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Synthesis of Thiazole Orange Derivatives (TO3-Biotin

):
1,4-Methylquinolinium iodide (2, 3)

A mixture of 4-methylquinoline (25.0 ml, 189 mmol, 1.0 equiv.) and iodomethane (25.0
ml, 402 mmol, 2.1 equiv.) in 1,4-dioxane (375 ml) was heated at reflux for 1 hr. On cooling to
ambient temperature the precipitate was collected by filtration and was washed with diethyl ether
(3 × 30 ml), hexanes (3 × 30 ml) and dried in vacuo to afford the title compound (51.3 g, 95%) as
a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 3.01 (s, 3H), 4.58 (s, 3H), 7.91 – 8.14 (m, 2H),
8.16 – 8.35 (m, 1H), 8.49 (d, J = 8.9 Hz, 1H), 8.54 (d, J = 8.5 Hz, 1H), 9.36 (d, J = 6.0 Hz, 1H).

4-[(E)-2-Anilinoethenyl]-1-methylquinolinium iodide (6)

A mixture of 1,4-methylquinolinium iodide (40.0 g, 140 mmol, 1.0 equiv.) and N,N′diphenylformamidine (55.1 g, 281 mmol, 2.0 equiv.) in ethanol (225 ml) was heated at reflux for
1 hr. On cooling to room temperature the precipitate was collected by filtration and was washed

 

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with ethanol (3 × 70 ml) and then dried in vacuo to afford the title compound (29.3 g, 54%) as a
dark red/brown solid. This material was used in the next step without further purification.
4-[(E)-2-(Acetylphenylamino)ethenyl]-1-methylquinolinium iodide (6)

A mixture of 4-[(E)-2-anilinoethenyl]-1-methylquinolinium iodide (29.0 g, 74.7 mmol,
1.0 equiv.) and acetic anhydride (115 ml, 1.21 mol, 16 equiv.) in pyridine (400 ml) was stirred at
room temperature overnight. Ethyl acetate (1.0 L) was then added and the precipitate was
collected by filtration which was washed with ethyl acetate (3 × 100 ml). The resultant solid was
suspended in dichloromethane (150 ml) and then stirred for 30 min at room temperature. A green
precipitate was removed by filtration and the filtrate was concentrated in vacuo. The resultant
residue was dissolved in dichloromethane (400 ml) and diethyl ether (700 ml) was added. The
precipitate was then collected by filtration and was washed with diethyl ether (3 × 70 ml) and
then dried in vacuo to afford the title compound (17.3 g, 55%) as a tan coloured solid. 1H NMR
(400 MHz, DMSO-d6) δ 2.08 (s, 3H), 4.47 (s, 3H), 6.04 (d, J = 14.0 Hz, 1H), 7.53 (d, 2H), 7.76 –
7.60 (m, 3H), 7.95 – 7.83 (m, 2H), 8.21 – 8.12 (m, 1H), 8.36 (dd, J = 7.7, 3.7 Hz, 2H), 8.89 (d, J
= 13.9 Hz, 1H), 9.16 (d, J = 6.6 Hz, 1H).

 

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TO3-Acetate (6)

Triethylamine (2.20 ml, 15.8 mmol, 0.5 equiv.) was added to a stirred solution of 2-(2methylbenzo[d]thiazol-3-ium-3-yl)acetate (11.5 g, 34.9 mmol, 1.0 equiv.) and 4-[(E)-2(acetylphenylamino)ethenyl]-1-methylquinolinium iodide (15.0 g, 34.9 mmol, 1.0 equiv.) in
dichloromethane (200 ml). After 14 hrs the reaction mixture was concentrated in vacuo and the
resultant residue was suspended in dichloromethane (800 ml). The precipitate was collected by
filtration and was washed with acetone (3 × 80 ml) and then dried in vacuo to afford the title
compound (7.7 g, 59%) as a dark blue solid. Exact mass calculated for [M+H+, C22H19N2O2S2+]
requires m/z 375.1162. Found 375.1156. HPLC: tR = 19.4 min.

 

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TO3-Biotin

Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU, 15.2 mg,
40.1 µmol, 15 equiv.) and N,N-diisopropylethylamine (DIPEA, 14.2 µl, 81.5 µmol, 30.5 equiv.)
were added to a solution of TO3-Acetate (1.0 mg, 2.7 µmol, 1.0 equiv.) and EZ-link amine-PEGbiotin (5.0 mg, 12 µmol, 4.5 equiv.) in N,N-dimethylformamide (250 µl). The reaction was
stirred overnight at ambient temperature. The final product was isolated and purified directly by
reverse-phase HPLC and identified by mass spectrometry. Exact mass calculated for [M+,
C40H51N6O6S2+] requires m/z 775.3306. Found 775.3304. HPLC: tR = 27 min.

 

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