CN109134545B - Mitochondrial targeting fluorescent probe and preparation method and application thereof - Google Patents
Mitochondrial targeting fluorescent probe and preparation method and application thereof Download PDFInfo
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- 230000008685 targeting Effects 0.000 title claims abstract description 5
- 238000002360 preparation method Methods 0.000 title claims description 9
- 230000002438 mitochondrial effect Effects 0.000 title claims description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 117
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 15
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- 150000001875 compounds Chemical class 0.000 claims description 23
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- 230000035484 reaction time Effects 0.000 claims description 14
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical group CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
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- 238000000034 method Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 7
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- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- 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"
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Abstract
The invention discloses a BODIPY fluorescent probe for targeted detection of formaldehyde in mitochondria, which takes BODIPY as a fluorophore and triphenyl phosphonium group as a targeting group, has the advantages of sensitive reaction, low detection limit, good specificity and good biocompatibility, and can be applied to targeted detection of formaldehyde in mitochondria.
Description
Technical Field
The invention relates to a fluorescent probe for detecting formaldehyde, in particular to a mitochondrial-targeted fluorescent probe and a preparation method and application thereof.
Background
The harm of formaldehyde existing in the environment to human health has been reported in a large amount, but endogenous formaldehyde generated in vivo and related diseases caused by the endogenous formaldehyde are relatively rarely researched. Formaldehyde is commonly found in various systems of the human body, particularly in the central nervous system, circulatory system, digestive system, urinary system, and the like. Formaldehyde can chemically react with some important biomolecules, such as proteins, DNA, RNA, neurotransmitters, etc., and affect their structure and function. Imbalances in anabolism and catabolism may be responsible for the accumulation and excess of formaldehyde in the body. Studies have shown that endogenous formaldehyde is closely associated with aging, nerve cell degeneration, division and proliferation of tumor cells, etc., and particularly neurodegenerative diseases ("endogenous formaldehyde and related human major diseases," pre-childhood, etc., natural scientific progress, stage 2008 11).
Normal formaldehyde concentrations can maintain the metabolic balance of the body, and elevated levels of formaldehyde and related reactive carbonyl species can cause a number of diseases including cancer, diabetes, chronic liver disease, neurodegenerative disease, and cardiovascular disease ("progress in the study of endogenous formaldehyde and alzheimer's disease pathogenesis," li weiwei, journal of chinese traditional medicine, stage 2012 20). Formaldehyde is an important signal factor in vivo and is involved in carbon cycle processes in vivo, wherein a typical example is that formaldehyde is involved in folate-regulated mitochondrial single carbon cycle processes. In the process, dimethylglycine generates glycine under the action of mitochondrial enzymes DMGDH and SARDH and releases two molecules of formaldehyde, the generated formaldehyde can generate 5, 10-methylenefolic acid with folic acid in mitochondria, and is used for further participating in the single carbon cycle process in cytoplasm and nucleus, and finally purine nucleotides, thymidylate, methionine, serine and other substances for maintaining normal physiological functions of cells are produced, and the abnormality of the single carbon cycle of cells is often accompanied with certain diseases such as dysplasia, cancer and the like. It has been shown that excessive formaldehyde triggers the mitochondrial caspase apoptosis process leading to cell death. However, the influence of fluctuation of the amount of formaldehyde in the human body, particularly in mitochondria, on the physiology and pathology of the human body is still not clear enough ("progress of research on chemical reaction involving formaldehyde in the organism," Korea, university of Wuhan-Han-Engineers, journal of 2018, phase 01). Therefore, the development of a fluorescent probe which is easy to synthesize, has good selectivity and can be used for quickly detecting the formaldehyde in the mitochondria is of great significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the BODIPY fluorescent probe for targeted detection of formaldehyde in mitochondria, and the probe has the advantages of sensitive reaction, low detection limit, good specificity and good biocompatibility.
Another objective of the present invention is to provide a method for synthesizing a BODIPY fluorescent probe for targeted detection of formaldehyde in mitochondria.
In order to achieve the purpose, the BODIPY fluorescent probe for targeted detection of formaldehyde in mitochondria provided by the invention takes BODIPY as a parent nucleus and triphenyl phosphonium as a targeting group, and has the following structural formula:
the preparation method of the BODIPY fluorescent probe is characterized in that the preparation route of the method is shown in the following reaction formulas (1) to (4).
In the reaction formula (1), the reaction conditions include that the molar ratio of 4-methyl-2-pyrrole carboxylic acid shown in the formula a to chloroacetyl chloride is 1.5-3: 1, preferably 2-2.2: 1; the reaction temperature is room temperature, and the reaction time is 10-120 min.
In the reaction formula (2), the compound of the formula b reacts with boron trifluoride diethyl etherate in the presence of triethylamine, the reaction temperature is controlled to be 25-30 ℃, and the reaction time is 2-12 hours.
In the reaction formula (3), the compound of the formula c and the compound of the formula d react in the presence of a dehydrating agent/catalyst, the reaction temperature is room temperature, and the reaction time is 12-24 hours.
In the reaction formula (4), dissolving the compound of the formula e in a tetrahydrofuran solution, slowly dropwise adding ammonia water (1-2 h), stirring at room temperature, and reacting for 6-24 h to obtain the fluorescent probe molecule shown in the formula f.
Preferably, in the reaction formula (1), the molar ratio of 4-methyl-2-pyrrole carboxylic acid shown in the formula a to chloroacetyl chloride is preferably 2-2.2: 1; the reaction time is preferably 30 min.
In the reaction formula (2), the compound of the formula b is reacted with boron trifluoride diethyl etherate in the presence of triethylamine, the reaction temperature is controlled at 25 ℃, and the reaction time is preferably 4 hours.
In the reaction formula (3), the dehydrating agent is Dicyclohexylcarbodiimide (DCC) or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI); the catalyst is 4-dimethylamino pyridine (DMAP) or 1-Hydroxybenzotriazole (HOBT), and the reaction time is preferably 12 hours.
In the reaction formula (4), the reaction time is preferably 12 hours with stirring at room temperature.
The application of the fluorescent probe molecule in detecting the formaldehyde content in a solution, a physiological environment and/or a cell.
The mechanism of the fluorescent probe molecule detection is as follows:
(1) when the BODIPY fluorescent probe is applied to detecting formaldehyde, as formaldehyde reacts with amino, probe molecules decay under an excitation state, and a fluorescent signal is weakened or quenched, so that the formaldehyde can be selectively detected. (2) The influence on the fluorescence intensity of the whole compound before and after the reaction is used as a detection signal for identification, the change of the fluorescence intensity along with the formaldehyde concentration is tested, and a linear fitting curve is obtained, so that the concentration of the formaldehyde in the sample to be detected is quantitatively detected. (3) Because the electron transfer chain on the inner mitochondrial membrane pumps protons out of the mitochondrial matrix to the outer mitochondrial membrane, so that the potential difference on the mitochondrial membrane is caused, the probe molecules are connected with the new lipid triphenyl phosphonium cation, and are targeted and gathered in the mitochondrial matrix by virtue of the potential difference on the inner mitochondrial membrane, so that the detection of the formaldehyde in the mitochondria is realized.
Compared with the prior art, the invention has the following beneficial effects:
(1) the probe molecule of the invention has novel structure, takes BODIPY as a parent body, takes amino as a detection group, and can be gathered in mitochondria in a targeted manner by introducing lipophilic cation triphenyl phosphonium group in the molecular structure design, thereby realizing the detection of formaldehyde in the mitochondria.
(2) The probe molecule can detect the content of formaldehyde with nanomolar concentration, has low detection limit, no toxic or side effect on cells, good cell permeability and good molecular water solubility, can be prepared into solution for testing, avoids the use of organic solvent, reduces tissue damage and has higher biocompatibility.
Detailed Description
The present invention is further illustrated by the following specific examples.
EXAMPLE 1 preparation of the Compound of formula b
Adding 2.1mmol of 4-methyl-2-pyrrole formic acid and 1mmol of chloroacetyl chloride into a 25mL round-bottom flask, adding 2mL of dichloromethane, magnetically stirring at room temperature for 30 minutes, detecting the reaction completion by TLC, concentrating under reduced pressure, and separating by silica gel column chromatography (ethyl acetate/petroleum ether is used as an elution solvent) to obtain the compound shown in the formula b. Yield (89%, calculated as chloroacetyl chloride).
EXAMPLE 2 preparation of Compound of formula c
Adding 1mmol of the compound shown in the formula b into a 25mL round-bottom flask, then adding 2mmol of triethylamine and 2mL of dichloromethane, slowly dropwise adding 0.5mL of boron trifluoride diethyl ether under stirring, controlling the reaction temperature to be 25 ℃, reacting for 6 hours, detecting that the compound shown in the formula b completely reacts through TLC, concentrating under reduced pressure to remove the solvent, and separating the residue through silica gel column chromatography (ethyl acetate/petroleum ether is used as an elution solvent) to obtain the compound shown in the formula c. Yield (85%).
EXAMPLE 3 Synthesis of Compound of formula d
In a 25mL round bottom flask, 1mmol of the compound of formula c and 2.1mmol of the compound of formula d are dissolved in dichloromethane, then a solution of DCC (1.5mmol) and DMAP (0.1mmol) in dichloromethane (1 mL each) is added dropwise, the reaction is stirred at room temperature overnight (about 12 hours), after completion of the reaction, the solvent is removed by concentration under reduced pressure, and the residue is separated by silica gel column chromatography (ethyl acetate/petroleum ether as eluting solvent) to obtain the compound of formula e. Yield (93%).
EXAMPLE 4 Synthesis of fluorescent Probe molecule Compound f
Adding 1mmol of the compound shown in the formula e into a 25mL round-bottom flask, adding 4mmol of DMSO, stirring for dissolving, slowly dropwise adding ammonia water (0.4mL, the concentration is 28%) at room temperature, reacting for 8 hours at room temperature, removing the solvent through decompression and concentration after the reaction is finished, and separating the residue through silica gel column chromatography (ethyl acetate/petroleum ether is used as an elution solvent) to obtain the fluorescent probe molecule shown in the formula f. Yield (84%).1H NMR(400MHz,DMSO-d6)8.90-9.23(1H),8.69-8.84(2H),8.17-8.33(1H),7.61(1H),7.29-7.43(30H),7.10(1H),6.93(1H),5.42(1H),3.18-3.32(6H),1.8(4H),1.3(4H)。
Example 5 variation of fluorescence intensity of Probe molecules at different Formaldehyde concentrations
Different concentrations of formaldehyde (0 to 250. mu.M, formaldehyde concentration: 0,10,20,40,60,80,100,120,150,200,250) were added to 5. mu.M PBS buffer solutions (pH 7.4, 5% DMSO) of the probe represented by formula f, and then the volume was fixed with 7.4 PBS buffer solution for one hour, and fluorescence spectrum measurement was performed (λ ex: 440nm), and the fluorescence intensity in each system was measured (maximum emission λ em: 528 nm).
The results show that the fluorescence intensity at 528nm of the system decreases with increasing concentration of OCHO. The linear regression constant of the linearly fitted curve was 0.9962, indicating that the probe was able to quantitatively determine the concentration of OCHO.
Example 6 Probe molecular mitochondrial localization for Formaldehyde assay
(1) The density is 3 x 105HeLa cells per mL were seeded onto sterilized 35mm petri dishes plated with glass slides (22 mm. times.22 mm) and cultured in a CO2 incubator (37 ℃, 5% CO2) until the cells attached.
(2) Adding 100 mu M formaldehyde into a cell culture solution, incubating for 20min, adding 5 mu M probe into a cell culture dish, incubating for 40min in a cell culture box, and adding 5 mu M mitochondrial localization dye (MitoTracker Deep Red) into the cell culture dish for incubating for 10 min;
(3) the samples were washed 3 times with PBS buffer and imaged under a fluorescence microscope after sampling. The Pearson correlation coefficient R is 0.9335 after testing and calculation. The probe molecule can be positioned in mitochondria to detect formaldehyde.
The embodiments described above are only preferred embodiments of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.
Claims (4)
1. A mitochondrial targeting formaldehyde detection fluorescent probe is characterized by having the following structure:
2. the method for preparing a fluorescent probe according to claim 1, wherein the preparation route is represented by the following reaction formulas (1) to (4):
in the reaction formula (1), the molar ratio of 4-methyl-2-pyrrole carboxylic acid shown in the formula a to chloroacetyl chloride is 1.5-3: 1, the reaction temperature is room temperature, and the reaction time is 10-120 min;
in the reaction formula (2), the compound of the formula b reacts with boron trifluoride diethyl etherate in the presence of triethylamine, the reaction temperature is controlled to be 25-30 ℃, and the reaction time is 2-12 hours;
in the reaction formula (3), the compound of the formula c and the compound of the formula d react in the presence of a dehydrating agent/catalyst, the reaction temperature is room temperature, and the reaction time is 12-24 hours;
in the reaction formula (4), dissolving the compound of the formula e in a tetrahydrofuran solution, slowly dropwise adding ammonia water for 1-2 h, and stirring at room temperature for reaction for 6-24 h to obtain the fluorescent probe molecule shown in the formula f.
3. The preparation method according to claim 2, wherein in the reaction formula (1), the molar ratio of 4-methyl-2-pyrrole carboxylic acid represented by the formula a to chloroacetyl chloride is 2-2.2: 1; the reaction time is 30 min;
in the reaction formula (2), the compound of the formula b reacts with boron trifluoride diethyl etherate in the presence of triethylamine, the reaction temperature is controlled at 25 ℃, and the reaction time is 4 hours;
in the reaction formula (3), the dehydrating agent is Dicyclohexylcarbodiimide (DCC), the catalyst is 4-Dimethylaminopyridine (DMAP), and the reaction time is 12 hours;
in the reaction formula (4), the reaction time was 12 hours with stirring at room temperature.
4. Use of the fluorescent probe according to claim 1 for targeted detection of formaldehyde in mitochondria for non-therapeutic or diagnostic purposes.
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| CN107043372A (en) * | 2017-05-05 | 2017-08-15 | 深圳大学 | A kind of flavones fluorescence probe of targetted mitochondria and preparation method and application |
| CN107417714A (en) * | 2017-07-06 | 2017-12-01 | 南开大学 | A kind of highly sensitive fluorescence probe and its synthetic method and application based on BODIPY |
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