Fluorescein-based amino acids for solid phase synthesis of fluorogenic protease substrates.
An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with approximately 30% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a approximately 2.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.
Burchak
Olga N
ON
Laboratoire Biopuces, Département Réponse et Dynamique Cellulaires, Commissariat à l'Energie Atomique, 17 rue des Martyrs, 38054 Grenoble, France.
Mugherli
Laurent
L
Chatelain
François
F
Balakirev
Maxim Y
MY
D000596:Amino Acids / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D010447:Peptide Hydrolases; D019793:Fluorescein