3.5. Synthesis

The synthesis of BOD-Gal is shown in Scheme 1. The starting material, compound 1, was synthesized by a routine procedure used in the construction of the BODIPY core [23]. The tetra-O-acetyl-galactose bromide 2 was obtained according to the previous method [24]. Compound 3 was synthesized with a 60% yield by Koenigs–Knorr glycosylation of compound 1 and 2. Zemplén deprotection in all the acetates with K₂CO₃/CH₃OH gave BOD-Gal in 88% yield. All intermediates and BDBH were well characterized by ¹H NMR spectroscopy, ¹³C NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

4-Hydroxybenzaldehyde (0.49 g, 4 mmol) and 2,4-dimethylpyrrole (0.76 g, 8 mmol) were dissolved in anhydrous CH₂Cl₂ (600 mL). Two drops of trifluoroacetic acid (TFA) were added and the resulting mixture was stirred in the dark for 12 h under N₂ at room temperature. After TLC showed the complete consumption of aldehyde, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) (1.09 g, 4.8 mmol) was added. After the mixture was stirred for 1 h, diisopropylethylamine (DIPEA, 5 mL) and BF₃·OEt₂ (5 mL) were added. The resulting mixture was further stirred for another 1 h, then concentrated and filtered. After the filtrate was washed twice with water and brine, the organic layer was collected, dried over anhydrous MgSO₄ and concentrated under reduced pressure. The obtained crude product was purified by column chromatography (R_f = 0.2, PE/EA = 3:1, eluent: PE/EA = 30/1–4/1, v/v) to give compound 1 (0.38 g, 28% yield) as a yellow-red powder. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.12 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 5.98 (s, 2H), 5.30–5.26 (m, 1H), 2.55 (s, 6H), 1.44 (S, 6H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 156.3, 155.3, 143.2, 141.8, 131.8, 129.4, 127.2, 121.2, 116.1, 14.6; ¹⁹F NMR (376 MHz, CDCl₃) δ (ppm): −146.06 (m, 2F). HRMS-ESI (m/z): [M]^ˉ Calc. for (C₁₉H₁₈BF₂N₂O), 339.1480, found: 339.1489.

Compound 1 (98 mg, 0.3 mmol), tetra-O-acetyl-α-D-galactose bromide 2 (148 mg, 0.36 mmol), and Ag₂O (104 mg, 0.45 mmol) were suspended in dry acetonitrile (5 mL). After the mixture was stirred for 6 h at r.t under argon and filtered, the solvent was removed in vacuum. The residue was purified by silica gel column (R_f = 0.5, PE/EA = 2:1, eluent: PE/EA =10/1–3/1, v/v) to give compound 3 (121 mg, 60% yield) as an orange solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): δ 7.19 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 5.97 (s, 2H), 5.54 to 5.52 (m, 1H), 5.48 (d, J = 3.2 Hz,1H), 5.16 to 5.12 (m, 2H), 4.28 to 4.23 (m, 1H), 4.18 to 4.09 (m, 2H), 2.53 (s, 6H), 2.18 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.39 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 170.36, 170.26, 170.17, 169.40, 157.46, 155.68, 143.08, 141.06, 131.70, 129.94, 129.52, 121.38, 117.61, 99.59, 71.36, 70.85, 68.73, 66.96, 61.45, 20.83, 20.71, 20.65, 14.68; ¹⁹F NMR (376 MHz, CDCl₃) δ (ppm): −146.17 (m,2F). HRMS-ESI (m/z): [M+H]⁺ Calc. for (C₃₃H₃₈BF₂N₂O₁₀), 671.2588, found: 671.2587.

Compound 3 (78 mg, 0.1 mmol) was dissolved in anhydrous methanol (3 mL), then K₂CO₃ (2 mg, 0.02 mmol) was added. The resulting mixture was stirred at r.t for 2 h and the pH was adjusted to 6~7with 1 M aqueous HCl. After the reaction mixture was filtered, the filtrate was concentrated in vacuum. The residue was purified by silica gel column (R_f = 0.3, DCM/MeOH = 5:1, eluent: DCM/MeOH = 15:1, v/v) to afford BOD-Gal (52 mg, 88% yield, m.p. 143–145 °C) as an orange powder. [α]D25 = +33.33. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): δ 7.26 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 6.17 (s, 2H), 5.23 (s, 1H), 4.90 (d, J = 8.0 Hz, 2H), 4.67 (s, 1H), 4.54 (s, 1H), 3.72 (s, 1H), 3.63–3.43 (m, 5H), 2.44 (s, 6H), 1.40 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 156.25, 154.66, 142.70, 142.01, 131.03, 128.98, 127.06, 121.26, 116.84, 101.11, 75.52, 73.23, 70.35, 68.06, 60.29, 14.19; ¹⁹F NMR (376 MHz, DMSO-d₆) δ (ppm): −143.57 (m,2F). HRMS-ESI (m/z): [M+Na]⁺ Calc. for (C₂₅H₃₀BF₂N₂O₆Na), 525.1984, found:525.1959.