Author: O, Wa Yi
Title: Chemoselective cysteine modification of peptides and proteins using 1H-isoindolium-based electron-deficient allenes
Advisors: Wong, Man-kin (ABCT)
Degree: M.Phil.
Year: 2021
Subject: Peptides
Proteins -- Chemical modification
Chemical tests and reagents
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: xx, 218 pages : color illustrations
Language: English
Abstract: Site-selective modification of peptides and proteins has been recognized as a powerful tool for biological studies. It is of ongoing interest to develop new modification strategies with high selectivity and efficiency under mild reaction conditions. Cysteine with low abundance and high nucleophilicity is an ideal residue for selective modification. A novel approach for cysteine modification of peptides and proteins using 1H-isoindolium-based electron-deficient allenes was developed. A novel electrophile-mediated cascade cyclization/iodination of propargylamine-based 1,6-diyne has been developed for the synthesis of a new class of spiro 1H-isoindoliums 3. Upon treatment with base, the alkyne moieties of 1H-isoindoliums were transformed to allene functionalities to give the corresponding allene products. The application of this new class of 1H-isoindolium-based electron-deficient allenes to cysteine modification of peptides and proteins was presented. By treatment of cysteine-containing peptides with 5 equivalents of allene reagents in aqueous media at 25 °C for 4 h, cysteine-modified peptides were obtained in good conversions up to 96% as confirmed by LC-MS/MS analysis. Allene reagents with different functional groups were compatible with this modification and the modified products were stable towards excessive thiol-containing reagents and reducing agents. Further application of this modification was demonstrated in protein modification and labeling. Fluorescent tags could be labeled on the cysteine-containing proteins by sequential modification using the azide-alkyne click reaction. Based on the success of this newly developed cysteine modification method, a one-step procedure for preparation of electron deficient allene reagents for bioconjugation was established to enhance the efficiency of the modification. By reaction of propargylamine-based 1,6-diynes with iodine monochloride for the formation of in situ generated 1H-isoindoliums, followed by the addition of 1 equivalent of reducing agent, stock solutions of in situ generated electron deficient allene reagents with high stability could be prepared for direct use in bioconjugation. With 1 equivalent of the in situ generated allene reagents, the modification of peptides could proceed with high efficiency up to 99% conversion in 4 h and the performance was comparable with the isolated allene reagents. The in situ generated allene reagents could be further applied to protein modification and labeling. The results demonstrated that this approach could serve as an alternative method for preparation of allene reagents and be utilized in the newly developed cysteine modification using electron-deficient allenes.
Rights: All rights reserved
Access: open access

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