Extended Data Fig. 6: Synthesis and properties of new HaloTag and SNAP-tag ligands based on 15–20 and 37–38.
From: A general method to optimize and functionalize red-shifted rhodamine dyes
a, Expanded synthetic scheme of HaloTag ligands 15HTL–20HTL and 37HTL–38HTL starting with nucleophilic aromatic substitution (SNAr) of 15–20 and 37–38 with masked acyl cyanide 34. b, Two-color montage image of fixed coronal slices with zoom-in regions from a mouse expressing HaloTag–GFP in neurons transduced by IV administration of the viral vector PHP-eB-Syn-HaloTag–GFP followed by IV administration of 15HTL (100 nmol), perfusion, and slicing; GFP signal in green and 15HTL signal in magenta; scale bar = 3 mm; experiment was duplicated with similar results. c, Structures of JF669–HaloTag ligand (15HTL), JF646–HaloTag ligand (5HTL), JF593–HaloTag ligand (20HTL), and JF570–HaloTag ligand (3HTL). d–e, Confocal image and line-scan from Fig. 2l showing live U2OS cells expressing Sec61β–HaloTag labeled with 2HTL (30 nM, 30 min, 3× wash) and SNAP-tag–histone variant H2A.Z labeled with 15STL (30 nM, 30 min, 3× wash); costained with Hoechst 33342 (1 μM, 30 min, 3× wash); d, Confocal image with white line indicating line-scan position; e, Line-scan profile; scale bar: 5 μm; experiment was duplicated with similar results. f, Two-photon absorption spectra of the HaloTag conjugates of HaloTag ligands 15HTL–20HTL, and 37HTL–38HTL. g, Plot of fluorescence from cells expressing HaloTag–H2B labeled with 2HTL (200 nM) or 19HTL (200 nM) over 30 bleach cycles; error bars indicate SE; n = 3 independent cellular samples. h, Plot of fluorescence from fixed U2OS cells expressing HaloTag–H2B labeled with 3HTL (200 nM) or 20HTL (200 nM) over 30 bleach cycles; error bars indicate SE; n = 3 independent cellular samples.
