CN114195797B - Near infrared fluorescent probe for mitochondrial marking - Google Patents

Near infrared fluorescent probe for mitochondrial marking Download PDF

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CN114195797B
CN114195797B CN202210045398.5A CN202210045398A CN114195797B CN 114195797 B CN114195797 B CN 114195797B CN 202210045398 A CN202210045398 A CN 202210045398A CN 114195797 B CN114195797 B CN 114195797B
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probe
near infrared
fluorescent probe
mitochondrial
fluorescent
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CN114195797A (en
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刘兴江
牛培鑫
张会
张文英
李禹函
魏柳荷
孙爱灵
刘华玉
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Henan Zhengdashan Zhipu Material Technology Co ltd
Zhengzhou University
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Henan Zhengdashan Zhipu Material Technology Co ltd
Zhengzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Materials Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention discloses a near infrared fluorescence probe of mitochondria based on a pyranoquinoline structure, which belongs to the technical field of chemical analysis and detection, and has the following molecular structural formula:

Description

Near infrared fluorescent probe for mitochondrial marking
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a mitochondrial near infrared fluorescent probe and application thereof in bioluminescence imaging.
Background
Mitochondria are an organelle found in most eukaryotic cells, known as the "cell power plant", and are the primary sites for oxidative phosphorylation and synthesis of Adenosine Triphosphate (ATP) within cells, primarily providing energy for cellular activities. Mitochondrial labeled fluorescent probes have become an effective tool in studying mitochondrial physiology and in the course of cases. At present, the commercial mitochondrial fluorescent probes are mainly red fluorescent probes and green fluorescent probes, and mitochondrial near infrared fluorescent probes are rarely reported. The near infrared fluorescent probe (emission wavelength: 700-2500 nm) has the advantages of good penetrability, small background interference, small damage to tissues and the like, and has wider application in the field of biological fluorescent imaging. In addition, fluorescent probes with large Stokes shift can reduce self-absorption to increase detection sensitivity. Therefore, it is significant to develop mitochondrial fluorescence probes that emit near infrared and have large stokes shifts.
Disclosure of Invention
To overcome the deficiencies of the prior art, the present invention is directed to a near infrared fluorescent probe for mitochondrial marking.
The fluorescent probe has the following structure:
the fluorescent probe in the invention is prepared by the following synthetic route:
(a) Bromopropyne, potassium carbonate, N-dimethylformamide, 25 ℃ for 10h; (b) P-aminobenzyl alcohol, cuprous iodide, N-dimethylformamide, 110 ℃ for 6h; (c) manganese dioxide, methylene chloride, 25 ℃ for 5h; (d) piperidine, acetonitrile, reflux, 6h.
The fluorescence probe mitochondria marking mechanism in the invention is as follows: because the probe has positive charges, the probe can be targeted and positioned by utilizing the membrane potential difference of mitochondria, thereby realizing the function of marking the mitochondria.
The fluorescent probe of the invention has near infrared fluorescence emission and large Stokes shift. Maximum absorption peak (lambda) in different solvents abs Nm), maximum emission peak (lambda) em Per nm), stokes shift (Deltass/nm), molar extinction coefficient (ε/L. Mu. Mol) -1 cm -1 ) Quantum yield (phi) f ) And fluorescence lifetime (τ/ns) are shown in the following table.
The fluorescent probe has lower cytotoxicity (MTT method test). After the HeLa cells and the probes are incubated for 24 hours at 37 ℃, the cell survival rate is more than 90% within the concentration of 15.0 mu M.
The fluorescent probe has good marking performance on mitochondria. The fluorescence imaging of HeLa cells is carried out by co-staining the fluorescent probe of the invention with a commercially available mitochondrial marking probe Mito-Tracker Green, and the pearson correlation coefficient of a red channel (fluorescence of the probe) and a Green channel (fluorescence of the Mito-Tracker Green) is obtained by analysis and is 0.97.
The fluorescent probe provided by the invention can be used for imaging zebra fish staining. After incubation with the probe, the zebra fish was stained with red fluorescence.
The probe has good light stability. At 20mW/cm 2 The light degradation rate is less than 5% when the xenon lamp is irradiated for 1 hour.
Drawings
FIG. 1 is a graph showing normalized UV-visible absorption spectra of fluorescent probes of the present invention in different solvents. The abscissa is wavelength and the ordinate is absorbance.
FIG. 2 is a normalized fluorescence spectrum of the fluorescent probe of the present invention in different solvents. The abscissa is wavelength and the ordinate is fluorescence intensity.
FIG. 3 is a toxicity test of the fluorescent probe of the present invention on cells. The abscissa indicates probe concentration, and the ordinate indicates cell viability.
FIG. 4 is a mitochondrial targeting fluorescence image of co-stained HeLa cells with fluorescent probe (5.0. Mu.M) of the invention and mitochondrial label probe Mito-Tracker Green (0.2. Mu.M). (a) green and red channels are superimposed; (b) the Green channel of Mito-Tracker Green dye; (c) a red channel of the probe; (d) Correlation of green fluorescence channel with red fluorescence channel.
FIG. 5 is a fluorescent image of zebra fish staining with fluorescent probes of the present invention (5.0. Mu.M).
FIG. 6 is a graph showing the light stability test of the fluorescent probe of the present invention. At 20mW/cm 2 The dye photodegradation rate is lower than 5% after 1 hour of irradiation under a xenon lamp.
Examples of the embodiments
Example 1: synthesis of Compound 2
Compound 1 (0.2173 g,1.0 mmol) and 3-bromopropyne (0.2380 g,2.0 mmol) were dissolved in 10.0mL DMF in a 50mL single port round bottom flask, anhydrous potassium carbonate (0.2764 g,2.0 mmol) was added, stirring was stopped at room temperature for 12 hours, the reaction was stopped, the filtrate was filtered and the filtrate was spun dry to give a solid crude product, and finally, compound 2, yellow solid, 0.2002g, yield 78.4% was isolated by column chromatography.
Example 2: synthesis of Compound 3
Compound 2 (4.6259 g,20.1 mmol) and para-aminobenzyl alcohol (2.4631 g,20.1 mmol) were added to a round bottom flask, dissolved in 5.0mL DMF, heated to 110 ℃ and stirred for 6 hours. After completion of the reaction, the mixture was cooled to room temperature, and the solvent was removed in vacuo and purified by column chromatography to give 3 (0.8693 g, 13%) as a brown solid. HRMS (ESI) m/z calcd.for C 21 H 23 N 2 O 2 [M+H] + 335.1760；found 335.1756. 1 H NMR(400MHz,CDCl 3 )δ8.38(d,J＝8.2Hz,1H),8.13(d,J＝8.2Hz,1H),7.69(d,J＝24.2Hz,2H),7.59(dd,J＝8.7,1.7Hz,1H),6.53(dd,J＝8.9,2.4Hz,1H),6.23(d,J＝2.5Hz,1H),5.30(s,1H),5.25(s,2H),4.83(s,2H),3.42(q,J＝7.1Hz,4H),1.22(t,J＝7.1Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ159.2,151.2,149.8,147.7,137.9,130.5,128.8,128.6,128.5,126.8,126.5,124.9,124.7,115.2,112.8,107.0,98.2,68.6,65.2,64.8,44.6,12.7.
Example 3: synthesis of Compound 4
Compound 3 (3.3417 g,10.1 mmol) was dissolved in 10mL DCM, manganese dioxide (1.7387 g,20.1 mmol) was added and stirred at room temperature for 5 hours. After completion of the reaction, it was filtered through celite, washed with methylene chloride, and the filtrate was collected. The solvent was removed in vacuo and purified by column chromatography to give a yellow solid (2.3268 g, 70%). HRMS (ESI) m/z calcd.for C 21 H 21 N 2 O 2 [M+H] + 333.1603；found 333.1600. 1 H NMR(400MHz,CDCl 3 )δ10.11(s,1H),8.28(d,J＝8.8Hz,1H),8.18(s,1H),8.09(s,2H),7.83(s,1H),6.51(dd,J＝8.9,2.2Hz,1H),6.22(d,J＝2.2Hz,1H),5.28(s,2H),3.42(q,J＝7.0Hz,4H),1.22(t,J＝7.1Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ191.5,159.7,151.9,132.9,132.7,131.4,129.6,127.4,127.1,126.1,125.6,123.3,113.7,107.2,98.0,68.4,44.7,31.5,30.2,29.7,12.7.
Example 4: synthesis of probes
Compound 4 (10 mmol) and 2, 3-dimethylbenzo [ d ]]The thiazole-3-onium (10 mmol) was dissolved in 10mL of acetonitrile, 10. Mu.L of piperidine was added thereto, stirred under reflux for 6 hours, and after completion of the reaction, the solvent was removed under vacuum and purified by column chromatography to give a probe in a black solid with a yield of 56%. HRMS (ESI) m/z calcd.for C 30 H 28 N 3 OS + [M] + 478.1948；found 478.1980. 1 H NMR(400MHz,DMSO-d 6 )δ8.44(d,J＝7.9Hz,1H),8.40(d,J＝6.4Hz,2H),8.35(s,1H),8.31(s,2H),8.26(d,J＝8.4Hz,1H),8.11(d,J＝8.9Hz,2H),8.01(d,J＝9.4Hz,1H),7.89(t,J＝7.8Hz,1H),7.81(t,J＝7.7Hz,1H),6.54(dd,J＝9.0,2.1Hz,1H),5.36(s,2H),4.40(s,3H),3.43(q,J＝7.0Hz,4H),1.14(t,J＝7.0Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ176.2,167.8,162.8,155.3,151.1,146.2,144.3,142.2,140.1,139.6,137.8,137.7,136.1,134.7,132.8,130.5,130.1,128.8,126.3,124.0,122.8,120.7,117.1,113.3,107.5,103.3,83.8,70.9,42.8,12.1.
Example 5: application of probes to mitochondrial labelling
HeLa cells were probed with a 5.0. Mu.M probe and a commercially available mitochondrial marking probe (MitoGreen,0.2 μm) was incubated at 37 ℃ for 15min, and laser confocal imaging experiments were performed after rinsing the cells with pbs buffer. Compared with the commercial mitochondrial marking probe, the pearson correlation coefficient reaches 97%, which shows that the mitochondrial marking probe has good mitochondrial marking performance.
Example 6: fluorescent staining of zebra fish by probe molecules
Zebra fish 3 days old were selected and incubated with the probe (5.0. Mu.M) for 30 minutes before fluorescence imaging. The probe can be used for carrying out fluorescence imaging on the zebra fish.

Claims (1)

1. A near infrared mitochondrial fluorescent probe, which is characterized by having the structural formula:
CN202210045398.5A 2022-01-15 2022-01-15 Near infrared fluorescent probe for mitochondrial marking Active CN114195797B (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN106928189A (en) * 2017-03-03 2017-07-07 济南大学 A kind of mitochondrial fluorescence probe of identification with larger Stokes displacements

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106928189A (en) * 2017-03-03 2017-07-07 济南大学 A kind of mitochondrial fluorescence probe of identification with larger Stokes displacements

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Title
吡喃喹啉类荧光染料的合成、性能研究及生物应用;宋相志 等;第十五届全国光化学学术讨论会会议论文;112-113 *

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