Development of Microplate Screening System

Home , Xanthone

DEVELOPMENT OF MICROPLATE SCREENING SYSTEM

AND CONSTRUCTION OF FLUORESCEIN-BASED

LIBRARY

FENG SUIHAN

A THESIS SUMBITTED

FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF CHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2010 Acknowledgements

First of all, I would like to express my deep gratitude to my supervisor, Associate Professor

Young-Tae Chang for his profound knowledge, invaluable guidance, constant support and inspiration throughout my graduate studies. The knowledge, both scientific and otherwise, that I accumulated under his supervision, will aid me greatly throughout my life.

Next, I would like to give my sincere thanks to Dr. Marc Vendrell and Dr. Hyung-Ho Ha, for their warm support during my entire graduate studies.

Besides, my sincere thinks goes to Miss Siqiang Yang, who helped me to set up the microplate screening platform.

Also, I would like to thank all the graduate students of Chang lab, Animesh, Duanting, Ghosh,

Jun-Seok, Yun-Kyung, Raj, for their cordiality and friendship. We had a great time together.

I also benefited from all other lab members, particularly but not limited to, Siti, Yee Ling,

Chew Yan, Xubin, Jeffrey, Shin Hui, Dr. Bi, Dr. Cho, Dr. Jun Li, Dr. Lees, Dr. Kang, Dr.

Kim, Dr. Park, and etc. Thanks for making the working place an enjoyable one.

I am grateful to Dr. Tan for recruiting me into NUS Medicinal Chemistry graduate program and I am also thankful to NUS for awarding me the research scholarship.

At last, I would like to express greatest thanks to my family, particularly my wife, Bai Yang, whose encourage and understanding help me to finish my graduate study in NUS.

Table of Contents

Chapter One Introduction 1

1.1 Chemical Genetics 1

1.1.1 Overview 1

1.2 Chemical Toolboxes 3

1.2.1 Diversity Oriented Synthesis 3

1.2.2 Tagged Library Approach 5

1.2.2.1 PNA Tagged Library 5

1.2.2.2 Click Chemistry Library Approach 6

1.2.2.3 Activity-Based Protein Profiling 8

1.2.2.4 Dynamic Combinatorial Library 9

1.3 Diversity Oriented Fluorescence Library Approach 10

1.3.1 Styryl Library 10

1.3.2 Hemicyanine Library 12

1.3.3 Rosamine Library 14

1.3.4 BODIPY library 15

1.4 Screening System 16

1.4.1 In vitro screening 16

1.4.2 Cell based screening 17

1.5 Conclusion and Perspectives 18

Reference 19

Chapter Two Development of microplate screening system and

discovery of a green DNA probe 27

2.1 Introduction 27

2.2 Development of the microplate screening system 28

2.3 Discovery of a Green DNA Probe for Live-Cell Imaging 31

2.4 Experimental Section 39

2.4.1 Development of the microplate screening system 39

2.4.1.1 Preliminary Test 39

2.4.1.2 Analytes Information 40

2.4.2 Discovery of a Green DNA Probe for Live-Cell Imaging 41

2.4.2.1 General Information 41

2.4.2.2 C61 Characterization 41

2.5 Conclusion 42

Reference 43

Chapter Three Synthesis of fluorescein-based library 46

3.1 Introduction 46

3.2 Results and discussion 47

3.2.1 Synthesis of CF library 47

3.2.2 Cell screening of CF library 51

3.3 Experimental Section 52

3.4 Conclusion 54

Reference 55

iii

Summary

Chemical genetics studies the structures and functions of genes at the molecular level using

small molecules. Probe development is thus of great interests in molecular biology,

particularly in cell imaging study. Due to the simplicity and straight forwardness,

fluorescence techniques are emerging recently to help elucidate and explain complicated

biological phenomena in cells and animals. Furthermore, the green fluorescein protein (GFP)

also facilitated the progress of fluorescence imaging research.

However, novel fluorescent sensor design is still difficult and time consuming, as little knowledge is well understood in this field. There is a pressing need to development new

approaches to design fluorescent sensors. Diversity Oriented Fluorescence Library Approach

(DOFLA) provided an alternative strategy to generate large number of fluorescent

compounds by combining solid phase chemistry and combinatorial chemistry. Although the

obstacle of generating diversified fluorophores could be achieved, it is crucial to design a

practical platform to select fluorescent sensors.

Chapter 2 describes a novel biological screening system to find potential sensors. Besides, one green DNA probe, C61, to stain live and dead cells is presented. Compare with available green fluorescent sensors, C61 showed dramatic DNA selectivity over RNA. Chapter 3 presents one chloro-fluorescein library synthesis and its biological exploration in melanoma and skill cancer cells.

List of abbreviation

CDCl3 Deuterated chloroform

DCM Dichloromethane

DIC N,N'-Diisopropylcarbodiimide

DMF N, N-Dimethylformamide

DMSO Dimethyl sulfoxide

DMSO-d6 Deuterated Dimethyl Sulfoxide

EA Ethyl acetate

Et2O Diethyl ether

EtOH Ethanol

HATU 2-(1H-7-Azabenzotriazol-1-yl)--1,1,3,3-tetramethyl uronium

hexafluorophosphate Methanaminium

HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HOBt Hydroxybenzotriazole

KOH Potassium hydroxide

MeOH Methanol

NMR Nuclear Magnetic Resonance

RBF Round bottom flask p-TsOH p-Toluenesulfonic acid

List of Figures

Figure 1.1 Forward genetics and reverse genetics 1

Figure 1.2 Forward chemical genetics and reverse chemical genetics 2

Figure 1.3 Diversity Oriented Synthesis (DOS) 4

Figure 1.4 The chemical structure of Peptide Nucleic Acid (PNA) 6

Figure 1.5 Activity-Based Protein Profiling (ABPP) probes 7

Figure 1.6 target identification using ABPP probes 8

Figure 1.7 Dynamic combinatorial libraries 9

Figure 1.8 Application of styryl library; (a) Images of representative localizations

(bar = 10 mm), (b) localization distribution of styryl sensors 11

Figure 1.9 Confocal images and chemical structures of amyloid sensors 12

Figure 1.10 Constructuion of hemicyanine library 13

Figure 1.11 Fluorescence spectra and CCD images of GTP Green 13

Figure 1.12 Chemical structures of heparin sensors and the corresponding

CCD images 13

Figure 1.13 Confocal images (A) and fluorescence emission spectra

(B) of Glucagon Yellow 16

Figure 2.1 The hydrogen peroxide sensor designed by cleavage

of carbon-arylboronate bond 27

Figure 2.2 Analyte map for high throughput screening 28

Figure 2.3 Liquid handling automated system 29

Figure 2.4 DOFL screening database 30

Figure 2.5 Absorbance and emission spectra of C61 in ethanol 32

Figure 2.6 (a) Fluorescence response of C61 (5 µM) with dsDNA (green line) vi

and total RNA (red line) at different concentrations.

(b) Fluorescence emission spectra (excitation: 420nm, cutoff: 495 nm)

of C61 (5 µM) with dsDNA at different concentrations

in HEPES buffer (20mM, pH 7.4) 33

Figure 2.7 DNA/RNA responses in vitro 34

Figure 2.8 Live and fixed cell staining of C61 in A549 and HeLa cell lines 35

Figure 2.9 The DNase and RNase digest experiments 36

Figure 2.10 DNA cell cycle analysis of C61 37

Figure 2.11 Cytotoxicity measurement 38

vii

List of Schemes

Scheme 1.1 Click chemistry reaction 6

Scheme 1.2 Synthesis of styryl library 10

Scheme 1.3 Synthetic routine of rosamine library 14

Scheme 1.4 Synthetic routine of BODIPY library 15

Scheme 1.5 The chemical structure of pluripotin 17

Scheme 2.1 The chemical structure of C61 31

Scheme 3.1 The chemical structure of modified fluorescein derivatives 46

Scheme 3.2 Synthetic routine of dichlorofluorsecein 47

Scheme 3.3 Synthesis routine of fluorescein-based library (CF) 48

Scheme 3.4 Chemical structures of amine build blocks 49

viii

List of Tables

Table 2.1 Fluorescence properties of C61 32

Table 3.1 Maximum absorbance and fluorescence wavelength of CF library 50 50

Chapter One

Introduction

1.1 Chemical Genetics

1.1.1 Overview

In molecular biology and cell biology, genetic study stresses on the structures and functions of genes at the molecular level. Based on the difference of investigative directions, genetic research can be divided into two approaches1: forward genetic and reverse genetics (Figure

1.1). Typically, forward genetic research starts from choosing the specific mutant, and then identifying the specific genes that are responsible for a particular phenotype of interest, which can be termed as ‘from phenotype to genotype’, while reverse genetics follows the opposite direction, i.e. from genotype to phenotype. Specifically, a target gene is first selected, and the consequences of the mutation are investigated after modification or deletion of this gene.

Figure 1.1 Forward genetics and reverse genetics1

Chemical genetics, first proposed by Schrieber et al2., was defined as genetic study using small molecules to elucidate the biological processes. Similarly, chemical genetics is divided into two major research fields: forward chemical genetics, and reverse chemical genetics

(Figure 1.2)2-3. In forward chemical genetics, typically the study starts from the phenotypes selection. With one specific phenotype, small molecules are screened under biological assays.

Once the compounds demonstrated interested results or phenomena, the next step work is to identify the target that the small molecule binds inside the phenotype. Due to the complicity of the biological system, identification of the binding site always takes more time, and is tedious sometimes. On the other hand, reverse chemical genetics study starts from the interested proteins or other biological component, then the ligands were screened with small molecule libraries to find the hit compounds. Phenotype assay is the next step to test the feasibility of this compound with the ligand in vivo. But unlike classical genetics, chemical genetics focuses on analyzing of proteins instead of genes.

Figure 1.2 Forward chemical genetics and reverse chemical genetics3a

In classical genetics, genetic techniques are relatively difficult to proceed and is time

consuming. But chemical genetics, by using libraries of compounds, avoids the shortcomings

of genetic techniques, and it can be conducted on the cellular, tissue, and animal models in a

relative short time. More importantly, small molecules that bind to proteins and affect their

activities could mimic the random mutations used in classical genetic screens. This is

particularly useful when the gene is crucial to the organism’s survival, since altering the gene

(knockout) may cause the organism to die quickly, and the chemical genetic approach, as an

alternative way, enables researchers to investigate the target protein without killing the organisms.

1.2 Chemical Toolboxes

To facilitate the development of chemical genetics, it is necessary to generate a large number of diversified small molecules. Nature has accumulated numerous of bioactive compounds from long periods of evolution, and some of them, such as colchicines, have been used for medical treatment over 2000 years.1a, 4 Nowadays, many new drugs are still designed based

on natural resources like terrestrial and marine organisms5. However, natural products are

always coexisted with other component in the mixture, and it is difficult to isolate and purify the single component from the extract. Therefore, the role of bioactive natural products is more similar as the template rather than the library itself. Indeed, libraries are often built up based on the target natural products by keeping the active sites and modifying the nonreactive motifs.

1.2.1 Diversity Oriented Synthesis

Proposed by Schreiber et al. in 1990s, diversity oriented synthesis (DOS)6 has recently emerged as a power tool in chemical genetics (Figure 1.3). DOS is a strategy which allows 3

quick access to molecule libraries with an emphasis on skeletal diversity and unexplored

regions of chemical spaces. Using this approach and small-molecule screening, Schreiber

helped illuminate the nutrient-response signaling network involving TOR proteins in yeast

and mTOR in mammalian cells. Besides, Schreiber and co-workers has demonstrated the

utility of using a densely functionalized β-amino alcohol to generate multiple scaffolds 2–8

via pairing reactions.7 In another example, Shair et al. demonstrated the power of DOS

combined with phenotypic screening in their synthesis of a library of compounds based upon the natural product galanthamine.8 From this library, they discovered a compound, secramine, which blocked protein trafficking from the Golgi apparatus to the plasma membrane.

Figure 1.3 Diversity Oriented Synthesis (DOS) 6

1.2.2 Tagged Library Approach

Diversity oriented synthesis (DOS) approach improved both the number and diversity of the small molecule libraries. After high throughput screening, bioactive compounds are selected

for structure-activity relationship (SAR) study and further optimization. In forward chemical genetics, the next step is protein identification of the ‘hit’ compound, which may bring more

synthetic modification if the compound cannot form covalent bond with the target protein or

macromolecules. One solution is tagged library approach, which has been designed to

overcome the problems of target identification. A tag here referred to a small moiety that

adds to the molecule scaffold for additional functions, which help to visualize, purify, and

identify the target macromolecules.3a

1.2.2.1 PNA Tagged Library

Peptide Nucleic Acids (PNA) are synthetic oligonucleotides with modified backbones

(Figure 1.4).9 In PNAs, the sugar backbone is replaced by peptide-like backbones. Since

PNAs can bind to both DNA and RNA targets to form stable double helical structure, PNA

tagged libraries have been developed for the spatially addressable localization and

identification of probes on the microarray surfaces. In the early work, Schultz and co-workers

demonstrated10 that PNA-encoded libraries of protein ligands can be screened against several

targets simultaneously by incubating the library with the various targets containing different

fluorophores. Also, based on PNA tagged libraries, Bradley et al. identified specific peptides

targeting different enzymes, including proteases11, Abelson tyrosine kinase12, and so on.

O B O HN B

O N O O P O O O B HN B O

N O O O P O O HN O B B O N O O O O P O HN O

DNA PNA

Figure 1.4 The chemical structure of Peptide Nucleic Acid (PNA)

1.2.2.2 Click Chemistry Library Approach

Another typical example of the tag approach is the azide - alkyne cycloaddition reaction

(click chemistry, Scheme 1.1). Sharpless et al. developed click chemistry13 to generate

substances quickly and reliably by joining small units together. So far, azide-alkyne

cycloaddition reaction (Huisgen reaction) is the most successful application of this concept.

Either azide or alkyne can be added into the scaffold as a tag, and this tagged molecule can be

identified by the counterpart in vivo under suitable conditions, and verse versa. Approaches

based on this reaction have been extensively used drug discovery, proteomics, cell imaging,

and so on.

Cu+ R' RN3 + R' N R N N

Click Chemistry

Scheme 1.1 Click chemistry reaction

In 2002, a femtomolar inhibitor of acetylcholinesterase (Kd = 77 fM) has been successfully

identified14 from an array of building blocks containing an azide group which undergo an

click chemistry reaction as a result of binding to the enzyme. Later on, many potential

enzyme inhibitors were reported based on the in situ click chemistry. For example, Omura et

al. reported one new and potential inhibitor (syn-7)15 against Serratia marcescens chitinase

(SmChi) B by click chemistry after rapid screening; Herczegh et al. discovered several

teicoplanin derivatives16 that showed good anti-bacteria or anti-influenza virus activity.

Furthermore, Bertozzi lab17 extended the application of the clycoaddition reaction by

avoiding copper catalyst, which is harmful for the living organisms. To date, Bertozzi and co-

workers successfully developed series of bioorthogonal probes based copper-free click

chemistry, and applied into the detection and modification of glycans18, glycoproteins19 and fatty acids20. In 2008, this Cu-free click chemistry method even applied into the in vivo imaging of zebrafish21 by adding one fluorescent probe on the membrane associate glycans.

Figure 1.5 Activity-Based Protein Profiling (ABPP) probes 22

1.2.2.3 Activity-Based Protein Profiling

As an emerging chemical proteomic method, ABPP uses probes to monitor and visualize

changes in protein functions or levels in the cell, which makes it particularly useful in the

investigation of enzymes activity and proteomics. Although overlapped with click chemistry

in many cases, ABPP should be stressed independently due to its promising future.

As shown in Figure1.5, an ABPP probe22 consists of three elements: one probe with reactive

electrophilic group, one spacer, and one tag. But not limited into click chemistry reaction, the

tags vary from fluorophores to biotin, alkyne, azide, etc. Compare with traditional proteomic

profiling, ABPP enables to monitor the availability of the enzyme active site directly (Figure

1.6). By expanding the ABPP libraries, probes have been developed for more than a dozen

enzyme classes, including proteases23, kinases24, phosphatases25, glycosidases26, and oxidoreductases27. In one example, Cravatt et al. reported that they could target glutathione

S-transferases (GST)28 both in vitro and in vivo in whole proteomes.

Figure 1.6 target identification using ABPP probes 22

1.2.2.4 Dynamic Combinatorial Library

Since Dynamic Combinatorial Chemistry (DCC, Figure 1.7) was independently reported by the groups of Sanders29 and Lehn30 in the mid-1990s, it has become a powerful tool to develop compounds with significant molecular recognition properties. In contrast to the traditional combinatorial libraries which are synthesized primarily by parallel or split-pool techniques, dynamic combinatorial libraries (DCLs) require only one step library construction. DCC bases on the generation of a mixture of compounds by connecting simple building blocks using a reversible reaction. Since the constituents are mixed by reversible reactions, the thermodynamic stability of DCLs is not only influenced by constituents themselves, but also by the external chemical and physical environments (e.g. temperature, pH, solvents, substrates, etc). The reversible characteristic of DCL makes it particularly useful in molecular recognition, and sensor development, although DCL has been used to identify protein ligands. For example, Lehn et al. has identified one potent HPr kinase/phosphatase inhibitor from the DCLs which contain up to 440 different constituents.31

Figure 1.7 Dynamic combinatorial libraries

1.3 Diversity Oriented Fluorescence Library Approach

The technical advances in imaging instruments promoted not only the development of cell biology and neurobiology, but also the progress of fluorescent small molecules. Fluorescent sensors, molecules that undergo changes in fluorescence upon recognition of the specific target, have drawn considerable attention for its application in analytical chemistry and macromolecules labeling. For example, DAPI and Hoechst are known to be the most popular dyes to label DNA in nucleus; Rhodamine and derivatives32 are extensively used as probes in mitochondria. Nevertheless, the design of fluorescent sensors is largely rely on the research experience rather than systematic study. Furthermore, the difficult of purification in synthesizing fluorophores, together with the limited class of fluorophores, hindered the development of novel fluorescent molecules. Thus, the fluorescent sensors are reported separately in a relative low speed.

To accelerate the development of fluorescent sensors, Chang and co-workers proposed the concept of diversity oriented fluorescence library approach (DOFLA).33 Using this approach, thousands of fluorescent small molecules, which cover all the visible colors, have been synthesized by combinatorial chemistry and solid-phase chemistry. Moreover importantly, fluorescent probes have an extra advantage over conventional probes in chemical genetics, as they are visible by fluorescence microscopy. Here, I list the representative libraries designed by the diversity oriented fluorescence library approach.

1.3.1 Styryl Library

H3C Microwave or heating R + R' CHO R N DMSO-EtOH, pyrrolidine N I I

Scheme 1.2 Synthesis of styryl library

Styryl compounds have been investigated as plasma membrane and mitochondrial labeling

probes. Since styryl dyes are compounds that connecting pyridinium or quinolinium moiety

with one or more aromatic groups via a double bond bridge, thus styryl libraries can be easily

synthesized by knoevenagel condensation reaction. As shown in Scheme 1.2, Rosania et al.

constructed the first styryl library34 by condensing 14 pyridinium and 41 commercial

aldehydes in the 96-well plate. This library has a broad color range good fluorescence

properties, and especially the potential to stain different cellular organelles, such as nucleus,

mitochondria, reticulum, vesicles, etc (Figure 1.8).

Figure 1.8 Application of styryl library; (a) Images of representative localizations (bar = 10

mm), (b) localization distribution of styryl sensors 35

Following modification of styryl library36 by Li et al. induced the solid-phase chemistry into

the styryl library, and the size was enlarged. In addition, this library found its application in

the detection of amyloid aggregation (Figure 1.9). Amyloid has been associated with a large

number of protein-misfolding diseases, including Alzheimer’s Parkinson’s, Huntington’s and

others. But the decomposition of β-amyloid (Aβ) aggregates in brain tissue cannot be

accurately detected by conventional methods. Thus, the discoveries of several amyloid sensors, and following reports, provided novel idea to detect Alzheimer’s disease (AD).

Figure 1.9 Confocal images and chemical structures of amyloid sensors 33

1.3.2 Hemicyanine Library

As one of the heimicyanine dyes, benzimidazolium dyes are widely used as sensitizers and additives in photographic industry. Wang and Chang reported37 the benzimidazolium library through both solution phase and solid phrase chemistry (Figure 1.10). With positively charged benzimidazolium scaffold, 96 derivatives were designed to specifically interact with negatively charged molecules. After screening, one GTP sensor (GTP Green, Figure 1.11) with green fluorescence emission color was discovered to selectively stain GTP over nucleotide analogues. Besides, due to the low quantum yield, GTP green was reported as a turn-on senor for the detection of GTP.

Figure 1.10 Constructuion of hemicyanine library 37

Figure 1.11 Fluorescence spectra and CCD images of GTP Green 37

As a negatively charged glycosaminoglycan, heparin has vital functions in physiological

research38, but only few assays have been established to monitor the heparin concentration in

physiological conditions39. The screening of benzimidazolium library also led to the

discovery of two heparin sensors40 (Figure 1.12), which demonstrated potential clinical

applicability through further validation experiment which detected heparin using

concentrations within the therapeutic range.

Figure 1.12 Chemical structures of heparin sensors and the corresponding CCD images40

1.3.3 Rosamine Library

Whereas low quantum yields of fluorescence dyes are preferable to develop turn-on sensors, brightness is one of the criteria to select fluorophores which suitable for fluorescence microscopy. In addition, other photophysical properties, such as extinction coefficient, photostability, pH-insensitivity and relative longer emission, are also need to be considered.

Rhodamine derivatives are one of the highly favorable dyes in bioimaging because they satisfy all of the criteria addressed above. However, the synthesis of rhodamine dyes is one of the most challenging tasks, as the low yield and difficulty of purification. To overcome this limitation, Ahn and Chang41 developed a novel solid-phase approach (Scheme 1.3). In this

method, the xanthone moiety was first synthesized, and then loaded onto the resin. The final

product was obtained by condensing the xanthone moiety with Grignard reagents on the resin.

Since the last step was performed in the resin, the purification problem was largely

circumvented. To date, more than 300 compounds were reported, and several probes to detect

different targets, including glutathione41, human serum albumin42, zebrafish neural system43, myogenic44, and etc have been reported.

Scheme 1.3 Synthetic routine of rosamine library 41

1.3.4 BODIPY library

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) has outstanding photophysical

properties as a fluorescent scaffold, and the chemical modification of it has been well

studied45. But a library based on BODIPY has not been thoroughly studied due to the

synthetic challenges46. Recently, Lee et al. reported the first BODIPY library47, which

contains 160 members, based on dimethyl BODIPY scaffold using solution phase chemistry

(Scheme 1.4). The dimethyl scaffold was coupled with different commercial aldehydes using

the Knoevenagel reaction. After that, the purification step was completed with the help of

preparative HPLC.

Scheme 1.4 Synthetic routine of BODIPY library 47

After the image-based screening, one glucagon sensor was identified as it selectively stained glucagon in AlphaTC1 cells. Following experiments confirmed the selectivity both in vitro and in vivo (Figure 1.13).

Figure 1.13 Confocal images (A) and fluorescence emission spectra (B) of Glucagon

Yellow47

1.4 Screening System

In chemical genetics, whereas target oriented synthesis is usually designed for one specific target, diversity oriented synthesis attempts to generate a large number of diverse compounds, which may suitable for multi-targets. Therefore, numerous screening systems have been developed in the last decades to maximize the opportunities to discover ‘hit’ compounds.

According to different purposes, the systems vary from in vitro screening, cell-based screening, yeast-based screening, to zebrafish and mouse model screens.

1.4.1 In vitro screening

To target one macromolecule of interest, in vitro screening is the simplest option to select.

Using purified proteins, especially enzymes, these assays aim to find inhibitors or binders with high affinity. This screening approach, due to the simplicity, is utilized as a standard tool to identify effective inhibitors. For instance, Saltiel reported48 the discovery of one selective inhibitor of the upstream kinase MEK that is orally active after the screening; after

synthesized a diverse purine combinatorial library, Schultz and co-workers49 screened them

for inhibition of cyclin-dependent kinase (CDK) activity, and identified two potent inhibitors.

In addition, Navre used a fluorescence-based assay to find potent and selective inhibitors of

collagenase-1 from a diketopiperazine combinatorial library.50

1.4.2 Cell based screening

In vitro screening provides a straightforward approach for identifying ligands for proteins,

but other factors that affected the real biological activity are not considered. Cell-based

assays, on the other hand, offer a more complex and more biological-friendly environment.

Furthermore, as a standard protocol in forward chemical genetics, cell-based assay are easier

to perform compare with more complicated model systems like zebrafish, elegan and mouse

systems.

H C 3 O CF N N N 3 N H N N N N O H H CH3

Scheme 1.5 The chemical structure of pluripotin

Equipped with high throughput screening platform and large arrayed chemical libraries, Ding

lab has been developing and integrating new chemical and functional genomic tools51 to

study stem cell biology and regeneration. In 2006, Ding et al. carried out a high-throughput

screen of 50,000 synthetic small molecules using a transgenic reporter mouse ES cell line

expressing green fluorescent protein (GFP) under control of the Oct4 gene (a gene

specifically expressed in pluripotent stem cells) regulatory elements. Following confirmation

and structure activity relationship (SAR) studies led to the discovery of pluripotin (Scheme

1.5),52 a small molecule activator of stem cell renewal that allows the propagation of OG2

mES cells for at least 10 passages in an undifferentiated state.

1.5 Conclusion and Perspectives

As an interdisciplinary research field, chemical genetics closely connects synthetic chemistry

and biological research. Using combinatorial chemistry approach, large numbers of libraries

have been synthesized to target specific biological challenges. On the other hand, probes,

inhibitors, and substrates have been identified and evaluated by the in vivo system after

screening. In this rapid moving field, many methodologies, such as protein microarray, have

been established and become standard tools for researchers. Besides, novel ideas and

technologies are emerging to optimize or replace existing approaches, to promote the

development of this field. Undoubtedly, chemical genetics becomes the frontier and one of

the most important research branches in chemical biology.

Fluorescent dyes have been extensively used in industrial and academic research due to the

unique characteristics. But recent progress in cell biology53, medicine54, and fluorescence

microscopy techniques55, proposed more challenges and opportunities for researchers

working in fluorescence chemistry. Fluorescent library approach, which has been used for probe development in chemical genetics, would be one of the solutions to resolve the new challenges.

Reference

1. (a) Darvas, F.; Dorman, G.; Krajcsi, P.; Puskas, L. G.; Kovari, Z.; Lorincz, Z.; Urge,

L., Recent advances in chemical genomics. Curr Med Chem 2004, 11 (23), 3119-45; (b)

Thorpe, D. S., Forward & reverse chemical genetics using SPOS-based combinatorial

chemistry. Comb Chem High Throughput Screen 2003, 6 (7), 623-47.

2. Schreiber, S. L., Chemical genetics resulting from a passion for synthetic organic

chemistry. Bioorg Med Chem 1998, 6 (8), 1127-52.

3. (a) Ahn, Y. H.; Chang, Y. T., Tagged small molecule library approach for facilitated chemical genetics. Acc Chem Res 2007, 40 (10), 1025-33; (b) Blackwell, H. E.; Zhao, Y.,

Chemical genetic approaches to plant biology. Plant Physiol 2003, 133 (2), 448-55; (c) Kim,

Y. K.; Chang, Y. T., Tagged library approach facilitates forward chemical genetics. Mol

Biosyst 2007, 3 (6), 392-7; (d) Walsh, D. P.; Chang, Y. T., Chemical genetics. Chem Rev

2006, 106 (6), 2476-530.

4. Breinbauer, R.; Vetter, I. R.; Waldmann, H., From protein domains to drug

candidates-natural products as guiding principles in the design and synthesis of compound

libraries. Angew Chem Int Ed Engl 2002, 41 (16), 2879-90.

5. (a) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P., Drug development

from marine natural products. Nat Rev Drug Discov 2009, 8 (1), 69-85; (b) Koehn, F. E.;

Carter, G. T., The evolving role of natural products in drug discovery. Nat Rev Drug Discov

2005, 4 (3), 206-20.

6. Schreiber, S. L., Target-oriented and diversity-oriented organic synthesis in drug

discovery. Science 2000, 287 (5460), 1964-9.

7. Kumagai, N.; Muncipinto, G.; Schreiber, S. L., Short synthesis of skeletally and stereochemically diverse small molecules by coupling petasis condensation reactions to cyclization reactions. Angew Chem Int Ed Engl 2006, 45 (22), 3635-8.

8. Pelish, H. E.; Westwood, N. J.; Feng, Y.; Kirchhausen, T.; Shair, M. D., Use of biomimetic diversity-oriented synthesis to discover galanthamine-like molecules with biological properties beyond those of the natural product. J Am Chem Soc 2001, 123 (27),

6740-1.

9. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O., Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 1991, 254

(5037), 1497-500.

10. Winssinger, N.; Harris, J. L.; Backes, B. J.; Schultz, P. G., From split-pool libraries to spatially addressable microarrays and its application to functional proteomic profiling. Angew

Chem Int Ed Engl 2001, 40, 3152-5.

11. Diaz-Mochon, J. J.; Bialy, L.; Bradley, M., Dual colour, microarray-based, analysis of

10,000 protease substrates. Chem Commun (Camb) 2006, (38), 3984-6.

12. Pouchain, D.; Diaz-Mochon, J. J.; Bialy, L.; Bradley, M., A 10,000 member PNA- encoded peptide library for profiling tyrosine kinases. ACS Chem Biol 2007, 2 (12), 810-8.

13. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical

Function from a Few Good Reactions. Angew Chem Int Ed Engl 2001, 40 (11), 2004-2021.

14. Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn,

M. G.; Sharpless, K. B., Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew

Chem Int Ed Engl 2002, 41 (6), 1053-7.

15. Hirose, T.; Sunazuka, T.; Sugawara, A.; Endo, A.; Iguchi, K.; Yamamoto, T.; Ui, H.;

Shiomi, K.; Watanabe, T.; Sharpless, K. B.; Omura, S., Chitinase inhibitors: extraction of the

active framework from natural argifin and use of in situ click chemistry. J Antibiot (Tokyo)

2009, 62 (5), 277-82.

16. Pinter, G.; Batta, G.; Keki, S.; Mandi, A.; Komaromi, I.; Takacs-Novak, K.;

Sztaricskai, F.; Roth, E.; Ostorhazi, E.; Rozgonyi, F.; Naesens, L.; Herczegh, P., Diazo

transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives

with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding

study. J Med Chem 2009, 52 (19), 6053-61.

17. (a) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I.

A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R., Copper-free click chemistry for dynamic in vivo

imaging. Proc Natl Acad Sci U S A 2007, 104 (43), 16793-7; (b) Johnson, J. A.; Baskin, J.

M.; Bertozzi, C. R.; Koberstein, J. T.; Turro, N. J., Copper-free click chemistry for the in situ

crosslinking of photodegradable star polymers. Chem Commun (Camb) 2008, (26), 3064-6;

Chem Soc Rev 2010, 39 (4), 1272-9.

18. (a) Laughlin, S. T.; Agard, N. J.; Baskin, J. M.; Carrico, I. S.; Chang, P. V.; Ganguli,

A. S.; Hangauer, M. J.; Lo, A.; Prescher, J. A.; Bertozzi, C. R., Metabolic labeling of glycans

with azido sugars for visualization and glycoproteomics. Methods Enzymol 2006, 415, 230-

50; (b) Laughlin, S. T.; Bertozzi, C. R., Metabolic labeling of glycans with azido sugars and

subsequent glycan-profiling and visualization via Staudinger ligation. Nat Protoc 2007, 2

(11), 2930-44.

19. Rabuka, D.; Hubbard, S. C.; Laughlin, S. T.; Argade, S. P.; Bertozzi, C. R., A

chemical reporter strategy to probe glycoprotein fucosylation. J Am Chem Soc 2006, 128 (37),

12078-9.

20. Yap, M. C.; Kostiuk, M. A.; Martin, D. D.; Perinpanayagam, M. A.; Hak, P. G.;

Siddam, A.; Majjigapu, J. R.; Rajaiah, G.; Keller, B. O.; Prescher, J. A.; Wu, P.; Bertozzi, C.

R.; Falck, J. R.; Berthiaume, L. G., Rapid and selective detection of fatty acylated proteins

using omega-alkynyl-fatty acids and click chemistry. J Lipid Res 2010, 51 (6), 1566-80.

21. Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R., In vivo imaging of

membrane-associated glycans in developing zebrafish. Science 2008, 320 (5876), 664-7.

22. Cravatt, B. F.; Wright, A. T.; Kozarich, J. W., Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 2008, 77, 383-414.

23. Speers, A. E.; Cravatt, B. F., A tandem orthogonal proteolysis strategy for high-

content chemical proteomics. J Am Chem Soc 2005, 127 (28), 10018-9.

24. Colman, R. F., Affinity labeling of purine nucleotide sites in proteins. Annu Rev

Biochem 1983, 52, 67-91.

25. Shreder, K. R.; Wong, M. S.; Nomanbhoy, T.; Leventhal, P. S.; Fuller, S. R.,

Synthesis of AX7593, a quinazoline-derived photoaffinity probe for EGFR. Org Lett 2004, 6

(21), 3715-8.

26. Vocadlo, D. J.; Bertozzi, C. R., A strategy for functional proteomic analysis of glycosidase activity from cell lysates. Angew Chem Int Ed Engl 2004, 43 (40), 5338-42.

27. Wright, A. T.; Cravatt, B. F., Chemical proteomic probes for profiling cytochrome p450 activities and drug interactions in vivo. Chem Biol 2007, 14 (9), 1043-51.

28. (a) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F., Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat Biotechnol 2002,

20 (8), 805-9; (b) Barglow, K. T.; Cravatt, B. F., Discovering disease-associated enzymes by proteome reactivity profiling. Chem Biol 2004, 11 (11), 1523-31.

29. Brady, P. A.; Bonar-Law, R. P.; Rowan, S. J.; Suckling, C. J.; Sanders, J. K. M.,

"Living" macrolactonisation: thermodynamically-controlled cyclisation and interconversion of oligocholates. Chem Commun (Camb) 1996, 35, 319-320.

30. Lehn, J. M.; Eliseev, A. V., Dynamic combinatorial chemistry. Science 2001, 291

(5512), 2331-2.

31. Bunyapaiboonsri, T.; Ramstrom, H.; Ramstrom, O.; Haiech, J.; Lehn, J. M.,

Generation of bis-cationic heterocyclic inhibitors of Bacillus subtilis HPr kinase/phosphatase from a ditopic dynamic combinatorial library. J Med Chem 2003, 46 (26), 5803-11.

32. Lakowicz, J. R., Principles of Fluorescence Spectroscopy. 4th Printing ed.; Springler:

2006.

33. Lee, J. S.; Kim, Y. K.; Vendrell, M.; Chang, Y. T., Diversity-oriented fluorescence library approach for the discovery of sensors and probes. Mol Biosyst 2009, 5 (5), 411-21.

34. Rosania, G. R.; Lee, J. W.; Ding, L.; Yoon, H. S.; Chang, Y. T., Combinatorial approach to organelle-targeted fluorescent library based on the styryl scaffold. J Am Chem

Soc 2003, 125 (5), 1130-1.

35. Lee, J. W.; Jung, M.; Rosania, G. R.; Chang, Y. T., Development of novel cell- permeable DNA sensitive dyes using combinatorial synthesis and cell-based screening. Chem

Commun (Camb) 2003, (15), 1852-3.

36. (a) Li, Q.; Lee, J. S.; Ha, C.; Park, C. B.; Yang, G.; Gan, W. B.; Chang, Y. T., Solid-

phase synthesis of styryl dyes and their application as amyloid sensors. Angew Chem Int Ed

Engl 2004, 43 (46), 6331-5; (b) Li, Q.; Min, J.; Ahn, Y. H.; Namm, J.; Kim, E. M.; Lui, R.;

Kim, H. Y.; Ji, Y.; Wu, H.; Wisniewski, T.; Chang, Y. T., Styryl-based compounds as potential in vivo imaging agents for beta-amyloid plaques. Chembiochem 2007, 8 (14), 1679-

87.

37. Wang, S.; Chang, Y. T., Combinatorial synthesis of benzimidazolium dyes and its

diversity directed application toward GTP-selective fluorescent chemosensors. J Am Chem

Soc 2006, 128 (32), 10380-1.

38. Boneu, B.; de Moerloose, P., How and when to monitor a patient treated with low

molecular weight heparin. Semin Thromb Hemost 2001, 27 (5), 519-22.

39. (a) Kitchen, S., Problems in laboratory monitoring of heparin dosage. Br J Haematol

2000, 111 (2), 397-406; (b) Francis, J. L.; Groce, J. B., 3rd, Challenges in variation and

responsiveness of unfractionated heparin. Pharmacotherapy 2004, 24 (8 Pt 2), 108S-119S.

40. Wang, S.; Chang, Y. T., Discovery of heparin chemosensors through diversity

oriented fluorescence library approach. Chem Commun (Camb) 2008, (10), 1173-5.

41. Ahn, Y. H.; Lee, J. S.; Chang, Y. T., Combinatorial rosamine library and application

to in vivo glutathione probe. J Am Chem Soc 2007, 129 (15), 4510-1.

42. Ahn, Y. H.; Lee, J. S.; Chang, Y. T., Selective human serum albumin sensor from the

screening of a fluorescent rosamine library. J Comb Chem 2008, 10 (3), 376-80.

43. Li, J.; Ha, H. H.; Guo, L.; Coomber, D.; Chang, Y. T., Discovery of novel zebrafish

neural tracers by organism-based screening of a rosamine library. Chem Commun (Camb)

2010, 46 (17), 2932-4.

44. Kim, Y. K.; Ha, H. H.; Lee, J. S.; Bi, X.; Ahn, Y. H.; Hajar, S.; Lee, J. J.; Chang, Y.

T., Control of muscle differentiation by a mitochondria-targeted fluorophore. J Am Chem Soc

2010, 132 (2), 576-9.

45. Loudet, A.; Burgess, K., BODIPY dyes and their derivatives: syntheses and

spectroscopic properties. Chem Rev 2007, 107 (11), 4891-932.

46. Lavis, L. D.; Raines, R. T., Bright ideas for chemical biology. ACS Chem Biol 2008, 3

(3), 142-55.

47. Lee, J. S.; Kang, N. Y.; Kim, Y. K.; Samanta, A.; Feng, S.; Kim, H. K.; Vendrell, M.;

Park, J. H.; Chang, Y. T., Synthesis of a BODIPY library and its application to the development of live cell glucagon imaging probe. J Am Chem Soc 2009, 131 (29), 10077-82.

48. Sebolt-Leopold, J. S.; Dudley, D. T.; Herrera, R.; Van Becelaere, K.; Wiland, A.;

Gowan, R. C.; Tecle, H.; Barrett, S. D.; Bridges, A.; Przybranowski, S.; Leopold, W. R.;

Saltiel, A. R., Blockade of the MAP kinase pathway suppresses growth of colon tumors in

vivo. Nat Med 1999, 5 (7), 810-6.

49. Chang, Y. T.; Gray, N. S.; Rosania, G. R.; Sutherlin, D. P.; Kwon, S.; Norman, T. C.;

Sarohia, R.; Leost, M.; Meijer, L.; Schultz, P. G., Synthesis and application of functionally

diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. Chem Biol 1999, 6 (6), 361-75.

50. Szardenings, A. K.; Antonenko, V.; Campbell, D. A.; DeFrancisco, N.; Ida, S.; Shi,

L.; Sharkov, N.; Tien, D.; Wang, Y.; Navre, M., Identification of highly selective inhibitors

of collagenase-1 from combinatorial libraries of diketopiperazines. J Med Chem 1999, 42 (8),

1348-57.

51. Xu, Y.; Shi, Y.; Ding, S., A chemical approach to stem-cell biology and regenerative

medicine. Nature 2008, 453 (7193), 338-44.

52. Chen, S.; Do, J. T.; Zhang, Q.; Yao, S.; Yan, F.; Peters, E. C.; Scholer, H. R.; Schultz,

P. G.; Ding, S., Self-renewal of embryonic stem cells by a small molecule. Proc Natl Acad

Sci U S A 2006, 103 (46), 17266-71.

53. Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y., Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 2002, 3 (12), 906-18.

54. Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G., Activatable photosensitizers for imaging and therapy. Chem Rev 2010, 110 (5), 2839-57.

55. (a) Huang, B.; Bates, M.; Zhuang, X., Super-resolution fluorescence microscopy.

Annu Rev Biochem 2009, 78, 993-1016; (b) Hell, S. W., Far-field optical nanoscopy. Science

2007, 316 (5828), 1153-8.

Chapter Two

Development of microplate screening system and discovery of a green DNA probe

2.1 Introduction

While different assay systems are widely used to select and identify inhibitors and probes1 in

chemical genetics, fluorescent chemosensor development is heavily rely on specific binding

modes. Particularly, reactions which could trigger the turn-on fluorescence are preferable in

designing novel sensors. For instance, Chang et al. developed series of probes to detect the

hydrogen peroxide and applied into the living cells.2 The hydrogen peroxide probes all shared one arylboronate motif (Figure 2.1), which breaks the bond with quenched fluorophore in the existence of hydrogen peroxide, and emits fluorescence.

Figure 2.1 the hydrogen peroxide sensor designed by cleavage of carbon-arylboronate bond.

As this strategy dominates the fluorescent sensor development, most of the sensors, including

metal sensors,3 pH sensors,4 and reactive oxygen species (ROS) sensors,5 are generated using

this approach. Due to the challenges of synthesis, however, sensors are reported individually

with a relative low efficiency. To facilitate the sensor development, Chang et al. proposed the

concept of diversity oriented fluorescence library approach (DOFLA),6 combing sensor

development with combinatorial chemistry. So far, series of sensors are identified after the

target-specific screening, and some of them are successfully applied in the in vivo biological system. However, different in vitro screening systems are utilized to evaluate different libraries, which restrict the comparison between libraries. Also, the number of analytes is limited, which reduces the opportunities to identify potential sensors. Therefore, designing of

novel in vitro screening system is of great concerns.

2.2 Development of the microplate screening system

To design a better high throughput screening method, more analytes were incorporated into

the screening. Since 96-well plate is widely used in the academic laboratories, we developed

a screening system containing 96 different analytes (Figure 2.2), which includes nucleosides

& nucleotides, proteins, peptides, DNAs & RNAs, pesticides, oxido-redox molecules, metal

ions, pH buffers, and other bioactive analytes, all of which were put according to this map in

the screening system.

Figure 2.2 Analyte map for high throughput screening

At the beginning of screening, all analytes solution are prepared in a 96 deep well plate, and

used within one day. Then, the mother plate is diluted and distributed into three different

concentrations in three deep well plates respectively. By using an automated liquid handling

system (Figure 2.3), analytes of four concentrations are distributed in a 384 well plate

accordingly. Once the 384-well plates are prepared, the dye in HEPES buffer is added into

plates; and the fluorescence emission spectra is measured by a fluorescent reader.

Figure 2.3 Liquid handling automated system

The raw data are collected and uploaded onto the online database, and compare with the

control. The fluorescence variation reflects the binding between analytes and fluorescent

compounds. Selectivity, fluorescence variation, and fluorescence properties are criteria to

select the potential sensors. To reflect the information of criteria, fluorescence foldchange is

labeled by different colors in the database. Specifically, green color indicates fluorescence

enhancement; red color indicates fluorescence quenching. Besides, corresponding color variation bar is shown in the database as the log2 value for comparison. With the color change of each compound, good sensor candidates are easily selected from the screening results. Furthermore, structure information, such as structure, molecule weight, formula, compound code, absorbance, and emission is also included in the database (Figure 2.4). 29

Figure 2.4 DOFL screening database

The BD library7 was first screened; in the next step we screened styryl library8, amyloid

library9, JC library10 and some Rosamine compounds11.

2.3 Discovery of a Green DNA Probe for Live-Cell Imaging

Nucleic acid-staining fluorophores are extensively used in biological assays including cellular

imaging and DNA quantification. Though many such probes are available,12 only limited

dyes such as Hoechst13 and DRAQ514, are suitable for live-cell imaging. Hoechst is the most commonly used probe, but it requires UV illumination, which is known to damage cellular

DNA. DRAQ5 emits fluorescence in the deep red region, maybe with low phototoxicity, but

shows high chemical cytotoxicity. Recently, BENA43515 was reported to stain the nucleus in

live cells, but still with emission located at the cyan region (lem = 485 nm). Thus, discovery

of DNA-selective probes with both low toxicity and various emission-colors are of interest.

After analyzing the in microplate screening data, dyes with distinct preference to dsDNA

over RNA were selected and subjected to cellular imaging to confirm their DNA-selectivity

in cells. As a result, C61 (Scheme 2.1) was selected as the best DNA selective dye in terms of

selectivity, vivid cellular image and low cytotoxicity at reasonably low concentration.

S S I N+

C61

Scheme 2.1 The chemical structure of C61

The detailed properties of C61 with and without DNA are summarized in Table 2.1 and

Figure 2.5. The maximum wavelength of absorbance was red-shifted from 399 nm to 425 nm,

and fluorescence emission was shifted to a slightly shorter wavelength. The fluorescence

quantum yield was increased 19.9 fold upon binding to dsDNA. 31

Table 2.1 Fluorescence properties of C61

λabs(nm) λem(nm) Ф ε

Buffer 399 547 0.0034 6.8 × 103

DNA 425 540 0.0675 6.9 × 103

* The dye concentration of C61 was 5µM in HEPES buffer (20mM, pH 7.4). Acridine

Orange was used as the reference for quantum yield.

Figure 2.5 Absorbance and emission spectra of C61 in ethanol

In addition, the selectivity of C61 to DNA over RNA was about 10times (Figure 2.6). This selectivity is much higher than DRAQ5, and is comparable to Hoechst and DAPI (Figure

2.7). Although several nucleic acid dyes, such as PicoGreen, YOYO, and SYBR dyes, show higher DNA selectivity, they are restricted to in vitro DNA quantification, and are not suitable for live-cell imaging due to their limited cell permeability.

Figure 2.6 (a) Fluorescence response of C61 (5 µM) with dsDNA (green line) and total RNA

(red line) at different concentrations. (b) Fluorescence emission spectra (excitation: 420nm,

cutoff: 495 nm) of C61 (5 µM) with dsDNA at different concentrations in HEPES buffer

(20mM, pH 7.4).

Figure 2.7 DNA/RNA response in vitro. For the measurement, all analytes (0.5 mg/ml) were

dissolved in HEPES buffer and mixed with 10 µM of each dye. Hoechst 33342 and DAPI

were excited at 350 nm and cutoff at 420 nm; C61 was excited at 420 nm and cutoff at 475

nm; DRAQ5 was excited at 640 nm and cutoff at 665nm.

The following cell-image staining experiments also confirmed that C61 stains nucleus clearly in both live and fixed cells, as shown in Figure 2.8.

Figure 2.8 Live and fixed cell staining of C61 in A549 and HeLa cell lines. The final

concentration of C61 was 5 µM. The images were taken under FITC channel. The scale bar

represented 10µm.

With this distinctive selectivity to dsDNA in vitro, other important biological properties of

C61 were further investigated in live cells. The in vivo selectivity of DNA over RNA was

evaluated by deoxyribonuclease (DNase) and ribonuclease (RNase) digest experiments. The

fluorescence signal of C61 diminished dramatically after the DNase digestion, but was not

influenced by RNase digestion (Figure 2.9). A similar pattern of fluorescence signal was observed with Hoechst, the conventional blue color DNA-selective probe. These results supported that C61 has prominent selectivity to DNA over RNA inside cells.

Figure 2.9 The DNase and RNase digest experiments. A549 cell line was used in this

experiment. The final concentration of Hoechst was 10 µM, and the images were taken under

DAPI channel; the concentration of C61 was 50 µM, and the images were taken under FITC

channel. The scale bar represented 10um.

Next, C61 was applied to quantitate DNA contents in the cells, since one of the most

common usages of DNA-specific dyes was the cell cycle analysis using flow cytometry. To examine DNA quantification of C61, we prepared two distinct cell populations: (i) normal cells (ii) G2-M arrested cells by nocodazole treatment. Flow cytometry results showed clear discrimination between two different cell cycle populations, and G2-M arrested cells showed a doubled fluorescence signal that verified the suitability of C61 for flow cytometric analysis of the cell cycle. (Figure 2.10)

Figure 2.10 DNA cell cycle analysis of C61, A549 cell line was used in this experiment. The

final concentration of C61 was 25um. The cells were compared between (a) normal cells, and

(b) Nocodazole-treated G2-M arrested cells.

Lastly, we assessed cytotoxicity of C61 with other DNA probes. Low cytotoxicity is one of the key criteria for DNA probes, since most DNA probes cause damage to the cells. Using a

MTS assay, we compared the cytotoxicity of C61 (GI50 16.0 µM), Hoechst, DAPI, and

DRAQ5 with different concentrations in A549 and Hela cells. The result showed that

DRAQ5 is most cytotoxic as reported previously, and the other three dyes had similar ranges of cytotoxicity (Figure 2.11). When we test the phototoxicity of those dyes, we observed a similar toxicity pattern as a chemical toxicity result. These results indicated that our green

DNA probe, C61, is comparable to conventional blue color DNA probes in terms of toxicity.

Figure 2.11 Cytotoxicity measurement. To compare cytotoxic effects of DNA-selective dyes, compounds were treated to cells at various concentrations covering their working concentration. After 6 hours of incubation cytotoxicity was measured by MTS assay

(Promega). Blue bar represented C61; orange bar represented Hoechst 33342, green bar represented DAPI, and red bar represented DRAQ5. Y axis indicates percentage of cell proliferation. The GI50 of C61 was 16.0um.

2.4 Experimental Section

Unless otherwise noted, all chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. HEPES buffer (20mM, pH 7.4) was used in all solution experiments.

2.4.1 Development of the microplate screening system

The Precision pH meter (Fisher Scientific, Singapore) was used to measure pH values; the

Microplate Pipetting System (Biotek, USA) was used to distribute prepared 384 well plate from 96 deep well plate; the Microplate Spectrophotometer (SpectraPlus from Molecualr

Devices, USA) was used to collect absorbance data; the Microplate Spectrofluorometer

(Gemini XS from Molecualr Devices, USA) was used to collect fluorescence data.

2.4.1.1 Preliminary Test

As the aim of this screening system is designed to identify sensor which is compatible with aqueous solution in terms of fluorescence, not all library compound is suitable for screening due to poor solubility, low fluorescence, and low stability. Therefore, all compounds are tested in this step.

Weigh 1mg of the testing fluorescent compound to make 10mM DMSO solution; if the sample is not fully dissolved, add more DMSO to make 1mM DMSO dye solution. If the sample is still not fully dissolved, then this sample is not suitable for the screening system.

For the qualified samples, take 1µl to make 10µM dye solutionin HEPES (20mM, pH = 7.4), check for any precipitant.

For the prepared sample in DMSO, dilute with HEPES buffer (20mM, pH = 7.4) to make

100µM, 10µM, and 1µM solution respectively. Measure the absorbance spectra and fluorescence spectra, only the compound with clear spectra are selected for the screening.

2.4.1.2 Analytes Information

The 96 analytes are classified into ten types, and the classification details are as follow.

1. Control: Distilled water, HEPES (20mM, pH = 7.4);

2. Viscosity: Glycerol;

3. pH: pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11;

4. Nucleotide & Nucleoside: Adenosine, AMP, ADP, ATP, Cytidine, CMP, CDP, CTP

Guanosine, GMP, GDP, GTP, Uridine, µMP, UDP, UTP, Sodium Phosphate, SodiµM

Pyrophosphate, Sodium Triphosphate, cAMP, cGMP;

5. DNA & RNA: ssDNA; dsDNA; tRNA; total RNA

6. Peptide: HA tag; HA12CA5 tag; His tag; HisG tag, VSV-G tag; V5 tag; IQ tag; AP tag;

Flag tag; cMyc tag,

7. Protein: IgG(human), IgG(bovine serµM), HSA, BSA, Transferrin (holo),

Transferrin(apo), Insulin, Histone, Hemoglobin, Ubiquitin, Myoglobin, Fibrinogen,

Cytochrome C

8. Metal Ion: NaCl, KCl, MgCl2, ZnCl2, FeCl2, CaCl2

9. Oxido-Redox: GSH, GSSG, NAD, NADH, Ascorbic acid, DTT, NaOCl, ROO, KO2,

Peroxide, Iron (II) with Peroxide, Pesticides Bitertanol, Buprofezin, Carbaryl, Diazinon,

Dimethoate, Ethoprophos, Hexythiazox, Phosalone;

10. Etc.: Histamine, Dopamine, Heparin, Caffeine, GABA, MSG, Glucose, Glucose phosphate, Fructose, Fructose phosphate.

2.4.2 Discovery of a Green DNA Probe for Live-Cell Imaging

2.4.2.1 General Information

A SpectraMax M2 microplate reader (Molecular Devices) was used to collect absorbance and fluorescence spectra of JN-C61. DNA cell cycle assay experiment was analyzed by BD LSR

II Bioanalyzer (BD). Greiner 96- or 384 well polypropylene plates were used in the experiments.

A549 and HeLa cells were grown on cell culture BD Falcon dishes in RPMI medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100ug/ml penicillin/streptomycin mixture) in a humidified atmosphere at 37ºC with 5% CO2.

All cell staining followed similar procedures as previous reported. Specifically, after adding dyes, cells were incubated at 37ºC with 5% CO2 for 30min. Then the medium were removed and changed with fresh medium. ImageXpress automated acquisition and analysis system

(Molecular Devices) was used in all the fluorescence image experiments. Cells were cultured

12-24 hours in the Greiner 96-well polystyrene cell culture plates before the fluorescence image experiments. Image brightness and contrast were adjusted to improve picture quality.

For the cell cycle assay experiment, A549 were cultured 48 hours in the dishes before fixation, Nocodazole was treated into the dish 24h before fixation with 1um final concentration. The cells were fixed by 10% formaldehyde in 20min, and then the dye was added, and incubated in 30min for flow cytometry experiment.

2.4.2.2 C61 Characterization

1H-NMR (300 MHz, DMSO-d6): 8.38 (t, 1H, J=8.2Hz), 8.24 (d, 2H, J=8.0Hz), 7.78 (d, 2H,

J=8.4Hz), 7.70 (d, 4H, J=15.9Hz), 7.63 (d, 2H, J=15.9Hz), 7.35 (d, 4H, J=8.5Hz), 4.25 (s,

3H), 2.52 (s, 6H). ESI-MS(m/z) calcd (found): 390.1 (390.0) for [M]+.

2.5 Conclusion

In summary, one practical in vitro screening system has been built up. Besides, we

discovered a green color DNA-selective probe, C61, capable of cell imaging, and also DNA

quantification in flow cytometry. It is noteworthy that C61 works both in live and fixed cells, which allows various experimental options for biological imaging by a simple chemical

method.

Reference

1. Stockwell, B. R., Frontiers in chemical genetics. Trends Biotechnol 2000, 18 (11),

449-55.

2. (a) Dickinson, B. C.; Chang, C. J., A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells. J Am Chem Soc 2008, 130 (30), 9638-

9; (b) Chang, M. C.; Pralle, A.; Isacoff, E. Y.; Chang, C. J., A selective, cell-permeable

optical probe for hydrogen peroxide in living cells. J Am Chem Soc 2004, 126 (47), 15392-3.

3. Que, E. L.; Domaille, D. W.; Chang, C. J., Metals in neurobiology: probing their

chemistry and biology with molecular imaging. Chem Rev 2008, 108 (5), 1517-49.

4. Han, J.; Burgess, K., Fluorescent indicators for intracellular pH. Chem Rev 2010, 110

(5), 2709-28.

5. Nagano, T., Bioimaging probes for reactive oxygen species and reactive nitrogen

species. J Clin Biochem Nutr 2009, 45 (2), 111-24.

6. Lee, J. S.; Kim, Y. K.; Vendrell, M.; Chang, Y. T., Diversity-oriented fluorescence

library approach for the discovery of sensors and probes. Mol Biosyst 2009, 5 (5), 411-21.

7. Lee, J. S.; Kang, N. Y.; Kim, Y. K.; Samanta, A.; Feng, S.; Kim, H. K.; Vendrell, M.;

Park, J. H.; Chang, Y. T., Synthesis of a BODIPY library and its application to the

development of live cell glucagon imaging probe. J Am Chem Soc 2009, 131 (29), 10077-82.

8. (a) Li, Q.; Kim, Y.; Namm, J.; Kulkarni, A.; Rosania, G. R.; Ahn, Y. H.; Chang, Y. T.,

RNA-selective, live cell imaging probes for studying nuclear structure and function. Chem

Biol 2006, 13 (6), 615-23; (b) Lee, J. W.; Jung, M.; Rosania, G. R.; Chang, Y. T.,

Development of novel cell-permeable DNA sensitive dyes using combinatorial synthesis and 43

cell-based screening. Chem Commun (Camb) 2003, (15), 1852-3; (c) Rosania, G. R.; Lee, J.

W.; Ding, L.; Yoon, H. S.; Chang, Y. T., Combinatorial approach to organelle-targeted

fluorescent library based on the styryl scaffold. J Am Chem Soc 2003, 125 (5), 1130-1.

9. (a) Lee, L. L.; Ha, H.; Chang, Y. T.; DeLisa, M. P., Discovery of amyloid-beta

aggregation inhibitors using an engineered assay for intracellular protein folding and

solubility. Protein Sci 2009, 18 (2), 277-86; (b) Li, Q.; Min, J.; Ahn, Y. H.; Namm, J.; Kim,

E. M.; Lui, R.; Kim, H. Y.; Ji, Y.; Wu, H.; Wisniewski, T.; Chang, Y. T., Styryl-based

compounds as potential in vivo imaging agents for beta-amyloid plaques. Chembiochem 2007,

8 (14), 1679-87; (c) Li, Q.; Lee, J. S.; Ha, C.; Park, C. B.; Yang, G.; Gan, W. B.; Chang, Y.

T., Solid-phase synthesis of styryl dyes and their application as amyloid sensors. Angew

Chem Int Ed Engl 2004, 43 (46), 6331-5.

10. (a) Wang, S.; Chang, Y. T., Discovery of heparin chemosensors through diversity

oriented fluorescence library approach. Chem Commun (Camb) 2008, (10), 1173-5; (b)

Wang, S.; Kim, Y. K.; Chang, Y. T., Diversity-oriented fluorescence library approach

(DOFLA) to the discovery of chymotrypsin sensor. J Comb Chem 2008, 10 (3), 460-5.

11. (a) Ahn, Y. H.; Lee, J. S.; Chang, Y. T., Selective human serum albumin sensor from

the screening of a fluorescent rosamine library. J Comb Chem 2008, 10 (3), 376-80; (b) Ahn,

Y. H.; Lee, J. S.; Chang, Y. T., Combinatorial rosamine library and application to in vivo

glutathione probe. J Am Chem Soc 2007, 129 (15), 4510-1.

12. (a) Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P., Characterization of

PicoGreen reagent and development of a fluorescence-based solution assay for double-

stranded DNA quantitation. Anal Biochem 1997, 249 (2), 228-38; (b) Glazer, A. N.; Rye, H.

S., Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence

detection. Nature 1992, 359 (6398), 859-61.

13. Crissman, H. A.; Hirons, G. T., Staining of DNA in live and fixed cells. Methods Cell

Biol 1994, 41, 195-209.

14. (a) Smith, P. J.; Wiltshire, M.; Errington, R. J., DRAQ5 labeling of nuclear DNA in live and fixed cells. Curr Protoc Cytom 2004, Chapter 7, Unit 7 25; (b) Smith, P. J.; Blunt,

N.; Wiltshire, M.; Hoy, T.; Teesdale-Spittle, P.; Craven, M. R.; Watson, J. V.; Amos, W. B.;

Errington, R. J.; Patterson, L. H., Characteristics of a novel deep red/infrared fluorescent cell- permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry 2000, 40 (4), 280-91; (c) Martin, R. M.; Leonhardt,

H.; Cardoso, M. C., DNA labeling in living cells. Cytometry A 2005, 67 (1), 45-52.

15. Erve, A.; Saoudi, Y.; Thirot, S.; Guetta-Landras, C.; Florent, J. C.; Nguyen, C. H.;

Grierson, D. S.; Popov, A. V., BENA435, a new cell-permeant photoactivated green

fluorescent DNA probe. Nucleic Acids Res 2006, 34 (5), e43.

Chapter Three

Synthesis of fluorescein-based library

3.1 Introduction

Fluorescein is characterized by large extinction coefficients (90 000 M-1cm-1), high quantum

yields (0.95), water solubility, biological tolerance, and the ability to be excited by the widely

used 488 nm line of the argon ion laser as applied in confocal fluorescence microscopy.1

Therefore, large number of fluorescein derivatives has been developed to detect metal ions,2 pH,3 NO,4 and label organelles5 inside cells and tissues, and so on.

However, the application of fluorescein dyes is restricted as fluorescein-based fluorophores

are pH sensitivity, and susceptible to photobleaching. To address the above problems,

different strategies have been used to modify the core structure of fluorescein (Scheme 3.1).

For example, halogen-based fluorescein has been designed to increase the photostability.

Recently, Nagano and co-workers developed novel fluorescein dye, Tokyo Green,6 by

replacing carboxylic acid with methyl group, which reduced the pH sensitivity.

-O O O -O O O -O O O -O O O

F F F F COO- COO-

Fluorescein Oregon Green Tokyo Green Pennsylvania Green

Scheme 3.1 The chemical structure of modified fluorescein derivatives

In addition, to enter the cells, fluorescein dyes need to be protected to form esters. After entering the cells, the ester bonds are cleaved by esterase, and the fluorescence starts to emit.

Although effective, the protecting step brings extra work, and limits the application.

In this chapter, one fluorescein-based library (CF library, 80 compounds) was synthesized, with the aim to identify fluorescein dyes which could selectively enter cell lines.

3.2 Results and discussion

3.2.1 Synthesis of CF library

OH OH HO OH

HO O OH DCM / Et2O Cl Cl O MeSO H Cl O 3 95%

COOMe 1

HO O O

1. p-TsOH, toluene, reflux Cl Cl

2. KOH, 2-propanol / H2O 28% COOH 2 Scheme 3.2 Synthetic routine of dichlorofluorsecein

It is reported that1 the dichloro-fluorescein was more photostable and less pH sensitive. Thus dichloro-fluorsecein (compound 2, Scheme 3.2) was chosen as the library scaffold. In addition, the carboxylic acid group was removed based on our previous experience in

Rosamine library.7 As shown in Scheme 3.2, the fluorescein scaffold (compound 2) was synthesized by following the reported approach.1, 8

HO O O O HO O O NO2 N Cl Cl H OH Cl Cl

DMF, HATU

COOH O O 2 3

HO O O R R' N H Cl Cl DMF

R' N O R 4

Scheme 3.3 Synthesis routine of fluorescein-based library (CF)

In the next step, the dichlorofluorescein (compound 2) was loaded onto nitro phenol resin,9 forming the active ester resin (compound 3). After adding the amine in DMF, the final product was obtained after simple workup (Scheme 3.3). Considering the poor solubility of the final product in non-protic solvents, DMF was used during the reaction, and removed in workup. In addition, since anilines were not reactive enough to form the amine bond, aliphatic amines were selected as build blocks, which consist of 80 amines with various chemical structures (Scheme 3.4). After synthesized the CF library, we also measured the absorbance and fluorescence, which clearly showed that most of the compounds had absorbance close to 535 nm, fluorescence close to 565 nm.

H N HN NH N NH2 O O NH2 NH 11 20 32 33 34 66

NH2 F

NH2 H NH2 NH2 N F H2N

74 77 78 80 95 100 Cl

F O F NH F 2 N NH2 H2N H2N Cl H 115 131 135 157 164

H NH O NH2 H2NO N NH2 H2N 166 167 171 175 177 180

H2N O O

NH2 O NH2 H N N 2 H HN 182 184 185 188 193 195

NH2 NH H H N NH2 N H2N 199 201 206 215 219 222 O

NH2 O N O N HN N O NH H O H2N 223 227 230 232 266 O

NH H2N NH H2N H2NO NH2 269 271 273 274 277 292

H NH2 N H N N NH2 H 311 335 343 279 349 O

N O NH2

H2N H2N NH2 H2N

357 373 377 382 384

NH2 O H N NH NH2 2 O NH2 H2N 387 395 419 422 424 462

NH2 NH2 NH2 H N H2N H2N 428 429 439 441 442 443

F Cl OO N NH2 NH2 NH2 NH2 H 477 478 480 599 426

NH2 N H H N 602 582 307 H H2N N 28 657

H2N H2N 55 65 Scheme 3.4 Chemical structures of amine build blocks

Table 3.1 Maximum absorbance and fluorescence wavelength of CF library

Code Abs(nm) Em(nm) Code Abs(nm) Em(nm) CF-28 535 568 CF-135 535 567 CF-32 535 569 CF-157 534 564 CF-55 535 568 CF-554 535 568 CF-66 535 566 CF-180 535 566 CF-77 535 567 CF-182 535 567 CF-80 535 570 CF-184 535 567 CF-115 535 568 CF-185 535 564 CF-164 535 567 CF-199 535 564 CF-166 535 568 CF-201 535 568 CF-171 535 568 CF-206 535 564 CF-175 535 568 CF-219 535 566 CF-177 535 567 CF-223 535 566 CF-188 535 566 CF-227 534 563 CF-193 535 569 CF-232 535 565 CF-195 535 568 CF-269 534 563 CF-215 535 566 CF-271 535 565 CF-222 535 569 CF-273 535 569 CF-230 535 568 CF-274 535 567 CF-266 535 569 CF-279 535 569 CF-277 535 568 CF-292 535 569 CF-349 468 569 CF-307 535 563 CF-384 535 569 CF-311 534 562 CF-387 535 569 CF-335 535 566 CF-395 535 568 CF-343 535 567 CF-428 535 564 CF-357 535 566 CF-429 535 568 CF-373 535 569 CF-442 535 568 CF-377 535 566 CF-443 535 568 CF-382 535 567 CF-478 535 568 CF-419 535 568 CF-582 535 563 CF-422 535 567 CF-11 535 567 CF-424 534 566 CF-20 535 565 CF-426 534 563 CF-33 535 565 CF-439 535 566 CF-34 535 565 CF-441 535 567 CF-65 535 565 CF-462 534 563 CF-74 535 565 CF-477 535 567 CF-78 535 568 CF-480 535 566 CF-95 535 568 CF-599 535 568 CF-100 535 568 CF-602 535 567 CF-131 535 568 CF-657 535 562

3.2.2 Cell screening of CF library

The cell screening system was designed to evaluate the cell permeability of CF library. As a preliminary test, five skin cancer and melanoma cell lines (H522, HeKaT, HEKa, M14, SK-

MEL-28) were selected; and normal human fibroblast (NHF) was used as control cells. After the cell screening, two compounds, CF-311 and CF-582, were discovered to selectively stain skin cancer cells, rather than NHF.

HO O O HO O O

Cl Cl Cl Cl

O N O N

CF311 CF582

Scheme 3.5 Chemical Structure of CF-311 and CF-582

3.3 Experimental Section

Unless otherwise noted, all chemicals and solvents were purchased from Sigma-Aldrich,

Acros, Nova-biochem, and used without further purification.

4-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (Compound 2, Scheme

3.2)

Put the chlororesorcinol (1.44 g, 10 mmol, 2 equiv.), and methyl 4-formylbenzoate (0.82 g, 5 mmol, 1eqiv.) into 100ml RBF, and add 50 ml of DCM / Ether (1 : 1). Add 5 ml of methanesulfonic acid into the reaction system and stir at ambient temperature under nitrogen atmosphere. Monitor the reaction by TLC (DCM : MeOH = 10 : 1). After 2 h, the starting material disappeared, quench the reaction by adding 20ml of water, and stir 5 min. After adding 100 ml of Ethyl Acetate, separate by separation funnel. Wash by water (20 ml × 2), saturated sodium bicarbonate solution (20 ml × 1), water (20 ml × 1). Dry by sodium sulfate, concentrate. Further dry under vacuum pump. 2.1 g was obtained (yield 96%). The crude was used directly for the next step without further purification.

Put compound 1 (440 mg, 1mmol, 1eqiv.), p-Toluenesulfonic acid (1.4 g, 8 equiv.), and 100 ml Toluene into a 250ml RBF. Reflux under nitrogen gas. Stop heating after 20 h. Remove the solvent, add 200 ml of ethyl acetate and 50ml of brine, filter. Collect the red powder.

Separate the liquid, and wash the organic layer by water (50 ml × 2). Concentrate to remove solvent, combine with the red powder, approximate 120 mg of crude is obtained. Add 20ml of iso-propanol, 20 ml of 2 M potassium hydroxide solution, stir at ambient temperature, leave it overnight. During this process, the solid is fully dissolved under basic conditions, and the deep red color is expected to see. Pour the reaction into a 250 ml beaker, adjust pH by adding concentrated HCl until the pH is less than 2. Then the red solid is generated. Filter,

wash by water, cold ether, and cold methanol. Dry under vacuum pump. 100 mg red solid

1 was obtained (Yield 25%). H NMR (300 MHz, DMSO-d6): 6.14-8.16 (d, 2H, J=8.4 Hz),

7.55-7.58 (d, 2H, J=8.2 Hz), 6.83 (s, 2H), 6.25 (s, 2H). ESI-MS (m/z) calcd (found): 401.0

(400.0) for [M] +

General procedures of synthesizing CF library

The nitro phenol active ester resin was prepared according to the reported method. Generally,

in a 50-mL polystyrene cartridge, to an amino polystyrene resin (1 g, 1.2 mmol) in DMF (15

mL) were added 4-hydroxy-3-nitrobenzoic acid (1a, 1 g, 5.5 mmol), HOBt (1 g, 7.4 mmol), and DIC (1 ml, 6.4 mmol). After overnight shaking, the reaction mixture was washed with

DMF (20 ml, 5 times), DCM, and methanol (20 ml, 5 times alternatively). To remove any

undesirable side product, DMF (5 ml) and piperidine (0.5 ml) were added to the cartridge and

allowed to shake for 1.5 h. The resin was filtered and washed with DMF (20 ml, 5 times).

The resulting piperidine salt was removed via the addition of a 10% HCl solution (in DMF,

20 ml) and was allowed to shake for 1.5 h. The resin was then filtered; washed with DMF,

methanol, and DCM (20 ml, 5 times each); and dried by nitrogen gas flow.

Nitro phenol resin (250mg, 0.25 mmol), HATU (190 mg, 0.5 mmol), and fluorescein acid

(200 mg, 0.5mmol) were put into the 50 ml cartridge, shake overnight after adding 20 ml

DMF. The resin was filtered and washed with DMF, methanol, and DCM (20 ml, 5 times

each); and dried by nitrogen gas flow.

In the last step, generally 20 mg (0.02 mmol) fluorescein active ester resin was mixed with

amine (0.05 mmol, 2 equiv.) in 1 ml DMF, heating 10 minutes under 50 °C. Add Ethyl

Acetate (15 ml) and water (5 ml), wash to remove the excess amine. The amine was further

monitored under ninhydrin test. All the library compounds were characterized by LC-MS,

and the purity was all above 85%. 53

3.4 Conclusion

In Summary, one fluorescein library was synthesized based on solid phase chemistry, and the biological screening identified two dyes, CF-311 and CF-582, which selectively penetrated the cell membrane of skin cancer cells. The above results suggest that properly modified fluorescein derivatives are able to enter cell directly without protection groups.

Reference

1. Mottram, L. F.; Boonyarattanakalin, S.; Kovel, R. E.; Peterson, B. R., The

Pennsylvania Green Fluorophore: a hybrid of Oregon Green and Tokyo Green for the construction of hydrophobic and pH-insensitive molecular probes. Org Lett 2006, 8 (4), 581-

2. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y., New strategies for fluorescent probe design in medical diagnostic imaging. Chem Rev 2010, 110 (5), 2620-40.

3. Han, J.; Burgess, K., Fluorescent indicators for intracellular pH. Chem Rev 2010, 110

(5), 2709-28.

4. (a) Nagano, T., Bioimaging probes for reactive oxygen species and reactive nitrogen species. J Clin Biochem Nutr 2009, 45 (2), 111-24; (b) Nagano, T.; Yoshimura, T.,

Bioimaging of nitric oxide. Chem Rev 2002, 102 (4), 1235-70.

5. Goncalves, M. S., Fluorescent labeling of biomolecules with organic probes. Chem

Rev 2009, 109 (1), 190-212.

6. Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T., Evolution of fluorescein as a platform for finely tunable fluorescence probes. J Am Chem Soc 2005, 127

(13), 4888-94.

7. (a) Ahn, Y. H.; Lee, J. S.; Chang, Y. T., Selective human serum albumin sensor from the screening of a fluorescent rosamine library. J Comb Chem 2008, 10 (3), 376-80; (b) Ahn,

Y. H.; Lee, J. S.; Chang, Y. T., Combinatorial rosamine library and application to in vivo glutathione probe. J Am Chem Soc 2007, 129 (15), 4510-1.

8. Mottram, L. F.; Maddox, E.; Schwab, M.; Beaufils, F.; Peterson, B. R., A concise

synthesis of the Pennsylvania Green fluorophore and labeling of intracellular targets with O6- benzylguanine derivatives. Org Lett 2007, 9 (19), 3741-4.

9. Lee, J. W.; Louie, Y. Q.; Walsh, D. P.; Chang, Y. T., Nitrophenol resins for facile amide and sulfonamide library synthesis. J Comb Chem 2003, 5 (3), 330-5.