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1 Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues Mayeul Collot,* Tkhe Kyong Fam, Pichandi Ashokkumar, restis Faklaris, Thierry Galli, Lydia Danglot, Andrey S. Klymchenko Supporting information 1) Synthesis and characterization 1 1 was synthesized according to a reported protocol. 1 F F B BF 3.Et 2 Ac Protocol adapted from Zyabrev et al. 2 To 1 (1.00 g, mmol) was added acetic anhydride (10 ml) and the heterogeneous solution was warmed at 95 C. To the hot heterogeneous solution was added BF 3.Et 2 (520 µl, mmol, 1.2eq), the solution became homogeneous and yellowish. After 30 minutes TLC showed that the reaction was completed. The solvents were evaporated, MeH was added and the white solid was filtered to give g of 2 as a white powder (Yield = 77%). Rf=0.34 (100% DCM). 1 H- MR (400 MHz, CDCl 3 ): δ 4.79 (m, 2H, CH- ), 2.84 (s, 3H, CH 3 ), (m, 4H, CH 2 CyHex), (m, 4H, CH 2 CyHex), (m, 6H, CH 2 CyHex), (m, 6H, CH 2 CyHex). 13- C MR (101 MHz; CDCl 3 ): δ (C enol), (C), (C urea), 148.2(C), 95.2 (Cq), 58.0 (CH- ), 55.4 (CH3), 29.2 (C CyHex), 28.7 (C CyHex), (C CyHex), (C CyHex), (C CyHex), 25.2, 24.9 (C CyHex). HRMS (ESI + ), calcd for C 18 H 26 BF a [M+H] , found S1

2 1 H MR spectrum of 2 13 C MR spectrum of 2 S2

3 HRMS spectrum of 2 I - I - Pentyn indoleninium iodide and pentyn benzoindoleninium iodide were synthesized according to a published protocol. 3 General procedure for the synthesis of the Statomerocyanines. To a solution of pentynyl indoleninium 1 or pentynyl benzoindoleninium 2 (1eq) in acetic anhydride (C=0.1 M) was added 1.1 equivalent of,ʹ - Diphenylformamidine, or malonaldehyde dianilide hydrochloride or glutaconaldehydedianil hydrochloride. The solution was heated at 100 C for 3 h before the solvents were evaporated. To the residue was added 1.1 equivalent and pyridine was added (C=0.1 M). The mixture was heated at 50 C for 2 h. The solvents were evaporated and the residue was extracted with EtAc, washed with HCl (1 M) and a saturated solution of ahc 3. The organic phase was dried over anhydrous MgS 4 filtered, evaporated and the crude was purified by column chromatography on silica gel. F F B SMCy-3 Conditions for column chromatography on silica gel: 99:1 to 8:2 heptane:etac. Rf=0.38 (7:3 heptane:etac). Pink powder, yield= 25%. MR spectra revealed the presence of - acetylaniline that could not be separated by chromatography nor by crystallization. 1 H- MR (400 MHz, CDCl 3 ): δ 8.70 (t, J = 13.4 Hz, 1H, H ethylene), 7.64 (d, J = 13.2 Hz, 1H, H ethylene), (m, 1H, H Ar), (m, 1H, H Ar), 7.20 (m, 1H), 7.09 (d, J = 8.0 Hz, 1H, H Ar), 6.05 (d, J = 13.5 Hz, 1H, H ethylene), (m, 2H, CH- ), (m, 2H, CH 2 - ), S3

4 2.33 (m, 6H), 2.21 (s, 1H, C CH), (m, 2H), (m, 4H), (m, 2H), (m, 10H, 2 CH3 indolenine + 4 CH), (m, 6H, CH CyHex). 13 C MR (101 MHz; CDCl 3 ): δ 175.4, 172.8, 172.4, 162.4, 160.4, 151.2, (C Ar), (C Ar), 14 (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), 90.3 (C Ar), 82.3 (C alkyne), 70.5 (CH alkyne), 57.0, 54.7, 48.8, 42.3, 31.9, 29.4, 28.84, 28.79, 26.48, 26.38, 25.6, 25.3, 25.1, 24.4, F MR (376 MHz; CDCl 3 ): δ HRMS (ESI+), calcd for C 35 H 42 BF a [M+a] , found H MR spectrum of SMCy- 3 S4

5 13 C MR spectrum of SMCy F MR spectrum of SMCy- 3 S5

6 HRMS spectrum of SMCy- 3 F F B SMCy-3.5 Conditions for column chromatography on silica gel: 8:2 to 5:5 to 0:1 heptane:dcm. Rf=0.46 (7:3 heptane:etac). Dark red powder, yield= 58%. 1 H- MR (400 MHz, CDCl 3 ): δ 8.82 (t, J = 13.3 Hz, 1H, H ethylene), 8.12 (d, J = 8.7 Hz, 1H), (m, 2H), (m, 2H), 7.48 (t, J = 7.4 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 6.10 (d, J = 13.6 Hz, 1H, H ethylene), 4.85 (s, 2H, CH- ), 4.14 (s, 2H, CH 2 - ), 2.40 (s, 6H), 2.25 (s, 1H, C CH), (m, 8H, 2 CH 3 indolenine + 2 CH), (m, 10H), (m, 6H). 13 C MR (101 MHz; CDCl 3 ): δ 174.9, 174.3, 165.2, 160.4, (CH Ar), 149.2, 139.5, 131.5, (CH Ar), (CH Ar), 128.3, , (CH Ar), (CH Ar), (CH Ar), (CH Ar), (CH Ar), 99.6 (CH Ar), 90.2, 82.2 (C alkyne), 7 (CH alkyne), 57.0, 54.7, 5, 31.9, 29.7, 29.4, 28.86, 28.81, 28.3, 26.50, 26.40, 25.84, 25.83, 25.4, 25.1, F MR (376 MHz; CDCl 3 ): δ HRMS (ESI + ), calcd for C 39 H 42 BF a [M+a] , found S6

7 1 H MR spectrum of SMCy C MR spectrum of SMCy- 3.5 S7

8 19 F MR spectrum of SMCy- 3.5 HRMS spectrum of SMCy- 3.5 F F B SMCy-5 Conditions for column chromatography on silica gel: 5:5 to 0:1 heptane:dcm. Rf=0.42 (7:3 heptane:etac). Dark blue amorphous solid, yield= 60%. 1 H- MR (500 MHz, CDCl 3 ): δ 8.24 (t, J = 13.1 Hz, 1H, H ethylene), 7.69 (m, 2H, 2 H Ar), (signal in the CDCl 3 peack, 2H), 7.13 (t, J = 7.4 Hz, 1H, H Ar), 6.98 (d, J = 7.9 Hz, 1H, H Ar), 6.40 (t, J = 12.8 Hz, 1H, H ethylene), 5.89 (d, J = 13.0 Hz, 1H, H ethylene), 4.83 (m, 2H, CH- ), 3.97 (t, J = 7.3 Hz, 2H, CH 2 - ), (m, 6H), 2.19 (t, J = 2.5 Hz, 1H, C CH), 1.98 (t, J = 6.9 Hz, 2H), 1.87 (m, 4H), 1.77 (d, J = 10.4 Hz, 2H), (m, 10H, CH CyHex, 2 CH 3 indolenine), 1.38 (m, 4H), 1.27 (m, 2H). S8

9 19- F MR (376 MHz; CDCl3): δ HRMS (ESI + ), calcd for C 37 H 45 BF [M+H] , found H MR spectrum of SMCy C MR spectrum of SMCy- 5 S9

10 19 F MR spectrum of SMCy- 5 HRMS spectrum of SMCy- 5 F F B SMCy-5.5 Conditions for column chromatography on silica gel: 5:5 to 0:1 heptane:dcm. Rf=0.45 (7:3 heptane:etac). Shiny dark turquoise solid, yield= 26%. 1 H- MR (400 MHz, CDCl 3 ): δ 8.28 (t, J = 13.1 Hz, 1H, H ethylene), 8.10 (d, J = 8.5 Hz, 1H, H Ar), 7.90 (2d, J = 8.5 Hz, 2H), 7.83 (t, J = 13.0 Hz, 1H, H ethylene), 7.68 (d, J = 13.7 Hz, 1H, H ethylene), 7.60 (t, J = 7.7 Hz, 1H, H Ar), 7.43 (t, J = 7.5 Hz, 1H, H Ar), 7.43 (t, J = 7.5 Hz, 1H, H Ar), 7.30 (signal in the CDCl 3 peack, 1H, H Ar), 6.43 (t, J = 12.7 Hz, 1H, H ethylene), 5.93 (d, J = 12.9 Hz, 1H, H ethylene), (m, S10

11 2H, CH- ), (m, 2H, CH 2 - ), (m, 6H, CH CyHex), 2.22 (s, 1H, C CH), (m, 12H, CH CyHex, 2 CH 3 indolenine), (m, 2H, CH CyHex), (m, 4H, CH CyHex), (m, 6H, CH CyHex). 13 C MR (101 MHz; CDCl 3 ): δ 175.7, 170.0, 165.4, 160.2, 155.9, 150.3, (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), (C Ar), 99.4 (C Ar), 90.9 (C Ar), 82.5 (C alkyne), 70.4 (CH alkyne), 57.1, 54.7, 50.0, 41.9, 31.9, 29.4, 29.01, 28.82, 27.8, 26.47, 26.42, 26.39, 25.8, 25.3, 25.1, F MR (376 MHz; CDCl 3 ): δ HRMS (ESI + ), calcd for C 41 H 46 BF a [M+a] , found The most abundant peak is the oxidized form, formed in the source [M+15] calcd for C 41 H 44 BF a [M- 2H+] , found H MR spectrum of SMCy- 5.5 S11

12 13 C MR spectrum of SMCy C MR spectrum of SMCy- 5.5 S12

13 HRMS spectrum of SMCy- 5.5 F F B SMCy-7 Conditions for column chromatography on silica gel: 5:5 to 0:1 heptane:dcm. Rf=0.52 (7:3 heptane:etac). Dark green solid, yield= 11%. Crystallization for X- ray crystallography: SMCy7 was dissolved in DCM and single crystals were obtained by slow solvents exchange with heptane. 1 H- MR (400 MHz, CDCl 3 ): δ 8.11 (t, J = 13.1 Hz, 1H, H ethylene), 7.76 (d, J = 14.0 Hz, 1H, H ethylene), 7.36 (t, J = 13.2 Hz, 1H, H ethylene), (m, 2H), 7.14 (t, J = 12.8 Hz, 1H, H ethylene), 7.04 (t, J = 7.4 Hz, 1H, H Ar), 6.89 (d, J = 7.8 Hz, 1H, H Ar), 6.48 (t, J = 13.0 Hz, 1H, H ethylene), 6.27 (t, J = 12.8 Hz, 1H, H ethylene), 5.72 (d, J = 12.7 Hz, 1H, H ethylene), 4.83 (q, J = 12.3 Hz, 2H, CH- ), 3.88 (t, J = 7.3 Hz, 2H, CH 2 - ), (m, 6H), 2.15 (s, 1H, C CH), (m, 6H), (m, 2H), 1.66 (m, 10H), 1.41 (m, 6H). 13 C MR (101 MHz; CDCl 3 ): δ 177.6, 165.9, 163.6, 159.9, 154.7, 152.9, 148.9, 143.4, 142.9, 139.5, 128.1, 126.4, 124.8, , , 117.0, 107.8, 98.7, 91.6, 82.8 (C alkyne), 69.9 (CH alkyne), 57.3, 54.9, 47.1, 41.5, 31.9, 29.4, 28.8, 28.4, 26.44, 26.35, 25.40, 25.30, 25.0, F MR (376 MHz; CDCl3): δ The most abundant peak is the oxidized form, formed in the source [M+15] calcd for C 39 H 45 BF [M- 2H+] , found X- ray analysis of single crystals of SMCy7 confirmed the structure. S13

14 1 H MR spectrum of SMCy C MR spectrum of SMCy- 7 S14

15 19 F MR spectrum of SMCy- 7 HRMS spectrum of SMCy- 7 F F B SMCy-7.5 Conditions for column chromatography on silica gel: 3:7 to 5:5 heptane:dcm. Rf=0.50 (7:3 heptane:etac). Dark shiny green solid, yield= 27%. The product was found to be insoluble at concentrations required for MR in several MR solvents (CDCl 3, MeH, DMS and mixtures). The most abundant peak is the oxidized form, formed in the source [M+15] calcd for C 43 H 47 BF [M- 2H+] , found S15

16 HRMS spectrum of SMCy- 7.5 F F B + P 3 Pyridine CuI 75 C F F B SMCy3 (3-azidopropyl)triphenylphosphonium bromide P SMCy3-M To a solution of SMCy- 3 (20 mg, µmol) and (3-azidopropyl)triphenylphosphonium bromide (15 mg, µmol, 1.08 eq) in pyridine (2 ml) was added CuI (8 mg, 42.1 µmol, 1.3 eq) ans the solution was allowed to stir at 75 C under argon for 2 h. The solvent was evaporated and the residue was extracted with DCM, washed with HCl (1 M) and a saturated solution of ahc 3. The organic phase was dried over anhydrous MgS 4 filtered, evaporated and the crude was purified by column chromatography on silica gel (98/2 to 9/1 DCM/MeH) to give 11 mg of SMCy3- M as dark pink amorphous solid (yield= 35%). MS (ESI + ), calcd for C 56 H 63 BF P [M] , found S16

17 1 H MR spectrum of SMCy- 3- M 19 F MR spectrum of SMCy- 3- M S17

18 MS spectrum of SMCy- 3- M Spectroscopy Absorbance SMCy Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) ormalized F.I Figure S1. Absorption and emission spectra of SMCy3 (1 µm) in various solvents with increasing polarity. Excitation wavelength: 490 nm S18

19 Table S1. Photophysical properties of SMCy3 in various solvents. Excitation wavelength: 490 nm. The quantum yields were determined using Rhodamine 6G as a reference. 4 Absorbance SMCy3.5 Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) ormalized F.I Figure S2. Absorption and emission spectra of SMCy3.5 (1 µm) in various solvents with increasing polarity. Excitation wavelength: 490 nm. S19

20 Table S2. Photophysical properties of SMCy3.5 in various solvents. Excitation wavelength: 490 nm. The quantum yields were determined using Rhodamine 6G as a reference. The quantum yields were determined using Rhodamine 6G as a reference. 4 SMCy5 Absorbance Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) Figure S3. Absorption and emission spectra of SMCy5 (200 nm) in various solvents with increasing polarity. Excitation wavelength: 570 nm. ormalized F.I S20

21 Table S3. Photophysical properties of SMCy5 (200 nm) in various solvents. Excitation wavelength: 570 nm. The quantum yields were determined using DID as a reference. 5 Absorbance SMCy5.5 Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) ormalized F.I Figure S4. Absorption and emission spectra of SMCy5.5 (200 nm) in various solvents with increasing polarity. Excitation wavelength: 590 nm. S21

22 Table S4. Photophysical properties of SMCy5.5 (200 nm) in various solvents. Excitation wavelength: 590 nm. The quantum yields were determined using DID as a reference. 5 Absorbance SMCy Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) ormalized F.I Figure S5. Absorption and emission spectra of SMCy7 (1 µm) in various solvents with increasing polarity. Excitation wavelength: 630 nm. S22

23 Table S5. Photophysical properties of SMCy7 (1 µm) in various solvents. Excitation wavelength: 630 nm. The quantum yields were determined using Rhodamine- 800 as a reference. 6 Absorbance SMCy7.5 Toluene Dioxane Ethyl Acetate THF EtH MeH DCM AC DMF DMS ormalized Absorbance Fluorescence Intensity (A.U.) ormalized F.I Figure S6. Absorption and emission spectra of SMCy7 (1 µm) in various solvents with increasing polarity. Excitation: 630 nm for toluene to THF and 690 nm for EtH to DMS. S23

24 Table S6. Photophysical properties of SMCy7.5 (1 µm) in various solvents. From toluene to THF: excitation wavelength was 630 nm, Rhodamine 800 was used to determine the quantum yields. 6 From EtH to DMS, excitation wavelength was 690 nm, Indocyanine Green was used to determine the quantum yields. 7 ormalized Fluorescence Intensity MC-540 SMCy3 DID (Cy5) SMCy Time (min) Figure S7. Photostability of SMCy3 compared to MC- 540 (orange lines, 200 nm in dioxane, excitation at 520 nm (0.95 mw/cm 2 ), fluorescence intensity monitored at 560 nm) and SMCy5 compared to DID (blue lines, 200 nm in dioxane, excitation at 600 nm (0.73 mw/cm 2 ), fluorescence intensity monitored at 640 nm). Excitation and emission slits were 10 and 1 respectively. S24

25 ormalized Fluorescence Intensity SMCy3 SMCy5 ile Red Bodipy 496/ Time (min) Figure S8. Decrease of fluorescence intensity of different LDs markers in labrafac (1 µm) under continuous excitation. Each dye was excited at the same molar extinction coefficient value: 40,000 M- 1.cm- 1. The excitation slit opening was 16 mm. Figure S9. Laser scanning confocal microscopy of KB cells incubated 3 hours in a starving medium (opti- MEM) in the presence of fluorophores (500 nm). Hoechst (5 µg. ml- 1) was used to stain the nucleus (blue). Green channel (left column) excitation at 488 nm emission collected: nm; Far- red channel (middle column) excitation at 635 nm, emission collected: nm. Right column is the merge of the green and red channel. This experiment showed that there was no cross talk of ile red in the far- red channel and therefore that the fluorescence observed in the lipid droplets was due to SMCy7. S25

26 Figure S10. Images of KB cells incubated with SMCy3 (1 µm) at 37 C for different amount of time. For fixed cells, the cells were first fixed with 4% PFA, washed and then incubated at 37 C in the presence of SMCy3 for different amount of time, the fluorescence intensity of images has been enhanced 4- fold compared to live cells. Each image is the max projection of Z- stack images in order to display a maximum of LDs. The nucleus of cells was stained with Hoechst (5 µg. ml- 1). Figure S11. Laser scanning confocal microscopy images of live KB cells right after addition of BDIPY 493/503 (A) and SMCy3 (B). Concentrations of the dyes were 500 nm and the nucleus was stained with Hoechst (5 µg. ml- 1). Laser excitation was 488 nm and emission range nm. S26

27 A B ormalized Absorbance µm 2 µm 1 µm 0.5 µm 0.2 µm 0.1 µm Soluble form in oil ormalized Absorbance µm 2 µm 1 µm 0.5 µm 0.2 µm 0.1 µm Soluble form in oil Figure S12. ormalized absorption spectra of BDIPY 493/503 (A) and SMCy5 (B) in water with increasing concentrations. The broadening of the spectra depicts the aggregation. For comparison, spectra of BDIPY 493/503 (1 µm) and SMCy5 (200 nm) in colza oil show the soluble states. Figure S13. DLS measurements of SMCy3 (A), SMCy3.5 (B) and SMCy5 (C) in water at 1 µm. S27

28 Figure S14. Laser scanning confocal microscopy images of live KB cells after incubation of BDIPY 493/503 (A) and SMCy3 (B) for 1 h. Concentrations of the dyes were 500 nm and the nucleus was stained with Hoechst (5 µg. ml- 1). Laser excitation was 488 nm and emission range nm. Conditions of illumination, recording and processing were identical. Figure S15. Laser scanning confocal microscopy images of live KB cells after incubation of BDPIY 493/503 (A) and SMCy5 (B) for 1 h in opti- MEM. C is the merge of the A and B. Concentrations of the dyes were 500 nm and the nucleus was stained with Hoechst (5 µg. ml- 1). Laser excitation was 488 nm and emission range nm for BDIPY 493/503 and 561 nm, for SMCy5. Pearson s coefficient depicting the goodness of colocalization was 4 ± 0.02 (5 different images). S28

29 ormalized Fluorescence Intensity BDP 493/503 SMCy Frame Figure S16.Photostability of green LD markers: BDIPY 493/503 and SMCy3 assessed by laser scanning confocal microscopy using live KB cells after incubation at 500 nm for 1h in opti- MEM. Laser excitation was 488 nm and emission range nm. Laser power was set to 100%. S29

30 Cytotoxicity Figure S17. Cytotoxicity assay of the SMCy probes after 3 h incubation quantified by the MTT assay. S30

31 Figure S18. 3D imaging of fixed (4% PFA) KB cells incubated 3 hours in opti- MEM with 500 nm (A) SMCy- 3, (B) SMCy- 3.5, (C) SMCy- 5, (D) SMCy- 5.5, (E) SMCy- 7, (F) SMCy The nuclei (gray) were stained with Hoechst (5 µg. ml- 1), the lipid droplets are depicted in rainbow colored spots. The images were obtained by reconstruction of laser scanning confocal microscopy Z stack images (21 µm depth Z- stack (300 nm stepwise) and were processed using icy software Slope = log I (output intensity) (a.u.) log I (input laser power) (a.u.) Figure S19. Quadratic intensity dependence plot for SMCy3 (6.04 µm; λ Ex =690 nm) S31

32 13.6 Slope = log I (output intensity) (a.u.) log I (input laser power) (a.u.) Figure S20. Quadratic intensity dependence plot for SMCy3.5 (2.08 µm; λ Ex = 760 nm) Slope = log I (output intensity) (a.u.) log I (input laser power) (a.u.) Figure S21. Quadratic intensity dependence plot for SMCy5 (1.06 µm; λ Ex = 780 nm) S32

33 13.4 Slope = 1.90 log I (output intensity) (a.u.) log I (input laser power) (a.u.) Figure S22. Quadratic intensity dependence plot for SMCy5.5 (285 nm; λ Ex = 820 nm) Figure S23. Crosstalk control for SMCy3- M. Laser scanning confocal microscopy of KB cells incubated for 1 h with Hoechst (5 µg/mg, blue color) and 200 nm of SMCy3- M. A is the green channel: excitation at 488 nm emission collected: nm; B is the red channel Far- red channel excitation at 561 nm, emission collected: nm. C is the merge of A and B. This experiment showed that there was no crosstalk of SMCy3- M in the red channel. Scale bar is 10 µm. References: (1) Bose, A. K.; Garratt, S. Tetrahedron 1963, 19, (2) Zyabrev, K.; Doroshenko, A.; Mikitenko, E.; Slominskii, Y.; Tolmachev, A. Eur. J. rg. Chem. 2008, 2008, (3) Gerowska, M.; Hall, L.; Richardson, J.; Shelbourne, M.; Brown, T. Tetrahedron 2012, 68, (4) Magde, D.; Rojas, G. E.; Seybold, P. G. Photochem. Photobiol. 1999, 70, (5) Texier, I.; Goutayer, M.; Da Silva, A.; Guyon, L.; Djaker,.; Josserand, V.; eumann, E.; Bibette, J.; Vinet, F. J. Biomed. pt. 2009, 14, (6) Alessi, A.; Salvalaggio, M.; Ruzzon, G. J. Lumin. 2013, 134, (7) Benson, R.; Kues, H. Phys. Med. Biol. 1978, 23, S33