螢光探針具有巨大的潛力，並被廣泛應用於化學以及生物醫學研究。螢光探針
可以大致分為兩個類別：第一類是標記型探針，該類型探針擁有離去基，使蛋白表面
上的親核基可與探針形成共價鍵結，達成標記目標蛋白的目的。此類型探針之缺點為
細胞內的非特異性標記難以被有效地去除。第二類型是螢光增益型探針，此類型探針
會與目標蛋白結合而產生螢光增益。但此類型探針較容易產生非特異性訊號，使螢光
訊雜比較差，難以進行準確地分析。因此，本論文結合了上述兩類螢光探針的特點，
發展出螢光增益標記探針。此種探針能同時間對目標蛋白進行螢光增益測試以及標記
分析，提供較為廣泛的應用，我們更用以偵測細胞內的人類碳酸酐酶的表達。
此外，我們也合成了一種高親水性膜蛋白標記探針。此探針產生之標記訊號能
夠利用凝膠螢光實驗進行分析，達到更準確的判斷。我們相信這些螢光探針能夠有效
地被應用於生醫分析以及疾病確診。

Fluorescent probes are widely used in chemical and biomedical researches. Currently,
there are two types of fluorescent probes to study protein expressions and locations. The first
type is the protein labeling probes which fluorescent dye can be added covalently to the target
protein. However, this type of labeling probes is not appropriate for labeling intracellular
proteins since the non-labeled intracellular signals are difficult to be removed. Another type is
the fluorogenic probes, which exhibit fluorescent enhancement upon non-covalent binding to
the target protein. Herein, we combined the concepts of these two kinds of fluorescent probes
to obtain fluorescent turn-on labeling probes (FTLPs). This novel concept is capable to be
applied on both fluorescent enhancement test and labeling analysis, which provides much wider
applications. We showed that these probes can be applied to study the expression level of
carbonic anhydrase proteins.
In addition, we also developed another protein-labeling probe to study transmembrane
CAs in living cells. The signals that are produced by this probe can be further analyzed by
using in-gel fluorescent SDS-PAGE to obtain more precise results. We believed that these
novel concepts could be applied in biomedical analysis and disease diagnosis.

摘要 ................................................................................................................................... I
Abstract............................................................................................................................. II
謝誌 ................................................................................................................................. III
Publications ....................................................................................................................... V
Table of Content ............................................................................................................... VI
Chapter 1 Introduction ..................................................................................................... 1
1.1 Proteins ............................................................................................................................... 1
1.1.1 Enzymes .............................................................................................................................................. 2
1.1.2 Proteins and Diseases ......................................................................................................................... 3
1.1.3 Cancer biomarkers .............................................................................................................................. 4
1.2 Methods for Protein Analysis ............................................................................................... 5
1.2.1 Mass Spectrometry ............................................................................................................................. 5
1.2.2 Immunoassays .................................................................................................................................... 7
1.3 Environment-Sensitive Fluorophores ................................................................................... 11
1.3.1 Polarity-Sensitive Fluorophore ......................................................................................................... 13
1.3.2 Viscosity-Sensitive Fluorophore ....................................................................................................... 14
1.3.3 pH-sensitive Fluorophore ................................................................................................................. 15
1.4 Carbonic Anhydrases ........................................................................................................... 16
1.4.1 Physiological Functions of the Carbonic Anhydrases ...................................................................... 17
1.4.2 Isozymes of Carbonic Anhydrases .................................................................................................... 18
Chapter 2 Literature Review ........................................................................................... 19
2.1 Reporter Gene as the Visualization Tools ............................................................................ 19
2.1.1 Green Fluorescent Protein (GFP) and other FPs .............................................................................. 19
2.1.2 Beta-Galactosidase-Based Techniques ............................................................................................ 21
2.1.3 Luciferase-Based Bioluminescence .................................................................................................. 22
2.2 Protein Tags, Peptide Tags and Enzymatic Labeling ............................................................. 25
2.2.1 HaloTag: Protein Labeling Technology for Cell Imaging .................................................................. 25
2.2.2 Biotinylation by Biotin Ligase (BirA) ................................................................................................ 27
2.2.3 Farnesyltransferase .......................................................................................................................... 29
2.3 Bio-orthogonal Chemistry ................................................................................................... 30
2.3.1 Unnatural Amino Acids Incorporation ............................................................................................. 30
2.3.2 Bio-Orthogonal Reactions ................................................................................................................ 31
2.4 Affinity-Based Protein Profiling (ABPP) ................................................................................ 32
2.4.1 High Affinity Fluorescent Probe for Proteinase-Activated Receptor 2 (PAR2) ............................... 33
2.4.2 Ligand-Directed Dibromophenyl Benzoate (LDBB) Designs ............................................................ 34
Chapter 3 Probe Design ................................................................................................... 35
3.1 The FTLP Design .................................................................................................................. 35
3.2 Fluorescent Probe 2 and 3 ................................................................................................... 36
Chapter 4 Results and Discussion ................................................................................... 37
4.1 Feasibility of FTLP ................................................................................................................ 37
4.1.1 Synthesis and Spectroscopic Properties of the Environment-Sensitive Fluorophore --- Mero4 ... 37
4.1.2 Synthesis of probe 2 and 3 ............................................................................................................... 39
4.1.3 Fluorescent Test in 96-Well Microplate of FTLPs ............................................................................. 40
4.1.4 Selectivity Test of 2 and 3 ................................................................................................................. 42
4.1.5 Stability Test of 2 and 3 .................................................................................................................... 43
4.2 In-Gel Fluorescence of the FTLP ........................................................................................... 44
4.2.1 In-Gel Selectivity Test ....................................................................................................................... 44
4.2.2 Sensitivity Test .................................................................................................................................. 46
4.3 Cell Imaging and Cytotoxicity of Probe 2 and 3 .................................................................... 48
4.3.1 Live Cells Imaging of Probe 2 and 3 .................................................................................................. 48
4.3.2 Cytotoxicity of Probe 2 and 3 ........................................................................................................... 50
4.4 Water-Solubility Improvement for TMCAs Labeling ............................................................. 52
4.4.1 Overexpressed CAII Labeling on HeLa Cells ..................................................................................... 53
4.4.2 Endogenous Transmembrane CAs Labeling ..................................................................................... 55
4.4.3 In-Gel Fluorescence Test of Probe 4 ................................................................................................. 57
4.4.4 Regulations of CAXII Expression by Hypoxic Conditions ................................................................. 58
4.4.5 Identification of CA Families by Western Blot ................................................................................. 61
Chapter 5 Conclusion ..................................................................................................... 63
Chapter 6 Experimental Section ..................................................................................... 64
6.1 Material and Instruments .................................................................................................... 64
6.1.1 Chemicals and Reagents ................................................................................................................... 64
6.1.2 Instruments ....................................................................................................................................... 64
6.1.3 Biological reagents ........................................................................................................................... 65
6.2 Protein Studies .................................................................................................................... 65
6.2.1 Recombinant hCAII Expression and Purification ............................................................................. 65
6.2.2 Gel Preparation ................................................................................................................................. 66
6.2.3 Protein Samples Preparation ........................................................................................................... 66
6.2.4 Protein Quantification Test .............................................................................................................. 66
6.2.5 Gel Electrophoresis ........................................................................................................................... 68
6.2.6 Western Blotting ............................................................................................................................... 68
6.2.7 Protein Marker ................................................................................................................................. 69
6.2.8 Quantification of Band Intensities ................................................................................................... 70
IX
6.3 Cell Experiments ................................................................................................................. 71
6.3.1 Cell Culture ........................................................................................................................................ 71
6.3.2 Cell Subculture .................................................................................................................................. 71
6.3.3 Cell Transfection ............................................................................................................................... 72
6.3.4 Live Cell Imaging ............................................................................................................................... 72
6.3.5 Cell Lysate Experiments .................................................................................................................... 73
6.3.6 MTT Assay for Cell Cytotoxicity ........................................................................................................ 73
6.4 Organic Synthesis Method and Protocol .............................................................................. 74
Reference ........................................................................................................................ 89
Appendices .................................................................................................................... 102

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