Development of Fluorescein Analogue, TokyoMagenta, as a Novel Scaffold. for Fluorescence Probes in Red Region

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1 Supplementary Material (ESI) for Chemical Communications Supporting Information for: Development of Fluorescein Analogue, TokyoMagenta, as a Novel Scaffold for Fluorescence Probes in Red Region Takahiro Egawa, a Yuichiro Koide, a,b Kenjiro Hanaoka, a,b Toru Komatsu, a Takuya Terai, a,b and Tetsuo Nagano*,a,b a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-, Hongo, Bunkyo-ku, Tokyo 3-33, Japan b CREST, Japan Science and Technology Agency, Sanbancho-blg, 5 Sanbancho, Chiyoda-ku, Tokyo, 2-75, Japan tlong@mol.f.u-tokyo.ac.jp S

2 Methods Supplementary Material (ESI) for Chemical Communications General Methods. General chemicals were of the best grade available, supplied by Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical Co., Alfa Aesar, Dojindo, GE Healthcare and Invitrogen, and were used without further purification. All solvents were used after appropriate distillation or purification. NMR spectra were recorded on a JEL JNM-LA3 instrument at 3 MHz for H NMR and at 75 MHz for 3 C NMR or a JEL JNM-LA4 instrument at 4 MHz for H NMR and at MHz for 3 C NMR. Mass spectra (MS) were measured with a JEL JMS-TLC AccuToF using ESI. δ values are given in ppm relative to tetramethylsilane. HPLC analysis was performed on an Inertsil DS-3 ( mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-98, JASC) and a detector (MD-25 or FP-225, JASC). Preparative HPLC was performed on an Inertsil DS-3 (. 25 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-28, JASC) and a detector (MD-25 or FP-225, JASC). UV-Vis Absorption and Fluorescence Spectroscopy. UV-visible spectra were obtained on a Shimadzu UV-65. Fluorescence spectroscopic studies were performed on a Hitachi F45. The slit width was 2.5 nm for both excitation and emission. The photomultiplier voltage was 7 V. Relative fluorescence quantum efficiency of 2-Me TM was obtained by comparing the area under the emission spectrum of the test sample excited at 54 nm with that of a solution of Rhodamine B in EtH, which has a quantum efficiency of.65, SR and 2-Me TM βgal was referred to fluorescein in. M NaH, whose quantum efficiency is.85. SR2 Fluorescence Imaging. A confocal imaging system (TCS-SP5; Leica) equipped with an argon laser and a white light laser was used. Fluorescence images were taken with monitoring of fluorescence in the 6-62 nm channel. The excitation wavelength was 58 nm (white light laser). HEK293 cells were cultured in Dulbecco s modified Eagle s medium (DMEM) (Invitrogen Corp.) supplemented with % (v/v) fetal bovine serum (Invitrogen Corp.), % penicillin and % streptomycin (Invitrogen Corp.) in a humidified incubator containing 5% C 2 in air. For fluorescence microscopy, HEK293 cells were plated in a 35-mm S2

3 Supplementary Material (ESI) for Chemical Communications PDL-coated glass-bottomed dish (MatTek Corporation) in Dulbecco s modified Eagle s medium (DMEM). For dye loading, the cells were incubated with μm 9 in DMEM for 3 min in an incubator. S3

4 Synthetic procedures and characterizations Supplementary Material (ESI) for Chemical Communications Elution in all HPLC analyses was done with a 2-min linear gradient from 2 % CH 3 CN /. % TFA aq. to 8 % CH 3 CN /. % TFA aq. (flow rate =. ml / min). UV, 46 nm. 3-Bromo-N,N-diallylaniline (2) Br N To a suspension of K 2 C 3 (22. g, 59 mmol) in MeCN were added 3-bromoaniline (8.7 ml, 8. mmol) and allyl bromide (23.7 ml, 28 mmol), and the mixture was stirred at 8 C for 4 h. After cooling, the reaction mixture was filtered through a Celite pad, washed with AcEt and evaporated to dryness. The residue was purified by column chromatography (silica gel, /4 AcEt/hexane) to give pure 2 (7. g, 67.9 mmol, 85% yield). H NMR (3 MHz, CDCl 3 ): δ (m, 4H), (m, 2H), (m, 2H), (m, 2H) 6.58 (dd, H, J = 2.2, 8. Hz), (m. 2H) 7. (t, H, J = 8.Hz); 3 C NMR (75 MHz, CDCl 3 ): δ 52.7,.8, 5., 6.3, 9., 23.3, 3.2, 33.2, 5.; HRMS (ESI + ): m/z Found , calculated for [M+H] + (+4. mmu). Bis(2-bromo-4-N,N-diallylaminophenyl)methane (3) Br Br N N To a solution of compound 2 (7. g, 67.9 mmol) in AcH (2 ml) was added 37% formaldehyde (.2 g, 34 mmol), and the mixture was stirred at 8 C for 75 min. After cooling to room temperature, the reaction mixture was neutralized with saturated NaHC 3 aq. and NaH aq., and extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was purified by column chromatography (silica gel, /3 AcEt/hexane) to give pure 3 (5.2 g, 29.5 mmol, 87% yield). H NMR (3 MHz, CDCl 3 ): δ (m, 8H), 3.96 (s, 2H), (m, 8H), (m, 4H), 6.54 S4

5 (dd, 2H, J = 2.9, 8.8 Hz), 6.8 (d, 2H, J =8. Hz), 6.9 (d, 2H, J = 2.9 Hz); 3 C NMR (75 MHz, CDCl 3 ): δ 39.7, 52.7,.7, 6., 6.2, 25.5, 26.9, 3.8, 33.5, 48.; HRMS (ESI + ): m/z Found , calculated for [M+H] + ( 2.3 mmu). Supplementary Material (ESI) for Chemical Communications N,N,N',N'-Tetraallyldiamino--xanthone (4) N N To a flame-dried flask flushed with argon, compound 3 (8.6 g, 5.8 mmol) and anhydrous THF (5 ml) were added. The solution was cooled to 78 C, M sec-buli (45 mmol) was added, and the mixture was stirred for 2 min. At the same temperature, a solution of Me 2 Cl 2 (2.9 ml, 3 mmol) in anhydrous THF (5 ml) was slowly added, and the mixture was warmed to room temperature, then stirred for h. The reaction was quenched by addition of 2 N HCl aq., then the mixture was neutralized with NaHC 3, and extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was dissolved in CH 3 CCH 3 (5 ml), and the solution was cooled to C. To this solution, KMn 4 (6.88 g, 43.5 mmol) was added in small portions over a period of 2 h with stirring. The mixture was stirred for another h at the same temperature, then diluted with CH 2 Cl 2 (2 ml), filtered through paper filter and evaporated to dryness. The residue was purified by column chromatography (silica gel, CH 2 Cl 2 ) to give pure 4 (2.23 g, 5.2 mmol, 33% yield). H NMR (3 MHz, CDCl 3 ): δ.4 (s, 6H), 4.2 (d, 8H, J = 5. Hz) (m, 8H), (m, 4H), (m, 4H), 8.34 (d, 2H, J = 8. Hz); 3 C NMR (75 MHz, CDCl 3 ): δ -., 52.8, 3.5, 4.8, 6.7, 3., 3.7, 33., 4.5, 5.2, 85.; HRMS (ESI + ): m/z Found , calculated for [M+H] + (.5 mmu). Diamino--xanthone (5) H 2 N NH 2 S5

6 Supplementary Material (ESI) for Chemical Communications To a flame-dried flask flushed with argon, Pd(PPh 3 ) 4 (35. mg,.33 mmol) and,3-dimethylbarbituric acid (69 mg,.8 mmol) were added. A mixture of compound 4 (99.2 mg,.23 mmol) dissolved in CH 2 Cl 2 ( ml) was further added and the solution was stirred at 35 C for 6 h, then evaporated to dryness. The residue was suspended in saturated Na 2 C 3 aq. and extracted with Cl 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was purified by column chromatography (silica gel, 4/3 AcEt/hexane) to give crude 5 (48.8 mg,.82 mmol, 79% yield). H NMR (3 MHz, CD 3 D): δ.4 (s, 6H), 6.76 (dd, 2H, J = 2.6, 8.4 Hz), 6.88 (d, 2H, J = 2.2 Hz), 8.3 (d, 2H, J = 8.8 Hz); 3 C NMR (75 MHz, CD 3 D): δ -.3, 6.6, 8.4, 3., 32.8, 42.6, 53., 87.5; HRMS (ESI + ): m/z Found: 269.8, calculated 269. for [M+H] + (.2 mmu). Dihydroxy--xanthone (6) H H A solution of compound 5 (48.8 mg,.82 mmol) in MeH / 6 N H 2 S 4 (45 ml) was cooled to C. A solution of NaN 2 (84.6 mg,.22 mmol) in H 2 (2 ml) was slowly added, and the mixture was stirred at the same temperature for h, then slowly added dropwise into boiling N H 2 S 4 (5 ml). The resulting mixture was refluxed for another min, then allowed to cool to room temperature, and extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated. The residue was purified by column chromatography (silica gel, /2 MeH/CH 2 Cl 2 ) to give crude 6 (32.9 mg,.22 mmol, 67% yield). H NMR (3 MHz, CD 3 D): δ.45 (s, 6H), 6.95 (dd, 2H, J = 2.2, 8.8 Hz), 7.7 (d, 2H, J = 2.2 Hz), 8.26 (d, 2H, J = 8.8 Hz); 3 C NMR (75 MHz, CD 3 D): δ -.5, 8.4, 2., 33.3, 33.8, 43., 62.2, 87.6; HRMS (ESI ): Found , calculated for [M-H] - (+4. mmu). S6

7 3,6-DiTBDMS--xanthone (7) Supplementary Material (ESI) for Chemical Communications TBDMS TBDMS To a solution of compound 6 (32.9 mg,.22 mmol) and imidazole (85.5 mg,.26 mmol) in CH 2 Cl 2 (2 ml) was slowly added TBDMSCl (85 mg,.23 mmol) dissolved in CH 2 Cl 2 (5 ml), and the mixture was stirred at room temperature for 4 h. H 2 was added, and the mixture was extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was purified by column chromatography (silica gel, /2 AcEt/hexane) to give pure 7 (52.8 mg,.6 mmol, 84% yield). H NMR (3 MHz, CDCl 3 ): δ.26 (s, 2H),.46 (s, 6H),. (s, 8H), 6.98 (dd, 2H, J = 2.2, 8.8 Hz), 7.4 (d, 2H, J = 2.9 Hz), 8.37 (d, 2H, J = 8.8 Hz); 3 C NMR (75 MHz, CDCl 3 ): δ -4.3, -.6, 8.3, 25.6, 2.8, 23.7, 32.3, 34.5, 4., 58.7, 86.; HRMS (ESI + ): m/z Found , calculated for [M+H] + ( 4. mmu). 2-Me TokyoMagenta (8) H To a flame-dried flask flushed with argon, 2-bromotoluene (2 μl,.6 mmol) and anhydrous THF (5 ml) were added. The solution was cooled to -78 C, M sec-buli (. mmol) was added, and the mixture was stirred for 2 min. At the same temperature, compound 7 (9.4 mg,.9 mmol) dissolved in anhydrous THF (5 ml) was slowly added. The mixture was warmed to room temperature then stirred for h, and 2 N HCl aq. ( ml) was added to it. Stirring was continued for 2 min, then the mixture was extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was purified by HPLC to give pure 2-Me TokyoMagenta (8) (4.5 mg,.3 mmol, 69% yield). H NMR (3 MHz, D 2 ): δ.46 (s 6H), 2. (s, 3H), 6.33 (dd, 2H, J = 2.9, 9.5 Hz), (m, 5H), (m, 3H); HRMS (ESI ): Found calculated for [M-H] ( 3.4mmu). HPLC S7

8 chromatogram after purification was as follows. Supplementary Material (ESI) for Chemical Communications Absorbance 2 3 time (min) ) Ac Ac Ac Ac Cs 2 C 3 MeCN r.t. Br H H H 2) NaMe MeH C H H Scheme S Synthetic scheme for 2-Me TokyoMagenta βgal (9) 2-Me TokyoMagenta βgal (9) H H H H A mixture of 2-Me TokyoMagenta (8) (4.6 mg,.3 mmol), 2,3,4,6-tetra--acetyl-α-galactopyranosyl bromide SR3 (8.8 mg,.97 mmol) and Cs 2 C 3 (29.6 mg,.99 mmol) in MeCN (3 ml) was stirred at room temperature under argon overnight. The inorganic precipitate was filtered off, and the filtrate was evaporated to dryness. The resulting residue was dissolved with MeH (3 ml) and the solution was cooled to C. Then 5 μl of 28 % NaMe in MeH was slowly added, and the mixture was stirred for h. The reaction was quenched by addition of.2 N HCl aq., and the mixture was extracted with CH 2 Cl 2. The organic layer was washed with brine, dried over Na 2 S 4 and evaporated to dryness. The residue was purified by HPLC to give pure 2-Me TokyoMagenta βgal (9) (2.4 mg,.47 mmol, 36% yield). H NMR (3.4 MHz, CD 3 D): δ.5 (d, 3H, J =.8 Hz),.52(d, 3H J =.8 Hz), 2.4 (s, 3H), 3.59 (dd, H, J = 3.7, 9.5 S8

9 Supplementary Material (ESI) for Chemical Communications Hz), (m, 5H), 4.98 (dd, H, J = 3.3, 7.7 Hz), 6.23 (dd, H, J = 2.2,.3 Hz), (m, 2H), (m, 3H), (m, 3H), 7.5 (d, H, J = 2.2 Hz); HRMS (ESI + ): m/z Found calculated for [M+H] + (.3 mmu). HPLC chromatogram after purification was as follows. Absorbance 2 3 time (min) S9

10 Supplementary Material (ESI) for Chemical Communications Normalized absorbance ph 3 ph 4 ph 5 ph 6 ph 7 ph 8 ph 9 ph ph Wavelength (nm) ph Fig. S ph-dependent change of absorbance spectra of μm 2-Me TM in. M sodium phosphate buffer containing % DMS (left). pk a Plot of 2-Me TM at 582 nm (right). The pk a value was 6.8. Normalized fluorescence ph 3 ph 4 ph 5 ph 6 ph 7 ph 8 ph 9 ph ph Wavelength (nm) ph Fig. S2 ph-dependent change of fluorescence spectra (Ex = 582 nm) of μm 2-Me TM in. M sodium phosphate buffer containing % DMS (left). Plot of the normalized maximum fluorescence intensity against ph (right). The pk a value was 6.8. Normalized fluorescence ph 3 ph 4 ph 5 ph 6 ph 7 ph 8 ph 9 ph ph Wavelength (nm) Fig. S3 ph-dependent change of fluorescence spectra (Ex = 47 nm) of μm 2-Me TM in. M sodium phosphate buffer containing % DMS. S

11 Supplementary Material (ESI) for Chemical Communications Normalized fluorescence ph 3 ph 4 ph 5 ph 6 ph 7 ph 8 ph 9 ph ph Wavelength (nm) Fig. S4 ph-dependent change of excitation spectra (Em = 64 nm) of μm 2-Me TM in. M sodium phosphate buffer containing % DMS. Normalized fluorescence min min 2 min 3 min Normalized fluorescence min min 2 min 3 min Wavelength (nm) Wavelength (nm) Fig. S5 Photobleaching tests. Normalized fluorescence spectra of μm 2-Me TM (left) and 2-Me TG (right) in. M sodium phosphate buffer (ph 7.4) containing % DMS. Samples were exposed to light (3 mw, nm for 2 Me TM and 3 mw, nm for 2-Me TG) for,, 2 and 3 min. S

12 Table S Fluorescence quantum yield of 2-Me TM βgal. Φ fl 2-Me TM βgal.5 Supplementary Material (ESI) for Chemical Communications The fluorescence quantum yield was determined with fluorescein in. M NaH aq. (Φ fl =.85) SR2 as a standard. 3 Fluorescence intensity (a.u.) 2 2-Me-4-Me TG βgal 2-Me TM βgal Time (min) Fig. S6 Comparison of time-dependent fluorescence changes between 2-Me TM βgal and 2-Me-4-Me TG βgal upon addition of β-galactosidase. Both experiments were performed at 37 C in. M sodium phosphate buffer (ph 7.4) containing μm 2-Me TM βgal or 2-Me-4-Me TG βgal and. % DMS as a cosolvent. unit (.3 μg) of β-galactosidase was added at 4 s. Supporting references SR R. F. Kubin and A. N. Fletcher, J. Luminescence, 982, 27, 455. SR2 Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose and T. Nagano, J. Am. Chem. Soc., 25, 27, SR3 J. Montero, J. Winum, A. Leydet, M. Kamal, A. Pavia, and J. Roque, Carbohydrate Research, 997, 297, 75. S2