Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes

Abstract

Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions of the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent and fluorogenic analogues of the classic fluorescein and rhodamine 110 fluorophores by replacement of the xanthene oxygen with a quaternary carbon. These anthracenyl "carbofluorescein" and "carborhodamine 110" fluorophores exhibit excellent fluorescent properties and can be masked with enzyme- and photolabile groups to prepare high-contrast fluorogenic molecules useful for live cell imaging experiments and super-resolution microscopy. Our divergent approach to these red-shifted dye scaffolds will enable the preparation of numerous novel fluorogenic probes with high biological utility.

Figure 1

(a) Chemical structures of xanthene dyes and carbon-containing isologues. (b) Existing synthetic strategy to carborhodamine dyes. (c) Divergent synthesis of carborhodamines through carbofluorescein intermediates.

Scheme 1. Synthesis of Carbofluoresceins

Figure 2

Properties of fluorescent dyes. (a) Spectral properties of dyes. (b) Normalized absorbance at λ_max versus pH for fluorescein (1) and carbofluorescein (4). Error bars show standard error (SE; n = 2). Determined values (±SE): compound 1, pK_a = 6.35 ± 0.02, Hill coefficient = 0.93 ± 0.02; compound 4, pK_a = 7.44 ± 0.01, Hill coefficient = 1.33 ± 0.03. (c) Absorption at λ_max versus dielectric constant for rhodamine 110 (2) and carborhodamine 110 (5). Error bars show SE (n = 2).

Figure 3

Confocal microscopy of live, unwashed HeLa cells incubated with esterase substrates 20 or 26 and counterstained with Hoechst 33342; scale bars = 10 μm. (a) Compound 20, 1 h incubation. (b) Compound 26, 24 h incubation.

Figure 4

PALM imaging of F-actin in fixed mouse embryonic fibroblasts. (a) Synthesis of caged carborhodamine 110–phalloidin conjugate 32. Reagents and conditions: (a) TFA/CH₂Cl₂, rt, 98%. (b) TSTU, DIEA, DMF, 74%. (c) i. H₂N-PEG₈-CO₂H, DIEA, DMF; ii. TSTU, DIEA, DMF, then phalloidin-NH₂, 69%, two steps. (b, c) PALM images for labeling with CRh₁₁₀–PEG₈–phallodin conjugate 32. (d, e) Labeling with mEos2–actin. (f, g) Labeling with AF647–phalloidin. Images are rendered as a heat map of localization probability/nm² with scale in lower right. For clarity, PALM images were plotted at a minimum localization precision of 22 nm (b, d, and f) and 11 nm (c, e, and g). Scale bars (lower left): 5 μm (b, d, and f) or 1 μm (c, e, and g).