CN112390859B - Self-assembly polypeptide probe for identifying Caspase protein, preparation method and application - Google Patents

Abstract

The invention relates to a self-assembly polypeptide probe for identifying Caspase protein, a preparation method and application thereof, and a prepared small molecular polypeptide compound Nap-GFF_PYDEVD-AFC and Nap-GFF_PYIETD-AFC, using small-molecule polypeptide Nap-GFF_PY is connected with recognition sequences DEVD and IETD of Caspase-3 and Caspase-8 proteins and a fluorophore AFC, the small molecular polypeptide compound is catalyzed by alkaline phosphatase to self-assemble to form nano hydrogel, the nano hydrogel can enter cells in an endocytosis mode, and the recognition sequences DEVD and IETD matched with the Caspase-3 and Caspase-8 proteins can be used for indicating the activity of the Caspase-3 and Caspase-8 proteins in the cells. Therefore, the nano hydrogel transmission system established by the invention not only solves the problems of low solubility and low cell entering efficiency of Ac-DEVD-AFC and Ac-IETD-AFC, but also can indicate the expression conditions of Caspase-3 and Caspase-8 proteins in apoptotic cells by using AFC.

Description

Self-assembly polypeptide probe for identifying Caspase protein, preparation method and application

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a self-assembly polypeptide probe for identifying Caspase protein, a preparation method and application thereof.

Background

Apoptosis, also known as programmed cell death, is a self-destruction mechanism that exists in cells. During apoptosis, caspases, play an important roleIn use, the process of apoptosis is actually a cascade of amplification reactions in which Caspase irreversibly hydrolyzes substrates. Caspase-3 and Caspase-8 play a very important role in the Caspase family. The current method for detecting the apoptosis proteins Caspase-3 and Caspase-8 is mainly realized by specific recognition sequences DEVD and IETD of Caspase-3 and Caspase-8. Ac-DEVD (IETD) -AFC is a commonly used detection method, but because of the problems of poor solubility and low efficiency of entering cells, the expression conditions of Caspase-3 and Caspase-8 proteins cannot be detected in real time in living cells according to fluorescence intensity. Rapid detection of the apoptotic proteins Caspase-3 and Caspase-8 in living cells undergoing apoptosis is a significant challenge. The living cell labeling technology requires high specificity, high labeling density, cell permeability of labeled molecules, and strong targeting of target proteins. To develop a simpler and more convenient platform, we take advantage of the self-assembly properties of small molecule polypeptides. Because of its advantages of good biocompatibility, no toxicity to human body, easy synthesis, etc. Small molecule hydrogel as a novel material has been widely studied by scientists at home and abroad due to good biocompatibility and good three-dimensional appearance in the aspects of biomedicine and biomaterials. To date, the connection of Nap-GFF to the N-terminal of small-molecule polypeptide has not been found_PDEVD (IETD) -AFC is connected with the Y end and the C end, nanofibers are formed through alkaline phosphatase catalysis self-assembly, and research reports of the expression conditions of Caspase-3 and Caspase-8 proteins in apoptotic cells can be indicated in real time.

Disclosure of Invention

The invention aims to solve the problems in the prior art and aims to solve the problems by using short peptide Nap-GFF_PY, Caspase-3/8 specific recognition sequence DEVD/IETD and AFC are connected to form Nap-GFF_PYDEVD-AFC and Nap-GFF_PThe micromolecular polypeptide compound of YIETD-AFC forms nano-fiber through alkaline phosphatase catalysis self-assembly, the nano-fiber can enter cells in an endocytosis mode, and the protein activities of Caspase-3 and Caspase-8 in the living cells which are subjected to apoptosis are detected according to the fluorescence intensity of AFC.

The self-assembly polypeptide probe for recognizing Caspase protein is used for recognizing Caspase-3Self-assembly polypeptide probe Nap-GFF of protein_PThe structural formula of YDEVD-R is as follows:

self-assembly polypeptide probe Nap-GFF for recognizing Caspase-8 protein_PThe structural formula of YIETD-R is as follows:

wherein R is a fluorescent functional group;

wherein the content of the first and second substances,

wherein-Nap represents a naphthylacetic acid hydrophobic end-capping group, -AFC represents a fluorophore formed by an aspartic acid derivative. Self-assembled short peptide Nap-GFF_PThe connection of Y with DEVD-AFC and IETD-AFC may be selected from 0 to 3 glycines.

The invention also provides a preparation method of the self-assembly polypeptide probe for recognizing Caspase protein, which is characterized by mainly comprising the following steps:

(1) the intermediate compound Fmoc-Asp (OH) -AFC was synthesized as follows:

i) taking Fmoc-Asp (OtBu) -COOH, coumarin 151, anhydrous pyridine and phosphorus oxychloride for reaction; after reaction, adding a hydrochloric acid solution, extracting and drying to obtain Fmoc-Asp (OtBu) -AFC;

ii) dissolving Fmoc-Asp (OtBu) -AFC in trifluoroacetic acid solution, and reacting under ice bath condition to obtain Fmoc-Asp (OH) -AFC;

(2)Nap-GFF_PYDEVD-AFC and Nap-GFF_PThe synthesis process of YIETD-AFC is as follows:

iii) connecting Fmoc-Asp (OH) -AFC synthesized in (1) to a solid-phase carrier 2-chlorotrityl chloride resin in a solid-phase synthesizer, and reacting for 1-3 hours by using dichloromethane as a solvent;

iv) removing a protecting group Fmoc by using a dimethyl formamide solution of piperidine, taking a polypeptide condensing agent HBTU, putting the polypeptide condensing agent HBTU into the solid phase synthesizer in the step iii), sequentially adding Fmoc-valine, Fmoc-glutamic acid, Fmoc-aspartic acid, Fmoc-phosphotyrosine, Fmoc-phenylalanine and Fmoc-glycine for reaction, and blocking the N end of the polypeptide by using naphthylacetic acid to obtain the Nap-GFF_PYDEVD-AFC; sequentially adding Fmoc-threonine, Fmoc-glutamic acid, Fmoc-isoleucine, Fmoc-phosphotyrosine, Fmoc-phenylalanine and Fmoc-glycine for reaction, and capping the N end of the polypeptide by using naphthylacetic acid to obtain the Nap-GFF_PYIETD-AFC。

v) cleaving the prepared self-assembled short peptide probe from the solid phase resin with 95% trifluoroacetic acid, and HPLC-cleaving the resulting Nap-GFF_PYDEVD-AFC and Nap-GFF_PAnd purifying and separating YIETD-AFC.

The invention also provides a method for applying the self-assembly polypeptide probe for identifying Caspase protein to a small molecule hydrogel nanofiber transmission system, and Nap-GFF_PThe YDEVD-AFC micromolecule compound is self-assembled to form nano-fibers under the action of alkaline phosphatase on the surface of a cell membrane, and the nano-fibers enter cells in an endocytosis mode; is recognized by Caspase-3 protein in apoptotic cells and is cut to release a fluorescent group AFC, thus playing a role in detecting the expression of Caspase-3 protein in live cells in real time;

Nap-GFF_Punder the action of alkaline phosphatase on the surface of a cell membrane, a YIETD-AFC small molecular compound self-assembles to form a nanofiber and enters cells in an endocytosis mode; is recognized by Caspase-8 protein in apoptotic cell and cut to release fluorescent group AFC, which plays the role of real-time detection of Caspase-8 protein expression in living cell.

It should be noted that all amino acids used in the present invention are natural amino acids in L configuration.

The invention has the following beneficial effects:

(1) the small molecular self-assembly polypeptide compound Nap-GFF prepared by the invention_PYDEVD-AFC and Nap-GFF_PYIETD-AFC, using small-molecule polypeptide Nap-GFF_PY is connected with recognition sequences DEVD and IETD of Caspase-3 and Caspase-8 proteins and a fluorophore AFC, the small molecular polypeptide compound is catalyzed by alkaline phosphatase to self-assemble to form nano hydrogel (nanofiber), the nano hydrogel can enter cells in an endocytosis mode, and the recognition sequences DEVD and IETD matched with the Caspase-3 and Caspase-8 proteins can be used for indicating the activity of the Caspase-3 and Caspase-8 proteins in the cells; namely, the problem of solubility of Ac-DEVD (IETD) -AFC is solved. The expression of Caspase-3(Caspase-8) protein in apoptotic cells can also be detected in real time in living cells.

(2) The small molecular polypeptide compound Nap-GFF of the invention_PYDEVD-AFC and Nap-GFF_PRaw materials used in the YIETD-AFC preparation process are various amino acids required by cultured cells every day, no toxic or side effect is generated on the cells, and the prepared Nap-GFF_PYDEVD-AFC and Nap-GFF_PThe MTT results of YIETD-AFC showed no inhibition of cell growth.

(3) The nano hydrogel prepared by the invention has good biocompatibility and good three-dimensional appearance (about 10nm nano-fiber), can more easily enter tumor cells, and can be widely applied to Caspase-3/8 activity detection.

Drawings

FIG. 1 is a scheme showing the chemical synthesis scheme of Fmoc-L-Asp (OtBu) -AFC;

FIG. 2 shows Nap-GFF_PYDEVD-AFC and Nap-GFF_PChemical synthesis scheme of YIETD-AFC;

FIG. 3 shows Nap-GFF_PA hydrogen spectrum of YDEVD-AFC;

FIG. 4 shows Nap-GFF_PHydrogen spectrum of YIETD-AFC;

FIG. 5 shows Nap-GFF_PYDEVD-AFC and Nap-GFF_PA three-dimensional topography observed by a transmission electron microscope of YIETD-AFC two kinds of small molecular hydrogel, wherein ALP is serum alkaline phosphatase;

FIG. 6 shows the protein Caspase-3 vs. Nap-GFF_PYDEVD-AFC probesInfluence of medium AFC fluorophore release;

FIG. 7 shows the protein Caspase-8 vs. Nap-GFF_PEffect of AFC fluorophore release in YIETD-AFC probe;

FIG. 8 shows Nap-GFF in Hela cells_PThe concentration of YDEVD-AFC is 50 mu mol/L, and the concentration is 1mmol/L H₂O₂Induces Hela cells to undergo apoptosis at the concentration of (a); the fluorescence intensity of AFC is gradually increased along with the increase of induction time (0min, 10min, 20min, 40min and 60 min);

FIG. 9 shows Nap-GFF in Hela cells_PThe concentration of YDEVD-AFC is 50 mu mol/L, and the concentration is 1mmol/L H₂O₂Induces Hela cells to undergo apoptosis at the concentration of (a); the fluorescence intensity of AFC was gradually increased with the increase of the induction time (0min, 10min, 20min, 40min, 60 min).

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified. The present invention will be described in detail with reference to examples.

EXAMPLE Synthesis of Fmoc-L-Asp (OH) -AFC

Fmoc-L-Asp (OtBu) -OH (10mmol, 4.11 g) and 7-amino-4-trifluoromethylcoumarin (10mmol, 2.29 g) were weighed into a 50 mL round-bottomed flask, 40 mL of anhydrous pyridine was added, and 10mmol of phosphorus oxychloride (3.06g) was added dropwise at minus 15 ℃. And (4) protecting the reaction product with nitrogen so as to avoid contacting moisture in the air, and reacting for 30 minutes at a constant temperature of minus 15 ℃. To the dark red viscous liquid was added 3mol/L hydrochloric acid solution until the PH of the solution was 2. Extracted three times with ethyl acetate, washed three times with saturated brine, and dried over anhydrous sodium sulfate for 2 hours. Ethyl acetate was removed by rotary evaporation. Purification by silica gel chromatography (ethyl acetate: petroleum ether ═ 1: 3). A total of 5.07 g of Fmoc-L-Asp (OtBu) -AFC was obtained as a white solid in 81.38% yield.

Fmoc-L-Asp (OtBu) -AFC (6.42mmol, 4g) was weighed out and dissolved in a solution of 95% trifluoroacetic acid (trifluoroacetic acid: dichloromethane 95%: 5%) and reacted for 60 minutes under ice-bath conditions. Trifluoroacetic acid and dichloromethane were removed by rotary evaporation and purified by silica gel chromatography (dichloromethane: methanol 75: 1) to give a total of 2.59 g of Fmoc-L-asp (oh) -AFC in 71.26% yield.

The synthesis steps of this example can be referred to in FIG. 1.

EXAMPLE DINap-GFF_PYDEVD-AFC and Nap-GFF_PChemical Synthesis of YIETD-AFC

Synthesis of Nap-GFF by solid phase synthesis method_PYDEVD-AFC and Nap-GFF_PThe YIETD-AFC comprises the following specific synthetic steps:

(1) 0.75mmol (700 mg) of the dichloro resin was weighed into a solid phase synthesizer, 20 ml of Dichloromethane (DCM) was added to immerse the resin, the resin was swollen for 20 minutes, DCM solvent was removed, 1mmol (566 mg) of Fmoc-L-Asp (OH) -AFC in DCM was added, and 800. mu.l of N, N-Diisopropylethylamine (DIPEA) was added. The reaction was carried out for 2 hours. After which it was washed five times with DCM for 2 minutes each. Using volume ratio DCM: methanol: DIPEA ═ 17: 2: 1 for 30 minutes. After that, the column was washed 5 times with 20 ml portions of DCM solution each time for 1 minute. After which the column was washed 5 times with 20 ml portions of DMF solution each time for 2 minutes.

(2) Nap-GFF was prepared on the basis of addition of 20 ml of 20% piperidine in DMF, reaction for 30 minutes, removal of Fmoc protecting group and exposure of amino group for the next reaction_PYDEVD-AFC and Nap-GFF_PYIETD-AFC。

Nap-GFF_PThe preparation process of YDEVD-AFC is as follows: to the solid phase synthesis tube, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-valine (339 mg) were added and reacted for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min, washed 5 times with 20 mL Dimethylformamide (DMF) solution for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-glutamic acid (426 mg) were added thereto, respectively, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-aspartic acid (412 mg) were added thereto, and the reaction was carried out for 120 minutesA clock. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-phosphotyrosine (484 mg) were added, respectively, and reacted for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg) and 1mmol of Fmoc-phenylalanine (388 mg) were added, respectively, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Fmoc-phenylalanine was added in duplicate and the reaction was carried out for 120 minutes. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-glycine (297 mg) were added thereto, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg) and 1mmol of naphthylacetic acid (186 mg) were added thereto, respectively, and the reaction was carried out for 120 minutes. After the reaction was complete, the reaction mixture was washed 5 times with 20 ml of DMF solution for 2 minutes each. The reaction mixture was washed 5 times with 20 ml of DCM solution each time for 1 minute. With 95% trifluoroacetic acid (TFA) (TFA: H)₂O: TIS 95%: 2.5%: 2.5%) for 1 hour to obtain Nap-GFF_PYDEVD-AFC。

Nap-GFF_PThe YIETD-AFC is prepared as follows: 1mmol of peptide condensing agent HBTU (379 mg) and 1mmol of Fmoc-threonine (339 mg) were added to a solid phase synthesis tube and reacted for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min, washed 5 times with 20 mL Dimethylformamide (DMF) solution for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-glutamic acid (426 mg) were added thereto, respectively, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-isoleucine (354 mg) were added, respectively, and reacted for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-phosphotyrosine (484 mg) were added, respectively, and reacted for 120 minutes. Then 20 percent of the total weight is usedThe Fmoc group was cleaved with piperidine for 30 min, washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg) and 1mmol of Fmoc-phenylalanine (388 mg) were added, respectively, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Fmoc-phenylalanine was added in duplicate and the reaction was carried out for 120 minutes. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg), 1mmol of Fmoc-glycine (297 mg) were added thereto, and the reaction was carried out for 120 minutes. The Fmoc group was then cleaved with 20% piperidine for 30 min and washed 5 times with 20 mL of DMF for 2 min each. Thereafter, 1mmol of peptide condensing agent HBTU (379 mg) and 1mmol of naphthylacetic acid (186 mg) were added thereto, respectively, and the reaction was carried out for 120 minutes. After the reaction was complete, the reaction mixture was washed 5 times with 20 ml of DMF solution for 2 minutes each. The reaction mixture was washed 5 times with 20 ml of DCM solution each time for 1 minute. With 95% trifluoroacetic acid (TFA) (TFA: H)₂O: TIS 95%: 2.5%: 2.5%) for 1 hour to obtain Nap-GFF_PYIETD-AFC。

(3) Washing with 1% TFA in dichloromethane 20 ml twice, collecting total cleavage solution, removing liquid by rotary evaporation, and separating polypeptide compound Nap-GFF with high performance liquid phase_PYDEVD-AFC and Nap-GFF_PYIETD-AFC separation;

in this step, the process of high performance liquid chromatography separation is as follows: passing through a C-18 reversed phase chromatographic column, and performing gradient elution by using water and methanol as eluent and 30-80% of methanol for 25 minutes; performing preparative HPLC on the eluent, and collecting fractions with retention time tR of 15.0-15.5 min to obtain Nap-GFF_PYDEVD-AFC; collecting the flow with retention time tR of 13.6-14.3 min under the same condition to obtain Nap-GFF_PYIETD-AFC。

The synthesis steps of this embodiment can be referred to fig. 2;

Nap-GFF_PYDEVD-AFC and Nap-GFF_PThe mass spectra of YIETD-AFC are shown in FIG. 3 and FIG. 4, respectively.

EXAMPLE three Small molecule hydrogel Nap-GFF_PYDEVD-AFC and Nap-GFF_PFormation of YIETD-AFC

The formation steps of the small molecule hydrogel are as follows:

(1) balance2.00mg of the obtained polypeptide compound was added to 1ml of PBS (pH 7.4 buffer), and the pH was adjusted to 7.4 with sodium carbonate to form a probe solution. Adding alkaline phosphatase solution (final concentration of enzyme is 2.0U/ml), standing overnight, and turning upside down to obtain Nap-GFF_PYDEVD-AFC and Nap-GFF_PYIETD-AFC gel-like substance.

(2) Sucking about 15 microliters of colloid by using a pipette gun, dropwise adding the colloid on a silicon wafer with a clean surface, spin-coating to ensure that the colloid is uniformly distributed on the silicon wafer, and observing the appearance by using a transmission electron microscope, wherein the specific figure is 5; the appearance of the nano-fiber is about 10nm of nano-fiber which is uniformly dispersed.

Example tetrapase-3 and Caspase-8 proteins promote the probe Nap-GFF, respectively_PYDEVD-AFC and Nap-GFF_PRelease of fluorophore AFC from YIETD-AFC.

(1) Taking Nap-GFF with initial concentration of 10mmol/L _P500. mu.l of a mother solution of YDEVD-AFC was prepared in the form of probe solutions having concentrations of 80. mu. mol/L, 40. mu. mol/L, 20. mu. mol/L, 10. mu. mol/L and 5. mu. mol/L. Caspase-3 protein is added at 25 deg.c and the mixture is set for 2 hr. The fluorescence intensity of AFC in the solution was then measured with a fluorescence photometer. See in particular fig. 6; the fluorescence intensity of the fluorescent probe is dependent on Nap-GFF_PThe increase in the concentration of YDEVD-AFC was gradually increased.

(2) Taking Nap-GFF with initial concentration of 10mmol/L_PThe mother solution of YIETD-AFC was prepared in 500. mu.l of probe solutions having concentrations of 80. mu. mol/L, 40. mu. mol/L, 20. mu. mol/L, 10. mu. mol/L and 5. mu. mol/L. Caspase-8 protein is added at 25 deg.c and the mixture is set for 2 hr. The fluorescence intensity of AFC in the solution was then measured with a fluorescence photometer. See in particular fig. 7; the fluorescence intensity of the fluorescent probe is dependent on Nap-GFF_PThe increase in YIETD-AFC concentration was gradually increased.

Example PenNap-GFF_PYDEVD-AFC and Nap-GFF_PBiological application of YIETD-AFC

(1) Normal culturing human cervical carcinoma Hela cell, uniformly spreading into 10 confocal small dishes, the cell number is 2.0x 10⁴And (4) respectively. Culturing at 37 deg.C under 5% carbon dioxide for 8 hr, and adding Nap-GFF_PYDEVD-AFC andNap-GFF_PYIETD-AFC, concentration set at 50 μmol/L. Cultured for four hours to allow Nap-GFF_PYDEVD-AFC and Nap-GFF_PYIETD-AFC entered the cells. Then at a concentration of 1mmol/L H₂O₂Under the action of the probe, setting time gradient (0min, 10min, 20min, 40min and 60min) to induce Hela cells to generate apoptosis, and shooting the fluorescence intensity of each group of cells by using a confocal microscope to further reflect the expression quantity of Caspase-3 and Caspase-8 proteins in the apoptotic cells. The results show that: with H₂O₂The induction time is prolonged, the expression quantity of the apoptotic proteins Caspase-3 and Caspase-8 is gradually increased, the quantity of AFC released by cutting is also gradually increased, the fluorescence intensity displayed by AFC is also gradually enhanced, and the result is shown in figures 8 and 9, which shows that the self-contained polypeptide probe designed by the invention can detect the apoptosis condition of cells in real time in living cells.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (4)

1. The self-assembly polypeptide probe for identifying Caspase protein is characterized in that the Caspase protein is Caspase-3 protein and Caspase-8 protein;

self-assembly polypeptide probe Nap-GFF for recognizing Caspase-3 protein_PThe structural formula of YDEVD-AFC is as follows:

self-assembly polypeptide probe Nap-GFF for recognizing Caspase-8 protein_PThe structural formula of YIETD-AFC is as follows:

wherein-Nap ═

2. The method for preparing the self-assembly polypeptide probe for recognizing Caspase protein according to claim 1, which is characterized by mainly comprising the following steps:

(1) the intermediate compound Fmoc-Asp (OH) -AFC was synthesized as follows:

i) taking Fmoc-Asp (OtBu) -COOH and coumarin 151, adding anhydrous pyridine, and dropwise adding phosphorus oxychloride to react at the temperature of minus 15 ℃; after reaction, adding a hydrochloric acid solution, extracting and drying to obtain Fmoc-Asp (OTBu) -AFC;

ii) dissolving Fmoc-Asp (OtBu) -AFC in trifluoroacetic acid solution, and reacting under ice bath condition to obtain Fmoc-Asp (OH) -AFC;

(2)Nap-GFF_PYDEVD-AFC and Nap-GFF_PThe synthesis process of YIETD-AFC is as follows:

iii) connecting Fmoc-Asp (OH) -AFC synthesized in (1) to a solid-phase carrier 2-chlorotrityl chloride resin in a solid-phase synthesizer, and reacting for 1-3 hours by using dichloromethane as a solvent;

iv) removing a protecting group Fmoc by using a dimethyl formamide solution of piperidine, putting a polypeptide condensing agent HBTU into the solid phase synthesizer in the step iii), sequentially adding Fmoc-valine, Fmoc-glutamic acid, Fmoc-aspartic acid, Fmoc-phosphotyrosine, Fmoc-phenylalanine and Fmoc-glycine for reaction, and blocking the N end of the polypeptide by using naphthylacetic acid to obtain Nap-GFFPYDEVD-AFC; sequentially adding Fmoc-threonine, Fmoc-glutamic acid, Fmoc-isoleucine, Fmoc-phosphotyrosine, Fmoc-phenylalanine and Fmoc-glycine, and blocking the N end of the polypeptide by using naphthylacetic acid to obtain Nap-GFFPYIETD-AFC;

v) cleaving the prepared short peptide from the resin with trifluoroacetic acid, and subjecting the resulting Nap-GFF_PYDEVD-AFC and Nap-GFF_PYIETD-AFC were separated separately.

3. The self-assembled polypeptide probe capable of recognizing Caspase protein according to claim 1The application of the nano-fiber transmission system for preparing the micromolecular hydrogel is characterized in that the nano-fiber transmission system is Nap-GFF_PThe YDEVD-AFC micromolecule compound is self-assembled to form nano-fibers under the action of alkaline phosphatase on the surface of a cell membrane, and the nano-fibers enter cells in an endocytosis mode; is recognized by Caspase-3 protein in apoptotic cell and cut to release fluorescent group AFC, which plays the role of real-time detection of Caspase-3 protein expression in living cell.

4. The application of the self-assembly polypeptide probe for recognizing Caspase protein in preparation of small molecule hydrogel nanofiber transmission system as claimed in claim 1, wherein the Nap-GFF_PUnder the action of alkaline phosphatase on the surface of a cell membrane, a YIETD-AFC small molecular compound self-assembles to form a nanofiber and enters cells in an endocytosis mode; is recognized by Caspase-8 protein in apoptotic cell and cut to release fluorescent group AFC, which plays the role of real-time detection of Caspase-8 protein expression in living cell.

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