal chemistry. Since then, I had been determined to change my field
for my Ph D. I joined USF in 2008 and looked for a medicinal chemistry lab. I met Dr.
Mark McLaughlin as a routine for a new graduate student. After a short conversation, I
decided to join to his lab and worked over five years. He has been a great mentor and
helper to give advices or insights for the research.
Along with Dr. Mark McLaughlin, my previous advisor, Dr. In Sook Han-Lee, is
another person who gives huge influence on my life. When I met her first time, I was
in sophomore year in college. At that time, I did not know what I want to do with my
life. Because I do not want to do what everybody does after graduation in my major.
One day at class, she encouraged people to explore and adventure the different world
not to follow the same route as everybody does after the graduation. I was intrigued by
her suggestion and started to work on studying abroad. If there had not been her advice,
I would have followed the same route as everybody does and felt unhappy with my life.
I also would like to my committee members, Dr. Wayne Guida, Dr. Jianfeng Cai, and
Dr. Juan Del Valle. Whenever I have a presentations, they have always taught me
something by asking questions related to my research. Thanks to Also, I want to say
thank you to Dr. Kumar Mohan to let me use the research facilities in his lab.
My previous group members, Dr. Laura Anderson, Dr. Melissa Topper, Dr. Mingzhou
Zhou, Dr. Phillip Murray, and Dr. David Badger, helped me to get used to the new
environment and taught new skills or knowledge for my research. I felt thankful to them.
Also, my current group members, Yi Liang, Josanne-dee Woodroffe, and Michael
Doligalski, helped and encouraged each other´ works, which had been motivations.
i
TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………...……………..iii
LIST OF FIGURES…………………………………………………...……................………….iv
LIST OF SCHEMES……………………………………………...…………………………..…vii
LIST OF ABBREVIATIONS………………….…………………………………………..…...viii
ABSTRACT………………………………...…………………………………………………...x
CHAPTER ONE: INTRODUCTION…………………………………………...…….…………..1
1.1 Amino acids, peptides, and proteins…………………………………………………..1
1.2 Protein structures…………………………………………………………………...…2
1.3 -Helix mimetics in drug discovery…………………………………………………..3
1.4 -Sheets, -turns, and -hairpins in drug discovery…………………………………12
1.5 Solid-phase peptide synthesis………………………………………………………..26
1.6 Solution-phase peptide synthesis…………………………………………………….32
1.7 Peptoids………………………………………………………………………………46
1.8 References……………………………………………………………………………54
CHAPTER TWO: DESIGN OF NON-PEPTIDIC OLIGO-PYRIMIDINES
MIMICKING i, i+4(3), i+7 POSITIONS OF -HLICES……………………………….96
2.1 Introduction………………………………………………………….…………….....96
2.2 Results and discussion……………………………………………………………….98
2.3 Experimental sect ion………………………………………………………..105
2.3.1 General comments ……………………………………….………….….105
2.3.2 Synthesis of monomeric pyrimidyl derivatives, la-lj……………….....105
2.3.3 Synthesis of dimeric pyrimidyl derivatives, 4a-4e………………..….......109
2.3.4 Synthesis of trimeric pyrimidyl derivatives, 7a-7d……………….............114
2.3.5 Synthesis of 8-10………………………………………………….............118
2.4 References…………………………………………………………………..............119
CHAPTER THREE: SOLUTION-PHASE SYNTHESIS OF -HAIRPIN PEPTIDE
INHIBITING MULTIPLE MYEOLMA CELLS ADHESION………………..............122
3.1 Introduction……………………………………………………………....................122
3.2 Results and discussion…………………………………………………...................127
3.3 Experimental section……………………………………………………..............…135
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3.3.1 General comments……………………………………………................135
3.3.2 -Turn promoter synthesis………………………………………..........…135
3.3.3 Recognition strand synthesis……………………………………..........….138
3.3.4 Synthesis of linker………………………………………………..........….143
3.3.5 Synthesis of non-recognition strand…………………………….............144
3.3.6 Cyclization of recognition strand and non-recognition strand……............148
3.3.7 Linear synthesis of MTI-101…………………………....…....…..............149
3.4 References..................................................................................................................155
CHAPTER FOUR: CONSTRUCTION OF PEPTOID-PEPTIDE HYBRIDS
ADOPTING -HAIRPIN STRUCTURE........................................................................159
4.1 Introduction…………………………………………………………………............159
4.2 Results and discussion…………………………………………………..............….162
4.3 Experimental section……………………………………………………............….165
4.3.1 General comments…………………………………………….............….165
4.3.2 General procedure…………………………………………….............…..165
4.3.3 Synthesis of acyclic and cyclic peptoid-peptide hybrids 1……….............166
4.3.4 Synthesis of acyclic and cyclic peptoid-peptide hybrids 2……….............168
4.3.5 Synthesis of acyclic and cyclic peptoid-peptide hybrids 3……….............169
4.3.6 NMR study of peptoid-peptide hybrids…………………………..............171
4.4 References…………………………………………………………………..............171
APPENDIX: 1H,
13C NMR SPECRA AND MASS SPECTRA………………………..............175
ABOUT THE AUTHOR………………………………………………………............…End page
iii
LIST OF TABLES
Table 1 Side Chain Orientation of Corner Residues in -Turns ................................................. 16
Table 2 Structures of Amylin (20-29), Peptoid 1, and Retropeptoid 2. ....................................... 53
Table 3 Percent Yield of Monomeric Pyrimidyl Derivatives Synthesis ................................... 100
Table 4 Percent Yield of Terpyrimidyl Dimers Synthesis ....................................................... 102
Table 5 Percent Yield of Terpyrimidine Trimers Analogues ................................................... 103
Table 6 Percent Yield of synthesis 8-10 .................................................................................. 104
Table 7 Comparison of Solid-phase Synthesis and Solution-phase Synthesis .......................... 126
Table 8 Percent Yield of Continuous Solution-phase Synthesis .............................................. 133
Table 9 Percent Yield of Continuous Solution-phase Synthesis .............................................. 133
iv
LIST OF FIGURES
Figure 1 The resonance structure of peptide bond ....................................................................... 3
Figure 2 Examples of helical stabilization: - interaction (1), salt-bridge (2), cation-
interaction (3), disulfide (4), lactam (5), azobenzene (6), olefin (7) ............................... 8
Figure 3 Schamatics of hydrocarbon stapling and hydrogen-bonding surrogates ......................... 8
Figure 4 Examples of type II mimetics ....................................................................................... 9
Figure 5 Examples of type III mimetics .................................................................................... 10
Figure 6 Hydrogen bonding in parallel and anti-parallel of -sheet ........................................... 13
Figure 7 -Turn ........................................................................................................................ 14
Figure 8 G1 -bulge type I turn ................................................................................................ 15
Figure 9 -Hairpin structure ..................................................................................................... 17
Figure 10 Structures of -hairpin sequences ............................................................................. 20
Figure 11 Structures of peptidomimetics .................................................................................. 21
Figure 12 -Hairpin inhibitor disrupting the HDM2-P53 interaction ......................................... 23
Figure 13 The template of -hairpin antibiotic L27-11 ............................................................. 24
v
Figure 14 The template of -hairpin antibiotic POL7080 .......................................................... 24
Figure 15 Structure of CXCR4 antagonist (POL 3026) ............................................................. 26
Figure 16 Synthesis of small peptide ........................................................................................ 28
Figure 17 The key process of solid-phase peptide synthesis ...................................................... 32
Figure 18 Merrifield resin ........................................................................................................ 33
Figure 19 Types of resin ........................................................................................................... 33
Figure 20 Two racemiztion mechanisms ................................................................................... 36
Figure 21 Structures of the Cbz and substituted Cbz groups ..................................................... 36
Figure 22 Amino moiety protecting groups .............................................................................. 38
Figure 23 C-terminus protecting groups ................................................................................... 39
Figure 24 Structures of carbodiimides ...................................................................................... 41
Figure 25 Two possible products via a mixed anhydride ........................................................... 42
Figure 26 Structures of the active esters ................................................................................... 44
Figure 27 Structures of Phosphonium, uronium, and guanidium salts ....................................... 46
Figure 28 Native chemical ligation for peptide synthesis .......................................................... 50
Figure 29 structures of peptide and peptoid .............................................................................. 50
Figure 30 The solid-phase submonomer method ....................................................................... 51
Figure 31 Peptoid secondary structure inducing side chains ...................................................... 51
Figure 32 Proposed model of intrahelix stabilizing hydeogen bond between ortho- and
para-substituted Nspe analogues (X=Cl, OCH3, etc.) ................................................ 52
vi
Figure 33 Peptoid hexamers 3, 4, and 5 .................................................................................... 54
Figure 34 A negative feedback loop between p53 and MDM2 .................................................. 97
Figure 35 Structure of HYD-1 ................................................................................................ 124
Figure 36 -Hairpin structure ................................................................................................. 125
Figure 37 Structure of MTI-101 ............................................................................................. 126
Figure 38 Peptoid-peptide hybrids with bioactivity ................................................................. 161
Figure 39 Structures of three sets of acyclic and cyclic peptoid-peptide hybrids ..................... 164
Figure 40 Structures of acyclic and cyclic peptoid-peptide hybrids 1 ...................................... 167
Figure 41 Structures of acyclic and cyclic peptoid-peptide hybrids 2 ...................................... 169
Figure 42 Structures of acyclic and cyclic peptoid-peptide hybrids 3 ...................................... 171
vii
LIST OF SCHEMES
Scheme 1 General synthesis procedure of terpyrimidyl derivatives ........................................... 99
Scheme 2 Synthesis of 1a-1j. ................................................................................................... 100
Scheme 3 General synthesis of terpyrimidyl dimers ............................................................... 101
Scheme 4 General synthesis of trimeric oligo-pyrimidine derivatives ..................................... 103
Scheme 5 Synthesis of 8-10 .................................................................................................... 104
Scheme 6 Synthesis of -turn promoter .................................................................................. 128
Scheme 7 Recognition strand synthesis of MTI-101 ............................................................... 129
Scheme 8 Synthesis of linker .................................................................................................. 130
Scheme 9 Non-recognition strand synthesis of MTI-101 ........................................................ 130
Scheme 10 Cyclization between recognition strand and non-recognition strand ...................... 131
Scheme 11 Synthesis of -turn promoter ................................................................................ 132
Scheme 12 Continuous solution-phase synthesis of MTI-101 ................................................. 134
Scheme 13 General procedure for peptoid-peptide hybrids in SPPS .................................. 166
viii
LIST OF ABBREVIATIONS
A = Amyloid beta
Boc = tert-Butyloxycarbonyl
Cbz = Carbobenzyloxy
CD = Circular dichoism
DBU = 1,8-Diazabicyclo[5.4.0]unec-7-ene
DCC = N, N´-Dicyclohexhexylcarbodiimide
DCM = Dichloromethane
DIC = N, N´-Diisopropylcarbodiimide
DIEA = N, N´-Diisopropylethylamine
DNA = Deoxyribonucleic acid
Fmoc = 9-Fluorenylmethoxycarbonyl
gB = Glycoprotein B
HCTU = N, N, N´, N´-Tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium
hexafluorophosphate
HDM2 = Human homologue of murine double minute 2
HF = Hydrofluoric acid
HOBt = 1-Hydroxybenzotriazole
HOSu = 1-Hydroxypyrrolidine 2,5-dione
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HPLC = High-performance liquid chromatography
MDM2 = Murine double minute 2
MM = Mutiple myeloma
NMR = Nuclear magnetic resonance
Nsch = N-(S)-(1-cyclohexylethyl)glycine
Nsna = N-(S)-(1-Naphthalenethyl)glycine
Nspe = N-(S)-(1-phenylethyl)glycine
Nrch = N-(R)-(1-cyclohexylethyl)glycine
PPIs = Protein-protein interactions
RNA = Ribonucleic acid
ROS = Reactive oxygen species
ROSEY = Rotating-frame overhauser effect spectroscopy
SN2 = Nucleophilic substitution bimolecular
SPPS = Solid-phase peptide synthesis
TEA = Triethylamine (Et3N)
TLC = Thin layer chromatography
TFA = Trifluoroacetic acid
THF = Tetrahydrofuran
T3P = 2, 4, 6-Tripropyl-1, 3, 5, 2, 4, 6-trioxatriphophorinane-2, 4, 6-trioxide
x
ABSTRACT
The protein-protein interactions (PPIs) occur when two or more proteins are bound
together. Also, this protein-protein interactions (PPIs) cause the various biological
processes in the body. Due to this reason, abilities of controlling or inhibiting PPIs can
give us promising advantages like (1) better understanding of biological systems, (2)
development of new diagnostic approaches for health or disease, and (3) establishment of
novel molecular therapeutics. Many proteins adopt the secondary structures, where most of
protein-protein interactions take place. -Helices and -sheets are the prevalent secondary
conformations, but there are extended secondary structures such as -hairpins, -turns, 310
helix, and so on. As a result, construction of molecules mimicking these protein
secondary structures is tractable target for drug design.
Moreover, in drug discovery, designing peptidomimetics or non-peptidic mimetics is a
popular strategy instead using peptides or truncated peptides because peptides or truncated
peptides are prone to proteolysis and degraded in the body. Also, peptidomimetics and
non-peptidic mimetics have not only the similar topology as peptides but also resistance
to proteolysis. Due to these advantages, in this study, peptidomimetics or non-peptidic
mimetics were synthesized and tested for different targets: (1) synthesis of non-peptidic
-helical mimetics for p53-MDM2 inhibition, (2) solution-phase synthesis of -hairpin
xi
peptide for the inhibition of multiple myeloma cells (MM) adhesion, and (3) synthesis of
-hairpin peptoid-peptide hybrids.
The synthesis in all three different studies was succeeded, but they still need some
improvements. For instance, non-peptidic -helical mimetics, terpyrimidyl derivatives,
were synthesized successfully, but they did not show any bioactivity against p53-MDM2.
Also, they have a solubility problem. Based on these results, it is necessary to improve
the pharmacokinetic properties and bioactivity by changing the substituents on the rings
or structures.
The -hairpin peptide for the second case already showed good bioactivity against
multiple myeloma (MM). For the next level of bio-study, the considerable amount of a
-hairpin peptide was demanded. In order to make the substantial -hairpin peptide, the
solution phase peptide synthesis was chosen instead of the solid phase peptide synthesis
because of the cost-effect. Two methodology were tried for the solution-phase peptide
synthesis: (1) segment ligation and (2) continuous synthesis. In the former case, the -
hairpin peptide synthesis was successful, but, in the latter case, it is necessary to
investigate the appropriate coupling reagents for each step.
Peptoid-peptide hybrids has been one of the popular peptidomimetics in the last two
decades. Also, mimicking the peptide secondary structure in peptoids has been studied
extensively these days. The combination of these two factors was the goal for the third
case. Because peptoid-peptide hybrids with a secondary structure can be recognizable by
native proteins and resistant to proteolysis. So far, three sets of peptoid-peptide hybrids
were synthesize and checked the secondary structure formation by using NMR. However,
there was no indication of the secondary structure formation in the three sets of peptoid-
xii
peptide hybrids. This result suggests that it is necessary to introduce the more
constrained components in peptoid-peptide hybrids.
In the above three chapters, it has been tried to find the new drug candidates by
synthesizing peptidomimetics or non-peptidic mimetics. Even though the synthesis was
successful, some intended results such as the bioactivity or the secondary structure
formation were not obtained. However, these results can give us the inspirations to
improve properties of peptidomimetics or non-peptidic mimetics for a certain purpose,
which leads to earn the intended results and eventually find new drug candidates.
1
CHAPTER ONE:
INTRODUCTION
1.1. Amino acids, peptides, and proteins
Proteins are the most abundant biological molecules in every cell. Proteins also have
diversity, not only thousands of different kinds, but also huge differences in size.
Moreover, their biological functions in the cells are the most important since their
outcomes have an effect on the biological pathways and genetic expressions. Despite
their key functions, combinations of relatively simple monomer subunits is the
fundamental element of protein structure. Proteins are built from a set of 20 amino acids
are covalently linked in linear sequences. Every amino acid is a -amino acid which has
carboxylic acid, amino group, and its side chain. The presence of carboxylic acid and
amino group makes amino acids act as acids and bases. Also, side chain of amino acids
gives different identity to each amino acid and side chains occur in a variety of
structure, size, and electric charge. Two amino acids can be covalently linked through a
peptide bond to obtain dipeptide. The peptide bond is formed by removal of water from
carboxylic acid of one amino acid and amino group of another amino acid. In a similar
way, tripeptide, tetra peptide, and so on are formed from amino acids. When a few
amino acids are involved in this fashion, the structure is termed as an oligopeptide.
When many peptides are involved, the product is termed as a polypeptides which has
2
molecular weight below 10,000. Proteins contain thousands of amino acid residues and
have larger molecular weights (1).
1.2. Protein structures
The primary structure of a protein is the linkage of individual amino acids which are
joined covalently through peptide bonds. The peptide bonds in protein is planar as a
result of resonance structure (Figure 1). This prevents the peptide bond rotation and gives
significant double bond character. Even though there are two possible configurations for
the peptide bond, trans configuration is frequently found out in proteins due to benefit
from steric hindrance.
The secondary structure of protein consists of the ordered regions adopted by
polypeptide chains. The secondary structure is normally about folding patterns of
polypeptide backbone. Moreover, most of proteins contain secondary structure regions.
There are three common types of secondary structures: the -helix, the -pleated sheet,
and the -turn.
The tertiary structure of proteins is the three-dimensional layout of all atoms. As the
length of a polypeptide gets longer, it will fold to be more ordered and stable. Because
amino acids which are apart can interact within folded protein structure through weak
bonding interactions or covalent bonds.
Some proteins such as hemoglobin consist of two or more subunits which may be
same or different types. Only proteins which contain more than two subunits are able to
have quaternary structures. The quaternary structure of proteins refers to the arrangement
of these subunits in three-dimensional complexes (1,2).
3
Figure 1. The resonance structure of peptide bond.
1.3. -helix mimetics in drug discovery
The -helix was first characterized by Linus Pauling in 1951 (3). A typical -helix
has 3.6 amino acids per turn, a rise 5.4 Å /turn, and backbone dihedral angles of = -50
and = -60 . The helix can be considered to have three distinct faces because the side
chains from certain residues are overlapped vertically every 3-4 residues. In other words,
side chains from helical residues at the position i, i + 3, i + 4, and i + 7 are positioned
along the same face of -helix. In -helix formation, there is a large entropy cost, but
this can be cancelled out due to intramolecular hydrogen bonds between the carbonyl
oxygen at the i position and the carboxamide hydrogen at the i + 4 position (4). The
principal character of -helix is the conformational hindrance of the side chains and
steric restriction. In numerous proteins, -helices are the bioactive regions which mediate
protein-protein interactions along with protein-DNA and protein-RNA interactions (5).
The protein-protein interactions (PPIs) plays a key role in aspects of biological processes.
Due to this reason, abilities of controlling or inhibiting PPIs can give us promising
advantages like (1) better understanding of biological systems, (2) development of new
diagnostic approaches for health or disease, and (3) establishment of novel molecular
therapeutics. Over 30% protein secondary conformation adopts -helices, allowing it the
most abundant secondary structure in nature. As a result, a number of PPIs are involved
in -helices and mimicking -helical templates is tractable target for drug design (6, 7).
4
Azzarito and co-workers classified three primary design strategies on the review paper
based on helix-mediated interactions and structural features (8): (1) type I mimetics which
are short oligomers and copy the local contour of -helical structural motif (9), (2) type
II mimetics which are small non-peptidic molecules but not always imitation of the -
helix structure of protein receptors, and (3) type III mimetics which are non-peptide
scaffolds and only interact with key residues of peptide receptors not the whole -helical
conformation (10, 11).
Type I mimetics are the derivatives of peptide backbone to replicate the local region
of -helical conformation when protein-protein interactions occur. Peptides are not
therapeutically appropriate forms because of their poor transport properties and easy
proteolytic degradation (12). However, due to their complex structures, small molecules
are not able to mimic completely their functions during the biological process (13).
Moreover, there is a study to show that synthetic peptides are not organized in solution
and adopt random conformation which delay the binding to the partner proteins (14).
Despite of these limitations, there have been several approaches for oligopeptides to
maximize their -helical properties and disrupt protein-protein interactions.
The first strategy is stabilization -helix conformation of short peptides by using salt
bridges, metal chelates, and covalent cyclization (Figure 2). Aldert and Hamilton studied
the restriction of helical structure of short peptides through hydrophobic interactions. This
can be accomplished by incorporating two -(3,5-dinitrobenzoyl)Lys residues at various
positions. They found that the helical stability is the highest for the peptide when a pair
of modified residues are positioned at the i and i + 4 (15). Scholtz and co-workers
showed Glu-Lys interactions at the position (i, i + 3) and (i, i + 4) stabilize -helical
5
structures regardless of orientation of the side chains (16). Tsou and co-workers found
-0.4 kcal/mole energy decrease for -helical peptide through Phe-Lys cation- interaction
(17).
The Mierke and Spatola groups have demonstrated the inhibition of disulfide-bridged
nonapeptide to estrogen receptor /co-activator interaction. Even though circular dichroism
spectra of the peptide indicated the minimal propensity of -helix in aqueous condition,
X-ray crystal structure confirmed disulfide-bridged peptide experienced the receptor-
induced conformational change and bound to receptor with -helix conformation (18, 19,
20). Rosenblatt and co-workers reported a parathyroid-hormone-related protein (PTHrP)
analogue (21) and Fairlie and co-workers demonstrated short -helical peptides (22). Both
of approaches utilized lactam link between lysine and aspartic acid in the i and i + 4
positions to stabilize -helical structure.
Despite of the stabilization of -helices through disulfide or lactam links, these
natively occurring components may be prone to cellular degradation. To overcome this
disadvantage, Grubbs group showed carbon-carbon bond restraint initially via a ring-
closing metathesis reaction from unnatural O-ally serine residues (hydrocarbon stapling)
(23). Arora and co-workers also exploited ring-closing metathesis to olefin bearing
residues at the i and i + 4 positions (hydrogen-bonding surrogates) (24). However, the
second approach is more attractive because the constraining element after ring-closing
metathesis does not block any faces of -helix conformation unlike the result of first
approach (Figure 3).
Even though all the above strategies have developed the promising peptide-based
inhibitors with -helix conformation, it is impossible to control the activity of these
6
restrained peptides. Alleman group established that external stimuli can govern the
protein-binding activity in a reversible way. This can be achieved by cis/trans
isomerization of azobenzene under radiation exposure. An azobenzene bridge was places
through cystein residues at the i, i + 4, i + 7 or i + 11 positions and this bridge makes
the peptides switch between random coil-like and -helical conformations under photon
emission (25).
Another approach for generation of type I mimetics is the use of foldamers. Gellman
and co-workers design -peptides by adding methylene unit to -amino acids to cure
human cytomegalovirus (HCMV, alternatively know as herpesvirus-5) infection. They
identified a 12-helical inhibitor derived from the HCMV protein gB with an IC50 of 30
M (26). The -peptides have not only better resistance to proteolysis but also more
favorable pharmacodynamic properties. This is because an additional degree of freedom
form extra methylene group makes -peptides more flexible and suitable for folding into
more defined secondary structures than -peptides (27). There are two well-known
secondary structures in -peptides, the 14-haelix and the 12-helix (named after the
number of backbone atoms per hydrogen-bond ring) (28). The group of Schepartz
showed 3-homodecamer 14-helical peptide originated from p53 with slightly lower
affinity than that of the original peptide to hDM2 (2.5-fold lower) (29). Other than -
peptide design, mixed /-peptides were used to mimic the -helical conformation.
Gellman and co-workers designed -foldamers for inhibition of anti-apoptotic and pro-
apoptotic members of the Bcl-2 family and gp41 (30, 31, 32, 33, 34, 35, 36). One of
examples is the development of gp41 inhibitors (35). The form of repetition was
prepared through the insertion of 3-residues into the -sequence of the C-terminal
7
heptads repeat domain (CHR) of gp41. Later, recurrent 3-residues were replaced by
constrained -residues to compensate the entropic penalty originated from the pre-
organization of more flexible analogues. This approach produced the structural mimetics
of 10 turns of an -helical CHR domain and the most potent peptide with a Ki of 9 nM
improved the ability of inhibition more (~380-fold) than that of the acyclic -peptide
analogue (35). Afterwards, the Gellman group transformed cyclic constraints to the ion
pairings on i, i + 3, and/or i, i + 4 positions to stabilize helical structures (36).
Type II mimetics are the small non-peptidic molecules which bind to the protein
receptors. However, molecules of this class are more like functional mimetics, not
necessarily mimicry of the original helix structure. Even though it is difficult to identify
inhibitors in this class due to different occupation or layout from traditional drug
molecules, there have been several discoveries of compelling inhibitors of PPIs (37-45)
(Figure 4).
One of examples is the finding of Nutlins, a family of tetra-substituted imidazoles
after a high-throughput screen. These compounds were optimized to inhibit p53-murine
double minute 2 (p53-MDM2) interactions and the most effective compound, Nutlin-3
showed IC50 value of 90 nM (37). Shaomeng Wang and co-workers demonstrated potent
inhibitors based on a spirooxindole template after virtual screening. They found
compounds from this family selectively bound to MDM2 protein (10,000-fold) over
mDMX (murind double minute X) as well as MI-219 is the most potent inhibitor (Ki =
5 nM) among this pool of compounds (39).
8
Figure 2. Examples of helical stabilization: - interaction (1), salt-bridge (2), cation-
interaction (3), disulfide (4), lactam (5), azobenzene (6), olefin (7).
Figure 3. Schematics of hydrocarbon stapling and hydrogen-bonding surrogates.
Abbott laboratories discovered the inhibitors of the Bcl-xL/Bak via fragment-based SAR
(structure-activity relationship), a modern drug discovery tool. Despite of the highest
affinity of ABT-737 (Ki = 0.6 nM) for Bcl-xL among all the derivatives (42), ABT-737
was not suitable for clinical use due to poor pharmacokinetic properties. Improvement of
oral availability resulted in the discovery of ABT-283 with the comparable affinity (Ki
1 nM) as that of ABT-737 (43). In a recent reports, optimized analogues of this inhibitor
9
showed suppression of cell growth in cancer cell lines with IC50 values of 60 - 90 nM
(44, 45)
Figure 4. Examples of type II mimetics.
Type III mimetics are the non-peptidic scaffolds which mimic the spatial orientation of
the critical recognition residues from the original helix instead of recapping the helical
structure like type I mimetics (46). Compounds of this class have not only a simple rod
shaped-like pharmacophore with side chains oriented in a similar way to that of a
native -helix but also the comparable protein-protein interaction areas unlike that of
type II mimetics.
The groups of Rees and Willems pioneered this concept by building trisubstituted
indanes as inhibitors of tachykinnin receptors (47, 48). However, the first true -helix
mimetic was built by the Hamilton group (49). They showed the synthesis and
conformational analysis of trisubstituted 3,2´,2´´-terphenyl derivatives via alternating alkyl
or aryl groups on ortho-positions of the aryl cores. The side chains on ortho-position
were projected in a similar way of i, i + 4, and i + 7 positions of the -helix. Analogues
10
of this family were shown inhibition to calmodulin / phosphodiesterase, Bcl-xL / Bak, and
gp41 interactions with IC50 values of nanomolar ranges (49 - 51).
Despite the potent inhibition results, derivatives of this series have a poor polarity.
In order to overcome this disadvantage, Hamilton and co-workers reported the
terephthalamide and 4,4-dicarboxamide templates (Figure 5a, b) which have easier
synthesis procedure and more drug-like property (52, 53). The inhibition results of these
derivatives against Bcl-xL / Bak interaction were close or lower than the terphenyl family
(Ki = 0.78 M 5a1, 1.8 M 5b2, and 0.11 M terphenyl).
Figure 5. Examples of type III mimetics.
The group of Rebek demonstrated a series of oxazole-pyridazine-piperazines with
hydrophobic side chains to mimic the i, i + 4, and i + 7 positions of the helix (Figure
5c). The incorporation of heterocyclic rings increased the hydrophilicity and this surface
would project to the solvent exposed area. This simultaneously allowed surface of
hydrophobic side chains to turn toward recognition area of protein receptor (54). In a
similar concept of pre-rigidity conformation, Hamilton and co-workers reported a
trispyridylamide scaffold by using aromatic oligoamides which are easy to synthesize via
11
amide bond formation (Figure 5d) (55). X-ray crystallography study confirmed this
template showed pre-organized conformation because of hydrogen-bonding among the NH
group of the amides, the ortho-alkoxy of pyridyl rings, and nitrogen on pyridyl rings.
This made side chains of the scaffold project to the same face as i, i + 4, and i + 7
positions of the helix (56).
Although structural rigidity is helpful to stabilize -helix conformation, a degree of
flexibility can improve interactions with the target protein via 'induced fit'. Based on
Hamilton`s original pyridyl carboxamide derivatives, several groups demonstrated a
oligobenzamide scaffold by replacing pyridine rings to benzene rings, which potentially
decrease the number of hydrogen-bonds within this structure of mimetics (57 - 60).
Fletcher and co-workers reported distribution of pyridine and benzene rings can control
intramolecular hydrogen bonding and inhibition activity against Bak (Figure 5e) (61).
The Wilson group described the first solid-phase synthesis for -helix mimetics. They
built N-alkylated oligobenzamides via this method (Figure 5f) and tested against to p53 /
hDM2 interaction (62, 63). The development of this strategy can bring the diversity
libraries for -helix mimetics with easy and effective way.
Although there have been significant progress for design and development of -helix
mediated protein-protein inhibitors, it is still essential to understand -helix mediated
interactions and generate therapeutically relevant molecules with higher affinity and
selectivity to protein receptors.
12
1.4. -sheets, -turns, and -hairpins in drug discovery
Although tertiary structures of folded proteins are diverse, only limited secondary
structures are observed with long-range order: helices ( and 310) and sheets (parallel
and anti-parallel) (64). As a result, it is critical to study the characteristic propensity of
helix and sheet conformational stability in order to predict protein folding preferences.
The -helix has been examined extensively to determine variables for helix stability such
as the effects of sequence, side chain - side chain interactions, solvent, length, and other
factors. This was possible because of the development of model system which adopts
-sheet
structure and stability are less well-documented due to the difficulty of model system
development in aqueous solution (66, 67). There was a first discovery of modest -
hairpin model system in 1993. This resulted in the development of recent well-defined -
-secondary structures (68).
A polypeptide with lack of side chains (poly-glycine) adopts a fully extended
conformation or "zig - zag" conformation that every peptide bond is located in a common
plane and a repeat distance between Ci and Ci+2 is 7.4 Å (69). However, polypeptides
with non-glycyl residues relieve the steric tension by rotating slightly in both the N – C
and C - C ́ bonds. A repetition of this fold in a regular fashion results in -structure,
one of the most common structures in proteins. In -structure, N – H and C = O of the
backbone are aligned approximately perpendicular to the direction of the chain and
involved in inter-strand hydrogen bonding. There are two layouts depending on hydrogen
bonding arrangement between strands of polypeptide. When hydrogen bonding interaction
between N – H and C = O occurs collinear, -strands are arrayed in an anti-parallel
13
fashion. However, deviation from co-linearity of hydrogen bonding leads to parallel
arrangement of strands (Figure 6). The determinant of parallel or anti-parallel
arrangement is the side chain interactions between strands (70). The side chains of -
conformation are toward to right angles alternatively above and below the plane and this
characteristic alignment results in a -sheet. Also, manipulating polar or non-polar
residues provides an amphiphilic property.
Figure 6. Hydrogen bonding in parallel and anti-parallel of -sheet.
When -strand changes its overall direction, a few residues are involved in a chain
reversal. The chain reversal, known as -turn is formed with two corner residues (i + 2
and i + 3) and hydrogen bonding between i and i + 3 (Figure 7). This hydrogen bond
constrains the incoming and outgoing -strands to maintain the -sheet. The -turn was
14
found initially in silk proteins by Geddes and co-workers (71) and defined
stereochemically by Venkatachalam group (72).
Based on the stereochemistry and conformation of -turns, they can be classified into
seven different types: type I, type II, type I , type II ,́ type III, type VI, and IG1 (G1
-bulge type I) (73 - 81). Type I, most frequently occurring in native proteins has L-
residues on both i + 1 and i + 2 positions (76, 77). However, proline favors to be located
on i + 2 position. In type II turns, proline as well as L-residue is inclined to be found
on position i + 1. For the i +2 position, glycine, small polar L-residue, or D-residue is
found because of steric hindrance caused by a side chain in the L-configuration. As a
result, the differences between type I and type II is the location of proline due to the
tension on the angle from the cyclic side chain.
Figure 7. -Turn.
Type I ́ and type II ́ are the mirror images of type I and type II and they have the
opposite stereochemistry on the equivalent positions. For example, D-proline instead of L-
proline would appear frequently on i + 1 for type I ́ or on position i + 2 for type II .́
Also, if residues with different chirality take up the corresponding sequence position,
their energy level is comparable.
15
There is a unique turn feature of 310 helix (82, 83). It still maintains hydrogen bonding
between i and i + 3, but the incoming and outgoing strands are twisted seriously as
regards to each other. The turn in this feature is called a type III turn.
Although the peptide bond of polypeptide prefers trans-configuration because of benefits
from steric effect, it is also able to accommodate cis-configuration. The type VI turn is
one of the instances. The type VI turn has hydrogen bond (i and i + 3) with ability to
link two -strands, but mediating peptide bond between i + 2 and i + 3 adopts cis
conformations.
The last turn type, as known G1 bulge type I, has three corner residues instead of
typical two corner residues (Figure 8). This results in the hydrogen bond between i and
i + 4 and L conformation in the i + 3 position (84). Sibanda and Thorton reported that
-bulge over a
tighter two-residue turn (75). Normally, -bulge turn is less related to cross-strand
interactions (85) which are crucial for peptides and proteins folding pattern (86). This
propensity considers -bulge turn type as an element of negative design (87).
Figure 8. G1 -bulge type I turn.
16
The stereochemical type of side chains in -turns can be defined in a similar way
as R-group arrangements on cyclohexyl rings (73). When side chain exists in the same
plane as polypeptide backbone, it has equatorial orientation. However, side chains exists
out of plane as polypeptide backbone, it can be defined as axial. Depending on turn
type and amino acids configuration in the turn position, orientation of side chains can be
predicted (Table 1).
Table 1. Side Chain Orientation of Corner Residues in -Turns (71)
Turn Type Position i + 1a Position i + 2
a
L-Residue D-Residue L-Residue D-Residue
I eq ax (down) ax (up) eq
I ́ ax (up) eq eq ax (down)
II eq ax (down) eq ax (down)
II ́ ax (up) eq ax (up) eq
III intermed intermed intermed intermed
VI (cis peptide bond) eq eq ax (down) ax (down)
a Eq: equatorial; ax: axial; intermed: intermediate.
-hairpin motifs are consisted of -strands and -turns and structurally diverse because
of variations in the hairpin constraints, variations in sequences, variations in the loop size,
and variations in the appearance of -bulges in the -strands (Figure 9). Also, -hairpins
are occurring widely not only in native polypeptides and proteins but also bioactive
cyclic acids such as gramicidin S and peptide hormones oxytocin and vasopressin.
17
Figure 9. -Hairpin structure.
There have been several studies about stability factors of isolated -hairpins (88 - 95)
and they proposed two key elements: cross-strand interactions and interactions within turn
residues. Cross-strand interactions include aromatic-aromatic (96), electrostatic (97), or
hydrophobic packing interactions (88, 98). The one of important interactions within turn
residues is combination of type I -turn and a G1 bulge which appears to stabilize in
many peptide hairpins (99 - 103). Recently, it is found that strands length of five or
more resides are required for -hairpin stability (104). Despite of these discoveries, many
studies are still undergoing to determine the relative contribution of these factors to -
hairpin stability.
Santiveri et al compared the contributions of turn, strand, and cross-strand side chain -
side chain interactions via mutations on both loop and strand and residue swapping
within a strand (105). They found that side chain - side chain interactions across the
strand may be more important for the hairpin stability. This result is crucial because
statistical analyses of protein structure have not considered these effects.
Perez-Paya group reported favor for an aromatic versus aliphatic residue at the i + 3
position in a type I ́ turn (106). They used the sequence Tyr - Asn - Gly - X as a type I ́
-hairpin most stable despite of the
18
appearance of tyrosine on the cross-strand from X. This result does not follow a typical
-sheet character, but the author explained this with context dependence. In another study,
Balaram and co-workers studied aromatic side chain - side chain interactions in organic
solvent in order to examine their importance without hydrophobic driving force (107).
They found that aromatic interactions contribute to hairpin stability through weak
electrostatic effects instead of the hydrophobic effects.
Hughes and Waters investigated the role of lysine methylation in mediating protein –
protein interactions within a -hairpin system (108). Methylation of lysine in histone tails
causes the binding of chromodomain proteins and eventually regulates chromatin
condensation. This can be achieved by packing the methylated lysine within an aromatic
pocket. Based on this interaction, the interaction of a trimethylated lysine and tryptophan
within -hairpin was investigated to resolve the nature of this type of cation -
interaction (Figure 10a). They found that trimethylation of lysine improves the interaction
with tryptophan by approximately 0.7 kcal/mol and hairpin structure shows the compelling
thermal stability. This interaction can contribute to structural stability for hairpin loops
within designed proteins.
The groups of Cochran, Wu, and Eidenschink suggested tryptophan zipper motif to
stabilize -hairpin structure (109 - 111). This motif can resolve the disadvantage of a
disulfide bridge constraint. Even though using a disulfide bridge has been valuable to
select specific peptide loop structures for certain protein targets in phage-display
technology (112), this bridge is susceptible to be cleaved in vivo in the presence of a
free thiol group. Also, the disulfide link is not able to stabilize -hairpin structure due
to many degrees of rational freedom which cost the loss of other stabilizing interactions
19
(113). However, tryptophan zipper motifs induce stacking interactions between
cross-strand tryptophans at non-hydrogen bonding positions and make -hairpins stable
(Figure 10c).
Many groups have investigated two-residue turn sequences to stabilize -hairpin
structures. For example, Asn – Gly, D-Pro – Gly, Aib – Gly, D-Pro – L-Pro, and Aib – D-
Pro turns promote the formation of -hairpins because of their character to form type I ́
or type II ́ turn (114, 115). Even though three-residue turn is considered as an element
of negative design (87), there have been a few successful three-residue turn design
recently in a -hairpin structure. One of examples is G1 bulge in a ubiquitin-based
hairpin sequence that resulted in a non-natural strand registry (116). However, Balaram
and co-workers reported a three-residue turn which maintains native strand registry in
methanol (Figure 10b). This can be achieved by insertion of D-Ala after L-Pro in a two-
residue type II -turn, D-Pro - L-Pro that leads to a left-handed helical conformation
(117).
So far, the factors which are naturally occurring and stabilizing -hairpin structure
have been discussed. Recently, many groups have investigated the possibility of
incorporation of peptidomimetics into -sheet or -hairpin structures because these
artificial elements may improve the proteolytic resistance. Many groups have examined
all aspects of -hairpin structure including the turn, strands, backbone, hydrogen bonding,
side chain - side chain interactions, and chirality and found the tolerance of -hairpins
from the a wide range of alterations.
20
Figure 10. Structures of -hairpin sequences.
Bartlett and co-workers have suggested an amide isostere consisting of a 1,6-dehydro-
3(2H)pyridinone ring for templation of -strands, known as @-tide (118 - 120). This @-
tide provides not only rigid template but also a characteristic CD signal at 280 nm
(Figure 11a). This signal produced by vinylogous amide is sensitive to conformational
changes that acts as an indicator of sequence and side chain effects on -hairpin folding.
Based on the CD signal at 280 nm, the authors determined intrinsic stability of each
residue and contribution of the side chain - side chain interaction (120).
Another example of a template b-sheet is the introduction of a guanidine-pyrrole
moiety at the N terminus of a tripeptide (Figure 11b). Schmuk and colleagues found that
21
this peptidomimetic model was bound to a tetra peptide in water through electrostatic and
hydrogen bonding between the guanidinopyrrole and the terminal carboxylate of tetra
peptide (121). This study showed sequence selectivity regarding charge pairing along the
strands.
Figure 11. Structures of peptidomimetics.
Kelly and co-workers have showed the replacement of strand residues in Apeptide
with alkene functionality. They switched Cand the NH to alkene moiety and
investigated the effect of backbone hydrogen bonding in protein aggregation (Figure 11c).
Even though formation of spherical aggregates was observed, they found that alkene
isostres on the Phe 19 and Phe 20 in A peptide prevent amyloid formation (122).
Last example of incorporation of peptidomimetic to -hairpin structure is the utilization
of a photoswithchable azobenzene as the turn sequence (Figure 11d). Two groups studied
and found -hairpin structure was maintained in the presence of cis-azobenzene
configuration (123-125). However, depending on the difference in the strand sequences,
two groups showed different result: the system designed by Hilvert and co-workers was
22
shown aggregation in the trans- configuration, but the model made by Renner and
colleagues was not shown any sign of aggregation in either configuration.
As discussed in the previous topic, -helix, the design of protein epitope mimetics is
considered as an innovative approach for the development of protein - protein and
protein - nucleic acid interaction inhibitors. This is because molecular recognition of
proteins is normally occurring on the surface exposed secondary structure motifs such as
-hairpins and -helices. There have been many studies about the -hairpin structure,
that contributed to the development of -hairpin motifs which can mimic the partial
contour of the restrained macro cyclic molecules (126, 127). Also, using parallel synthetic
chemistry in solid-phase promotes the diversity of -hairpin mimetics as well as
optimization of their propensities (128).
The group of Fasan found that the distance between the Catoms of two residues i
and i + 2 along one strand of a -hairpin is comparable to the -helical side chains at
the i and i + 4 residues (129). In order to test this design against HDM2, a series of -
hairpins with different peptide sequences was prepared. After a surface Plasmon
resonance (SPR) assay, -hairpin with a sequence of Phe1, Trp3, and Leu4 showed
inhibition of p53 binding to HDM2 (IC50 = 125 8 M) (Figure 12). For further
investigation, Phe or Trp residue was replaced by Ala and it was found that -hairpin
lost the inhibition ability of p53 binding. This result indicated the significance of the
side - chain complementarily.
In many organisms, it is found that the large family of cationic host-defense peptide is
involved in anti-infective defense mechanism (130). These short cationic peptides
typically have 10-50 residues and a net positive charge of +2 to +10. They have
23
amphipathic properties, but different sequences and secondary structures. Due to their
participation in immune response, this family of peptides has been considered as a
potential pool for development of novel antibiotics.
Figure 12. -Hairpin inhibitor disrupting the HDM2-P53 interaction.
Among many cationic peptides, PG-1 was taken as a initial model for -hairpin
mimetic design (131). Because PG-1 not only adopts -hairpin structure via two cross-
strand disulfide bridges but also displays wide spectrum of antimicrobial activity in
micromolar range. However, due to its weak potency, it is necessary to modify
sequences or propensity of the peptide. Recently, a family of protein epitope mimetic
(PEM) based on PG-1 was demonstrated that show the improvement of their potency
including the lead compound L27-11 (Figure 13) which shows nanomolar range activity
(132). These PEM compounds have the stabilized -hairpin structure through a D-Pro-L-
Pro turn template and attract to the negatively charged bacterial surface because of their
positive charges in the strands. After discovery of L27-11, another optimization was
followed to improve drug-like properties such as the plasma stability, target stability, and
24
toxicology. This resulted in the discovery of compound POL7001 (Figure 14) and the a
clinical lead candidate POL7080 (132). The safety of POL7080 is under testing in
healthy humans in a phase I clinical study, but it is already considered as the first in a
new class of antibiotics against Gram-negative bacteria (133).
Figure 13. The template of -hairpin antibiotic L27-11.
Figure 14. The template of -hairpin antibiotic POL7080.
25
CXCR4 is characterized as the class-A family of G-protein coupled receptors (GPCRs)
and plays a role as a co-receptor for CD4-dependent HIV infection of human T-cells as
well as a helper in hematopoietic stem cells (HSCs) mobilization from the bone marrow
(BM) to peripheral blood. Since CXCR4 appears on the surface of the majority of HSCs
and interacts with its native ligand SDF-1 in BM, CXCR4 antagonist are considered as
promising novel therapeutic for the efficient mobilization and harvest peripheral blood
HSCs by interruption of the SDF-1/CXCR4 interaction (134) both in cancer therapy as
antimetastatic agents (135) and in inflammation and tissue repair (136).
Polyphemusin II isolated from the American horseshoe crab (Limulus polyphemus) has
-hairpin structure with 18-amino acid residues and shows inhibition to CXCR4. This
peptide was taken as a starting point and a few of PEM molecules were designed and
optimized in biological assays. For example, POL3026 (Figure 15), POL5551, and
POL6326 are highly potent and selective CXCR4 antagonists (127, 137). POL6326
showed very limited or no mobilization of tumor cells in the leukapheresis and has
already moved into a phase II clinical trial for the transplantation of autologous stem
cells in newly diagnosed multiple myeloma patients.
Also, based on the recent X-ray structure, the -hairpin is bound to CXCR4 through
residues in the inward-facing projecting walls of the seven transmembrane helical bundle,
a few extracellular loops, and the N-terminal segment (138). For the selective high
affinity, a network of polar, hydrogen bonding, and hydrophobic interactions is important
between the ligand and the receptor. According to the structural similarity between this
bound ligand and POL3026, the macro cyclic b-hairpin mimetic may interact with
CXCR4 in a fundamentally identical way.
26
Figure 15. Structure of CXCR4 antagonist (POL 3026).
The fundamental understanding of -hairpin structure and folding has been improved
with the discovery of well-defined -hairpin model systems. Based on this improvement,
protein epitope mimetics (PEM) with -hairpin structures have come to the light in drug
discovery. With some early success, this field is now well on its way to discovering
well-folded -hairpin structure with high therapeutic potency against diverse disease.
1.5. Solid-phase peptide synthesis
The first synthesis of peptides was tried by Theodor Curtius in 1882 (139). He made
a dipeptide, Bz-Gly-Gly, by mixing benzoyl chloride and the silver salt of glycine.
However, Emil Fisher was generally considered as the first scientist who demonstrated
the total chemical synthesis of proteins (140). In 1907, Fisher reported an
octadecapeptide comprising of fifteen glycine and three leucine residues (141). Another
improvement of peptide synthesis after Fischer`s era was built by Bergmann and Zervas
27
(142). They suggested a reversible protecting groups for the -amino groups in early
1930s. This discovery allowed to control the sequences in peptide synthesis as well as to
avoid the racemization of amino acid in the amide coupling. With the carbodiimide
coupling methods (143) along with protecting group strategies, small peptides were able
to be synthesized in solution-phase such as glutathione (144) and carnosine (145). The
group of du Vigneaud showed the synthesis of oxytocin in 1954 (Figure 16) (146).
Although the above approaches of peptides and proteins have been performed in
solution-phase, the majority of peptide synthesis have been accomplished in solid-phase
by using the solid support such as resin bead. This is because solid-phase synthesis has
several advantages in comparison with the solution-phase methods (147). First, since the
synthetic intermediate is always bound to the resin, the excess reagents or unbound by-
products can be washed away easily. This leads to minimize the handling losses and
avoid further purification. Second, the use of large excess of reagents can force the
reaction to the completion. As a result, the couplings are fast and close to quantitative.
Last, depending on cleavage conditions and appropriate anchor or linker groups, the
polymeric resins are occasionally able to be regenerated and recycled.
Solid phase peptide synthesis (SPPS) was introduced by Merrifield first in 1963 (148).
In SPPS, the C-terminal amino acid of the intended peptide is bound covalently to a
solid support. The elimination of N()-protecting group is followed in the attached
peptide and the next amino acid in N()-and side-chain protected, carboxyl-activated
form is added after washing the resin-bound amino acid (Figure 17). Once the expected
peptide bond is formed successfully, the excess reagents and by-products are removed via
filtration and washing. These steps are repeated until the desirable peptide is formed on
28
the resin. In the last step, every protecting group is eliminated in the presence of
hydrofluoric acid (HF) or trifluoroacetic acid (TFA). During this process, the linker
bound to the solid support covalently is also cleaved to give the crude peptide which
needs to be purified through diverse methods.
Figure 16. Synthesis of small peptide.
Although the synthesis of peptides containing about 40-50 amino acids is still limited
through the most optimized SPPS, the invention of SPPS has changed the history of
peptides and protein chemistry. Even the SPPS is extended its application for the
synthesis of small non-peptide compounds. Despite of its useful usage, a few of essential
conditions are required for the successful solid phase peptide synthesis (149).
The first requirement is cross-linked insoluble polymeric supports such as resin beads
are stable to the synthetic conditions. The initial resin form used by Merrifield was
29
polystyrene beads where the styrene is fractionally cross linked with 1% divinylbenzene
and the chloromethyl group acts as an anchor or a linker (Figure 18). The next amino
acid can be coupled to the linker through an ester group. This ester group is stable to
the reaction conditions during the peptide synthesis and cleaved by using harsh acidic
condition at the last step of the synthesis. One disadvantage of polystyrene beads is its
hydrophobicity. Because the growing peptide is hydrophilic, the growing peptide is not
solvated well and folds itself to form internal hydrogen bonds. This eventually prevents
the next amino acid from accessing to the end of growing peptide chains. To resolve
this problem, it is necessary to develop more polar solid supports such as Sheppard`s
polyamide resin. In addition, the conditions for the non-peptide synthesis are different
from those of the peptide synthesis. This demands for the development of new class of
resins like Tentagel resin consisting of 80 % polyethylene glycol implanted to cross-
linked polystyrene. Regardless which polymer resin is chosen for the synthesis, they have
to be swelling well and robust to the synthetic conditions for the better synthesis
efficiency.
The second requirement is about a linker or an anchor which is bound to the
polymer chain of the solid support. It should contain the reactive functional group to the
starting material in the intended synthesis and the resulting linker should be inert
throughout the whole synthetic process. However, it can be cleaved readily to give the
final molecule. Depending on the functional groups on the starting material or the final
product, different linkers are used. The Wang resin has the suitable linker for the
addition and release of carboxylic acids. An N-protected amino acid is bound to the
30
linker via an ester link and this link is robust to the coupling and deprotection steps
during the peptide synthesis.
Once the synthesis is completed, the final peptide is cleaved from the bead by using
trifluoroacetic acid (Figure 19a). However, starting materials with carboxylic acid are
bound to the Rink resin through an amide link. After all the aimed reactions are
finished, the final product is released with a primary amide group instead of the original
carboxylic acid under the TFA treatment (Figure 19b). If a starting material has the
primary or secondary alcohol group, a linker on a dihydropyran derivatized resin is
suitable. The alcohol group can be linked to the resin in the presence of pyridinium 4-
toluenesulfonate in dichloromethane and the cleavage of final product will be done with
the treatment of TFA (Figure 19c).
The last requirement is protecting functional groups, which are not involved in any
synthetic reactions during SPPS. The selection of appropriate protecting groups is very
crucial. Not only are protecting groups stable to the reaction conditions during the whole
synthetic process but also they can be removed efficiently under the mild conditions.
Regarding the peptide synthesis, two primary protecting group strategies are used. The
first strategy is the use of Boc/benzyl protecting pair useful for the Merrifield resin
(Figure 18). The Boc-N()-protected amino acid is attached to the growing peptide chain
in the synthesis and the Boc (tert-butyloxycarbonyl) group is deprotected with TFA to
make amino group available for the next coupling with another Boc-N()-protected
amino acid. Other functional groups on the amino acid residues also need to be
protected during the synthesis. However, this protecting group should be robust to TFA.
Normally, benzyl-type protecting groups are used because they are stable to TFA but
31
susceptible to hydrofluoric acid. In addition, the bond between the growing peptide chain
and the linker is affected by hydrofluoric acid. As a result, treatment with hydrofluoric
acid cleaves the final product as well as deprotects functional groups on the residues at
the last step. However, this strategy has one major disadvantage which is the use of
hydrofluoric acid. Because hydrofluoric acid is a corrosive chemical which might
jeopardize human health. To avoid the serious health risk, Fmoc/t-Bu strategy is used as
an alternative. In this case, N()-terminus of every amino acid involved in couplings to
the growing peptide chain is protected by 9-fluorenylmethoxycarbonyl (Fmoc) group
instead of Boc-group. Because Fmoc-group can be deprotected with a mild base such as
piperidine. Other functional groups on amino acid residues are protected by t-butyl group
which can be removed easily with TFA rather than hydrofluoric acid. Moreover, the
suitable resin (e.g. Wang resin) is chosen where the bond between linker and the
growing peptide is cleaved with TFA.
Protein synthesis has been challenging subject in organic chemistry, but it is a very
critical topic in synthetic biology. There have been numerous modifications and changes,
such as invention of SPPS for the generation of proteins. Despite of huge efforts, the
efficiency of protein synthesis is still relatively low. To resolve this problem, it is
necessary to develop more efficient and flexible peptide chemical synthesis which
contributes to the further study in biology and drug discovery.
32
Figure 17. The key process of solid-phase peptide synthesis.
1.6. Solution-phase peptide synthesis
As mentioned in the previous chapter, Curtius and Fisher are the first pioneers in
simple peptide chemical synthesis in early twentieth century. Since then, the chemistry
33
has been developed based on selection of protecting groups for amino acids, their
deprotection, and peptide bond formation. These days, two main peptide synthesis
approaches are applied. One is solution-phase peptide synthesis and the other is solid-
phase peptide synthesis that have the fundamentally same principles. In this chapter,
principles for the peptide synthesis in solution will be covered.
Figures 18, 19. Merrifield resin (a) and types of resins (b, c, d).
It is important to prepare optically pure products, but racemization can occur during
amino acid deprotection as well as amino acid activation steps. Two racemization
mechanisms were considered: (1) direct proton abstraction (150) and (2) racemization
through oxazol-5(4h)-ones (151, 152). In the former case, it is a very rare situation such
as the rapid racemization of phenylglycine derivatives. The strong base triggers the
racemization by abstracting the -proton on amino acid (Figure 20). This can be solved
with the application of suitable conditions like the use of a tertiary amine (153, 154). In
the latter case, the formation of oxazol-5(4H)-ones is involved in racemization mechanism
34
(151). When the carbonyl moiety of N-acyl amino acids (acyl = acetyl, benzoyl, piperidyl,
and so on) is activated, oxazol-5(4H)-ones is formed which gives chirally unstable
intermediates through tautomerization of the oxazol-5(4H)-ones (Figure 20). In order to
avoid the formation of oxazol-5(4H)-ones, urethane-protected amino acids (e.g.
benzyloxycarbonyl, tert-butyloxycarbony etc) has been used for the peptide chemical
synthesis because of their resistance to racemization during activation and coupling.
As mentioned earlier, suitable protecting groups on amino acids is critical to avoid
recamization. Not only that, they are also important to control the peptide formation.
N()-protected amino acid and C()-protected amino acid are used to control the peptide
bond formation. After peptide formation, protected dipeptide can be isolated, purified, and
characterized. For the next coupling, N()-protecting group is removed and another N()-
protected amino acid is coupled to this dipeptide. As a result, in order to obtain desired
peptide, appropriate protecting groups should be selected for the N()-amino, carboxyl
moiety, or side-chain functional groups. In the following chapters, different kinds of
protecting groups and their deprotection remedy are described in detail.
Bergmann and Zervas introduced Cbz-group (Figure 21) for the protection of amino
groups (155). Z-amino acid can be prepared via the reaction between benzyloxycarbonyl
chloride and amino acid under the Schotten-Bauman condition. This protecting group can
be removed by hydrogenolysis, reduction with sodium in liquid ammonia, liquid HF and
so on. However, hydrogenolysis (155) and acidolysis with HBr (156) are the most
conventionally used procedures. However, if a peptide chain accomdates sulfur-containing
amino acids such as Cys and Met, the hydrogenolysis of Cbz-group fails due to catalyst
poisoning. To overcome this problem many attempts were made, for example use an
35
additive (e.g. BaSO4 (157), boron trifluoride etherate (158)) or a proton donor (e.g. 1,4-
cyclohexadiene (159), ammonium formate (160), cyclohexene (161)). This was reported to
improve hydrogenolysis only for the Met-containing peptides. Kuromizu and Meienhofer
demostraned catalytic hydrogenolysis of Cbz-group from Cys-containing peptide in liquid
ammonia (162). This method was applied for the somatostatin synthesis (163). There are
a few protecting groups which structurally similar to Cbz-group, but more stable in
acidic condition (Figure 21). The incorporation of an electron-donating group on the
aromatic ring improves the acid liability of the Cbz-group. (164 - 170). These groups
can be removed by hydrogenolysis or mild acid.
McKay and Albertson was introduced Boc group (Figure 22) as another amino
protecting group (171). Di-tert-butyl bicarbonate is preferably used to prepare for Boc-
amino acids (172, 173) and the Boc group can be removed under mild acidic conditions
like TFA. During the Boc cleavage by acidic treatment, tert-butyl cation is formed which
causes many side reactions such as alkylation. To prevent the side reaction, anisole is
used as a cation scavenger.
When Lys(Boc), Glu(OtBu), and Asp(OtBu) as well as sulfur-containing amino acids
(Met and Cys) are involved in the peptide chain, both Cbz- and Boc-protecting groups
cannot be applied for -amino groups. In order to avoid the dilemma, triphenylmethyl
(trityl : Tri) group (174 - 176) can be employed as a -amino protecting group because
this group is able to be removed with milder acids such as dilute acetic acid or 1
equivalent hydrochloric acid (Figure 22).
36
Figure 20. Two racemiztion mechanisms.
Figure 21. Structures of the Cbz and substituted Cbz groups.
.
37
Protecting groups removed by strong bases are not suitable for the peptide synthesis
due to possibility of recamization. However, protecting groups removable under mild
basic conditions is very valuable because selective cleavage of Bpoc or Tri group is
especially difficult in the presence of Lys (Boc). Among diverse base-labile protecting
groups, the 9-fluorenylmethyloxycarbonyl (Fmoc) group (Figure 22) is the most well-
known protecting group in solid phase synthesis (177). Fmoc-amino acid can be
prepared by reaction between free amino acid and Fmoc-Cl, but a side product,
Fmoc-dipeptides, can be also formed in the course of this reaction (178). This
resulted in the development of several additional Fmoc reagents such as Fmoc-OSu
(179, 180), Fmoc-OPCP (179), and Fmoc-OBt (179). Deprotection of Fmoc group
occurs easily by various amines like piperidine (181, 182) via -elimination
mechanism. The cleavage of Fmoc group by piperidine leads formation of a fulvene-
piperidine adduct difficult to remove in reaction mixture in solution-phase synthesis.
In order to resolve the disadvantage, polymer-bonded amines (183 - 186) have been
use to remove Fmoc group in solution.
The allyloxycarbonyl (Alloc) group can also provide an effective N()-protectin
(187, 188). The Alloc group (Figure 22) can be removed in the presence of
palladium catalyst in neutral conditions and the tBu group and fluorine-9-ylmethyl
(Fm) groups are intact during the deprotection process. The 2,2,2-
trichloroethoxycarbonyl (Troc) group (Figure 22) is also one of very useful N()-
protecting groups (189 - 192) because it is stable in both acid and base presence. It
can be removed by mild conditions such as zinc dust in acetic acid.
As described earlier, protecting the C-terminus on amino acids is also important as
much as protecting the N-terminus due to controlling peptide bond formation.
Regarding C-terminal protection, there are major difference between solid-phase
38
synthesis and solution phase synthesis. In the former, the insoluble polymeric support
may act as a C-terminal protecting group, whereas in the latter, more typical
protecting groups are used. The most commonly used C-terminus protecting groups
are alkyl ester, aryl ester, hydrizides, or protected hydrazides.
Figure 22. Amino moiety protecting groups.
Methyl ester (-OMe) and ethyl ester (-OEt) (Figure 23) can be prepared by two
steps: (1) activation alcohol (e.g. methanol, ethanol) by thionyl chloride and (2)
reaction between a free amino acid and a previously activated species. Methyl and
ethyl ester can be removed by saponification and modified to different functional
groups for further couplings. Preparation of amino acid tBu esters (Figure 23) can be
proceeded by a couple of different methods (193 - 198). For example, reaction
between amino acids and isobutylene can provide amino acid tBu ester in the
presence of sulfuric acid as a catalyst (193). tBu esters are inert to base-catalyzed
hydrolysis, hydrogenolysis, and nucleophiles. However, they can be removed by
acidolysis with moderately strong acid such as TFA or solutions of HCl in organic
solvents. The 1-adamantyl esters of amino acids has the similar characters as tBu and
it can be also deblocked by TFA.
39
The benzyl ester (-OBzl) of amino acids is prepared by a couple of mild
conditioned methods (199 - 201). Cleavage of the benzyl ester (Figure 23) can be
proceeded through acidolysis with strong acid or hydrogenolysis (202). Amino acid
phenacyl ester (Figure 23) are obtained by the treatment of N-protected amino acid
carboxylates with bromoacetophenone (203). The phenacyl group is very stable to
acidic condition even to liquid HF. Due to this propensity, the phenacyl group is
used along with the Boc group as N-protection. The phenacyl group can be removed
with zinc in acetic acid (203) or sodium thiophenoxide in an inert solvent (204).
Peptidic hydrazides are prepared by reacting peptide methyl, ethyl, or benzyl esters
to hydrazine hydrates (205, 206). The hydrazide can act as a C-terminal protecting
group as well as maybe be transferred into the acyl azide with suitable treatments
(207, 208). However, if the protected peptide alkyl ester contains Arg(NO2),
Asp(OBzl), Asp(OtBu), or Glu(OBzl), treatment of hydrazine hydrates causes side
reactions like hydrazinolysis of side chain functional groups. In order to suppress this
side reactions, it is more suitable to synthesize protected peptide hydrazides from the
comparable substituted hydrazide.
Figure 23. C-terminus protecting groups.
40
Some native amino acids have a third functional groups. As a result, in the
synthesis of peptides, it is necessary to protect the -amino group of Lys (or -amino
group of Orn) in either carbonyl or amino component and the -mercapto group of
Cys in either component. However, this rule is not applied to some functional groups
on amino acids such as the carbonyl groups of Asp and Glu, the guanidine group of
Arg, the phenolic hydroxyl group of Tyr, the aliphahatic hydroxyl group of Ser and
Thr, the imidazole of His, the primary carboxamide of Asn and Gln, the aliphatic
thioether of Met, and the indole ring of Trp. The group of Okada demonstrated the
synthesis of eglin c containing 70 amino acid residues by following the minimum
protection method (209, 210). In contrast, Sakakibara recommended “solution synthesis
of peptides by the maximum protection procedure" via the DCC coupling method and
a HF final deprotection method (211). "Maximum protection" strategy represents
protecting maximum numbers of functional groups to avoid the side reactions. Despite
less occurrence of side reaction, it is challenging to build highly soluble segments
with full protection. Nakao and co-workers showed the synthesis of osteocalcin
containing 40 amino acid residues by the maximum protection approach (212).
There are two main strategies to construct peptide molecules in solution: (1)
stepwise elongation and (2) segment condensation. Even though their approaches are
different, the chemical methods used on both are fundamentally similar.
It is critical to prevent epimerization during the peptide synthesis. Stepwise
elongation from the C-terminal by one amino acid at a time using urethane-protected
amino acids such as Z-amino acids and Boc-amino acids is beneficial to avoid
epimerization while the peptide bond is dormed (212 - 214). The stepwise elongation
41
method is suitable for small peptide synthesis and preparation peptide segments to
build larger peptides and proteins.
In 1952, Khorana used DCC for nucleotide synthesis (215) and Sheehan and Hess
employed DCC for peptide synthesis in 1955 (216). Since then, DCC (Figure 24) has
been the most effective and attractive coupling reagent both in solid phase peptide
synthesis and solution phase peptide synthesis because of its high reactivity and high
efficiency within short time. The defect of the use of DCC is the production of the
insoluble dicyclohexylurea during activation/coupling. To overcome this disadvantage,
diisopropylcarbodiimide (DIPCDI) has been utilized and is comparably effective and
forms a more soluble urea by-product (216).
Figure 24. Structures of carbodiimides.
Another principle applied to stepwise elongation is mixed anhydride method which
was reported by Curtius over 100 years ago, but Wieland was the first to use this
approach for peptide synthesis (217). This method has an regioselective problem due
to the use a mixed anhydride, containing two carbonyl groups, formed from protected
amino acids or peptides and carboxylic acids. In order to obtain the desired product,
the amino component has to attack to the correct carbonyl group (Figure 25). As a
result, the success of this approach depends on the reduced the possibility of
undesired attack on the carbonyl group. This can be achieved by using sterically
hindered carboxylic acids or their analogues. The most effective coupling reagents for
42
this case is containing isovaleryl (218) or pivaloyl residues (219). Also, another
popular type of mixed anhydrides is formation of carbonic acids rather than
carboxylic acids as intermediates. Generally, ethyl chloroformate (220, 221) and
isobutyl chloroformate (222) are used. The advantage of this approach is easy
removal of by-products made, carbon dioxide and the corresponding alcohol, from the
reaction mixture.
Based on the principle of the mixed anhydride method, Wieland and colleagues
showed an amino acid thiophenyl ester (223), activated the ester moiety that has a
tendency to form the peptide bond under mild conditions (224). The fundamental
concept of this principle is making the carbonyl carbon more vulnerable to the amino
component attack by increasing the electron-withdrawing character of the alcohol
moiety of the ester. This can be achieved by using phenols with electron withdrawing
group at ortho or para positions such as p-nitrophenyl ester (223), 2,4,5-
trichlorophenyl ester (225), pentachlorophenyl ester (226), or penafluorophenyl ester
(227). Anderson group demonstrated N-hydroxysuccinimide (HOSu) ester as one
example of N-hydroxylamine type active ester (Figure 26) (228). The advantages of
the N-hydroxylamine derivative are the suppression of epimerization and the easy
removal with water. Another N-hydroxylamine analogue is N-hydroxybenzotriazole
(HOBt) which is widely used in peptide synthesis (Figure 26) (229, 230).
Figure 25. Two possible products via a mixed anhydride.
43
The group of Kenner first postulated the use of acylphosphonium salts as reactive
intermediates for the amide bond formation (231). However, this method was not used
widely until Castro developed (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) reagent (232). Since then, phosphonium salts has been
used widely in peptide chemistry. BOP (Figure 27) is easy to handle and promotes
fast coupling, but produces the toxic byproduct, hexamethylphosphorotriamide (HMPA).
In order to avoid the toxic byproduct formation, Castro replaced dimethylamine by
pyrrolidine and developed (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP) (Figure 27) (233, 234).
The related uronium salts have also been employed in solid phase peptide synthesis.
The use of uronium salts reduces the occurrence of side reaction such as
epimerization (235). The group of Dourtoglou introduced 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) reagent (Figure 27) (234, 236).
Subsequently, a few other uronium salts were developed. Knorr and coworkers
showed the 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(HBTU) (Figure 27) (235). Later, the aza derivative of HBTU, 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (Figure
27) was reported (237). For years, HBTU and HATU were considered as uronium
salts, but X-ray crystallography analysis confirmed their true structure as guanidium
salts (238).
The condensation of peptide segments is an attractive approach for the larger
peptides or proteins synthesis in solution. The separation of the peptide from
truncated or deleted sequences is relatively easy. The main problem of this method is
the epimerization of C-terminal residue of carboxylic segment except glycine or
proline. As a result, the most critical requirement for coupling is reducing
44
epimerization of the C-terminal amino acid of the carboxyl moiety. The appropriate
coupling methods to diminish epimerization in segment condensation are: (1) the azide
procedure, (2) DCC methodology using an acidic additive, and (3) native chemical
ligation (239).
Figure 26. Structures of the active esters.
The azide procedure is favorably applied when the C-terminus does not contain a
Gly or Pro residue. With certain precautions, the amount of epimerization can be
maintained at very low levels (240). Another advantage of this approach is minimum
protection principle can be employed because the azides do not acylate hydroxyl
groups. However, its major defect is the low efficiency of coupling which leads to
the rearrangement reaction to give carbamide peptides difficult to remove (208). The
group of Shioiri introduced diphenylphosphoryl azide (DPPA) as an acyl azide
forming reagent that constructs a peptide bond without any side chain functional
groups protection as well as produces a low level of the epimeric peptide (241 -
243).
It was found that the coupling of protected peptide segments experienced a
significant epimerization of the C-terminal amino acid when carbodiimides were used
45
alone (244). The group of Weygand reported the segment coupling with the
combination of DCC and HOSu dramatically reduced epimerization (less than 1%)
(245, 246) and Wuensch and colleagues employed this approach to the synthesis of
glucagon (247 - 249). Later, Koenig and Geiger demonstrated a more powerful
reagent than HOSu, as known as HOBt (230) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,4-
benzotriazine (HOOBt) (250, 251). The DCC/HOBt procedure was successfully applied
to the synthesis of human adrenocorticotropic hormone (ACTH) (252).
Native chemical ligation is the most useful method to produce long peptide chains.
Native chemical ligation is remarkably chemoselective reaction between unprotected
peptides containing a C-terminal thioester and N-terminal Cys residue under mild
conditions in aqueous solution (253) (Figure 28). After ligation, treatment of selective
desulfurization with H2/metal reagents converts Cys to Ala (254). Since Ala residue is
one of the common amino acids in proteins, ligation at Ala residues can be utilized
broadly.
The final step of peptide synthesis is removing all protecting groups. As mentioned
earlier, all protecting groups on -amino, carboxyl component or side chain of amino
acids have characteristic propensities. As a result, it is important to choose suitable
deprotecting reagents to remove every protecting group. The final deprotection
procedures are mainly: (1) catalytic hydrogenolysis with palladium (152, 255), (2)
sodium treatment in liquid ammonia (256 - 258), (3) TFA treatment (259, 260), (4)
HF treatment (261, 262), or (5) the "hard-soft acid-base" remedy (HSAB)" (263 -
270).
Solution phase peptide synthesis has been a classical approach in peptide synthesis.
Even though solid phase peptide synthesis has replaced in most labs, it is still useful
to produce peptide in large-scale for industrial purposes.
46
1. 7. Peptoids
There have been many cases that short peptide and nucleic acid oligomers with
great affinity to various molecular targets suffered from poor pharmacokinetic
properties. As a result, developing peptidomimetics close to poly peptide structure
with the chemical diversity, spacing of side chains, a polar backbone, and the
resistance to proteolysis is one of crucial tasks in drug discovery (271). In late 1980s,
a new class of peptidomimetics was introduced as known as pepoids where
connectivity of side chain point was moved from the alpha carbon to the amide
nitrogen (272). In other words, the resulting structures are repetition of N-substituted
glycine units, essentially regioisomer of peptides (Figure 29). This slight change of
structures leads to the loss of a hydrogen bond donor (the NH group) and chiral
center at the -carbon and the gain of flexibility. Also, it gave peptoids a defiance to
the proteolysis.
Figure 27. Structures of Phosphonium, uronium, and guanidium salts.
47
The initial approach of peptoid synthesis was Merrifield method of solid-phase
peptide synthesis (273) by using N-substituted monomer units. The monomer units of
peptoids have secondary amines, which is more hindered than the primary amines of
peptides, so their coupling reaction time is longer in SPPS. Also, preparation of the
monomer units is challenging because SPPS demands the considerable amount of N-
substituted glycine. In order to resolve these problems, submonomer method was
employed for peptoid synthesis. The submonomer concept was originally utilized by
Emil Fischer who conceived peptide synthesis by using ammonia to displace
haloalkylamides (274), but the group of Kent actually inspired the application of
submonomer method for the peptoid synthesis. They demonstrated the ligation and
cyclization of peptide fragments based on coupling nucleophiles to peptidic
bromoacetamides (275). Based on the suggestion, the idea of peptoid synthesis was to
grow the main chain by an acylation reaction with a haloacetic acid and then attach
the side chain with a primary amine (Figure 30). This approach avoids the
preparation of fully protected monomer units and uses smaller submonomer units
instead. The first step is acylation of a secondary amine, which can be slower as
compared to a primary amine in peptide synthesis. However, using an activating agent
such as DIC can promote the reaction speed. The second step is a simple SN2
reaction to incorporate the peptoid side chain functionality. The advantages of this
step are that primary amines have not only good reactivity but also commercially-
available diversity.
-helices and -sheets are the most prevalent secondary structures for proteins, but
various other secondary structures are also well-defined for proteins, including
alternative types of helices (-helix, -helix, 310 helix, polyproline-helix, and collagen
helix), extended structures (-sheets, -hairpins, and -bulges), and different kinds of
48
turns (-turn, -turn, -turn, and -turn) (276). These secondary structures are
normally characterized according to one of two ordinary methodologies: (1) hydrogen
bonding pattern (277) and (2) the values of the three bond angles of the peptide
backbone (, , and ) (278). In peptoids, it is impossible to define the secondary
structure based on the former criteria due to the lack of the amide hydrogen, but
assessing constrained backbone , , and angles and the side chain conformations
can contribute to the classification of peptoid secondary structures. The secondary
structure of peptoid is generally evaluated by circular dichroism (CD) spectroscopy
which enables rapid analysis relative to characterization of NMR. Moreover, due to
the flexible structure of peptoids, their crystallization has been very difficult.
Excessive CD studies has been established in order to correlate relationship between
CD spectra and bioactivity changes and evaluate the peptoid structures. However, it is
careful when comparing CD data for peptoids because different side chain
compositions, especially aromatic side chains, affect on the CD spectra shape.
In peptoids, the polyproline-type-I-like helix is the most commonly reproductive
secondary structure component by using the chiral and steric side chains, as already
predicted by computational considerations (279). There are two available structures of
this type of peptoid helix (280, 281) and their pitch differs by 10%. With the
absence of the regular backbone hydrogen bonding effect in peptoids, the variation of
the helical pitch may be attributed by the different side chain used (Nspc and Nrch)
(Figure 31). Since the length of the peptoid backbone is fixed, a difference in helical
pitch directly affect on varying numbers of residues/turn.
Despite of the original effect of sterically bulky side chains such as Nspe- and
Nsch- (Figure 31) on the structure generating properties, further studies reported
depending on side chains, n- * interactions can affect on the cis/trans amide ratios
49
(282, 283). For example, one side chain (Nsna, Figure 31) preferred the cis-amide
conformation, that likely results in more highly structured peptoid helices (282). In
contrast, incorporation of aniline in peptoid side chains forces cis/trans amide
equilibrium to over 90 % trans according to the measurement of NMR spectroscopy
(284). By using these side chains and a monomer capable of backbone-side chain
hydrogen bonding, the characteristic acyclic peptoid reverse turn have been generated
recently (285).
Besides backbone constraints, Nspe derivatives with para-substitutents can create the
contacts (e.g. disulfide bonds, salt bridges, and hydrogen bonds) which are able to
stabilize specific tertiary and quaternary structures (286). Alternatively, Nspe analogues
with ortho- and para-substitutents can stabilize the peptoid helix via intramolecular
i i+3 interactions. For instance, Wetzler and Barron proposed that the a pyridine
Nspe analogue on position i of a peptoid helix would be protonated at close to pH 7
because hydrogen bond acceptor on the para-position of a substituted Nspe at i+3
position could form hydrogen bonding with the protonated pyridine ring (276, Figure
32).
More recently, turn components have been revealed that can be achieved either by
macrocyclization (287) or by utilization of heterocyclic turn-inducing unit (288).
Kirshenbaum and colleagues showed head-to-tail macrocyclization of achiral peptoid
oligomers and found macrocyclic hexamer or octamer peptoids are alike as peptide -
turns (287). The configurations of the turn region in each macrocycle were cis and
the rest of amides were trans. The superposition of hexamer and octamer peptoids
are highly similar to each other in the turn regions as well as the original peptide -
turns. Appella and co-workers demonstrated a triazole monomer acting as a turn
mimic in 2007 (288). The triazole moiety is able to constrain the peptoid backbone
50
similar to a cis double bond that leads to a rigid turn in the peptoid structures. This
outcome can facilitate the design of biomimetic peptoids using turn motif stabilization.
Figure 28. Native chemical ligation for peptide synthesis.
Figure 29. structures of peptide and peptoid.
51
Figure 30. The solid-phase submonomer method.
Figure 31. Peptoid secondary structure inducing side chains.
Even though peptides and proteins are involved in important biological processes,
they have not been developed for clinical use due to their poor oral bioavailability,
short half-life in the body, and an immune response induction (289). These
disadvantages of peptides gave the great opportunity for the peptoid therapeutics
because peptoids are resistant to proteolysis and prepared in large scale with low cost
relative to -peptides. Also, as mentioned earlier, peptoids can adopt the folded
conformations such as helices (290), loops (291, 292), and turns (287, 288) in
peptoids. Many researchers have taken these advantages of peptoids and developed
diverse peptoid mimetics.
The group of Liskamp showed peptoid mimetics of peptide amylin which is
involved in the beginning of type II diabetes (293). Amylin generates amyloid fibrils
and easily aggregates in the insulin-forming islet -cells (294) through cross -sheet
topology (295). Peptoid 1 and retropeptoid 2 (residues in reverse order) as analogs of
52
the amylin core region (20-29) were synthesized and studied their structures and
inhibition of amylin aggregation (Table 2). In order to evaluate the structures of
amylin (20-29), peptoid 1, and retropeptoid 2, CD spectroscopy was used: amylin (20-
29) showed characteristic CD spectrum of -sheet, but peptoid 1 and retropeptoid 2
displayed weak or random CD spectra indicating no defined secondary structure.
Based on these CD spectra difference, the ability of the peptoids was studied whether
peptoids can inhibit -sheet and amyloid fibril formation in amylin (20-29). A 1:1
(w/w) mixture of amylin (20-29) and peptoid 1 did not show any unique CD
spectrum of -sheet, that support peptoid 1 was able to disrupt the -sheet formation
of amylin (20-29). Also, this mixture was ~20 % opaque in a comparison of that of
amylin (20-29) alone, that represents inhibition of amyloid aggregation. However, the
retropeptoid 12 showed moderate inhibition of aggregation by displaying ~50 %
turbidity in a mixture (w/w) of amylin (20-29) and retropeptoid. This may be
attributed from the supramolecular assemblies of retropeptoid 2 which interfered the
effective inhibition of aggregation.
Figure 32. Proposed model of intrahelix stabilizing hydrogen bond between ortho- and
para-substituted Nspe analogues (X=Cl, OCH3, etc.).
Kodadeck and colleagues found out three peptoids (3-5) bound to the coactivator
CREB-binding protein (CBP) in vitro with a low micromolar range among a library
53
of ~100,000 peptoid hexamers (296, 297). This coactivator protein plays a key role in
mammalian genes transcription. After further studies, among three peptoids, only
peptoid 3 was selective for CBP while peptoids 4 and 5 were selective for bovine
serum albumin (Figure 33). In other words, peptoid 4 and 5 failed to induce
transcription, but peptoid 3 managed to activate transcription by serving as an
activation domain (EC50 = 8 ). Moreover, the cell permeability of all three peptoids
was evaluated. The peptoid 3 showed good cell permeability, which correlated well to
its relative hydrophilicity. Surprisingly, peptoid 4 is most hydrophobic among three
peptoids, but its cell permeability was moderate. Although peptoid 5 has intermediate
hydrophobicity, it showed the poor cell permeability. Based on this permeability result,
the researchers explained the reason of the transcription induction failure by peptoid 5
due to no accumulation inside cells (Figure 33).
Peptoid mimetics play an important role in drug discovery because of the fast
synthesis and structural similarity to polypeptides. Many researchers already have
worked on the design and application of peptoid mimics of bioactive molecules and
will continue to pursue the same goal
Table 2. Structures of Amylin (20-29), Peptoid 1, and Retropeptoid 2289
Oligomer Sequence (N- to C-terminus)
Amylin (20-29) SNNFGAILSS
Peptoid 1 NSer-NAsn-NAsn-NPhe-Gly-NAla-NIle-NLeu-Nser-NSer
Retropeptoid 2 NSer-NSer-NLeu-Nile-NAla-Gly-NPhe-NAsn-NAsn-NSer
54
Figure 33. Peptoid hexamers 3, 4, and 5.
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96
CHAPTER TWO:
DESIGN OF NON-PEPTIDIC OLIGO-PYRIMIDINES
MIMICKING i, i+4(3), i+7 POSITIONS OF -HELICES
2. 1. Introduction
P53 is a tumor suppressor which is inactive or mutated in tumor cells (1). In
unstressed normal cells, p53 is expressed in low levels by MDM2 (murine double
minute protein 2) through two ways (2). P53 is a transcription factor inhibited by
MDM2. This can be achieved by binding to N-terminal domain of p53 and
preventing transcriptional activity. P53 can be also decreased via ubiquitin-dependent
proteasome pathway. MDM2 promotes ubiquitination of lysine residues of p53 C-
terminal region by acting as an ubiquitin E3 ligase. However, stresses such as DNA
damage, ultra violet light, and oncogenes induce the activation of wild type of p53
protein (3, 4). This results in the presence of target genes in different regulatory
regions. These target genes lead to various effects like inhibition of angiogenesis, cell
cycle arrest, or apoptosis (Fig 1). Hence, over expression of MDM2 makes wild-type
p53 disabled and tumor cells elongate their lives (5).
Due to the negative feedback between MDM2 and p53, MDM2 is an attractive
therapeutic target for anti-cancer treatment. Two initial approaches were validated as
cancer targets, MDM2,. MDM2 expression was inhibited by antisense oligonucleotides.
This results in strong activation of p53 and cell cycle arrest or apoptosis (6, 7). Also,
97
injecting a monoclonal antibody to MDM2 leads to activation of p53 and cell growth
arrest by disrupting interaction between p53 and MDM2 (8). These studies promote
development of antagonists of MDM2, leading p53 active.
Figure 34. A negative feedback loop between p53 and MDM2.
Prior to antagonists synthesis, it is important to check key interactions between p53
and MDM2. According to the crystallography, the region of MDM2 that binds to
NH2-terminal peptide of p53 showed characteristic interactions between these two
proteins. Two pairs of -helices from p53 and MDM2 form a hydrophobic cleft
where p53 binds like a pocket-ligand (9). Stabilization of p53-MDM2 complex is
achieved through interactions between hydrophobic cleft and hydrophobic residues
(Phe19, Trp23, Leu26) along with one-faced p53 helical peptide. Further experimental
(10, 11) and computational (12, 13) studies have confirmed the important binding
98
residues of p53-MDM2 complex and this leads to study the possibility that small
molecules can be an anti-cancer alternative by competing for MDM2 binding (14).
Several strategies have been studied to develop MDM2 antagonists. Peptides which
can inhibit MDM2 were discovered after phage display at the 100 nmol/L of IC50
level in vitro (15). A recent study showed an improved peptide which has better
binding affinity and p53 activation (15). A high-throughput screen of chemical
libraries has been used to identify Nutlins, a group of cis-imidazoline derivatives
which were able to inhibit p53-MDM2 binding with high affinity (16). This study has
promoted development of small molecules that act as p53-MDM2 inhibitors.
Recently, the group of Dr. Hamilton has showed terphenyl scaffold which is small
non-peptidic molecules and mimics of the NH2-terminal -helix of p53 (17). In order
to make -helix structure of terphenyl derivatives, side chains on the phenyl rings are
projected like i, i+4, and i+7 fashion. This mimic of structure can be achieved by
substituting appropriate alkyl or aryl groups on ortho-position of each phenyl ring.
These terphenyl analogues also showed inhibition of p53-MDM2 interaction in vitro
and p53 activation in cell culture. This study showed the possibility to develop a
novel -helical mimetics as MDM2 antagonists..
Here, terpyrimidine derivatives which have a -helical mimetic structure and better
polarity based on terphenyl scaffold.
2. 2. Results and discussion
From the previous study, terphenyl scaffold with mimic of -helical structure
showed inhibition p53-MDM2 interaction and activation p53. Despite of the potential,
these terphenyl derivatives have poor solubility or polarity. Not only improving this
disadvantage but also finding better potency, a new group of -helical mimetics of
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the NH2-terminal p53 -helix was developed by using pyrimidyl rings instead of
phenyl rings. The terpyrimidyl derivatives project their side chains in a similar
geometry of those of a-helices. Also, containing nitrogens on pyrimidyl rings results
in the increase of polarity (Scheme 1).
Scheme 1. General synthesis procedure of terpyrimidyl derivatives.
100
Initially, various R groups (e.g. -Ph, -NH2, -S-CH3, -S-CH2-C6H5) were used to
make terpyrimidyl derivatives. Every terpyrimidyl product was obtained as a
precipitate except when R is phenyl group. Due to easy purification, more
terpyrimidyl libraries were built for the case of NH2, S-CH3, S-CH2-C6H5 as R groups.
Monomeric pyrimidyl analogues (1a-1j) were prepared by Michael addition (Scheme
2; Table 1). Pyrimidyl monomers were obtained as a precipitate.
Scheme 2. Synthesis of 1a-1j.
Table 3. Percent Yield of Monomeric Pyrimidyl Derivatives Synthesis
Entry Compound R R1 Yield
1 1a S-CH3 iPr 51%
2 1b S-CH3 Ph 92%
3 1c S-CH3 iBu 13%
4 1d S-CH2-C6H5 1-Naphthalene 13%
5 1e NH2 iPr 29%
6 1f NH2 1-Naphthalene 90%
7 1g NH2 tBu 55%
101
8 1h NH2 iBu 21%
9 1i NH2 Ph 88%
10 1j NH2 Benzyl 50%
Dimeric pyrimidyl derivatives were synthesized from compounds, 1a-1j (Scheme 3).
Depending on which atom of hydroxylamine is involved in SN2 reaction, monomeric
pyrimidines (1) were transformed into mixture of 2 and 2´. Under controlled condition,
derivatives of 2 were only reduced to compounds 3. The Michael addition of 3 with
different Michael acceptors followed and afforded a novel group of terpyrimidyl
dimers, 4a-4j (Table 2). Dimeric terpyrimidyl analogues were formed as a precipitate.
Scheme 3. General synthesis of terpyrimidyl dimers.
Table 4. Percent Yield of Terpyrimidyl Dimers Synthesis
102
Entry Compound R R1 R2 Yield
1 4a S-CH3 iPr tBu 13%
2 4b S-C6H5 1-Naphthalene iBu 86%
3 4c NH2 iBu Benzyl 25%
4 4d NH2 Benzyl 1-Naphthalene 8%
5 4e NH2 iBu 1-Naphthalene 58%
Nucleophilic substitution reaction (SN2) of dimeric terpyrimidyl derivatives with
hydroxylamine hydrochloride afforded the mixture of 5 and 5´. Analogues of 5 were
transformed into compounds 6 when reduction reaction was carried out in methanol
using potassium formate, acetic anhydride, and 10% palladium/carbon as reducing
reagents. Addition reaction with Michael donors 6 in the presence of Michael
acceptors and triethylamine occurred and afforded trimeric oligo-pyrimidyl derivatives
7a-7d (Scheme 4). Oligo-pyrimidyl trimers 7a-7d were precipitated after reaction was
done (Table 3).
Terminal groups (-CN, and -NH2) of oligo-pyrimidyl analogues were transformed
into carboxylic acid derivatives in order to elevate their polarity and dissolve better in
aqueous solution (Scheme 5). Terminal cyano group was hydrolyzed in methanol
(reflux) using NaOH. Another terminal group -NH2 was involved in nucleophilic
substitution reaction (SN2) when succinic anhydride was employed in THF. Products
after hydrolysis or SN2 reaction were obtained as precipitation (Table 4).
In conclusion, terpyrimidyl compounds were synthesized successfully after SN2
reaction, reduction, and Michael addition. Despite of nitrogen atoms on pyrimidyl
103
rings, oligo-pyrimidyl derivatives have solubility problems in aqueous solution. Based
on this result, development of novel MDM2 antagonists based on different scaffolds
need modifications which are in a compliance with therapeutic properties.
Scheme 4. General synthesis of trimeric oligo-pyrimidine derivatives.
Table 5. Percent Yield of Terpyrimidine Trimers Analogues
Entry Compound R R1 R2 R3 Yield
1 7a NH2 iBu 1-
Naphthalene
iBu 57%
2 7b NH2 Benzyl 1-
Naphthalene
iBu 28%
3 7c NH2 iBu 1- iPr 51%
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Naphthalene
4 7d NH2 iBu Benzyl iBu 37%
Scheme 5. Synthesis of 8-10.
Table 6. Percent Yield of synthesis 8-10
Entry compound R R1 R2 R3 Yield
1 8 NH2 iBu Benzyl - 5%
2 9 - iBu Benzyl - 37%
3 10 NH2 iBu Benzyl iBu 14%
105
2. 3. Experimental section
2. 3. 1. General comments
The chemicals were purchased from Aldrich-Sigma and Acros. 1H (400 MHz) and
13C (100 MHz) NMR spectra were obtained in deuterated solvents using Inova-400.
For high resolution mass spectrometry (HRMS) spectra, Agilent 1100 series was used
in the ESI-TOF mode.
2. 3. 2. Synthesis of monomeric pyrimidyl derivatives, 1a-1j
4-isopropyl-2-(methylthio)pyrimidine-5-carbonitrile (1a)
A mixture of Michael acceptor (6.02 mmol, 1 g), NaOEt ( 6.02 mmole, 0.45g) and 2-
methyl-2-thiopseudourea sulfate (2.04 mmol, 1.67 g) in methanol (absolute, 200 proof,
10mL) was located under microwave conditions until the TLC indicated completion of
the reaction. The mixture was brought to room temperature and the formed precipitate
was filtered by vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 51%. 1H
NMR (CDCl3, 400 MHz): 1.32-1.34 (d, 6H, J = 0.02), 2.60 (s, 3H), 3.33-3.38 (m, 1H),
8.60 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 14.61, 21.15, 29.93, 159.83. HR-MS: m/z
= 194 [M+H]+.
2-(methylthio)-4-phenylpyrimidine-5-carbonitrile (1b)
A mixture of Michael acceptor (4.99 mmol, 1 g), triethylamine ( 6.48 mmole, 0.9 ml)
and 2-methyl-2-thiopseudourea sulfate (9.98 mmol, 1.39 g) in methanol (absolute, 200
proof, 10mL) was stirred under refluxing conditions until the TLC indicated
completion of the reaction. The mixture was brought to room temperature and the
formed precipitate was filtered by vacuum and rinsed with ice cold methanol (5 mL x
3). Yield: 92%. 1H NMR (CDCl3, 400 MHz): 2.66 (s, 3H), 7.54-7.59 (m, 3H), 8.09-8.11
106
(m, 2H), 8.78 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 14.75, 129.19, 132.41, 161.56.
HR-MS: m/z = 228.05 [M+H]+.
4-isobutyl-2-(methylthio)pyrimidine-5-carbonitrile (1c)
A mixture of Michael acceptor (11.1 mmol, 2 g), triethylamine ( 14.4 mmole, 2.0 ml)
and 2-methyl-2-thiopseudourea sulfate (11.1 mmol, 1.54 g) in methanol (absolute, 200
proof, 10mL) was stirred under refluxing conditions until the TLC indicated
completion of the reaction. The mixture was brought to room temperature and the
formed precipitate was filtered by vacuum and rinsed with ice cold methanol (5 mL x
3). Yield: 13%. 1H NMR (CDCl3, 400 MHz): 0.91-0.93 (d, 6H), 2.17 (m, 1H), 2.62-
2.64 (d, 2H), 3.49 (s, 3H), 8.25 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 8.87 29.95,
44.59, 45.77, 121.60. HR-MS: m/z = 208 [M+H]+.
2-(benzylthio)-4-(naphthalene-1-ylmethyl)pyrimidine-5-carbonitrile (1d)
A mixture of Michael acceptor (6.43 mmol, 1.70 g), triethylamine ( 9.64 mmole, 1.3 ml)
and 2-benzyl-2-thiopseudourea sulfate (9.64 mmol, 1.96 g) in methanol (absolute, 200
proof, 10mL) was stirred under refluxing conditions until the TLC indicated
completion of the reaction. The mixture was brought to room temperature and the
formed precipitate was filtered by vacuum and rinsed with ice cold methanol (5 mL x
3). Yield: 13%. 1H NMR (CDCl3, 400 MHz): 4.17 (s, 2H), 2.21 (s, 2H), 7.10-8.17 (m,
12H), 8.64 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 44.59, 45.58, 123.98, 125.53,
125.91, 126.28, 126.48, 127.40, 129.03, 134.94, 159.91, 160.16, 161.62, 162.53,
168.76, 172.27, 176.63. HR-MS: m/z = 383 [M+NH4]+.
107
2-amino-4-isopropylpyrimidine-5-carbonitrile (1e)
A mixture of guanidine hydrochloride (7.23 mmol, 0.69 g) and potassium carbonate
(7.23 mmol, 1 g) in ethanol (5 mL) was added Michael acceptor (4.82 mmol, 0.8 g)
suspended in ethanol (1 mL). The reaction mixture was placed on a microwave reactor
for 25 min at 125 ºC. A colorless crystal-like precipitate formed in the solution upon
cooling to rt. The precipitate was filtered and rinsed with ice cold ethanol (3 mL x 2)
to isolate compound. Yield: 29%. HR-MS: m/z = 163.09 [M+H]+.
2-amino-4-(naphthalene-1-ylmethyl)pyrimidine-5-carbonitrile (1f)
To a mixture of Michael acceptor (3.03 mmol, 0.8 g) and guanidine hydrochloride
(4.55 mmol, 0.43 g) in ethanol (absolute, 200 proof, 12 mL) was added triethylamine
(4.55 mmol, 0.6 ml). The mixture was stirred under refluxing conditions until the
TLC indicated the complete consumption of starting materials. The mixture was
brought to room temperature and the formed precipitate was filtered by vacuum and
rinsed with ice cold methanol (5 mL x 3). Yield: 90%. HR-MS: m/z = 261.11 [M+H]+.
2-amino-4-(tert-butyl)pyrimidine-5-carbonitrile (1g)
A mixture of Michael acceptor (5.56 mmol, 1 g), triethylamine ( 8.34 mmole, 1.2 ml)
and guanidine hydrochloride (8.34 mmol, 0,8 g) in methanol (absolute, 200 proof,
10mL) was stirred under refluxing conditions until the TLC indicated completion of
the reaction. The mixture was brought to room temperature and the formed
precipitate was filtered by vacuum and rinsed with ice cold methanol (5 mL x 3).
Yield: 55%. 13
C NMR (CDCl3, 400 MHz):
HR-MS: m/z = 177.11 [M+H]+.
108
2-amino-4-isobutylpyrimidine-5-carbonitrile (1h)
A mixture of guanidine hydrochloride (8.91 mmol, 0.85 g) and triethylamine (8.91
mmol, 1.2 ml) in ethanol (5 mL) was added Michael acceptor (5.94 mmol, 1.07 g)
suspended in ethanol (1 mL). The reaction mixture was placed on a microwave reactor
for 25 min at 125 ºC. A colorless crystal-like precipitate formed in the solution upon
cooling to rt. The precipitate was filtered and rinsed with ice cold ethanol (3 mL x 2)
to isolate compound. Yield: 20%. HR-MS: m/z = 177.11 [M+H]+.
2-amino-4-phenylpyrimidine-5-carbonitrile (1i)
Microwave-assisted reactions were also done in the presence of potassium carbonate
(6.00 mmole, 0.83g) to obtain the desired pyrimidines. Michael acceptor (4.00 mmol, 0.8
g) and guanidine hydrochloride (6.00 mmol, 0.57 g) was mixed in ethanol (5 mL). The
reaction mixture was placed on a microwave reactor for 40 min at 120 ºC. A colorless
crystal-like precipitate formed in the solution upon cooling to rt. The precipitate was
filtered and rinsed with ice cold ethanol (3 mL x 2) to isolate compound 1i as needle-
like crystals. Yield: 88%. 1H NMR (CDCl3, 400 MHz):6.89 (s, 2H), 7.42-7.60 (m, 5H),
7.87 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 128.30, 128.45, 131.76, 159.60. HR-MS:
m/z = 197.08 [M+H]+.
2-amino-4-benzylpyrimidine-5-carbonitrile (1j)
To a mixture of Michael acceptor (3.92 mmol, 0.8 g) and guanidine hydrochloride
(5.88 mmol, 0.56 g) in ethanol (absolute, 200 proof, 12 mL) was added triethylamine
(5.88 mmol, 0.8 ml). The mixture was stirred under refluxing conditions until the
TLC indicated the complete consumption of starting materials. The mixture was
109
brought to room temperature and the formed precipitate was filtered by vacuum and
rinsed with ice cold methanol (5 mL x 3). Yield: 50%. HR-MS: m/z = 211.09 [M+H]+.
2. 3. 3. Synthesis of dimeric pyrimidyl derivatives, 4a-4e
4-(tert-butyl)-4´-isobutyl-2´-(methylthio)-[2,5´-bipyrimidine]-5-carbonitrile (4a)
A mixture of compound 1a (2.07 mmol, 0.4 g) , hydroxylamine (5.18 mmol, 0.36 g) and
triethylamine (5.18 mmol, 0.7 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and evaporate every ethanol under the
reduced pressure. White solid are formed. With this solid 2 (0.36 mmol, 0.11 g), start
reduction reaction. Dissolve solid 2 with methanol and add acetic anhydride (0.47 mmol,
0.04 ml). After five minutes, add potassium formate (1.44 mmol, 0.12g) and
palladium/carbon (0.11 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 3 can be observed. With compound 3, start another coupling reaction.
Dissolve solid 3 with methanol and add acetic anhydride (0.47 mmol, 0.04 ml). After
five minutes, add potassium formate (1.44 mmol, 0.12g) and palladium/carbon (0.11 g).
Heat the reaction solution and keep stirring overnight. By using diatomaceous earth,
filter the reaction solution and evaporate every solvent. White compound 3 can be
observed. With compound 3, start another coupling reaction. A mixture of compound 3 and
2´ (1.90 mmol, 0.4 g) and Michael acceptor (2.85 mmol, 0.29 g) in ethanol (absolute, 200
proof, 10mL) was stirred under refluxing conditions until the TLC indicated
110
completion of the reaction. The mixture was brought to room temperature and the
formed precipitate was filtered by vacuum and rinsed with ice cold methanol (5 mL x
3). Yield: 13%. 1H NMR (CDCl3, 400 MHz): 1.30 (d, 6H), 1.57 (s, 9H), 2.64 (s, 3H),
4.05 (m, 1H), 8.98 (s, 1H), 9.14 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 14.53, 22.05,
29.93, 32.24, 40.28, 104.28, 116.77, 121.65, 159.96, 162.34, 164.54, 174.27, 175.47,
179.55. HR-MS: m/z = 328 [M+H]+.
2´-(benzylthio)-4-isobutyl-4´-(naphthalen-1-ylmethyl)-[2,5´-bipyrimidine]-5-carbonitrile (4b)
A mixture of compound 1d (0.76 mmol, 0.28 g) , hydroxylamine (1.90 mmol, 0.13 g)
and triethylamine (1.90 mmol, 0.3 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and remove every ethanol under the
reduced pressure. White solid are formed. With this solid 2 (0.75 mmol, 0.13 g), start
reduction reaction. Dissolve solid 2 with methanol and add acetic anhydride (0.98 mmol,
0.1 ml). After five minutes, add potassium formate (3.00 mmol, 0.25g) and
palladium/carbon (0.30 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 3 can be observed. With compound 3, start another coupling reaction.
Dissolve solid 3 with methanol and add acetic anhydride (0.98 mmol, 0.1 ml). After
five minutes, add potassium formate (3.00 mmol, 0.25g) and palladium/carbon (0.30 g).
Heat the reaction solution and keep stirring overnight. By using diatomaceous earth,
filter the reaction solution and evaporate every solvent. White compound 3 and 2 ́
can be observed. With mixture of 3 and 2 ,́ start another coupling reaction. A mixture
of compound 3 and 2 ́ (0.75 mmol, 0.288 g), triethylamine (1.13 mmole, 0.2 ml) and
111
another Michael acceptor (1.13 mmol, 0.23 g) in ethanol (absolute, 200 proof, 10mL) was
stirred under refluxing conditions until the TLC indicated completion of the reaction.
The mixture was brought to room temperature and the formed precipitate was filtered
by vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 86%. 1H NMR (CDCl3,
400 MHz): 0.79-0.81 (d, 6H), 1.98 (m, 1H), 2.67-2.69 (d, 2H), 4.15 (s, 2H), 5.12 (s,
2H), 6.98-7.50 (m, 12H), 8.86 (s, 1H), 9.32 (s, 1H). 13
C NMR (CDCl3, 400
MHz):22.36, 28.89, 35.34, 40.17, 45.55, 115.19, 121.61, 124.16, 125.18, 125.54,
125.87, 126.25, 126.64, 127.31, 127.40, 128.60, 128.97, 129.12, 132.44, 133.96, 134.73,
160.09 . HR-MS: m/z = 502 [M+H]+.
2´-amino-4-benzyl-4´-isobutyl-[2,5´-bipyrimidine]-5-carbonitrile (4c)
A mixture of compound 1h (2.09 mmol, 0.368 g) , hydroxylamine (5.23 mmol, 0.36 g)
and triethylamine (5.23 mmol, 0.7 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and evaporate every ethanol under the
reduced pressure. White solid are formed. With this solid 2 (2.09 mmol, 0.44 g), start
reduction reaction. Dissolve solid 2 with methanol and add acetic anhydride (2.72 mmol,
0.3 ml). After five minutes, add potassium formate (8.36 mmol, 0.70g) and
palladium/carbon (0.44 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 3 can be observed. With compound 3, start another coupling reaction.
A mixture of compound 3 (2.09 mmol, 0.403 g), triethylamine (3.13 mmole, 0.4 ml) and
Michael acceptor (3.13 mmol, 0.64 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
112
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). 1H NMR (CDCl3, 400 MHz):
0.79 (d, 6H), 1.98 (m, 1H), 2.97 (d, 2H), 4.26 (s, 2H), 5.30 (s, 2H), 7.22-7.34 (m, 5H),
8.83 (s, 1H), 9.00 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 22.72, 28.66, 43.35, 45.05,
127.68, 129.16, 129.51, 160.41, 162.29. HR-MS: m/z = 345.18 [M+H]+.
2´-amino-4´-benzyl-4-(naphthalene-1-ylmethyl)-[2,5´-bipyrimidine]-5-carbonitrile (4d)
A mixture of compound 1j (1.90 mmol, 0.40 g) , hydroxylamine (7.60 mmol, 0.52 g)
and triethylamine (7.60 mmol, 1.1 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and remove every ethanol under the
reduced pressure. White solid are formed. With this solid 2 (1.90 mmol, 0.46 g), start
reduction reaction. Dissolve solid 2 with methanol and add acetic anhydride (2.47 mmol,
0.2 ml). After five minutes, add potassium formate (7.60 mmol, 0.64g) and
palladium/carbon (0.44 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 3 can be observed. With compound 3, start another coupling reaction.
A mixture of compound 3 (1.90 mmol, 0.43 g), triethylamine (2.85 mmole, 0.4 ml) and
Michael acceptor (2.85 mmol, 0.60 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). 1H NMR (CDCl3, 400 MHz):
.21 (s, 2H), 4.69 (s, 2H), 5.25 (s, 2H), 6.82-8.07 (m, 12H), 8.82 (s, 1H), 8.93 (s, 1H).
13C NMR (CDCl3, 400 MHz): 40.41, 42.11, 124.19, 125.72, 126.13, 126.32, 126.69,
128.33, 128.67, 128.79, 129.12, 162.60. HR-MS: m/z = 429 [M+H]+.
113
2´-amino-4´-isobutyl-4-(naphthalene-1-ylmethyl)-[2,5´-bipyrimidine]-5-carbonitrile (4e)
A mixture of compound 1h (1.02 mmol, 0.18 g) , hydroxylamine (2.55 mmol, 0.18 g)
and triethylamine (2.55 mmol, 0.4 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and evaporate every ethanol. White solid
are formed. With this solid 2 (1.02 mmol, 0.21 g), start reduction reaction under the
reduced pressure. Dissolve solid 2 with methanol and add acetic anhydride (1.33 mmol,
0.1 ml). After five minutes, add potassium formate (4.08 mmol, 0.34g) and
palladium/carbon (0.21 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 3 can be observed. With compound 3, start another coupling reaction.
A mixture of compound 3 (1.02 mmol, 0.19 g), triethylamine (1.33 mmole, 0.2 ml) and
Michael acceptor (1.33 mmol, 0.35 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). 1H NMR (CDCl3, 400 MHz):
0.71 (d, 6H), 1.91 (m, 1H), 2.85 (d, 2H), 4.80 (s, 2H), 5.28 (s, 2H), 7.50-8.10 (m, 7H),
8.91 (s, 1H), 8.93 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 2.58, 28.58, 40.56, 44.79,
124.33, 125.76, 126.17, 126.69, 128.68, 128.75, 129.12, 132.09, 132.17, 134.22,
160.54. HR-MS: m/z = 395 [M+H]+.
114
2. 3. 4. Synthesis of trimeric terpyrimidyl derivatives, 7a-7d
2 ´ ´ -amino-4,4 ´ ´ -isobutyl-4 ´ -(naphthalene-1-ylmethyl)-[2,5 ´ :2 ´ ,5 ´ ´ -terpyrimidine]-5-
carbonitrile (7a)
A mixture of compound 4e (0.73 mmol, 0.288 g) , hydroxylamine (2.92 mmol, 0.20 g)
and triethylamine (2.92 mmol, 0.4 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and remove every ethanol under the
reduced pressure. White solid are formed. With this solid (0.73 mmol, 0.31 g), start
reduction reaction. Dissolve solid with methanol and add acetic anhydride (0.95 mmol,
0.1 ml). After five minutes, add potassium formate (2.92 mmol, 0.25g) and
palladium/carbon (0.31 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 6 can be observed. With compound 6, start another coupling reaction.
A mixture of compound 6 (0.73 mmol, 0.30 g), triethylamine (2.92 mmole, 0.4 ml) and
Michael acceptor (1.09 mmol, 0.20 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 57%. 1H NMR (CDCl3,
400 MHz): 0.71-0.77 (d, 6H), 0.77-0.81 (d, 6H), 1.85-2.00 (m, 2H), 2.68-2.70 (d, 2H),
2.89-2.91 (d, 2H), 5.17 (s, 1H), 5.24 (s, 1H), 6.97-8.03 (m, 7H), 8.90 (s, 1H), 8.91 (s, 1H),
9.53 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 22.37, 22.62, 28.77, 28.96, 40.15, 44.60,
45.58, 115.11, 123.98, 125.91, 126.28, 126.48, 129.03, 132.27, 134.94, 159.91, 160.16,
161.63, 162.53, 164.63, 168.75, 172.26, 173.63. HR-MS: m/z = 529 [M+H]+.
115
2 ´ ´-amino-4 ´ ´-benzyl-4-isobutyl-4 ´-(naphthalene-1-ylmethyl)-[2,5 ´:2 ´ ,5 ´ ´-terpyrimidine]-5-
carbonitrile (7b)
A mixture of compound 4d (0.11 mmol, 0.05 g) , hydroxylamine (0.44 mmol, 0.03 g)
and triethylamine (0.44 mmol, 0.1 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and evaporate every ethanol under the
reduced pressure. White solid are formed. With this solid 5 (0.11 mmol, 0.05 g), start
reduction reaction. Dissolve solid 5 with methanol and add acetic anhydride (0.14 mmol,
0.01 ml). After five minutes, add potassium formate (0.44 mmol, 0.04g) and
palladium/carbon (0.05 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 6 can be observed. With compound 6, start another coupling reaction.
A mixture of compound 6 (0.11mmol, 0.05 g), triethylamine (0.28 mmole, 0.04 ml) and
Michael acceptor (0.17 mmol, 0.03 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 28%. 1H NMR (CDCl3,
400 MHz): 0.82 (d, 6H), 2.05 (m, 1H), 2.73 (d, 2H), 4.29 (s, 2H), 5.15 (s, 2H), 5.21 (s,
2H), 6.98-7.97 (m, 12H), 8.91 (s, 1H), 9.01 (s, IH), 9.52 (s, 1H). 13
C NMR (CDCl3, 400
MHz): 22.40, 28.96, 40.16, 41.46, 45.60, 104.99, 105.36, 107.25, 115.12, 116.34,
121.60, 123.99, 125.44, 125.86, 126.12, 126.77, 128.24, 129.03, 129.27, 132.28,
133.99, 134.84, 141.20, 154.44, 159.90, 160.18, 162.11, 162.79, 164.61, 168.85,
170.71, 173.61, 173.88. HR-MS: m/z = 563 [M+H]+.
116
2´´-amino-4´´-isobutyl-4-isopropyl-4´-(naphthalene-1-ylmethyl)-[2,5´:2´,5´´-terpyrimidine]-5-
carbonitrile (7c)
A mixture of compound 4e (0.40 mmol, 0.158 g) , hydroxylamine (1.60 mmol, 0.11 g)
and triethylamine (1.60 mmol, 0.2 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and remove every ethanol under the
reduced pressure. White solid are formed. With this solid 5 (0.40 mmol, 0.17 g), start
reduction reaction. Dissolve solid 5 with methanol and add acetic anhydride (0.52 mmol,
0.05 ml). After five minutes, add potassium formate (1.60 mmol, 0.26g) and
palladium/carbon (0.17 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 6 can be observed. With compound 6, start another coupling reaction.
A mixture e of compound 6 (0.40 mmol, 0.164 g), triethylamine (1.60 mmole, 0.2 ml) and
Michael acceptor (0.52 mmol, 0.086 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 51%. 1H NMR (CDCl3,
400 MHz): 0.68-0.69 (d, 6H), 1.21-1.24 (d, 6H), 1.91 (m, 1H), 2.83-2.85 (d, 2H), 3.42
(m, 1H), 5.19 (s, 4H), 7.03-7.86 (m, 7H), 8.85 (s, 1H), 8.92 (s, 1H), 9.54 (s, 1H). 13
C
NMR (CDCl3, 400 MHz): 21.16, 22.58, 28.71, 35.21, 40.06, 124.14, 125.54, 125.58,
126.27, 126.95, 127.50, 128.99, 159.95, 160.49. HR-MS: m/z = 515 [M+H]+.
117
2´´-amino-4´-benzyl-4,4´´-diisobutyl-[2,5´:2´,5´´-terpyrimidine]-5-carbonitrile (7d)
A mixture of compound 4c (0.43 mmol, 0.15 g) , hydroxylamine (1.72 mmol, 0.12 g)
and triethylamine (1.72 mmol, 0.2 ml) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and evaporate every ethanol under the
reduced pressure. White solid are formed. With this solid 5 (0.43 mmol, 0.16 g), start
reduction reaction. Dissolve solid 5 with methanol and add acetic anhydride (0.56 mmol,
0.1 ml). After five minutes, add potassium formate (1.72 mmol, 0.15g) and
palladium/carbon (0.16 g). Heat the reaction solution and keep stirring overnight. By
using diatomaceous earth, filter the reaction solution and evaporate every solvent.
White compound 6 can be observed. With compound 6, start another coupling reaction.
A mixture of compound 6 (0.43 mmol, 0.15 g), triethylamine (1.72 mmole, 0.2 ml) and
Michael acceptor (0.65 mmol, 0.12 g) in ethanol (absolute, 200 proof, 10mL) was stirred
under refluxing conditions until the TLC indicated completion of the reaction. The
mixture was brought to room temperature and the formed precipitate was filtered by
vacuum and rinsed with ice cold methanol (5 mL x 3). Yield: 37%. 1H NMR (CDCl3,
400 MHz): 0.83 (d, 6H), 1.00 (d, 6H), 1.61 (s, 2H), 2.01 (m, 2H), 2.91 (d, 2H), 3.15 (d,
2H), 4.72 (s, 2H), 5.27 (s, 2H), 7.82 (m, 2H), 7.86 (m, 3H), 8.99 (s, 1H), 9. 02 (s, 1H),
9.44 (s, 1H). 13
C NMR (CDCl3, 400 MHz): 14.10, 14.81, 22.79, 22.83, 25.80, 43.92,
48.82, 53.45, 121.61, 128.60, 129.29, 155.52, 157.58. HR-MS: m/z = 479 [M+H]+.
118
2. 3. 5. Synthesis of 8-10
2´-amino-4-benzyl-4´-isobutyl-[2,5´-bipyrimidine]-5-carboxylic acid (8)
A mixture of compound 4c (0.58 mmol, 0.2 g) and sodium hydroxide (5.8 mmol, 0.23
g) in methanol (absolute, 200 proof, 10mL) was stirred under refluxing conditions.
Check TLC and remove every solvent under the reduced pressure. Dissolve the white
solid with water and add 1M HCl until pH reaches to 2~3. Extract the product with
ethyl acetate and recrystalize the product with ether and ethyl acetate. The yield is
5 %. 1H NMR (MeOH, 400 MHz): 0.73-0.77 (d, 6H), 1.90-1.94 (m, 1H), 2.94-2.98 (d,
2H), 4.39 (s, 2H), 4.61 (s, 2H), 7.17-7.32 (m, 5H), 8.79-8.85 (t, 2H), 9.19 (s, 1H). 13
C
NMR (MeOH, 400 MHz): 21.57, 28.56, 40.93, 41.37, 43.99, 44.15, 104.98, 126.34,
126.51, 128.26, 128.36, 129.23, 129.32, 155.86, 156.01, 164.51. HR-MS: m/z = 364
[M+H]+.
4-benzyl-2´-(3-carboxypropanamido)-4´-isobutyl-[2,5´-bipyrimidine]-5-carboxylic acid (9)
Dissolve compound 8 (0.13 mmole, 0.05 g) with THF add succinic anhydride (0.13
mmole, 0.013 g) and triethylamine ( 0.26 mmole, 0.1 ml) into the same flask. Reflux the
mixture and check TLC. Evaporate every solvent under the reduced pressure and
perform the column to purify the compound 9. (ethyl acetate -> MeOH : ethyl acetate
(1:4)). The yield is 37 %. 1
H NMR (MeOH, 400 MHz): 0.66-0.67 (d, 6H), 0.82-0.84 (d,
2H), 1.01-1.03 (d, 2H), 1.80-1.85 (m, 1H), 2.84-2.86 (d, 2H), 4.54 (s, 2H), 6.93 (s, 2H),
7.13-7.29 (m, 5H), 8.68 (s, 1H), 8.94 (s, 1H). 13
C NMR (MeOH, 400 MHz): 22.93,
23.16, 28.56, 29.82, 30.63, 32.82, 128.79, 129.90, 151.75, 174.72, 202.97, 203.02,
204.18, 205.50, 205.57, 205.59, 206.21. HR-MS: m/z = 464 [M+H]+.
119
2´´-amino-4´-benzyl-4,4´´-diisobutyl-[2,5´:2´,5´´-terpyrimidine]-5-carboxylic acid (10)
A mixture of compound 7d (0.43 mmol, 0.2 g) and sodium hydroxide (5.8 mmol, 0.23
g) in methanol (absolute, 200 proof, 10mL) was stirred under refluxing conditions.
Check TLC and remove every solvent under the reduced pressure. Dissolve the white
solid with water and add 1M HCl until pH reaches to 2~3. Extract the product with
ethyl acetate and recrystalize the product with ethyl acetate. The yield is 14 %. 1H
NMR (MeOH, 400 MHz): 0.73-0.77 (m, 12H), 0.91-0.93 (d, 2H), 1.08-1.13 (m, 1H),
1.29-1.30 (d, 2H ), 1.89-1.99 (m, 1H), 4.72-4.76 (d, 2H), 4.39 (s, 1H), 4.61 (s, 1H), 7.13-
7.31 (m, 5H), 9.21 (s, 1H), 9.31 (s, 1H), 9.35 (s, 1H). 13
C NMR (MeOH, 400 MHz):
20.92, 21.57, 21.69, 28.53, 28.55, 28.60, 40.94, 41.28, 41.32, 41.39, 43.81, 43.98,
44.16, 126.22, 126.35, 126.50, 128.26, 128.35, 128.97, 129.00, 129.22, 129.32, 159.57,
160.80, 165.35. HR-MS: m/z = 498.27 [M+H]+.
2. 4. References
1. Hollstein M., Sidransky D., Vogelstein B., Harris CC. p53 mutations in human
cancers. Science. 1991; 253: 49-53.
2. Zhang Y., Xiong Y. Control of p53 Ubiquitination and nuclear export by MDM2
and ARF. Cell. Growth. Differ. 2001; 12: 175-86.
3. Meek DW. P53 Induction: phosphorylation sites cooperate in regulating. Cancer.
Biol. Ther. 2003; 1: 284-6.
4. Prives C., Hall PA. The p53 pathway. J Pathol. 1999; 187: 112-6.
120
5. Chen J., Wu X., Lin J., Levine AJ. Mdm-2 inhibits the G1 arrest and apoptosis
functions of the p53 tumor suppressor protein. Mol. Cell. Biol. 1996; 16: 2445-52.
6. Chen L., Agrawal S., Zhou W., Zhang R., Chen J. Synergistic activation of p53 by
inhibition of mdm2 expression and DNA damage. Proc. Natl. Acad. Sci. U S A 1998;
95: 195-200.
7. Chen L., Lu W., Agrawal S., Zhou W., Zhang R., Chen J. Ubiquitous induction of
p53 in tumor cells by antisense inhibition of MDM2 expression. Mol. Med. 1999; 5:
19-32.
8. Blaydes J.P., Gire V., Rowson J.M., Wynford-Thomas D. Tolerance of high levels of
wild-type p53 in transformed epithelial cells dependent on auto-regulation by mdm-2.
Oncogene, 1997; 14: 1859-68.
9. Kussie P.H., Gorina S, Marechal V, et al. Structure of the MDM2 oncoprotein
bound to the p53 tumor suppressor transactivation domain. Science. 1996; 274: 948-53.
10. Z. Lai, K. R. Auger, C. M. Manubay, R. A. Copeland. Thermodynamics of p53
binding to hdm2 (1-126) : effects of phosphorylation and p53 peptide length. Arch.
Biochem. Biophys. 2000; 381: 278-84.
11. O. Schon, A. Friedler, S. Freund, A. R. Fersht. Molecular mechanism of the
interaction between MDM2 and p53. J. Mol. Biol. 2002; 323; 491-501.
121
12. L. Massova, P. A. Kollman. Computational alanine scanning to probe protein-protein
interactions: a novel approach to evaluate binding free energies. J. Am. Chem. Soc.
1998; 121; 8133-43.
13. H. Zhong, H. A. Carlson. Computational studies and peptidomimetic design for the
human p53-MDM2 complex. Proteins. 2005; 58: 222-34.
14. P. Chene. Inhibiting the p53-MDM2 interaction: an important target for cancer
therapy. Nat. Rev. Cancer. 2003; 3; 102-109.
15. Bottger V., Bottger A., Howard S. F., et al. Identification of novel mdm2 binding
peptides by phage display. Oncogene. 1996; 13: mm2141-7.
16. Vassilev L. T., Vu B. T., Graves B., et al. In vivo activation of the p53 pathway by
small-molecule antagonists of MDM2. Science. 2004; 303: 844-848.
17. L. Chen, H. Yin, B. Farooqi, et al. p53 -helix mimetics antagonize p53/MDM2
interaction and activate p53. Mol. Cancer Ther. 2005; 4: 1019-1025.
122
CHAPTER THREE:
SOLUTION-PHASE SYNTHESIS OF -HAIRPIN PEPTIDE
INHIBITING MULTIPLE MYELOMA CELLS ADHESION
3. 1. Introduction
Many classes of blood cells are formed from blood stem cells in bone marrow.
Plasma cells, a type of white cell, can make antibodies which fight against infections.
Plasma cells follow the normal cell cycle. When genes of plasma cells are changed,
they turn into myeloma cells In contrast to plasma cells, myeloma cells do not follow
normal cell cycle. Overtime, myeloma cells outgrow plasma cells and form a mass of
myeloma cells which is called a solitary plasma cytoma in bone marrow. This can
result in invading bone tissue and spreading throughout the body.
Multiple myeloma (MM) is a cancer of plasma cells. Even though treatment for
multiple myeloma (MM) has been highly improved, the disease still remains incurable
because myeloma cells can develop drug-resistance by being exposed to chemotherapy
over long periods of time. There have been numerous studies of both solid and
hematological cancer cells from different tissues showed drug resistance (1-8). Even
though dominant or multiple factors are involved in multidrug resistance, a few drug
transporters (e.g. P-glycoprotein, multidrug resistance-associated protein, breast cancer
resistant protein) and proteins which control or regulate cell growth or cell death (e.g.
Bcl-2 family) as cellular drug-resistant factors were studied intensively (9). Despite of
123
the fact that current standard therapy for cancers, including MM, is targeting apoptotic
pathway, these studies suggested that activating multiple cell death pathways may
avoid cancer drug resistance and cure cancers better (10). Also, Jason et al found out
cell-adhesion mediated drug resistance (CAM-DR) of multiple myeloma cells is not
related to two well-known factors of drug resistance, active drug transport and
increased expression of Bcl-2 family members which activate the apoptotic pathway
(11).
There are three different types of cell death based on the morphological appearance
(12). According to characteristic morphological aspects, apoptosis (type 1 cell death)
shows nuclear pyknosis (chromatin condensation) and karyorthexis (nuclear
fragmentation) (13). Autophagy (type 2 cell death) includes the massive autophagic
vacuolization and the digestion of partial cytoplasm by lysosomal hydrolases (14).
Necrosis (type 3 cell death) is characterized by rupture of the plasma membrane by
deflation of cell volume and the random dismantle of swollen organelles. Currently,
most conventional anticancer drug discovery strategy is inducing apoptosis because of
plethora knowledge of molecular mechanism of apoptosis regulation (15). A delicate
balance between proapoptotic and antiapoptotic proteins of apoptosis pathways plays
an important role to control the survival of long-lived cells and termination of short-
lived cells in a number of tissues, including the bone marrow. However, imbalance of
proapoptotic and antiproptotic regulators causes various diseases. Especially, for
cancers, cells which facilitate neoplasia and malignance can prolong their survival by
thwarting action of antiapoptotic proteins. Although in a variety of accumulating
evidences validate that apoptosis induction can be a potential drug discovery strategy,
it is necessary to optimize selectivity of compounds which interact with more than
two members of apoptosis protein families along with improving pharmacokinetic
124
properties. This disadvantage is another reason to induce alternative cell death for
anti-cancer therapy.
Previously, it was reported that HYD-1 (kikmviswkg), a synthetic D-amino acid
peptide, can act as a ligand mimetic and block tumor cell adhesion (16). This
cascades preventing migration of prostate tumor cells on laminin 322 (laminin 5) and
changing the cellular signals coming from a laminin 322 (laminin 5) matrix (17, 18).
Later, Rajesh et al discovered HYD-1 can block binding 41 integrin-dependent
multiple myeloma cells to extracellular matrix, fibronectin, as well as induce cell
death in H929, 8226, and U266 multiple myeloma (MM) cell lines but not in
CD34+ normal hematopoietic progenitor cells or surrounding blood mono-nuclear
cells (19). HYD-1 induced necrosis (type 3 cell death) in MM cells that is different
from apoptosis or autophagy by observing the loss of Δψm, total cellular ATP
decrease, increased ROS expression. However, because of modest antitumor activity
of HYD-1 (IC50 = 33 µM) in vivo, it was necessary to modify HYD-1 to improve
bioavailablity or antitumor activity (e.g. PEGylation, cyclization).
Figure 35. Structure of HYD-1.
125
Hazlehurst and co-workers discovered that MVISW were the minimal elements of
HYD-1 for bioactivity after creating N- and C-terminal deletion peptides and
replacement isoleucine to valine increased bioactivity. Based on these findings, first
modification was designing -hairpin structure by applying MVVSW in the
recognition strand and KLKLK in the non-recognition strand (20). Advantages of -
hairpin design that contribute to better bioactivity include: (1) amphiphilic
functionality and (2) rigid conformational structure. After a structure-activity
relationship (SAR) study, methionine (M) was changed to norleucine and serine (S)
was replaced by alanine (A). For further optimization, conformational search and
energy minimization for -turn promoters were studied by using the macromodel
and the GLIDE software distributed by Schrodinger, Inc.. This result indicated that
ether-peptidomimetic proline derivative at one turn and methylsulfonamide amino
ethyl glycine at the other turn showed an impressive bioactivety. By applying every
optimization condition, a cyclic peptide referred as MTI-101 showed 1.08 µM
(=IC50) bioactivity after TOPRO3 assay.
Figure 36. -Hairpin structure.
Due to the promising bioactivity of MTI-101 in vitro, it is necessary to produce a
massive amount of MTI-101 to investigate whether the same effect will reproduce in
126
vivo. The mass production of peptides can be carried out through either continuous
solid-phase synthesis or conventional solution-phase synthesis. Each method has its
own disadvantages, for example, classic solution-phase synthesis needs to isolate and
purify intermediates and continuous solid-phase synthesis demands to handle large
amount of pricy resins or large excess of reagents. In order to scale up the
production of the biologically active peptides successfully, it seems better to combine
the strong aspects of both methods to overcome their disadvantages (21).
In this study, solution-phase synthesis which has advantages of solution-phase and
solid-phase methods was examined for the production of MTI-101. We approached in
two different ways: (1) combination of recognition strand and non-recognition strand
and (2) continuous solution-phase synthesis. The data suggested the possibility of
larger-scale production for MTI-101 with simple purification and less use of reactants
or reagents in solution phase.
Figure 37. Structure of MTI-101.
Table 7. Comparison of Solid-phase Synthesis and Solution-phase Synthesis
Advantages
Disadvantages
127
Solid –phase
synthesis
- Rapid purifiction
- Fast synthesis
- Limited workable reactions for
coupling or linking
- Difficulty of monitoring the
reaction process
- Solvation problems
Solution-phase
synthesis
- Many documented solution-phase
synthesis
- Many commercially available
protecting groups
- More economical for mass
production
Purification problems
3. 2. Results and discussion
Previously, continuous solution methods have utilized Fmoc- chemistry to build
small-scaled short peptides which consist of six to eight amino acids (22). Because of
inconvenience of by-product removal after Fmoc- deprotection, in this study, Cbz- or
Boc- protecting groups were used. In addition, in contrast to solid-phase approach, the
reaction can be monitored and analyzed at any stage. MALDI-TOF and LC-MS
techniques were used for the analysis.
128
Synthesis of MTI-101 was approached in two different ways. One is recognition
strand and non-recognition strand were synthesized separately and combined later. The
other is the whole peptide structure was built in a continuous way. We investigated
the former case first since short peptides of six to eight amino acid units were
synthesized successfully in solution (22).
Before building the recognition strand, -turn promoter was synthesized. Scheme 6
shows synthesis procedures of -turn promoter, derivative of ether-peptidomimetic
proline. Boc-D-proline was reduced to Boc-D-prolinol by using NaBH4. SN2 reaction
was followed to convert Boc-D-prolinol to Boc-D-pyrrolidine derivative. For the next
coupling, Boc- group was deprotected selectively under 0 °C. The sequence of
recognition strand (WAVVN) was attached to the -turn promoter. Three different
coupling reagents were used for the better yield. In order to proceed the next coupling,
Cbz- group was deprotected in the presence of 10 % Pd/C and high N2 (55 psi). After
each coupling, the product was purified by column chromatography. The final
deprotection step was completed via treatment with TFA (Scheme 7).
Scheme 6. Synthesis of -turn promoter.
Building non-recognition strand was started with the synthesis of methylsulfonamide
amino ethyl glycine linker. Excess of 1,2-ethylenediamine was reacted to di-tert-butyl
dicarbonate to give one side protected product. In the next step, the unprotected amino
129
group attacked sulfur atom of methanesulfonyl chloride in a fashion of SN2 reaction.
Due to slightly more neucleophilic property of nitrogen of methylsulfonamide side,
nucleophilic substitution reaction occurred only one side in the presence of methyl
bromoacetate and NaH. The Boc- group was deprotected for the next coupling
(Scheme 8). Scheme 9 shows all the coupling and deprotection steps for non-
recognition strand. Five amino acids were coupled to the linker by using T3P in an
order of K(Boc)LK(Boc)LK(Boc). Boc- protected lysine was used to avoid side
reactions in coupling and deprotection steps. Deblocking Cbz- group was processed
under 10 % Pd/C and H2 (55 psi). After every coupling, the pure product was obtained
through recrystallization in ether (Scheme 9).
Scheme 7. Recognition strand synthesis of MTI-101.
130
Scheme 8. Synthesis of linker.
Scheme 9. Non-recognition strand synthesis of MTI-101.
The last step for MTI-101 synthesis is cyclization of recognition strand and non-
recognition strand in solution. Linear peptide was formed between -NH2 from non-
recognition strand and -COOH from recognition strand under the condition of T3P,
DIEA, and anhydrous THF. For the final cyclization, Cbz- deprotection was proceeded
earlier than that of methyl ester group. Because free carboxylic acid was transformed
back into methyl ester in the presence of excess methanol which is solvent for the
Cbz- deprotection step. After Cbz- deprotection, methyl ester group was deblocked by
using 5 % lithium hydroxide. Cyclization of linear peptide occurred via treatment with
T3P. The pure product was obtained as precipitation in ether. The final Boc-
deprotection of MTI-101 was completed with TFA/DCM (Scheme 10).
131
Scheme 10. Cyclization between recognition strand and non-recognition strand.
Even though cyclization of recognition strand and non-recognition strand was
successful in solution, this approach suffered from economical problem. Because
recognition strand synthesis demands column chromatograph purification after every
coupling. This would eventually cost more for the large-scale peptide synthesis. In
order to overcome this deficiency, continuous solution-phase synthesis was examined
since every product of non-recognition strand was able to be recrystallized in ether.
Before launching continuous solution-phase synthesis, it is necessary to modify
protecting group from previously synthesized -turn promoter due to involvement of
Boc- protected lysine. Instead of Boc-D-proline, Cbz-D-proline was reduced to Cbz-D-
prolinol in the presence of N-methylmorpholine, isobutyl chloroformate, and NaBH4.
Cbz-D-prolinol was transformed into Cbz-pyrrolidine derivative by using tert-butyl
132
bromoacetate. tert-Butyl group was deprotected for the coupling in two different ways:
(1) TFA/DCM and (2) H3PO4/toluene. Even though the combination of TFA and DCM
is the typical method for deblocking Boc- group, coupling reagent, T3P, enabled
trifluoroacetic acid active and caused uncontrolled side reaction. Moreover, this side
product was impossible to remove after recrystallization (Scheme 11).
Scheme 11. Synthesis of -turn promoter.
In the beginning, continuous solution-phase synthesis followed the same procedure
of non-recognition strand synthesis. A sequence of K(Boc)LK(Boc)LK(Boc) was
coupled to the linker by using combination of N-methylmorpholine and
isobutylchloroformate or T3P as coupling reagents. Except percent yield of dipeptide 2,
the most of percent yields were comparable for both coupling reagents. This is
because the linker contained trifluoroacetic acids as counter ions after Boc-
deprotection and they were activated by T3P. This resulted in undesirable reactions
and decreased percent yield. Between coupling steps, Cbz- group was deprotected
under the condition of Pd/C and H2 or Pd/C and triethylsilane. A combination of
Pd/C and triethylsilane made Cbz- deprotection faster, however, the efficiency of
deblocking completion decreased as the peptide became longer. After hexapeptide 6,
133
two more couplings (-turn promoter and trypthophan) were proceeded. However,
because of facing a couple of problems, for example, side reactions or low percent
yield, it is implausible to proceed the rest of coupling and cyclization. (Scheme 12,
Table 6, 7)
Table 8. Percent Yield of Continuous Solution-phase Synthesis
Entry Compound Yield
1 2a1 45%
2 3c1 89%
3 4a1 95%
4 5c1 88%
5 6a1 96%
6 7d -
*T3P as coupling reagent
Table 9. Percent Yield of Continuous Solution-phase Synthesis
Entry Compound Yield
1 2a2 98%
2 3c2 95%
3 4a2 89%
134
4 5c2 86%
5 6a2 91%
6 7d 73%
7 8e 51%
*N-methylmorpholine and isobutylchloroformate as coupling reagent
Scheme 12. Continuous solution-phase synthesis of MTI-101.
In summary, it is shown the possibility of MTI-101 synthesis in solution by using
two different approaches: (1) cyclization of recognition strand and non-recognition
strand and (2) continuous synthesis. In the former case, synthesis of MTI-101 was
successful in solution, however, column chromatography purification was necessary for
135
recognition strand that causes more cost for the large-scale synthesis. In the latter
case, even though products after each coupling were recrystallized in ether, synthesis
of MTI-101 was unable to be completed due to side reactions or low yield. Large-
scale synthesis of MTI-101 would be refined by using appropriate coupling reagents
or methods.
3. 3. Experimental section
3.3.1. General comments
All peptides were identified by mass spectral analysis (Bucker) and all new
compounds showed consistent 1H,
13C NMR (INOVA-400), and high resolution mass
spectral data (Agilent 1100 series).
3.3.2. -turn promoter synthesis
3.3.2.1. Tert-butyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-carboxylate
N-Boc-D-prolinol (1 g, 4.96 mmol) was dissolved in 20 ml of toluene and cooled
down to 0 °C. Tetrabutylammonium iodide (0.92 g, 2.49 mmol), 30 % NaOH solution (12
ml, 90 mmol), and tert-butylbromoacetate (1.46 ml, 9.93 mmol) were added subsequently
into the cooled solution. The reaction mixture was brought to room temperature after
5 hours and left overnight. 20 ml of toluene was added and the mixture was extracted.
The organic layer was washed with 1 N HCl (15 ml) and brine (15 ml). The organic
layer was concentrated after being dried over MgSO4. Purification by flash column
chromatography (Hexane-EtOAc, 3:1) gave a light yellow colored oil (1.41 g, 90 %). 1H
NMR (400 MHz, CDCl3): 1.46 (s, 9 H), 1.47 (s, 9H), 1.80-1.82 (m, 1H), 1.91-1.93
(m, 2H), 2.05-2.07 (m, 1H), 3.32-3.34 (m, 2H), 3.52-3.54 (m, 1H), 3.60-3.63 (m, 1H),
136
3.87-3.89 (m, 1H), 3.96 (s, 2H); 13
C NMR (400 MHz, CDCl3): 27.86, 28.04, 28.34,
28.76, 56.58, 69.25, 175.3; HRMS: m/z calcd for C16H29NO5 [M + H+], 316.2046; found,
316.2007.
3.3.2.2. Benzyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-carboxylate
N-methylmorpholine (1.3 ml, 12.1 mmol) and isobutylchloroformate (1.6 ml, 12.1
mmol) were added to a solution of Cbz-proline (3.01 g, 12.1 mmol) in 30 ml at 0 .
After 10 minutes, a white precipitation was removed by filteration and filtrate was
transferred to three neck flask. Solution of NaBH4 (0.69 g, 18.1 mmol) in 10 ml water
was mixed slowly to the cool filtrate while purging Ar gas. TLC indicated the
completion of reduction after l hour. THF was removed under reduced pressure and
water was added to destroy excess of NaBH4. Product was extracted with ethyl
acetate (3 10 ml), washed with brine, and dried over Na2SO4. After evaporation of
ethyl acetate, light yellow oil was obtained. N-Cbz-D-prolinol (2.84 g, 12.1 mmol) was
dissolved in 20 ml of toluene and cooled down to 0 °C. Tetrabutylammonium iodide
(2.23 g, 6.05 mmol), 30 % NaOH solution (29 ml, 218 mmol), and tert-butylbromoacetate
(3.6 ml, 24.2 mmol) were added subsequently into the cooled solution. The reaction
mixture was brought to room temperature after 5 hours and left overnight. 20 ml of
toluene was added and the mixture was extracted. The organic layer was washed with
1 N HCl (15 ml) and brine (15 ml). The organic layer was concentrated after dried over
MgSO4. Purification by flash column chromatography (Hexane-EtOAc, 4:1) gave a light
yellow colored oil (2.96 g, 70 %). 1H NMR (400 MHz, CDCl3): 1.47 (s, 9H), 1.81-
1.84 (m, 1H), 1.90-1.96 (m, 2H), 2.07-2.11 (m, 1H), 3.39-3.42 (m, 2H), 3.57-3.58 (m,
1H), 3.58-3.59 (m, 1H), 3.89 (s, 1H), 3.97 (s, 1H), 4.01-4.02 (m, 1H), 5.08-5.18 (m,
137
2H), 7.28-7.38 (m, 5H); 13
C NMR (400 MHz, CDCl3): 23.15, 24.08, 28.17, 28.34,
28.94, 46.98, 47.26, 56.59, 57.24, 66.81, 67.02, 68.70, 69.24, 71.76, 72.38, 76.73,
76.94, 77.04, 77.26, 77.36, 77.46, 77.57, 81.76, 82.10, 128.01, 128.15, 128.68, 137.16,
155.19, 169.92; HRMS: m/z calcd for C16H29NO5 [M + H+], ; found, .
3.3.2.3. Tert-butyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-1-carboxylic acid
Tert-butyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-carboxylate (1.41g, 4.47
mmol) in 10 ml of DCM was cooled down to 0 °C and 3 ml of trifluoroaceticacid was
added to the cooled solution. Reaction mixture was kept at 0 °C for 5 hours. Mixture
was extracted by DCM (10 ml x 3) after excess TFA was neutralized by 6 M KOH.
The combined organic layers were washed with brine (10 ml) and dried over MgSO4.
After concentration, light yellow oil was obtained (0.91g, 95 %).
3.3.2.4. 2-((1-((benzyloxy)carbonyl)pyrrolidin-2-yl)methoxy)acetic acid
i) Benzyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-carboxylate (2.96g, 8.46
mmol) in 10 ml of DCM was cooled down to 0 °C and 3 ml of trifluoroaceticacid was
added to the cooled solution. Reaction mixture was kept at 0 °C for 5 hours. After
concentration, a light yellow oil was obtained. 1H NMR (400 MHz, CDCl3): 1.54-
1.58 (m, 1H), 1.79-2.00 (m, 3H), 3.33-3.47 (m, 4H), 4.05 (s, 2H), 4.24-4.28 (m, 1H),
4.96-5.09 (dd, J = 0.05, 2H), 7.27-7.35 (m, 5H); 13
C NMR (400 MHz, CDCl3): 24.02,
27.89, 29.97, 46.71, 56.79, 67.74, 68.33, 75.31, 76.93, 77.24, 77.56, 114.15, 117.02,
127.90, 128.37, 128.70, 136.14, 157.23, 161.89, 162.28, 162.27, 163.06, 175.80;
HRMS: m/z calcd for C15H19NO5 [M + H+], 294.1336; found, 294.1344.
138
ii) Benzyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-carboxylate (0.70 g, 1.99
mmol) in 1 ml of toluene was added to l ml of 85 % phosphoric acid of another
flask. Reaction mixture was kept overnight. 5 ml of water added to reaction mixture
and product was extracted with ethyl acetate. The combined organic layer was washed
with brine and dried over Na2SO4. After concentration and flush column
chromatography (Hexane:ethyl acetate=1:2), a viscous light yellow oil was obtained
(0.41 g, 70%).
3.3.3. Recognition strand synthesis
3.3.3.1. -turn promoter(O-t-Bu)-Trp-NH(Cbz)
Solution of Tert-butyl-2-((2-tert-butoxy-2-oxoethoxy)methyl)pyrrolidine-1-carboxylic
acid (1.19 g, 5.53 mmol) and DIEA (2.3 ml, 13.83 mmol) was prepared in anhydrous
DCM (10 ml) under argon gas. N-Cbz-Trp-OH (1.87 g, 5.53 mmol) and
Propylphosphonic Anhydride (T3P, 3.52 g, 11.06 mmol) were added to previously
prepared solution. Leave the reaction mixture at room temperature overnight while
purging argon gas. The reaction was quenched with water (10 ml) and extracted with
DCM (10 ml x 3). The combined organic layers were washed with brine (10 ml), dried
over MgSO4, and concentrated. After flash column chromatography (Hexane-EtOAc,
1:1), a light yellow crystal was obtained (2.375 g, 80 %). 1H NMR (400 MHz, CDCl3):
1.44 (s, 9 H), 1.66-1.73 (m, 2H), 1.82-1.93 (dd, J = 0.09, 0.02, 2H), 2.48-2.55 (dd, J
= 0.05, 0.02, 1H), 3.06-3.12 (dd, J = 0.05, 0.02, 1H), 3.12-3.19 (m, 2H), 3.44-3.47 (m,
1H), 3.58-3.59 (m, J = 0.01, 0.01, 1H), 3.66-3.68 (m, 1H), 3.89 (s, 2H), 4.67-4.73 (q, J =
0.02, 1H), 5.10 (s, 2H), 7.04-7.38(m, 10H); 13
C NMR (400 MHz, CDCl3): 23.63, 2.51,
28.33, 42.25, 47.07, 53.96, 57.18, 66.97, 69.10, 70.90, 76.68, 81.78, 110.93, 111.26,
139
111.37, 119.90, 122.47, 128.21, 128.28, 128.33, 128.71, 136.19, 136.26, 169.89,
170.91; HRMS: m/z calcd for C16H29NO5 [M + H+], 536.2682; found, 536.2755.
3.3.3.2. -turn promoter(O-t-Bu)-Trp-NH2
Lin-Trp-NH(Cbz) (2.375 g, 4.43 mmol) and palladium carbon powder (10 %) was
added to methanol (20 ml). Leave mixture under high H2 pressure (48 psi) for 20
hours. Excess palladium carbon powder was removed over diatomaceous earth. White
crystal was obtained after methanol removal under reduced pressure (1.677 g, 94 %).
3.3.3.3. -turn promoter(O-t-Bu)-Trp-Ala-NH(Cbz)
Lin-Trp-NH2 ( 2.375 g, 4.43 mmol) was dissolved in 10 ml of anhydrous DCM under
argon gas. Subsequently, Cbz-Ala-OH (0.99 g, 4.43 mmol), DIEA (1.8 ml, 11.08 mmol),
and Propylphosphonic Anhydride (T3P, 5.64 g, 8.86 mmol) were added to previous
solution. Reaction mixture was stirred for 22 hours while purging argon gas. The
crude product was extracted with DCM (10 ml x 3) after 10 ml of water was added
for quenching excess T3p. The combined organic layers were washed with brine (10
ml), dried over MgSO4, and concentrated. Purification by flash column
chromatography (Hexane-EtOAc, 1:2) gave a light yellow crystal (1.935 g, 72 %). 1H
NMR (400 MHz, CDCl3): = 1.33-1.35 (d, J = 0.02, 3H), 1.44 (s, 9 H), 1.66-1.73 (m,
2H), 1.82-1.93 (dd, J = 0.09, 0.02, 2H), 3.16-3.18 (d, J = 0.02, 2H), 3.28-3.30 (m, 2H),
3.50-3.51 (m, 1H), 3.56-3.57 (m, 1H), 3.89 (s, 2H), 3.99-4.01 (m, 1H), 6.96-7.36 (m,
10H); 13
C NMR (400 MHz, CDCl3): 23.63, 2.51, 28.33, 42.25, 47.10, 47.12, 52.51,
57.24, 69.04, 70.82, 81.82, 110.87, 111.28, 118.95, 119.89, 122.46, 123.28, 128.42,
128.76, 136.15, 170.28 ; HRMS : m/z calcd for C33H42N4O7 [M + H+], 607.3053;
found, 607.3120.
140
3.3.3.4. -turn promoter(O-t-Bu)-Trp-Ala-NH2
Lin-Trp-Ala-NH2 ( 1.935 g, 3.19 mmol) and 10 % palladium carbon powder were
added to methanol and kept under high H2 pressure (53 psi) overnight. Excess
palladium carbon powder was filtered over diatomaceous earth. Concentration of
filtrate gave a white crystal (1.357 g, 90 %).
3.3.3.5. -turn promoter(O-t-Bu)-Trp-Ala-Val-NH(Cbz)
Solution of Lin-Trp-Ala-NH2 ( 2.68 g, 5.67 mmol) was prepared in anhydrous DCM
(10 ml) while purging argon gas. Cbz-Val-OH (1.14 g, 4.54 mmol), DIEA (1.5 ml, 9.08
mmol), and Propylphosphonic Anhydride (T3P, 5.77 g, 9.08 mmol) were added to
previously prepared solution. Reaction mixture was under dry condition overnight and
quenched with water (10 ml). Crude product was extracted with DCM (15 ml x 3),
washed with brine (15 ml), and dried over Na2SO4. Purification by column flash
chromatography (pure ethylacetate) gave a white crystal (3.207 g, 80 %). 1H NMR (400
MHz, CDCl3): = 0.89-0.91 (d, J = 0.02, 6H), 1.44 (s, 9 H), 2.05-2.07 (d, J = 0.02, 3H),
2.62-2.68 (m, 2H), 3.01-3.03 (m, 1H), 3.16-3.17 (d, J = 0.01, 2H), 3.29-3.31 (m, 2H),
3.50-3.51 (m, 1H), 3.89 (s, 2H), 3.95-4.01 (m, 2H), 4.48-4.52 (t, J = 0.02, 1H), 4.87-
4.92 (q, J = 0.02, 1H), 5.12 (s, 2H), 5.44-5.46 (d, J = 0.02, 1H), 7.06-7.36 (m, 10H); 13
C
NMR (400 MHz, CDCl3): 19.50, 23.77, 27.23, 28.33, 29.02, 31.44, 47.12, 49.15,
52.52, 57.25, 60.53, 67.34, 69.03, 70.84, 76.68, 81.84, 110.68, 111.35, 118.85, 119.84,
122.43, 123.38, 128.29, 128.41, 128.77, 136.18, 169.88, 170.17, 171.38; HRMS : m/z
calcd for C38H51N5O8 [M + H+], 706.3771; found, 706.3975.
141
3.3.3.6. -turn promoter(O-t-Bu)-Trp-Ala-Val-NH2
Lin-Trp-Ala-Val-NH(Cbz) ( 3.207 g, 4.54 mmol) was dissolved in methanol (20 ml)
and palladium carbon powder was added. The reaction mixture was left under H2 (53
psi) overnight and filtered over diatomaceous earth. A white crystal ( 2.464 g, 95 %)
was obtained after filtrate was concentrated.
3.3.3.7. -turn promoter(O-t-Bu)-Trp-Ala-Val-Val-NH(Cbz)
Lin-Trp-Ala-Val-NH2 ( 2.169 g, 3.80 mmol) and DIEA (1.3 ml, 7.60 mmol) were
dissolved in 10 ml of anhydrous DCM under argon gas. Cbz-Val-OH (0.95 g, 3.80
mmol) and Propylphosphonic Anhydride (T3P, 4.84 g, 7.60 mmol) were added to
previously prepared mixture. The reaction was left overnight while purging argon gas.
Excess Propylphosphonic Anhydride (T3P) was quenched with water (15 ml) and crude
product was extracted with DCM (15 ml x 3). The combined organic layers were
washed with brine (15 ml), dried over NaSO4, and concentrated. Purification by flash
column chromatography (ethylacetate-methanol; 20:1) gave a white crystal (2.326 g,
76 %). 1H NMR (400 MHz, CDCl3): = 0.90-0.96 (d, J = 0.02, 12H), 1.43 (s, 9 H),
1.46-1.48 (d, J = 0.02, 3H), 1.84-1.93 (m, 2H), 2.08-2,16 (m, 2H), 2.58-2.60 (m, 1H),
3.15-3.17 (d, J = 0.02, 2H), 3.22-3.29 (m, 1H), 3.41-3.45 (m, 2H), 3.54-3.61 (m, 2H),
3.55 (s, 2H), 3.99-4.01 (m, 1H), 4.11-4.18 (m, 1H), 4.34-4.38 (m, 1H), 4.74-4.76 (m,
1H), 4.91-4.95 (q, J = 0.02, 1H), 5.10 (s, 1H), 7.04-7.53 (m, 10H); 13
C NMR (400 MHz,
CDCl3): 18.32, 18.34, 18.47, 18.48, 19.48, 19.49, 23.69, 24.43, 27.18, 28.33,
29.28, 31.40, 47.12, 48.46, 56.53, 57.18, 58.81, 60.84, 67.13, 67.21, 69.00, 69.20,
70.88, 76.67, 81.79, 110.68, 111.31, 119.80, 122.37, 127.71, 128.17, 128.33, 128.74,
136.14, 136.62, 156.78, 169.87, 170.35, 170.84, 171.62, 171.97, 172.00; HRMS : m/z
calcd for C43H60N6O9 [M + H+], 805.4455; found, 805.4518.
142
3.3.3.8. -turn promoter(O-t-Bu)-Trp-Ala-Val-Val-NH2
The solution of pentamer (2.558 g, 3.18 mmol) was prepared in methanol and
palladium carbon powder was added. The reaction mixture was left under H2 (53 psi)
for one day and filtered over diatomaceous earth. Concentration of filtrate gave a
white crystal (1.937 g, 91 %).
3.3.3.9. -turn promoter(O-t-Bu)-Trp-Ala-Val-Val-Nlle-NH(Cbz)
Lin-Trp-Ala-Val-Val-NH2 (1.937 g, 2.89 mmol) and DIEA (7.23 mmol, 1.2 ml) were
added to 10 ml of anhydrous DCM under argon gas. Cbz-Nlle-OH (0.77 g, 2.89
mmol) and Propylphosphonic Anhydride (T3P, 1.83.7 g, 5.78 mmol) were added to
previously prepared solution. The reaction mixture was kept overnight while purging
argon gas. Excess T3P was quenched with water (15 ml) and crude product was
extracted with DCM (15 ml x 3). The combined organic layers were washed with
brine (15 ml), dried over Na2SO4, and concentrated. Recrystallization with Et2O gave
a white crystal (2.406 g, 91 %). HRMS : m/z calcd for C49H71N7O10 [M + H+],
918.5296; found, 918.6535.
3.3.3.10. -turn promoter(OH)-Trp-Ala-Val-Val-Nlle-NH(Cbz)
Lin(O-t-Bu)-Trp-Ala-Val-Val-Nlle-NH(Cbz) ( 2.406 g, 2.62 mmol) and
trifluoroaceticacid (TFA, 3 ml) were added to 10 ml of DCM. The mixture was kept
overnight, neutralized with 6 M KOH, and extracted with DCM (15 ml x 3). The
combined organic layers were washed with brine (15 ml) and dried over Na2SO4.
Evaporation of DCM gave a white crystal (2.123 g, 94 %). C45H63N7O10 [M - H+],
860.4564; found, 860.4544.
143
3.3.4. Synthesis of linker
3.3.4.1. Tert-butyl-2-aminoethylcarbamate
Ethylenediamine (55.6 mL, 831 mmol) was dissolved in THF (200 mL). A solution of
Boc2O (20 g, 91.6 mmol) in 200 ml of THF was added dropwisely at 0°C overnight.
The resulting solution was concentrated, dissolved in DCM (100 mL), and extracted
water (3x20 mL). The combined water layer was back-extracted with DCM (100 mL),
and the all of organic layers were combined and dried over anhydrous Na2SO4. After
concentrated, a yellow oil was left (14.52 g, 99%).
3.3.4.2. Tert-butyl (2-(methylsulfonamido)ethyl)carbamate
Tert-butyl-2-aminoethylcarbamate (11.19 g, 69.8 mmol) was dissolved in 100 ml of
THF. Triethylamine (11.68 mL, 83.8 mmol), and methanesulfonyl chloride (5.41 mL, 69.8
mmol) were added and kept at room temperature for one hour. The reaction mixture
was concentrated, extracted in ethyl acetate (EtOAc, 3 25 mL), and washed with brine
(10 mL). The organic layer was concentrated, resulting in an off-white solid.
Recrtstallization in diethyl ether gave white solids (15.11 g, 90.8%). 1H NMR (400
MHz, CDCl3): = 1.40 9s, 9H), 2.93 (s, 3H), 3.18-3.27 (m, 4H); 13
C NMR (400
MHz, CDCl3): 28.58, 40.35, 40.80, 40.95, 43.67, 79.62, 79.98, 156.74; HRMS :
m/z calcd for C43H60N6O9 [M + H+], 239.106; found, 239.105.
3.3.4.3 Methyl 2-(N-(2-(tert-butoxycarbonylamino)ethyl)methylsulfonamido)acetate
Tert-butyl-(2-(methylsulfonamido)ethyl)carbamate (15.11 g, 63.4 mmol) was dissolved
in 100 ml of anhydrous THF. 60% sodium hydride (2.54 g, 63.4 mmol) was added to
the previous prepared solution while purging Ar gas. After 15 minutes, addition of
methyl bromoacetate (5.83 mL, 63.4 mmol) was followed and the solution was stirred
144
for one hour. The solution was quenched with water (10 mL). The resulting solution
was concentrated, dissolved in EtOAc (75 mL), and extracted five times with water
(10 mL). The organic layer was concentrated, and the crude product was
recrystallized in EtOAc:Hexane (1:1), producing a white solid (14.96 g, 76.0%). 1H
NMR (400 MHz, CDCl3): = 1.38 (s, 9H), 3,21 (s, 3H), 3.22-3.25 (m, 2H), 3,32-
3.35 (m, 2H), 3.71 (s, 3H), 4.10 (s, 2H); 13
C NMR (400 MHz, CDCl3): 28.55,
38.89, 39.79, 47.81, 48.57, 52.63, 79.72, 156.27, 170.54; HRMS : m/z calcd for
C43H60N6O9 [M + H+], 311.127; found, 311.128.
3.3.4.4. Methyl-N-(2-aminoethyl)-N-(methylsulfonyl)glycinate
Methyl-N-(2-aminoethyl)-N-(methylsulfonyl)glycinate (2.00 g, 6.44 mmol) was
dissolved in 10 ml of DCM. Trifluoroacetic \acid (3 mL) was added and allowed to stir
overnight. The reaction mixture was concentrated and resulting in a yellow oil (2.02
g, 96.5%).
3.3.5. Synthesis of non-recognition strand (Contributed by Dr. Philip Murray)
3.3.5.1. Linker(OMe)-Lys(Boc)-NH(Cbz)
The deprotected linker (2.02 g, 6.22 mmol) was dissolved in anhydrous DCM (40
mL) under argon. Cbz-lysine(Boc)-OH (2.37 g, 6.22 mmol), 50% propanephosphonic
anhydride (7.92 mL, 12.4 mmol) and DIEA (2.57 mL, 16.0 mmol) were added
subsequently. The reaction mixture was allowed to stir overnight. The resulting
solution was quenched with water (10 mL), concentrated and dissolved in ethyl acetate
(50 mL). The filtrate was extracted three times with water (10 mL). The organic layer
was concentrated, and the crude product was recrystallized in ether (10 mL), producing
a white solid (1.93 g, 54.6%). m/z [M+H]+ 573.1.
145
3.3.5.2. Linker(OMe)-Lys(Boc)-NH2
Linker(OMe)-Lys(Boc)-NHCbz (1.93 g, 3.38 mmol) was dissolved in methanol. 10 %
Palladium carbon powder was added, and the solution was placed under high H2
pressure (55 psi) overnight. The palladium carbon powder was filtered over
diatomaceous earth, and the resulting solution was concentrated, forming a white solid.
3.3.5.3. Linker(OMe)-Lys(Boc)-Leu-NH(Cbz)
The deprotected dimer (1.48 g, 3.38 mmol) was dissolved in anhydrous DCM under
argon. DIEA (1.40 mL, 8.44 mmol), Z-Leu-OH (0.896 g, 3.38 mmol), and 50%
propanephosphonic anhydride (T3P, 4.30 mL, 6.75 mmol) were added and allowed to
stir overnight. The reaction mixture was quenched with water, concentrated and
dissolved in EtOAc (50 mL). The organic layer was washed three times with water
(10 mL), the organic layer was concentrated, and the resulting crude product was
recrystallized in ether (10 mL) overnight, producing a white solid (1.99 g, 86.3%). m/z
[M+H]+ 686.34.
3.3.5.4. Linker(OMe)-Lys(Boc)-Leu-NH2
Linker(OMe)-Lys(Boc)-Leu-NH(Cbz) (1.99 g, 2.91 mmol) was dissolved in methanol.
10 % Palladium carbon powder was added, and the solution was placed under H2
pressure (55 psi) overnight. The palladium carbon powder was filtered over
diatomaceous earth, and the resulting solution was concentrated, forming a white
solid.
146
3.3.5.5. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz)
The deprotected product (1.60 g, 2.91 mmol) was dissolved in anhydrous DCM (10
mL) under argon. Z-Lys(Boc)-OH (1.11 g, 2.91 mmol), 50% propanephosphonic
anhydride (T3P, 3.70 mL, 5.81 mmol), and DIEA (1.20 mL, 7.27 mmol) were added at
0°C and allowed to stir overnight. The reaction mixture was quenched with water (5
mL) for 1 hr, then concentrated and dissolved in EtOAc (50 mL). The organic
solution was extracted three times with water (10 mL). The organic layer was dried
over Na2SO4 and concentrated. The crude product was recrystallized in ether (10 mL)
overnight, producing a white solid (2.19 g, 82.7%). m/z [M+H]+ 914.49.
3.3.5.6. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-NH2
Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz) (2.19 g, 2.40 mmol) was dissolved in
methanol (100 mL). Palladium (10%) carbon powder (0.4 g) was added, and the solution
was placed under H2 pressure (55 psi) overnight. The palladium carbon powder was
filtered over diatomaceous earth, and the resulting solution was concentrated, forming
a white solid.
3.3.5.7. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-NH(Cbz)
The deprotected product (1.87 g, 2.40 mmol) was dissolved in anhydrous DCM (15
mL) under argon. Z-Leu- OH (0.638 g, 2.40 mmol), propanephosphonic anhydride (T3P,
3.06 mL, 4.79 mmol), and DIEA (1.04 mL, 6.00 mmol) were added at 0°C and allowed
to stir overnight. Water (5 mL) was added to quench the excess of T3P for 1 hour.
The resulting solution was concentrated and dissolved in EtOAc (50 mL). The solution
was extracted three times with water (10 mL). The organic layer was dried over
147
Na2SO4 and concentrated.. The crude product was recrystallized in ether (10 mL)
overnight, producing a white solid (1.83 g, 74.5%). m/z [M+H]+ 1027.57.
3.3.5.8. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-NH2
Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-NH(Cbz) (1.83 g, 1.78 mmol) was dissolved
in methanol (100 mL). Palladium (10%) carbon powder (0.4 g) was added, and the
solution was placed under H2 pressure (55 psi) overnight. The palladium carbon
powder was filtered over diatomaceous earth, and the resulting solution was
concentrated, forming a white solid.
3.3.5.9. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz)
The deprotected product (1.59 g, 1.78 mmol) was dissolved in anhydrous DCM (15
mL) under argon. Z-Lys(Boc)-OH (0.678 g, 1.78 mmol), propanephosphonic anhydride
(T3P, 2.27 mL, 3.56 mmol), and DIEA (0.736 mL, 4.45 mmol) were added at 0°C and
allowed to stir overnight. The resulting solution was quenched with water (5 mL) for
1 hour, then concentrated and dissolved in EtOAc (50 mL). The solution was extracted
three times with water (10 mL). The organic layer was dried over Na2SO4 and
concentrated. The crude product was then recrystallized in ether (10 mL) overnight,
producing a white solid (2.00 g, 89.5%). m/z [M+H]+ 1255.72.
3.3.5.10. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)-NH2
Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz) (2.00 g, 1.59 mmol) was
dissolved in methanol (100 mL). Palladium (10%) carbon powder (0.4 g) was added,
and the solution was placed under H2 pressure (55 psi) overnight. The palladium
148
carbon powder was filtered over diatomaceous earth, and the resulting solution was
concentrated, forming a white solid.
3.3.6. Cyclization of recognition strand and non-recognition strand
(Contributed by Dr. Priyesh Jain)
3.3.6.1. Synthesis of linear peptide
Hexameric recognition strand (0.14 mg, 0.125 mmol) and non-recognition strand (0.11
g, 0.125 mmol) were dissolved in anhydrous THF (10 ml). Solution of 50%
propanephosphonic anhydride (T3P, 0.16 mL, 0.25 mmol) was added to the previous
solution at 0°C. The reaction was allowed to stir overnight at room temperature.
Excess T3P was quenched with water (2 mL), concentrated, and dissolved in EtOAC.
The solution was extracted three times with water. The organic layer was dried over
Na2SO4 and concentrated. The crude product was recrystallized in ether (10 mL)
overnight, producing a white solid (fully protected linear peptide, 0.17 g, 68%). The
coupled peptide was validated by MALDI-TOF after deprotecting all Boc groups. m/z
[M+H]+ 1664.71
3.3.6.2. Deprotection of linear peptide
Protected linear peptide (0.17 g, 0.086 mmol) was dissolved in methanol (30 mL).
Palladium (10%) carbon powder (0.4 g) was added and the solution was placed under
H2 pressure (55 psi) overnight. The palladium carbon powder was filtered over
diatomaceous earth. The resulting solution was concentrated and white solid was
formed (0.14 g, 88%). Cbz-deprotected linear peptide (0.14 g, 0.076 mmol) was dissolved
in THF:H2O (1:1, 5 mL). 5% LiOH solution (2 mL) was added to the previous solution
at 0oC. The reaction was carried out for 30 minutes until the TLC showed complete
consumption of the starting material. The reaction contents were concentrated and
149
freeze dried to give peptide as a white solid. Deprotected linear peptide was validated
by MALDI-TOF after deprotecting all Boc groups. m/z [M+H]+ 1516.47.
3.3.6.3. Cyclization of linear peptide
Deprotected linear peptide (0.11 g, 0.076 mmol) was dissolved in anhydrous THF (10
mL). Solution of 50% propanephosphonic anhydride (0.10 mL, 0.15 mmol) was added
at 0°C. The reaction was allowed to stir overnight at room temperature. Excess T3P
was quenched with water (2 mL), concentrated, and dissolved in EtOAC. The solution
was then extracted three times with water. The organic layer was dried over Na2SO4
and concentrated. The crude product was recrystallized in ether (10 mL) overnight, a
white solid was formed. The cyclized peptide was validated by MALDI-TOF after
deprotecting all Boc groups. m/z [M+H]+ 1499.43.
3.3.7. Linear synthesis of MTI-101
3.3.7.1. Linker(OMe)-Lys(Boc)-NH(Cbz)
Cbz-N-Lys(Boc)-OH (0.77 g, 2.03 mmol) was dissolved in anhydrous DCM (10 ml)
and cool down at 0 . N-methylmorpholine (0.22 ml, 2.03 mmol) and
isobutylchloroformate (0.27 ml, 2.03 mmol) were added to the cooled solution. Reaction
mixture was allowed to stir for 15 minutes. Deprotected linker (0.43 g, 2.03 mmol)
and triethylamine (0.57 ml, 4.06 mmol) were mixed to the previous solution. The
reaction mixture was brought to room temperature and stirred overnight. The product
was extracted with DCM (3 10 ml). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The white solid was obtained after
recrystallization in ether (1.09 g, 98 %). 1H NMR (400 MHz, CDCl3): = 1.31-1.32 (d,
2H), 1.37 (s, 9H), 1.61-1.62 (m, 1H), 1.79-1.82 (m, 1H), 2.96 (s, 3H), 3.03-3.05 (m,
150
2H), 3.33-3.37 (m, 4H), 3.70 (s, 2H), 4.10 (s, 3H), 4.81-4.83 (m, 1H), 5.05 (s, 2H),
7.26-7.30 (m, 5H); 13
C NMR (100 MHz, CDCl3): = 12.21, 17.56, 18.79, 22.74,
28.63, 29.72, 32.12, 37.49, 39.61, 40.12, 42.18, 47.32, 48.37, 52.69, 53.86, 55.21,
67.19, 77.04, 77.36, 77.68, 79.23, 128.33, 128.55, 128.70, 136.49, 156.43, 156.52,
170.51, 172.72; HRMS : m/z calcd for C25H40N4O9S [M + H+], 573.258; found, 573.260.
3.3.7.2. Linker(OMe)-Lys(Boc)-NH2
Protected dimeric peptide (1.09 g, 1.90 mmol) was dissolved in 100 ml of methanol
and palladium carbon powder (10 %) was mixed. Triethylsilane (1.52 ml, 9.51 mmol)
was added slowly under the hydrogen gas. The completion of reaction was monitored
by TLC. Palladium carbon powder was removed over diatomaceous earth and the
resulting solution was concentrated under reduced pressure.
3.3.7.3. Linker(OMe)-Lys(Boc)-Leu-NH(Cbz)
Cbz-N-Leu-OH (0.50 g, 1.90 mmol) was dissolved in anhydrous DCM (10 ml) and
cool down at 0 . N-methylmorpholine (0.21 ml, 1.90 mmol) and isobutylchloroformate
(0.25 ml, 1.90 mmol) were added to the cooled solution. Reaction mixture was
allowed to stir for 15 minutes. Deprotected dimeric peptide (0.83 g, 1.90 mmol) and
triethylamine (0.53 ml, 3.80 mmol) were mixed to the previous solution. The reaction
mixture was brought to room temperature and stirred overnight. The product was
extracted with DCM (3 10 ml). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The white solid was obtained after
recrystallization in ether (1.24 g, 95 %). 1H NMR (400 MHz, CDCl3): = 0.82-0.84 (t,
6H, J = 0.02), 1.18-1.21 (m, 2H), 1.34 (s, 9H), 1.40-1.42 (m, 2H), 1.46-1.49 (m, 1H),
1.56-1.59 (m, 2H), 2.84-2.85 (m, 2H), 2.97 (s, 3H), 3.22-3.24 (m, 4H), 3.39-3.41 (m,
151
8H), 3.64 (s, 3H), 4.08 (s, 2H), 4.01-4.13 (m, 2H), 5.01 (s, 2H), 7.29-7.34 (m, 5H); 13
C
NMR (100 MHz, CDCl3): = 22.04, 23.33, 23.75, 24.85, 28.94, 29.87, 32.40, 38.04,
39.50, 39.66, 39.71, 39.92, 40.12, 40.33, 40.44, 40.54, 40.75, 41.21, 47.50, 48.98,
52.66, 53.17, 53.83, 66.06, 78.01, 127.67, 128.32, 128.45, 129/01, 137.68, 156.20,
156.64, 170.76, 172.45, 172.87; HRMS : m/z calcd for C31H51N5O10S [M + H+],
686.342; found, 686.345.
3.3.7.4. Linker(OMe)-Lys(Boc)-Leu-NH2
Protected trimeric peptide (1.24 g, 1.81 mmol) was dissolved in 100 ml of methanol
and palladium carbon powder (10 %) was mixed. Triethylsilane (1.44 ml, 9.03 mmol)
was added slowly under the hydrogen gas. The completion of reaction was monitored
by TLC. Palladium carbon powder was removed over diatomaceous earth and the
resulting solution was concentrated under reduced pressure.
3.3.7.5. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz)
Cbz-N-Lys(Boc)-OH (0.69 g, 1.81 mmol) was dissolved in anhydrous DCM (10 ml)
and cool down at 0 . N-methylmorpholine (0.20 ml, 1.81 mmol) and
isobutylchloroformate (0.24 ml, 1.81 mmol) were added to the cooled solution. Reaction
mixture was allowed to stir for 15 minutes. Deprotected trimeric peptide (1.00 g, 1.81
mmol) and triethylamine (0.50 ml, 3.62 mmol) were mixed to the previous solution.
The reaction mixture was brought to room temperature and stirred overnight. The
product was extracted with DCM (3 10 ml). The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated. The white solid was obtained
after recrystallization in ether (1.47 g, 89 %). 1H NMR (400 MHz, CDCl3): = 0.88-
0.93 (dd, 6H), 1.32-1.45 (m, 4H), 1.40 (s, 9H), 1.55-1.61 (m, 5H), 1.79-1.85 (m, 3H),
152
2.98 (s, 3H), 3.05-3.10 (m, 4H), 3.38-3.48 (m, 4H), 3.72 (s, 3H), 4.14 (s, 2H), 4.28-
4.36 (m, 1H), 4.80-4.83 (m, 1H), 5.07 (s, 2H), 7.30-7.35 (m, 5H); 13
C NMR (100 MHz,
CDCl3): = 21.95, 23.11, 23.26, 23.55, 24.51, 28.69, 29.66, 31.94, 32.08, 37.79,
39.24, 39.44, 39.65, 39.86, 40.07, 40.18, 40.28, 40.49, 40.91, 47.24, 48.71, 51.40,
52.42, 53.02, 55.17, 65.81, 77.80, 127.33, 128.10, 128.20, 128.76, 137.40, 155.99,
166.43, 170.52, 172.14, 172.25, 172.43; HRMS : m/z calcd for C42H71N7O13S [M + H+],
914.490; found, 914.494.
3.3.7.6. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-NH2
Protected tetrameric peptide (1.47 g, 1.61 mmol) was dissolved in 100 ml of
methanol and palladium carbon powder (10 %) was mixed. Triethylsilane (1.28 ml,
8.04 mmol) was added slowly under the hydrogen gas. The completion of reaction
was monitored by TLC. Palladium carbon powder was removed over diatomaceous
earth and the resulting solution was concentrated under reduced pressure.
3.3.7.7. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-NH(Cbz)
Cbz-N-Leu-OH (0.43 g, 1.61 mmol) was dissolved in anhydrous DCM (10 ml) and
cool down at 0 . N-methylmorpholine (0.18 ml, 1.61 mmol) and isobutylchloroformate
(0.21 ml, 1.61 mmol) were added to the cooled solution. Reaction mixture was
allowed to stir for 15 minutes. Deprotected tetrameric peptide (1.26 g, 1.61 mmol) and
triethylamine (0.45 ml, 3.22 mmol) were mixed to the previous solution. The reaction
mixture was brought to room temperature and stirred overnight. The product was
extracted with DCM (3 10 ml). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The white solid was obtained after
recrystallization in ether (1.42 g, 86 %). 1H NMR (400 MHz, CDCl3): = 0.87-0.95
153
(m, 6H), 1.25-1.47 (m, 10H), 1.39 (s, 9H), 1.57-1.60 (m, 8H), 1.82-1.95 (m, 2H), 2.97
(s, 3H), 2.99-3.08 (m, 4H), 3.38-3.40 (m, 4H), 3.71 (s, 3H), 4.16 (s, 2H), 4.20-4.34 (m,
2H), 4.75-4.89 (m, 2H), 5.09 (s, 2H), 7.29-7.40 (m, 5H); 13
C NMR (100 MHz, CDCl3):
= 14.51, 21.88, 23.46, 23.56, 28.69, 39.45, 39.66, 39.87, 40.08, 40.29, 47.23, 48.70,
52.41, 52.84, 53.58, 60.20, 65.79, 77.77, 127.40, 128.05, 128.19, 128.76, 137.44,
155.96, 156.37, 170.51, 170.78, 171.92, 172.11, 172.13, 172.80; HRMS : m/z calcd for
C48H82N8O14S [M + H+], 1027.574; found, 1027.576.
3.3.7.8. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-NH2
Protected pentameric peptide (1.42 g, 1.38 mmol) was dissolved in 100 ml of
methanol and palladium carbon powder (10 %) was mixed. Triethylsilane (1.11 ml,
6.92 mmol) was added slowly under the hydrogen gas. The completion of reaction
was monitored by TLC. Palladium carbon powder was removed over diatomaceous
earth and the resulting solution was concentrated under reduced pressure.
3.3.7.9. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)-NH(Cbz)
Cbz-N-Lys(Boc)-OH (0.52 g, 1.38 mmol) was dissolved in anhydrous DCM (10 ml)
and cool down at 0 . N-methylmorpholine (0.15 ml, 1.38 mmol) and
isobutylchloroformate (0.18 ml, 1.38 mmol) were added to the cooled solution. Reaction
mixture was allowed to stir for 15 minutes. Deprotected pentameric peptide (1.23 g,
1.38 mmol) and triethylamine (0.38 ml, 2.76 mmol) were mixed to the previous
solution. The reaction mixture was brought to room temperature and stirred overnight.
The product was extracted with DCM (3 10 ml). The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated. The white solid was obtained
154
after recrystallization in ether (1.58 g, 91 %). MALDI-TOF : m/z [M + H+] 955.387.
(MALDI-TOF was validated after deprotecting all Boc groups)
3.3.7.10. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)-NH2
Protected hexameric peptide (1.58 g, 1.26 mmol) was dissolved in 100 ml of
methanol and palladium carbon powder (10 %) was mixed. The reaction mixture was
located overnight under the high pressure of hydrogen gas. Palladium carbon powder
was removed over diatomaceous earth and the resulting solution was concentrated
under reduced pressure.
3.3.7.11. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)--turn promoter-NH(Cbz)
Deprotected -turn promoter (0.51 g, 1.26 mmol) was dissolved in anhydrous DCM
(10 ml) and cool down at 0 . N-methylmorpholine (0.14 ml, 1.26 mmol) and
isobutylchloroformate (0.16 ml, 1.26 mmol) were added to the cooled solution. Reaction
mixture was allowed to stir for 15 minutes. Deprotected hexameric peptide (1.41 g,
1.26 mmol) and triethylamine (0.35 ml, 2.52 mmol) were mixed to the previous
solution. The reaction mixture was brought to room temperature and stirred overnight.
The product was extracted with DCM (3 10 ml). The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated. The white solid was obtained
after recrystallization in ether (1.28 g, 73 %). MALDI-TOF : m/z [M + H+] 1096.709.
(MALDI-TOF was validated after deprotecting all Boc groups)
3.3.7.12. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)--turn promoter-NH2
Protected heptameric peptide (1.28 g, 0.92 mmol) was dissolved in 100 ml of
methanol and palladium carbon powder (10 %) was mixed. The reaction mixture was
155
located overnight under the high pressure of hydrogen gas. Palladium carbon powder
was removed over diatomaceous earth and the resulting solution was concentrated
under reduced pressure.
3.3.7.13. Linker(OMe)-Lys(Boc)-Leu-Lys(Boc)-Leu-Lys(Boc)--turn promoter-NH(Cbz)
Cbz-N-Trp-OH (0.31 g, 0.92 mmol) was dissolved in anhydrous DCM (10 ml) and
cool down at 0 . N-methylmorpholine (0.10 ml, 0.92 mmol) and isobutylchloroformate
(0.12 ml, 0.92 mmol) were added to the cooled solution. Reaction mixture was
allowed to stir for 15 minutes. Deprotected heptameric peptide (1.16 g, 0.92 mmol)
and triethylamine (0.26 ml, 1.84 mmol) were mixed to the previous solution. The
reaction mixture was brought to room temperature and stirred overnight. The product
was extracted with DCM (3 10 ml). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The white solid was obtained after
recrystallization in ether (0.2 g, 23 %). MALDI-TOF : m/z [M + H+] 1282.758.
(MALDI-TOF was validated after deprotecting all Boc groups)
3. 4. References
1. Choi, C.H.. ABC transporters as multidrug resistance mechanisms and the
development of chemo sensitizers for their reversal. Cancer Cell Int. 2005; 5: 30-42.
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3. Szakacs G., Paterson J.K., Ludwig J.A., Booth-Genthe C., Gottesman M.M..
Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006; 5: 219-234.
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and ABCG transporters: participation in a chemoimmunity defense system. Physiol.
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5. Deeley R.G., Westlake C., Cole S.P.. transmembrane transport of endo- and
xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol.
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6. Viktorsson K., Lewensohn R., Zhivotosky B.. Apoptotic pathways and therapy
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7. Brown J.M., Attardi L.D.. The role of apoptosis in cancer development and
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8. Doyle L.A., Yang W., Abruzzo L.V., Krogmann T., Gao A.K., Rishi A.K., Ross
D.D.. A multidrug resistance transporter for human MCF-7 breast cancer cells. Proc.
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9. Lowe S.W., Lin A.W.. Apoptosis in cancer. Carcinogenesis. 2000; 21: 485-495.
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10. Hu X., Xuan Y.. Bypassing cancer drug resistance by activating multiple death
pathways – a proposal from the study of circumventing cancer drug resistance by
induction of necroptosis. Cancer Lett. 2008; 259: 127-137.
11. Damiano J.S., Cress A.E., Hazlehurst L.A., Shtil A.A., Dalton W.S.. Cell adhesion
mediated drug resistance (CAM-DR): Role of integrins and resistance to apoptosis in
human myeloma cell lines. Blood. 1999; 93: 1658-1667.
12. Galluzzi L., Maiuri M.C., Vitale I., et al. Cell death modalities: classification and
pathophysiological implication. Cell Death Differ. 2007; 14: 1237-1243.
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15. Reed J.C., Pellecchia M.. Apoptosis-based therapies for hematologic malignancies.
Blood. 2005; 106: 408-418
16. DeRoock I.B., Pennington M.E., Sroka T.C., Lam K.S., Bowden G..T., Bair E.L.,
Cress A.E., Synthetic peptides inhibit adhesion of human tumor cells to extracellular
matrix proteins. Cancer Res. 2001; 61: 3308-3313.
17. Sroka T.C., Pennington M.E., Cress A.E.. Synthetic D-amino acid peptide inhibits
tumor cell motility on laminin-5. Carcinigenesis. 2006; 27: 1748-1757.
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18. Sroka T.C., Marik J., Pennington M.E., Lam K.S., Cress A.E.. The minimum
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19. Nair R.R., Emmons M.F., Cress A.E., Argilagos R.F., Lam K. Kerr W.T., Wang
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159
CHAPTER FOUR:
CONSTRUCTION OF PEPTOID-PEPTIDE HYBRIDS
ADOPTING -HAIRPIN STRUCTURE
4. 1. Introduction
Peptides play an important role in biological functions and therefore promising
candidates in therapeutic development. However, their natural instability to proteases
has led to numerous research in peptidomimetics recently (1). Peptidomimetics not
only have similar functions and interactions to the intended target as those of natural
peptides but also resist to the proteolysis. In other words, peptidomimetics would have
improved stability and bioavailability.
In late 1990s, the Gellman group introduced the field of foldamers (2). Foldamers
are defined as unnatural oligomers which can fold in a certain fashion and copy the
behaviors known from biopolymers (3 - 7). One example of a type of foldamer is
hybrid peptoid-peptide ligands. Goodman and colleagues reported the first cases of
peptoid-peptide hybrids in 1994 (8). They synthesized and characterized collagen
mimicry with peptide repetition H2N-Gly-[Gly-Pro-Hyp]n-, which were covalently
attached to the Kemp tricarboxylic acid (9 - 11). In addition to this, similar
assemblies containing the N-isobutylglycine (NIeu) peptoid residue instead of
hydroxyproline (Hyp) were investigated in a series of the study (12 - 16).
160
Hamy and co-workers showed hybrid peptoid-peptide ligands with the ability to
disrupt the interactions between HIV-1 transactivation protein (Tat) and the
transactivation response (TAR) RNA domain, which following HIV-1 replication in
primary human lymphocytes (17 - 19). After screening in a pool of 3.2 106
ligands, a inhibitor of the Tat-TAR RNA complex with nanomolar activity was found
(Figure 1a). The inhibitor was further studied and proved to bind TAR RNA
specifically by using NMR analysis and molecular modeling (16). In addition, the
group of Rocchi reported the receptor targeting case by using peptoid substituted
compounds based on dermorphin, deltorphin, and endomprohin as opioid receptor
agonists (Figure 1b) (20 - 22).
Most types of peptidomimetics, including peptoid-peptide hybrids, are investigated as
membrane active cationic anti-microbial peptide (AMP) derivatives. There are two
major strategies: (1) replacing certain residues in a known natural product or peptide
and (2) constructing the de novo design of an amphiphilic template. Both of these
strategies have been applied to develop AMP analogues with peptoid-peptide hybrids.
Robinson and co-workers displayed a small series of peptoid-containing analogues of
the antimicrobial -hairpin structure protegrin I by using the first strategy (Figure 1c)
(23). In a preliminary study, the disulfide bridges connecting native hairpin were
replaced by D-Pro-L-Pro moiety through macrocyclization. Two substituted compounds
were designed, synthesized, and evaluated (23). After deuterium exchange and 2D
ROESY NMR spectroscopy experiment along with the constrained molecular modeling,
a lead compound (Figure 1c) maintains a relatively stable -hairpin conformation in
aqueous solution. Also, this lead compound has slightly better potency than that of its
nonpeptoid-containing -peptides against the tested microorganisms, and more
161
improtaltly, it showed much more resistance to hemolysis the natural product lead
protegrin I (23).
Figure 38. Peptoid-peptide hybrids with bioactivity.
162
Ryge, Hansen, and colleagues reported de novo designed amphiphilic peptoid-
peptide hybrids as AMP mimetics and optimized their design via combinatorial
libraries (24 - 26). Their most powerful molecule showed low micromolar range for
the IC50 against a broad spectrum of both Gram-positive and Gram-negative bacteria
(Figure 1d). In order to investigate the transmembrane segment mimetics, peptoid-
peptide hybrids interacting membrane have also been studied (27, 28).
Since Goodman and co-workers introduced the first examples of peptoid-peptide
hybrid, the -amino acid combined peptoid residues have been engaged in numerous
biologically active ligands. As discussed earlier, ligands for inhibition protein-protein
interactions and antimicrobial activities are the examples as related research fields.
The research of peptoid-peptide hybrid structures has been thrived in development of
peptidomimetics and will provide more ligands with interesting bioactivities in the
future.
4. 2. Results and discussion
N-substituted glycine oligomers, or peptoids, are an important class of foldamers
built by tertiary amide linkages (29). The extensive chemical diversity of the peptoid
side chains promotes the design strategies which can be applied to the various fields
such as enenatioselective catalysis, molecular recognition, and intracellular delivery (30
- 32). Moreover, numerous studies have been dedicated to development of folded
peptoids with the distinct secondary structures by changing side chains which control
the backbone conformational ordering. Recently, two well-ordered peptoid secondary
structures were built using triazole linkages (33, 34). Even though this discovery
opens up the possibility of the peptoid design with similar structure as small folded
proteins, we seek the capability to craft functional peptoid-peptide hybrids adopting
163
the -hairpin structure. -hairpin structure is one of the extended peptide secondary
structures, that two functional strands are connected through a linkage. Depending on
side chains, -hairpin can improve the target recognition ability as well as the
pharmacokinetic properties.
Peptoid oligomers can be prepared efficiently via submonomer solid-phase synthesis
strategy. The strategy is to elongate the main chain by an acylation reaction with a
haloacetic acid and then attach the side chain with a primary amine. Because of its
high efficiency, the similar approach was applied in this experiment. The whole
peptoids were constructed on 2-chlorotrityl chloride resins. Before the synthesis was
initiated, the resins were reactivated by using thionyl chloride in DCM. For the
acylation step, bromoacetic anhydride was used instead of haloacetic acid due to its
high reactivity. Also, 2,4-dimethoxybenzyl amine was selected rather than ammonia to
introduce the secondary amide, hydrogen bonded to the carbonyl group on the
opposite strand,, because ammonia can cleave the peptoids on the resins. Also, 2,4-
dimethoxytoluene was able to be removed with TFA at the last stage of synthesis.
One of critical elements in -hairpin structure is turn promoter. For this experiment,
D-Pro-L-Pro template, type II´ turn, was chosen as a -turn promoter.
In order to observe the structure changes or differences between acyclic and cyclic
peptoid-peptide hybrids, the resins were divided in half before the cleavage from the
resins. The divided resins were deprotected separately and processed in different ways.
For the construction of acyclic peptoid-peptide hybrids, the resulting compounds after
deprotection were purified by HPLC. To build the cyclic peptoid-peptide hybrids, the
resulting compounds after deprotection were cyclized in the presence of HCTU in
DCM and then purified by HPLC. Based on this strategy, three sets of acyclic and
164
cyclic peptoid-peptide hybrids were prepared (Figure 2). Three sets of peptoid-peptide
hybrids were different in sizes and the number of turn promoters.
NMR study was employed to check the formation of -hairpin structures based on
the hypothesis: if the -hairpin structure is formed, glycine -protons of an acyclic
peptoid-peptide hybrid in the 4 ppm ranges will shift to 5 ppm ranges after
cyclization. Deturated acetonitrile or chloroform were used as NMR solvents. After
NMR studies, there was no indication of the -hairpin structure formation from three
sets of peptoid-peptide hybrids because there was no chemical shift changes from -
protons in acyclic and cyclic peptoid-peptide hybrids.
Figure 39. Structures of three sets of acyclic and cyclic peptoid-peptide hybrids.
In this study, the possibility of -hairpin structure in peptoid-peptide hybrids was
investigated. NMR studies were performed based on the hypothesis: glycine -protons
165
of an acyclic peptoid-peptide hybrid in the 4 ppm ranges will shift to 5 ppm ranges
after cyclization due to the -hairpin structure formation. However, the NMR study
results displayed that there was no indication of the -hairpin structure formation in
peptoid-peptide hybrids. These results suggested that the peptoid-peptide hybrid
structures were still flexible despite of the introduction of intra hydrogen bonding and
-turn promoters. According to this finding, it is necessary to develop the components
which provide the more rigid structure in peptoid-peptide hybrids such as
incorporations of ring moieties in the main chain or development of staple peptoid-
peptide hybrids.
4. 3. Experimental section
4.3.1. General comments
All reaction intermediates were monitered by mass spectral analysis (Agilent). The
complete peptoid-peptide hybrids were purified by using HPLC (DIONEX) and
purified compounds were studied via 1H NMR (INOVA-400).
4. 3. 2. General procedure
All the cyclic peptoid-peptide hybrids were prepared using solid-phase peptide
synthesis (Figure 2). On the 2-chlorotrityl chloride resin, Fmoc-D-Proline was attached
via SN2 reaction and typical submonomer strategy was performed to add different
peptoid residues. The treatment with TFA provided not only cleavage of linear
peptoid-peptide hybrids on the resin but also deprotection of 2,4-dimethoxytoluene,
transferring tertiary amine into secondary amine. The resulting peptoid-peptide hybrids
were cyclized in solution and purified by HPLC for further study.
166
Scheme 13. General procedure for peptoid-peptide hybrids in SPPS.
4. 3. 2. Synthesis of acyclic and cyclic peptoid-peptide hybrids 1
2-Chlorotrityl chloride resins (0.3 g; capacity: 0.79 mmol/g) were swelled with 3
ml of anhydrous DCM at room temperature for 20 minutes. Fmoc-D-proline (0.1 g,
0.95 mmol) and DIEA (0.28 ml, 1.58 mmol) in 2 ml of DCM were added to the
synthesis reaction vessel. After 30 minutes, the solution was drained and the same
washing step was repeated. The beads were washed with anhydrous DCM. Then the
beads were deprotected using 2 ml of 2 % DBU in DMF for 5 minutes at room
temperature. This step was repeated, and the beads were washed with DCM. The
acylation step was carried out at room temperature for 20 minutes. All the beads
were incubated in a mixture of bromoacetic anhydride (0.41 g, 1. 58 mmol), DIEA
(0.56 ml, 3.16 mmol), and 2 ml of DCM. After, that step the beads were washed with
DCM. Depending on the side chain sequences, different primary amines (3. 16 mmol)
in 2 ml of DCM were added to the beads and the reaction mixture was stirred at
room temperature for 20 minutes before the beads were washed with DMF. These
acylation and SN2 by primary amines (e.g. 2,4-dimethoxybenzyl amine, isopropyl
167
amine, benzyl amine and et al) were repeated until the intended sequences are done.
In order to complete -turn template (D-Pro-L-Pro), Fmoc-L-Proline (1.07 g, 3.16
mmol), HCTU (1.31 g, 3.16 mmol), and DIEA (1.12 ml, 6.32 mmol) in 3 ml of
DCM were mixed for 1 hour, and this step was repeated. The beads were washed
with DCM. Before the linear peptoids were cleaved from the resin, Fmoc group was
deprotected using 2 % DBU in DMF and the beads were washed with DCM. Based
on the mass, the resins were divided by in half for the preparation of acyclic and
cyclic peptoids. Both of peptoids on the resins were cleaved with TFA and the
resulting solution was collected in the flask. For the complete deprotection (2,4-
dimethoxytoluene), the resulting solution was stirred for 2 days. After removing TFA
under the reduced pressure, one of batches were purified using HPLC to collect the
acyclic peptoid (Figure 2). The peptoids in the other batch were cyclized in the
mixture of HCTU (0.65 g, 1.58 mmol), DIEA (0.56 ml, 3.16 mmol) in 5 ml of
DCM. After proper work-up, the cyclic peptoids were obtained after HPLC
purification (Figure 2). HRMS: m/z [M+H]+ (acyclic version). HRMS: m/z [M+H]
+
(cyclic version).
Figure 40. Structures of acyclic and cyclic peptoid-peptide hybrids 1.
168
4. 3. 3. Synthesis of acyclic and cyclic peptoid-peptide hybrids 2
2-Chlorotrityl chloride resins (0.3 g; capacity: 0.79 mmol/g) were swelled with 3
ml of anhydrous DCM at room temperature for 20 minutes. Fmoc-D-proline (0.1 g,
0.95 mmol) and DIEA (0.28 ml, 1.58 mmol) in 2 ml of DCM were added to the
synthesis reaction vessel. After 30 minutes, the solution was drained and the same
step was repeated. The beads were washed with anhydrous DCM. The beads were
deprotected using 2 ml of 2 % DBU in DMF for 5 minutes at room temperature.
This step was repeated, and the beads were washed with DCM. The acylation step
was carried out at room temperature for 20 minutes. All the beads were incubated in
a mixture of bromoacetic anhydride (0.41 g, 1. 58 mmol), DIEA (0.56 ml, 3.16
mmol), and 2 ml of DCM. The beads were washed with DCM. Depending on the
side chain sequences, different primary amines (3. 16 mmol) in 2 ml of DCM were
added to the beads and the reaction mixture was stirred at room temperature for 20
minutes before the beads were washed with DCM. These acylation and SN2 by
primary amines (e.g. 2,4-dimethoxybenzyl amine, isopropyl amine, benzyl amine and
et al) were repeated until the intended sequences are done. In order to complete -
turn template (D-Pro-L-Pro), Fmoc-L-Proline (1.07 g, 3.16 mmol), HCTU (1.31 g,
3.16 mmol), and DIEA (1.12 ml, 6.32 mmol) in 3 ml of DCM were mixed for 1
hour, and this step was repeated. The beads were washed with DCM. Before the
linear peptoids were cleaved from the resin, Fmoc group was deprotected using 2 %
DBU in DMF and the beads were washed with DCM. Based on the mass, the resins
were divided by in half for the preparation of acyclic and cyclic peptoids. Both of
peptoids on the resins were cleaved with TFA and the resulting solution was
collected in the flask. For the complete deprotection (2,4-dimethoxytoluene), the
resulting solution was stirred for 2 days. After removing TFA under the reduced
169
pressure, one of batches were purified using HPLC to collect the acyclic peptoid
(Figure 3). The peptoids in the other batch were cyclized in the mixture of HCTU
(0.65 g, 1.58 mmol), DIEA (0.56 ml, 3.16 mmol) in 5 ml of DCM. After proper
work-up, the cyclic peptoids were obtained after HPLC purification (Figure 3).
MALDI-TOF: m/z [M+H]+ (acyclic version). MALDI-TOF: m/z [M+H]
+ 929.57 (cyclic
version).
Figure 41. Structures of acyclic and cyclic peptoid-peptide hybrids 2.
4. 3. 3. Synthesis of acyclic and cyclic peptoid-peptide hybrids 3
2-Chlorotrityl chloride resins (0.3 g; capacity: 0.79 mmol/g) were swelled with 3
ml of anhydrous DCM at room temperature for 20 minutes. Fmoc-D-proline (0.1 g,
0.95 mmol) and DIEA (0.28 ml, 1.58 mmol) in 2 ml of DCM were added to the
synthesis reaction vessel. After 30 minutes, the solution was drained and the same
step was repeated. The beads were washed with anhydrous DCM. The beads were
deprotected using 2 ml of 2 % DBU in DMF for 5 minutes at room temperature.
This step was repeated, and the beads were washed with DCM. The acylation step
was carried out at room temperature for 20 minutes. All the beads were incubated in
a mixture of bromoacetic anhydride (0.41 g, 1. 58 mmol), DIEA (0.56 ml, 3.16
mmol), and 2 ml of DCM. The beads were washed with DCM. Depending on the
side chain sequences, different primary amines (3. 16 mmol) in 2 ml of DCM were
170
added to the beads and the reaction mixture was stirred at room temperature for 20
minutes before the beads were washed with DCM. These acylation and SN2 by
primary amines (e.g. 2,4-dimethoxybenzyl amine, isopropyl amine, benzyl amine and
et al) were repeated until the intended sequences are done. In order to complete -
turn template (D-Pro-L-Pro), Fmoc-L-Proline (1.07 g, 3.16 mmol), HCTU (1.31 g,
3.16 mmol), and DIEA (1.12 ml, 6.32 mmol) in 3 ml of DCM were mixed for 1
hour, and this step was repeated. The beads were washed with DCM. The Fmoc
group was deprotected by using 2 % DBU in DMF and the beads were washed with
DCM. In order to construct the other strand, the same procedure such as SN2
reaction with Fmoc-D-Proline, acylation, SN2 reaction with primary amines, and SN2
reaction with Fmoc-L-Proline was performed. Before the linear peptoids were cleaved
from the resin, Fmoc group was deprotected using 2 % DBU in DMF and the beads
were washed with DCM. Based on the mass, the resins were divided by in half for
the preparation of acyclic and cyclic peptoids. Both of peptoids on the resins were
cleaved with TFA and the resulting solution was collected in a flask. For the complete
deprotection (2,4-dimethoxytoluene), the resulting solution was stirred for 2 days.
After removing TFA under the reduced pressure, one of batches were purified using
HPLC to collect the acyclic peptoid (Figure 4). The peptoids in the other batch were
cyclized in the mixture of HCTU (0.65 g, 1.58 mmol), DIEA (0.56 ml, 3.16 mmol)
in 5 ml of DCM. After proper work-up, the cyclic peptoids were obtained after
HPLC purification (Figure 4). MALDI-TOF: m/z [M+H]+ 1677.99 (acyclic version).
MALDI-TOF: m/z [M+H]+ 1659.97 (cyclic version).
171
Figure 42. Structures of acyclic and cyclic peptoid-peptide hybrids 3.
4. 3. 4. NMR study of peptoid-peptide hybrids
In order to investigate the chemical shift change of -protons in acyclic and cyclic
peptoid-peptide hybrids, the proton NMR study was performed (INOVA-400 and
INOVA-500). Compounds were dissolved in deturated chloroform and acetonitrile.
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6. Huc I. Eur. J. Org. Chem. 2004; 17-29.
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16. Melacini G., Feng Y., Goodman M. Biochemistry. 1997; 36: 8725-8732.
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19. Hamy F., Felder E. R., Heizmann G., Lazdins J., Aboul-ela F., Varani G., Karn J.,
Klimkait T. Proc. Natl. Acad. Sci. USA. 1997; 94: 3548-3553.
20. Biodi L., Giannini E., Negri L., Melchiorri P., Lattanzi R., Rosso F., Ciocca L.,
Rocchi R. Int. J. Pep. Res. Ther. 2006; 12: 145-151.
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2004; 10: 578-587.
22. Biondi L., Giannini E., Filira F., Gobbo M., Marastoni M., Negri L., Scolaro B.,
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23. Shankaramma S. C., Moehle K., James S., Vrijbloed J. W., Obrecht D., Robinson
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25. Ryge T. S., Hansen P. R. J. Pep. Sci. 2005; 11: 727-734.
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175
APPENDIX: 1H, 13C NMR SPECTRA, MASS SPECTRA
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
(The sample was checked after three Boc-groups were deprotected with TFA.)
262
(The sample was checked after three Boc-groups were deprotected with TFA.)
263
264
(The sample was checked after three Boc-groups were deprotected with TFA.)
265
(The sample was checked after three Boc-groups were deprotected.)
266
(The sample was checked after three Boc-groups were deprotected.)
267
Acyclic (top) and cyclic (bottom) peptoid-peptide hybrid 1
268
Acyclic (top) and cyclic (bottom) peptoid-peptide hybrid 2
878.0 910.4 942.8 975.2 1007.6 1040.0
Mass (m/z)
4.2E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 Reflector Spec #1[BP = 947.5, 42414]
947.4
961
969.4
780
985.4
486
991.4
608
963.4
833
8 7 4 .0 9 0 0 .6 9 2 7 .2 9 5 3 .8 9 8 0 .4 1 0 0 7 .0
Mass (m/z)
2 .4 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Inte
nsity
4700 Reflector Spec #1[BP = 929.6, 23786]
92
9.5
73
4
95
1.5
59
7
91
2.1
46
2
96
7.5
35
3
94
7.5
83
1
98
7.5
87
1
89
0.5
48
6
90
2.5
20
3
269
Acyclic (top) and cyclic (bottom) peptoid-peptide hybrid 3
1 2 2 6 1 4 4 3 1 6 6 0 1 8 7 7 2 0 9 4 2 3 1 1
Mass (m/z)
2 .3 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Inte
nsity
4700 Reflector Spec #1[BP = 1678.0, 22654]
16
77
.99
43
16
99
.97
60
16
28
.93
88
17
75
.05
96
15
80
.91
52
16
60
.57
06
14
72
.83
40
1 3 6 6 .0 1 4 9 3 .8 1 6 2 1 .6 1 7 4 9 .4 1 8 7 7 .2 2 0 0 5 .0
Mass (m/z)
4 .4 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Inte
nsity
4700 Reflector Spec #1[BP = 2228.6, 46481]
16
81
.97
18
16
59
.96
92
16
42
.53
76
15
27
.87
40
16
97
.94
41
16
77
.98
62
15
05
.86
80
18
29
.29
99
19
00
.34
97
ABOUT THE AUTHOR
Ms. Hyun Joo Kil received dual bachelors` degrees in the general science education
and the chemistry education from Kangwon National University, South Korea in 2005.
She started the masters` program in physical organic chemistry at Kangwon National
university in 2005. She received her masters degree in 2007. She pursued her doctorate
degree at Kangwon National university in 2007 and transferrd to University of South
Florida in 2008. She has worked in Dr. Mark McLaughlin` lab to find drug candidates
to the various targets.