General Synthetic Method for Si-Fluoresceins and Si-Rhodamines

Abstract

The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compounds. Extensive exploration of these xanthene dyes has yielded general structure-activity relationships where the development of new probes is limited only by imagination and organic chemistry. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging experiments. Nevertheless, efforts to tune the chemical and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient preparation of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3-5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in solution, and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biological imaging.

Figure 1

Synthetic strategies for Si-fluoresceins and Si-rhodamines. (a) Cross-coupling synthesis and lactone–zwitterion equilibrium of JF₆₄₆ (3). (b) Two general approaches for the preparation of Si-fluoresceins and Si-rhodamines.

Scheme 1. Bis(5-methoxymethoxy-2-bromophenyl)silane Syntheses

Figure 2

Synthesis and properties of Si-fluoresceins. (a) Synthesis of Si-fluoresceins 1 and 24–28 via Li/Br exchange, transmetalation to magnesium, electrophile addition, and MOM deprotection. (b) Normalized absorbance spectra of 1 and 24–27 in 0.1 N NaOH. (c) Normalized absorbance at λ_max versus pH for 1 and 24–27. Dashed line indicates pH 7.4. Error bars show standard error (SE; n = 2). (d) Spectroscopic data for Si-fluoresceins 1 and 24–27.

Scheme 2. Extension of the Dibromide Approach to Fluorinated Rhodamines

Figure 3

Cellular imaging with thioether derivatives of JF₆₆₉. (a) Synthesis of thioethers 61 and 64 via S_NAr of JF₆₆₉ (35) with thiols. (b–d) Confocal maximum projection images of live, washed U2OS cells expressing HaloTag-H2B, incubated with JF₆₆₉-thio-HaloTag ligand 61 and counterstained with Hoechst 33342: (b) JF₆₆₉, red; (c) Hoechst 33342, blue; (d) merge. Scale bars = 10 μm. (e–f) Confocal images of fixed COS7 cells with immunolabeled microtubules (red), counterstained with Hoechst 33342 (blue): (e) JF₆₆₉-antibody conjugate from 64; (f) Alexa Fluor 660-antibody conjugate. Scale bars = 20 μm. (g) Photostability of JF₆₆₉ and AF660, as represented by the normalized decrease in fluorescence signal after repeated bleaching cycles. Performed on COS7 cells immunolabeled with dye-antibody conjugates.

Figure 4

Bis(azepanyl)rhodamines (“Potomac” dyes) as internal membrane stains. (a) Structures and λ_max/λ_em for Potomac Yellow (65), Potomac Orange (66), Si-rhodamine 34, and Potomac Red (38). (b–e) Confocal microscopy of fixed COS7 cells stained with (b) Potomac Yellow (65), (c) Potomac Orange (66), (d) Si-rhodamine 34, and (e) Potomac Red (38). Images d and e were taken under the same microscopy settings; scale bars = 20 μm.