CN103030682A - Enzyme-sensitive supramolecular hydrogel nano material, gelator and preparation method of gelator - Google Patents
Enzyme-sensitive supramolecular hydrogel nano material, gelator and preparation method of gelator Download PDFInfo
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Abstract
The invention discloses an enzyme-sensitive supramolecular hydrogel nano material which is easy to synthesize, a gelator and a preparation method of the gelator. The enzyme-sensitive supramolecular hydrogel nano material is characterized in that the gelator is synthesized by simple solid-phase polypeptide synthesis and conventional liquid-phase condensation reaction; gelatinization and gel dissolution are controlled in a manner of regulating pH; and meanwhile, the gelator comprises an oligopeptide sequence for identifying specificity of caspase 3, and thus, the gelator can be cut by the caspase 3 generated in the cell apoptosis process and survival time of cells can be prolonged. Compared with a conventional supramolecular hydrogel nano material, the enzyme-sensitive supramolecular hydrogel nano material disclosed by the invention has the advantages that complex reaction conditions are not required, the gelator is simple to synthesize and the gelatinization condition is easy to control; and the enzyme-sensitive supramolecular hydrogel nano material is sensitive to the specific caspase 3 generated in the cell apoptosis process, has excellent biocompatibility for cells and can be used for detecting activity of a specific enzyme related to apoptosis on the living cell plane and assisting cells to live in the cell culture.
Description
Technical field
The invention belongs to the supramolecular hydrogel technical field of nano material, be specifically related to can be used for the supramolecular hydrogel of enzyme sensitivity of helper survival and gelator and preparation method thereof.
Background technology
The oligopeptides of fluorenylmethyloxycarbonyl protection once saw magazine " the american Journal of the Chemical Society " (J.Am.Chem.Soc. of American Chemical Society as the supramolecular hydrogel of gelator; 2009; 131; report 11286-11287); this technology has been used the one-tenth glue process that Phosphoric acid esterase is regulated; bioprocess is integrated wherein, but further do not detected this material in the Biology Applications of active somatic cell aspect, having no relevant report aspect biocompatibility and the degradation property.The applied research of Caspase-3 in the supramolecular hydrogel degradation process sees magazine " chip lab " (the Lab Chip of RSC, 2010,10, report 1946-1951), wherein employed supramolecular hydrogel is formed as the monomer polymerization reaction take place by the polyoxyethylene glycol of acroleic acid esterification and the polypeptide fragment of corresponding modification, relate to numerous compounds, building-up process is complicated and wayward, although studied the degradation property of formed supramolecular hydrogel in the literary composition, had no the report of its biocompatibility.
The oligopeptides of the fluorenylmethyloxycarbonyl protection that employing can be degraded by Caspase-3 is as the supramolecular hydrogel of gelator, and is active and the research work of the biocompatibility aspect of cell be it is not yet seen bibliographical information for detection of the relevant certain enzyme of apoptosis.
Summary of the invention
The objective of the invention is to propose the gelator and preparation method thereof of the supramolecular hydrogel nano material of class of enzymes sensitivity, to obtain the supramolecular hydrogel nano material of enzyme sensitivity, overcome the complicated and uppity shortcoming of existing relevant supramolecular hydrogel material preparation process, thus can be further in the active and helper survival in cell cultures for detection of the relevant certain enzyme of apoptosis of active somatic cell aspect.
The preparation method of the gelator of the supramolecular hydrogel nano material of enzyme sensitivity of the present invention is characterized in that:
Synthesize respectively first two sections oligopeptides sequences, press: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the protecting group of glycine, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 4th amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acid of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, cut the protecting group of aspartic acid, the 1.6 mmole five amino acid α-amino-isovaleric acids that add activation reacted 2-3 hour, cut the protecting group of α-amino-isovaleric acid, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, cut the protecting group of last amino acid aspartic acid, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic peptide section from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation is also toppled over and is removed the upper strata ether, and gained white solid powder was peptide sequence after the ether volatilization was done---Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid; Press again: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the protecting group of glycine, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 4th the amino acid α-amino-isovaleric acid of 1.6 mmoles that adds activation reacted 2-3 hour, cut the protecting group of α-amino-isovaleric acid, 1.6 mmole five amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of adding activation-4-tert-butyl ester reacted 2-3 hour, cut the protecting group of aspartic acid, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, cut the protecting group of last amino acid aspartic acid, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic peptide section from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation and the upper strata ether that inclines, gained white solid powder was the oligopeptides sequence after the ether volatilization was done---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid;
With above-mentioned synthetic oligopeptides sequence---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the first head product X
1
With above-mentioned synthetic peptide sequence---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the second head product X
2
The first head product X with above-mentioned preparation
1Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the first pure compound Y
1With the second head product X
2Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the second pure compound Y
2
Wherein the synthetic used amino acid of solid-phase peptide all is with 9-fluorenylmethyloxycarbonyl
As the alpha-amino group protecting group, and the side chain carboxyl group of aspartic acid and L-glutamic acid is by 4-tert-butyl ester base
Protection; Wherein the reagent of activated amino acid is I-hydroxybenzotriazole and the benzotriazole-N with the amino acid equimolar amount, N, N ', N '-tetramethyl-urea hexafluorophosphate; When whenever connecting an amino acid and cutting the 9-fluorenylmethyloxycarbonyl protecting group, all adopt Caesar to test (Kaiser Test) reagent and detect amino whether the existence: if positive, aobvious blueness namely shows to have removed the 9-fluorenylmethyloxycarbonyl protecting group; If negative, displaing yellow shows that then amino acid connects.
Above-mentioned synthetic can be used for detects the active gelator that cell is had the supramolecular hydrogel nano material of good biocompatibility, i.e. the first pure compound Y of reaching of the relevant certain enzyme of apoptosis
1With the second pure compound Y
2, it is characterized in that structure is respectively:
The first pure compound Y wherein
1Can be by Caspase-3 specific recognition and the compound of shearing, the second pure compound Y
2Thereby be to be identified the control compound that can not be sheared by Caspase-3, their differences structurally only are that aspartic acid is different from the position of α-amino-isovaleric acid in the oligopeptides sequence.
The supramolecular hydrogel nano material of another kind of product enzyme sensitivity of the present invention is characterized in that being adjusted to glue according to following proportioning: with the first pure compound Y of 1 milligram
1The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the first pure compound Y
1Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material; The second pure compound Y with 1 milligram
2The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the second pure compound Y
2Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material.
Compare with traditional supramolecular hydrogel nano material, the remarkable advantage of the responsive supramolecular hydrogel nano material of enzyme of the present invention is that gelator is synthetic simple, become the adhesive tape part to control easily, responsive to the certain enzyme that produces in the apoptosis process simultaneously, can be used for detecting the active of certain enzyme and to the Study on biocompatibility of cell; Gelator of the present invention is synthetic by the synthetic and conventional liquid phase carboxylic ammonia condensation reaction of simple solid-phase polypeptide, wetting ability is fine, preparation easily, can be controlled to glue and colloidal sol by the mode of regulating pH, because gelator contains the oligopeptides sequence of Caspase-3 specific recognition, responsive to the Caspase-3 that apoptosis process produces simultaneously, can be sheared by this enzyme, stop cell to enter apoptosis program, prolong the survival time of cell.Therefore, supramolecular hydrogel nano material of utilizing enzyme sensitivity of the present invention and preparation method thereof can realize forming by the simple method synthesized gel rubber factor and control gelator self-assembly the supramolecular hydrogel nano material of enzyme sensitivity, finished simultaneously gelator for detection of the active of certain enzyme and to the research of the biocompatibility of cell.The inventive method has overcome traditional supramolecular hydrogel nano material need to pass through the shortcoming of the complicated chemical reaction synthesized gel rubber factor, and has realized that further detecting the relevant certain enzyme activity of apoptosis in the active somatic cell aspect reaches helper survival in cell cultures.
Description of drawings
Fig. 1 is the first pure compound Y synthetic among the embodiment 1
1The hydrogen nuclear magnetic resonance spectrogram;
Fig. 2 is the first pure compound Y synthetic among the embodiment 1
1Carbon-13 nmr spectra figure.
Fig. 3 is the second pure compound Y synthetic among the embodiment 1
2The hydrogen nuclear magnetic resonance spectrogram;
Fig. 4 is the second pure compound Y synthetic among the embodiment 1
2Carbon-13 nmr spectra figure.
Fig. 5 is the first pure compound Y among the embodiment 1
1The dynamic frequency scanning collection of illustrative plates of the storage modulus of the supramolecular hydrogel nano material that forms (G ') and loss modulus (G ");
Fig. 6 is the second pure compound Y among the embodiment 1
2The dynamic frequency scanning collection of illustrative plates of the storage modulus of the supramolecular hydrogel nano material that forms (G ') and loss modulus (G ").
Fig. 7 is the first pure compound Y among the embodiment 1
1The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes;
Fig. 8 is the second pure compound Y among the embodiment 1
2The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes.
Fig. 9 is the first pure compound Y among the embodiment 2
1Before and after finishing the identification of enzyme and shear, external environment carries out the result of efficient liquid phase chromatographic analysis;
Figure 10 is the second pure compound Y among the embodiment 2
2Before and after finishing the identification of enzyme and shear, external environment carries out the result of efficient liquid phase chromatographic analysis.
Figure 11 is the first pure compound Y among the embodiment 3
1The survival rate statistical graph of HepG2 cell after under different concns, jointly hatching different time with the HepG2 cell;
Figure 12 is the second pure compound Y among the embodiment 3
2The survival rate statistical graph of HepG2 cell after under different concns, jointly hatching different time with the HepG2 cell.
Figure 13 is the first pure compound Y among the embodiment 3
1With the second pure compound Y
2Respectively after jointly hatching 72 hours with the HepG2 cell under 400 micro-molar concentrations poly-ADP-ribose polymerase of shearing substrate of the intracellular Caspase-3 of HepG2, corresponding Caspase-3 precursor, Caspase-3 and shear after the western blotting comparison diagram of poly-ADP-ribose polymerase, wherein negative control group is not for adding the blank group of above-mentioned two kinds of compounds, and positive controls adds the camptothecine of 10 micro-molar concentrations for not adding above-mentioned two kinds of compounds.
Embodiment
The first pure compound Y is provided in the following examples 1
1With the second pure compound Y
2Synthetic, embodiment 2 be the active test experience of vitro enzyme, embodiment 3 is that the cell biological consistency is tested.The first pure compound Y
1The compound that can be identified and shear by enzyme spcificity, the second pure compound Y
2Thereby be to be identified the control compound that can not be sheared by enzyme.
Embodiment 1:
Adopt first the solid phase method of peptide synthesis that Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid and Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-two oligopeptides fragments of aspartic acid are synthesized respectively.
The first pure compound Y in the present embodiment
1(Fmoc-EDA-Gly-Gly-Gly-Asp-Val-Glu-Asp-Acetyl) synthetic route is as follows:
Adopt first solid phase method of peptide synthesis synthetic oligopeptide sequence Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid, press: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, the 4th amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acid of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of aspartic acid, the Kaiser test is aobvious blue, the 1.6 mmole five amino acid α-amino-isovaleric acids that add activation reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of α-amino-isovaleric acid, the Kaiser test is aobvious blue, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of L-glutamic acid, the Kaiser test is aobvious blue, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of last amino acid aspartic acid, the Kaiser test is aobvious blue, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic oligopeptides sequence from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation is also toppled over and is removed the upper strata ether, and gained white solid powder was the oligopeptides sequence after the ether volatilization was done---Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid; With above-mentioned synthetic oligopeptides sequence---Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the first head product X
1The first head product X with above-mentioned preparation
1Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the first purified product Y
1
The second pure compound Y
2(Fmoc-EDA-Gly-Gly-Gly-Val-Asp-Glu-Asp-Acetyl) synthetic route is as follows:
Adopt again solid phase method of peptide synthesis synthetic oligopeptide sequence Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of glycine, the Kaiser test is aobvious blue, the 4th the amino acid α-amino-isovaleric acid of 1.6 mmoles that adds activation reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of α-amino-isovaleric acid, the Kaiser test is aobvious blue, 1.6 mmole five amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of adding activation-4-tert-butyl ester reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of aspartic acid, the Kaiser test is aobvious blue, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of L-glutamic acid, the Kaiser test is aobvious blue, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, Kaiser tests displaing yellow, cut the fluorenylmethyloxycarbonyl protecting group of last amino acid aspartic acid, the Kaiser test is aobvious blue, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic oligopeptides sequence from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation is also toppled over and is removed the upper strata ether, and gained white solid powder was the oligopeptides sequence after the ether volatilization was done---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid; Again with above-mentioned synthetic oligopeptides sequence---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the second head product X
2Again with the second head product X
2Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the second purified product Y
2
Adopt German Brooker company (bruker) Brooker nuclear-magnetism software to resolve two kinds of synthetic in present embodiment compounds and obtain as shown in Figures 1 to 4 two kinds of nmr spectrums:
Fig. 1 is the first pure compound Y synthetic in the present embodiment
1The hydrogen nuclear magnetic resonance spectrogram; Fig. 2 is the second pure compound Y
2The hydrogen nuclear magnetic resonance spectrogram.As seen from Figure 1, the first pure compound Y
1Proton nmr spectra (d
6-methyl-sulphoxide, 300MHz): 8.24-8.34 (d, 1H), 8.13-8.21 (d, 1H), 7.96-8.07 (t, 3H), 7.91-7.97 (d, 1H), 7.84-7.91 (d, 2H), 7.75-7.84 (t, 1H), 7.66-7.75 (d, 2H), 7.59-7.66 (s, 1H), 7.36-7.47 (t, 2H), 7.27-7.36 (t, 2H), 7.21-7.27 (t, 1H), 4.45-4.61 (m, 2H), 4.26-4.32 (d, 2H), 4.15-4.26 (d, 2H), 4.06-4.15 (t, 1H), 3.68-3.86 (d, 6H), 3.62-3.68 (d, 4H), 3.06-3.16 (t, 2H), 2.98-3.06 (t, 2H), 2.85-2.92 (s, 1H), 2.66-2.77 (m, 2H), 2.12-2.34 (m, 2H), 1.87-2.04 (m, 2H), (1.78-1.87 s, 3H), 1.66-1.78 (m, 1H), 0.72-0.91 (t, 6H); As seen from Figure 2, the first pure compound Y
1Carbon-13 nmr spectra (d
6-methyl-sulphoxide, 300MHz): 174.07,171.80,171.69,171.04,170.99,170.87,170.81,169.50,169.13,168.98,168.80,156.16,143.84 (2C), 140.69 (2C), 127.58 (2C), 127.04 (2C), 125.11 (2C), 120.08 (2C), 65.32,57.60,51.92,49.63,49.49,46.65,42.33,42.09,41.95,35.91,35.64,34.30,30.43,29.97,27.02,22.44,19.07,17.91 (2C).
As seen from Figure 3, the second pure compound Y
2Proton nmr spectra (d
6-methyl-sulphoxide, 300MHz): 8.21-8.29 (d, 2H), 8.16-8.21 (m, 1H), 8.07-8.16 (t, 2H), 7.92-7.99 (d, 2H), 7.89-7.93 (s, 1H), 7.80-7.89 (t.1H), 7.68-7.76 (d.2H), 7.61-7.68 (d, 1H), 7.41-7.50 (t, 2H), 7.33-7.49 (t, 2H), 7.26-7.33 (t, 1H), 4.51-4.69 (m, 2H), 4.31-4.39 (d, 2H), 4.22-4.30 (m, 2H), 4.13-4.21 (t, 1H), 3.74-3.92 (d, 6H), 3.67-3.75 (d, 4H), 3.11-3.19 (t, 2H), 3.02-3.11 (t, 2H), 2.77-2.84 (d, 1H), 2.70-2.78 (t, 1H), 2.64-2.70 (d, 1H), 2.20-2.34 (m, 2H), 1.09-2.11 (m, 2H), (1.85-1.90 s, 3H), 1.69-1.85 (m, 1H), 0.77-0.95 (t, 6H); As seen from Figure 4, the second pure compound Y
2Carbon-13 nmr spectra (d
6-methyl-sulphoxide, 300MHz): 174.06,171.93 (2C), 171.17,171.09,170.86,170.42,169.71,169.29,169.14,168.94,156.29,143.95 (2C), 140.86 (2C), 127.66 (2C), 127.15 (2C), 125.22 (2C), 120.14 (2C), 65.43,57.89,51.94,49.68,49.54,46.82,42.21,42.08,42.04,35.97,35.60,30.75,30.45,29.88,27.34,22.54,19.20,17.89 (2C).
The first pure compound Y
1The compound that can be identified and shear by enzyme spcificity, the second pure compound Y
2Thereby be to be identified the control compound that can not be sheared by enzyme; The first pure compound Y
1With the second pure compound Y
2All become glue to obtain the supramolecular hydrogel nano material for pH regulator, in external test experience and the helper survival experiment of carrying out corresponding enzymic activity.
The supramolecular hydrogel nano material of enzyme sensitivity of the present invention is adjusted to glue according to following proportioning: with the first pure compound Y of 1 milligram
1The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the first pure compound Y
1Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material; The second pure compound Y with 1 milligram
2The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the second pure compound Y
2Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material.In the aforementioned one-tenth glue process, the use of deionized water be in the water other ions and impurity for the impact that becomes glue, the use of concentrated hydrochloric acid is to add hydrochloric acid to the impact of liquor capacity in order to reduce, and uses in principle the original-pack analytical pure concentrated hydrochloric acid that dispatches from the factory of not diluted.
The material that adopts aforesaid method to prepare except being used for the gel rheology property detection of supramolecular hydrogel material, comprises that also the microscopic appearance of gel is observed.
Fig. 5 is the first pure compound Y in the present embodiment
1The dynamic frequency scanning collection of illustrative plates of the storage modulus of the supramolecular hydrogel nano material that forms (G ') and loss modulus (G "); Fig. 6 is the second pure compound Y in the present embodiment
2The dynamic frequency scanning collection of illustrative plates of the storage modulus of the supramolecular hydrogel nano material that forms (G ') and loss modulus (G ").Fig. 7 has provided the first pure compound Y in the present embodiment
1The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes; Fig. 8 has provided the second pure compound Y in the present embodiment
2The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes.
Wherein Fig. 5 is the dynamic frequency scanning collection of illustrative plates of the storage modulus (G ') of supramolecular hydrogel nano material that the inventive method preparation is formed and loss modulus (G "); can be found out with the square curve a of frequency change and the loss modulus circle point curve b with frequency change by storage modulus among the figure; adopt the storage modulus (G ') of the supramolecular hydrogel nano material of the inventive method preparation to be approximately five times of loss modulus (G "), show that resulting materials is the supramolecular hydrogel material.Fig. 6 is the second pure compound Y in the present embodiment
2The dynamic frequency scanning collection of illustrative plates of the storage modulus of the supramolecular hydrogel nano material that forms (G ') and loss modulus (G "); can be found out with the square curve c of frequency change and the loss modulus circle point curve d with frequency change by storage modulus among the figure; its storage modulus (G ') is approximately five times of loss modulus (G "), shows that resulting materials is the supramolecular hydrogel material.Fig. 7 has provided the first pure compound Y in the present embodiment
1The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes, and shows that gained supramolecular hydrogel material is that the nanofiber of 6.1 ± 1.2 nanometers forms by diameter; Fig. 8 has provided the second pure compound Y in the present embodiment
2The freezing transmission electron microscope of the supramolecular hydrogel nano material that forms characterizes, and shows that gained supramolecular hydrogel material is that the nanofiber of 7.7 ± 2.2 nanometers forms by diameter.
Embodiment 2: the active test experience of vitro enzyme
Adopt concentration to be the first pure compound Y of every liter of 0.28 mmole in the present embodiment experiment in vitro
1Containing the 4-hydroxyethyl piperazine ethanesulfonic acid that concentration is every liter of 50 mmole, 0.1% 3-[(3-cholesterol aminopropyl) dimethylamino]-cumulative volume of the Caspase-3 that the ethylenediamine tetraacetic acid (EDTA) of every liter of sodium-chlor, 10 mmole of every liter of 1-propanesulfonic acid, 50 mmoles and 5 unit volumes are 50 microlitres is in the solution of 60 microlitres, hatches identification and the shearing of finishing enzyme in 100 minutes under 37 degrees centigrade; Adopt concentration to be the second pure compound Y of every liter of 0.28 mmole
2Containing the 4-hydroxyethyl piperazine ethanesulfonic acid that concentration is every liter of 50 mmole, 0.1% 3-[(3-cholesterol aminopropyl) dimethylamino]-cumulative volume of the Caspase-3 that the ethylenediamine tetraacetic acid (EDTA) of every liter of sodium-chlor, 10 mmole of every liter of 1-propanesulfonic acid, 50 mmoles and 5 unit volumes are 50 microlitres is in the solution of 60 microlitres, hatches identification and the shearing of finishing respectively enzyme in 100 minutes under 37 degrees centigrade.
Fig. 9 has provided the first pure compound Y in the present embodiment
1After external environment is finished the identification of enzyme and sheared, carry out high performance liquid chromatography and follow the tracks of the characterization result that detects; Figure 10 is the second pure compound Y in the present embodiment
2After external environment is finished the identification of enzyme and sheared, carry out high performance liquid chromatography and follow the tracks of the characterization result that detects.
Wherein Fig. 9 is the first pure compound Y to the inventive method preparation
1Carry out high performance liquid chromatography and follow the tracks of the characterization result that detects before and after external environment is finished the identification of enzyme and sheared, curve e is the first pure compound Y
1Do not hatch the stratographic analysis result who obtains, its chromatographic peak e with Caspase-3
1Namely represent the first pure compound Y
1, curve f is the first pure compound Y
1Hatch the stratographic analysis result who obtains, its medium and small chromatographic peak f with Caspase-3
1Chromatographic peak e with curve e
1Identical retention time is arranged, namely represent the first pure compound Y
1Still have remnants after identification is sheared through enzyme, and emerging large chromatographic peak f
2Then have shorter retention time, detect through the ground substance assistant laser flight time mass spectrum that dissociates simultaneously, it really is the first pure compound Y
1Fragment by after the enzyme identification shearing shows the first pure compound Y
1In external environment, can be identified and shear by Caspase-3; Figure 10 is the second pure compound Y to the inventive method preparation
2Carry out high performance liquid chromatography and follow the tracks of the characterization result that detects before and after external environment is finished the identification of enzyme and sheared, curve g is the second pure compound Y
2Do not hatch the stratographic analysis result who obtains, its chromatographic peak g with Caspase-3
1Namely represent the second pure compound Y
2, curve h is the second pure compound Y
2Hatch the stratographic analysis result who obtains, its chromatographic peak h with Caspase-3
1Chromatographic peak g with curve g
1Identical retention time is arranged, namely represent the second pure compound Y
2Still exist after shearing through enzyme identification, detect through the ground substance assistant laser flight time mass spectrum that dissociates simultaneously, it really is the second pure compound Y
2, do not sheared by enzyme identification, show the second pure compound Y
2In external environment, can not be identified and shear by Caspase-3.
Embodiment 3: the experiment of cell biological consistency
At first carry out the cultivation of human liver cancer cell HepG2: be 5%CO containing volumetric concentration
237 degrees centigrade of incubators of air ambient in use the DMEM culture medium culturing HepG2 cell lines of the bovine serum albumin that contains volumetric concentration 10%; Be in the cell three times of logarithmic phase with 0.01 mole every liter aseptic PBS damping fluid washing, then the trypsinase with mass body volume concentrations 0.25% digests it get off; Divide the DMEM rinse of dish cell, then count, and cell concn is diluted to every milliliter 3 * 10
4Individual cell.
Then use human liver cancer cell HepG2 kind plate: HepG2 cell kind is entered in the microwell plate in 96 holes every hole 3 * 10
3Individual cell is 5%CO containing volumetric concentration
237 degrees centigrade of incubators of air ambient in use the DMEM culture medium culturing 24 hours of the bovine serum albumin that contains volumetric concentration 10%.
Test respectively the first pure compound Y
1With the second pure compound Y
2Biocompatibility to human liver cancer cell HepG2: remove carefully the substratum in the microwell plate, adding freshly prepared volume in the hole respectively is 100 microlitres, and concentration is the first pure compound Y of every liter of 50 every liter of micromole, 100 every liter of micromole, 200 every liter of micromole and 400 micromole
1Serial dilutions; Do not adding the first pure compound Y
1In the hole of processing, remove carefully the substratum in the microwell plate, adding freshly prepared volume in the hole respectively is 100 microlitres, and concentration is the second pure compound Y of every liter of 50 every liter of micromole, 100 every liter of micromole, 200 every liter of micromole and 400 micromole
2Serial dilutions; The diluent of each concentration is done 9 hole parallel laboratory tests, does not add compound in the blank group and adds the aseptic PBS damping fluid of 100 microlitres and organize in contrast; Then be 5%CO containing volumetric concentration
237 degrees centigrade of incubators of air ambient in cultivate, make the compound effects time of each concentration all comprise three time spans, namely act on 24 hours 3 holes, 48 hours 3 holes and 72 hours 3 holes, amount to 9 holes; For the hole of finishing action time, adding volume is that 10 microlitres, concentration are the PBS solution of 5 milligrams every milliliter MTT, continues to cultivate 4 hours; Remove again substratum, and Xiang Kongzhong adds respectively the first Za crystal of PBS solution to generate in the dissolving hole of 100 microlitre 10%SDS; Record the absorption intensity of dissolving De Jia Za crystal in wavelength 570 nanometers and 680 nanometers afterwards.
Cell survival rate is calculated by following formula:
The absorption intensity that the absorption intensity of the absorption intensity of cell survival rate (%)=dosing group * 100/ control group, the absorption intensity here are corresponding 570 nanometers deducts the absorption intensity of 680 nanometers.
At last, test the content situation of the apoptosis-related protein enzyme in the human liver cancer cell HepG2 after two kinds of compound effects, verify that two kinds of compounds are to the biocompatibility of cell: human liver cancer cell HepG2 is divided into four groups, except negative control group did not add compound, its excess-three group added respectively the first pure compound Y of every liter of ultimate density 400 micromole
1Cultivate the second pure compound Y of every liter of 72 hour, ultimate density 400 micromoles
2Cultivating the camptothecine of every liter of 72 hour, ultimate density 10 micromoles cultivated 6 hours; After the end, with the cultured human liver cancer cell HepG2 of aseptic PBS buffered soln rinse once, with the abundant suspendible of freezing lysis buffer that contains the mixed protein enzyme inhibitors of 5 times of volumes, boil after 5 minutes and carry out sds polyacrylamide gel electrophoresis again, then change PVDF membrane over to; Film is placed among 4 ℃ the TBST that contains 5% skimmed milk after the incubated overnight with TBST rinse 20 minutes, then at room temperature hatched 1 hour with primary antibody; Gained marking figure is again with being compounded with the secondary antibody mark of alkaline phosphatase, uses at last the Western Blue for alkaline phosphatase of Promega company to stablize substrate and makes target protein visual.
Figure 11 is the first pure compound Y in the present embodiment
1The survival rate statistical graph of HepG2 cell after under different concns, jointly hatching different time with the HepG2 cell; Figure 12 is the second pure compound Y in the present embodiment
2The survival rate statistical graph of HepG2 cell after under different concns, jointly hatching different time with the HepG2 cell; Figure 13 is the first pure compound Y in the present embodiment
1With the second pure compound Y
2Respectively after jointly hatching 72 hours with the HepG2 cell under every liter of concentration of 400 micromoles the poly-ADP-ribose polymerase of the shearing substrate of the intracellular Caspase-3 of HepG2, Caspase-3 precursor, Caspase-3 and shear after the western blotting figure of poly-ADP-ribose polymerase, wherein negative control group is not for adding the blank group of above-mentioned two kinds of compounds, and positive controls adds the camptothecine group of every liter of concentration of 10 micromoles for not adding above-mentioned two kinds of compounds.
As shown in Figure 11, the top square curve i is the first pure compound Y
1Hatch 24 hours cell survival rate curve with human liver cancer cell HepG2, middle circle point curve j is the first pure compound Y
1Hatch 48 hours cell survival rate curve with human liver cancer cell HepG2, bottom trigonometric curve k is the first pure compound Y
1Hatch 72 hours cell survival rate curve with human liver cancer cell HepG2;
As shown in Figure 12, the top square curve l is the second pure compound Y
2Hatch 24 hours cell survival rate curve with human liver cancer cell HepG2, middle circle point curve m is the second pure compound Y
2Hatch 48 hours cell survival rate curve with human liver cancer cell HepG2, bottom trigonometric curve n is the second pure compound Y
2Hatch 72 hours cell survival rate curve with human liver cancer cell HepG2.According to Figure 11 and Figure 12, human liver cancer cell HepG2 with low concentration, namely two kinds of compounds of every liter of 50 every liter of micromole and 100 micromole were hatched 24 hours, the cell survival rate after 48 hours and 72 hours is all without significant difference.Comparatively speaking, human liver cancer cell HepG2 with the first pure compound Y of every liter of concentration 200 micromole
1Hatch together that cell survival rate has 96.3% after 72 hours, and human liver cancer cell HepG2 with the second pure compound Y of every liter of concentration 200 micromole
2Hatch together that cell survival rate is 82.0% after 72 hours, when compound concentration is brought up to every liter of 400 micromole, human liver cancer cell HepG2 with the first pure compound Y
1Hatch together that cell survival rate still has 85.9% after 72 hours, and human liver cancer cell HepG2 with the second pure compound Y
2Hatch together that cell survival rate drops to 49.1% after 72 hours.This comparative illustration the first pure compound Y
1With the second pure compound Y
2Compare and demonstrate lower cytotoxicity, be i.e. the first pure compound Y
1Cell is demonstrated good biocompatibility, the second pure compound Y
2In long-time situation of cultivating, cell is demonstrated lower biocompatibility.
As shown in figure 13, negative control group is listed as visible Caspase-3 precursor band o
1, the matrix poly of caspase (ADP-ribose) polysaccharase band o
2Reach the phosphoglyceraldehy-de dehydrogenase band o as interior reference
3The first pure compound Y
1Treatment group is listed as visible Caspase-3 precursor band p
1, caspase matrix poly (ADP-ribose) polysaccharase band p
2Reach the phosphoglyceraldehy-de dehydrogenase band p as interior reference
3The second pure compound Y
2Treatment group is listed as visible Caspase-3 precursor band q
1, the Caspase-3 band q after the shearing
2, the Caspase-3 band q after the shearing
3, caspase matrix poly (ADP-ribose) polysaccharase band q
4, poly (ADP-ribose) the polysaccharase band q after the shearing
5Reach the phosphoglyceraldehy-de dehydrogenase band q as interior reference
6The camptothecine treatment group is listed as visible Caspase-3 precursor band r
1, the Caspase-3 band r after the shearing
2, the Caspase-3 band r after the shearing
3, caspase matrix poly (ADP-ribose) polysaccharase band r
4, poly (ADP-ribose) the polysaccharase band r after the shearing
5Reach the phosphoglyceraldehy-de dehydrogenase band r as interior reference
6In these four groups, the interior reference phosphoglyceraldehy-de dehydrogenase band of each group is all suitable, illustrates that four groups of protein contents that cell extracts are suitable, can mutually compare between data; Negative control group only has matrix poly (ADP-ribose) the polysaccharase band of Caspase-3 precursor band, caspase and as the phosphoglyceraldehy-de dehydrogenase band of interior reference, as the contrast benchmark of cell normal growth; Matrix poly (ADP-ribose) the polysaccharase band of the visible Caspase-3 precursor of camptothecine treatment group band, the Caspase-3 band after shearing, the Caspase-3 band after shearing, caspase, poly (ADP-ribose) the polysaccharase band after shearing and as the phosphoglyceraldehy-de dehydrogenase band of interior reference, as the comparison model of cell apoptosis; The first pure compound Y
1Caspase-3 band after the treatment group row do not have to shear and poly (ADP-ribose) the polysaccharase band after the shearing, and its Caspase-3 precursor band p
1Caspase-3 precursor band o with negative control group
1Quite, the first pure compound Y is described
1The Caspase-3 precursor for the treatment of group cell is obviously activation not, does not produce the Caspase-3 of the shearing that can survey and poly (ADP-ribose) polysaccharase of shearing, cell normal growth; The second pure compound Y
2Treatment group is shown Caspase-3 band and poly (ADP-ribose) the polysaccharase band after the shearing after the obvious shearing and can be compared with camptothecine group respective strap, and its Caspase-3 precursor band q
1Caspase-3 precursor band r with the camptothecine group
1Quite, compare and will lack with front two groups Caspase-3 precursor band, simultaneously, its poly (ADP-ribose) polysaccharase band q
4Poly (ADP-ribose) polysaccharase band r with the camptothecine group
4Quite, compare and to lack with front two groups poly (ADP-ribose) polysaccharase band, the second pure compound Y is described
2The Caspase-3 precursor for the treatment of group cell has produced obvious activation, produces the Caspase-3 of shearing and poly (ADP-ribose) polysaccharase of shearing, and existing a considerable amount of cells enter apoptosis program.Above data show, than the second pure compound Y
2, the first pure compound Y
1Have better cell biological consistency, be more suitable for helper survival in cell cultures.
Result by above-described embodiment and test experience thereof is as can be known: the responsive supramolecular hydrogel nano material of enzyme of the present invention can be controlled to glue and colloidal sol by the mode of regulating pH, and gelator is synthetic by the synthetic and conventional liquid phase carboxylic ammonia condensation reaction of simple solid-phase polypeptide, wetting ability is good, preparation easily, simultaneously because gelator contains the oligopeptides sequence of Caspase-3 specific recognition, can be sheared by the Caspase-3 that apoptosis process produces, can prolong the survival time of cell.Gelator of the supramolecular hydrogel nano material of synthetic simple enzyme sensitivity of the present invention and preparation method thereof can overcome the complicated and uppity shortcoming of supramolecular hydrogel material preparation process, and cell is had good biocompatibility.To sum up, adopt the inventive method can synthesize by simple chemical reaction the gelator of supramolecular hydrogel nano material, obtain it to the biocompatibility of cell, and finish the active and helper survival in cell cultures of the relevant certain enzyme of detection apoptosis in the active somatic cell aspect.
Claims (4)
1. the preparation method of the gelator of the supramolecular hydrogel nano material of class of enzymes sensitivity is characterized in that:
Synthesize respectively first two sections oligopeptides sequences, press: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the protecting group of glycine, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 4th amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acid of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, cut the protecting group of aspartic acid, the 1.6 mmole five amino acid α-amino-isovaleric acids that add activation reacted 2-3 hour, cut the protecting group of α-amino-isovaleric acid, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, cut the protecting group of last amino acid aspartic acid, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic peptide section from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation is also toppled over and is removed the upper strata ether, and gained white solid powder was peptide sequence after the ether volatilization was done---Gly-Gly-Gly-aspartic acid-α-amino-isovaleric acid-L-glutamic acid-aspartic acid; Press again: with 1 mmole 2-chlorine trityl chloride resin at 2-3 milliliter N, in the dinethylformamide after swelling 4-8 minute, add 2 mmole N-fluorenylmethyloxycarbonyl-glycine, add again 2 mmole N, the N-diisopropylethylamine, react after 2-3 hour, with 100 microliter methanol reaction 5-10 minute, cut the protecting group of glycine, second amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 3rd amino acid glycine reactant of 1.6 mmoles of adding activation 2-3 hour, cut the protecting group of glycine, the 4th the amino acid α-amino-isovaleric acid of 1.6 mmoles that adds activation reacted 2-3 hour, cut the protecting group of α-amino-isovaleric acid, 1.6 mmole five amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of adding activation-4-tert-butyl ester reacted 2-3 hour, cut the protecting group of aspartic acid, the 6th amino acid N-fluorenylmethyloxycarbonyl-Pidolidone of 1.6 mmoles of adding activation-4-tert-butyl ester reacted 2-3 hour, 1.6 mmole seven amino acid N-fluorenylmethyloxycarbonyl-L-Aspartic acids of at last adding activation-4-tert-butyl ester reacted 3-4 hour, cut the protecting group of last amino acid aspartic acid, add 2-3 mmole aceticanhydride reaction 20-30 minute, be that the dichloromethane solution of 1% trifluoroacetic acid downcuts synthetic peptide section from this resin with volumetric concentration at last, make its Precipitation with ether, frozen centrifugation is also toppled over and is removed the upper strata ether, and gained white solid powder was the oligopeptides sequence after the ether volatilization was done---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid;
With above-mentioned synthetic oligopeptides sequence---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the first head product X
1
With above-mentioned synthetic peptide sequence---Gly-Gly-Gly-α-amino-isovaleric acid-aspartic acid-L-glutamic acid-aspartic acid is got 0.1 mmole and is dissolved in 2 milliliters of N, in the dinethylformamide, add 0.12 mmole N, the N-diisopropylethylamine, 0.12 mmole benzotriazole-N, N, N ', after N '-tetramethyl-urea hexafluorophosphate and 0.12 mmole I-hydroxybenzotriazole activate half an hour, add 0.12 mmole N-fluorenylmethyloxycarbonyl-quadrol, stirring at room reaction 2-3 hour, through the high performance liquid chromatography separating-purifying, be collected in the component that ultraviolet band 300 nanometers have characteristic absorbance, be the second head product X
2
The first head product X with above-mentioned preparation
1Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the first pure compound Y
1With the second head product X
2Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, through the high performance liquid chromatography separating-purifying, being collected in ultraviolet band 300 nanometers has the component of characteristic absorbance to be the second pure compound Y
2
Wherein the synthetic used amino acid of solid-phase peptide all is with 9-fluorenylmethyloxycarbonyl
As the alpha-amino group protecting group, and the side chain carboxyl group of aspartic acid and L-glutamic acid is by 4-tert-butyl ester base
Protection; Wherein the reagent of activated amino acid is I-hydroxybenzotriazole and the benzotriazole-N with the amino acid equimolar amount, N, N ', N '-tetramethyl-urea hexafluorophosphate; When whenever connecting an amino acid and cutting the 9-fluorenylmethyloxycarbonyl protecting group, all adopt Caesar to test (Kaiser Test) reagent and detect amino whether the existence: if positive, aobvious blueness namely shows to have removed the 9-fluorenylmethyloxycarbonyl protecting group; If negative, displaing yellow shows that then amino acid connects.
2. the gelator of two kinds of supramolecular hydrogel nano materials of the described method of claim 1 preparation, i.e. the first pure compound Y
1With the second pure compound Y
2, it is characterized in that structure is respectively:
The first pure compound Y wherein
1Can be by Caspase-3 specific recognition and the compound of shearing, the second pure compound Y
2Thereby be to be identified the control compound that can not be sheared by Caspase-3, their differences structurally only are that aspartic acid is different from the position of α-amino-isovaleric acid in the oligopeptides sequence.
3. the gelator with the supramolecular hydrogel nano material of claim 1 method preparation is the first pure compound Y
1The supramolecular hydrogel nano material of the enzyme sensitivity that is modulated into is characterized in that being adjusted to glue according to following proportioning: with the first pure compound Y of 1 milligram
1The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the first pure compound Y
1Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material.
4. the gelator with the supramolecular hydrogel nano material of claim 1 method preparation is the second pure compound Y
2The supramolecular hydrogel nano material of the enzyme sensitivity that is modulated into is characterized in that being adjusted to glue according to following proportioning: with the second pure compound Y of 1 milligram
2The resistivity that is dissolved in 100 microlitres be in the deionized water of 18.3 Europe rice after, add yellow soda ash pressed powder regulator solution pH to the second pure compound Y
2Dissolving forms colourless transparent solution fully, adds concentrated hydrochloric acid regulator solution pH to 1 again, and vibration solution makes it to mix, to forming the supramolecular hydrogel nano material.
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| CN113995849A (en) * | 2021-11-01 | 2022-02-01 | 东南大学 | Gel factor precursor and gel material for loading autophagy inhibitor and chemotherapeutic drug, preparation method and application |
| CN114948860A (en) * | 2022-05-06 | 2022-08-30 | 东南大学 | Supermolecule hydrogel nano material, gelator, preparation method and application thereof |
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