CN103788941B - A kind of novel mercapto fluorescence probe, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a novel sulfhydryl fluorescent probe, a preparation method and an application thereof. The novel fluorescent probe has a structure as shown in formula 1: Formula 1, where R=NH ₂ or COOH, can be used for quantitative analysis or location tracking of polysaccharides, peptides, proteins and nucleic acids.

Description

A novel sulfhydryl fluorescent probe, its preparation method and its application

technical field

The invention belongs to the technical field of biological detection, and in particular relates to a novel sulfhydryl fluorescent probe, a preparation method and an application thereof.

Background technique

Fluorescent labeling technology refers to the use of some fluorescent substances to covalently bind or physically adsorb on a certain group of the molecule to be studied, and use its fluorescence characteristics to provide information about the research object. Fluorescent labeling technology originated in the 1940s by using fluorescently labeled antibodies to detect corresponding antigens. With the development of modern medicine and molecular biology, and the application of various advanced fluorescent detection technologies and instruments, fluorescent labeling as a non-radioactive label The technology has received a lot of attention and has achieved rapid development. It is widely used in the detection of intracellular and extracellular substances, the detection of nucleic acids, and the early diagnosis of diseases.

Fluorescent labeling reagents commonly used in fluorescent labeling technology include fluoresceins and rhodamines, and other fluorescent labeling reagents are polycyclic aromatic hydrocarbon compounds, aromatic heterocyclic compounds, and chelates of some rare earth elements. Among them, fluorescein labeling reagents mainly include fluorescein isothiocyanate, hydroxyl fluorescein, tetrachlorofluorescein, etc.; rhodamines mainly include R101, tetraethylrhodamine, and carboxytetramethylrhodamine, etc.; other fluorescent labels Reagents include polycyclic aromatic hydrocarbons such as naphthalene, anthracene, and pyreneindole, aromatic heterocyclic compounds such as acridine, coumarin, and phenanthrene indole, lanthanide rare earth chelates, and phycoerythrin.

Although these traditional fluorescent reagents have a wide range of applications, there are still some defects. For example, there are few reports on the fluorescent labeling of polysaccharides. Most of the existing labeling methods lack selectivity and cannot well reflect the biological properties of polysaccharides. Existing fluorescent reagents cannot rationally label proteins. Some fluorescent reagents have weak fluorescent signals and unstable fluorescent signals, which cannot satisfy the research on the biological properties of biological macromolecules.

Therefore, it has become a research focus to synthesize a polysaccharide molecule that can specifically label polysaccharide molecules and rationally target proteins that need to be studied.

Contents of the invention

The purpose of the present invention is to address the above existing problems and provide a novel sulfhydryl fluorescent probe, preparation method and application thereof, which can be used for specific labeling of polysaccharides, peptides, proteins, and nucleic acids. The fluorescent probe of the present invention has good fluorescence stability and intensity, not only can perform qualitative and quantitative analysis on polysaccharides, peptides, proteins and nucleic acids, but also can establish a fast and simple trace method to detect the above substances in vivo metabolic kinetics studies.

Concrete technical scheme of the present invention is as follows:

A novel sulfhydryl fluorescent probe with formula 1:

where R = _NH2 or COOH.

When R=NH ₂ , the above-mentioned fluorescent probe is 2-(2-mercaptoethylamino) benzamide, The molecular formula is C ₉ H ₁₂ N ₂ OS. After being dissolved in ethanol, the compound will emit an emission wavelength of 440nm under excitation light of 340nm.

When R=COOH, the above-mentioned fluorescent probe is 2-(2-mercaptoethylamino)benzoylformic acid, The molecular formula is C ₁₀ H ₁₁ NO ₃ S. After being dissolved in ethanol, the compound has an emission wavelength of 450nm under excitation light of 350nm.

The -SH of the compound of formula 1 in the present invention can form a disulfide bond with the amino acid containing -SH in the peptide or protein, or specifically bind with the αβ unsaturated carbonyl structure of polysaccharides, or the maleimide structure , so it can be targetedly combined with these macromolecular substances. The conjugated structure of the benzene ring and the carbonyl group of the compound of formula 1 is a fluorescent light-emitting group, which can make the labeled substance generate fluorescence.

Based on the technical concept of the present invention, compounds with the following formula can also be used for specific labeling of polysaccharides, peptides, proteins, and nucleic acids:

Wherein, A is any substituted phenyl group, polycyclic aromatic group or aromatic heterocyclic group, and the substituents are selected from halogen, C1-C6 alkane group, nitro group, amino group, hydroxyl group, C1-C6 alkane group, ether group, etc., R is NH ₂ , OH, SH, COOH, C1-C6 alkane group, OCOR ₁ , or COOR ₂ , wherein R ₁ or R ₂ is C1-C6 alkane group.

The present invention also provides a preparation method for the above-mentioned novel mercapto fluorescent probe, comprising the following steps:

(1) sodium hydrosulfide and chloroacetaldehyde are reacted to obtain mercaptoaldehyde;

(2) mercaptoacetaldehyde prepared in step (1) is reacted with 2-aminobenzamide or 2-aminobenzamide by catalyst to obtain 2-(2-mercaptoethylamino)benzamide dimer;

(3) reducing the 2-(2-mercaptoethylamino) benzamide dimer obtained in step (2) with a reducing agent to obtain 2-(2-mercaptoethylamino) benzamide;

Or, (4) catalyzed reaction of mercaptoaldehyde and 2-aminobenzoic acid obtained in step (1) to obtain 2-(2-mercaptoethylamino)benzoylformic acid dimer;

(5) Reducing the 2-(2-mercaptoethylamino)benzoylformic acid dimer obtained in step (4) with a reducing agent to obtain 2-(2-mercaptoethylamino)benzoylformic acid.

The synthetic schematic diagram of the above-mentioned sulfhydryl fluorescent probe preparation method is as follows:

Above-mentioned step (1) reaction temperature is preferably 0-4 ℃, preferably adjusts solution with alkali to be acidic, and reaction solvent is the mixed solution of water or water and organic solvent, preferably water, and organic solvent is the solvent that can miscible with water, as methanol , ethanol, ether, acetone, etc.

Specifically, chloroacetaldehyde can be dissolved in water to obtain a chloroacetaldehyde solution, cooled in an ice bath, and the pH of the chloroacetaldehyde solution is adjusted using Na ₂ CO ₃ solution, preferably pH = 3-5, and then sodium hydrosulfide is dissolved in water to obtain Sodium hydrosulfide solution, cooled in an ice bath and added dropwise to the chloroacetaldehyde solution, while continuously stirring the chloroacetaldehyde solution, a white solid precipitated out of the solution, after the sodium hydrosulfide was added, suction filtered and vacuum-dried to obtain mercapto Acetaldehyde;

The catalyst in the above step (2) or step (4) is preferably zinc chloride and/or sodium borohydrocyanide, preferably a dehydrating agent is further used, more preferably the dehydrating agent is 3A molecular sieve. The reaction temperature is room temperature, and the reaction solvent is a polar organic solvent, preferably methanol or ethanol.

The reduction reaction described in the above step (3) or step (5) is a reaction condition commonly used in the art, the reducing agent is preferably dithiothreitol, the reaction temperature is room temperature, and the reaction solvent is preferably dioxythreitol.

The 2-(2-mercaptoethylamino)benzamide obtained in the above step (3) can be further purified by reverse phase chromatography, the sample is dissolved in DMSO solution, and the sample is loaded at a ratio of 1:50, using 30% acetonitrile The solution was eluted isocratically, and the components with a retention time of 10 min were collected. After rotary evaporation of acetonitrile, the white powder was obtained by lyophilization, which was the target product 2-(2-mercaptoethylamino)benzamide.

The 2-(2-mercaptoethylamino)benzoylformic acid obtained in the above step (5) can be further purified by reverse phase chromatography, the sample is dissolved in DMSO solution, eluted isocratically with 50% acetonitrile solution, collected and retained After 15 minutes, the acetonitrile was evaporated by rotary evaporation, and then freeze-dried to obtain a white powder, which was the target product 2-(2-mercaptoethylamino)benzoylformic acid.

The present invention also provides the application of the above-mentioned sulfhydryl fluorescent probe in the quantitative analysis or location tracing of polysaccharides, peptides, proteins and nucleic acids. The fluorescent probe of the present invention can specifically bind to the non-reducing end of low molecular weight heparin, and can prepare fluorescent double-labeled low molecular weight heparin, which provides a better detection method for studying the expression of heparanase in tumor cells At the same time, it can specifically label the target protein, which is simple, rapid and does not affect the function of the protein. It provides a novel detection method for studying the conformational changes of protein molecules and the interaction between proteins.

The fluorescent probe of the present invention provides a better research method for studying sulfhydryl-related biological signaling pathways, and the significance and diagnosis of sulfhydryl in some diseases or in vivo processes.

Fluorescent probe of the present invention compares with prior art, and its positive effect is:

1) Good stability, high fluorescence intensity, and can be stored for a long time;

2) Can be specifically combined with polysaccharides, peptides, proteins, etc., for fluorescent labeling;

3) It can easily enter living cells and living tissues, and specifically react with sulfhydryl groups in cells or proteins;

4) The Stokes shift is large, the fluorescence background is weak, and the signal-to-noise ratio is good.

Description of drawings

Fig. 1 is the mass spectrogram of 2-(2-mercaptoethylamino) benzamide.

Fig. 2 is the nuclear magnetic resonance figure of 2-(2-mercaptoethylamino) benzamide: H spectrum, C spectrum.

Figure 3 is the excitation spectrum and emission spectrum of 2-(2-mercaptoethylamino)benzamide.

FIG. 4 shows the use of fluorescent double-labeled heparin to indicate the expression of heparanase in tumor cells.

Figure 5 shows the change of FRET value of fluorescent double-labeled heparin in aqueous solution.

Fig. 6 is the result of quantitative detection of MMP3 gene content in colorectal cancer cells and normal cells using 2-(2-mercaptoethylamino) benzamide.

detailed description

The specific steps of the present invention are illustrated below through the examples, but not limited by the examples.

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only for illustration of the present invention, but not to limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The preparation of embodiment 12-(2-mercaptoethylamino) benzamide

1) Dissolve 19ml of chloroacetaldehyde solution with a mass percentage of 40% in 40ml of aqueous solution, cool to 0°C in an ice bath to obtain diluted chloroacetaldehyde solution, add dropwise a sodium carbonate solution with a concentration of 2Mol/L, and adjust the pH to 3 ;

2) Dissolve 9.9g of sodium hydrosulfide in 33ml of water to obtain an aqueous solution of sodium hydrosulfide, cool it to 0°C in an ice bath, and add it dropwise to the chloroacetaldehyde solution at a rate of 2-3 drops per second, and at the same time, at 200 rpm Stir the chloroacetaldehyde solution at a constant speed, and a white solid precipitates out in the solution. After the sodium hydrosulfide is added, filter it with suction and dry it in vacuum to obtain mercaptoaldehyde;

3) Dissolve 434mg of mercaptoaldehyde in 120ml of methanol solution, stir until dissolved at room temperature, add 1.5g of 2-aminobenzamide and stir until dissolved, add 80g of 3A molecular sieve, 250mg of zinc chloride, 500mg of borohydrocyanide Sodium chloride, stirred at 20-25°C for 16 hours, centrifuged to remove the precipitate, 10% NaOH solution was added dropwise to the supernatant solution at a rate of 2-3 drops per minute, a light yellow precipitate appeared, and stood for 1- After 2 hours, wash the filter cake with water for 2-3 times after suction filtration, and obtain 2-(2-mercaptoethylamino)benzamide dimer after drying;

4) Add 300mg of 2-(2-mercaptoethylamino)benzamide dimer to the dioxide sulfoxide solution, stir until dissolved at room temperature, add 236mg of dithiothreitol, stir and react at room temperature for 4 hours , the solution after the reaction is directly purified on a reversed-phase silica gel column, 30% acetonitrile isocratic elution, HPLC detects the fractions, collects the corresponding fractions, and after the acetonitrile is rotary evaporated, freeze-drying to obtain a white powder is the target product 2-( 2-Mercaptoethylamino)benzamide. The mass spectrum is shown in Figure 1, and the NMR spectrum H and C spectra are shown in Figure 2.

Use a fluorescence detector to detect 2-(2-mercaptoethylamino)benzamide, its excitation wavelength is 340nm, and its emission wavelength is 440nm, as shown in Figure 3.

The preparation of embodiment 22-(2-mercaptoethylamino) benzoylformic acid

1) 28.5ml mass percentage is that 40% chloroacetaldehyde solution is dissolved in 60ml aqueous solution, is cooled to 0 ℃ in ice bath, obtains the diluted chloroacetaldehyde solution, drips the sodium carbonate solution that concentration is 2Mol/L, adjusts pH to be 3 or so;

2) Dissolve 15g of sodium hydrosulfide in 50ml of water to obtain an aqueous solution of sodium hydrosulfide, cool it to 0°C in an ice bath, and add it dropwise to the chloroacetaldehyde solution at a rate of 2-3 drops per second, at the same time at a rate of 200 rpm Constantly stirring the chloroacetaldehyde solution, a white solid precipitates out in the solution, and after the sodium hydrosulfide is added, it is suction filtered and vacuum-dried to obtain mercaptoaldehyde;

3) Dissolve 108 mg of mercaptoaldehyde in 50 ml of methanol solution, stir at room temperature until dissolved, add 304 mg of 2-aminobenzoylformic acid and stir until dissolved, add 125 mg of sodium borohydrocyanide, stir at room temperature for 5 hours, and 10% NaOH solution was added dropwise into the supernatant solution at a rate of 2-3 drops per minute, and a white precipitate appeared, which was allowed to stand for 1-2 hours. After suction filtration, the filter cake was rinsed with water for 2-3 times, and after drying, 2 -(2-Mercaptoethylamino)benzoylformic acid dimer;

4) Add 100mg of 2-(2-mercaptoethylamino)benzoylformic acid dimer to the dioxide sulfoxide solution, stir until dissolved at room temperature, add 136mg of dithiothreitol, stir and react at room temperature for 4 hours, put the reacted solution directly on a reverse-phase silica gel column for purification, 30% acetonitrile isocratic elution, HPLC detects the fractions, collects the corresponding fractions, spins out the acetonitrile, and lyophilizes to obtain a white powder that is the target product 2- (2-Mercaptoethylamino)benzoylformic acid.

Use a fluorescence detector to detect 2-(2-mercaptoethylamino)benzoylformic acid, its excitation wavelength is 350nm, and its emission wavelength is 450nm.

Example 3 Using the compound of the present invention to specifically label the non-reducing end of heparin

1. Use 2-(2-mercaptoethylamino)benzamide to specifically label the non-reducing end of heparin

Add 500mg of enoxaparin into 3.6ml of water, stir to dissolve, and add 1.27mg of benzethonium chloride into 1.1ml of water, slowly drop the benzethonium chloride solution into the low molecular weight heparin sodium solution, and stir for 1h , washed with water three times, filtered, and dried under reduced pressure to obtain 240 mg of benzethon chloramine salt.

Take 100mg of benzethon chloramine salt and add 2ml of dichloromethane, stir to dissolve, add 110ul benzyl chloride, stir at room temperature for 24h, add sodium acetate-methanol solution after the reaction, stir for 30min, remove the supernatant, wash with methanol for 3 times, filtered, dried under reduced pressure, and freeze-dried to obtain low molecular weight heparin benzyl ester.

Add 45mg of low molecular weight heparin benzyl ester into 10ml of formamide solution, heat to dissolve, take 55mg of 2-(2-mercaptoethylamino)benzamide, add 20mg of boric acid into the formamide solution, and react at 50°C 24h.

Add the reacted solution into a dialysis bag with a cut-off space of MW500, dialyze in water for 24 hours, and freeze-dry the dialysate to obtain heparin benzamide labeled with 2-(2-mercaptoethylamino)benzamide at the non-reducing end. base ester.

Take 100mg of heparin benzyl ester of fluorescent 2-(2-mercaptoethylamino)benzamide, dissolve it in 3ml of water, heat to 62°C, add 15mg of NaOH, stir and react for 1h, cool to room temperature, adjust the pH to 6.0 with HCl, Add 300 mg of NaCl to a final concentration of 10%, add methanol while stirring, precipitate out, filter and wash, and lyophilize to obtain heparin with a non-reducing terminal bonded to 2-(2-mercaptoethylamino)benzamide.

Use 2-(2-mercaptoethylamino)benzoylformic acid to specifically label the non-reducing end of heparin. Add 200mg of enoxaparin to 2ml of water, stir to dissolve, and add 250mg of benzethonium chloride to 3ml of water. , the benzethonium chloride solution was slowly added dropwise to the low molecular weight heparin sodium solution, stirred for 1 h, washed with water three times, filtered, and dried under reduced pressure to obtain 300 mg of benzethonium chloride salt.

Take 100mg of benzethon chloramine salt and add 2ml of dichloromethane, stir to dissolve, add 110ul benzyl chloride, stir at room temperature for 24h, add sodium acetate-methanol solution after the reaction, stir for 30min, remove the supernatant, wash with methanol for 3 times, filtered, dried under reduced pressure, and freeze-dried to obtain low molecular weight heparin benzyl ester.

Add 40mg of low molecular weight heparin benzyl ester into 5ml of formamide solution, heat to dissolve, take 25mg of 2-(2-mercaptoethylamino)benzoylformic acid, add 15mg of boric acid into the formamide solution, and heat at 50°C Reaction 24h.

Add the reacted solution into a dialysis bag with a cut-off space of MW500, dialyze in water for 24 hours, and freeze-dry the dialysate to obtain heparin labeled with 2-(2-mercaptoethylamino)benzoylformic acid at the non-reducing end Benzyl esters.

Take 100mg of heparin benzyl ester of fluorescent 2-(2-mercaptoethylamino)benzoylformic acid, dissolve it in 3ml of water, heat to 62°C, add 15mg of NaOH, stir for 1h, cool to room temperature, adjust the pH to 6.0 with HCl , add NaCl300mg to a final concentration of 10%, add methanol while stirring, precipitate out, filter and wash, freeze-dry to obtain heparin with non-reducing terminal bonded 2-(2-mercaptoethylamino)benzoylformic acid.

Example 4 Fluorescent double-labeled heparin is used for rapid diagnosis of tumor cell metastasis:

1) Take 12mg of heparin labeled with 2-(2-mercaptoethylamino)benzamide, add 560ul of water to dissolve, take 4mg of 2-aminoacridone and add it to 158ul3/17 acetic acid/DMSO solution, mix the two and add 25mg of sodium borohydrocyanide was reacted at 37°C for 16h.

2) Add the reacted solution into a dialysis bag with a molecular weight cut-off of 500, dialyze in the aqueous solution for 24 hours, and freeze-dry the dialysate to obtain fluorescent double-labeled heparin, whose reducing end is labeled with 2-aminoacridone, non- The reducing end is labeled with 2-(2-mercaptoethylamino)benzamide.

3) Culture liver cancer cells, pancreatic cancer cells, cervical cancer cells, normal liver cells, pancreatic cells, and cervical cells respectively, collect 10 million cells respectively, and add 1ml protein extraction reagent to extract cell protein.

4) Take 3mg of double-labeled fluorescein heparin, add PBS (50mM, PH7.4PBS-Na) to a final concentration of 1mg/ml, take a black 96-well plate, add 100ul of double-labeled fluorescein heparin at a concentration of 1mg/ml, and then Add 100ul of the cell protein extracted above, and incubate at 37°C for 24h. Detect the change of fluorescence value with excitation wavelength 340nm and emission wavelength 538nm. Double-labeled fluorescein heparin can specifically bind to heparanase in tumor cells. Since heparanase is highly expressed in a variety of malignant tumors and may play an important role in the invasion and metastasis of malignant tumor cells, it can The metastasis of tumor cells can be judged by the expression of heparanase labeled with double-labeled fluorescent heparin in tumor cells, that is, the level of fluorescence value. The results are shown in Figure 4. The results showed that the FRET fluorescence values of the cancer cell groups decreased to varying degrees, with pancreatic cancer cells decreased by 50%, cervical cancer cells decreased by 30%, and liver cells decreased by 20%. The reduction degree of the maximum reaches 50%, while the FRET fluorescence value of the normal cell group does not change, indicating that the migration ability of pancreatic cancer cells is stronger, and the other two cancer cell lines also have migration ability.

Example 5 uses 2-(2-mercaptoethylamino)benzamide to study the conformational change of heparin in aqueous solution:

1) Take 3mg of the fluorescent double-labeled heparin prepared in Example 4, add it to 3ml of water, and make a fluorescent double-labeled heparin solution with a concentration of 1mg/ml, take 200ul and add it to a black 96-well plate, and detect the excitation wavelength at 340nm. The change of the fluorescence value at the emission wavelength of 440nm is monitored in real time, and the change curve is drawn. The result is shown in Figure 5. The fluorescence value rises from 8000bps to 15000bps, then slowly decreases to 9000bps and then rises again. Repeatedly, it shows that the conformation of heparin in aqueous solution is gradually curled up from the long chain to the maximum degree, and then returns to the long chain.

Example 6 uses 2-(2-mercaptoethylamino)benzamide to study protein-protein interaction.

1) Query the gene sequence of calmodulin from NCBI GenBank: M27319.1, and at the same time change the codon ACC of the 6th amino acid to TGC, and change threonine to cysteine. The synthesized gene sequence is shown as SEQ ID NO.1 As shown, the gene sequence was introduced into PET28a vector and expressed in Escherichia coli, cultured at 37°C for 2 hours, added IPTG to a final concentration of 0.5mM, induced expression at 20°C for 8h, collected at 5000rpm, and stored at -20°C.

2) Query the gene sequence NM_006888.4 of calmodulin kinase 1 from NCBI, and at the same time change the 56-position amino acid codon AGT to UGC, and serine to cysteine. The synthesized gene sequence is shown in SEQ ID NO.2. The gene sequence was introduced into the PET28a vector and expressed in Escherichia coli, cultured at 37 degrees Celsius for 2 hours to an OD value of 0.6-1, added IPTG to a final concentration of 0.5mM, induced at 25 degrees for 12 hours, collected at 5000 rpm, and stored at -20 degrees Celsius .

3) Take 1g of bacteria from steps (1) and (2) and add 10ml PBS buffer solution to dissolve, crush under high pressure at 30Kpsi, centrifuge at 12000rpm to remove cell debris, use 5mlDEAE column, 50mMPH6.0PBS-Na buffer elution, respectively for the two The protein was purified by column, the pure protein was collected, verified by SDS-PAGE, and concentrated by centrifugation at 1000rpm for 30 minutes with a 10KD protein concentration tube.

4) Add 100ul of calmodulin into 100ul of PBS buffer, add 2mg of 2-(2-mercaptoethylamino)benzamide at the same time, place it overnight at 4 degrees, and dialyze to remove unbound 2-(2-mercaptoethyl) Amino)benzamide to form 2-(2-mercaptoethylamino)benzamide-labeled calmodulin.

5) Add 100ul of calmodulin kinase 1 to 80ul of PBS buffer, add 20ul of TFA at the same time, weigh 4mg of fluorescein-5-maleamide, place it overnight at 4 degrees, and dialyze out unbound fluorescein-5- Maleamide, forming fluorescein-5-maleamide-tagged calmodulin kinase 1.

6) Take 10ul 2-(2-mercaptoethylamino)benzamide-labeled calmodulin, 10ul fluorescein-5-maleimide-labeled calmodulin kinase 1 into a black 96-well plate, and use a fluorescence detector for real-time Detect the change of FRET value, the excitation wavelength is 340nm, and the emission wavelength is 510nm. It is found that with the prolongation of time, the FRET value gradually increases, and the FRET value reaches the maximum at 5 minutes, indicating that calmodulin and calmodulin kinase will be close to each other , interacting with each other.

Example 7 uses 2-(2-mercaptoethylamino)benzamide for quantitative detection of nucleotide content

1) Query the MMP-3 gene sequence GenBank: U51914.1 in Genebank, and design and synthesize primers through primerpremier5.0. The primer sequences are as follows:

5'-CTGTTTGACA-3'SEQ ID NO.3

3'-CCTTGCTGTCTT-5'SEQ ID NO.4

2) Dissolve 1 mg of adenine in 100 ul of water, add 5 mg of 2-(2-mercaptoethylamino) benzamide, add 1 mg of boric acid, react at 37 degrees for 2 hours, and mark 2-(2-mercaptoethylamino) in the preparation chromatography Adenine of benzamide, lyophilized to give 0.8 mg.

3) Dissolve 1 mg of cytosine in 100 ul of water, add 2 mg of dabcyl, add 4 mg of DCC, condense at 37 degrees for 4 hours, prepare cytosine marked with dabcyl by chromatographic separation, and freeze-dry to obtain 0.5 mg.

4) Synthesize the probe sequence of the MMP-3 gene, as shown in SEQ ID NO.5, the 3' end is cytosine containing the quenching group dabcyl, and the 5' end is containing the reporter group 2-(2-mercaptoethylamino) Adenine from benzamide:

3'dabcyl-CTTCCCTTTA-2-(2-mercaptoethylamino)benzamide 5'SEQ ID NO.5.

5) Use the Trizol RNA extraction kit to extract total RNA from colorectal cancer tissue and adjacent normal tissue, quantify it using an ultraviolet spectrophotometer, and take 1% of it for reverse transcription to obtain cDNA, and store it at -20°C.

6) PCR reaction system using real-time quantitative PCR, GAPDH and MMP-3: total reaction system 50ul, probe 10ul, upstream primer 0.5ul, downstream primer 0.5ul, dNTP 0.5ul, Taq enzyme 1ul, positive template DNA 5ul, ddH ₂ 032.5ul. Gently flick the bottom of the tube, briefly centrifuge at 6000rpm, the reaction conditions are: 93°C for 2 minutes, then 93°C for 1 minute, 55°C for 2 minutes, a total of 40 cycles.

7) The results are shown in Figure 6. The results show that both normal colorectal tissue and colorectal cancer tissue express MMP-3, but the expression level in cancer tissue is significantly higher than that in normal tissue.

Example 8 Use 2-(2-mercaptoethylamino)benzamide to detect the distribution and metabolic process of captopril in vivo

1) Dissolve 3g of captopril in 5ml of water, add 1g of 2-(2-mercaptoethylamino)benzamide, and react at room temperature for 4h to obtain Catopol labeled with 2-(2-mercaptoethylamino)benzamide Pulley.

2) Use a reverse-phase silica gel column CH-C18-100-40/75 (SMB), elute with 50% acetonitrile, collect the pure product 2-(2-mercaptoethylamino)benzamide captopril, and remove acetonitrile by rotary evaporation Afterwards, freeze-dried and stored at -20°C.

3) Dissolve 100mg of 2-(2-mercaptoethylamino)benzamide captopril in 1ml of water, give the mice a gavage, and use fluorescence detection at 15min, 30min, 1h, 3h, 4h, 6h, 9h, 12h The device scans the mice to observe its distribution and metabolic changes in the mice. It can be seen in most parts of the stomach 15 minutes after gavage to mice, it can be widely distributed in the blood at 1 hour, it can be seen in a large amount in the liver at 3 hours, it can be distributed in a large amount in the kidney at 4 hours, and the metabolism in the body is basically completed after 9 hours .

Claims (7)

1. one kind has the novel mercapto fluorescence probe be shown below:

wherein, R=NH ₂or COOH.

2. a preparation method for novel mercapto fluorescence probe as claimed in claim 1, is characterized in that comprising the steps:

(1) Sodium sulfhydrate and monochloroacetaldehyde are obtained by reacting mercapto-acetaldehyde;

(2) mercapto-acetaldehyde obtained for step (1) and 2-aminobenzamide are obtained by reacting 2-(2-mercaptoethyl is amino) benzamide dimer through catalyst;

(3) 2-(2-mercaptoethyl is amino) benzamide dimer reductive agent reduction obtained for step (2) is obtained 2-(2-mercaptoethyl is amino) benzamide;

Or mercapto-acetaldehyde obtained for step (1) and 2-amino-benzoyl formic acid are obtained by reacting 2-(2-mercaptoethyl is amino) benzoyl formic acid dimer through catalyst by (4);

(5) 2-(2-mercaptoethyl is amino) benzoyl formic acid dimer reductive agent reduction obtained for step (4) is obtained 2-(2-mercaptoethyl is amino) benzoyl formic acid;

Described step (2) or step (4) catalyzer are zinc chloride and/or boron hydrocyanation sodium.

3. preparation method as claimed in claim 2, it is characterized in that described step (1) temperature of reaction is 0-4 DEG C, reaction solvent is the mixing solutions of water or water and organic solvent, and solution is alkalescence.

4. preparation method as claimed in claim 2, is characterized in that described step (2) or step (4) also can use dewatering agent further.

5. preparation method as claimed in claim 4, is characterized in that described dewatering agent is 3A molecular sieve.

6. preparation method as claimed in claim 2, is characterized in that described step (3) or step (5) reductive agent are dithiothreitol (DTT).

7. the application of mercapto fluorescence probe as claimed in claim 1 in the quantitative analysis or location spike of polysaccharide, peptide, protein and nucleic acid.