CN104017572A - Extremely acidic pH fluorescent probe, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an extremely acidic pH fluorescent probe, and a preparation method and application thereof. The pH fluorescent probe is constructed by using butadiene bridged indole or derivatives thereof and 3-[3-(4-fluorophenyl)-1-alkyl-1H-indole on the basis of intramolecular charge transfer effect. The preparation method comprises the following steps: in an inert gas protective atmosphere, dissolving 2,3,3-trimethylindole or 1,1,2-trimethyl-1H-benzo[e]indole and 3-[3-(4-fluorophenyl)-1-alkyl-1H-indolyl-2-yl]-acraldehyde in N,N-dimethylformamide, adding KOH solid, stirring at room temperature, standing over night, concentrating under reduced pressure to obtain a crude product, and separating through a silica gel column to obtain the pure product. The probe has the advantages of lower pKa value, higher sensitivity for H<+>, favorable selectivity and large Stocks displacement. The probe has obvious color variation before and after combination with H<+>, and thus, can be conveniently identified by naked eyes. The probe can penetrate the cytomembrane, and is suitable for intracellular pH value detection and cell imaging.

Description

A kind of extreme acid pH fluorescent probe and its preparation method and application

Technical field

The present invention relates to fluorescent probe, specifically belong to a kind of extreme acid pH fluorescent probe and preparation method thereof, and the application of this probe in cell imaging.

Background technology

PH value not only has obvious impact to the physico-chemical property of material and reactivity worth thereof, and many important physiological processs of cell and crganelle etc. are also all closely related with pH value.Acid and alkalescence is crossed and can be caused by force the heart, tuberculosis to become or neural class disease, when serious, even can be in peril of one's life.Under normal physiological condition, H in enchylema ⁺concentration be about 40nmol/L (pH=7.40), its amplitude of fluctuation is generally in 0.1～0.2Ge pH unit.In cell, also having a pH value scope is acid range, and if lysosomal pH value scope is 4.5-5.5, the pH value scope of endosome is 5.4-6.2, is called acid organoid.Extremely sour environment (pH<4) is fatal to most of organisms.The substantial connection of intracellular ph value and cell function means can provide key message for studying the little physiology to individual cells device and pathological process to the accurate measurement of intracellular ph value.So the dynamic change of monitoring intracellular ph value has important effect for understanding the mechanism of many physiological functions in cell.

Life system requires to detect small pH value and changes.Measure pH value general by glass electrode, but electrochemistry is disturbed owing to existing, the possible defects such as physical abuse are unsuitable for live body pH monitoring.Adopt in addition colorimetric method for determining pH value, but colorimetry also exists obvious shortcoming, cannot be applied to life system and carry out active somatic cell radiography.In addition, the sensitivity of colorimetry is lower.Fluorescent probe has quick real-time response, to cell not damaged, the advantage such as response sensitivity is high and selectivity is single-minded, utilize fluorescent probe to change and detect the broad interest that causes people intracellular ph value.At present many pH fluorescent probes of report concentrate on detect in cell neutral, i.e. the acid organoid pH value such as the pH value of enchylema and lysosome and endosome; Measure extremely environment, considerably less as the fluorescent probe report of pH<4 or pH>9.Therefore, design synthetic there is large Stokes displacement, to H ⁺the fluorescent probe that ion is extremely sensitive, selectivity good, can measure pH under environment extremely all has potential using value at aspects such as biological and chemicals.

Summary of the invention

One of object of the present invention be to provide a kind ofly there is large Stocks displacement, extremely sensitive to pH, selectivity is high, can measure the fluorescent probe of pH value under acidic conditions extremely; Two of object is to provide the preparation method of this probe, and the method technique is simple, with low cost; Three of object is to provide the purposes of this probe, and this probe detects intracellular ph value as fluorescent indicator.

The pH fluorescent probe of a kind of extreme acidity provided by the invention, its structural formula is:

Wherein, R ₁, R ₂for hydrogen atom or alkyl.

The synthetic route of the pH fluorescent probe of extreme acidity provided by the invention is as follows:

Its preparation method, comprises the steps:

(1) under protection of inert gas, by 2,3,3-tri-methyl indole or 1,1,2-trimethylammonium-1H-benzo [e] indoles and 3-[3-(4-fluorophenyl)-1-alkyl-1H-indoles-2-yl]-propenal in molar ratio 1 ︰ 1.0-1.5 be dissolved in N, dinethylformamide, by 2,3,3-tri-methyl indole or 1,1,2-trimethylammonium-1H-benzo [e] indoles meter, adds the KOH solid of 5 times of mole numbers, stirring at room, reaction is spent the night;

(2) remove by filter KOH solid, to the H that adds 1-2 times of volume in reaction solution ₂o, uses CH ₂cl ₂extract 2-3 time; Merge organic phase, use anhydrous Na ₂sO ₄after dry, underpressure distillation obtains crude product;

(3) crude product is separated through silicagel column, obtains sterling.

Probe of the present invention has good cytolemma penetrance, can be used for detection and the cell imaging of intracellular ph value.

Compare with existing pH fluorescent probe, the synthetic probe of the present invention has the following advantages: the Stokes displacement that (1) is large, is conducive to reduce the interference of exciting light in angiographic procedure; (2) low pKa value, can detect the pH value under extreme acidic conditions; (3) good selectivity, this probe is to H ⁺response be not subject to the interference of common metal ion; (4) this probe shifts (ICT) principle design based on intramolecular charge, and in the process of protonated and deprotonation, Absorption and emission spectra changes, and under natural light, solution colour obviously changes, can be by visual inspection.(5) this probe has good cytolemma penetrance, utilizes laser confocal imaging technology can carry out detection and the cell imaging of internal pH; (6) synthesis step of this probe is simple, with low cost, has larger practical value.

Accompanying drawing explanation

Fig. 1. the uv absorption spectra that the embodiment of the present invention 1 middle probe changes with pH value.

Fig. 2. the embodiment of the present invention 1 middle probe under natural light in conjunction with H ⁺front and back colour-change, color becomes a piece redness from glassy yellow.

Fig. 3. the fluorescence emission spectrogram that the embodiment of the present invention 1 middle probe changes with pH value.

Fig. 4. the sigmoidal matched curve that the fluorescence intensity of the embodiment of the present invention 1 middle probe changes with pH value.

Fig. 5. the response condition of the embodiment of the present invention 1 middle probe to common metal ion.

Fig. 6. the embodiment of the present invention 1 middle probe and human renal carcinoma cell 7860 are jointly hatched the laser confocal imaging figure of 30min under the condition of pH value 7.4, and cell sends bright green glow (a) and ruddiness (b).

Fig. 7. the embodiment of the present invention 1 middle probe and human renal carcinoma cell 7860 are jointly hatched the laser confocal imaging figure of 30min under the condition of pH value 4.2, and the fluorescence of cell is obviously to weaken for 7.4 o'clock compared with pH value.

Embodiment

Embodiment 1

A kind of preparation of the extreme acid pH fluorescent probe based on benzindole:

(1) under protection of inert gas, by 0.283g (1.35mmol) 1,1,2-trimethylammonium-1H-benzo [e] indoles and 0.454g (1.48mmol) 3-[3-(4-fluorophenyl)-1-sec.-propyl-1H-indoles-2-yl]-propenalbe dissolved in 6mL DMF, add 0.378g (6.75mmol) KOH solid, stirring at room, reaction is spent the night.

(2) remove by filter KOH solid, in reaction solution, add H2O (6mL), mixing solutions CH ₂cl ₂extraction (10ml * 3).The organic phase merging is through anhydrous Na ₂sO ₄after dry, underpressure distillation obtains crude product.

(3) product crude product is separated through silicagel column, and methylene dichloride is eluent, obtains orange/yellow solid. ¹H?NMR(300MHz,DMSO-d ₆),δ:1.556(s,6H),1.648-1.671(d,6H),5.009-5.055(m,1H),6.607-6.658(m,2H),7.037-7.085(m,1H),7.178-7.258(m,2H),7.309-7.364(t,2H),7.414-7.570(m,4H),7.596-7.691(m,1H),7.691-7.755(t,3H),7.917-7.946(d,1H),7.999-8.025(d,1H),8.125-8.153(d,1H).

Embodiment 2

Concentration and probe concentration in embodiment 1 is remained on to 5 μ mol/L, and the HCl with 1mol/L in ethanol/water (volume ratio is 2:1) system regulates pH value, records its absorption spectrum (Fig. 1).Along with the reduction of pH value, the absorption peak at short wavelength 426nm place declines gradually, and the absorption peak at long wavelength 546nm place significantly strengthens, and has an isobestic point at 450nm place.The color of solution has also become a piece redness (Fig. 2) from original glassy yellow.

Embodiment 3

Concentration and probe concentration in embodiment 1 is remained on to 5 μ mol/L, and the HCl with 1mol/L in ethanol/water (volume ratio is 2:1) system regulates pH value, take 426nm as excitation wavelength, records its fluorescence emission spectrum (Fig. 3).Along with the reduction of pH value, the fluorescence peak at 571nm place weakens gradually.Utilize the fluorescence intensity at 571nm place to the mapping of pH value, and utilize sigmoidal to carry out matching (Fig. 4), the pKa value that obtains the probe in embodiment 1 is 2.89.

Embodiment 4

Concentration and probe concentration in embodiment 1 is remained on to 5 μ mol/L, investigate respectively the response condition of this probe to common metal ion.As shown in Figure 5, this probe, to almost not response of metal ion, proves that this probe is to H ⁺the selectivity having had.In Fig. 5, the order of material and concentration are followed successively by: 1. probe; 2.Na ⁺(30mM); 3.K ⁺(30mM); 4.Mg ²⁺(25mM); 5.Ca ²⁺(25mM); 6.Ba ²⁺(25mM); 7.Fe ³⁺(60 μ M); 8.Fe ²⁺(1mM); 9.Al ³⁺(1mM); 10.Cu ²⁺(1mM); 11.Zn ²⁺(25mM); 12.Mn ²⁺(25mM); 13.Ni ²⁺(15mM); 14.Co ²⁺(4mM); 15.Cr ³⁺(1 μ M); 16.Pb ²⁺(0.5mM); 17.Cd ²⁺(0.1mM); 18.Hg ²⁺(0.1mM); 19.Ag ⁺(0.1mM).

Embodiment 5

By the probe in cultured human renal carcinoma cell 7860 and embodiment 1 respectively under pH value 7.4 and 4.2 conditions, in 37 ℃, the incubator of 5%CO2, jointly hatch 30min, then with the phosphate buffer soln of corresponding pH value, wash 3 times, under laser confocal microscope, observe, fixedly excitation wavelength is 426nm, the fluorescence sending at green glow and ruddiness passage collecting cell respectively.Experimental result shows, the cell of pH value 7.4 send comparatively bright green (cell green fluorescence in Fig. 6 a) and ruddiness (b in Fig. 6) fluorescence, and during pH4.2 (and in Fig. 7 a) and red fluorescence (b in Fig. 7) all obviously weaken.

Claims (4)

1. an extremely acid pH fluorescent probe, is characterized in that structural formula is:

Wherein, R ₁, R ₂for hydrogen atom or alkyl.

2. the preparation method of the pH fluorescent probe of a kind of extreme acidity as claimed in claim 1, is characterized in that, comprises the steps:

(1) under protection of inert gas, by 2,3,3-tri-methyl indole or 1,1,2-trimethylammonium-1H-benzo [e] indoles and 3-[3-(4-fluorophenyl)-1-alkyl-1H-indoles-2-yl]-propenal in molar ratio 1 ︰ 1.0-1.5 be dissolved in N, dinethylformamide, by 2,3,3-tri-methyl indole or 1,1,2-trimethylammonium-1H-benzo [e] indoles meter adds the KOH solid of 5 times of mole numbers, stirring at room, and reaction is spent the night;

(2) remove by filter KOH solid, to the H that adds 1-2 times of volume in reaction solution ₂o, uses CH ₂cl ₂extract 2-3 time; Merge organic phase, use anhydrous Na ₂sO ₄after dry, underpressure distillation obtains crude product;

(3) crude product is separated through silicagel column, obtains sterling.

3. the pH fluorescent probe of a kind of extreme acidity as claimed in claim 1 is as the indicator that detects intracellular ph value.

4. the application of the pH fluorescent probe of a kind of extreme acidity as claimed in claim 1 in cell imaging.