![Author ORCID: We display the ORCID iD icon alongside authors names on our website to acknowledge that the ORCiD has been authenticated when entered by the user. To view the users ORCiD record click the icon. [opens in a new tab]](https://chemrxiv.org/engage/assets/public/chemrxiv/images/logos/orcid.png)
Abstract
We report N-alkylpyridinium reagents for site-selective dual modification of proteins via a regioselective 1,6-thiol addition reaction. The N-alkylpyridinium derivatives can be syn-thesized in two reaction steps and demonstrate high labelling efficiency and chemose-lectivity towards cysteine residues. This reaction is combined with strain-promoted azide-alkyne click and inverse-electron-demand Diels−Alder reactions to achieve dual functionalization of proteins in a sequential one-pot reaction. A Rho-inhibiting enzyme is functionalized with a cancer cell-targeting peptide and a fluorescent dye for successful in vitro uptake imaging and concomitant inhibition of specific Rho-mediated cellular pathways. The ease of synthesis, fast kinetics, high solubility and regioselectivity make N-alkylpyridinium reagents unique for protein/peptide modification to increase their functional diversities for medical applications.
Supplementary materials
Title
1,6 Thiol Addition of Trifunctional N Alkylpyridinium for Site selective Dual Functionalization of Proteins
Description
The Supporting Information contains 69 pages, Section 1 – 12 with Figures S3.1–S11.34, Table S1. Section 1 provides further information on the materials and methods, Section 2 describes the chemical synthesis of N-alkylpyridinium derivatives, Section 3 shows the cLogP prediction of bioconjugation derivatives, Section 4 evaluates the chemoselectivity and the bioconjugation reaction kinetics and Section 5 describes the stability study of N-alkylpyridinium reagents. Section 6 contains the NMR characterization of key product 9. Section 7 describes the computational methods for the reagent design and frontier molecular orbital (FMO) analysis, the mechanism elucidation using Density functional theory (DFT) calculations. Sections 8 and 9 report the procedures used site-selective modification of peptides and proteins, respectively, as characterization of the obtained bioconjugates. Section 10 summarizes detailed in vivo experiments, including confocal microscopy, intoxication assay and Western Blot analysis.
Actions
