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Synthesis and Characterization of SN38-Linker-Trastuzumab Conjugates via Click Chemistry

  • 배현영 (아주대학교 일반대학원)

초록/요약

Antibody-based therapies offer a targeted strategy by recognizing distinct antigens expressed on the surfaces of cancer cells through an antigen-antibody immune response. While these therapeutics help reduce the off-target cytotoxicity associated with conventional chemotherapy, their efficacy is often limited by patient variability in therapeutic outcomes. Antibody-drug conjugates (ADCs), which combine cytotoxic drugs with monoclonal antibodies, have been proposed to overcome these limitations. The efficacy and stability of ADCs are largely determined by the biochemical properties of the conjugate that binds the antibody to the drug. In this study, a spacer linker was synthesized using two-click reagents. Thiol-Michael addition and dibenzocyclooctyne-mediated azide-alkyne cycloaddition were used for the click reactions. These reactions were used to couple thiol- and azide- functionalized reagents to the spacer linker. The cytotoxic drug SN38 was conjugated to the spacer linker using a thiol/aza-Michael addition reaction. Polyethylene glycol (PEG) was also added to the linker to increase solubility and minimize aggregation to improve the poor physical properties due to the hydrophobic nature of SN38. The conjugated SN38 is characterized by a bystander effect, resulting in higher cytotherapeutic efficacy. These spacer linkers could be used to produce dual-payload ADCs that can bind fluorescent agents or other additives, potentially enhancing the efficacy of the ADCs through selective click reactions, with additional potential applications in drug delivery systems such as liposomes, peptides, and polymers. _____________________________________________________________ Keywords: Antibody-drug conjugate, Click chemistry, Linker, Payload

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목차

1. Introduction 1
2. Materials and Methods 13
2.1 Materials 13
2.2 Synthesis of (R)-methyl 2-(5-azidopentanamido)-6-((tert-butoxycarbonyl)amino)hexanoate [N3-PEG3-Lys(Boc)-PEG3-OMe] [With-PEG, 1a] 15
2.3 Synthesis of (R)-methyl 2-(5-azidopentanamido)-6-((tert-butoxycarbonyl)amino)hexanoic acid [N3-PEG3-Lys(Boc)-PEG3-OH] [With-PEG, 1b] 16
2.4 Synthesis of (R)-tert-butyl (5-(5-azidopentanamido)-6-((6-mercaptohexyl)amino)-6-oxohexyl)carbamate [N3-PEG3-Lys(Boc)-PEG3-SH] [With-PEG, 1c] 18
2.5 Synthesis of tert-butyl ((5R)-5-(5-azidopentanamido)-6-((6-((1-(6-(((S)-1-(((S)-1-((4-((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)-2,5-dioxopyrrolidin-3-yl)thio)hexyl)amino)-6-oxohexyl)carbamate [N3-PEG3-Lys(Boc)-PEG3-SN38] [With-PEG, 1d] 20
2.6 Synthesis of 4-((2S)-2-((2S)-2-(6-(3-((6-((R)-6-amino-2-(5-azidopentanamido)hexanamido)hexyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate [N3-PEG3-Lys-PEG3-SN38] [With-PEG, 1e] 21
2.7 Synthesis of 4-((2S)-2-((2S)-2-(6-(3-((6-((R)-2-(5-azidopentanamido)-6-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxamido)hexanamido)hexyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate [N3-PEG3-Lys(SMCC)-PEG3-SN38] [With-PEG, 1f] 22
2.8 Production and Purification of Trastuzumab 23
3. Results 24
3.1 Synthesis of compound (1a and 2a) 24
3.2 Synthesis of compound (1b and 2b) 27
3.3 Synthesis of compound (1c and 2c) 29
3.4 Synthesis of compound (1d and 2d) 31
3.5 Synthesis of compound (1e) 33
3.6 Synthesis of compound (1f) 35
4. Discussion 36
5. Conclusion 39
References 40
Appendix 45
-국문요약- 51

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