Phosphorylated 3‐Heteroarylcoumarins and Their Use in Fluorescence Microscopy and Nanoscopy

Abstract

Photostable and bright fluorescent dyes with large Stokes shifts are widely used as markers in far-field optical microscopy, but the variety of useful dyes is limited. The present study introduces new 3-heteroaryl coumarins decorated with a primary phosphate group (OP(O)(OH)(2)) attached to C-4 in 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused with the coumarin fluorophore. The general synthetic route is based on the Suzuki reaction of 3-bromocoumarines with hetarylboronic acids followed by oxidation of the methyl group at the C=C bond with SeO(2) (to an aldehyde), reduction with NaBH(4) (to an alcohol), and conversion into a primary phosphate. The 4 position in the coumarin system may be unsubstituted or bear a methyl group. Phosphorylated coumarins were found to have high fluorescence quantum yields in the free state and after conjugation with proteins (in aqueous buffers). In super-resolution light microscopy with stimulated emission depletion (STED), the new coumarin dyes provide an optical resolution of 40-60 nm with a low background signal. Due to their large Stokes shifts and high photostability, phosphorylated coumarins enable to combine multilabel imaging (using one detector and several excitation sources) with diffraction unlimited optical resolution.

Bright Building Blocks for Chemical Biology

“…The extended conjugation in the dihydroquinoline moiety and the 3-pyridyl substituent also contribute to the useful spectral properties observed with this dye. 43 …”

“…46 This reaction can be performed on highly functionalized molecules such as the reaction of dihydroquinoline 15 and 2-pyridylacetic acid 16 to afford highly soluble coumarin 10 . 43 …”

Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2‐Diazo‐1‐Indanone Caging Group: Synthesis, Photophysics, and Light Microscopy Applications

“…[17] Compounds 7-H,SO 3 H,Y are readily available by sulfonation of the corresponding precursors 7-H,H,Y (Y = H, CO 2 Et), which, in turn, can be easily synthesized from phenol 5-H,TBDMS [18] (Scheme 4; TBDMS = tert-butyldimethylsilyl). The appropriate protecting groups should be compatible with the conditions of the diazoketone synthesis.…”

Synthesis of a Fluorescent‐Labeled Bisbenzamidine Containing the Central (6,7‐Dimethoxy‐4‐coumaryl)Alanine Building Block

“…Its synthesis is depicted in Scheme . Coumarin 343 is a representative of rigidified 7‐aminocoumarins, characterized by their bathochromic shift of both absorption and emission . It has been reported that coumarin 343 gives a high fluorescence quantum yield (ϕ = 0.7) in water and also in organic solvents, i.e., ethanol (ϕ = 0.63) .…”

“…Miscellaneous types of fluorophores have been employed for manifold purposes. Besides BODIPY (boron‐dipyrromethene) and fluorescein derivatives, coumarins represent a widely used class of fluorescent dyes and are valued for their small molecular size and large Stokes shifts . Structure–fluorescence relationships of the coumarin chemotype revealed high fluorescence as a result of the introduction of an electron‐donating substituent at the 7‐position.…”