A kind of wash-free snap-tag probe excited by 488nm and preparation method thereof

A technology of probes and synthesis methods, applied in the field of protein labeling, can solve the problems of inability to obtain imaging results, increase of false signals, and inability to control the number and position, and achieve the effects of maintaining accuracy, low-cost synthetic materials, and simple and general methods.

Active Publication Date: 2022-03-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, organic small molecule dyes are exogenous species, and the problem is that they cannot be derived from cells like fluorescent proteins, so the number and position in cells cannot be controlled
Especially in today's research on the function of a single protein, single-molecule and super-resolution imaging technologies put forward high requirements for the stability and brightness of fluorescent dyes, and at the same time put forward higher requirements for the authenticity of fluorescent signals, and an increase in the background will cause false signals increase, unable to obtain reliable imaging results

Method used

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  • A kind of wash-free snap-tag probe excited by 488nm and preparation method thereof
  • A kind of wash-free snap-tag probe excited by 488nm and preparation method thereof
  • A kind of wash-free snap-tag probe excited by 488nm and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthesis of SNAP-DAze:

[0046] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0047]

[0048] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 4-aminomethylbenzyl alcohol (500 mg, 3.60 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 (petroleum ether: dichloromethane=3:1-dichloromethane:methanol=200:1, V / V) to obtain 480 mg of off-white solid. Yield 35%. Its nuclear magnetic spectrum hydrogen spectrum data are as follows:

[0049] 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).

[0050] Synthesis of BA-DAze

[0051]

[0052] BA-NBr (300 mg, 0.68 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 300 mg of azetidine was added th...

Embodiment 2

[0065] Synthesis of SNAP-DAzo

[0066] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0067]

[0068] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 4-aminomethylbenzyl alcohol (2.00 g, 14.4 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 (petroleum ether: dichloromethane=3:1-dichloromethane:methanol=200:1, V / V) to obtain 880 mg of off-white solid. Yield 66%.

[0069] Synthesis of intermediate BA-DAzo

[0070]

[0071] BA-NBr (100 mg, 0.23 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and tetrahydropyrrole 300 mg was added thereto. The reaction solution was slowly heated to 100°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:...

Embodiment 3

[0079] Synthesis of SNAP-DMEDA

[0080] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0081]

[0082] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 80 mL of ethanol, and 4-aminomethylbenzyl alcohol (1.00 g, 7.2 mmol) was added 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-dichloromethane:methanol=200:1, V / V) to obtain 573 mg of off-white solid, Yield 43%.

[0083] Synthesis of BA-DMEDA

[0084]

[0085] BA-NBr (150 mg, 0.34 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 200 mg of N,N'-dimethylethylenediamine was added thereto. The reaction solution was slowly heated to 100°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=...

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Abstract

The invention provides a wash-free SNAP-tag probe excited at 488nm and a preparation method thereof. The wash-free SNAP-tag probe introduces a spiro ring rigid structure into a disubstituted naphthalimide dye at the 4,5-position And design and synthesize a series of wash-free SNAP-tag probes that can be used for 488nm excitation, and its structural formula is shown in (1). The introduction of the rigid structure not only limits the intramolecular torsion, improves the stability and brightness of the fluorescent molecules, but also increases the planarity of the probe molecules. This causes the SNAP‑tag probe molecules to form a strong π‑π interaction in water, resulting in a quenching of the fluorescence. After combining with SNAP-tag, the fluorescence of the probe molecule is released, which can be enhanced up to 28 times. This series of probes can achieve specific labeling of SNAP-tag in living cells and achieve wash-free fluorescence imaging. In addition, due to the improvement of stability and brightness, this series of probes can also be used in SIM (Structured Light Illumination Microscopy), STED (Stimulated Emission Depletion) and other super-resolution fluorescence imaging fields.

Description

technical field [0001] The invention belongs to the field of protein labeling, and in particular relates to a 488nm-excited wash-free SNAP-tag probe and a preparation method thereof. Background technique [0002] Fluorescence imaging technology has gradually become a powerful tool for studying protein function from the cellular level to the individual level. Due to the advantages of small size, broad fluorescence emission spectrum, and a variety of fluorescent colors, organic small molecule fluorescent dyes have gradually become a substitute for fluorescent proteins in the field of protein labeling. However, organic small molecule dyes are exogenous species, and the problem is that they cannot be derived from cells like fluorescent proteins, so the number and position in cells cannot be controlled. In order to solve this problem, chemists have developed a variety of bioorthogonal methods to covalently attach small molecule dyes to target proteins, so that the location and f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D401/14C07D401/06C07D473/18C07D519/00C09K11/06G01N21/64A61K49/00
CPCC07D401/14C07D401/06C07D473/18C07D519/00C09K11/06G01N21/6428G01N21/6486G01N21/6456G01N21/6458A61K49/0021C09K2211/1007C09K2211/1029C09K2211/1044G01N2021/6432
Inventor 徐兆超乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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