Synthesis of a dihalogenated pyridinyl silicon rhodamine for mitochondrial imaging by a halogen dance rearrangement

Background: Since their first synthesis, silicon xanthenes and the subsequently developed silicon rhodamines (SiR) gained a lot of attention as attractive fluorescence dyes offering a broad field of application. We aimed for the synthesis of a fluorinable pyridinyl silicon rhodamine for the use in multimodal (PET/OI) medical imaging of mitochondria in cancerous cells. Results: A dihalogenated fluorinatable pyridinyl rhodamine could be successfully synthesized with the high yield of 85% by application of a halogen dance (HD) rearrangement. The near-infrared dye shows a quantum yield of 0.34, comparable to other organelle targeting SiR derivatives and absorbs at 665 nm (εmax = 34 000 M−1cm−1) and emits at 681 nm (τ = 1.9 ns). Using colocalization experiments with MitoTracker® Green FM, we could prove the intrinsic targeting ability to mitochondria in two human cell lines (Pearson coefficient >0.8). The dye is suitable for live cell STED nanoscopy imaging and shows a nontoxic profile which makes it an appropriate candidate for medical imaging. Conclusions: We present a biocompatible, nontoxic, small molecule near-infrared dye with the option of subsequent radiolabelling and excellent optical properties for medical and bioimaging. As a compound with intrinsic mitochondria targeting ability, the radiolabelled analogue can be applied in multimodal (PET/OI) imaging of mitochondria for diagnostic and therapeutic use in, e.g., cancer patients.


General remarks
Not otherwise described, reactions requiring exclusion of oxygen and moisture were carried out in heat-gun flasks dried under argon gas atmosphere using the Schlenk technique.
All chemicals and solvents were used from Sigma-Aldrich Laborchemikalien GmbH, abcr GmbH, Acros Organics and were used as received without other purification. Deuterated solvents were used from Deutero GmbH. NMR spectra were recorded at room temperature on a 400 MHz Bruker Avance III spectrometer. Chemical shifts are reported in δ units relative to methanol-d4 (δH = 3.31; δC = 49.0). 1 Analyses followed first order and the following abbreviations were used throughout: s = singlet, d = doublet, t = triplet, dd = doublet of doublet etc., m = multiplet.
Coupling constants (J) are given in Hz and refer to H,H-couplings. Assignment (Cquart, CH, CH2, CH3) was conducted with DEPT and HSQC experiments.

Synthesis of SiR dye 15
In a flame-dried flask, 3-bromo-2-chloropyridine 19 (249 mg, 1.29 mmol, 20 equiv) was dissolved in 3.0 mL anhydrous THF under argon. The solution was cooled to −78 °C, then tert-BuLi (1.7 M in pentane, 2.59 mmol, 1.52 ml, 40 equiv, CAUTION: solutions of tert-butyllithium react explosively with water and may ignite in moist air) was added drop-wise and the brownred mixture was stirred for 30 min. At the same temperature, silicon xanthone 17 2 dissolved in anhydrous THF (2.0 mL) was added drop-wise. The mixture went deep red. The mixture was warmed to room temperature after 2 min and the mixture turned dark. The reaction was stirred for 4 hours, then water and DCM were added. Some drops of 1 N HCl were added and the organic phase turned deep blue. The organic phase was separated and the water phase was extracted with DCM several times. The organic phase was dried with brine and sodium sulfate and after filtration, the solvents were evaporated in vacuo. Column chromatography (silica gel, 1% to 10% methanol in DCM) afforded 15 as a deep blue solid (29.5 mg, 0.0551 mmol) in 85% yield.

Fluorescence properties 4.1 Absorption and emission
Absorbance spectra were recorded on a Cary 5000 UV-vis-NIR spectrophotometer, emission spectra on a Cary Eclipse Fluorescence spectrophotometer with excitation at 600 nm using standard 1 cm quartz cuvettes. The stock solution of dye 15 (4.67 mM in MeCN) was diluted in either PBS pH 7.4 or in MeCN to 15.5 µM for UV-vis/NIR absorbance measurements or to 4.7 µM for fluorescence emission measurements. The extinction coefficient was calculated according to Lambert-Beer for the absorbance maximum.  Eclipse Fluorescence spectrophotometer with excitation at 600 nm in the 610-800 nm interval.

Fluorescence quantum yield
All dilutions were measured using the same parameters. Absorbance at 600 nm was plotted versus the integrated fluorescence intensity and fitted by linear regression ( Figure S2). The fit values (Table S1)   using Nile Blue as reference standard.  is shown in red.

Live cell imaging
Samples were imaged on a custom-built STED microscope similar to the one published by

Assessment of cytotoxicity
Potential cytotoxicity of SiR dye 15 was assessed via analyzing duration and frequency of cell division using holographic time-lapse imaging with a HoloMonitor ® M4 cytometer. One day prior to the experiment, U2OS (human bone osteosarcoma epithelial) cells were seeded in a 24-well cell culture plate. The medium of one half of the wells was replaced with medium containing 1 µM SiR dye 15 and the other half with fresh medium without dye. Incubation was done for 1 h at 37 °C and 5% CO2. All wells were washed three times with medium before holographic time-lapse imaging was started using PHI HoloLids TM to ensure optimal image quality. Images were recorded for 14.5 h with one image of each well every 30 min. Data analysis was done with the HstudioM4 Software including cell segmentation, tracking of dividing cells ( Figure S7), cell counting and confluency measurement ( Figure S8). The results confirm the absence of cytotoxicity during the experiment.