WO2013075285A1

WO2013075285A1 - One-class bi-benzyl pentamethyl cyanine fluorescent dye, and preparation method and application thereof

Info

Publication number: WO2013075285A1
Application number: PCT/CN2011/082619
Authority: WO
Inventors: 樊江莉; 李·安德鲁; 彭孝军; 强新新; 王倩
Original assignee: 大连理工大学
Priority date: 2011-11-22
Filing date: 2011-11-22
Publication date: 2013-05-30

Abstract

A one-class bi-benzyl pentamethyl cyanine fluorescent dye, and a preparation method and application thereof. The fluorescent dye has the structure of a general formula I. In the formula I, X^- is selected from a halogen anion, ClO₄ ^-, PF₆ ^-, CF₃ ^-, BF₄ ^-, R₁CO₂ ^-, R₂SO₃ ^- or OTs^-, wherein R₁ and R₂ are respectively and independently selected from C_1-12 alkyl or aryl. The compound can be used for the fluorescent specificity marker of living cell mitochondria or fixed cell mitochondria.

Description

Bis-benzyl pentamethine cyanine fluorescent dye, preparation method and application thereof

Technical field

The present invention relates to a class of bisbenzylpentaphthalocyanine fluorescent dyes, a process for their preparation and their use as specific fluorescent probes in living cells and fixed cell markers. Background technique

Mitochondria are organelles that are ubiquitous in eukaryotic cells. Most of the energy required for various life activities is provided by ATP synthesized in mitochondria. Therefore, it is known as the "power plant" of cells. Currently available commercial mitochondrial fluorescent probes are: JC-1, Rhodamine 123 and MitoTracker® series of probes. Among them, JC-1 is the most widely used. When the concentration or mitochondrial membrane potential is low, it exists as a monomer with an excitation wavelength of 490 nm and an emission wavelength of 527 nm, which is green fluorescent. When the concentration is increased or the lineage of the mitochondrial membrane is increased, JC-1 forms a J-mer with orange fluorescence, and the excitation wavelength is 490 nm and the emission wavelength is 590 nm. The most important application of JC-1 is to detect membrane potential in mitochondria in living cells and to track changes in mitochondria in apoptotic cells. Rhodamine 123 is a fluorescent probe that penetrates into cells and carries cations. It has an excitation wavelength of 505 nm and an emission wavelength of 534 nm. Living cells uptake Rhodamine 123 reaches equilibrium at a faster rate. It takes only 5 minutes to observe the mitochondria stained green by Rhodamine 123 by laser scanning confocal microscopy. When cells are washed repeatedly, Rhodamine 123 is usually not retained by cells, but many cancer cells retain this dye for a long time, so it contributes to the diagnosis of certain cancers. However, once the membrane potential of the mitochondria is lost, they are easily washed away. This limits some applications. The MitoTracker® range of probes, designed by Invitrogen for mitochondria, is cell-permeable and passively penetrates the plasma membrane and accumulates in active mitochondria. Although some MitoTracker probes can achieve mitochondrial labeling in fixed cells, the entire cell is evenly stained in a short period of time, so these dyes are recommended only for living cells.

Therefore, current mitochondrial fluorescent probes have different disadvantages, such as being controlled by mitochondrial membrane potential, and unable to label mitochondria in fixed cells. More importantly, the absorption and emission wavelengths of these probes are in the visible region (490-530 nm), and the biological sample has strong absorption and fluorescence emission in this region, which causes the fluorescence detection efficiency to be greatly reduced.

With the rapid development of biotechnology and fluorescent labeling technology, since the cyanine dye has a large molar extinction coefficient, the absorption and emission wavelengths are adjustable with the length of the dye conjugated chain, and the fluorescence is stronger after binding with biomolecules, and the cytotoxicity is small. It has become the main fluorescent probe used in analytical assays such as DNA, proteins and nucleic acids. The absorption and emission wavelength of the pentamethine cyanine dye Cy5 is above 600 nm, close to the near-infrared region, and is the shortest wavelength near-infrared Cyanine dyes, as a new generation of commercial fluorescent labeling agents, have been widely used in protein labeling, DNA sequencing, ion neutral small molecule recognition, cell and living tissue imaging. However, to date, no pentamethine cyanine dye has been reported for fluorescence-specific labeling of intracellular mitochondria.

Summary of the invention

SUMMARY OF THE INVENTION An object of the present invention is to provide a probe molecule based on pentamethine cyanine dye which has a spectral range in the near-infrared region, which is capable of fluorescently labeling mitochondria in living cells and fixed cells, and having a simple structure and excellent performance.

One of the objects of the present invention is a class of bisbenzylpentaphthalocyanine fluorescent dyes having the following structural formula I:

In the formula I, X- is selected from the group consisting of halogen anions, ClO ₄ \ PF ₆ " , CF ₃ -, BF ₄ R!C , R2SO or OTs",

Wherein and R ₂ are each independently selected from a fluorenyl or aryl group of d- ₁₂ .

Preferred technical scheme, and the each independently selected from alkyl with ₆ _.

Still more preferably, in the formula I, X- is selected from the group consisting of halogen anions, most preferably Cl-, Br-, and fluorene.

Another object of the present invention is to provide a process for producing the above-mentioned bisbenzylpentaphthalocyanine fluorescent dye of the present invention, which comprises reacting a compound of the formula II with a compound III (condensing agent malondialdehyde aniline salt) in the presence of a base. The reaction solvent is an acid anhydride, and the reaction temperature is 20 to 150 ° C, and the reaction time is 10 to 100 minutes. The preferred reaction time is 30 to 50 minutes.

Wherein X is selected from the group consisting of halogen anions, ClO ₄ \ PF ₆ " , CF ₃ -, BF ₄ -, R!C, R ₂ SO ₃ - or OTs- preferably halogen anions, most preferably Cl-, Br\ Γ.

Wherein R n are each independently selected from a fluorenyl or aryl group of d ₁₂ , preferably a fluorenyl group of d ₆ .

The molar ratio of the compound II to the compound III is 1: 0.1 to 1;

The molar ratio of the compound II to the base is 1:0.5 to 2. In the above production method of the present invention, the molar ratio of the compound II to the compound III is preferably 1:0·4 to 0.6.

In the above process for producing a bisbenzylpentaphthalocyanine fluorescent dye of the present invention, the condensation reaction of the compound II and the compound III is carried out in the presence of a base. The same type of reaction has been described in the prior art, and therefore, those skilled in the art can generally determine the amount of base used in the reaction and its amount. In a preferred embodiment of the present invention, sodium acetate, potassium acetate, sodium phosphate, sodium formate, sodium propionate, potassium propionate, sodium oxalate or potassium oxalate is selected as the base. Most preferred is sodium acetate. The amount of the base is 0.9 to 1.1 times the molar amount of the compound II.

In the above preparation method of the bisbenzylpentaphthalocyanine fluorescent dye of the present invention, the condensation reaction of the compound II and the compound III is carried out in a reaction system in which an acid anhydride is a solvent, and preferred acid anhydrides as a solvent include acetic anhydride, propionic anhydride, and dibutyl hydride. Anhydride and glutaric anhydride. Most preferred is acetic anhydride.

Fluorescent dyes prepared by the above preparation methods can be recovered by separation and purification techniques well known in the art to achieve the desired purity. For example, after completion of the reaction, it is cooled, poured into a saturated aqueous solution of NaCl, and stirred to precipitate blue particles. Filter, wash with ether, dry a blue solid. Purification of the product can be carried out by normal phase silica gel column chromatography (e.g. petroleum ether: ethyl acetate = 1:10).

The bisbenzylpentaphthalocyanine dye of the structural formula I provided by the above invention has the most absorption in DMSO at 654/662 nm and the maximum emission spectrum at 672 nm; the maximum absorption and maximum emission spectra in water are respectively 647 Nm and 662 nm; specific fluorescent labeling of mitochondria in fixed cells, independent of mitochondrial membrane potential, is therefore particularly useful for pathogenic cells; cell viability IC _{5 ()} after incubation for 24 hours at room temperature with 10 nM concentration I Can reach 60%. Mitotracker Green ^TM I and mitochondria in living cells staining position identical mitochondria in living cells by specific fluorescent label.

Accordingly, it is still another object of the present invention to provide the use of the above-described bisbenzylpentaphthalocyanine fluorescent dye for mitochondrial fluorescence-specific labeling. The mitochondria described therein include living cell mitochondria and fixed cell mitochondria. Similar to the prior art in the art, fluorescent labeling with the bisbenzylpentaphthalocyanine fluorescent dye of the present invention necessarily includes a compound of one of the above dyes of the present invention, a conjugate thereof, or a dye containing the same. A composition of a composition in an effective concentration at a dose in sufficient contact with the cells to be labeled under suitable conditions. More specific conditions, those skilled in the art can select a suitable solution for a specific sample to be dyed according to the prior art. DRAWINGS

Figure 7 of the accompanying drawings,

Figure 1 (a) is the absorption spectrum of dye 1-1 in DMSO and water, respectively. The abscissa is the wavelength and the ordinate is the absorption intensity; Figure 1 (b) is the fluorescence emission spectrum of dye 1-1 in DMSO and water, respectively. The abscissa is the wavelength (nm). The ordinate is the relative fluorescence intensity. The instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453 _; fluorescence spectrophotometer, model: FP-6500. The concentration of the dye 1-1 was 5 μΜ. Figure 2 is a fluorescent staining image of live cell staining of different concentrations of dye 1-1 in MCF-7:

The final concentrations of dye 1-1 in the medium were a) 10 nM _; b) 100 nM; c) 500 nM and d) ΙμΜ; dye and MCF-7 live cells at 37 ° C, 5% CO ₂ After incubation for 30 minutes, photographs were taken using a fluorescence inverted microscope (Olympus 1X81), Cy5 filter (excitation wavelength: 628/40 nm), magnified 20 times. Figure 3 (a) is a picture of live cell staining of Mito-Tracker GreenTM in MCF-7;

Figure 3 (b) is a photograph of live cell staining of dye 1-1 in MCF-7;

Figure 3 (c) is a superimposed photograph of a and b;

Figure 3 (d) is an enlarged view of the fluorescent staining area of the box portion in Figure c; Figure 4 is the result of analyzing Example 5 using the Slidebook (Olympus) software, i.e., staining of living cells by the two dyes in Figure 3d. The yellow line indicates the range of cell analysis selected by the software. The abscissa is micron, the left ordinate is the relative fluorescence intensity of dye 1-1, and the right ordinate is the relative fluorescence intensity of the dye Mito-Tracker GreenTM. Instrument used: Fluorescence inverted microscope (Olympus 1X81), respectively using a Cy5 filter (excitation wavelength: 628/40 nm) to excite probe dye 1-1, GFP filter (excitation wavelength: 472/30 nm) to stimulate Mitotracker Green TM, adjust the corresponding exposure time (500 亳 - 1000 亳 seconds) with an oil mirror (60x). Figure 5 is a counterstained picture of dye 1-1 and DAPI in MCF-7 fixed cells in Example 6; Figure 6 is a random selection of a cell in Example 6, and analysis of DAPI and dye 1 using Slidebook (Olympus) software -1 in the stained area of the fixed cells. Gray lines represent DAPI and black lines represent dye 1-1. Instrument used: Fluorescence inverted microscope (Olympus 1X81), using Cy5 filter (excitation wavelength: 628/40 nm) to excite probe dye 1-1, DAPI filter (excitation wavelength: 377/50 nm), adjust the corresponding The exposure time (500 sec - 1000 sec) was observed with an oil mirror (60x). Figure 7 is the cell viability of dye 1-1 in the presence of MCF-7 cells. The final concentration of the dye in the medium was 0 M (TCP), 0.01 μΜ, 0.1 μΜ, 1 μΜ and 10 μΜ, 5% CO ₂ cells at 37 ° C. Incubator for 24 hours after addition of CellTiter-Blue ^TM, fluorescent cells were counted using a fluorescence counter (FL600, Bio-Tek), 560 EM / 590 EX fluorescence was recorded. detailed description

The following non-limiting examples are provided to enable those of ordinary skill in the art to understand the invention.

Example 1. Synthesis of Dye 1-1:

Accurately weigh 524 mg (2 mmol) of intermediate 11-1, 258 mg (1 mmol) of the condensing agent malondialdehyde aniline hydrochloride (III-1) and mix and pour into a 10 ml single-necked flask. Add 164 mg (2 mmol) of sodium acetate, dissolve in 5 ml of acetic anhydride, stir with heating, and reflux for 40 min, then stop heating. After cooling, it was poured into a saturated aqueous solution of NaCl, and after stirring, blue particles were precipitated. Filtration, washing with diethyl ether, dried and weighed 520 mg of a blue solid. The product was purified by normal phase silica gel column chromatography (petroleum ether: ethyl acetate = 1 : 10). The nuclear magnetic and high resolution mass spectral data of dye 1-1 are as follows:

1H hire R (400 MHz, DMSO) δ 8.35 (t, 2H, CH=CH), 7.66 (d, J = 7.4 Hz, 2H, Ar-H), 7.56 (d, J = 7.9 Hz, 2H, Ar- H), 7.42-7.20 (m, 12H, Ar-H), 7.01 (t, J = 7.4 Hz, 2H, Ar-H), 6.38 (d, J = 13.6 Hz, 3H, CH=CH), 5.39 ( s, 4H, N-CH ₂ ), 1.73 (s, 12H, C(CH ₃ ) ₂ ). ¹³ C NMR (100 MHz, DMSO) δ 173.85, 168.71, 142.68, 141.49, 139.79, 135.55, 129.44, 129.10, 128.97, 128.25, 127.01, 125.40, 123.41, 123.03, 1 19.44, 1 1 1.77, 104.36, 49.52, 27.70, 24.45. HRMS-ESI: m/z, calc. 535.3108 for C ₃₉ H ₃₉ N ₂ +; found 535.3109. Example 2. Synthesis of Dye 1-2:

Accurately weigh 524 mg (2 mmol) of intermediate 11-2, 258 mg (1 mmol) of the condensing agent malondialdehyde aniline hydrobromide (ΠΙ-2) and mix and pour into a 10 ml single-necked flask. Add 164 mg (2 mmol) of sodium acetate, dissolve in 5 ml of acetic anhydride, stir with heating, and reflux for 40 min, then stop heating. After cooling, it was poured into a saturated aqueous solution of NaCl, and after stirring, blue particles were precipitated. Filtration, washing with diethyl ether, dried and weighed 430 mg of a blue solid. The crude yield was 70%. The product was purified by normal phase silica gel column chromatography (petroleum ether: ethyl acetate = 1 : 10). The nuclear magnetic and high resolution mass spectral data of dye 1-1 are as follows:

1H hire R (400 MHz, DMSO) δ 8.35 (t, 2H, CH=CH), 7.66 (d, J = 7.4 Hz, 2H, Ar-H), 7.56 (d, J = 7.9 Hz, 2H, Ar- H), 7.42-7.20 (m, 12H, Ar-H), 7.01 (t, J = 7.4 Hz, 2H, Ar-H), 6.38 (d, J = 13.6 Hz, 3H, CH=CH), 5.39 ( s, 4H, N-CH ₂ ), 1.73 (s, 12H, C(CH ₃ ) ₂ ). ¹³ C NMR (100 MHz, DMSO) δ 173.85, 168.71, 142.68, 141.49, 139.79, 135.55, 129.44, 129.10, 128.97, 128.25, 127.01, 125.40, 123.41, 123.03, 1 19.44, 1 1 1.77, 104.36, 49.52, 27.70, 24.45. HRMS-ESI: m/z, calc. 535.3108 for C ₃₉ H ₃₉ N ₂ +; found 535.3109. Example 3. Absorption and Emission Spectra of Dye 1-1 in DMSO and Water

Dye 1-1 was dissolved in DMSO and water, respectively, to prepare a solution having a final concentration of 5 μM.

The test results are shown in Figure 1. The maximum absorption of dye 1-1 in DMSO is 654/662 nm, and the maximum emission spectrum is 672 nm _. The maximum absorption and maximum emission spectra in water are at 647 nm and 662 nm, respectively. It can be seen that the maximum absorption and emission spectra of dye 1-1 are in the near-infrared region. It can also be seen from Figure 1 that the probe absorbs and emits more in a hydrophobic environment than in an aqueous environment, so when the probe interacts with mitochondrial lipids, it exhibits strong fluorescence.

The instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500. Example 4. Observation of dyes under fluorescence microscope 1-1 staining of MCF-7 live cells at different concentrations

The passaged MCF-7 cells were seeded in a 12-well plate, cultured in a cell culture incubator at 37 ° C, 5% CO ₂ for 24 hours, and dye 1-1 solution (dissolved in DMSO) was added. Incubate for 10 min at 37 nC for 10 nM, 100 nM, 500 nM and 1 μΜ, aspirate the medium, rinse twice with PBS, and add fresh medium without phenol red, observe under a fluorescent inverted microscope (Olympus 1X81) Cell morphology.

At the same exposure time, a Cy5 filter (excitation wavelength: 628/40 nm) was used to structure the dye, which was amplified (20x) and repeated three times.

It can be seen from the four pictures of &, b, c and d in Figure 2 that different concentrations of dye 1-1 can be used for MCF-7. The cells were fluorescently stained, and as the concentration of the dye increased, the fluorescence was enhanced, and the stained region was restricted to the cytoplasm. Example 5 Observation of in vivo staining of MCF-7 by dye 1-1 and Mitrotracker GreenTM under a fluorescence microscope

Mitotracker GreenTM is a commercial mitochondrial green fluorescent probe that can be used for mitochondrial-specific fluorescent staining in live cells. The structural 1-1 dye and Mitotracker GreenTM were respectively stained for MCF-7 live cells, and their staining effects on living cells were compared to further determine the specific fluorescent labeling of DBCy5 on mitochondria.

The passageable MCF-7 cells were seeded in a 12-well glass well plate (the thickness of the well plate was about 0.13-0.17 亳m), and cultured in a cell culture incubator at 37 ° C, 5% CO ₂ for 24 hours, respectively. Structure 1-1 dye and Mitotracker GreenTM, final concentration 500 nM, incubate at 37 °C for 30 minutes, aspirate the medium, rinse 2 times with PBS, and add fresh medium without phenol red in an inverted microscope (Olympus 1X81) Under observation of cell morphology. The Cy5 filter (excitation wavelength: 628/40 nm) was used to excite the 1-1 dye, and the GFP filter (excitation wavelength: 472/30 nm) was used to excite the Mitotracker GreenTM, and the corresponding exposure time was adjusted (500 sec-1000 Leap seconds) Observe with oil mirror (60x) and repeat three times.

Figure 3a is a photograph of live cell staining of Mito-Tracker GreenTM (green), Figure 3b is a photograph of live cells staining of dye 1-1 (red), and Figure 3c is a superimposed photograph of Figures 3a and 3b. As can be seen from these three pictures, Mito-Tracker GreenTMiP Dye 1-1 completely overlaps the staining position of living cells. Figure 3d is an enlarged view of the partially fluorescent stained area in Figure 3c (inside the white box), and the position of the staining of the living cells by the two dyes in Figure 3d was analyzed using the Slidebook (Olympus) software. The results are shown in Fig. 4. It can be seen that the two dyes have exactly the same fluorescent staining of mitochondria in living cells, and quantitatively demonstrate the specific fluorescent labeling of dye 1-1 on mitochondria in living cells. Example 6. Observation of dye 1-1 and DAPI counter-staining of MCF-7 fixed cells under fluorescence microscope

DAPI is a blue fluorescent dye that penetrates cell membranes. When combined with double-stranded DNA, it produces more than 20-fold more fluorescence than DAPI itself, so it is often used directly to fix cell nuclei in cells or tissues. The DAPI dye and dye 1-1 were counterstained on fixed cells to observe the staining area of dye 1-1 in the cells.

The passageable MCF-7 cells were seeded on coverslips and cultured in a cell culture incubator at 37 ° C under 5% CO ₂ for 24 hours. Add dyes 1-1 and DAPI at the same time, the final concentration is 500 nM, incubate at 37 °C for 30 min, aspirate the medium, rinse 2 times with PBS, fix the cells with 10% formaldehyde solution for 2 min, aspirate the formaldehyde solution, rinse 3 times with PBS. . Immediately unfold, seal, and observe cell morphology under an inverted microscope (Olympus 1X81). The C5 filter (excitation wavelength: 628/40 nm) was used to excite the probe dye 1-1, and the DAPI filter (excitation wavelength: 377/50 nm) was used to excite the DAPI dye. Observe with oil mirror (60x) and repeat three times. As can be seen from Figure 5, The gray (blue) fluorescent region indicates DAPI stained MCF-7 nuclei, and the white (red) fluorescent region is dye 1-1 specific fluorescent labeled mitochondria. As shown in Figure 6, a cell was randomly selected, and the staining of DAPI and Dye 1-1 on the fixed cells was analyzed by Slidebook (Olympus) software. It was found that the two dyes had no cross-over area for cell staining, indicating that 1-1 dye can be Specific fluorescent labeling of mitochondria in fixed cells is independent of mitochondrial membrane potential and is therefore particularly useful for pathogenic cells. Example 7 Cellular Activity of Dye 1-1 in MCF-7 Cells

CellTiter-BlueTM Cell Activity Assays A homogeneous, fluorescent method is used to estimate the number of active cells in a cell population. This system comprises a CellTiter-Blue ^TM reagent, i.e. resazurin was dissolved in a buffer solution of high purity. Resazurin is a redox indicator that can be added directly to cell culture. The cells convert the dark blue oxidative dye (Resazurin) into a red, reduced dye (Querone). Since the inactive cells quickly lose their metabolic ability and cannot restore resazurin, they cannot produce a fluorescent signal, so the system specifically detects active cells. The experimental results can be recorded using a fluorometer or a spectrophotometer.

The same number of cells were seeded into 96-well plates at a final volume of 100 L per well. Each group of dyes was inoculated with 3 wells at a cell density of 7 x 10 ³ cells/well, 37 ° C, and 5% CO ₂ in the cells. Incubate for 24 h in the incubator. Add 1-1 dye of DMSO stock solution to the final concentration of each group of dyes 0 μΜ (Τ Ρ), 0.01 μΜ, 0.1 μΜ, Ι μΜ and 10 μΜ, shake at room temperature for 10 s, at 5% at 37 ° C After incubating for 24 h in a CO ₂ cell incubator, the cells were removed from the cell culture incubator, and 20 L of CellTiter-BlueTM was added to each well, shaking at room temperature for 10 s, at 5% CO ₂ at 37 ° C. Incubate for 2 h in a cell culture incubator; fluorescence cells were counted using a fluorescence counter (FL600, Bio-Tek) and fluorescence was recorded at 560 _EM /590 _EX . This test was repeated four times for statistical analysis. As can be seen from Fig. 7, the cell viability IC _5Q reached 60% after the concentration of the dye of the structural formula 1 at 10 nM.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention. As a fluorescent dye is a use of the novel compounds of the present invention, it is not believed that the compounds of the present invention are used only for fluorescent dyes, and the same effect of the compounds of the present invention as fluorescent dyes is known to those of ordinary skill in the art to which the present invention pertains. Under the consideration of the mechanism, a number of simple inferences can also be made, and other application uses of the compounds of the present invention are considered to be within the scope of the present invention.

Claims

Claim

1. A class of bisbenzylpentazone I:

The bisbenzylpentaphthalocyanine fluorescent dye according to claim 1, wherein the sum is independently selected from a fluorenyl group of C^ ₆ .

3. A fluorescent dye according to claim 2, characterized in that said X is preferably a halogen anion.

The method for preparing a fluorescent dye according to claim 1, which is obtained by reacting a compound of the formula II with a compound hydrazine (condensing agent malondialdehyde aniline salt) in the presence of a base, the reaction solvent is an acid anhydride, and the reaction temperature is 20~ 150 ° C, reaction time 10

Wherein, selected from the group consisting of halogen anions, CV, PF ₆ - , CF ₃ -, BF ₄ \ CH ₃ COO"i3⁄4 OTs"; the molar ratio of the compound II to the compound III is 1: 0.1~1;

The molar ratio of the compound II to the base is 1:0.5 to 2.

The method according to claim 4, characterized in that the molar ratio of the compound II to the compound III is from 1:0 to 4·6.

6. The method of claim 4 wherein said base is selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate, Sodium formate, sodium propionate, potassium propionate, sodium oxalate and potassium oxalate.

7. Process according to claim 6, characterized in that the molar ratio of the compound II to the base is 1:0·9~1·1.

8. Process according to claim 4, characterized in that the anhydride is selected from the group consisting of acetic anhydride, propionic anhydride, succinic anhydride and glutaric anhydride.

9. The use of the bisbenzylpentaphthalocyanine fluorescent dye according to claim 1 in a mitochondrial fluorescence specific labeling.

10. Use according to claim 9, characterized in that said mitochondria comprise living cell mitochondria and fixed cell mitochondria.