488 nm excited no-clean Halo-tag probe as well as synthesis and biological application thereof

A 488nm probe technology, applied in the field of protein labeling, can solve the problems of poor signal-to-noise in fluorescence imaging, low cell permeability, and fluorescence signal errors, and achieve the effects of high photostability, simple purification, and high brightness

Active Publication Date: 2020-06-26
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The luminescent form of fluorescein is negative ion form, which not only has poor stability, but also has low cell permeability; while rhodamine-based dyes overcome the problems of cell perme

Method used

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  • 488 nm excited no-clean Halo-tag probe as well as synthesis and biological application thereof
  • 488 nm excited no-clean Halo-tag probe as well as synthesis and biological application thereof
  • 488 nm excited no-clean Halo-tag probe as well as synthesis and biological application thereof

Examples

Experimental program
Comparison scheme
Effect test

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 hours 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, 12...

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 24h. Ethylene glycol methyl ether was removed under reduced press...

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 ...

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Abstract

The invention provides a 488 nm excited no-clean Halo-tag probe as well as synthesis and biological application thereof. The probe is based on naphthalimide dye, and the structural formula of the probe is as shown in (1) shown in the specification. According to the probe, inhibition on intramolecular torsion is realized through azetidine with strong ring tension, so that the Halo-tag probe can keep high quantum yield and stability. In addition, the maximum excitation of the probe in water is 482 nm, and the probe is suitable for fluorescence imaging under 488 nm excitation. The probe can realize specific labeling of target protein fused with Halo-tag in living cells, achieves no-clean fluorescence imaging, and has a good application prospect in the fields of single molecule detection, super-resolution fluorescence imaging and the like.

Description

technical field [0001] The invention belongs to the field of protein labels, and in particular relates to a 488nm excited non-washing Halo-tag probe and its synthesis and biological application. Background technique [0002] Organic small-molecule fluorescent dyes have the advantages of small size, broad fluorescence emission spectrum, and a variety of fluorescent colors, which also make them gradually become substitutes for fluorescent proteins and are widely used in protein fluorescent labeling. The emergence of tagged proteins enables small-molecule fluorescent dyes to be covalently linked to tagged proteins through specific enzymatic reactions, thereby indirectly achieving stable labeling of target proteins. Among them, Halo-tag, as a variant of non-human dehalogenase, is favored by many researchers. Halo-tag can specifically react with fluorescent dyes containing halogenated aliphatic hydrocarbons, so that the two can pass through Stable ester linkage. [0003] The ap...

Claims

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Application Information

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IPC IPC(8): C07D401/14C09B57/08C09K11/06G01N21/64A61K49/00
CPCC07D401/14C09B57/08C09K11/06G01N21/6486A61K49/0021C09K2211/1029
Inventor 徐兆超乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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