Target-selective fluorescent "switch-on" protein labeling by 6π-azaelectrocyclization

Katsunori Tanaka, Masataka Kitadani, Koichi Fukase
2011 Organic and biomolecular chemistry  
General Procedures. All commercially available reagents were used without further purification. Dichloromethane were refluxed over and distilled from CaH 2 . Anhydrous DMF was purchased from Aldrich, and anhydrous THF was purchased from Kanto Chemicals, Tokyo. Preparative separation was usually performed by column chromatography on silica gel (FUJI silysia LTD, and by thin layer chromatography on silica gel (Merck, 20 x 20 cm, Silica gel 60 F 254 , 1 mm). 1 H NMR spectra was recorded on a JEOL
more » ... NM-LA 500 spectrometer and chemical shifts were represented as δvalues relative to the internal standard TMS. MALDI-TOF-mass spectra were measured on PerSeptive Biosystems, Voyager RP-DE/H and SHIMADZU AXIMA-CFR mass spectrometers equipped with a nitrogen laser (λ = 337 nm). UV-vis spectra were recorded on a JASCO V-530 spectrophotometer and reported as λ max [nm] (ε max [Lmol -1 cm -1 ]). Fluorescence emission spectra were measured either on a JASCO FP-6500 spectrofluorometer. 2-{2-{4-[4-(Dimethylamino)phenylazo]benzoyl}aminoethoxy}ethyl (E,E)-4-tertButyldiphenylsilanylhydroxy-2-(6-aminostyryl)but-2-enoate (3). To a solution of 2 (30 mg, 40 µmol) in CH 2 Cl 2 (3.0 mL) was added TFA (750 µL) at 0 °C and the mixture was stirred at this temperature for 20 min. The solution was neutralized with 1 N aqueous NaOH (3.0 mL) and extracted with CH 2 Cl 2 . The organic layers were combined, washed with brine, dried over Na 2 SO 4 , filtrated, and concentrated in vacuo to give the crude diamine, which was subjected to the acylation without further purification. To a solution of the diamine (35% out of the crude products obtained above (14 µmol)) in dry CH 2 Cl 2 (1.0 mL) was added 4-[4-(dimethylamino)phenylazo]benzoic acid succinimidyl ester (5.2 mg, 14 µmol) at room temperature and stirred overnight at this temperature. After the solution was concentrated in vacuo, the residue was purified by preparative TLC on silica gel (60% ethyl acetate in hexane) to give 3 as a bright red solid (7.2 mg, 62 % for 2 steps); MALDI-TOF-MS m/z calcd for C 47 H 53 N 5 O 5 Si (M+Na) + 818.4, found 818.3; HRESI-MS m/z calcd for C 47 2-{2-{4-[4-(Dimethylamino)phenylazo]benzoyl}aminoethoxy}ethyl (E,E)-4-tertButyldiphenylsilanylhydroxy-2-{6-[7-(diethylamino)coumarin-3-carboxamide]styryl}but-2enoate. To a solution of 3 (1.5 mg, 1.9 µmol) in dry DMF (500 µL) were added HATU (1.1 mg, 2.8  µmol) and 7-(dimethylamino)coumarin-3-carboxylic acid (590 µg, 2.3 µmol) at room temperature. After the mixture was stirred at this temperature for 10 min, triethylamine (530 nL, 3.8 µmol) was added to this solution and stirred at room temperature overnight. The resulting mixture was concentrated in vacuo to give the crude product, which was directly purified by preparative TLC on silica gel (9 % MeOH in CHCl 3 ) to give the coupling product as a bright red solid (1.5 mg, 76 %): MALDI-TOF-MS m/z calcd for C 61 H 66 N 6 O 8 Si (M+Na) + 1061.5, found 1061.6; HRESI-MS m/z calcd for C 61 H NMR (500 MHz, CDCl 3 ) δ 10.9 (s, 1H), 8.76 (s, 1H), 7.86 (d, 2H, J = 8.5 Hz), 7.85 (d, 2H, J = 9.2 Hz), 7.80 (d, 2H, J = 8.6 Hz), 7.68-7.65 (m, 4H), 7.64 (d, 2H, J = 8.6 Hz), 7.46 (d, 1H, J = 9.0 Hz), 7.42-7.30 (m, 6H), 7.24 (d, 2H, J = 8.6 Hz), 6.86 (t, 1H, J = 5.9 Hz), 6.73 (d, 2H, J = 9.0 Hz), 6.69 (d, 1H, J = 16.3 Hz), 6.67 (dd, 1H, J = 2.5, 8.9 Hz), 6.58 (d, 1H, J = 16.3 Hz), 6.53 (d, 1H, J = 2.1 Hz), 4.53 (d, 2H, J = 6.0 Hz), 4.42 (t, 2H, J = 4.7 Hz), 3.81 (t, 2H, J = 4.7 Hz), 3.72 (t, 2H, J = 4.6 Hz), 3.70 (t, 2H, J = 4.6 Hz), 3.46 (q, 4H, J = 7.1 Hz), 3.07 (s, 6H), 1.26 (t, 6H, J = 7.2Hz), 1.05 (s, 9H); 13 C NMR (125 MHz, CDCl 3 ): Due to Electronic Supplementary Material (ESI)
doi:10.1039/c1ob05320e pmid:21691664 fatcat:4ogam5nrrzcvrf4skyuzpf3qdy