Development of Microplate Screening System
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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. i 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 ii 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. iv 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 v 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 ix 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 1 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 2 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.