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A kind of highly stable free-washing snap-tag probe and its preparation method and application

A high-stability, probe technology, applied in the field of fluorescence imaging, can solve the problems of rhodamine and cyanine dyes that cannot achieve wash-free imaging, difficult to achieve long-term real-time imaging, poor photostability of cyanine dyes, etc. Accuracy, simple and general method, good photostability

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, most of the current commercial SNAP-tag probes are based on rhodamine and cyanine dyes, which have little change in fluorescence before and after binding to SNAP-tag proteins, and rhodamine and cyanine dyes tend to aggregate in mitochondria, making wash-free imaging impossible
In addition, cyanine dyes have poor photostability, making it difficult to achieve long-term real-time imaging

Method used

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  • A kind of highly stable free-washing snap-tag probe and its preparation method and application
  • A kind of highly stable free-washing snap-tag probe and its preparation method and application
  • A kind of highly stable free-washing snap-tag probe and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Synthesis of SNAP-tag probe SNAP-DAze.

[0041] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0042]

[0043] 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%. The specific data of its nuclear magnetic spectrum hydrogen spectrum are as follows:

[0044] 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).

[0045] Synthesis of BA-DAze:

[0046]

[0047] BA-NBr (300 mg, 0.68 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 500 mg of azetidine was added t...

Embodiment 2

[0062] Synthesis of SNAP-tag probe SNAP-DAze.

[0063] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0064]

[0065] 4-Bromo-5-nitro-1,8-naphthalimide (1.50 g, 4.66 mmol) was dissolved in 30 mL of ethanol, and 4-aminomethylbenzyl alcohol (0.75 g, 5.5 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 656 mg of off-white solid with a yield of 32%.

[0066] Synthesis of BA-DAze:

[0067]

[0068] BA-NBr (420 mg, 0.95 mmol) was dissolved in 21 mL of ethylene glycol methyl ether, and azetidine (420 mg, 7.37 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 (dichloromethane:methanol=60:1, V / V) to obtain ...

Embodiment 3

[0075] Synthesis of SNAP-tag probe SNAP-DAze.

[0076] Synthesis of intermediate N-(4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr):

[0077]

[0078] 4-Bromo-5-nitro-1,8-naphthalimide (1.25 g, 3.88 mmol) was dissolved in 25 mL of ethanol, and 4-aminomethylbenzyl alcohol (5.0 g, 36.5 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 479 mg of off-white solid with a yield of 28%.

[0079] Synthesis of BA-DAze:

[0080]

[0081] BA-NBr (500 mg, 1.13 mmol) was dissolved in 25 mL of ethylene glycol methyl ether, and azetidine (500 mg, 8.77 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 (dichloromethane:methanol=60:1, V / V) to obtain 58 ...

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Abstract

The invention provides a high-stability wash-free SNAP-tag probe and its preparation method and application. The probe is based on a 4,5-position disubstituted naphthalimide dye, and benzylguanine is used as a targeting group. group, its structural formula is shown in (1). The azetidine structure at the 4,5-position of the probe largely inhibits the generation of TICT (twisted intramolecular charge transfer), allowing the naphthalimide molecule to maintain high stability while being resistant to polarity changes. not sensitive. The probe is able to complete in 2 minutes (t 1 / 2 =8s) specifically binds to SNAP-tag, and achieves an 8-fold fluorescence enhancement after binding, realizing wash-free fluorescence imaging of SNAP-tag in living cells.

Description

technical field [0001] The invention belongs to the technical field of fluorescence imaging, and in particular relates to a high-stability wash-free SNAP-tag probe and a preparation method and application thereof. Background technique [0002] 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. With the emergence of tagged proteins, small-molecule fluorescent dyes can undergo specific enzymatic covalent linkage reactions with target proteins fused with tagged proteins, thereby realizing the specific linking of small-molecule fluorescent dyes to target proteins. Among them, the most widely used tag protein is SNAP-tag, which can react with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives so that SNAP-tag can be labeled with artificially synthesized probes. [0003] With ...

Claims

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Application Information

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