학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 화학부, 2018. 2. 김병문.Factors affecting cellular phenomena comprise a series of
complicated and elaborate signal transductions. Due to the complexity
of biomolecules, direct observation of a specific biomolecule is
extremely difficult. Therefore conjugation of functionality onto the
biomolecule is often implemented for the purpose of monitoring a
bio-target. Since hydrophobic interaction between a biomolecule and a
ligand is usually not strong enough to be monitored by common
analytical tools, covalent bond formation of an artificial tag molecule
has been developed.
After Huisgens discovery of 1,3-dipolar cycloaddition between an
azide and an alkyne, Sharpless group reported that the reaction can be
catalyzed by a Cu(I) species. Furthermore, introduction of
azidophenylalanine into proteins through expanded genetic code renders
possible the conjugation of an artificial functionality such as
fluorescence, and anti-cancer activity, etc. It is of particular note that
the fluorescence attachment makes the spatiotemporal observation of
bio-target possible. As a consequence, both the development of a new
bioconjugation method, and the synthesis of functional molecules to be ligated have become significant research subjects in chemical biology
field.
This dissertation includes research results of two parts, 1) developing
synthetic method of fluorescent molecules which can be used as a
biosensor, and 2) discovery of novel tyrosine bioconjugation chemistry.
The first part shows one of our approaches in utilizing novel metal
assisted 1,3-cycloaddition and Pd-mediated C-H activation for efficient
syntheses of indolizinone fluorophores containing a small molecule
library. Since the photophysical property of indolizinone is regulated by
the electronic characteristics of C7 and C9 substituents, diversification
of those substituents was crucial for the construction of a fluorescent
molecule library. Through the new synthetic route, synthesis of a
number of fluorescent molecules was accomplished, as diversification of
C9 substituent occurs at the late stage of the synthesis. Moreover,
several biosensors were synthesized, which had been previously
infeasible.
In part 2, tyrosine selective conjugation method through sulfate click
reaction is described. Since SuFEx (sulfur(VI) fluoride exchange)
chemistry was known as the reaction between aryl fluorosulfate and
aryl silyl ether, aryloxy anion has scarecely been recognized as a
useful reaction partner for SuFEx. In this part, a reaction condition to
allow for the reaction of aryl fluorosulfate with tyrosine in the
presence of several nucleophilic amino acid residues was carefully
selected, and a feasibility test of a new site-specific biomolecule
modification was successfully carried out on a model peptide, TAT
47-57, and a physiologically important protein, erythropoietin.SuFEx 2
1.4 References 3
Chapter 2. Developing Synthetic Method of Indolizin-one based Fluorescent Molecules. 5
2.1 Introduction. 5
2.2 Results and Discussion. 7
2.2.1 Synthesis of Fluorophore through 1,3-Dipolar Cycloaddition 7
2.2.2 Coupling Reaction of the Fluorophore and Aryl Iodide through Pd-mediated C-H Activation 8
2.2.3 Fluorescence Quenching through PeT. 11
2.2.4 Synthesis of Hydrogen Peroxide Sensor 16
2.2.5 Heck type Coupling Reaction of the Fluorophore and Styrene Derivatives. 18
2.3 Conclusion. 21
2.4 Experimental Section 22
2.4.1 Materials and Methods 22
2.4.2 Preparations of Compounds 24
2.4.3 Cell Culture and Imaging 40
2.5 References 41
Chapter 3. Discovery of Tyrosine Selective Bioconjugation. 43
3.1 Introduction. 43
3.2 Results and Discussion. 45
3.2.1 SuFEx Reaction of Aryloxy Anion and Aryl Fluorosulfate. 45
3.2.2 Chemoselectivity of SuFEx Reaction among Various Nucleophilic Amino Acid Model Compounds. 47
3.2.3 Fluorescence Tagging on TAT 47-57 Peptide 49
3.2.4 PEGylation of Erythropoietin 52
3.3 Conclusion. 58
3.4 Experimental Section 59
3.4.1 Materials and Methods 59
3.4.2 Preparations of Compounds, Peptides and Proteins. 60
3.4.3 Cell Culture and Imaging 70
3.4.4 In Vivo Mice Experiment 71
3.5 References 78
Abstract in Korean 75Cahpter 1. Introduction. 1
1.1 Bioconjugation of Protein 1
1.2 Fluorescencetagging and biosensor 2
1.3 Sulfur (VI) Fluoride ExchangeDocto
