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A class of highly stable wash-free halo-tag probes and their synthesis and biological applications

A technology with high stability and synthesis method, which is applied in the field of fluorescent labeling, can solve the problems of insufficient stability and brightness, and achieve the effect of good photostability and high brightness

Active Publication Date: 2022-08-09
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

At present, the stability and brightness of existing Halo-tag probes are still insufficient, and it is still necessary to develop multi-band highly stable Halo-tag probes

Method used

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  • A class of highly stable wash-free halo-tag probes and their synthesis and biological applications
  • A class of highly stable wash-free halo-tag probes and their synthesis and biological applications
  • A class of highly stable wash-free halo-tag probes and their synthesis and biological applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthesis of Halo-DAze

[0046] Synthesis of Intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-5-nitro-1,8-naphthalimide (OAN-NBr)

[0047]

[0048] 4-Bromo-5-nitro-1,8-naphthalimide (2.00 g, 6.24 mmol) was dissolved in 40 mL of ethanol, and diethylene glycolamine (2.0 g, 19.0 mmol) was added dropwise thereto. After 10 h at 40°C, the solvent was distilled off under reduced pressure, and the residue was separated by silica gel column (petroleum ether:dichloromethane=3:1-0:1, V / V) to obtain 996 mg of off-white solid with a yield of 39%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

[0049] 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). 13 C NMR (101MHz, CDCl 3 / DMSO)δ167.48,166.76,155.77,140.86,137.11,136.25,135.21,130.44,128.84,128.34,127.29,125.60,77.28,72.13,65.73.

[0050] Its high-resolu...

Embodiment 2

[0063] Synthesis of Halo-DAC

[0064] Synthesis of Intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-5-nitro-1,8-naphthalimide (OAN-NBr)

[0065]

[0066] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.12 mmol) was dissolved in 80 mL of ethanol, and diethylene glycolamine (0.5 g, 9.5 mmol) was added dropwise thereto. After 1 h at 90°C, the solvent was distilled off under reduced pressure, and the residue was separated on a silica gel column (petroleum ether:dichloromethane=3:1-0:1, V / V) to obtain 677 mg of off-white solid with a yield of 53%.

[0067] Synthesis of N-dodecyl-4,5-(1,2-cyclohexanediamino)-1,8-naphthalimide (DDAN-DAC)

[0068]

[0069] OAN-NBr (100 mg, 0.24 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 100 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100 °C and reacted for 24 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel...

Embodiment 3

[0082] Synthesis of Halo-Aze

[0083] Synthesis of Intermediate N-(2-(2-hydroxy)-ethoxy)ethyl-4-bromo-1,8-naphthalimide (OAN-Br)

[0084]

[0085] 4-Bromo-1,8-naphthalene anhydride (1.0 g, 3.6 mmol) and diethylene glycol amine 1000 mg were placed in 80 mL of ethanol, and slowly heated to 40°C. Heating was stopped after 10 h, and the reaction solution was cooled to room temperature. The off-white solid 1.1 g was obtained by filtration, and the yield was 85%.

[0086] Synthesis of OAN-Aze

[0087]

[0088] OAN-Br (200 mg, 0.55 mmol) was dissolved in 15 mL of ethylene glycol methyl ether, and 300 mg of azetidine was added thereto. The reaction solution was slowly heated to 140 °C and reacted for 10 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V / V) to obtain 122 mg of a yellow solid with a yield of 61%. Its nuclear magnetic spectrum hydrogen spectrum and car...

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Abstract

The invention provides a class of highly stable disposable Halo-tag probes, a synthesis method and biological application thereof, and the probes are designed and synthesized based on naphthalimide dyes. Probe, whose structural formula is shown in (1), through the inhibition of intramolecular torsion by rigid cyclic amines, this type of Halo-tag probe can maintain high quantum yield and stability, and the quantum yield in water reaches more than 0.80 . In addition, this type of probe can realize the specific labeling of target proteins fused with Halo-tag in living cells, and realize wash-free fluorescence imaging. Such probes have good application prospects in the fields of single-molecule detection and super-resolution fluorescence imaging.

Description

technical field [0001] The invention belongs to the field of fluorescent labeling, and particularly relates to a class of highly stable disposable Halo-tag probes and a synthesis method and biological application thereof. Background technique [0002] The method of fusing a target protein with a functional tag protein has become an important technology for protein detection and analysis. The introduction of fluorescent proteins can realize the fluorescent labeling of target proteins, so that the distribution, quantity and function of proteins can be monitored in situ and in real time through the collection of fluorescent signals. However, the transformation of fluorescent proteins is relatively complex and the colors are not rich, which brings great difficulties to many researchers. The synergistic development of tagged proteins such as SNAP-tag and Halo-tag and organic small molecule fluorescent probes has largely overcome this problem. Through the rational design of orga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D221/14C07D401/14C07D471/06C09B57/08C07D401/04C09K11/06G01N21/64A61K49/00
CPCC07D221/14C07D401/14C07D471/06C07D401/04C09B57/08C09K11/06G01N21/6486G01N21/6456G01N21/6458A61K49/0021C09K2211/1007C09K2211/1029C09K2211/1044Y02P20/55
Inventor 徐兆超乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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