Self-assembled polypeptide probe for recognizing caspase protein, preparation method and application
A self-assembly, fmoc-asp technology, applied in the field of biomedicine, to achieve the effect of good three-dimensional shape and good biocompatibility
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Embodiment
[0037] Synthesis of Embodiment 1 Fmoc-L-Asp(OH)-AFC
[0038] Weigh Fmoc-L-Asp(OtBu)-OH (10mmol, 4.11g) and 7-amino-4-trifluoromethylcoumarin (10mmol, 2.29g) into a 50ml round bottom flask, add 40 Milliliters of anhydrous pyridine, under the condition of negative 15 degrees Celsius, was added dropwise 10 mmol of phosphorus oxychloride (3.06 g). Use nitrogen protection to avoid contact with moisture in the air, and react at a constant temperature of minus 15 degrees Celsius for 30 minutes. Add 3mol / L hydrochloric acid solution to the dark red viscous liquid until the pH of the solution=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. Purify by silica gel chromatography (ethyl acetate:petroleum ether=1:3). A total of 5.07 g of white solid Fmoc-L-Asp(OtBu)-AFC was obtained with a yield of 81.38%.
[0039] Weigh Fmoc-L-Asp(OtBu)-AFC (6.42mm...
Embodiment 2
[0041] Embodiment 2 Nap-GFF P YDEVD-AFC and Nap-GFF P Chemical Synthesis of YIETD-AFC
[0042] Synthesis of Nap-GFF by Solid Phase Synthesis P YDEVD-AFC and Nap-GFF P YIETD-AFC, the specific synthesis steps are as follows:
[0043] (1) Weigh 0.75 mmol (700 mg) of dichloro resin, add it to a solid-phase synthesizer, add 20 ml of dichloromethane (DCM) to immerse the resin, swell the resin for 20 minutes, remove the DCM solvent, add 1 mmol (566 mg) of Fmoc -L-Asp(OH)-AFC solution in DCM, add 800 microliters of N,N-diisopropylethylamine (DIPEA). React for 2 hours. Then wash five times with DCM for 2 minutes each. Use a blocking solution with a volume ratio of DCM:methanol:DIPEA=17:2:1 to block for 30 minutes. Then wash with DCM solution 5 times, each time with 20 ml of DCM solution, for 1 minute each time. Then wash with DMF solution 5 times, each time with 20 ml of DMF solution, each time for 2 minutes.
[0044] (2) Add 20 ml of 20% piperidine in DMF, react for 30 minute...
Embodiment 3
[0051] Example Three Small Molecule Hydrogel Nap-GFF P YDEVD-AFC and Nap-GFF P Formation of YIETD-AFC
[0052] The formation steps of small molecule hydrogel are as follows:
[0053] (1) Weigh 2.00 mg of the obtained polypeptide compound, add 1 ml of PBS (pH=7.4 buffer solution) and adjust the pH to 7.4 with sodium carbonate to form a probe solution. Then add alkaline phosphatase solution (the final concentration of the enzyme is 2.0U / ml), after standing overnight, you can see a jelly-like colloid by inverting the vial, which is to obtain Nap-GFF P YDEVD-AFC and Nap-GFF P YIETD-AFC gel-like substance.
[0054] (2) Use a pipette gun to absorb about 15 microliters of colloid and drop it onto a silicon wafer with a clean surface, spin coating to make the colloid evenly distributed on the silicon wafer, and observe the morphology with a transmission electron microscope. For details, see Figure 5 ; Its morphology is about 10nm nanofibers dispersed uniformly.
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