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

Design and synthesis of coumarin-based fluorescent probe2.1 Development of a coumarin-based fluorescent probe 9 2.2 Practical synthesis of N-alkylated maleimides and maleimidocarboxylic

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

To my parents

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

Design and synthesis of coumarin-based fluorescent probe

2.1 Development of a coumarin-based fluorescent probe

9 2.2 Practical synthesis of N-alkylated

maleimides and maleimidocarboxylic esters

NHS-11

2.4 Proof-of-Principle studies with coumarin probe

18 2.4.1 Labelling RGDC with coumarin 18

2.4.2 Antibody-labelling, cell-sorting and

cell culture study

fatty acid synthase inhibitors

61

4.4 Summary of tetrahydrolipstatin syntheses 64

5.1 Development of a solid-supported strategy to

5.5.2 In situ and in vitro proteome profiling 93

5.5.3 Target identification and validation 95

6 EXPERIMENTAL 101

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

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

D C C … … …

N

N H

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

γ-hydroxy group was introduced via Keck allylation or resolution of the homoallylic

alcohol as its (R)-O-acetylmendelic ester

O

C 6 H 13

O OHCHN

THL

O

C 6 H 13

O O

O

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 O

8

O O

H N

8 O

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)

were identified to possess similar anti-proliferative activity against HepG2 cells as compared to THL

THL dialkyne analogs

O

O O

O OHCHN

N

N N R

structural diversity

O NHCHO

O

N

N N Br

O NHCHO

O

N

N N NH

O

N

N N

NH S O O

LIST OF TABLES

2.1 Convenient synthesis of N-alkylated

maleimides

13 2.2 One-pot preparation of maleimido-carboxylic

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

LIST OF SCHEMES

SCHEME

NO

2.1 Attempted synthesis of maleimide 2.4

following the literature methods

11 2.2 Esterification of mixture of maleimide 2.4 12

5.1 First-generation strategies to the synthesis of

5.12 Synthesis of R- and S-β-hydroxy aldehydes 85

5.13 Synthesis of thiopyridyl ketene acetal 5.68 86

7.5 Proposed mechanism of Keck asymmetric

allylation

157

LIST OF FIGURES

FIGURES

NO

1.1 Commercially available carbocynine dyes with

reactive N-hydroxysuccinimide groups for

fluorescence labelling purposes

3

1.3 Outline of the Ser/Thr protein phosphatase

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

4.1 Transition state of Ti-mediated condensation 49

4.2 Romo’s diversification strategies to

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

5.5 1,3-asymmetric induction model for TMAL 81

5.6 Δδ (δs-δR) 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

COSY correlated spectroscopy

DACA 7-dimethylaminocoumaric acid

DAGLα diacylglycerol lipase α

DBPO di-tert-butylperoxyoxalate

d doublet

DEPT distortionless enhancement of polarisation transfer

DIC 2-diisopropylaminoethyl chloride hydrochloride

ESI electrospray ionization

HMBC heteronuclear multiple bond correlation

MTPA α-methoxy-α-(trifluoromethyl)phenylacetic acid

o-NBSH o-nitrobenzenesulfonyl hydrazide

LIST OF APPENDICES

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 9th International Symposium for Chinese Organic Chemists (ISCOC-9), Singapore, 18-20 December 2006

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 6th

Singapore International Chemical Conference (SICC-6), Singapore, 15-18

December 2009

Design and Synthesis of Coumarin-Based

Fluorescent Probe

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

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

O O

(Cy3, Cy5, Cy5.5, Cy7, 1.5-1.8) are commercially available

N N

O O N O

O

n

O O N O

O

O O N O

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

N N

HN

PEI O

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

O

2

N H

O

N H 2

N

N N NH O

NH2

NIR2-folate (1.10)

Cheng and co-workers reported the design of oligomeric

arginine-glycine-aspartic acid (RGD) peptides for imaging the αvβ3 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

Thornberry et al have synthesized coumarin conjugated peptides for apoptosis imaging The probe consisting of 7-amino-4-methylcoumarin (AMC) as the

fluorophore and tetrapeptide Asp-Glu-Val-Asp (DEVD) as the ligand (1.11).8

N H Ac-DVED

1.11 Giner and co-workers synthesized fluorescent derivatives (1.17-1.19) of the

potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin The fluorophores

used in this study were NBD-sarcosine (1.14) and 7-dimethylamino coumarin-4-acetic acid (1.15).9

O O

H O

O

O MeO

O O

O

O O

1.13 R = OAc 1.18 R = 1.14 1.19 R = 1.15

O R

R O

Figure 1.2: Structure of PI 3-kinase inhibitors

A fluorescent derivative of phorboxazole A has been synthesized by Forsyth

and co-workers for in vivo intracellular localization and target elucidation studies.10

An abiotic N,N-dimethyl-7-aminocoumarin (DMC) was used as the label because it

analogue 1.20 was synthesized via Sonogashira coupling between the corresponding

vinyl iodide and C46 terminal alkyne

OH

N

O O

N Sonogashira

1.20

46

Ueda et al reported the synthesis of fluorescence-labeled probes based on

phyllanthurinolactone 1.21, which is a leaf-closing substance of phyllanthus urinaria

The fluorophore, 7-amino-4-methyl-3-coumaric acid (AMCA) was conjugated to the

hydroxyl group at the C6′ position at the sugar moieties of 1.21 to form the fluorescence probe 1.22 The fluorescence study showed that the target cell for 1.21 is

a motor cell.11

O O

O

O HO

HO

HO OH

1.21

O O

O

O HO

HO

HO

O

O H N O 5

Seto and co-workers developed an assay for Ser/Thr protein phosphatases

based on tripeptides incorporating a coumarin fluorophore 1.23 and 1.24.12 Figure 1.3 shows an outline of the assay Hydrolysis of the phosphate ester by a phosphatase

generates the free serine alcohol group, which induces a cyclization on the N-terminal

carbamate and releases the 7-hydroxycoumarin fluorophore

Tripeptides 1.23 and 1.24 were tested against alkaline phosphatase (ALP),

bacteriophage λ protein phosphatase (λ-PPase), and vaccinia H1 related phosphatase

(VHR) Tripeptides 1.23 and 1.24 are excellent substrates for ALP, although tripeptide 1.23 shows 6-fold preference over tripeptide 1.24 for λ-PPase Tripeptide 1.23 is also

a substrate for VHR, but tripeptide 1.24 does not react with VHR, even at high

concentrations and over extended reaction times

H N

N H

NH 2

N Me O

H N

N H

NH2N

Me O

O O

O

P O

phosphatase

H N N Me O O

O O

HO

OH O

O

H N O peptide

O N O Me

fluorescent non-fluorescent

Figure 1.3: Outline of the Ser/Thr protein phosphatase assay.

References

1 Kai, L Topics Curr Chem 2002, 222, 1

2 Ballou, B.; Ernst, L A.; Waggoner, A S Curr Med Chem 2005, 12,

5 Moon, W K.; Lin, Y.; O’Loughlin, T.; Tang, Y.; Kin, D.-E.; Weissleder, R.;

Tung, C.-H Bioconjugate Chem 2003, 14, 539-545

6 Cheng, Z.; Wu, Y.; Xiong, Z.; Gambhir, S S.; Chen, X Bioconjugate

Chem.2005, 16, 1433-1441

7 Trenor, S R.; Shultz, A R.; Love, B J.; Long, T E Chem Rev 2004, 104,

3059-3077

8 Zeng, W.; Miao, W Anti-Can Agents Med Chem 2009, 9, 986

9 Giner, J.-L.; Kehbein, K A.; Cook, J A.; Smith, M C.; Vlahos, C J.; Badwey,

J A Bioorg Med Chem 2006, 16, 2518-2521

10 Chen, J.; Ying, L.; Hansen, T M.; Engler, M M.; Lee, C S.; La Clair, J J.;

Forsyth, C J Bioorg Med Chem 2006, 16, 901-904

11 Kato, N.; Inada, M.; Sato, H.; Miyatake, R.; Kumagai, T.; Ueda, M

Tetrahedron, 2006, 62, 7307-7318

12 Xue, F.; Seto, C T Org Lett., 2010, 12, 1936-1939

CHAPTER 2

RESULTS AND DISCUSSION

2.1 Development of a coumarin-based fluorescent probe

In our study towards optical imaging and ligand targeting, we developed a thiol-reactive probe for conjugation to thiol-containing peptides and antibodies The reactive thiol group of cysteine was chosen for several reasons First, the thiol group

of cysteine is more nucleophilic than amines and generally is the most reactive functional group in a biomolecule Second, thiols are reactive at neutral pH, therefore, thiols can be conjugated to molecular imaging probes selectively in the presence of amines, which are typically only reactive above pH 8.1 Third, in the derivatization of proteins, the fluorescence-labeling of cysteine residues are not cleaved by commonly available proteases, hence their modification leaves peptide maps unaltered by mass spectrometry.2 Among thiol-reactive reagents, the maleimide and haloacetyl derivatives are most widely used in biomolecular labeling These reagents react rapidly at neutral pH Maleimides are more selective than iodoacetamides because they do not react with histidine, methionine, or thionucleotides.3,4 In addition, maleimides are more photostable as compared with iodoacetamides, which are not

photostable.1 With this in mind, we designed our optical imaging probe based on the maleimide scaffold

The design of our fluorescent probe is shown in Figure 2.1; the probe consists

of a coumarin as the fluorophore, a polyethylene glycol linker and a maleimide reactive group Our choice of 7-dimethylaminocoumarin-4-acetic acid5 as the fluorophore stems from several favourable characteristics: it possesses desirable photophysical properties, such as a large Stoke shift and visible excitation and

emission wavelength (λex= 370 nm, ε = 22,000 M-1 cm-1, λem = 459 nm, Ф = 0.1-0.4),6

lacks biological activity,7 is small8 and is water soluble;9 it does not localize within cells and can be readily removed by media washing or dialysis By further possessing immunoaffinity-fluorescent (IAF) properties, one additional facet of this fluorophore

is that it allows biomolecule (typically protein) identification and isolation by co-immunopreciptation with its own specific antibody partner.8 The PEG linker serves

as a spacer and increases the water solubility of the probe

N

N H

unit

Biomolecule HS

Figure 2.1: Design of coumarin-based fluorophore

2.2 Practical synthesis of N-alkylated maleimides and maleimidocarboxylic

NHS-esters

Maleimides are widely used in the area of bioconjugate chemistry Commercially available maleimide containing probes are expensive The reported syntheses also have shortcomings such as harsh conditions,10 the use of large amounts

of toxic reagents, and low yields.11 We decided to determine a facile method for the

synthesis of substituted maleimides and maleimido-carboxylic

N-hydroxysuccinimide (NHS) esters

We first targeted the maleimide derivative 2.4 following the literature reported

procedure (Scheme 2.1).10b Maleic anhydride was first treated with glycine in acetic acid at room temperature for 15 h, followed by reflux in acetic acid for 8 h To our

dismay, only uncyclized amide 2.3 was isolated In a second set of attempts, maleic

anhydride and glycine was refluxed in a mixture of acetic acid and toluene in the presence of a catalytic amount of acetic anhydride.12 Again, to our dismay, this reaction only resulted in an inseparable complex mixture

O

O NH

Scheme 2.1: Attempted synthesis of maleimide 2.4 following literature methods.10b,12

In order to facilitate the isolation of reaction products in the second attempts,

compounds were isolated after careful chromatography The desired methyl ester 2.5 was isolated in 59% Ring opening products of maleic anhydride (2.6-2.9) were also

detected in the esterification mixture, presumably due to the harsh and acidic conditions of the reported procedure

OMe O

MeO O

2.6

OMe O

OMe O N

OMe O O

OMe O

2.8

O

H N O

Scheme 2.2: Esterification of mixture of maleimide 2.4

Hampered by the complexity of the previous attempts, we turned our synthesis

to a stepwise approach Maleic anhydride was first converted to the maleamic acid 2.3 and then cyclized to maleimide 2.4a according to the procedure of Rich and co-

workers.11b The intermediate, maleamic acid 2.3 was synthesized by ring opening of

maleic anhydride with glycine in acetic acid at room temperature The white precipitate formed was filtered and washed with water and the residue was recrystallized from water The 1H NMR of the recrystallized product showed two singlets at 3.67 ppm and 6.08 ppm with equal intergration, and the 13C NMR showed four signals at 40.3, 135.4, 167.5, and 169.4 The 1H and 13C NMR data showed a symmetrical structure for the olefinic protons (δH 6.08, δC 135.4) Asymmetric

1H and 13C data, we concluded that maleamic acid 2.3 cyclized to maleimide 2.4a

upon heated in water during the process of recrystallization With this observation in hand, we envisaged that maleimides could be synthesized by heating its corresponding uncyclized maleamic acid in water to induced cyclization In order to investigate this proposition, maleic anhydride was treated with various linear amino acids in acetic

acid at room temperature to form the maleamic acids 2.3 (Table 2.1) The resulting

precipitate was heated in water for 30 minutes, and the reaction mixture was cooled

down to give the maleimides 2.4a-e in good yield (68% to 87%)

Table 2.1: Convenient synthesis of N-alkylated maleimides. a

2.4 2.1

O

O NH OH R

2.3

O OH O

carefully, the olefinic protons of cyclized products showed singlets at 6.01-6.04 ppm, which were upfield as compared to the signal at 7.00 ppm of the authentic maleimide

In addition the methylene protons adjacent to nitrogen group of 2.4b showed a

chemical shift at 3.00 ppm, which was upfield as compared to 3.63 ppm of the

authentic maleimide 2.10b This showed that the methylene group is adjacent to an

amine group instead of an amide group Based on these data, we conclude that

maleamic acids 2.3 were hydrolyzed to the corresponding hemi-maleate salts 2.10a-e

Due to the need of making the activated ester of 2.4, we next developed a

one-pot method to sequentially couple maleic anhydride, an amino acid and

N-hydroxylsuccinimide (NHS) to generate 2.12 (Table 2.2) Eventually we found the

following protocol to be optimal: maleic anhydride and the amino acid were reacted in DMF at room temperature for 2 hours, cooled to 0 °C, DCC and NHS were added, and then the reaction mixture was allowed to stir at room temperature overnight The solid was filtered off, and the filtrate was poured onto ice, producing a white to pale yellow

solid The solid was collected and dried to obtain the desired NHS-ester 2.12 in good

Table 2.2: One-pot preparation of maleimido-carboxylic NHS-esters.a

O

X C O

O N

O

O

0oC to rt, overnight NHS, DCC

2.1

O

O NH OH X

O OH

to excellent yield (61% to 90%) Alternatively, after pouring onto ice, the solid could

be collected by dissolving in CH2Cl2, washed and extracted, dried over Na2SO4 and

concentrated to provide 2.12 Upon a more detailed literature search, this method was

found to be developed previously.14 However, our method give higher yields and facile workup (precipitation from ice-water, instead of extraction) as compared to the reported method

2.3 Synthesis of coumarin probe

With the maleimide activated ester in hand, we focused on the synthesis of

coumarin-based probe 2.20 Coumarin 2.15 was synthesized by reacting

m-dimethylaminophenol (2.13) with ethyl acetonedicarboxylate (2.14) in the presence of

ZnCl2 in refluxing absolute ethanol; the von Pechmann reaction (Scheme 2.3).7c,15 It

was found that m-dimethylaminophenol was not stable and commercial samples

contained impurities The yield of coumarin 2.15 was improved when the starting

phenol was purified by vacuum liquid chromatography prior to use.16

The yield of formation of the coumarin 2.15 was 63%, which was higher than

the reported yield of 20%.17 The ethyl ester of coumarin 2.15 was hydrolyzed with lithium hydroxide to give 7-dimethylaminocoumaric acid (DACA, 2.16) in 91% yield

as a yellow solid DACA (2.16) was then coupled to the Boc-protected polyethyleneglycol linker 2.17 [synthesized by mono-selective Boc-protection of 1,8- diamino-3,6-dioxaoctane (2.21) with 1-ethyl-3-(3′-diethylaminopropyl)carbodiimide (EDC) and DMAP (Scheme 2.4)] to give compound 2.18 in 77% yield The Boc-

O N

O N

O N

O N

O O

O

O N

O N

O

N H

2

O

N O

O

3 9

Scheme 2.3: Synthesis of coumarin-probe 2.20 Reagents and conditions: a) 2.14 (1.1

equiv), ZnCl2 (1.2 equiv), EtOH, reflux, 16 h; b) 1M LiOH (2 equiv), THF:H2O (3:1),

0 °C to rt, 2 h; c) 2.17 (1.2 equiv), EDC (1 equiv), DMAP (0.2 equiv), CH2Cl2:DMF,

0 °C to rt , 12 h; d) TFA (excess), CH2Cl2, 0 °C to rt, 30 min; e) 2.12a (2.0 equiv),

NMM (2.5 equiv), DMF, rt, 5 h

used without further purification Treatment of the TFA salt 2.19 with the activated

ester 2.12a in the presence of N-methylmorpholine (NMM) afforded the fluorescent

probe 2.20 in 88% yield over two steps

Scheme 2.4: Synthesis of mono-Boc linker Reagents and conditions: a) (Boc)2O (7.0 equiv), CH2Cl2, 0 °C to rt, 12 h

The fluorescent probe 2.20 was obtained as light yellowish oil The HRMS of 2.20 showed an [M + Na]+ peak at m/z 551.2101, which correlated to C26H32N4O8 [M + Na]+ The 1H NMR spectrum showed the presence of the 1,2,4-trisubstituted benzene ring moiety (δH 7.50, d, J = 9.5 Hz; δH6.70, dd, J = 8.8, 2.5 Hz; 6.54, d, J =

2.5 Hz) (Table 2.3) The 1H and 13C NMR spectra showed the presence of three olefinic hydrogens: δH 6.00, s, H-3 and δH 6.99, s, H-11’, H-12’; δc 126.3 (C-3) and 134.5 (C-11’ and C-12’) The singlet at δH 3.01 was assigned to the dimethylamino (NMe2) group The observed HMBC correlations from NH(2) to C-7’ and from H-9’

to C-7’ and C-10’ indicated the attachment of the maleimide group to the polyethylene glycol linker

Table 2.3: 1H, 13C, COSY and HMBC data of fluorescent probe 2.20

O O N

N H

1

3 5

(1)

(2)

Figure 2.2: Selected HMBC correlations of fluorescent probe 2.20

The fluorescent probe 2.20 gave consistent excitation-emission spectra (λex

370 nm, λem 457 nm, Figure 2.3), which was found comparable to the free

coumarin acid 2.16 in ethanol The fluorescence data shows that 2.20 is suitable

for biomolecule labelling and imaging work due to its large Stoke shift (87 nm)

Figure 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

2.4 Proof-of-principle studies with coumarin probe

2.4.1 Labelling RGDC with fluorescent probe 2.20

The αvβ3 integrin is overexpressed on the surface of tumor and endothelial

binds extracellular matrix molecules such as vitronectin, which contains the amino acid sequence Arg-Gly-Asp (RGD).19 Integrin αvβ3 imaging is important in tumor diagnosis, understanding the tumor biology of angiogenesis, and facilitates integrin targeted therapy.20b For these reasons, many molecular fluorescent probes containing the RGD motif, such as the cyanine dyes, have been developed for targeted imaging of integrin αvβ3 expression.20 We decided to synthesize a coumarin-RGDC (Arg-Gly-Asp-Cys) conjugate and test the effectiveness of a sulphur-Michael addition of the

thiol group in RGDC to the maleimide group in 2.20 The probe 2.21 was readily synthesized by stirring coumarin 2.20 and RGDC in MeOH for 1 h at room temperature (Scheme 2.5) After removal of the solvent in vacuo, the conjugate 2.21 was verified by LC-MS and HPLC The HRMS of conjugate 2.21 showed an [M +

2H]+ at m/z 979.4043, which analyzed well for C41H59N11O15S [M + 2H]+, calcd 979.4069

H CO2H

S N O

O

Fluor

2.20

2.21 R-G-D-C

Scheme 2.5: Synthesis of fluorescent RGDC conjugate Reagents and conditions: 2.20

(1.0 equiv), MeOH, rt, 1 h, quantitative

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

We also tested the application of the fluorescent probe to label an antibody (#130) specific to the human leukocyte antigen A2 (HLA-A2).21 Antibody #130 was