Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Page 15 of 83 Supplementary Tables   Supplementary Table 1. Confocal and STED imaging parameters.     Preparative flash chromatography was performed on Biotage Isolera Spektra One flash purification system (Biotage AG, Sweden) using the Puriflash Silica HP 30µm series flash cartridges from Interchim and solvent gradients as indicated.
For photoactivation of the dyes on a semipreparative scale, Photoreactor m1 (Penn PhD, USA) was used together with a custom home-built 405 nm LED unit (described in [1]). Photobleaching quantum yields were calculated as previously described [1].

Confocal and STED (stimulated emission depletion) microscopy
Confocal and STED images were acquired using two Abberior Expert Line (Abberior Imaging and image processing was done with ImSpector software (v. 16.3.13367; Abberior Instruments GmbH, Göttingen, Germany), and all images are displayed as raw data unless otherwise noted.
Particular imaging conditions are given in Supplementary Table 1.

Photoactivated localization microscopy (PALM)
Images were acquired on a custom-built setup [1], equipped with a 473 nm (500 mW),  Details on antibodies used can be found in the Supplementary Table S2.

Compound 2a-CF
Compound 2a (10 mg, 24.6 µmol) was placed in a 10 mL pear-shaped flask equipped with a stirring bar, dissolved in degassed methanol (5 mL) and irradiated in a Penn OC Photoreactor m1 using a custom 405 nm LED light source (20% power) until the reaction was found complete by HPLC analysis (50 min total time). Trifluoroacetic acid (10 µL) was added, the resulting bright red solution was evaporated, the product was isolated by preparative HPLC  (2 mL) under argon, and the resulting dark blue solution was stirred at rt for 20 min. It was then transferred dropwise into the stirred mixture of S-3 (compound "Me-Hydrazine" in [12]; 58 mg, 0.2 mmol, 2 equiv.), 2,6-lutidine (58 µL, 0.5 mmol, 5 equiv.) and dry CH2Cl2 (1 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 °C for 1 h, poured into sat. aq. NaHCO3 (50 mL), the product was then extracted with ethyl acetate (3×25 mL) and the combined extracts were dried over Na2SO4. The product was isolated by flash column chromatography (12 g Interchim SiHP 30 µm cartridge, gradient 0% to 50% EtOAc/CH2Cl2) and freeze-dried

Compound 3a
Compound S-5 (35 mg, 75.3 µmol) was dissolved in the mixture of trifluoroacetic acid (0.2 mL) and CH2Cl2 (1 mL) and stirred at rt for 2 h. Toluene (2 mL) was added, the solvents were evaporated and the residue was chased with toluene-CH2Cl2 (2×2 mL). The residue was then

3-Halo
A solution of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP; The organic solvents were removed in vacuo, and the product was isolated by preparative