Chapter 2 - Azide-Specific Labeling of Biomolecules by Staudinger–Bertozzi Ligation: Phosphine Derivatives of Fluorescent Probes Suitable for Single-Molecule Fluorescence Spectroscopy

Abstract

We describe the synthesis of phosphine derivatives of three fluorescent probes that have a brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labeling through Staudinger-Bertozzi ligation, as well as procedures for the quantitation of labeling specificity and labeling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labeling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy.