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Heptamethine cyanine active fluorescent probe and preparation method and application thereof

A technology of fluorescent probes and cyanines, which is applied in the field of active fluorescent probes of heptamethine cyanines and its preparation and application, can solve the problems of increased synthesis complexity and difficulty of separation and purification, a single type of active site, and inability to meet the requirements of proteins. etc. to simplify the preparation and purification process, reduce complexity, and increase fluorescence intensity

Inactive Publication Date: 2018-05-15
INST OF BIOMEDICAL ENG CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the application of near-infrared long-wave heptamethine indole fluorescent probes in bioluminescent analysis is still limited by some problems.
[0006] First of all, the fluorescent probes of heptamethine, which can be fluorescently labeled through chemical reactions, usually have carboxyl or succinimide active esters, and the active site is located on the indoline aromatic core. The overall structure is asymmetric, which greatly increases Synthetic complexity and difficulty of separation and purification
[0007] Secondly, the current commercial Qijiachuan fluorescent dyes have a single type of active site, which cannot meet the labeling needs of proteins, DNA and other biologically active molecules, polymers or inorganic nanocarriers.
[0008] Thirdly, the current commercial heptamethine fluorescent probe is expensive, and it is necessary to optimize the synthesis route and purification process

Method used

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  • Heptamethine cyanine active fluorescent probe and preparation method and application thereof
  • Heptamethine cyanine active fluorescent probe and preparation method and application thereof
  • Heptamethine cyanine active fluorescent probe and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 contains cyclohexene chlorine bridging parent dye 1-a synthesis

[0035] According to the synthesis route shown in the figure below, the parent dye 1-a containing cyclohexene chlorine bridge is synthesized, and the specific steps are as follows:

[0036]

[0037] (1) Intermediate N-ethyl-2,3,3-trimethyl-3H-indoline

[0038]Add 2,3,3-trimethyl-3H-indoline (25mL, 24.8g, 156mmol) in a 250mL round bottom flask, disperse in 50mL anhydrous toluene, add iodoethane (30g, 192mmol) dropwise Anhydrous toluene solution was refluxed for 12 h under the protection of argon, and the reaction was terminated after heating was stopped. The solid product was obtained by filtration, washed three times with anhydrous diethyl ether and dried in vacuo to obtain a pink solid with a yield of 79%.

[0039] (2) Synthesis of condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene

[0040] Under ice-bath conditions, add 10 mL of phosphorus oxychloride dropwise to 10 mL of an...

Embodiment 2

[0043] Synthesis and characterization of embodiment 2 target fluorescent probe 2-a-a

[0044]

[0045] Add 200 mg of parent dye 1-a and 1.5 times the molar amount of nucleophile N-(3-aminopropyl) methacrylamide hydrochloride and 3.0 times the molar amount of DIPEA in a 50 mL round bottom flask, and use 20 mL of anhydrous Disperse with methanol, and react in the dark at 50°C for 12 hours. After the reaction solution is cooled to room temperature, it is dropped into 200 mL of anhydrous ether, and a large amount of solid precipitates are precipitated. After filtration, it is vacuum-dried. Using dichloromethane / methanol as the eluent, the 2-a-a dye was obtained by medium-pressure flash preparative chromatography with a yield of 75%.

[0046] fluorescent probe 2-a-a, 1 H NMR (400MHz, DMSO-d6): δ = 8.73 (s, 1H, NH); 8.45 (s, 1H, NH); 8.36 (d, 2H, CH); 7.40 (d, 2H, ArH); 7.37 ( t,ArH,2H); 7.37(t,2H,ArH); 7.16(d,2H,ArH); 6.25(d,2H,CH); 5.72(s,1H,CH); ); 4.24(q,CH 2 ,4H); 3.85(t...

Embodiment 3

[0047] Synthesis and characterization of embodiment 3 target fluorescent probe 2-a-b

[0048]

[0049] The synthesis method is the same as in Example 2, and the nucleophile uses 2-(2-pyridyldithio)ethylamine hydrochloride.

[0050] fluorescent probe 2-a-b, 1 H NMR (400MHz, DMSO-d6): δ=8.73 (s, 1H, NH); 8.36 (d, 2H, CH); 8.25 (s, 1H, CH); 7.00-8.00 (m, 11H, ArH); 6.25(d,2H,CH); 4.24(q,CH 2 ,4H); 3.49(d,2H,CH 2 ); 3.09 (dd,2H,CH 2 ); 2.85(dd,4H,CH 2 ); 2.45(t,1H,CH,); 1.70(s,12H,CH 3 ); 1.56(m,2H,CH 2 ); 1.44(t,CH 3 ,6H). MS(ESI): m / z calculated: 661.34; found: 661.42.

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Abstract

The invention relates to a heptamethine cyanine active fluorescent probe and a preparation method and application thereof. The structural formula of the heptamethine cyanine active fluorescent probe is as shown in the specification, wherein X=II-IX; each of R1 and R2 is (CH2)mCH3, (CH2)nOH, (CH2CH2O)pCH3 and CH2C6H5; each of R3 and R4 is H, SO3H, SO3Na and SO3K; each of a, b, c, d, e, f and g is 2-8; each of n, m and p is 1-10. The heptamethine cyanine active fluorescent probe has the advantages that the heptamethine cyanine active fluorescent probe is based on near-infrared long-wave heptamethine cyanine dye, indoline is selected as the aroma parent nucleus to increase fluorescence intensity, and methenyl chain intermediate cyclohexene rigid bridging enhances stability; nitrogen derivatives with chemical reactivity sites are used to perform nucleophilic substitution on the meso-position of the heptamethine cyanine parent dye, and accordingly Stokes shift and active chemical groups areincreased greatly to facilitate the fluorescent labeling of various substances; the fluorescent probe is of a symmetrical structure, preparation and purification processes are simplified, and cost islowered favorably; the probe can be used as the fluorescent labeling probe of biological molecules such as high-sensitivity protein, sugar and DNA and nano carriers to perform cell or living-body horizontal fluorescence imaging, and the like.

Description

technical field [0001] The present invention relates to a heptamethine active fluorescent probe and its preparation method and application, in particular to a fluorescent label suitable for biomolecules and nanocarriers such as highly sensitive proteins, sugars, DNA, etc., with a Stokes shift greater than 100 nm Fluorescent probes for cellular or in vivo fluorescence imaging. Background technique [0002] Fluorescent labeling of biomolecules is a rapid and convenient biomolecular analysis method following isotope labeling. With the development of life science, there is an urgent need for high-sensitivity analysis and detection methods. In medicine, enzyme-catalyzed immunoassay and fluorescence polarization immunoassay with fluorescence technology as the core have become the standard methods of medical diagnosis. In molecular biology, laser confocal microscopy is used to track the position and movement of intracellular components. In addition, flow cytometry uses fluoresce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D209/14C07D401/14C07D403/14C09K11/06C08F220/34C08F220/60C08F220/36C08F2/38G01N21/64
CPCC07D209/14C07D401/14C07D403/14C08F2/38C08F220/34C08F220/36C08F220/60C09K11/06C09K2211/1007C09K2211/1029C09K2211/1466G01N21/6428G01N2021/6432
Inventor 黄平升王伟伟秦怡博孔德领
Owner INST OF BIOMEDICAL ENG CHINESE ACAD OF MEDICAL SCI
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