Synthesis of Clickable Fluorescent Probes

We prepared AHA and the triazole ligand as described previously47 (link). We prepared the TexasRed–PEO₂–Alkyne by dissolving TexasRed–PEO₂–propionic acid succinimidyl ester (Biotium Inc.) in excess neat propargylamine (Sigma–Aldrich). After 30 min, we added the solution dropwise to anhydrous diethyl ether. We collected the resulting precipitate by centrifugation (5 min, 10°C, 10.000 × g). Then, we washed the precipitate three times with anhydrous diethyl ether, dried and characterized it by ion spray MS to confirm the formation of the product with a molecular weight of 802.90 g/mol (Supplementary Fig. 1a). We synthesized the azide–bearing fluorescein tag in a similar way using the amine–reactive 5–carboxyfluorescein–PEO₈–propionic acid succinimidyl ester (Biotium Inc.) and 3–azidopropan–1–amine20 (link) to yield a product with a molecular weight of 881.20 g/mol (Supplementary Fig. 1b).