Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED

Abstract

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

Keywords: dyes/pigments; fluorescence; living cells; optical microscopy; rhodamines.

Figure 1

Membrane‐permeant and fluorescent rhodamines (R), carbopyronines (CP), silico‐ (SiR) or germanorhodamines (GeR) designed for STED microscopy of living cells (in free dyes, R=OH; in dye‐ligand conjugates, R=NH‐ligand or NH‐linker‐ligand). H in names stands for hydroxylated fluorophores.

Scheme 1

a) Synthesis of the bis‐hydroxylated rhodamine dye 530RH (an analog of Rh_Q‐CO₂H). b) Synthesis of hydroxylated ROX dyes (6‐COOH‐X‐Rhodamines) 575RH (13 a) and 13 b. Alternative schemes indicate better yielding sequences. PPSE=trimethylsilyl polyphosphate.

Scheme 2

Synthesis of GeR and SiR dyes. a) nBuLi, −78 °C, then Me₂GeCl₂ or Me₂SiCl₂; b) NBS; c) nBuLi or tBuLi, −78 °C, then Me₂NCOCl; d) 20, tBuLi (2 equiv), −78 °C to RT, then HCl; e) 21, nBuLi, THF‐pentane, −100 °C to RT; f) TBAF, then Tf₂O, pyridine; g) 3‐(tert‐butylsilyloxy)azetidine, cat. Pd₂(dba)₃/XPhos, K₂CO₃, dioxane, 100 °C, then TBAF, then TFA.

Figure 2

Normalized extinction ϵ/ϵ _max at λ _max of the dyes from Table 1 versus dielectric constant D of dioxane–water mixtures (575RH and 6‐ROX are unresponsive and are not included). The D _0.5 values correspond to the intersection of interpolated graphs with ϵ/ϵ _max=0.5 line.1a.

Figure 3

Two‐color STED image (raw data) of vimentin‐HaloTag fusion protein (green; labeled with 1 μm 575RH‐Halo) and endogeneous tubulin (magenta; labeled with 5 μm GeR‐tubulin) in living HeLa cells, simultaneous incubation time 20 min, followed by 10 min washing. (a) STED image with confocal part in bottom left corner. (b) and (c): zoomed (confocal and STED) views of the region marked in (a) in separate colors. Scale bars 2 μm; pixel dwell time: 12 μs for both color channels; pixel size: 28 nm for STED and confocal image.

Figure 4

An example of normal cell division (2 min between images) in the presence of 640SiRH bound to CID‐SNAP and 580R bound to CAL1‐Halo after incubation with 0.4 μm 580R‐Halo1a (4 h) followed by addition of 0.4 μm 640SiRH‐BG and imaging for 12 h. Cell viability in the presence of dyes in the centromeres was verified by measuring cell morphology of Drosophila S2 cells using a holographic microscope (Holomonitor M4). For the corresponding time‐lapse movie, see supplementary Movie.

Scheme 3

Three reference dyes (A, M, N) are chosen in such a way that M and N differ from the unknown dye X in one structural element. Dye A has one common structural element with dye M, and another common element with dye N. The known changes in properties between A and M (or N) allow estimation of λ and D _0.5 for X. Assuming Δλ(AN)≈Δλ(MX) and Δλ(AM)≈Δλ(NX) (in which Δλ(AN)=λ(N)–λ(A), valid for both absorption and emission maxima; similarly for ΔD _0.5), the estimated values for 560CP are: λ _abs(560CP)=λ _abs(580CP)+Δλ _abs(520R,500R)=582+(500–521) nm=561 nm; D _0.5(560CP)=D _0.5(580CP)+ΔD _0.5(520R,500R)=34.6+(32.5–7.5)=59.6. For details, see Figures S20, S21 and Table S2 in the Supporting Information.