A 488nm-excited wash-free halo-tag probe and its synthesis and biological application
A 488nm, probe technology, applied in the field of protein labeling, can solve the problems of poor signal-to-noise, low cell permeability, and fluorescent signal error in fluorescence imaging, and achieve the effect of high signal-to-noise ratio, simple purification, and high brightness
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Embodiment 1
[0041] Synthetic method of Halo-tag probe (Halo-DAze).
[0042] Synthesis of intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-5-nitro-1,8-naphthalimide (OAN-NBr):
[0043]
[0044] 4-Bromo-5-nitro-1,8-naphthalimide (2.00 g, 6.24 mmol) was dissolved in 80 mL of ethanol, and diglycolamine (1.97 g, 18.7 mmol) was added dropwise thereto. After 2 h at 70°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 3-1:1, V / V) to obtain 996 mg of off-white solid with a yield of 39%. Its nuclear magnetic spectrum proton spectrum and carbon spectrum data are as follows:
[0045] 1 H NMR (400MHz, CDCl 3 / DMSO -d 6 )δ8.70(d,J=7.8Hz,1H),8.50(d,J=7.9Hz,1H),8.28(d,J=7.9Hz,1H),8.13(d,J=7.8Hz,1H) ,4.37(t,J=5.9Hz, 2H),4.21(s,1H),3.78(t,J=5.9Hz,2H),3.58(s,4H).
[0046] 13 C NMR (101MHz, CDCl 3 / DMSO)δ167.48, 166.76, 155.77, 140.86, 137.11, 136.25, 135.21, 130.44, 128.84, 128.34, 127.29, 125.60...
Embodiment 2
[0067] Synthetic method of Halo-tag probe (Halo-DAze).
[0068] Synthesis of intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-5-nitro-1,8-naphthalimide (OAN-NBr):
[0069]
[0070] 4-Bromo-5-nitro-1,8-naphthalimide (1.75 g, 5.45 mmol) was dissolved in 35 mL of ethanol, and diglycolamine (0.58 g, 5.55 mmol) was added dropwise thereto. After 10 h at 40°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether:dichloromethane=3-1:1, V / V) to obtain 756 mg of off-white solid with a yield of 34%.
[0071] Synthesis of intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4,5-diazetidinyl-1,8-naphthalimide (Halo-OH):
[0072]
[0073] The intermediate OAN-Br (75 mg, 0.18 mmol) was dissolved in 7.5 mL of ethylene glycol methyl ether, and azetidine (25 mg, 0.44 mmol) was added thereto. The reaction solution was slowly heated to 50 °C and reacted for 24 h. Ethylene glycol methyl ether was removed under reduced pre...
Embodiment 3
[0080] Synthetic method of Halo-tag probe (Halo-DAze).
[0081] Synthesis of intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-5-nitro-1,8-naphthalimide (OAN-NBr):
[0082]
[0083] 4-Bromo-5-nitro-1,8-naphthalimide (2.25 g, 7.02 mmol) was dissolved in 225 mL of ethanol, and diglycolamine (9.0 g, 85.2 mmol) was added dropwise thereto. After 1 h at 90°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether:dichloromethane=3-1:1, V / V) to obtain 1.044 g of off-white solid with a yield of 31%.
[0084] Synthesis of intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4,5-diazetidinyl-1,8-naphthalimide (Halo-OH):
[0085]
[0086] The intermediate OAN-Br (100 mg, 0.24 mmol) was dissolved in 60 mL of ethylene glycol methyl ether, and azetidine (900 mg, 15.7 mmol) was added thereto. The reaction solution was slowly heated to 140°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pre...
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