Author: Lai, Chun Him Nathanael
Title: N-terminal modification of peptides and proteins using 2-ethynylbenzaldehydes via isoquinolinium formation
Advisors: Wong, Man-kin (ABCT)
Degree: M.Phil.
Year: 2018
Subject: Bioconjugates
Amino acids
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: x, 187 pages : color illustrations
Language: English
Abstract: Bioconjugation is an enabling technology for studies of fundamental biological events through selective modification and labeling of biomolecules that is of great application potential in the development of protein therapeutics. To assemble structurally well-defined bioconjugates, it is of importance to develop novel site-selective bioconjugation reactions that are compatible with complex biological systems under mild reaction conditions. However, common approaches for bioconjugation, such as lysine modification, generally give heterogeneous bioconjugate mixtures due to the difficulties in controlling the number of modifications of the multiple lysine residues on the protein surface. Note that the α-amino group of N-terminal residues stand out as a unique reactive site rendering site-selective bioconjugation possible, as proteins usually contain one N-terminal α-amino residue. Thus, the development of efficient N-terminal α-amino group modification reaction is a promising strategy for site-selective bioconjugation. Along with our long-term interest in selective N-terminal bioconjugation, a novel method for highly selective N-terminal α-amino group modification of peptides and proteins via isoquinolinium formation by using easily accessible 2-ethynylbenzaldehydes was developed in this thesis. A series of 2-ethynylbenzaldehydes (2a-2q) were prepared from Sonogashira coupling reaction and deprotection of trimethylsilyl group in good yields. Treatment of a model peptide YTSSSKNVVR 1a with 20 equivalents of 2-ethynylbenzaldehyde 2a under mild reaction conditions (50 mM PBS buffer (pH 6.3) at 37°C for 16 h) afforded stable isoquinolinium-peptide conjugates in moderate N-terminal selectivity (the ratio of modified N-terminal α-amino group/modified lysine ε-amino group = 21:1) analyzed by LC-MS/MS. A model reaction study by treatment of (S)-2-amino-N-benzylpropanamide 1b with 2-ethynylbenzaldehyde 2a afforded the corresponding isoquinolinium product 1ba in 30% isolated yield, as confirmed by NMR analysis. Screening of 2-ethynylbenzaldehydes (2a-2q) with YTSSSKNVVR 1a gave the corresponding isoquinolinium-peptide conjugates in low to excellent conversion (8-86%) and moderate to excellent N-terminal selectivity (12:1 to >99:1). Among the 2-ethynylbenzaldehydes (2a-2q) examined, reaction of peptide 1a with 2-ethynyl-5-hydroxy-4-methoxybenzaldehyde 2b was found to afford good conversion (86%) and excellent N-terminal selectivity (>99:1) in the peptide modification. Studies on the reactions of peptide 1a with 2b in different reaction media with a range of pH values (pH 5.0-9.0 of 50 mM PBS) revealed that the reaction provided excellent N-terminal selectivity (>99:1) in slightly acidic reaction medium (pH 6.3). Studies of the N-terminal selectivity of different N-terminal amino acid residues was conducted by treatment of 2-ethynyl-5-hydroxy-4-methoxybenzaldehyde 2b with a peptide library of XSKFR (X = 20 amino acids). Excellent N-terminal selectivity (>99:1) was achieved in 13 out of the 20 peptides with good to excellent conversions (32-93%). Proteins including insulin, lysozyme, ribonuclease A (RNaseA), bovine serum albumin (BSA), human serum albumin (HSA), and a therapeutic protein BCArg were subjected to the newly developed N-terminal bioconjugation reaction at 37°C using 2-ethynyl-5-hydroxy-4-methoxybenzaldehyde 2b to give good conversions (44-71%).
Rights: All rights reserved
Access: open access

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