Design, synthesis and biological studies of coumarin based probes and tetrahydrolipstatin analogs 1

DESIGN, SYNTHESIS AND BIOLOGICAL STUDIES OF
COUMARIN-BASED PROBES AND
TETRAHYDROLIPSTATIN ANALOGS








NGAI MUN HONG
(MSc., Universiti Teknologi Malaysia)









A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY




NATIONAL UNIVERSITY OF SINGAPORE





2010



i

To my parents
















ii



ACKNOWLEDGEMENTS



I would like to express my sincere gratitude and appreciation to my research
advisor, Assistant Prof. Dr. Martin J. Lear, for his guidance, support, encouragement
and patience throughout the completion of this work.

Grateful acknowledgements go to Assoc. Prof. Dr. Yao Shao Qin for his
advice and guidance.
My sincere appreciation also extends to Mr. Yang Pengyu and Mr. Liu Kai
from Prof. Yao lab for performing the biological assays for THL libraries.
I wish to extend my gratitude to Assistant Prof. Dr. Paul MacAry, Ms. Song
Zhenying from the Department of Microbiology and Dr. Song Hongyan for their help
in carrying out the bioimaging experiments.
I would like to thank Ms. Cheong Wei Fun and Mr. Shareef Mohideen Ismail
for their assistance with HRMS.
I wish to express my indebtedness to all current and former members of the
Lear group for their support and friendship. I am particularly thankful to Dr. Bastien
Reux and Mr. Oliver Simon for expanding my world view and vocabulary.
Finally, I wish to thank lab technicians and others who have provided
assistance at various occasions.





iii



TABLE OF CONTENTS



CHAPTER TITLE PAGE

TITLE PAGE

DEDICATION
i
ACKNOWLEDGEMENTS
ii
TABLE OF CONTENTS
iii
SUMMARY
vi
LIST OF TABLES
ix
LIST OF SCHEMES
x
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xiv
LIST OF APPENDICES
xvii
LIST OF PUBLICATIONS
xviii

Design and synthesis of coumarin-based fluorescent probe


1 INTRODUCTION
1

References 8

2 RESULTS AND DISCUSSION
9
2.1 Development of a coumarin-based
fluorescent probe

9
2.2
Practical synthesis of N-alkylated
maleimides and maleimidocarboxylic NHS-
esters

11
2.3 Synthesis of coumarin probe

15
2.4 Proof-of-Principle studies with coumarin
probe

18
2.4.1 Labelling RGDC with coumarin
probe 2.20

18



iv
2.4.2 Antibody-labelling, cell-sorting and
cell culture study

19
2.5 Conclusion

24
References

25



3 EXPERIMENTAL
29

References

42




Synthesis and biological studies of tetrahydrolipstatin
analogs





4 INTRODUCTION
43
4.1 Isolation and biological activities of (-)-

tetrahydrolipstatin

43
4.2 Previous syntheses of (-)-tetrahydrolipstatin

44
4.3 Studies of tetrahydrolipstatin derivatives as
fatty acid synthase inhibitors

61
4.4 Summary of tetrahydrolipstatin syntheses

64
4.5 Objectives

65
References

66



5 RESULTS AND DISCUSSION
69
5.1 Development of a solid-supported strategy to
THL analogues

69
5.1.1 Mechanism of tandem Mukiyama
aldol-lactonization (TMAL)

78
5.2 Synthesis of THL-L analogues

81
5.3 Synthesis of THL-R analogues

85
5.4 Synthesis of THL-T analogues

89
5.5 Biological studies of THL probes

91
5.5.1 Effects on cell proliferation,
phosphorylation of eIf2 and activation
of caspase-8

91
5.5.2 In situ and in vitro proteome profiling

93
5.5.3 Target identification and validation

95
5.6 Conclusion

96
References

97






v
6 EXPERIMENTAL
101


7 RESULTS AND DISCUSSION
149

7.1 Introduction to click chemistry 149

7.1.1 Applications of click chemistry

151

7.2 Synthesis of THL analogs by click chemistry 152

7.2.1 Mechanism of Keck asymmetric
allylation

156

7.3 Synthesis of azides 158

7.4 Biological evaluation of THL analogs 160

7.5 Conclusion 162

References 163


8 EXPERIMENTAL
167

References

190



APPENDICES
191







vi



SUMMARY

A coumarin-based fluorescent probe containing a thiol-reacting maleimide
group was synthesized. A cysteine containing tetrapeptide Arg-Gly-Asp-Cys was
successfully labelled with the coumarin probe. In addition, an antibody was labelled
with the coumarin probe via the selenol-promoted reduction of the native disulfide
bond. Besides that, one-pot synthesis of maleimido-carboxylic N-hydroxysuccinimide
(NHS) esters were accomplished by cyclized maleamic acids with NHS and
DCC.……………………………………………………………….

O ON
N
H
O
O
O
H
N
N
O
O
O
fluorophore
linker
activating
unit
Biomolecule
HS

Tetrahydrolipstatin (THL) is an FDA-approved anti-obesity drug that
selectively inhibits gastrointestinal lipases and fatty acid synthases (FAS). Our study
focuses on the synthesis of alkyne-modified THL analogs for identification of new
cellular targets. Terminal alkyne group was introduced at the left or right side chains,
and also at the amino ester group of THL. The β-lactone moiety of THL analogs was
synthesized by ZnCl
2
-mediated tandem Mukaiyama aldol-lactonization (TMAL). The



vii
γ-hydroxy group was introduced via Keck allylation or resolution of the homoallylic
alcohol as its (R)-O-acetylmendelic ester.
O O
O
C
6
H
13
O
OHCHN
THL
O
C
6
H
13
O
OO

OHCHN R
8
TMAL
Mitsunobu
Keck asymmetric allylation
or resolution

Three THL probes, THL-L, THL-R and THL-T were tested for potential
biological activities. All three compounds showed antiproliferation activity against
HepG2 cells. Besides the known FAS, eight new proteins (GAPDH, β-tubulin,
Hsp90AB1, ANXA2, RPL7a, RPL14, RPS9 and an unnamed protein) were identified
through activity-based proteome profiling approach.

O
O
OO
R NHCHO
8
O
O
OO
R NHCHO
8
O
O
OO
H
N
8
O

THL-L analogs
THL-R analogs
THL-T


Another approach for the synthesis of THL analogs was developed by
introducing diversity by click chemistry. The β-lactone was reacted with eighteen
aliphatic and aromatic azides with different electronic properties under click reaction
conditions to give THL dialkyne analogs after desilylation. Four analogs (7.22k/m/r/s)



viii
were identified to possess similar anti-proliferative activity against HepG2 cells as
compared to THL.
THL dialkyne analogs
O
O
OO
OHCHN
N
N
N
R
structural diversity

O
O
O
NHCHO

O
N
N
N
Br
O O
O
NHCHO
O
N
N
N
O
2
N
O O
O
NHCHO
O
N
N
N
NH
SMeO
O
O O O
O
NHCHO
O
N

N
N
NHS
O
O
7.22k
7.22m
7.22r
7.22s





ix



LIST OF TABLES



TABLE NO. TITLE PAGE

1.1 Optical properties of Keio-fluors 2
2.1 Convenient synthesis of N-alkylated
maleimides
13
2.2 One-pot preparation of maleimido-carboxylic
NHS-esters

14
2.3
1
H,
13
C, COSY and HMBC data of
fluorescence probe 2.20
17
5.1 Heathcock-Evans anti-aldol reactions 74
5.2 Attempted TBDPS deprotection 78
5.3 Synthesis of tetrahydrolipstatin derivatives 84
5.4 Synthesis of tetrahydrolipstatin derivatives
(THL-R)
87
5.5 Synthesis of tetrahydrolipstatin derivatives 88
5.6 Proteins identified by pull down and mass
spectrometry
96
7.1 Optimization of click chemistry 154
7.2 Click chemistry 155
7.3 Synthesis of azides 158








x

LIST OF SCHEMES



SCHEME
NO.
TITLE PAGE
2.1 Attempted synthesis of maleimide 2.4
following the literature methods
11
2.2 Esterification of mixture of maleimide 2.4 12
2.3 Synthesis of coumarin-probe 2.20 16
2.4 Synthesis of mono-Boc linker 16
2.5 Synthesis of flurescent RGDC conjugate 19
4.1 Schneider’s synthesis of THL 45
4.2 Hoffmann-La Roche second generation
synthesis of THL.
45
4.3 Uskoković’s synthesis of THL 47
4.4 Kocienski’s synthesis of THL 48
4.5 Hanessian’s synthesis of THL 49
4.6 Ghosh’s asymmetric synthesis of THL 50
4.7 Mcleod’s synthesis of THL 52
4.8 Laglois’ synhesis of THL 53
4.9 Romo’s synthesis of THL 53
4.10 Hoffmann-La Roche enantiosynthesis of THL 54
4.11 Kumaraswamy’s synthesis of THL 56

4.12 Synthesis of β-hydroxy aldehyde 4.71 57
4.13 Davies’ synthesis of THL 57
4.14 Raghavan’ synthesis of THL 59
4.15 Hanson’ synthesis of THL 60
4.16 Synthesis of chiral aldehydes via Noyori
reduction
62
4.17 Cross-metathesis strategy 62
4.18 Synthesis of tetrahydrolipstatin analogues 64




xi
5.1 First-generation strategies to the synthesis of
THL analogue
71
5.2 Synthesis of S-aldehyde 5.17 73
5.3 Synthesis of (R)-imide 5.18 75
5.4 TMAL strategies in the synthesis of
tetrahydrolipstatin analogues
76
5.5 Synthesis of R-aldehyde 5.29 76
5.6 Tandem Mukaiyama aldol-lactonisation
(TMAL)
77
5.7 Proposed mechanism of TMAL 79
5.8 synthesis of unprotected alkyne 82
5.9 Synthesis of Mosher esters 82
5.10 Synthesis of β-lactone 5.51 83

5.11 Synthesis of THL-L antipode 5.58 84
5.12 Synthesis of R- and S-β-hydroxy aldehydes 85
5.13 Synthesis of thiopyridyl ketene acetal 5.68 86
5.14 TMAL (THL-R) 87
5.15 TMAL (THL-R) 88
5.16 TMAL (THL-T) 89
5.17 Synthesis of THL-T 89
5.18 Synthesis of alkyne-leucine derivatives 5.79 and
5.80
90
7.1 Diversity of left wing sidechain by click
chemistry
149
7.2 Proposed mechanism of click chemistry 150
7.3 Synthesis of antifreeze glycopeptides 151
7.4 Synthesis of dialkyne 7.20 153
7.5 Proposed mechanism of Keck asymmetric
allylation
157





xii



LIST OF FIGURES

FIGURES
NO.
TITLE PAGE

1.1 Commercially available carbocynine dyes with
reactive N-hydroxysuccinimide groups for
fluorescence labelling purposes
3
1.2 Structure of PI 3-kinase inhibitors 5
1.3 Outline of the Ser/Thr protein phosphatase
assay
7
2.1 Design of coumarin-based fluorophore 10
2.2 Selected HMBC correlations of fluorescence
probe 2.20
18
2.3 Fluorescence spectrum of coumarin 2.20
(normalised). Excitation and emission spectra
are shown by black (left) and blue (right) lines,
respectively. The spectra were taken with 10
µM of 2.20 in ethanol
18
2.4 Illustration of selenol-mediated reduction of
antibody disulfide bonds and labelling with
fluorophore 2.20
20
2.5 UV absorption spectra of coumarin, antibody,
coumarin labelled antibody and calculated

coumarin labelled antibody
21
2.6 (a) Flow cytometric analysis of CIR A2 cells
after incubation with coumarin 2.20 labeled
antibody #130. A significant detection of
fluorescence was observed on CIR A2 cells,
which express the HLA-A2 protein (blue).
CIR cells, which do not express HLA-A2,
were used as a negative control (red). (b)
Fluorescence microscopy. There was a
significant detection of fluorescence on CIR
A2 cells (bottom-left) compared to the
negative control CIR cells (top-right)
23
2.7 FACS dot plot of

abeled CIR A2 cells (top-
right segment comprises 70.16% of

abeled
cells)
23



xiii
4.1 Transition state of Ti-mediated condensation 49
4.2 Romo’s diversification strategies to
tetrahydrolipstatin derivatives
61

5.1 Synthesis of THL-L, THL-R, THL-T and THL
analogues
70
5.2 Initial strategies for THL derivatives synthesis 71
5.3 Non-bonding interactions in the transition state
of (E)-ketene acetal 5.41 and 5.42
80
5.4 Non-bonding interactions in the transition state
of (Z)-ketene acetal 5.43 and 5.44
80
5.5 1,3-asymmetric induction model for TMAL 81
5.6 Δδ (δs-δ
R
) data for the S- and R-MTPA Mosher
esters 5.50S and 5.50R
83
5.7 Overall strategy for activity-based proteome
profiling of potential cellular targets of THL
91
5.8 93
5.9 94
7.1 Corey’s stereochemical model 157
7.2 161





xiv

LIST OF ABBREVIATIONS



2-AG 2-arachidoglycerol
AMCA 7-amino-4-methyl-3-coumaric acid
BAIB bis(acetoxy)iodobenzene
BDP boron-dipyrromethane
BINOL 1,1’-bi-2,2’-naphthol
BIPHEP biphenylphosphine
BOPCl bis(2-oxo-3-oxazolidinyl)phosphinic chloride
br broad
calcd calculated
CI chemical ionisation
COSY correlated spectroscopy
DACA 7-dimethylaminocoumaric acid
DAGLα diacylglycerol lipase α
DBPO di-tert-butylperoxyoxalate
d doublet
dd doublet of doublets
DEPT distortionless enhancement of polarisation transfer
DIC 2-diisopropylaminoethyl chloride hydrochloride
DIEA diisopropylethylamine
DMAP 4-(N,N-dimethylamino)pyridine



xv

DMC N,N-dimethyl-7-aminocoumarin
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
dr diastereomeric ratio
ee enantiomeric excess
ESI electrospray ionization
HMBC heteronuclear multiple bond correlation
IAF immunoaffinity-fluorescent
IBX 2-iodoxybenzoic acid
Im imidazole
i-Pr isopropyl
IR infrared
J coupling constant
m multiplet
m/z mass-to-charge ratio
MS mass spectrometry
MTPA α-methoxy-α-(trifluoromethyl)phenylacetic acid
o-NBSH o-nitrobenzenesulfonyl hydrazide
NIR near infrared
NMM N-methylmorpholine
NMR nuclear magnetic resonance
PPTS pyridinium 4-toluenesulphonate
py pyridine
q quartet
rt room temperature



xvi
s singlet

sext sextet
TBAF tetrabutylammonium fluoride
TBDPS t-butyldiphenylsilyl
TBS tert-butyldimethylsilyl
TFA trifluoroacetic acid
THF tetrahydrofuran
THL tetrahydrolipstatin
TLC thin-layer chromatography
TMAL tandem Mukaiyama aldol-lactonization
Ts 4-toluenesulphonyl
t triplet
tt triplet of triplets
VLC vacuum liquid chromatography
δ chemical shift



xvii



LIST OF APPENDICES



APPENDIX TITLE PAGE
Spectral data for Chapter 2 191
Spectral data for Chapter 5 227
Spectral data for Chapter 7 333

xviii



LIST OF PUBLICATIONS

1. Song, H. Y.; Ngai, M. H.; Song, Z. Y.; MacAry, P. A.; Hobley, J.; Lear, M. J.
Practical synthesis of maleimides and coumarin-linked probes for protein and
antibody labelling via reduction of native disulfides. Org. Biomol. Chem., 2009, 7,
3400-3406. (featured with inside cover-page)

2. Yang, P Y.; Liu, K.; Ngai, M. H.; Lear, M. J.; Wenk, M. R.; Yao, S. Q. Activity-
based proteome profiling of potential cellular targets of orlistat − an FDA-approved
drug with anti-tumor activities. J. Am. Chem. Soc. 2010, 132, 656-666.

3. Ngai, M. H.; Yang, P Y.; Liu, K.; Shen, Y.; Wenk, M. R.; Yao, S. Q.; Lear, M. J.
Click-based synthesis and proteomic profiling of lipstatin analogues. Chem. Commun.
2010, 46, 8335-8337 (featured with front-cover)

Conferences:

1. Ngai, M. H.; Song, H. Y.; Lear, M. J. Development of coumarin-based fluorescent
bioimaging agent, Poster presented at 9
th
International Symposium for Chinese
Organic Chemists (ISCOC-9), Singapore, 18-20 December 2006.




xix
2. Ngai, M. H.; Song, H. Y.; Song, Z. Y.; MacAry, P. A.; Lear, M. J. Development of
a coumarin-based maleimide reagent for the labeling of peptides and antibodies,

Poster presented at 10
th
Tetrahedron Symposium, Paris, France, 23 – 26 June
2009.

3. Ngai, M. H.; Yang, P Y. Liu, K.; Yao, S. Q.; Lear, M. J. Analogue total synthesis
and biological study of tetrahydrolipstatin-based probes, Poster presented at 6
th
Singapore International Chemical Conference (SICC-6), Singapore, 15-18
December 2009.





Design and Synthesis of Coumarin-Based
Fluorescent Probe



CHAPTER 1



INTRODUCTION



Optical imaging is an important technique in bioimaging because of its high
sensitivity and convenience without the use of ionizing radiation, as well as providing

a facile method for studying diseases at the cellular and molecular level.
1
A variety of
different probes for the optical detection of diseases have been synthesized and
characterized for their potential to engender disease-specific optical signals within
cells and tissues. Presently, cyanine dyes, fluorescein, oxazine dyes and coumarin
dyes are widely used as molecular probes for bioimaging purposes. For this chapter,
we shall briefly highlight some typical and emerging imaging modalities relevant to
our studies.

The optimal properties of fluorescence probes should include:
2
(1) A high absorption coefficient and high quantum yield;
(2) Chemical and photochemical stability;
(3) Minimal chemical and phototoxicity to cells;
(4) High water solubility.




2
Suzuki et al. designed and synthesized a series of boron-dipyrromethane
(BDP) dyes dubbed Keio-fluors (1.1-1.4). These display useful optical performance
such as high quantum yields (up to 0.98), high extinction coefficients (up to 288 000
M
-1
cm
-1
), high photostabilities, and sharp fluorescent spectra bands as compared to
quantum dots (Table 1.1). With these good properties, Keio-fluors have the potential

to replace or complement existing, commercially available fluorescent dyes as
bioimaging agents.
3

Table 1.1: Optical properties of Keio-fluors.
N
B
N
OO
R
1
R
1
F F
R
2
Keio-Fluors
1.1
KFL-1 : R
1
=Me,R
2
=H
1.2 KFL-2 : R
1
=Me,R
2
=CF
3
1.3 KFL-3 : R

1
=4-MeO-Ph,R
2
=H
1.4
KFL-4 : R
1
=4-MeO-Ph,R
2
=CF
3

dye λ
abs
(nm) λ
flu
(nm) ε (M
-1
cm
-1
) ϕ
KFL-1 579 583 202 000 0.96
KFL-2 613 620 185 000 0.98
KFL-3 673 683 288 000 0.86
KFL-4 723 738 253 000 0.56

Near-infrared (NIR, 700-850 nm) fluorophores offer several advantages over
visible or UV fluorophores. NIR probes have better tissue penetration (>500 μm to
cm), less autofluorescence, and large Stokes shifts.
4

The most common organic NIR
fluorophores are cyanine dyes. Among this class of compounds, the CyDye series
(Cy3, Cy5, Cy5.5, Cy7, 1.5-1.8) are commercially available.




3
N N
-
O
3
S SO
3
-
Na
+
O
O
N
O
O
n
N N
O
O
N
O
O
O

O
N
O
O
-
O
3
S
SO
3
-
SO
3
-
SO
3
-
3K
+
1.5
n=1:Cy3
1.6
n=2:Cy5
1.7 n=3:Cy7
1.8 Cy5.5

Figure 1.1: Commercially available carbocyanine dyes with reactive N-
hydroxysuccinimide groups for fluorescence labelling purposes. Cy3 (absorption
maximum 550 nm, fluorescence maximum 570 nm), Cy5 (650 nm/670 nm), Cy7 (743
nm/767 nm) and Cy5.5 (675 nm/694 nm).

The general method to improve the accumulation of contrast agents at the
target site is to conjugate the fluorophore to a ligand that binds to a specific molecular
target. Ligands can be small molecules, peptides, proteins and antibodies. High
selectivity and affinity of receptor ligands result in a high signal to noise ratio, while
enabling low (picomolar and nanomolar) concentrations of the compounds.
1


Masotti and co-workers synthesized a NIR indocyanine dye-polyethylenimine
(PEI) (IR820-PEI) conjugate for DNA delivery imaging in vivo. The conjugate has
high chemical stability and a large Stokes shift (115 nm). IR820-PEI is able to form
complexes with DNA, and the delivery process can be monitored in vivo with non-
invasive optical imaging techniques.
4





4
N N
SO
3
-
SO
3
-
Na
+

HN
PEIO
N
N
NH
2
NH
NH
N
NH
H
N
PEI (25 kDa)
IR820-PEI (1.9)

Tung and co-workers synthesized a NIR folate receptor (FR) imaging probe
for detection of FR-positive cancers. The probe consists of a NIR fluorophore and
folic acid.
5


N N
SO
3
-
K
+
SO
3
-

KO
3
S N
H
O
O
N
H
O
2
N
H
O
N
H
2
N
N
N
NH
O
NH
2
NIR2-folate (
1.10
)

Cheng and co-workers reported the design of oligomeric arginine-glycine-
aspartic acid (RGD) peptides for imaging the α
v

β
3
integrin receptor in xenografted
tumors in vivo. They synthesized monomeric, dimeric and tetrameric cyclic RGD
units and conjugated them to Cy5.5. The Cy5.5-RGD tetramer displayed the highest
tumor uptake and tumor-to-background fluorescence ratio.
6

Since coumarin was first reported and isolated in the 1820s, more than 1000
derivatives have been developed from naturally existing coumarin derivatives.
7

Coumarins have been long utilized as fluorescence probes in molecular imaging.