Developing new fluorophores for applications in protease detection and protein labeling

DEVELOPING NEW FLUOROPHORES FOR APPLICATIONS IN
PROTEASE DETECTION AND PROTEIN LABELING

LI JUNQI

NATIONAL UNIVERSITY OF SINGAPORE
2010


DEVELOPING NEW FLUOROPHORES FOR APPLICATIONS IN
PROTEASE DETECTION AND PROTEIN LABELING

LI JUNQI

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2010


ACKNOWLEGEMENTS

This thesis is not the result of a sole experimenter working in isolation, but the
culmination of efforts of all who have supported the individual in her search for
greater knowledge. The journey as a graduate student in NUS may have ended, but it
is the beginning of a path leading to a boundless world of scientific pursuits. My
utmost gratitute to the following people who have made it possible:

Prof Yao Shao Qin – supervisor, mentor, teacher and a friend in need – has
been instrumental in shaping my development both as a scientist and as an individual.

The years spent under his tutelage have had the most profound impact on my life as a
student of science. It is with his enthusiasm and insight in scientific research, as well
as confidence in my abilities that have led me to my accomplishments.

My parents and my brother have been a silent pillar of support, showing their
care and concern in their own ways even when I hardly spent time with them
throughout the course of this degree. I can only reciprocate their love by dedicating to
them every small accomplishment I make, including this thesis.

The members of the Yao Lab, both past and present, have guided and
accompanied me throughout these years. I thank particularly the following people:
Jinzhan, Souvik and Candy for being great friends who shared my frustrations;
Mingyu and Jingyan who have been great companions in the lab; and Mahesh and
Wang Jun who have mentored me when I was learning the ropes of research.

i


I thank my old pals Aileen, Ke Ming and Zhiying who have not forgotten me
during the time I disappeared into the lab. It is certainly comforting to know that our
friendship has weathered these years.

Last but not least, I thank National University of Singapore for funding my
studies through the research scholarship, and the President’s Graduate Fellowship.

ii


TABLE OF CONTENTS

Acknowledgements

i

Table of Contents

iii

List of Figures

vii

List of Schemes

ix

List of Tables

x

Index of Abbreviations

xi

List of Amino Acids

xiv

List of Publications

xv

Abstract

xvii

Chapter 1 INTRODUCTION
1.1 Detecting Enzyme Activity

1

1.2 Small Molecule-Based Fluorogenic Enzyme Substrates

Chapter 2

1.2.1

FRET and Internally Quenched Substrates

3

1.2.2

Fluorophore Release after Enzymatic Cleavage

6

1.2.3

Fluoromorphic Probes

11

1.2.4

Fluorescence Detection of Binding Events

12

DEVELOPING A NEW FLUOROGENIC PROBE FOR
PROTEASE ACTIVITY

2.1 Fluorogenic Protease Substrates for Detecting Protease Activity

15

on the Microarray and in Live Cells
2.2 Design of a New Fluorophore for Microarray and Bioimaging

iii

18


Applications
2.3 Chemical Synthesis of SG and SG-Conjugated Peptides

20

2.4 Profiling Protease Activity on the Microarray

32

2.5 Imaging Caspase-3 and -7 Activities in Live Cells

38

2.6 Conclusions

39

Chapter 3 FLUOROGENIC PROBES FOR DETECTING
PROTEASE ACTIVITY AT SUBCELLULAR
LOCATIONS
3.1 Targeted Delivery of Molecules into Intracellular Locations

41

3.2 Design of Cell-Permeable Protease Substrates Targeting

46

Different Organelles
3.3 Chemical Synthesis of Peptide Substrates and Localization

51

Peptides
3.4 Bioimaging of Control Peptides

61

3.5 Current Work

65

Chapter 4 DISCOVERY AND DEVELOPMENT OF
FLUOROGENIC LABELS FOR BIOMOLECULES
4.1 Fluorogenic Labeling of Biomolecules

69

4.2 Combinatorial Discovery of Fluorophores

74

4.3 Design of Xanthone- and Xanthene-Based Fluorophores

76

4.4 Chemical Synthesis of Xanthone- and Xanthene-based “Click”

77

Fluorophores
4.5 Spectroscopic Analysis of the “Click” Fluorophore Library

iv

87

4.6 Conclusions

97

Chapter 5 EXPERIMENTAL SECTION
5.1 General Information

98

5.2 Solution-Phase Synthesis of Fluorophores, Linkers and Azides

99

5.2.1

Synthesis of SG1 and SG2 and related derivatives

5.2.2

Synthesis of alkynes A – F

111

5.2.3

Synthesis of Linkers

123

5.2.4

Synthesis of Azides

128

5.3 Solid-Phase Synthesis of Peptides and SG-Peptide Conjugates

99

132

5.3.1

General Information

128

5.3.2

General Procedures

128

5.3.3

Synthesis of Ac-DEVD-SG1

129

5.3.4

Synthesis of SG2-Peptide Conjugates

130

5.3.5

Synthesis of Alkyne-Functionalized SG2-Based

132

Substrates
5.3.6

Synthesis of Azido-Peptides and Control Peptides

133

5.4 Synthesis of Fluorophores Using “Click” chemistry

135

5.5 Spectroscopic Analysis

140

5.5.1

General Information

140

5.5.2

Determination of Molar Extinction Coefficients and

140

Quantum Yields
5.6 Microplate-Based Fluorescence Assays

141

5.6.1

General Information

141

5.6.2

Enzymatic Assays with SG-Peptide Conjugates

142

v

5.6.3

Fluorescence Analysis of “Click” Fluorophores

142

5.7 Microarray Experiments

143

5.8 Bioimaging

145

5.8.1

General Information

145

5.8.2

Detecting Caspase-3 and -7 Activity in Live HeLa

146

Cells
5.8.3

Evaluating the subcellular locations of the localization

146

peptides

148

Chapter 7 REFERENCES

vi


LIST OF FIGURES

Figure

Page

1.1

Enzyme assays with fluorescence detection methods

3

2.1a

Protease and protease substrate nomenclature

16

2.1b

2 common types of synthetic peptide substrates

16

2.2

Structures of common fluorophores used in fluorogenic peptide

19

substrates
2.3

Resonance stabilization of phenolate anion resulting from TBS

21

deprotection
2.4

The 2 major resonance structures of the asymmetric xanthene

21

2.5

Formation of the undesired N-acylurea from Fmoc-Asp-SG1

24

and DIC
2.6

LC-MS profile of Ac-DEVD-SG1

25

2.7

LC-MS profiles of the 10 SG2-peptide conjugates

29

2.8a

Fluorescence spectra of SG1

33

2.8b

Fluorescence increase from cleavage of Ac-DEVD-SG1

33

2.9

Detecting protease activity on the microarray

34

2.10a

Enzyme “fingerprints” obtained

36

2.10b

Time-dependent kinetic profiles from microarray

36

2.11

Selected kinetic data from microplate and microarray

37

enzymatic assays
2.12

Detecting caspase-3/-7 activity in live HeLa cells with Ac-

39

DEVD-SG1
3.1

Overall strategy for imaging protease activity in subcellular

vii

47


organelles
3.2

Acylation of resin-bound secondary amine by Fmoc-SG2-

52

COOH and possible side reaction
3.3

General structures and LC-MS profiles of desired peptides and

54

side products
3.4

LC-MS profiles of azido-localization peptides and control

57

peptides

3.5

Fluorescent images of control peptides and corresponding

63

organelle stains
4.1

Fluorophore types which have been synthesized using “click”

75

chemistry
4.2

Design of xanthone- and xanthene-based “click” fluorophores

77

4.3

Undesired products obtained during the nucleophilic aromatic

79

substitution of 2b and 1ii with different nucleophiles
4.4

Structures of azides used in this study

81

4.5

Selected LC-MS profiles of “click” fluorophores

83

4.6

Emission spectra of selected fluorophores from microplate-

89

based fluorescence screening
4.7

Heat map showing fluorescence intensities of each “click”

91

product
4.8

Structures of fluorophores selected for quantitative fluorescence

93

analysis

4.9

Excitation and emission spectra of “hit” fluoropohores and their
corresponding alkynes

viii

94


LIST OF SCHEMES

Scheme

Page

2.1

Initial proposed synthesis of SG

20

2.2

Synthesis of SG1 and SG2

22

2.3

Derivatization of SG1 and solid-phase synthesis of Ac-DEVD-

25

SG1
2.4

Synthesis of Fmoc-SG2-CHO for peptide synthesis

26

2.5

Solid-phase synthesis of aldehyde-functionalized SG2-peptide

28

conjugates
2.6

Functionalization of glass slides with alkyoxyamines

35

3.1

Synthesis of Fmoc-SG2-COOH (3-1) and Fmoc-SG2-COCl (3-

51

2)
3.2

Solid-phase synthesis of alkyne-functionalized substrates, Ac-

53

X-SG2-alkyne
4.1

General synthetic strategy towards alkynes A, B, D and E

78

4.2

Synthesis of 4-2a and 4-2b from 4-1

78

4.3

Synthesis of alkynes C and F

80

4.4

Synthesis of aromatic azides from anilines

81

4.5

“Click” assembly of fluorophores

82

5.1

Synthesis of linker 2-12 used in the preparation of SG2

124

5.2

Synthesis of azide z15

127

ix


LIST OF TABLES

Table

Page

2.1

Peptide sequences synthesized and their target proteases

35

3.1

Alkyne-functionalized SG2-based substrates and their target

49

enzymes
3.2

Azide-functionalized localization peptides selected and their

50

target organelles
4.1

λex and λem for each “click” fluorophores

88

4.2

Summary of spectroscopic properties of “hit” fluorophores

93

5.1

Reagent concentrations and volumes used per “click” reaction

135

5.2

Volumes of solvents used for scale-up “click” chemistry

136

5.3

Concentrations and buffers for proteases used in microarray

144

experiments

x


INDEX OF ABBREVIATIONS

ABP

Activity-based probe

ACC

7-Aminocoumarin-4-acetic acid

AMC

7-Amino-4-methylcoumarin

aq.

Aqueous

Boc

t-Butoxy carbonyl

br

Broad

CPP

Cell-penetrating peptide

dd

Doublet of doublets

DIC

N,N′-Diisopropylcarbodiimide (as a reagent) / Differential interference
contrast (in bioimaging)

DIEA

N,N′-Diisopropylethylamine

DCE

1,2-Dichloroethane

DCM

Dichloromethane

DMAP

4-Dimethylaminopyridine

DMF

Dimethylformamide

DMP

Dess-Martin Periodinane

EA

Ethyl acetate

EDT

1,2-ethanedithiol

equiv

Equivalent

ESI

Electron spray ionization

Et

Ethyl

EtOH

Ethanol

FLIP

Fluorescence loss in photobleaching

xi


Fmoc

9-Fluorenylmethoxycarbonyl

FP

Fluorescent protein

FRAP

Fluorescence recovery after photobleaching

g

Gram

GFP

Green fluorescent protein

HeLa

Human cervical adenocarcinoma

HBTU

2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetrauroniumhexafluorophosphate

HOBt

N-hydroxybenzotriazole

Hz

Hertz

h

Hours

λem

Wavelength of excitation maximum

λex

Wavelength of emission maximum

LC-MS

Liquid chromatography-mass spectrometry

M

Molar

MeOH

Methanol

m

Multiplet

mg

Milligram

min

Minute

mM

Millimolar

µM

Micromolar

mmol

Millimole

MMP

Matrix metalloproteases

NLS

Nuclear localization sequences

NMR

Nuclear magnetic resonance

nM

Nanomolar

xii


OTf

Trifluoromethane sulfonyl / Triflate

OTs

p-Toluenesulfonyl / Tosylate

PDC

Pyridinium dichromate

Ph

Phenyl

PL-FMP

Polystyrene – 4-formyl-3-methoxyphenoxy resin

ppm

Parts per million

PTD

Protein transduction domain

PyBrOP

Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate

q

Quartet

RFP

Red fluorescent protein

SG

Singapore Green

SMM

Small molecule microarray

s

Singlet

sat.

Saturated

SV40

Simian virus 40

t

Triplet

TBS/TBDMS tert-Butyldimethylsilyl
tBuOH

tert-Butyl alcohol

TFA

Trifluoroacetic acid

THF

Tetrahydrofuran

TIS

Triisopropylsilane

TLC

Thin layer chromatography

TMS

trimethylsilyl

UV

Ultraviolet

xiii


LIST OF AMINO ACIDS

One Letter

Three Letter

Amino Acid

A

Ala

Alanine

C

Cys

Cysteine

D

Asp

Aspartic acid

E

Glu

Glutamic acid

F

Phe

Phenylalanine

G

Gly

Glycine

H

His

Histidine

I

Ile

Isoleucine

K

Lys

Lysine

L

Leu

Leucine

M

Met

Methionine

N

Asn

Asparagine

P

Pro

Proline

Q

Gln

Glutamine

R

Arg

Arginine

S

Ser

Serine

T

Thr

Threonine

V

Val

Valine

W

Trp

Tryptophan

Y

Tyr

Tyrosine

r

D-Arg

D-Arginine

Fx

-

Cyclohexylalanine

xiv


LIST OF PUBLICATIONS

1. Li, J.; Hu, M.; Yao, S. Q. Rapid synthesis, screening and identification of
xanthone- and xanthene-based fluorophores using click chemistry. Org. Lett. 2009,
11, 3008-3011.

2. Li, J.; Yao, S. Q. “Singapore Green” – a new fluorescent dye for microarray and
bioimaging applications. Org. Lett. 2009, 11, 405-408.

3. Hu, M.; Li, J.; Yao, S. Q. In situ “click” assembly of small molecule matrix
metalloprotease inhibitors containing zinc-chelating groups. Org. Lett. 2008, 10,
5529-5539

4. Uttamchandani, M.; Li, J.; Sun, H.; Yao, S. Q. Activity-based profiling: new
developments and directions in protein fingerprinting. Chembiochem 2008, 9,
667-675

5. Srinivasan, R.; Li, J.; Ng, S. L.; Kalesh, K. A.; Yao, S. Q. Methods of using click
chemistry in the discovery of enzyme inhibitors. Nat. Protocols 2007, 2, 26652664.

6. Lee, W. L.; Li, J.; Uttamchandani, M.; Sun, H.; Yao, S. Q. Inhibitor fingerprinting
of metalloproteases using microplate and microarray platforms – an enabling
technology in Catalomics. Nat. Protocols 2007, 2, 2126-2138.

xv


7. Uttamchandani, M.; Wang, J.; Li, J.; Hu, M.; Sun, H.; Chen, K. Y.-T.; Liu, K.;
Yao, S. Q. Inhibitor fingerprinting of matrix metalloproteases using a
combinatorial peptide hydroxamate library. J. Am. Chem. Soc. 2007, 129, 1311013117.

8. Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S. Q. “Click” synthesis of
small molecule probes for activity-based fingerprinting of matrix metalloproteases.
Chem. Commun. 2006, 3783-3785

9. Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S. Q. Rapid assembly of matrix
metalloproteases (MMP) inhibitors using click chemistry. Org. Lett. 2006, 8,
3821-3824

xvi


ABSTRACT

The design and synthesis of a new bi-functional fluorophore with emission
and excitation wavelengths similar to fluorescein, and the utility of the fluorophore in
microarray and bioimaging applications are described herein. We demonstrate the
compatilibity of the fluorophore to solid-phase peptide synthesis for the assembly of
various fluorophore-peptide conjugates which are used fluorogenic substrates for
detecting protease activity on the microarray and in live cells. With the objective of

expanding the bioimaging applications of the fluorophore to detecting protease
activity in specific organelles, we synthesized, via solid phase synthesis, peptide
conjugates functionalized with an alkyne which can be attached to cellular
localization sequences via “click chemistry”. The use of a single fluorophore for these
applications obviates the need for re-designing and synthetic evaluation of peptide
conjugates for potetntial substrate profiling on the microarray and the live-cell
imaging of enzyme activity separately.

Based on the scaffold of our new fluorophore, we designed and synthesized a
panel of new fluorophores with emission wavelengths from blue to yellow region by
the “click” reaction of alkyne-functionalized xanthones and xanthenes with various
azides. Screening of these fluorophores led to the identification of “hit” fluorophores
which showed a fluorescence increase upon triazole formation. These “click”activated fluorogenic dyes could potentially be used for bioconjugation and
bioimaging purposes.

xvii


CHAPTER 1 INTRODUCTION

1.1

Detecting Enzyme Activity

Enzymes – macromolecular catalysts in biological reactions – are the life force
of the cell, providing it with energy and function. Numerous pathological conditions
are caused by aberrant enzymatic activity, leading researchers to seek the “magic
bullet” for the specific inhibition or activation for each disease-associated enzyme [1].
These enzymes constitute more than twenty percent of the drug targets [2],
underscoring the importance of finding small molecule modulators with either the aim

of gaining a fundamental understanding of enzyme function or with the ultimate
purpose of drug discovery. The development of enabling tools that could
quantitatively assess the efficacy of these modulators in a reliable fashion is thus of
tantamount importance. In vitro assays for various classes of enzymes have evolved
from the labor-intensive, use of liquid chromatography and radio-labeled enzyme
substrates to operationally simple methods allowing high-throughput and image-based
analysis. In vivo tracking of enzymatic activity has advanced rapidly from the
landmark discovery and applications of the green fluorescent protein (GFP), a
milestone development in molecular biology that was awarded the Nobel Prize in
2008.

Assays employing fluorescence detection methods have seen widespread use
in both the academics and the industry. The appeal of fluorescence methods stems
from their compatibility in both in vivo and in vitro settings, as well as their suitability
for both quantitative analyses for real-time monitoring of enzyme kinetics and for

1


visual tracking of enzymatic activity. The proven utility of these assays has driven
active research in designing and/or modifying fluorescent proteins, inorganic
nanoparticles and small molecule organic fluorophores for use in these assays.
Enzyme assays with fluorescence-based detection methods are based on a common
principle – the synthetic substrate containing a fluorophore or pro-fluorophore is acted
upon by the enzyme which results in a significant change in the fluorescence property
of the substrate. This change could be achieved with the following mechanisms: 1)
fluorescence resonance energy transfer (FRET) between a donor and acceptor
fluorophore and other fluorophore-fluorophore interactions leading to quenching; 2) a
fluorogenic dye which displays no or low fluorescence until enzymatic action on the
substrate; and 3) the use of a metal sensitive-fluorogenic dye which is fluorescent

only when chelated to metals, or an environment-sensitive fluorophore which display
different spectral properties in different media (Figure 1.1). A formidable arsenal of
organic fluorophores that display fluorescence changes through these mechanisms has
been developed.

Coupled with their amenability to structural changes through

chemical synthesis, organic fluorophores now constitute an important component of
the fluorescent toolbox. Their versatility has led to the development of synthetic
substrates for enzymes that are not readily assayed using genetically encoded
biosensors assembled from fluorescent proteins. The following section surveys the
strategies in designing small molecule-based fluorogenic substrates for detecting
enzyme activity.

2


a)

b)

c)

Figure 1.1. Enzyme assays with fluorescence detection methods. a) In FRET substrates,
fluorescence emission is observed from the acceptor fluorophore (red) when excited at the
donor excitation wavelength until enzymatic cleavage of the substrate separates the donor
and acceptor. Thereafter, emission is observed at the donor emission wavelength. b) the
fluorogenic substrate is not fluorescent with the enzyme recognition head is attached. Upon
enzymatic cleavage which removes the recognition head, fluorescence is restored. c) Addition
of a phosphate group to the substrate by a kinase allows chelation of a metal ion by the

fluorophore and phosphate group. The fluorescence is enhanced by the chelation event.

3


1.2

Small Molecule-Based Fluorogenic Enzyme Substrates

1.2.1 FRET and internally quenched substrates

These fluorogenic substrates have fluorophores that are quenched by the
interaction with an adjacent fluorophore or a fluorescently silent acceptor. While both
types of interactions result in the decrease of the parent fluorophore, quenching and
fluorescence resonance energy transfer are mechanistically distinct [4]. Quenching
arises from the interaction of the electron cloud of the fluorophore and the quencher,
and since molecular contact falls off rapidly with distance, most quenching
mechanisms are operative only at short distances. This phenomenon was utilized in
the design of synthetic graft polymers for selective tumor imaging by the Weissleder
group [5]. The polymer consists of poly-L-lysine, which contains Cy5.5 (a nearinfrared cyanine dye) conjugated to some of the lysine residues, with the remaining
residues either bearing free amines or protected with methoxypolyethylene glycol. In
the intact polymer, the cyanine dyes are held in close proximity relative to each other
and are quenched. The biocompatible polymer is known to accumulate in tumor cells
and is internalized by fluid-phase endocytosis. Following endocytosis, endosomal
proteases such as the cathepsins which are upregulated in tumor cells rapidly cleave
the polymer by virtue of enzymatic recognition of the free lysine residues. Upon
cleavage, the polymer backbone disintegrates and the Cy5.5 dyes are separated
spatially. The static quenching is disengaged and the tumors are illuminated with the
resultant fluorescence. This enzyme-responsive, selective tumor imaging probe was
also successful in the in vivo imaging of matrix metalloprotease 2 (MMP2) - secreting

tumor cells by modification of the polymer side chain to include an MMP2 substrate

4


[6]. More significantly, the fluorogenic polymer was used to assess the in vivo MMP
inhibition of known inhibitors by directly detecting MMP activity in tumors. The
work by Weissleder and co-workers is considered an important advance in clinical
molecular imaging and set the stage for developing similar imaging strategies and
techniques targeting other enzymes.

In contrast to quenching, fluorescence resonance energy transfer (FRET) is a
result of long range dipole-dipole interaction between the donor and acceptor,
resulting in the excess energy from the excited donor fluorophore being transferred to
an acceptor in the ground state without emission of a photon during the transfer. The
transfer efficiency is dependent on the distance between the donor and acceptor, the
extent of overlap of the donor emission spectrum and the acceptor absorption
spectrum, and the relative orientation between the donor and acceptor. FRET is
usually efficient up to 100 Å between the donor and acceptor. The acceptor may or
may not be fluorescent. The use of a fluorescent acceptor results in a construct that
absorbs at the donor excitation wavelength and emits at the acceptor wavelength when
the two fluorophores are in close proximity, enabling a ratiometric fluorescence
response to the distance separating the fluorophores. While enzyme substrates
utilizing fluorescent donors and acceptors are typically not termed as fluorogenic
substrates, enzymatic action does result in a fluorescence change in both the donor
and acceptor emission wavelengths. If a non-fluorescent acceptor is used (“dark
quencher”), the substrate is optically silent until an enzymatic event causes the
departure of the quencher from the fluorophore, giving rise to a fluorescence increase.
This class of substrates have emerged to become the most widely used and versatile in
design among the different classes of enzymatic substrates used.

5


The first FRET substrate which was developed by Matayoshi and co-workers
targeted the human immunodeficiency virus-1 (HIV-1) protease [7]. The FRET
substrate, (DABCYL)-SQNYPIVQ-(EDANS), contains the 8-amino acid peptide
sequence that is known to be cleaved by the HIV-1 protease, and a fluorophore
EDANS which is quenched by the dark quencher DABCYL. Upon cleavage by the
protease, the fluorophore is separated from the quencher, providing a direct read-out
of enzymatic activity which could be monitored in a real-time fashion. This seminal
work establishes a general design of fluorogenic substrates for other proteases, many
of them are commercially available.

Recent developments have focused on the use of FRET for the design of nonpeptidic, small molecule-based substrates. One of the first small molecule-based
FRET substrate was designed for β-lactamases by Tsien and co-workers, with the aim
of using enzymatically-amplified fluorescence readout for gene expression [8].
Mammalian cells which were stably transfected with the TEM-1 β-lactamase gene
regulated by a promoter rapidly gave blue fluorescence from the β-lactamasecatalyzed hydrolysis of the FRET substrate when the promoter was added which led
to upregulated gene expression. It was found that the fluorescence intensities
correlated well with the number of β-lactamases expressed per cell, which could
enable quantification of the readout. The group also showed that this β-lactamase
reporter system could also be used for flow cytometry in engineering cell lines with
targeted patterns of gene expression, and for screening drug candidates which affect
gene expression.

6