CN104672300A - Double enzyme-sensitive fluorescent probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a dual-enzyme-sensitive fluorescent probe, that is, a fluorescent probe capable of simultaneously detecting matrix metalloproteinases and apoptotic enzymes. Metalloproteases specifically recognize polypeptide sequences and two pairs of molecular fluorescence for energy resonance transfer. The fluorescent probe can selectively interact with one of the enzymes in the presence of matrix metalloproteinases and apoptotic enzymes, that is, specifically recognize the corresponding polypeptide sequence and cut it off, so that the quenched fluorescence can be restored. In order to realize the detection of the two enzymes; it can realize the rapid non-invasive detection of the complex microenvironmental system where matrix metalloproteinases and apoptotic enzymes coexist, with good selectivity and high sensitivity, and has great significance for the early detection of tumors and the evaluation of therapeutic effects. significance and broad application prospects.

Description

A dual-enzyme sensitive fluorescent probe and its preparation method and application

technical field

The invention relates to the fields of chemical analysis, biological analysis and clinical medical detection, and belongs to the technical field of organic small molecule fluorescent probes.

Background technique

Matrix metalloproteinases are a class of proteases of extracellular matrix components, which play an important role in the pathological damage of diseases. The expression and activity of matrix metalloproteinases are strictly regulated by cytokines, hormones, etc., and are inhibited by their natural inhibitors. Overexpression of matrix metalloproteinases can cause diseases such as tumor invasion and metastasis. Therefore, for matrix metalloproteinases The detection and identification of its activity are beneficial to the diagnosis and detection of diseases such as early tumors.

Apoptase is a cysteine hydrolase that plays an important role in the initiation and execution of apoptosis signaling. Apoptase exists in an inactive form in the cell. When it encounters an apoptotic stimulus signal, it realizes its transformation into a physiologically active structure through intracellular proteolysis, thereby regulating cell apoptosis. At the same time, most tumor therapeutic drugs have apoptosis-related mechanisms. Therefore, the detection of apoptotic enzymes is beneficial to the evaluation of early tumor treatment effects, advancement and optimization of tumor treatment plans.

Energy resonance transfer is that the donor fluorophore in the excited state transfers energy to the adjacent acceptor fluorophore in a non-radiative process through dipole interaction, and converts it into other energy. When the energy is not released in the form of fluorescence emission, the fluorescence Quenched. But when the distance between the fluorophores exceeds the range of energy resonance transfer When , the energy resonance transfer phenomenon disappears, thereby restoring the fluorescence emitted when the excited state donor returns to the ground state. Because of the distance-sensitive characteristic of energy resonance transfer, it is widely used in the monitoring of biological macromolecules and dynamic processes.

Polypeptide is a kind of substance with good biocompatibility and specific biological activity. The structure composed of certain amino acids according to a certain sequence can be specifically recognized and cut by relevant enzymes. It is widely used in the detection of biomolecules and the diagnosis of diseases. Diagnosis and delivery of medicines. Polypeptide solid-phase synthesis technology can realize effective synthesis and separation of target molecules, and diversified amino protection can realize multifunctional modification of polypeptides, so as to meet different detection and analysis requirements.

Currently, fluorescent probes targeting apoptotic enzymes or matrix metalloproteinases are widely designed and utilized. However, the commonly used fluorescent probes are only designed for a certain enzyme. Due to the interference of the complexity of the biological system on the detection of specific enzymes, the ability of enzymes to recognize specific polypeptide sequences is limited, and the evaluation of tumor diagnosis and treatment effects at this stage The demand for integrated multifunctional fluorescent probes, fluorescent probes designed for dual-enzyme sensitivity have great application value, and the dual energy resonance transfer fluorescence quenching process can effectively quench the fluorescence of fluorescent molecules, thereby improving The signal-to-noise ratio of the probe can greatly increase the specific detection ability and application range of the fluorescent probe.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a fluorescent probe for detecting matrix metalloproteinase and apoptosis enzyme, its synthesis method and application.

The fluorescent probe for detecting matrix metalloproteinases and apoptosis enzymes provided by the present invention, that is, a dual-enzyme sensitive fluorescent probe, comprises apoptotic enzyme-specific recognition polypeptide sequences, matrix metalloproteinase-specific recognition polypeptide sequences and two pairs of energy resonance transfer pair of molecular fluorescence.

The donors of the two pairs of energy resonance transfer molecular fluorescence pairs are the same donor.

The molecular fluorescence pair is a fluorescence quenching molecular fluorescence pair with fluorescein as the same donor and different dimethylaminoazobenzenes as acceptors.

The specific recognition polypeptide sequence of apoptosis enzyme is a polypeptide sequence comprising amino acid-amino acid-amino acid-aspartic acid; the specific recognition polypeptide sequence of matrix metalloproteinase is proline-leucine-glycine-valine Acid-Arginine, Proline-Leucine-Glycine-Leucine-Alanine-Glycine, Proline-Leucine-Glycine-Cysteine-Alanine-Glycine, Glycine- One of Proline-Proline-Glycine-Valine-Valine-Glycine-Glutamic Acid-Lysine-Glycine-Glutamic Acid-Glutamine.

The polypeptide sequence comprising amino acid-amino acid-amino acid-aspartic acid is aspartic acid-glutamic acid-valine-aspartic acid or tryptophan-glutamic acid-histidine-aspartic acid acid.

Further, the dual-enzyme-sensitive fluorescent probe is preferably chemically named dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine-lysine Acid (fluorescein)-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene, the chemical structure is as shown in formula (I):

The preparation method of the dual-enzyme sensitive fluorescent probe of structure shown in formula (I), comprises the following steps:

(A) Synthesis of dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine, fluorescence

Vein-lysine-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene;

(B) Dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine and fluorescein-lysine-serine-aspartic acid-glutamic acid -Valine-aspartic acid-serine-lysine-dimethylaminoazobenzene undergoes fragment condensation to obtain the fluorescent probe for detecting matrix metalloproteinase and apoptosis enzyme shown in formula (I).

In described step (A), dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine and fluorescein-lysine-serine-aspartic acid The synthesis of acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene adopts the polypeptide solid-phase synthesis method;

The synthesis of the step (B) adopts the method of solid phase fragment condensation.

The resin used in the solid-phase synthesis of peptides is one of 2-chloro-trityl chloride resin, Rink Amide resin, and MBHA resin.

In the described step (A), (i) the synthesis of dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine specifically includes the following steps:

(1) Swell 2-chloro-trityl chloride resin in redistilled N,N-dimethylformamide for 0.5-2 hours, and remove the solvent;

(2) Dissolve FMOC-protected glycine and N,N-diisopropylethylamine together in N,N-dimethylformamide, react at room temperature for 1-2 hours, remove the solvent, and then use N,N-diisopropylethylamine Wash with methylformamide 2 to 4 times;

(3) Capping the unreacted active site: Methanol, N,N-dimethylformamide, N,N-diisopropylethylamine according to the volume of 0.5～2:9～11:0.5～2 Prepare a mixed solution, react at room temperature for 0.5 to 1 hour, remove the solvent, and then wash with N,N-dimethylformamide for 2 to 4 times;

(4) Cut-off of FMOC protecting group: add piperidine/N,N-dimethylformamide mixed solution with a volume fraction of 10-50% to react for 1-15 minutes, repeat the operation twice, and use N,N - Washing with dimethylformamide for 2 to 4 times;

(5) Amino acids protected by FMOC, benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropyl Dissolve ethylamine together in N,N-dimethylformamide, react at room temperature for 1-2 hours, remove the solvent, and then wash with N,N-dimethylformamide for 2-4 times;

(6) Use the ninhydrin/methanol solution of 10mg/mL to test, if the reaction is not complete, then repeat step (5); if the reaction is complete, then carry out the reaction of step (7); other amino acids according to step (4) (5) (6) conduct;

(7) Add dimethylaminoazobenzoic acid, benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-di Dissolve isopropylethylamine in N,N-dimethylformamide together, react at room temperature for 1-2 hours, remove the solvent, and then wash with N,N-dimethylformamide for 2-4 times;

(8) washing with methanol for 2 to 4 times, and washing with dichloromethane for 2 to 4 times;

(9) Cut off: 0.5-4% trifluoroacetic acid/96-99.5% dichloromethane, once every 1-10 minutes, cut 4-10 times;

(10) Collect the cut liquid, spin evaporate, and vacuum dry;

(ii) the synthesis of fluorescein-lysine-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene specifically comprises the following steps:

(1) Swell the FMOC-protected Rink Amide resin in redistilled N,N-dimethylformamide for 0.5 to 1 hour, and remove the solvent;

(2) Cut-off of FMOC protecting group: add piperidine/N,N-dimethylformamide mixed solution with a volume fraction of 10-50% to react for 1-15 minutes, repeat the operation twice, and use N,N - Washing with dimethylformamide for 2 to 4 times;

(3) FMOC-protected lysine, benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diiso Dissolve propylethylamine in N,N-dimethylformamide, react at room temperature for 1-2 hours, remove the solvent, and wash with N,N-dimethylformamide for 2-4 times;

(4) Capping: unreacted amino groups are reacted with a mixed solution containing 2-6% 2,6-lutidine/2-5% acetic anhydride/N,N-dimethylformamide for 10-60 minutes , wash with N,N-dimethylformamide for 2 to 4 times after the reaction;

(5) Removal of FMOC According to the reaction step (2), after removing and washing, the FMOC-protected lysine, benzotriazole-N,N,N'N'-tetramethyluronium hexafluoro Phosphate, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide, reacted at room temperature for 1-2 hours, and the solvent was removed, N,N-di Wash with methylformamide 2 to 4 times;

(6) To verify the degree of progress of the reaction process in the previous step, use 0.3-1 mL of ninhydrin/methanol solution of 10 mg/mL for inspection. If the reaction is not complete, the previous step will be repeated, and the next step will be carried out if the reaction is complete;

(7) Carry out the reaction of remaining amino acid according to step (5), step (6);

(8) 5(6)-carboxyfluorescein, 2-(7-azobenzotriazole)-N,N,N'N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzo Dissolve triazole and N-methylmorpholine together in N,N-dimethylformamide, react at room temperature for 2 to 12 hours, remove the solvent, and wash with N,N-dimethylformamide for 2 to 4 times;

(9) Dimethylaminoazobenzene, 2-(7-azobenzotriazole)-N,N,N'N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole Dissolve oxazole and N-methylmorpholine together in N,N-dimethylformamide, react at room temperature for 2-12 hours, remove the solvent, and wash with N,N-dimethylformamide for 2-4 times;

(10) Wash with N,N-dimethylformamide for 2 to 4 times, methanol for 2 to 4 times, dichloromethane for 2 to 4 times, and dry.

Described step (B) specifically comprises the following steps:

(1) Place the Rink Amide resin comprising the sequence of fluorescein-lysine-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene Swell in N,N-dimethylformamide for 0.5-2 hours;

(2) Use trifluoroacetic acid/dichloromethane mixed solution with a volume ratio of 0.5-4:96-99.5 to remove methyltrityl side groups. After removal, wash with dichloromethane 2-4 times, N , N-dimethylformamide washing 2 to 4 times;

(3) Dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine, 2-(7-azobenzotriazole)-N,N ,N'N'-Tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, and N-methylmorpholine were dissolved in N,N-dimethylformamide, and reacted at room temperature for 2 to 10 hours. Then remove the solvent, and wash with N,N-dimethylformamide for 2 to 4 times;

(4) Cutting off: Use trifluoroacetic acid/water mixture solution with a volume ratio of 83-95:5-17 to cut off for 1-3 hours, filter under reduced pressure and collect the filtrate, and concentrate the filtrate by rotary evaporation under reduced pressure; Drop by drop into frozen anhydrous ether, centrifuge, decant the supernatant, dry in vacuum overnight, and it is ready.

The application of the dual-enzyme-sensitive fluorescent probe in the detection of matrix metalloproteinase and apoptosis enzyme activity in chemical systems and biological systems and the screening of apoptosis enzyme inhibitors and matrix metalloproteinase inhibitors.

The fluorescent probe of the present invention has the largest fluorescence recovery under the joint action of apoptotic enzyme and matrix metalloproteinase, and the recovery of luciferin fluorescence realizes the detection of apoptotic enzyme and matrix metalloproteinase, as shown in FIG. 2 .

The fluorescent probe of the present invention has the same effect as the simultaneous action of matrix metalloproteinase and apoptosis enzyme after acting on matrix metalloproteinase and then acting on apoptosis enzyme, as shown in FIG. 3 .

The fluorescent probe of the present invention has weak fluorescence recovery under the action of caspase inhibitor, as shown in FIG. 4 .

The fluorescent probe of the present invention has weak fluorescence recovery under the action of matrix metalloproteinase inhibitors, as shown in FIG. 5 .

The present invention has the following advantages and beneficial effects:

1) The whole synthesis process adopts solid-phase synthesis technology and fragment condensation method, and the yield is high and the purification is simple.

2) The fluorescent probe has dual specific recognition ability of apoptosis enzyme and matrix metalloproteinase.

3) Under the condition that only the apoptotic enzyme and the matrix metalloproteinase act together, the fluorescence of the probe has a great recovery.

4) The probe has practical value for evaluating the complex microenvironment of tumor and the effect of cancer treatment.

Description of drawings

Figure 1: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of fluorescent probes.

Figure 2: Fluorescence recovery diagram of the fluorescent probe under the joint action of apoptase and matrix metalloproteinase.

Figure 3: Fluorescence recovery over time when fluorescent probe interacts with matrix metalloproteinase and apoptotic enzyme, and fluorescence recovery over time when fluorescent probe interacts with matrix metalloproteinase and apoptotic enzyme sequentially.

Figure 4: Fluorescence recovery diagram of fluorescent probes under the action of caspase inhibitors.

Figure 5: Fluorescence recovery diagram of fluorescent probes under the action of matrix metalloproteinase inhibitors.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

Synthesis of dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine:

(1) Weigh 0.5g of 2-chloro-trityl chloride resin (1.05mmol/g), swell in re-distilled N,N-dimethylformamide (DMF) for 0.5~2h, and remove DMF ;

(2) Dissolve the first amino acid—FMOC-protected glycine and N,N-diisopropylethylamine (DIEA) in DMF, add to the reactor to react at room temperature for 1-2 hours, remove the solvent, and then wash with DMF 2 to 4 times;

(3) Capping the unreacted active site: prepare a mixed solution of methanol, DMF, and DIEA at a volume ratio of 0.5-2:9-11:0.5-2 and add it to the reactor, react at room temperature for 0.5-1 hour, pump Remove the solvent, and then wash with DMF 2-4 times.

(4) Cut-off of FMOC protecting group: add 10-50% piperidine/DMF solution, react for 1-15min, react like this twice, wash the resin with DMF for 2-4 times after the reaction;

(5) FMOC-protected amino acid, benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HoBt), DIEA dissolved In DMF, add to the reactor at room temperature to react for 1-2 hours, remove the solvent, and then wash with DMF for 2-4 times.

(6) Verify the degree of progress of the previous step reaction process, check with 0.3-1 mL of ninhydrin/methanol solution of 10 mg/mL, if the reaction is not complete, then repeat step (5); if the reaction is complete, then proceed to step (7) )Reaction.

Other amino acids are carried out according to steps (4)(5)(6).

(7) Dissolve dimethylaminoazobenzoic acid, HBTU, HoBt, and DIEA in DMF together, add to the reactor to react at room temperature for 1-2 hours, remove the solvent, and then wash with DMF for 2-4 times.

(8) Methanol washes 2 to 4 times, and dichloromethane washes 2 to 4 times.

(9) Cut off: 0.5-4% trifluoroacetic acid/96-99.5% dichloromethane, once every 1-10 minutes, cut 4-10 times.

(10) Collect the cut liquid, spin evaporate, dry in vacuum, and store at -20°C in the dark.

Example 2

Synthesis of fluorescein-lysine-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene:

(1) Weigh 0.5g of FMOC-protected Rink Amide resin (0.7mmol/g), swell in re-distilled DMF for 0.5-1h, and extract DMF;

(2) Cut off the FMOC protecting group, use 10-50% piperidine/DMF solution, react for 1-15min, react like this twice, wash the resin with DMF for 2-4 times after the reaction;

(3) FMOC-protected lysine, HBTU, HoBt, DIEA were dissolved in DMF, added to the reactor at room temperature for 1-2 hours, the solvent was removed, and DMF was washed 2-4 times;

(4) Capping, unreacted amino groups are reacted with 2-6% 2,6-lutidine/2-5% acetic anhydride/DMF solution for 10-60 minutes, and washed 2-4 times with DMF after the reaction;

(5) Removal of FMOC According to the reaction step (2), after removing and washing, dissolve the FMOC-protected amino acid, HBTU, HoBt, and DIEA in DMF, add them to the reactor for 1-2 hours at room temperature, and remove the solvent , DMF washing 2 to 4 times;

(6) To verify the degree of progress of the reaction process in the previous step, use 0.3-1 mL of ninhydrin/methanol solution of 10 mg/mL for inspection. If the reaction is not complete, the previous step will be repeated, and the next step will be carried out if the reaction is complete;

(7) The reaction of other amino acids is carried out according to step (5) (6);

(8) 5(6)-Carboxyfluorescein (FAM), 2-(7-azobenzotriazole)-N,N,N'N'-tetramethyluronium hexafluorophosphate (HATU), HoBt, N-methylmorpholine, dissolved in DMF, added to the reactor at room temperature for 2 to 12 hours, the solvent was removed, and DMF was washed 2 to 4 times;

(9) Dimethylaminoazobenzene (Dabcyl) is carried out according to the condensation method of step (8);

(10) Wash 2-4 times with DMF, 2-4 times with methanol, 2-4 times with dichloromethane, dry, and store in the dark at -20°C.

Example 3

Dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine-lysine (fluorescein)-serine-aspartic acid-glutamic acid-valine Synthesis of acid-aspartic acid-serine-lysine-dimethylaminoazobenzene (such as formula I):

(1) Take the one containing fluorescein-lysine-serine-aspartic acid-glutamic acid-valine-aspartic acid-serine-lysine-dimethylaminoazobenzene sequence in Example 2 Rink Amide resin, swell in DMF for 0.5-2 hours;

(2) Use trifluoroacetic acid/dichloromethane mixed solution with a volume ratio of 0.5-4:96-99.5 to remove methyltrityl side groups. After removal, wash 2-4 times with dichloromethane, DMF Wash 2 to 4 times;

(3) Dissolve dimethylaminoazobenzene-glycine-proline-leucine-glycine-valine-arginine-glycine, HATU, HoBt, N-methylmorpholine (NMM) in DMF , add to the reactor at room temperature to react for 2-10 hours, remove the solvent, and wash with DMF for 2-4 times;

(4) Cut off: the volume fraction is 83-95%: 5-17% trifluoroacetic acid/water mixed solution cut off for 1-3 hours, vacuum filtration and collect the filtrate, the filtrate is concentrated by rotary evaporation under reduced pressure, and the concentrated The liquid was dropped into frozen anhydrous ether, centrifuged, and the supernatant was decanted, dried overnight in vacuum, and stored at -20°C.

(5) Detection of target molecules: a small amount of sample was dissolved in methanol and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The chemical structure of the target molecule was characterized by MALDI-TOF-MS, as shown in Figure 1.

Example 4: Fluorescent spectra before and after the simultaneous action of fluorescent probes with matrix metalloproteinases and apoptotic enzymes

Dissolve the fluorescent probe in phosphate buffered solution (PBS) with a pH of 7.4, prepare a 1 μM stock solution, add 200 pM caspase (Caspase-3) and 200 ng matrix metalloproteinase (MMP-2) and the corresponding PBS buffer solution, so that the concentration of the fluorescent probe in the working solution was 0.5 μM, reacted at 37 ° C, and detected the change of the fluorescence emission spectrum with time by a fluorescence spectrometer, the excitation wavelength was 465 nm, and the slit width was 5 nm, as shown in Figure 2, the fluorescence probe The needle has a fluorescence intensity change of more than 18.5 times at 520 nm before and after cutting.

Example 5

Fluorescent spectra of fluorescent probes interacting with matrix metalloproteinases and apoptotic enzymes and comparisons of fluorescent probes interacting with matrix metalloproteinases and apoptotic enzymes at the same time

Prepare the fluorescence recovery comparison group according to the same method as in Example 4. At the same time, add 200ngMMP-2 to the prepared stock solution and dilute the concentration of the fluorescent probe to 0.5μM with PBS buffer solution of pH 7.4 to prepare a working solution at 37°C. For the next reaction, the fluorescence emission in the working solution was detected by a fluorescence spectrometer, the excitation wavelength was 465nm, and the slit width was 5nm. When the fluorescence intensity of the working solution tends to balance with time, divide the working solution into two parts, add 200pMcaspase-3 enzyme to one part, add the same volume of PBS to the other part, and add the same volume of PBS buffer to the control group to maintain The three probes had the same concentration, reacted at 37°C, and recorded the emission intensity of fluorescence at 520nm as a function of time by a fluorescence spectrometer, with an excitation wavelength of 465nm and a slit width of 5nm. As shown in Figure 3, under the simultaneous action of 200ng MMP-2 and 200pM Caspase-3, there was a 17.7-fold fluorescence enhancement after 10.5h, while only a 5.9-fold fluorescence enhancement under the condition of only 200ng MMP-2 action. Moreover, under the condition of only 200ng MMP-2, the reaction time increased to 25.1h, and the fluorescence intensity was not further enhanced compared with the same concentration of MMP-2 enzyme for 10.5h. However, after 200ng MMP-2 for 10.5h After that, it was treated with 200pM caspase-3 enzyme for 25.1h, and the fluorescence intensity was further enhanced compared with that of 200ngMMP-2 for the same time without adding 200pM caspase-3 enzyme, and its fluorescence intensity was the same as that added at the same time from the initial The MMP-2 and Caspase-3 have the same fluorescence intensity.

Example 6

Taking Ac-DEVD-CHO as an example, using probes to detect the inhibitory effect of caspase inhibitors on caspase activity

React 200pM caspase-3 enzyme with 50μM caspase inhibitor Ac-DEVD-CHO for 2h, then add fluorescent probe to make working solution, and make the concentration of fluorescent probe in the working solution 0.5μM, react at 37℃ , the change of fluorescence intensity at 520nm was recorded by a fluorescence spectrometer, the excitation wavelength was 465nm, and the slit width was 5nm. As shown in Figure 4, after the addition of caspase inhibitor, the fluorescence intensity at 520nm was only increased by 1.1 times after the fluorescent probe reacted with caspase for 6.6 hours, indicating that the inhibitor specifically recognizes caspase It has a significant inhibitory effect on the specific polypeptide sequence in the cut-off probe. On the contrary, the probe can be used to screen the corresponding enzyme inhibitor through the change of fluorescence before and after the interaction with the target inhibitor.

Example 7

Taking 1,10-phenanthroline as an example, using a probe to detect the inhibitory effect of matrix metalloproteinase inhibitors on matrix metalloproteinase activity

200ng of MMP-2 enzyme was reacted with 1,10-phenanthroline for 2 hours, and then added a fluorescent probe to prepare a working solution, and the concentration of the fluorescent probe in the working solution was 0.5 μM, reacted at 37°C, and passed the fluorescence spectrometer Record the change of fluorescence intensity at 520nm, the excitation wavelength is 465nm, and the slit width is 5nm. As shown in Figure 5, adding matrix metalloproteinase inhibitors, after the fluorescent probe reacted with matrix metalloproteinases for 4.6h, the fluorescence intensity at 520nm was only enhanced by 1.8 times, indicating that the inhibitors specifically recognize matrix metalloproteinases And cut off the peptide sequence in the probe has a significant inhibitory effect. On the contrary, the probe can be used to screen the corresponding enzyme inhibitor through the change of fluorescence before and after the interaction with the target inhibitor.

Claims (10)

1. a two enzyme responsive type fluorescent probe, is characterized in that: the molecular fluorescence pair comprising apoptosis enzyme spcificity Recognition polypeptide sequence, matrix metalloproteinase specific recognition peptide sequence and two pairs of resonance energy transfers.

2. according to claim 1 pair of enzyme responsive type fluorescent probe, is characterized in that: what two pairs of described resonance energy transfer molecular fluorescences were right is same as body to body.

3. according to claim 2 pair of enzyme responsive type fluorescent probe, is characterized in that: described molecular fluorescence is to being be same as body and the fluorescence quencher molecule fluorescence pair that is acceptor with different dimethyl amino-azo-benzene with fluorescein.

4. the two enzyme responsive type fluorescent probes according to claim 1 or 3, is characterized in that: described apoptosis enzyme spcificity Recognition polypeptide sequence is the peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid; Described matrix metalloproteinase specific recognition peptide sequence is the one in proline-leucine-glycine-α-amino-isovaleric acid-arginine, proline-leucine-Gly-Leu-Ala-Gly, proline-leucine-glycine-halfcystine-Ala-Gly, Gly-Pro-proline(Pro)-glycine-α-amino-isovaleric acid-α-amino-isovaleric acid-glycine-glutaminic acid-LYS-GLY-Glu-Gln.

5. according to claim 4 pair of enzyme responsive type fluorescent probe, is characterized in that: the described peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid is Asp-Glu-valine aspartic acid or tryptophane-L-glutamic acid-histidine-asparate.

6. according to claim 5 pair of enzyme responsive type fluorescent probe, it is characterized in that: chemistry dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine-Methionin (fluorescein)-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene by name, chemical structure is as shown in formula I:

7. a preparation method for according to claim 6 pair of enzyme responsive type fluorescent probe, is characterized in that: comprise the following steps:

(A) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine, fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene is synthesized;

(B) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene are carried out condensation, namely obtain the fluorescent probe of the detection matrix metalloproteinase shown in formula I and apoptosis enzyme.

8. the preparation method of according to claim 7 pair of enzyme responsive type fluorescent probe, is characterized in that:

In described step (A), the synthesis of dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene adopts Solid-phase synthesis peptides method;

Described step (B) adopts the method for solid phase fragment condensation.

9. the preparation method of according to claim 8 pair of enzyme responsive type fluorescent probe, is characterized in that: the resin adopted in Solid-phase synthesis peptides is the one in the chloro-trityl chloride resin of 2-, Rink Amide resin, mbha resin.

10. the application of the two enzyme responsive type fluorescent probes described in any one of claim 1-6 in the screening of chemical system, living things system matrix metalloproteinase and apoptosis Enzyme assay and apoptosis enzyme inhibitors and matrix metallo-proteinase inhibitor.