Fluorescent probe for rapidly and specifically labeling SNAP-tag as well as preparation and biological application of the fluorescent probe
A fluorescent probe and specific technology, applied in the field of protein fluorescent labeling, can solve the problem of low binding rate of cell permeability, achieve the effect of simple and general method, good photostability, and low price of synthetic raw materials
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Embodiment 1
[0041] Synthesis method of SNAP-tag probe SNAP-DMEDA.
[0042] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):
[0043]
[0044]4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 4-aminomethylbenzyl alcohol (853 mg, 6.22 mmol) was added thereto. After 10 h at 80°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V / V) to obtain 480 mg of off-white solid with a yield of 35%. Its nuclear magnetic spectrum hydrogen spectrum data are as follows:
[0045] 1 H NMR (400MHz, DMSO-d 6 )δ8.69(d, J=8.1Hz, 2H), 8.50–8.39(m, 2H), 7.35(d, J=8.1Hz, 2H), 7.25(d, J=7.9Hz, 2H), 5.23( s,2H),5.13(t,J=5.8Hz,1H),4.45(d,J=5.5Hz,2H).
[0046] Synthesis of intermediate BA-DMEDA:
[0047]
[0048] BA-NBr (150 mg, 0.34 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 200 mg of N,N'-dimethylet...
Embodiment 2
[0061] Synthesis method of SNAP-tag probe SNAP-DMEDA.
[0062] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):
[0063]
[0064] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 20 mL of ethanol, and 4-aminomethylbenzyl alcohol (500 mg, 3.65 mmol) was added thereto. After 9 hours at 40°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V / V) to obtain 437 mg of off-white solid with a yield of 32%.
[0065] Synthesis of intermediate BA-DMEDA:
[0066]
[0067] BA-NBr (300 mg, 0.68 mmol) was dissolved in 15 mL of ethylene glycol methyl ether, and N,N'-dimethylethylenediamine (300 mg, 3.4 mmol) was added thereto. The reaction solution was slowly heated to 50°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichl...
Embodiment 3
[0073] Synthesis method of SNAP-tag probe SNAP-DMEDA.
[0074] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):
[0075]
[0076] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 100 mL of ethanol, and 4-aminomethylbenzyl alcohol (4.0 g, 29 mmol) was added 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 (dichloromethane:methanol=200:1, V / V) to obtain 425 mg of off-white solid with a yield of 31%.
[0077] Synthesis of intermediate BA-DMEDA:
[0078]
[0079] BA-NBr (150 mg, 0.34 mmol) was dissolved in 22.5 mL of ethylene glycol methyl ether, and N,N'-dimethylethylenediamine (600 mg, 6.8 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 pressure, and the residue was separated through a silica gel column (dichloro...
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