Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

2 Photostable, Amino Reactive and Water-soluble Fluorescent Labels Based on Sulfonylated Rhodamine with a Rigidized Xanthene Fragment Vadim P. Boyarskiy, Vladimir N. Belov,* Rebecca Medda, Birka Hein, Mariano Bossi and Stefan W. Hell

3 1. Synthesis 1,2,3,4-Tetrahydro-7-nitroquinoline (5): 1 1,2,3,4-Tetrahydroquinoline 4 (26 g, 0.20 mol) was slowly added to conc. H 2 SO 4 (90 ml) at 0 7 C with stirring and cooling with an ice bath. After 15 min, a precooled mixture of conc. H 2 SO 4 (40 ml) and 100% HNO 3 (13 g, 0.21 mol) was added at 0 9 C with vigorous stirring and intensive cooling with an ice-salt bath. The reaction mixture was stirred at 0 0 C for 3 h, poured onto crushed ice (about 1 kg), and carefully neutralized to ph = 7 8 with cooled concentrated aqueous NaOH solution. The red precipitate was filtered off, the filter cake was washed with water, and the product was crystallized from a mixture of MeOH (200 ml) and water (50 ml). On the next day the formed red crystals were filtered off and air-dried. Yield of the nitro compound: 18 g (50%), m.p. = 62 C (lit. 1 m.p. = C). 1,2,3,4-Tetrahydro-7-hydroxyquinoline (7): 1 A 0.5 L round-bottom flask fitted with a dropping funnel and a reflux condenser with nitrogen inlet and outlet (on the top) was charged under N 2 with compound 5 (18 g, 0.1 mol), MeOH (100 ml) and Ra-Ni (5 g, suspended in MeOH). The reaction mixture was warmed-up to 60 C (bath), and a solution of N 2 H 4 *H 2 O (20 ml, 0.4 mol) in 120 ml of MeOH was added gradually with stirring at reflux during 5 h. After complete conversion of the starting compound (TLC-control), the reaction mixture was filtered, the filtrate was evaporated in vacuo, and the crude product was dissolved in 85 % H 3 PO 4 (100 ml) under N 2. The mixture was refluxed under N 2 at 150 C (bath) for 14 h, until the starting compound reacted fully (TLC-control). Then the reaction mixture was poured onto crushed ice (about 1 kg) with NaOH (180 g). ph-value of the obtained mixture was found to be about 13. After cooling, the precipitate of Na 3 PO 4 was filtered off, the filtrate was washed with CH 2 Cl 2 (3 200 ml), and carefully neutralized to ph = 7 8 with cold aqueous HCl (18%). The title product was extracted with CH 2 Cl 2 (3 200 ml), the organic layer was washed with sat. aq. NaHCO 3, brine, dried with Na 2 SO 4, and filtered through SiO 2. The title compound was eluted with EtOAc. After evaporation of the solvent in vacuo, compound 7 was obtained (12 g, 85%), m.p. = 90 C (lit. 2 m.p. = C). 1 H NMR (CDCl 3, 300 MHz, ppm), δ = 1.90 (m, 2H, H-3), 2.67 (t, J = 6.3, 2H, H-4), 3.25 (t, J = 5.4, 2H, H-2), 5.96 (d, J = 2.4, 1H, H-8), 6.10 (dd, J = 2.4 and 8.1, 1H, H-6), 6.79 (d, J = 8.1, 1H, H-5). Rhodamine 8: 3 A mixture of the finely ground compound 7 (2.0 g, 13 mmol) and phthalic anhydride (3.0 g, 20 mmol) was heated at 170 C for 3 h. Then an additional portion of 7 (2.0 g, 13 mmol) and 85% aq. H 3 PO 4 (6.5 ml) were added to the cooled reaction mixture and heating was continued at 170 C for 3 h. After cooling, the reaction mixture was stirred and

4 refluxed with methanol (40 ml) for several minutes, cooled, and diluted with CH 2 Cl 2 (80 ml). The precipitate was filtered off and dried in vacuo to yield 2.5 g (46%) of the title compound 8 with m.p. = 285 C (dec.). UV (MeOH):? max = 538 nm (ε = 84000),? em = 558 nm, Φ fl = HPLC: t R = 25.6 min (area 100%). 1 H NMR ([D 6 ]DMSO, 300 MHz, ppm), δ = 1.77 (br. s, 4H, (CH 2 )CH 2 (CH 2 )), 2.60 (br. s, 4H, ArCH 2 ), 3.37 (br. s, 4H, CH 2 N), 6.52 (s, 2H, H-4/5), 6.61 (br. s, 2H, H-1/8), 7.33 (d, J = 6.9, 1H, H-3'), (m, 5H, H-4'/5'/6' and NH). 13 C NMR ([D 6 ]DMSO, 75.5 MHz, ppm), δ = 20.0 ((CH 2 )CH 2 (CH 2 )), 26.1 (ArCH 2 ), 96.9 (CH), (C), (C), (CH), (CH), (CH), (C), (C), (CH), (CH), (C), (C), (C), (CO). ESI-MS, positive mode: m/z (rel. int., %) = 411 (100) [M+H] +, 433 (13) [M+Na] Microscopy 2.1 Experimental setup for STED microscopy STED-images were recorded in a homebuilt stage-scanning fluorescence microscope. The excitation light was provided by a diode laser (PicoTA, Picoquant, Berlin, Germany) emitting 100-ps pulses at 532 nm. The STED-pulses were generated by an optical parametric amplifier (APE, Berlin, Germany) pumped by a Ti:Sapphire laser (Mai Tai HP, Spectra Physics, Mountain View, CA, USA) operating at 80 MHz, which also triggered the excitation source. The STED pulses were stretched to approximately 300 ps in a glass fiber und then converted into a doughnut shaped focal spot (see figure 5S) with a custom-designed phase plate. 4 Both beams were coupled into an oil immersion objective (HXC PL APO, 100, Leica Microsystems, Mannheim, Germany) with a custom-designed dichroic mirror. The fluorescence signal was collected by the same objective lens and imaged onto a photon counting avalanche photodiode (SPCM-AQR-13-FC, Perkin Elmer, Waltham, MA, USA) with an aperture of size corresponding to 0.71 times the magnified Airy disk of the fluorescence spot. Typical values used for the pixel-size and the pixel-dwell-time were nm 2 and 200 µs, respectively. 2.2 Confocal and STED images

5 Figure S1. Confocal image of a dividing mammalian PtK2 cell, recorded in a Leica TCS SP5 confocal microscope (excitation: 514 nm; detection: nm). Microtubules were immunostained with compound 3f-AB. Figure S2. Imaging of the microtubules of a mammalian PtK2 cell immunostained with compound 3b-AB: confocal (left) and subdiffraction STED (center). The plot to the right shows the line profiles along the dotted line indicated in the images (black line: confocal image; red line: STED image). Two fibers spaced 182 nm apart were discerned in the STED image, while they appear as a single object in the confocal counterpart, because the distance is below the diffraction barrier (~230 nm).

6 Figure S3. Subdiffraction imaging of the microtubules of a mammalian PtK2 cell immunostained with compound 3c-AB. Confocal (left) and STED (right) images are shown. Figure S4. Line profiles across single tubuline fibers extracted from the images in Figure 5. Figure S5. Excitation (532 nm) and depletion (630 nm) Point Spread Functions (PSF) measured in the focal plane of the STED microscope. The lower plot display the profiles along the lines indicated in the images (the same profile is expected in the other direction). The depletion PSF at 640 nm was very similar to the one displayed here at 630 nm. 3. Spectroscopic Data 3.1 HPLC-traces and 13 C-NMR spectra of the new compounds

7 3a 3b

8 3c

9 3d 3e 3f

10 3g 9

11 13 15

12 Literature [1] G. Field, P. R. Hammond (Dep. of Energy, USA), US Patent ( ). [2] R. L. Atkins, D. E. Bliss, J. Org. Chem. 1978, 43, [3] P. R. Hammond (Dep. of Energy, USA), US Patent ( ). [4] K. I. Willig, J. Keller, M. Bossi, S. W. Hell, New J. Phys. 2006, 8, 106.