Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy.
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.
Chakraborty
Anirban
A
Department of Chemistry and Chemical Biology, Waksman Institute, Rutgers University, Piscataway, New Jersey, USA.
Wang
Dongye
D
Ebright
Yon W
YW
Ebright
Richard H
RH
D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D015394:Molecular Structure; D010720:Phosphines / Q000138:chemical synthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D001386:Azides; D005456:Fluorescent Dyes; D010720:Phosphines; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP; C044646:phosphine