Development of novel analytical and synthetic method for DNA-encoded library synthesis
- Abstract
- DNA-encoded library (DEL) technology is a powerful platform for the discovery of drugs in biological and pharmaceutical interest. In DEL synthesis, DNA tags must be exposed to various conditions, which can unexpectedly modify or destroy encoded chemical information. In this study, the fluorescence-based DNA damage assay method was developed for the DEL synthesis. The fluorescence change of PicoGreen, which is widely used a fluorescent nucleic acid stain for quantitating dsDNA, in respond to dsDNA enable quantification of that, it allowed easy access to overall integrity of DNA. For the various DEL synthesis conditions, the assay was validated through cross-checking with HPLC analysis. Especially, the fact which is not previously well known, the usage of specific ratio of organic solvent could critically induce the DNA damage was confirmed. Furthermore, the applicability of this assay was confirmed through the screening of DNA-damaging condition of on-DNA amide coupling and Pd-catalyzed on-DNA N-arylation reaction. Given its simplicity, and assay speed, this assay could be applicable in the high-throughput screening of multiple reaction conditions at an early stage in DEL synthesis.
Additionally, DEL synthesis has only a few reactions show direct or potential applicability to on-DNA reactions because of DNA stability or functionally. To overcome this issue, we designed and synthesized detergent type-based novel molecules in which a PEG group was introduced into DNA intercalators such as naphthalimide and phenanthridine. We assumed the PEG-intercalator could be combined the base pairs of DNA and 1) the protection of DNA is allowed steric hindrance to prevent the reactant from accessing the DNA and 2) DNA could be soluble in the organic solvents. This strategy is expected development of useful on-DNA reactions will continue to future drug discovery.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Gyung A Kim
- Department
- 대학원 화학과
- Advisor
- Han, Min Su
- Table Of Contents
- Abstract
1. Introduction 1
1.1. DNA-encoded library (DEL) technology 1
1.2. DNA-compatible reaction in DEL synthesis 3
1.3. DNA damage analysis in DEL synthesis 5
1.3.1. qPCR-based DNA damage analysis 6
1.3.2. Electrophoresis-based DNA damage analysis 6
1.3.3. Sanger sequencing-based DNA damage analysis 6
1.3.4. LC-MS based DNA damage analysis 6
1.4. Development of novel analytical method for DEL synthesis. 8
1.4.1. Fluorescence-based simple and practical assay method for DNA damage analysis 8
1.4.2. Fluorescence-based dsDNA-selective dye 9
1.5. Development of novel synthetic method for DEL synthesis 10
2. General information 13
2.1. Materials and instruments 13
2.2. Buffers 14
3. Fluorescence-based simple and practical assay method for DNA damage analysis in DEL synthesis 15
3.1. Calculation of molar concentration of PicoGreen (PG) 15
3.2. AOP-HP DNA titration 15
3.2.1. AOP-HP DNA titration to PG 16
3.2.2. AOP-HP DNA titration to Hoechst 33258 17
3.3. DNA damage analysis for pH effect 18
3.3.1. DNA damage reaction 18
3.3.2. Fluorescence-based DNA damage analysis using PG 18
3.3.3. Fluorescence-based DNA damage analysis using a Hoechst 33258 22
3.3.4. HPLC-based DNA damage analysis 23
3.3.5. Evaluation of pH effect to DNA damage 29
3.4. DNA damage analysis for solvent effect 30
3.4.1. DNA damage reaction 30
3.4.2. Fluorescence-based DNA damage analysis using a PG 30
3.4.3. HPLC-based DNA damage analysis 31
3.4.4. Evaluation of solvent effect to DNA damage 33
3.5. DNA damage analysis for metal ions 35
3.5.1. DNA damage reaction 35
3.5.2. Fluorescence-based DNA damage analysis using a PG 36
3.5.3. HPLC-based DNA damage analysis 36
3.5.4. Evaluation of metal ion effect to DNA damage 41
3.6. Reaction screening 42
3.6.1. On-DNA amide coupling reaction for prep. (DEL-1)32, 33 42
3.6.2. On-DNA amide coupling reaction for DNA damage analysis32,33 44
3.6.3. Pd-catalyzed on-DNA C-N cross coupling reaction32 45
3.6.4. Fluorescence-based DNA damage analysis using a PG 45
3.6.5. HPLC-based DNA damage analysis 46
3.6.6. Application for DNA damage analysis method in DEL synthesis 50
4. Development of DNA protection technology using a DNA-intercalator based-detergent molecules in DEL
synthesis 52
4.1. Design and synthesis of DNA-intercalator based-detergent molecules 52
4.1.1. Synthesis of naphthalimide derivatives 53
4.1.2. Synthesis of phenanthridine derivatives 57
4.2. Binding study of DNA-intercalator based-detergent molecules with dsDNA 59
4.2.1. Absorption and fluorescence properties of PEG-intercalators 59
4.2.2. Fluorescence-based interaction study of the PEG-intercalator with dsDNA 61
4.3. On-DNA reaction study 63
4.3.1. Pd-catalyzed on-DNA C-N cross coupling reaction32 63
4.3.2. Pd-catalyzed on-DNA C-N cross coupling reaction with PEG-intercalator 63
4.3.3. HPLC analysis for on-DNA C-N cross coupling reaction 66
4.3.4. On-DNA amide coupling reaction for Chan-Lam reaction (DEL-2)33 72
4.3.5. Cu-catalyzed on-DNA Chan-Lam reaction (CL-01~04)42 74
4.3.6. HPLC analysis for on-DNA Chan-Lam reaction (CL-01~04) 76
4.3.7. Cu-catalyzed on-DNA Chan-Lam reaction with PEG-intercalator (CL-n1~n3) 77
4.3.8. HPLC analysis for on-DNA Chan-Lam reaction (CL-n1~n3) 78
4.3.9. Summary for on-DNA reaction with PEG-intercalators 80
5. Conclusion and outlook 81
6. References 82
7. Supplementary Information 87
- Degree
- Master
- Appears in Collections:
- Department of Chemistry > 3. Theses(Master)
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