Preparation method, characterization method and intracellular non-equilibrium state construction method of polypeptide compounds

Abstract

The present application relates to a method for preparing a polypeptide compound, a characterization method and a method for constructing a non-equilibrium state in a cell, wherein the polypeptide compound preparation method of the present application uses amino acids for solid phase synthesis to prepare a tripeptide, which can enter the mitochondria in the cell and can control the non-equilibrium state in the cell by illumination. A peptide whose self-assembly state can be controlled by illumination is successfully synthesized from aromatic amino acids (tyrosine, tryptophan and phenylalanine substituted with phenyl para-nitro), lysine and spiropyran. In in vitro experiments, the present application determined that this type of polypeptide has different assembly states when there are different aromatic substituents. When conducting cell experiments, this type of polypeptide was successfully used to realize the construction of a light-controlled non-equilibrium state in the cell. Compared with the prior art, the present application uses light as the energy source for artificial non-equilibrium state in the cell for the first time, and successfully realizes the migration of peptides from mitochondria to the nucleus under non-equilibrium state.

Description

Technical Field

[0001] The present application relates to the technical field of intracellular non-equilibrium states, and in particular to a method for preparing and characterizing a polypeptide compound and a method for constructing an intracellular non-equilibrium state. Background Art

[0002] To date, most artificial intracellular assembly structures are thermodynamically controlled, that is, once the precursor molecules enter the cell, the resulting supramolecular structure is in thermodynamic equilibrium. This is in stark contrast to the natural assembly structures in living systems, which exist in a unique non-equilibrium state and require a continuous influx of energy to maintain life activities. This dynamic property provides spatiotemporal control over the formation of the cytoskeleton and allows for complex regulation of its biological functions. Therefore, the development of non-equilibrium assembly systems in cells can provide an ideal platform for understanding the basic principles that control life and serve as a stepping stone to synthetic life. In addition, non-equilibrium states will provide additional levels of control for intracellular assemblies and may endow them with new properties.

[0003] However, strategies to develop intracellular assembly systems that mimic the nonequilibrium features of natural biological assemblies are currently lacking. Summary of the invention

[0004] In view of this, the present application proposes a method for preparing a polypeptide compound, a method for characterizing the polypeptide compound, and a method for constructing a non-equilibrium state in a cell to solve the above problems.

[0005] According to one aspect of the present application, a method for preparing a polypeptide compound is provided, comprising the following preparation steps:

[0006] The first raw material is shown in Formula 1, the second raw material is shown in Formula 2, the first raw material and the second raw material are refluxed in ethanol, and purified by silica gel column chromatography to obtain a first product, as shown in Formula 3;

[0007] Formula 1:

[0008]

[0009] Formula 2:

[0010]

[0011] Formula 3:

[0012]

[0013] Adding any one of the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or the third raw material to the amino-functionalized resin, and sequentially performing a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection to obtain a second product; and the third raw material is as shown in Formula 4;

[0014] Formula 4:

[0015]

[0016] The structural formula of the Fmoc-Trp-OH is as follows:

[0017]

[0018] The structural formula of the Fmoc-Tyr-OH is as follows:

[0019]

[0020] The first product and PyBop are dissolved in DMF, iPrNEt2 is added and mixed, and then added to the second product, and reacted and washed in sequence to obtain a third product;

[0021] After the third product is mixed and shaken in a preset cleavage reagent, the filtrate is collected and concentrated under a N2 flow, and after adding cold Et2O, the suspension is centrifuged, and the corresponding solid is dissolved in a preset mixed solvent, and lyophilized and purified in sequence to obtain peptides 5, 6, and 7;

[0022] The structural formula of the peptide 5 is as follows:

[0023]

[0024] The structural formula of peptide 6 is as follows:

[0025]

[0026] The structural formula of the peptide 7 is as follows:

[0027]

[0028] In a possible implementation, the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials is added to an amino-functionalized resin, and a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection are sequentially performed to obtain a second product, including:

[0029] Dissolve Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials and PyBop in DMF, and add iPrNEt2 for activation;

[0030] The structural formula of the PyBop is as follows:

[0031]

[0032] After adding the activated Fmoc-Trp-OH, the Fmoc-Tyr-OH or any one of the third raw materials to the amino-functionalized resin, the amino-functionalized resin is washed with DMF and CH2Cl2 to perform a first round of N-terminal coupling, and a 20% piperidine DMF solution is added to cover the amino-functionalized resin, and the suspension is shaken to perform a first round of Fmoc deprotection;

[0033] The amino-functionalized resin is subjected to a second round of N-terminal coupling and a second round of Fmoc deprotection in the same manner as the first round of N-terminal coupling and the first round of Fmoc deprotection, respectively, to obtain a second product.

[0034] In a possible implementation, the first product and PyBop are dissolved in DMF, iPrNEt2 is added and mixed, and then added to the second product, and reacted and washed in sequence to obtain a third product, including:

[0035] Dissolving the first product and the PyBop in DMF, adding iPrNEt2, to obtain a first mixture;

[0036] The first mixture was added to the second product, and after shaking, the mixture was washed with DMF and CH2Cl2 to obtain a third product.

[0037] In a possible implementation, the preset cutting reagent is a mixture of TFA, H2O and TIS, and TFA:H2O:TIS=95:2.5:2.5.

[0038] In a possible implementation, the preset mixed solvent is H2O and MeCN, and H2O:MeCN=1:1.

[0039] According to another aspect of the present application, a method for characterizing a polypeptide compound is provided, which is used to characterize a polypeptide compound prepared by any of the above polypeptide compound preparation methods, comprising:

[0040] The peptides 5, 6 and 7 are tested by at least one of UV test, circular dichroism test, TEM test, particle size test and Zeta potential test.

[0041] In a possible implementation, when the peptides 5, 6, and 7 are tested by the UV test, the method includes:

[0042] preparing aqueous solutions of the peptides 5, 6 and 7 at different concentrations respectively;

[0043] After the aqueous solutions of the peptides 5, 6, and 7 with different concentrations are subjected to ultraviolet testing in a first preset wavelength band under dark conditions, they are illuminated in a second preset wavelength band and the aqueous solutions of the peptides 5, 6, and 7 with different concentrations are tested.

[0044] In a possible implementation, when the peptides 5, 6, and 7 are tested by UV testing, the method further includes:

[0045] Draw the self-assembly curve, and draw the ε curve according to the self-assembly formula to determine the self-assembly form.

[0046] In a possible implementation, the first preset wavelength band is 200 nm-600 nm, and the second preset wavelength band is 405 nm.

[0047] In another aspect, the present application provides a method for constructing a non-equilibrium state in a cell, which is implemented by using a polypeptide compound prepared by any of the methods for preparing a polypeptide compound described above, comprising:

[0048] adding any one of the peptides 5, 6, and 7 to the incubation matrix to obtain a peptide dilution solution;

[0049] The polypeptide dilution is added to the cells and incubated for a preset time to construct a non-equilibrium system in the cells.

[0050] Beneficial effects of this application:

[0051] The method for preparing a polypeptide compound of the present application uses amino acids for solid phase synthesis to prepare a tripeptide, which can enter the mitochondria in cells and control the non-equilibrium state in cells by illumination. A peptide whose self-assembly state can be controlled by illumination is successfully synthesized from aromatic amino acids (tyrosine, tryptophan, and phenylalanine substituted with phenyl para-nitro), lysine, and spiropyran. In in vitro experiments, the present application determined that this type of polypeptide has different assembly states when there are different aromatic substituents. When conducting cell experiments, this type of polypeptide was successfully used to achieve the construction of a light-controlled non-equilibrium state in cells. Compared with the prior art, the present application uses light as the energy source for artificial non-equilibrium state in cells for the first time, and successfully achieves the migration of peptides from mitochondria to the nucleus under non-equilibrium state.

[0052] Other features and aspects of the present application will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the present application and, together with the description, serve to explain the principles of the present application.

[0054] Figure 1 The peptide 5 compound of the present application example is shown 1 HNMR spectra;

[0055] Figure 2 The ESI-MS diagram of the peptide 5 compound of the present application example is shown;

[0056] Figure 3 The peptide 6 compound of the present embodiment is shown 1 HNMR spectra;

[0057] Figure 4 The ESI-MS diagram of the peptide 6 compound of the present application example is shown;

[0058] Figure 5 The peptide 7 compound of the present application example is shown 1 HNMR spectra;

[0059] Figure 6 The ESI-MS diagram of the peptide 7 compound of the present application example is shown;

[0060] Figure 7 The UV absorption results of peptides 5, 6, and 7 of the examples of the present application under dark conditions and light conditions are shown;

[0061] Figure 8 ε curves of peptides 5, 6, and 7 of the present application examples under light-free conditions and blue light conditions are shown;

[0062] Fig. 9 Shown are CD results of peptides 5, 6, and 7 of the present application examples before and after illumination;

[0063] Fig.10 The Zeta potential results of peptides 6, 7, and 5 of the present application example before and after illumination are shown;

[0064] Fig.11 Shown are the morphological images of peptide 5 of the present application example before and after illumination;

[0065] Fig.12 Shown are the morphological images of peptide 6 of the present application example before and after illumination;

[0066] Fig.13 Shown are the morphological images of peptide 7 of the present application example before and after illumination;

[0067] Fig.14Shown are the MTT structures of peptides 5, 6, and 7 of the present application examples;

[0068] Fig.15 The figure shows the mitochondrial staining result of peptide 6 of the present application example;

[0069] Fig.16 The figure shows the mitochondrial staining result of peptide 7 of the present application example;

[0070] Fig.17 The figure shows the mitochondrial staining result of peptide 5 of the present application example;

[0071] Fig.18 The figure shows the result of peptide 5 migrating from mitochondria to nuclei;

[0072] Fig.19 The figure shows the result of peptide 6 migrating from mitochondria to nuclei;

[0073] Fig. 20 The figure shows the result of peptide 6 migrating from mitochondria to nuclei. DETAILED DESCRIPTION

[0074] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0075] Among them, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application or simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0076] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0077] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0078] In addition, in order to better illustrate the present application, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0079] At present, synthetic supramolecular materials with equilibrium properties are usually developed using chemical dyes or light. Compared with the non-equilibrium system of chemical dyes, the light-mediated method provides additional space and time for system operation and does not produce toxic wastes often encountered in chemical dye systems, but it has not yet been implemented in a complex closed environment such as cells. This application develops a new strategy to construct non-equilibrium intracellular self-assembly, a method for intracellular peptide assembly that can transition from equilibrium to non-equilibrium state by light, and proves that the dynamic, non-equilibrium nature of peptide assembly can effectively regulate its localization in cells, and observes obvious migration behavior between mitochondria and cytoplasm / nucleus.

[0080] According to one aspect of the present application, a method for preparing a polypeptide compound is provided, comprising the following preparation steps:

[0081] S100, the first raw material is as shown in Formula 1, the second raw material is as shown in Formula 2, the first raw material and the second raw material are refluxed in ethanol, and purified by silica gel column chromatography to obtain a first product as shown in Formula 3;

[0082] Formula 1:

[0083]

[0084] Formula 2:

[0085]

[0086] Formula 3:

[0087]

[0088] In this step, firstly, the first product needs to be synthesized by reflux the first raw material (5 mmol) and the second raw material (5 mmol) in ethanol for 8 h. After the monitored reaction is completed, silica gel column chromatography is performed using dichloromethane / methanol (10 / 1, v / v) as a developing agent to obtain the first product.

[0089] S200, adding any one of the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or the third raw material to the amino-functionalized resin, and sequentially performing a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection to obtain a second product; and the third raw material is as shown in Formula 4;

[0090] Formula 4:

[0091]

[0092] The structural formula of the Fmoc-Trp-OH is as follows:

[0093]

[0094] The structural formula of the Fmoc-Tyr-OH is as follows:

[0095]

[0096] In this step, Fmoc-Trp-OH, Fmoc-Tyr-OH or the third raw material is used as the raw material, and after activation, it is added to the amino-functionalized resin and sequentially subjected to the first round of N-terminal coupling, the first round of Fmoc deprotection, the second round of N-terminal coupling and the second round of Fmoc deprotection to obtain the second product. Wherein, whether it is the first round of N-terminal coupling or the second round of N-terminal coupling, the completion of the coupling step is monitored by the chloramine test and repeated when necessary. And whether it is the first round of Fmoc deprotection or the second round of Fmoc deprotection, DMF (5x) and CH2Cl2 (5x) are used to wash the resin after Fmoc deprotection to remove residual impurities and obtain the second product.

[0097] S300, dissolving the first product and PyBop in DMF, adding iPrNEt2 and mixing, and then adding to the second product, reacting and washing in sequence to obtain a third product;

[0098] In this step, the first product obtained in step S100 is used as a raw material. After being activated by PyBop, iPrNEt2 is added, and then added to the second product and shaken well. The mixture is washed with DMF (3×) and CH2Cl2 (3×). The completion of the reaction is monitored by a chloramine test, and the test is repeated if necessary to obtain a third product.

[0099] S400, mixing and shaking the third product in a preset cleavage reagent, collecting the filtrate and concentrating it under a N2 flow, adding cold Et2O, centrifuging the suspension, dissolving the corresponding solid in a preset mixed solvent, and lyophilizing and purifying it in sequence to obtain peptides 5, 6, and 7;

[0100] The structural formula of the peptide 5 is as follows:

[0101]

[0102] The structural formula of peptide 6 is as follows:

[0103]

[0104] The structural formula of the peptide 7 is as follows:

[0105]

[0106] In this step, the third product resin obtained in step S300 is shaken and mixed in a cutting reagent, and the filtrate is collected and concentrated under a N2 flow. After adding cold Et2O, the suspension is centrifuged, and the supernatant is poured out and discarded. The remaining solid is then dissolved in a preset mixed reagent, lyophilized to obtain a yellow powder, and purified by semi-preparative high performance liquid chromatography to obtain the final peptide compound, including peptide 5, peptide 6 and peptide 7.

[0107] In a possible implementation, the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials is added to an amino-functionalized resin, and a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection are sequentially performed to obtain a second product, including: dissolving Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials and PyBop in DMF, and adding iPrNEt2 for activation;

[0108] The structural formula of the PyBop is as follows:

[0109]

[0110] After adding the activated Fmoc-Trp-OH, the Fmoc-Tyr-OH or any one of the third raw materials to the amino-functionalized resin, the amino-functionalized resin is washed with DMF and CH2Cl2, and a first round of N-terminal coupling is performed, and a 20% piperidine DMF solution is added to cover the amino-functionalized resin, and the suspension is shaken to perform a first round of Fmoc deprotection; the amino-functionalized resin is subjected to a second round of N-terminal coupling and a second round of Fmoc deprotection in the same manner as the first round of N-terminal coupling and the first round of Fmoc deprotection to obtain a second product.

[0111] Specifically, after the first product is generated, Fmoc-Trp-OH or Fmoc-Tyr-OH or the third raw material (3 equivalents) and PyBop (2.9 equivalents) are dissolved in a small amount of DMF, and iPrNEt2 (6 equivalents) are added. After 2 minutes, the activated Fmoc-Trp-OH or Fmoc-Tyr-OH or the third raw material is added to the amino-functionalized resin (same as the load of the resin). The resin is shaken for 2 hours and then washed with DMF (3x) and CH2Cl2 (3x). The completion of the coupling step is monitored by the chloramine test and repeated if necessary.

[0112] Further, Fmoc deprotection was performed by covering the resin with 20% piperidine in DMF and shaking the suspension for 10 min. This step was repeated and the resin was then washed with DMF (5x) and CH2Cl2 (5x).

[0113] Furthermore, a second round of N-terminal coupling was performed in the same manner as the first round of N-terminal coupling, i.e., by dissolving Fmoc-Trp-OH or Fmoc-Tyr-OH or 4 (3 equiv.) and PyBop (2.9 equiv.) in a small amount of DMF and adding iPrNEt2 (6 equiv.). After 2 minutes, the mixture was added to the resin, shaken for 2 hours, and then washed with DMF (3×) and CH2Cl2 (3×). The completion of the reaction was monitored by the chloramine test and repeated if necessary.

[0114] Further, a second round of Fmoc deprotection was performed in the same manner as the first round of Fmoc deprotection, i.e., 20% piperidine DMF solution was added to cover the resin and the suspension was shaken for 10 minutes. This step was repeated, and then the resin was washed with DMF (5x) and CH2Cl2 (5x) to obtain the second product.

[0115] In one possible implementation, the first product and PyBop are dissolved in DMF, iPrNEt2 is added and mixed, and then added to the second product, and reacted and washed in sequence to obtain a third product, including: dissolving the first product and PyBop in DMF, adding iPrNEt2 to obtain a first mixture; adding the first mixture to the second product, shaking well, and washing with DMF and CH2Cl2 to obtain a third product.

[0116] Specifically, the first product (3 equiv.) and PyBop (2.9 equiv.) were dissolved in a small amount of DMF, and iPrNEt2 (6 equiv.) was added to obtain a first mixture. After 2 minutes, the first mixture was added to the resin, shaken for 2 hours, and then washed with DMF (3×) and CH2Cl2 (3×). The completion of the reaction was monitored by the chloramine test, and the test was repeated if necessary to obtain the third product.

[0117] Subsequently, the third product resin is shaken in a preset cleavage reagent for 2.5 hours. In one possible implementation, the preset cleavage reagent is a mixture of TFA, H2O and TIS, and TFA:H2O:TIS=95:2.5:2.5. The filtrate is collected and concentrated under a stream of N2. After adding cold Et2O, the suspension is centrifuged, the supernatant is poured out and discarded. The remaining solid is dissolved in a preset mixed solvent. In a possible implementation, the preset mixed solvent is H2O and MeCN, and H2O:MeCN=1:1. After freeze-drying, a yellow powder is obtained, which is purified by semi-preparative high performance liquid chromatography to finally obtain peptides 5, 6, and 7.

[0118] like Figure 1 As shown, the peptide 5 compound is shown 1 HNMR spectrum. 1 H NMR (600MHz, DMSO-d6) δ8.25-8.19(m,2H),8.14-8.12(d,J=8.5Hz,2H),7.96-7.91(m,2H),7.70-7.58(m,4H),7.51-7.49(d,J=8.1Hz, 2H),7.44-7.42(d,J=8.3Hz,2H),7.21(s,1H),7.14-7.10(m,1H),7.07-7.05(dd,J=10.4,2.8Hz,1H),6.80-6.74(m,2H),6.60-6.58(d ,J=7.7Hz,1H),5.87-5.85(d,J=10.2Hz,1H),4.60-4.49(m,2H),4.32-4.30(m,1H),3.18-3.11(m,3H),2.97-2.88(m,3H),2.74-2.67( m,4H),2.62-2.61(p,J=1.9Hz,1H),2.52-2.49(m,1H),2.39-2.38(m,1H)1.60-1.45(m,5H),1.23-1.21(m,5H),1.05(d,J=2.3Hz,3H).

[0119] like Figure 2As shown, it is the ESI-MS diagram of peptide 5 compound, the corresponding molecular ion peak is 411.2113, and the relative molecular mass is 411.2113.

[0120] like Figure 3 As shown, the peptide 6 compound is shown 1 HNMR spectrum. 1 H NMR(600MHz,DMSO-d6)δ9.16-9.11(d,J=28.3Hz,2H),8.25-8.24(d,J=7.8Hz,1H),7 .87-7.85(m,1H),7.75-7.74(d,J=2.2Hz,1H),7.67-7.61(m,3H),7.26(d,J=2.6Hz, 1H),7.14-7.05(m,3H),6.99-6.94(m,4H),6.80-6.76(m,2H),6.64-6.54(m,4H),5. 85(d,J=10.2Hz,1H),4.39-4.31(m,3H),2.88-2.83(m,2H),2.76-2.61(m,6H),1.68 -1.60(m,2H),1.52-1.48(m,2H),1.32-1.29(m,1H),1.22(s,4H),1.11(s,3H).

[0121] like Figure 4 As shown, it is the ESI-MS diagram of peptide 6 compound, the corresponding molecular ion peak is 388.1958, and the relative molecular mass is 388.1958.

[0122] like Figure 5 As shown, the peptide 7 compound is shown 1 HNMR spectrum. 1H NMR (600MHz, DMSO-d6) δ10.79-10.78(dd,J=6.6,2.4Hz,2H),8.23-8.22(d,J=7.6Hz,1H),8.02-8.01(d,J=7.6Hz,1H),7.88- 7.87(d,J=8.0Hz,1H),7.72-7.52(m,7H),7.32-7.27(m,3H),7.13-7.07(m,8H),6.97-6.91(m,2H),6.80-6.74(m,2H),6.58( d,J=7.7Hz,1H),5.84(d,J=10.2Hz,1H),4.54-4.46(m,2H),4.36-4.33(m,1H),3.13-3.09(m,2H),3.01-2.94(m,2H),2.73-2 .69(q,J=6.8Hz,2H),2.65(d,J=3.5Hz,3H),1.66-1.59(m,2H),1.50-1.46(q,J=6.7Hz,3H),1.29-1.21(m,6H),1.11(s,3H).

[0123] like Figure 6 As shown, it is the ESI-MS diagram of peptide 7 compound, the corresponding molecular ion peak is 417.1859, and the relative molecular mass is 417.1859.

[0124] According to another aspect of the present application, a method for characterizing a polypeptide compound is provided, which is used to characterize a polypeptide compound prepared by any of the above polypeptide compound preparation methods, comprising:

[0125] The peptides 5, 6 and 7 are tested by at least one of UV test, circular dichroism test, TEM test, particle size test and Zeta potential test.

[0126] The polypeptide compound characterization method of the present application characterized peptides 5, 6, and 7, and characterized the polypeptide compounds in vitro through UV testing, circular dichroism testing, and TEM testing, and determined that this type of polypeptide can form a non-equilibrium system when light is used as an energy source.

[0127] In a possible implementation, when the peptides 5, 6, and 7 are tested using the ultraviolet test, the method includes: preparing aqueous solutions of the peptides 5, 6, and 7 of different concentrations respectively; conducting an ultraviolet test of the aqueous solutions of the peptides 5, 6, and 7 of different concentrations in a first preset band under dark conditions, irradiating the solutions with light under a second preset band, and testing the aqueous solutions of the peptides 5, 6, and 7 of different concentrations.

[0128] Specifically, aqueous polypeptide solutions with concentrations of 1000 μM, 900 μM, 850 μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 50 μM, 25 μM and 1 μM are prepared respectively; then, the aqueous polypeptide solutions with different concentrations are subjected to UV testing under dark conditions, wherein the wavelength band during the UV testing is preferably 200 nm-600 nm, and then the peptide solutions with different concentrations are tested after irradiation at 405 nm.

[0129] In a possible implementation, when the peptides 5, 6, and 7 are tested by UV testing, the method further includes: drawing a self-assembly curve, and drawing an ε curve according to a self-assembly formula to determine the self-assembly form.

[0130] In a possible implementation, the first preset wavelength band is 200 nm-600 nm, and the second preset wavelength band is 405 nm.

[0131] This application is based on the peptide compound characterization method. The peptide compound is characterized in vitro by UV, circular dichroism and TEM, and it is determined that when light is used as an energy source, this type of peptide can form a non-equilibrium system.

[0132] The following examples are provided for detailed description.

[0133] Example 1 UV-Vis Spectroscopy - Study of Polymers Using UV-Vis Dilution Experiments

[0134] UV-Vis measurements were performed on semi-micro cuvettes with frosted interior. Spectra were recorded in 1 cm quartz cuvettes at 25°C with the following parameters: range = 190-900 nm, sensitivity = standard, data spacing = 0.5 nm, bandwidth = 2 nm. The corresponding solutions were freshly prepared in the indicated concentrations and buffers.

[0135] like Figure 7 As shown, Figure 7 A, B, and C in the figure are the UV absorption of peptides 5, 6, and 7 at concentrations of 1000 μM, 900 μM, 850 μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 50 μM, 25 μM, and 1 μM, respectively;

[0136] Figure 7 D, E, and F in the figure are the UV absorption of peptides 5, 6, and 7 at concentrations of 1000 μM, 900 μM, 850 μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 50 μM, 25 μM, and 1 μM, respectively, under 405 nm illumination;

[0137] The aggregation model of peptide assembly was determined by dilution experiments monitored by UV-visible spectroscopy. Peptides were prepared in water at the specified concentrations and their absorption spectra were recorded. A handheld LED lamp (3W, 405-410nm) was used to irradiate the samples for 15s. The extinction coefficient at 245nm was calculated as 1 for each concentration and the extinction coefficient graph was plotted for the corresponding concentrations.

[0138] For the synergistic growth model, the obtained data is fitted using formula (1):

[0139]

[0140] Wherein, ε(c) is the extinction coefficient measured at each concentration; C is the total compound concentration; K is the binding constant; σ is the degree of synergy; εf and εa are the extinction coefficients of the free state and the aggregated state, respectively.

[0141] For the isokinetic growth model, the obtained data were fitted using formula (2):

[0142]

[0143] like Figure 8 As shown, Figure 8 A is the ε curve of peptide 6 at 245nm under no light conditions, B is the ε curve of peptide 7 at 245nm under no light conditions, and C is the ε curve of peptide 5 at 254nm under no light conditions; D is the ε curve of peptide 6 at 245nm under blue light conditions, E is the ε curve of peptide 7 at 245nm under blue light conditions, and F is the ε curve of peptide 5 at 280nm under blue light conditions.

[0144] That is, peptide 6 self-assembles at a constant rate before illumination and at a constant rate after illumination, and peptide 7 self-assembles at a constant rate before and after illumination. The reason is that the electron-withdrawing effect of the nitro group makes the π-π stacking effect of the benzene ring part worse, resulting in the failure to form a "core" for nucleation self-assembly. Peptide 5 self-assembles at a constant rate before and after illumination, and its indole ring has a stronger π-π stacking effect.

[0145] Example 2 Circular dichroism test

[0146] CD spectra were recorded at 25°C using a J-1000 CD spectrometer in a 10 mm quartz cuvette with the following parameters: range = 260-190 nm, sensitivity = standard (100 mdeg), scan speed = 200 nm / min, data spacing = 1 nm, bandwidth = 1 nm, response = 1 sec. For measurements with and without light irradiation, solutions of the peptide (200 μM) were freshly prepared in H2O.

[0147] like Fig. 9 As shown, Fig. 9The CD results of peptide 6 in Figure A before and after illumination (100) are quite different. In the absence of light, there are two negative peaks, the maximum negative peak is located at about 230nm, the other negative peak is located at about 198nm, and the maximum positive peak is located at 205nm, which is similar to a pseudo-α-helix structure. After illumination (200), the maximum negative peak is located at about 218nm, and the maximum positive peak is located at about 200nm, which is an irregular curled structure. The difference between peptide 7 in Figure B before and after illumination is not obvious. Its maximum negative peak before illumination (300) is located at about 224nm, and the maximum negative peak after illumination (400) is located at about 222nm. It is a β-sheet structure before and after illumination. Figure C has two negative peaks before illumination (500), with the largest negative peak at 228nm, another negative peak at 200nm, and the largest positive peak at 214nm. After illumination (600), there are two negative peaks, with the largest negative peak at 227nm, another negative peak at 203nm, and the largest positive peak at 210nm. Both before and after illumination are pseudo-α-helix structures.

[0148] Example 3 Zeta potential experiment

[0149] All measurements were performed in a capillary sample cell (DTS1017) purchased from Malvern at 25°C. Peptides were prepared in water at a concentration of 300 μM and filtered through a syringe filter before measurement. The autocorrelation function of the backscattered light fluctuations was analyzed using the zeta potential provided by the ZSXPLORER software from Malvern.

[0150] like Fig.10 As shown, Fig.10 In Figure A, the Zeta potential of peptide 6 before illumination was 6.948 mV, and the Zeta potential after illumination was 17.060 mV. In Figure B, the Zeta potential of peptide 7 before illumination was 31.602 mV, and the Zeta potential after illumination was 40.01 mV. In Figure C, the Zeta potential of peptide 5 before illumination was 16.770 mV, and the Zeta potential after illumination was 19.11 mV.

[0151] Example 4 Transmission Electron Microscopy (TEM)

[0152] A drop of freshly prepared peptide solution (300 μM in H2O) was placed on a 400 mesh carbon-coated copper grid. Approximately 10 minutes after deposition, the grid was tapped with filter paper to remove residual solvent from the surface. A drop of uranyl acetate ethanol solution (2%) was added to the copper grid for negative staining. After 1 minute, the liquid on the grid surface was removed by tapping with filter paper. The samples were prepared under dim light or blue light until dry.

[0153] like Fig.11 As shown, Fig.11 Figure AB is the morphology of peptide 5 before illumination, and Figure CD is the morphology of peptide 5 after illumination; Fig.12As shown, Figures A and B are the morphologies of peptide 6 before illumination, and Figures C and D are the morphologies of peptide 6 after illumination; Fig.13 As shown, Figures A and B are the morphologies of peptide 7 before illumination, and Figures C and D are the morphologies of peptide 7 after illumination.

[0154] In another aspect, the present application provides a method for constructing a non-equilibrium state in a cell, which is implemented by using a polypeptide compound prepared by any of the methods for preparing a polypeptide compound described above, comprising:

[0155] adding any one of the peptides 5, 6, and 7 to the incubation matrix to obtain a peptide dilution solution;

[0156] The polypeptide dilution is added to the cells and incubated for a preset time to construct a non-equilibrium system in the cells.

[0157] The method for constructing a non-equilibrium state in a cell of the present application is to add a polypeptide compound to an incubation matrix to obtain a dilution of the polypeptide, then add the polypeptide dilution to the cells, and incubate for 5-20 minutes. Through the cell experiment of the present application, it can be confirmed that a non-equilibrium system is also constructed in the cell, and the migration of the polypeptide from the mitochondria to the nucleus is achieved through the non-equilibrium process.

[0158] It should be noted that the polypeptide concentration in this application is 1mM, and the amount added is preferably 0.1-5μL, more preferably 1.5μL; the incubation matrix type is preferably water, PBS or serum-free culture medium (DMEM, Opti-MEM, etc.), more preferably water. At the same time, the incubation matrix volume is preferably 5-50μL, more preferably 25μL after the addition of the polypeptide. The incubation time in this application is preferably 30-40min, more preferably 60min.

[0159] Example 5 MTT (cytotoxicity test)

[0160] One day before the experiment, 10,000 HeLa cells per well were suspended in DMEM (200 μL, 10% FCS), seeded into 96-well plates, and allowed to adhere overnight. The cells were washed once with PBS (200 μL) and incubated with the indicated concentrations of DMEM (200 μL, 10% FCS) at 37°C for 24 h. The cells were then washed with PBS (3 × 100 μL), and MTT solution (100 μL, 5 mg / ml in PBS) was added and incubated at 37°C for 3 h. The supernatant was discarded and DMSO (200 μL) was added. The samples were stirred and the absorbance was measured at 570 nm using a plate reader after 30 min. The background absorption was subtracted. Each measurement was repeated three times.

[0161] The structure of MTT shows Fig.14The A peptide 5 shown has no obvious cytotoxicity at a concentration of 200 μM, but has strong cytotoxicity at concentrations exceeding 200 μM. Fig.14 The cytotoxicity of peptide 6 in B is still not obvious at a concentration of 500 μM. When the concentration exceeds 500 μM, it has strong cytotoxicity. Fig.14 C-peptide 7 has strong cytotoxicity when the concentration exceeds 50μM.

[0162] Example 6 Cellular localization of polypeptides

[0163] Prior to measurement, HeLa cells were seeded at a density of 10,000 cells / well in ibidi 8-well plates and cultured in DMEM (10% FCS) at 37°C for 1 day. The cells were then washed with PBS and fresh DMEM (10% FCS) was added. The peptide solution was added to the cells to a final concentration of 20 μM and incubated at 37°C for 1 h or 16 h. The cells were washed with PBS, incubated with Hoechst 33342 (2 μM in DMEM) for 5 min at 37°C, and subsequently incubated with Mito-Tracker Green (20 nM in DMEM) for 20 min at 37°C. After washing the live cells, they were immediately examined on an Olympus IXploreSpinSR laser scanning confocal microscope with a 60x super-resolution mode objective. The laser lines were 405 nm (for Hoechst 33342), 561 nm (for 1), and 488 nm (for Mito-Tracker Green).

[0164] like Fig.15 Shown is the result of mitochondrial staining of peptide 6. The experimental concentration of peptide 6 is 50 μM. The red part is peptide 6, the green part is mitochondria, the blue part is the nucleus, and the scale bar is 10 μm.

[0165] like Fig.16 Shown is the result of mitochondrial staining of peptide 7. The experimental concentration of peptide 7 is 50 μM. The red part is peptide 7, the green part is mitochondria, the blue part is the nucleus, and the scale bar is 10 μm.

[0166] like Fig.17 As shown, it shows the mitochondrial staining result of peptide 5. The experimental concentration of peptide 5 is 50 μM. The red part is peptide 5, the green part is mitochondria, the blue part is the nucleus, and the scale bar is 10 μm.

[0167] Example 7 Peptide Circulation in Cells Irradiation

[0168] Prior to measurement, HeLa cells were seeded as described above. Peptides were added to the cells to a final concentration of 20 μM and incubated at 37°C for 1 h. Staining was performed with Hoechst 33342 (2 μM in DMEM) for 5 min at 37°C. Then, the live cells to be measured were periodically illuminated for 3 min using a 405 nm laser line (Olympus IXploreSpinSR) and then recovered in the dark for 7 min. Confocal images were taken using a 100x objective lens, and super-resolution images were taken using a 100x super-resolution mode objective lens. Afterwards, based on the confocal images at different times, the initial nuclear fluorescence intensity was used as the reference. Under this parameter, the nuclear fluorescence intensity was used as the ordinate and the time was used as the abscissa to construct a nuclear fluorescence intensity coordinate system to confirm the rate at which the peptide enters the nucleus under illumination.

[0169] like Fig.18 As shown, it is the result of peptide 5 migrating from cell mitochondria to cell nucleus. Wherein, Y = (1-exp(-k*X))*A, k = 0.01314, R 2 =0.9729. The peptide started to enter the cell at 22 minutes and ended at 46 minutes. The process was consistent with the fitting results of the first-order kinetic formula. After calculation, the half-life from entering the cell nucleus was 52.75 minutes, that is, from the beginning of light exposure, it took 72.75 minutes for the peptide to enter the cell nucleus.

[0170] like Fig.19 As shown, it is the result of peptide 6 migrating from cell mitochondria to cell nucleus, where Y = (1-exp(-k*X))*A, k = 0.04528, R 2 =0.9488. The peptide started to enter the cell from 54 minutes to 66 minutes, and the process was consistent with the fitting results of the first-order kinetic formula. After calculation, the half-life from entering the cell nucleus was 15.31 minutes, that is, from the beginning of light exposure, it took 67.31 minutes for the peptide to enter the cell nucleus.

[0171] like Fig. 20 As shown, it is the result of peptide 7 migrating from cell mitochondria to cell nucleus, where Y = (1-exp(-k*X))*A, k = 0.1257, R 2 =0.9789. The peptide started to enter the cell at 56 minutes and ended at 72 minutes. The process was consistent with the fitting results of the first-order kinetic formula. After calculation, the half-life from entering the cell nucleus was 5.51 minutes, that is, from the beginning of light exposure, it took 59.51 minutes for the peptide to enter the cell nucleus.

[0172] Kinetic analysis experiments showed that these peptides are likely to enter the cell nucleus through nuclear surface channels (nuclear pore transporters) rather than in the form of free diffusion (0-order kinetics), which provides a verification of the way in which peptides enter the cell nucleus.

[0173] According to the results of kinetic analysis, we can get a guess about the speed of movement of this type of peptide in the cell. Under the condition of spiropyran as a fluorescent and nucleus-directed group, we can draw a conclusion that different substituents have different movement speeds in the cell by comparing the results of kinetic analysis, that is, in a self-assembly system with two adjacent aromatic rings as the core, the movement speed of the aromatic ring with electron-withdrawing groups is faster than that with electron-donating groups. Because they all start from the mitochondria and end at the nucleus, each peptide has gone through the same journey. From the analysis of kinetic data, the smaller the half-life, the easier it is to enter the nucleus, that is, the substance is more easily transported by the nuclear pore. The half-life of peptide 7 (5.51min)>peptide 6 (15.31min)>peptide 5 (52.75min), and the electron-withdrawing group on the aromatic ring is more conducive to nuclear pore transport.

[0174] In summary, the method for preparing a polypeptide compound of the present application uses amino acids for solid phase synthesis to prepare a tripeptide, which can enter the mitochondria in cells and can control the non-equilibrium state in cells by illumination. A peptide whose self-assembly state can be controlled by illumination is successfully synthesized from aromatic amino acids (tyrosine, tryptophan, and phenylalanine substituted with phenyl para-nitro), lysine, and spiropyran. In in vitro experiments, the present application determined that this type of polypeptide has different assembly states when there are different aromatic substituents. When conducting cell experiments, this type of polypeptide was successfully used to achieve the construction of a light-controlled non-equilibrium state in cells. Compared with the prior art, the present application uses light as the energy source for artificial non-equilibrium state in cells for the first time, and successfully achieves the migration of peptides from mitochondria to the nucleus under non-equilibrium state.

[0175] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a polypeptide compound, characterized in that: The method comprises the following preparation steps: The first raw material is shown in Formula 1, the second raw material is shown in Formula 2, the first raw material and the second raw material are refluxed in ethanol, and purified by silica gel column chromatography to obtain a first product, as shown in Formula 3; Formula 1: Formula 2: Formula 3: Adding any one of the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or the third raw material to the amino-functionalized resin, and sequentially performing a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection to obtain a second product; and the third raw material is as shown in Formula 4; Formula 4: The structural formula of the Fmoc-Trp-OH is as follows: The structural formula of the Fmoc-Tyr-OH is as follows: The first product and PyBop are dissolved in DMF, iPrNEt2 is added and mixed, and then added to the second product, and reacted and washed in sequence to obtain a third product; After the third product is mixed and shaken in a preset cleavage reagent, the filtrate is collected and concentrated under a N2 flow, and after adding cold Et2O, the suspension is centrifuged, and the corresponding solid is dissolved in a preset mixed solvent, and lyophilized and purified in sequence to obtain peptides 5, 6, and 7; The structural formula of the peptide 5 is as follows: The structural formula of peptide 6 is as follows: The structural formula of the peptide 7 is as follows: The peptide 5, peptide 6 and peptide 7 can all migrate from the cell mitochondria to the cell nucleus.

2. The method for preparing a polypeptide compound according to claim 1, characterized in that: Adding the activated Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials to the amino-functionalized resin, and sequentially performing a first round of N-terminal coupling and Fmoc deprotection, and a second round of N-terminal coupling and Fmoc deprotection to obtain a second product, including: Dissolve Fmoc-Trp-OH, Fmoc-Tyr-OH or any one of the third raw materials and PyBop in DMF, and add iPrNEt2 for activation; The structural formula of the PyBop is as follows: After adding the activated Fmoc-Trp-OH, the Fmoc-Tyr-OH or any one of the third raw materials to the amino-functionalized resin, the amino-functionalized resin is washed with DMF and CH2Cl2 to perform a first round of N-terminal coupling, and a 20% piperidine DMF solution is added to cover the amino-functionalized resin, and the suspension is shaken to perform a first round of Fmoc deprotection; The amino-functionalized resin is subjected to a second round of N-terminal coupling and a second round of Fmoc deprotection in the same manner as the first round of N-terminal coupling and the first round of Fmoc deprotection, respectively, to obtain a second product.

3. The method for preparing a polypeptide compound according to claim 1, characterized in that: The first product and PyBop are dissolved in DMF, iPrNEt2 is added and mixed, and then added to the second product, and reacted and washed in sequence to obtain a third product, including: Dissolving the first product and the PyBop in DMF, adding iPrNEt2, to obtain a first mixture; The first mixture was added to the second product, and after shaking, the mixture was washed with DMF and CH2Cl2 to obtain a third product.

4. The method for preparing a polypeptide compound according to claim 1, characterized in that: The preset cutting reagent is a mixture of TFA, H2O and TIS, and TFA:H2O:TIS=95:2.5:2.

5.

5. The method for preparing a polypeptide compound according to claim 1, characterized in that: The preset mixed solvent is H2O and MeCN, and H2O:MeCN=1:

1.

6. A method for characterizing a polypeptide compound, used for characterizing a polypeptide compound prepared by the method for preparing a polypeptide compound according to any one of claims 1 to 5, characterized in that: include: The peptides 5, 6 and 7 are tested by at least one of UV test, circular dichroism test, TEM test, particle size test and Zeta potential test.

7. The method for characterizing a polypeptide compound according to claim 6, characterized in that: When the peptides 5, 6, and 7 are tested using the UV test, the method includes: preparing aqueous solutions of the peptides 5, 6 and 7 at different concentrations respectively; After the aqueous solutions of the peptides 5, 6, and 7 with different concentrations are subjected to ultraviolet testing in a first preset wavelength band under dark conditions, they are illuminated in a second preset wavelength band and the aqueous solutions of the peptides 5, 6, and 7 with different concentrations are tested.

8. The method for characterizing a polypeptide compound according to claim 7, characterized in that: When the peptides 5, 6, and 7 are tested using UV testing, the following steps are also included: Draw the self-assembly curve, and draw the ε curve according to the self-assembly formula to determine the self-assembly form.

9. The method for characterizing a polypeptide compound according to claim 7, characterized in that: The first preset wavelength band is 200nm-600nm, and the second preset wavelength band is 405nm.

10. A method for constructing a non-equilibrium state in a cell, which is achieved by using a polypeptide compound prepared by the method for preparing a polypeptide compound according to any one of claims 1 to 5, characterized in that: include: adding any one of the peptides 5, 6, and 7 to the incubation matrix to obtain a peptide dilution solution; The polypeptide dilution is added to the cells and incubated for a preset time to construct a non-equilibrium system in the cells.

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