Fluorescent Labeling of Protein Using

Supplementary Material

Fluorescent Labeling of Protein Using

Blue-Emitting 8-Amino-BODIPY Derivatives

Dokyoung Kim,* Donghee Ma, Muwoong Kim, Yuna Jung, Na Hee Kim, Chiho Lee,

Seo Won Cho, Sungnam Park, Youngbuhm Huh, Junyang Jung, Kyo Han Ahn

* Corresponding author. E-mail:

Scheme S1. Scheme for the preparation of 8-SMe-BODIPY.

Scheme S2. Scheme for the preparation of compound 3.

Scheme S3. Scheme for the preparation of compound 5.

Scheme S4. Scheme for the conjugation of BP2 and BSA or Lysozyme.

Figure S1. Absorption and fluorescence emission spectra of BP1 in various solvents.

Figure S2. Absorption and fluorescence emission spectra of BP2 in various solvents.

Figure S3. Absorption and fluorescence emission spectra of BP3 in various solvents.

Figure S4. Absorption and fluorescence emission spectra of BP4 in various solvents.

Figure S5. Absorption and fluorescence emission spectra of BP1–4 in dichloromethane.

Figure S6. Absorption and fluorescence emission spectra of BP1–4 in ethyl acetate.

Figure S7. Absorption and fluorescence emission spectra of BP1–4 in methanol.

Figure S8. Absorption and fluorescence emission spectra of BP1–4 in acetonitrile.

Figure S9. Absorption and fluorescence emission spectra of BP1–4 in dimethylsulfoxide.

Figure S10. Absorption and fluorescence emission spectra of BP1–4 in deionized water.

Figure S11. Time-gated fluorescence emission spectra of BP1 and BP2.

Figure S12. MALDI-TOF spectra of BSA and the BP2-labeled BSA product.

Synthesis

Scheme S1. Scheme for the preparation of 8-SMe-BODIPY.

Bis-(1H-pyrrol-2-yl)-methanethione (1): A solution of pyrrole (1.02 g, 1.05 mL, 15.1 mmol) in anhydrous diethyl ether (20 mL) under argon atmosphere was added dropwise to a vigorously stirred solution of thiophosgene (0.9 g, 0.6 mL, 7.83 mmol) in anhydrous toluene (20 mL) at 0 °C. After 10 min, aqueous methanol (10% methanol) (ca. 25 mL) was added and the mixture stirred for further 30 min at room temperature (25 °C). The solvents were removed in vacuo and the residue was purified by neutral alumina column chromatography (column diameter = 4 cm, height of neutral alumina = ca 15 cm, eluent: dichloromethane) to afford compound 1 as a crystalline dark-red solid (1.01 g, 76%). 1H NMR (CDCl3, 300 MHz, 293K): δ 6.40 (2H, m), 7.05 (2H, m), 7.20 (2H, m), 9.77 (2H, s). 13C NMR (CDCl3, 75 MHz, 293K): δ 112.5, 114.8, 127.7, 138.4, 193.2.

2-[Methylsulfanyl-(1H-pyrrol-2-yl)-methylene]-2H-pyrrolium iodide (2): To a solution of compound 1 (0.88 g, 5.0 mmol) in anhydrous dichloromethane (15 mL) under argon atmosphere was added methyl iodide (12.77 g, 5.6 ml, 90.0 mmol) at room temperature (25 °C). The reaction mixture was stirred for 15 h (with TLC monitoring). The solvent was removed under reduced pressure to obtain compound 2 as a brownish and gummy solid (1.63 g). The compound 2 was used for the next reaction without further purification.

8-(Thiomethyl)4,4-difluoro-4-bora-3a,4a-diaza-sindacene (8-SMe-BODIPY): To a solution of compound 2 (1.63 g) in anhydrous dichloromethane (35 mL) under argon atmosphere at room temperature (25 °C) was added triethylamine (3.03 g, 4.2 mL, 30.0 mmol). After being stirred for 30 min, BF3·Et2O (6.39 g, 5.6 mL, 45.0 mmol) was added to the mixture. The mixture was stirred for 12 h at room temperature (25 °C). The solvent was evaporated under vacuum, and the residue was filtered on silica gel (column diameter = 3 cm, height of silica gel = ca 10 cm, eluent: dichloromethane) to yield 8-SMe-BODIPY as a crystalline dark red solid (840 mg, 71% from two steps). 1H NMR (CDCl3, 300 MHz, 293K): δ 7.80 (2H, m), 7.26–7.42 (2H, m), 6.50–6.51 (2H, m), 2.91 (3H, s). 13C NMR (CDCl3, 75 MHz, 293K): δ 154.0, 140.8, 133.4, 127.3, 117.6, 20.0.

Scheme S2. Scheme for the preparation of compound 3.

4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid (3): To a solution of 4-aminobenzoic acid (1.0 g, 7.29 mmol) in tetrahydrofuran (15 mL) was added a solution of maleic anhydride (0.72 g, 7.29 mmol) in tetrahydrofuran (15 mL), and the mixture was stirred for 1 h at room temperature (25 °C). The solvent was evaporated under vacuum, and the residue was washed two times with diethyl ether. The residue was washed with diethyl ether on filter paper (Whatman®, Z274844) to afford maleic acid compound as a white solid (quant.). The crude maleic acid compound was dissolved in 20 mL of acetic anhydride, and 0.6 g of sodium acetate was added at room temperature (25 °C). The mixture was heated to 80 °C for 3 h. After 3 h, the reaction was cooled to room temperature (25 °C), and deionized H2O (20 mL) was added for reaction quenching. The aqueous layer was extracted with ethyl acetate (3 ´ 20 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4 and concentrated to afford compound 3 as a white solid (1.52 g, 96%). 1H NMR (CDCl3, 300 MHz, 293K): δ 6.40 (2H, m), 7.05 (2H, m), 7.20 (2H, m), 9.77 (2H, s). 13C NMR (DMSO-d6): δ 169.5, 166.7, 135.5, 134.9, 129.9, 129.5, 126.1.

Scheme S3. Scheme for the preparation of compound 5.

1,5-Diazido-pentane (4): Sodium azide (0.975 g, 15.0 mmol) was added to a solution of the 1,5-dibromopentane (1.36 ml, 10.0 mmol) in N,N-dimethylformamide (15.0 mL). The mixture was stirred for 10 h at 60 °C. After 10 h, deionized H2O (100.0 mL) was added to the mixture, and the product was extracted with diethyl ether (3 ´ 10 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluent: n-hexane) to afford the compound 4 as a colorless liquid (1.46 g, 95%). 1H NMR (CDCl3, 300 MHz, 293K): δ 1.40 (2H, m), 1.68 (4H, m), 6.7 (4H, t). 13C NMR (CDCl3, 75 MHz, 293K): δ 51.5, 28.9, 26.5.

5-Azido-pentylamine (5): To a solution of compound 4 (160 mg, 1.04 mmol) in diethyl ether (1 mL) were added to mixture of ethyl acetate (1 mL) and 5% HCl aqueous solution (1.8 mL). To the resulting mixture at 0 °C was added PPh3 (1.9 g, 7.2 mmol, 1.0 equiv) in small portions over 1 h. The mixture was stirred at room temperature for 24 h. After 24 h, HCl solution (1 M, 10 mL) was added, and the organic layer was discarded. The aqueous layer was washed with dichloromethane (2 ´ 5 mL), which was discarded, and neutralized with 6 M NaOH until the pH reached 12. The basic aqueous layer was saturated with sodium chloride and extracted with dichloromethane (4 ´ 5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated to afford compound 5 as a colorless oil (106 mg, 82%): 1H NMR (CDCl3, 300 MHz, 293K): δ 3.28 (t, 2), 2.73 (t, 2), 2.12 (br, 2), 1.67−1.58 (m, 2), 1.55−1.45 (m, 2), 1.48−1.38 (m, 2). 13C NMR (CDCl3, 75 MHz, 293K): δ 51.3, 41.7, 32.7, 28.6, 24.0.

Scheme S4. Scheme for the conjugation of BP2 and BSA or Lysozyme.

BP2 (0.4 mg, 0.0015 mmol), N′-ethylcarbodiimide hydrochloride (0.3 mg, 0.0018 mmol), and N-hydroxysuccinimide (0.2 mg, 0.0016 mmol) were added in distilled H2O (1.5 mL), and the mixture was stirred for 4 h at room temperature (25 °C). In the reaction mixture, 100 µL of activated ester was subtracted, and then 15 µL of BSA or lysozyme (0.1 g, 1.5 mL PBS buffer, pH 7.4), 885 µL of distilled H2O were added at room temperature (25 °C) and incubated for 24 h at 37 °C. The resulting product was lyophilized. Lysozyme: Cat. No. 10837059001, Sigma-Aldrich, USA.

Supplementary Figures

Fig. S1. (a) absorption and (b) fluorescence emission spectra of BP1 in various solvents. All measurements were carried out at 25 °C for a solution of the BP1 (10 µM) in the given solvent. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S2. (a) absorption and (b) fluorescence emission spectra of BP2 in various solvents. All measurements were carried out at 25 °C for a solution of the BP2 (10 µM) in the given solvent. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S3. (a) absorption and (b) fluorescence emission spectra of BP3 in various solvents. All measurements were carried out at 25 °C for a solution of the BP3 (10 µM) in the given solvent. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S4. (a) absorption and (b) fluorescence emission spectra of BP4 in various solvents. All measurements were carried out at 25 °C for a solution of the BP4 (10 µM) in the given solvent. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S5. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in dichloro-methane. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S6. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in ethyl acetate. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S7. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in methanol. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S8. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in acetonitrile. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S9. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in dimethyl-sulfoxide. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S10. (a) absorption and (b) fluorescence emission spectra of BP1–4 (10 µM) in deionized H2O. All measurements were carried out at 25 °C. The fluorescence emission spectra were recorded under excitation at the maximum absorption wavelength.

Fig. S11. Time resolved fluorescence (TRF) signals of BP1/BP2 (10 µM) in deionized H2O. Experiments were carried out using a time-correlated single-photon counting (TCSPC) method (PicoHarp 300, Picoquant, Germany). The sample solutions were excited by a 375 nm pulse (LDH-P-C-375, Picoquant, Germany) and the TRF signals from the sample solutions were collected at 460 nm. The fluorescence from the sample solutions was measured by a single photon detector (ID100, ID Quantique, Switzerland). The instrumental response function (IRF) of our TCSPC setup was about 0.08 ns in FWHM (full width at half maximum). The intensities were normalized to 1.0. TRF signals, P(t), were fitted from 0.1 ns by a bi-exponential function, i.e., P(t) = A1exp(-t/T1) + A2exp (-t/T2) with A1 + A2 = 1. The average fluorescence lifetime, Tavg = A1T1/(A1+A2) + A2T2/(A1+A2), was calculated and used for comparison.

Fig. S12. MALDI-TOF spectra of (a) BSA and the (b) BP2-labeled BSA product. Inset number indicated the mass of BSA (~66 kDa) and BP2-labeled BSA product (~66 kDa + 548 (M.W. of BP2=279.05)).

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