SUPPLEMENTARY INFORMATION

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1 A dendritic single-molecule fluorescent probe that is monovalent, photostable and minimally blinking Si Kyung Yang 1, Xinghua Shi 2,3,4, Seongjin Park 2, Taekjip Ha 2,3,4,5 and Steven C. Zimmerman 1 * 1 Department of Chemistry, 2 Department of Physics, 3 Institute for Genomic Biology, 4 Howard Hughes Medical Institute, and 5 Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA tjha@illinois.edu, sczimmer@illinois.edu Table of Contents General methods... 2 Synthetic procedures... 2 Microtubule-12 Prep... 4 Single molecule image of 12 with Trolox and oxygen scavenging system... 5 Intensity traces of 50 representative molecules of PGD-KFL 12, BODIPY 650/665X, Cy5, KFL 3, PGD-BODIPY 650/665X, and PGD-Cy Microtubule network images in cells H NMR spectra References Supplementary Information Video: Video showing a side-by-side comparison of the blinking of Cy5 and PGD-KFL 12 molecules recorded at 10 frames per second over a period of 100 seconds. NATURE CHEMISTRY 1

2 General Methods. All reagents were purchased from Advanced ChemTech, Alfa Aesar, or Sigma-Aldrich, and used without further purification unless otherwise noted. NMR spectra were recorded using a Varian Unity 400 or 500 MHz spectrometer. Chemical shifts are reported in ppm and referenced to the corresponding residual proton in deuterated solvents. Mass spectral analyses were provided by the Mass Spectrometry Laboratory, School of Chemical Science, University of Illinois, using ESI on a Waters Micromass Q-Tof spectrometer, or MALDI-TOF on an Applied Biosystems Voyager-DE STR spectrometer. UV-Vis absorbance spectra were recorded on a Shimadzu UV- 2501PC spectrophotometer. Fluorescence spectra were recorded on a Horiba Jobin Yvon Fluoromax-3 or -4 spectrophotometer and quantum yields (Φ) were calculated from the following equation: Φ = Φ r * [A r /A] * [F/F r ] * [n/n r ] 2 where A is the absorbance at the excitation wavelength, F is the integrated area under the fluorescence emission spectrum and n is the refractive index of the solvent. DOTCI (Φ = 0.63 in DMSO) 1 was used as a reference. Dialysis was performed using dialysis tubing (Sigma-Aldrich MWCO 1200) against water at 25 C. Compounds 1 2, 4 3, and 6 4 were synthesized according to the previously published procedures. KFL 3. To a mixture of 4-formylbenzoic acid (92 mg, 0.61 mmol) and 1 (0.35 g, 1.3 mmol) was added TFA (5 ml). After the mixture was stirred at 50 C for 1 h under a nitrogen atmosphere, POCl 3 (1.5 ml) was added and the solution was stirred for 10 min at 50 C and precipitated into cold water. The precipitated 2 was collected by filtration and used without further purification. A mixture of 2 (0.34 g, 0.61 mmol), N- hydroxysuccinimide (0.35 g, 3.0 mmol), EDCI (0.59 g, 3.1 mmol), and DMAP (35 mg, 0.29 mmol) was dissolved in DMF (5 ml), stirred at 25 C for 15 h under a nitrogen atmosphere, and then precipitated into cold water. The precipitate was collected by filtration and dried under high vacuum. To a solution of the crude 2-NHS in dichloromethane (20 ml) were added triethylamine (1 ml) and BF 3 OEt 2 (1 ml). After the mixture was stirred at 25 C for 1 h under a nitrogen atmosphere, water was added and the mixture was extracted with dichloromethane. The combined organic layers were washed with water, dried over MgSO 4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using dichloromethane/ethyl acetate (50:1) as eluent to yield 3 (0.13 g, 30%). 1 H NMR (CDCl 3 ): δ 8.30 (d, J = 8.5 Hz, 2H), 7.76 (m, 6H), 6.99 (d, J = 8.5 Hz, 4H), 6.87 (s, 2H), 6.14 (s, 2H), 3.88 (s, 6H), 2.96 (s, 4H). 13 C NMR (CDCl 3 ): δ 169.6, 169.0, 162.1, 161.9, 154.1, 150.5, 141.4, 139.2, 139.2, 131.6, 130.8, 127.7, 126.5, 122.7, 115.0, 102.7, 94.1, 55.9, MS-ESI (m/z): [M+H] + calcd for C 38 H 27 BF 2 N 3 O 8, ; found, Trifunctional Core 5. 4 (0.30 g, 1.3 mmol), Fmoc-Lys(Boc)-OH (0.71 g, 1.5 mmol), and HATU (0.62 g, 1.6 mmol) were dissolved in anhydrous DMF (5 ml) under a nitrogen atmosphere. DIPEA (0.54 ml) was added and the mixture was stirred at 25 C for 15 h. After the solvent was removed under reduced pressure, water was added and the mixture was extracted with dichloromethane. The combined organic layers were washed with water, dried over MgSO 4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using hexanes/ethyl acetate (5:3) as eluent to yield 5 (0.80 g, 92%). 1 H NMR (CDCl 3 ): δ 7.77 (d, J = 7.4 Hz, NATURE CHEMISTRY 2

3 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 6.06 (s, 1H), 5.49 (br, 1H), 4.62 (br, 1H), 4.38 (d, J = 7.2 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H), 4.12 (s, 6H), 3.83 (m, 6H), 3.12 (m, 3H), 2.44 (s, 3H), 1.82 (m, 2H), 1.66 (m, 2H), 1.43 (m, 11H). 13 C NMR (CDCl 3 ): δ 171.3, 156.0, 143.8, 141.3, 127.7, 127.1, 125.1, 120.0, 79.4, 74.8, 68.8, 68.3, 67.0, 59.4, 58.6, 55.1, 47.2, 40.1, 32.7, 29.7, 28.4, MS-ESI (m/z): [M+H] + calcd for C 39 H 48 N 3 O 8, ; found, PGD 7. To a solution of compounds 5 (0.11 g, 0.16 mmol) and 6 (0.64 g, 0.73 mmol) in DMF (3 ml) were added CuSO 4 5H 2 O (40 mg, 0.16 mmol) and sodium ascorbate (64 mg, 0.32 mmol). The mixture was stirred at 25 C for 18 h and then filtered. Subsequently, the filtrate was concentrated under reduced pressure and precipitated into cold water. The precipitated crude product was purified by column chromatography on silica gel using dichloromethane/methanol (20:1) as eluent to afford 7 (0.53 g, 99%). 1 H NMR (CDCl 3 ): δ (br, 11H), 6.94 (br, 1H), (m, 25H), (m, 48H), (br, 169H), 3.05 (m, 3H), (br, 15H). MALDI-TOF (m/z): [M+K] + calcd for C 174 H 272 KN 12 O 50, ; found, PGD 9. To a solution of 7 (85 mg, 25 μmol) in dichloromethane (8 ml) was added TFA (0.5 ml) at 0 C. The mixture was stirred at 25 C for 2 h and then evaporated under reduced pressure. The residue was redissolved in dichloromethane and N- methylmorpholine (0.1 ml) was added. The mixture was stirred at 25 C for 10 min and evaporated under reduced pressure to obtain 8 which was used without further purification. To a solution of the crude 8 in acetone/water/t-butanol (5:5:1) were added K 2 OsO 4 2H 2 O (catalytic amount), N-methylmorpholine-N-oxide(0.28 g, 2.4 mmol), and citric acid (0.12 g, 0.60 mmol). After the mixture was stirred at 25 C for 15 h, excess Smopex-105 (an osmium scavenger) was added and the solution was stirred at 25 C for 24 h. The mixture was filtered to remove the osmium scavenger and the filtrate was evaporated under reduced pressure to afford the crude 9 that was further purified by dialysis against water at 25 C for 2 d (0.10 g, 97%). 1 H NMR (CD 3 OD): δ (br, 13H), (br, 249H). MALDI-TOF (m/z): [M+Na] + calcd for C 169 H 312 N 12 NaO 96, ; found, PGD 10. To a solution of 9 (40 mg, 9.9 μmol) in DMF (1.5 ml) was added biotin NHS ester (67 mg, 0.20 mmol). The mixture was stirred at 25 C for 18 h, concentrated under reduced pressure, and then precipitated into dichloromethane. Subsequently, the precipitate was dissolved in water and dialyzed against water at 25 C for 1 d to afford 10 (30 mg, 71%). 1 H NMR (CD 3 OD): δ (br, 14H), (br, 262H). MALDI- TOF (m/z): [M+Na] + calcd for C 179 H 326 N 14 NaO 98 S, ; found, PGD 11. To a solution of 10 (30 mg, 7.0 mmol) in DMF (2 ml) was added piperidine (0.4 ml). After the mixture was stirred at 25 C for 1 h, the solvent was removed under reduced pressure and the residue was dissolved in water and filtered off. The filtrate was dialyzed against water at 25 C for 1 d to obtain 11 (21 mg, 74%). 1 H NMR (CD 3 OD): δ (br, 5H), (br, 259H). MALDI-TOF (m/z): [M+Na] + calcd for C 164 H 316 N 14 NaO 96 S, ; found, NATURE CHEMISTRY 3

4 PGD-KFL 12. To a solution of 11 (1.0 mg, 0.25 mmol) and 3 (2.0 mg, 2.9 mmol) in DMF (0.2 ml) was added triethylamine (30 ml). After the mixture was stirred at 25 C for 7 d, the solvent was removed under reduced pressure and the residue was dissolved in water and filtered off to remove excess 3. The filtrate was washed with dichloromethane and dialyzed against water at 25 C for 1 d to afford 12 (1.0 mg, 87%). 1 H NMR (CD 3 OD): δ (br, 22H), (br, 265H). MALDI-TOF (m/z): [M+Na] + calcd for C 198 H 337 BF 2 N 16 NaO 101 S, ; found, UV-Vis (H 2 O): l max (e) = 685 nm ( M -1 cm -1 ). Fluorescence (H 2 O): l max (Φ) = 705 nm (0.57). PGD-BODIPY 650/665X. PGD-BODIPY 650/665X (1.0 mg, 89%) was obtained from 11 (1.0 mg, 0.25 mmol) and BODIPY 650/665X NHS ester (1.6 mg, 2.5 mmol) according to the synthetic procedure employed for compound H NMR (CD 3 OD): δ (br, 22H), (br, 271H). MALDI-TOF (m/z): [M+Na] + calcd for C 193 H 343 BF 2 N 18 NaO 99 S, ; found, PGD-Cy5. To a solution of 11 (0.5 mg, 0.12 mmol) and Cy5 NHS ester (0.8 mg, 1.0 mmol) in DMF (0.2 ml) was added triethylamine (15 ml). After the mixture was stirred at 25 C for 2 d, the solvent was removed under reduced pressure and the residue was dissolved in water. Excess Cy5 NHS ester was removed using an Amicon Ultra-4 centrifugal filter (MWCO 3000, Millipore) to obtain PGD-Cy5 (0.3 mg, 52%). 1 H NMR (CD 3 OD): δ (br, 17H), (br, 286H). MALDI-TOF (m/z): [M+Na] + calcd for C 197 H 353 KN 16 NaO 103 S 3, ; found, Microtubule-12. Mouse embryonic fibroblast (MEF) cells were grown about 2 d in a 8- well chamber (lab-tek), fixed by 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X in PBS for 15 min, blocked with 3% BSA in PBS for 1 h, and incubated with primary antibody against β-tubulin ( , Invitrogen) for 1 h. Then the cells were incubated with biotinylated secondary antibody (sc-2098, santa cruz biotech) for 1 h, followed by neutravidin (20 μg/ml) incubation for 30 min and final incubation with 12 (100 nm) for 30 min. All procedures were performed at room temperature. Control cells were incubated in the absence of neutravidin. NATURE CHEMISTRY 4

5 Figure S1. Single-molecule fluorescence image of immobilized 12 in the presence of both Trolox and an oxygen scavenging system. NATURE CHEMISTRY 5

Figure S2. Fluorescence intensity traces of 50 representative molecules of PGD-KFL 12 in the presence of an oxygen scavenging system without Trolox.

6 Figure S2. Fluorescence intensity traces of 50 representative molecules of PGD-KFL 12 in the presence of an oxygen scavenging system without Trolox. Y-axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 6

7 Figure S3. Fluorescence intensity traces of 50 representative molecules of BODIPY 650/665X attached to D27-5Am DNA in the presence of an oxygen scavenging system without Trolox. Y-axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 7

8 Figure S4. Fluorescence intensity traces of 50 representative molecules of Cy5 attached to D27-5Am DNA in the presence of an oxygen scavenging system without Trolox. Y- axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 8

9 Figure S5. Fluorescence intensity traces of 50 representative molecules of KFL 3 attached to D27-5Am DNA in the presence of an oxygen scavenging system without Trolox. Y-axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 9

10 Figure S6. Fluorescence intensity traces of 50 representative molecules of PGD- BODIPY 650/665X in the presence of an oxygen scavenging system without Trolox. Y- axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 10

Figure S7. Fluorescence intensity traces of 50 representative molecules of PGD-Cy5 in the presence of an oxygen scavenging system without Trolox.

11 Figure S7. Fluorescence intensity traces of 50 representative molecules of PGD-Cy5 in the presence of an oxygen scavenging system without Trolox. Y-axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 11

12 Figure S8. Fluorescence intensity traces of 50 representative molecules of PGD-KFL 12 in the presence of an oxygen scavenging system without Trolox, with 50 mw power at 640 nm. Y-axis values correspond to fluorescence intensity (Counts / 100 ms) as a function of time (s, X-axis). NATURE CHEMISTRY 12

13 Figure S9. Fluorescence labeling of microtubule network in mouse embryonic fibroblast (MEF) cells. (a) Bright-field and fluorescence images of cells labeled with 12 after incubation in the presence of neutravidin. (b) As a control, cells were labeled without neutravidin. Scale bars = 5 μm. NATURE CHEMISTRY 13

14 Figure S10. 1 H NMR spectrum of 3 in CDCl 3. Figure S11. 1 H NMR spectrum of 5 in CDCl 3. Figure S12. 1 H NMR spectrum of 7 in CDCl 3. NATURE CHEMISTRY 14

15 Figure S13. 1 H NMR spectrum of 8 in CDCl 3. Figure S14. 1 H NMR spectrum of 9 in CD 3 OD. Figure S15. 1 H NMR spectrum of 10 in CD 3 OD. NATURE CHEMISTRY 15

16 Figure S16. 1 H NMR spectrum of 11 in CD 3 OD. Figure S17. 1 H NMR spectrum of 12 in CD 3 OD. Figure S18. 1 H NMR spectrum of PGD-BODIPY 650/665X in CD 3 OD. NATURE CHEMISTRY 16

17 Figure S19. 1 H NMR spectrum of PGD-Cy5 in CD 3 OD. References. (1) Verdree, V. T., Pakhomov, S., Su, G., Allen, M. W., Countryman, A. C., Hammer, R. P. & Soper, S. A. Water soluble metallo-phthalocyanines: the role of the functional groups on the spectral and photophysical properties. J. Fluoresc. 17, (2007). (2) Umezawa, K., Nakamura, Y., Makino, H., Citterio, D. & Suzuki, K. Bright, colortunable fluorescent dyes in the visible near-infrared region. J. Am. Chem. Soc. 130, (2008). (3) Schlick, K. H., Morgan, J. R., Weiel, J. J., Kelsey, M. S. & Cloninger, M. J. Clusters of ligands on dendrimer surfaces. Bioorg. Med. Chem. Lett. 21, (2011). (4) Elmer, S. L., Man, S. & Zimmerman, S. C. Synthesis of polyglycerol, porphyrincored dendrimers using click chemistry. Eur. J. Org. Chem. 2008, (2008). NATURE CHEMISTRY 17

Supplementary Figure 2. Full power on times. Histogram showing on times of bursts with 100 pm 1, 100 pm 2 and 1 nm Et 3 N at full laser power.

Supplementary Figure 2. Full power on times. Histogram showing on times of bursts with 100 pm 1, 100 pm 2 and 1 nm Et 3 N at full laser power. S1 Supplementary Figures Supplementary Figure 1. Time-correlated still frame images. Expanded still frames images from TIRFM video of CuAAC of 1 and 2 and corresponding intensity trajectory of a single