“Reduced” Coumarin Dyes with an <i>O</i>‐Phosphorylated 2,2‐Dimethyl‐4‐(hydroxymethyl)‐1,2,3,4‐tetrahydroquinoline Fragment: Synthesis, Spectra, and STED Microscopy

Abstract

Large Stokes-shift coumarin dyes with an O-phosphorylated 4-(hydroxymethyl)-2,2-dimethyl-1,2,3,4-tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N-substituted and O-protected 1,2-dihydro-7-hydroxy-2,2,4-trimethylquinoline was oxidized with SeO2 to the corresponding α,β-unsaturated aldehyde and then reduced with NaBH4 in a "one-pot" fashion to yield N-substituted and 7-O-protected 4-(hydroxymethyl)-7-hydroxy-2,2-dimethyl-1,2,3,4-tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes ("reduced coumarins") and 1,2-dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The "reduced coumarins" were found to be more photoresistant and brighter than their 1,2-dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N-hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red-emitting coumarin dye bearing a betaine fragment at the C-3-position showed excellent performance in stimulation emission depletion (STED) microscopy.