Peptidyl crystal with in-situ adjustable pH response and preparation method thereof

Abstract

The invention belongs to the technical field of biological materials, and provides a pH response in-situ adjustable peptidyl crystal and a preparation method thereof. Chiral dipeptide with positive charges is mixed with anions, and formation of chiral micro-rolls is induced through co-assembly, so that the peptidyl crystal with in-situ adjustable pH response is prepared. The chiral dipeptide is 9-fluorenyl methoxy carbonyl-tyrosine-lysine (Fmoc-YK), and the anion is a sulfate ion (SO4 < 2->). Through a simple co-assembly mode, the two-dimensional (2D) nanosheet is induced to be curled, and the curled structure can greatly amplify the supramolecular chirality of the peptide assembly; and embedding and separation of SO4 < 2-> are controlled through pH, so that in-situ circulation of morphology transformation from the micron roll to the micron ribbon to the micron roll is realized for more than three times. The micron roll and the micron belt have opposite supermolecule chirality, so that the supermolecule chirality of the co-assembly system can be reversed for three times in situ. The method disclosed by the invention is simple and strong in operability, and promotes the development of chiral peptide nano materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and relates to a pH-responsive in-situ tunable peptide-based crystal and a preparation method thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Self-assembled chiral nanostructures are the basis of various biological processes. Inspired by these biological chiral structures, biomolecules such as sugars, nucleobase chains, and polypeptides have been widely used to construct supramolecular chiral materials. These materials have shown great application potential in asymmetric synthesis, chiral recognition, separation, molecular switches, optoelectronics, etc. Among biomolecules, peptides have become an important component due to their inherent molecular chirality, programmable synthesis, easy modification, sequence-dependent functions, and most importantly, highly tunable assembly processes. By adjusting factors such as pH, temperature, and aging time to regulate non-covalent interactions, peptide assemblies can be designed to control chiral expression, transfer, and inversion.

[0004] Interestingly, certain achiral additives profoundly affect the non-covalent interactions of polypeptides, thus changing the synthesized chiral structures. For example, the co-assembly of N-Fmoc-L-phenylalanine dipeptide (L-FmocFF) with achiral pyridine derivatives produces left-handed or right-handed helices due to additive-induced H- or J-aggregation, thereby changing the microenvironment around the chiral center. Despite these advances, precisely regulating the process of chiral expression, amplification, and inversion from molecules to supramolecules remains a challenge. Therefore, there is a need to further develop methods for precisely controlling the chiral structures of peptide-based materials. Summary of the Invention

[0005] To solve the above problems, the present invention provides a pH-responsive in-situ tunable peptide-based crystal and a preparation method thereof, and completes the pH-triggered in-situ chiral inversion in a dipeptide co-assembly through supramolecular engineering, providing a simple and efficient strategy for the development of intelligent responsive chiral materials.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a preparation method of a pH-responsive in-situ tunable peptide-based crystal is provided, including:

[0008] Mix the Fmoc-YK solution and the sulfate solution evenly, and statically assemble to form micron rolls under the condition of pH 0-4.0, thus obtaining.

[0009] Among them, the structural formula of the Fmoc-YK is as follows:

[0010]

[0011] The terminal lysine residue in the dipeptide plays a crucial role in controlling the structure of the assembly and supramolecular chirality. Therefore, in the present invention, the charge of the terminal lysine is changed by controlling the change of pH in the assembly environment, and the structure of the assembly and supramolecular chirality are dynamically changed by causing changes in electrostatic interactions.

[0012] In some embodiments, the sulfate is selected from at least one of sodium sulfate, magnesium sulfate, ferrous sulfate, potassium sulfate, and ammonium sulfate.

[0013] In some embodiments, the pH value is 2.0 - 3.0.

[0014] In some embodiments, the molar ratio of Fmoc-YK to the sulfate is <5:2, preferably 5:8 - 5:4, and more preferably 5:5.

[0015] In some embodiments, the volume ratio of Fmoc-YK to SO 4 2- is >5:2, preferably 10:1 - 5:2, and more preferably 5:1.

[0016] In some embodiments, the static assembly temperature is 4 - 80°C, preferably 25 - 37°C.

[0017] In some embodiments, the static assembly time is 10 - 60 min, preferably 60 min.

[0018] More specifically, it includes:

[0019] Fmoc-YK is dissolved in ddH 2 O, sonicated until clear, then the sulfate solution is added, and it is immediately mixed evenly by vortex oscillation, and statically assembled to form micro-rolls.

[0020] In the second aspect of the present invention, a peptide-based crystal prepared by the above method is provided.

[0021] In the present invention, a simple co-assembly method of the positively charged 9-fluorenylmethoxycarbonyl-tyrosine-lysine (Fmoc-YK) and the anion SO 4 2- is used to induce the curling of two-dimensional (2D) nanosheets to form micro-rolls, and the embedding and extraction of SO 4 2- are controlled by pH to achieve in-situ cyclic transformation of the morphology from micro-rolls to micro-belts and then back to micro-rolls more than 3 times, and in-situ inversion of supramolecular chirality 3 times.

[0022] To Fmoc-YK / SO 4 2- Alternately add OH to the micron coils - and H + , enabling in-situ cyclic morphology and in-situ inversion of supramolecular chirality more than 3 times. Therefore, in the third aspect of the present invention, there is provided a method for in-situ alternate transformation of the above-mentioned peptide-based crystal, including:

[0023] By controlling the embedding and extraction of sulfate in the peptide-based crystal through pH, realizing the morphological transformation from micron coils to micron belts, or from micron belts to micron coils;

[0024] Among them, when an alkaline substance is added to the micron coils and the pH is 4.0 - 10.3, micron belts are formed; while the amino group remains protonated, the carboxyl group will be deprotonated, and Fmoc-YK exists in the form of zwitterions and is in a charge balance state by itself. At this time, Fmoc-YK will not co-assemble with SO 4 2- but tends to self-assemble to form micron belts.

[0025] When an acidic substance is added to the micron belts and the pH ≤ 4.0, micron coils are formed. The amino group is protonated, and the molecule exists in the form of a cation. To maintain the overall charge balance, Fmoc-YK tends to co-assemble with the SO 4 2- anion to form micron coils.

[0026] In the present invention, OH is added to the micron coil system - , SO 4 2- is extracted from the micron coils, and Fmoc-YK self-assembles to form micron belts. When H is added to the micron belt system + , SO 4 2- re-assembles with Fmoc-YK to form micron coils again. With the alternate addition of OH - and H + , in-situ cyclic morphology and supramolecular chirality are achieved more than 3 times.

[0027] In some embodiments, when the pH is 6.0 - 8.0, micron belts are formed;

[0028] In some embodiments, when the pH is 2.0 - 3.0, micron coils are formed;

[0029] In some embodiments, the alkaline substance is selected from at least one of NaOH, KOH, NaHCO 3 ;

[0030] In some embodiments, the acidic substance is selected from H2 SO 4 、HCl、CH 3 COOH、HNO 3 at least one of

[0031] In some embodiments, the assembly time for the formation of the micro-rolls into micro-belts is 1 - 2 h; preferably, it is 2 h.

[0032] In some embodiments, the assembly time for the formation of the micro-belts into micro-rolls is 5 - 20 min, preferably, it is 20 min.

[0033] Advantages of the present invention

[0034] The present invention provides a method for preparing a pH-responsive in-situ tunable peptide-based crystal using an aqueous solvent without using organic solvents, which has little environmental pollution, a simple method, strong operability, and promotes the development of chiral peptide nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0036] Figure 1 : a is the SEM image of Fmoc-YK / SO 4 2- micro-rolls, b is the TEM image of Fmoc-YK / SO 4 2- micro-rolls.

[0037] Figure 2 For Fmoc-YK / SO 4 2- SEM images of the dynamic evolution of the micro-roll structure.

[0038] Figure 3 : a is the SEM image of 3 in-situ cycles of pH response, b is the screenshot of the microscope video of the transformation from micro-rolls to micro-belts and then back to micro-rolls.

[0039] Figure 4 For Fmoc-YK / SO 4 2- CD spectra of the micro-rolls and micro-belts.

[0040] Figure 5 For Fmoc-YK / SO 4 2- Schematic diagram of the assembly mechanism of the micro-rolls and micro-belts. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0042] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are interpretations rather than limitations of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; unless otherwise specified, the reagents used in the following embodiments are all commercially available.

[0044] The dipeptide described in the present invention is positively charged Fmoc-YK, which is synthesized by Shanghai Gil Biochemical Co., Ltd. with a purity greater than 98%. The structural formula is as follows:

[0045]

[0046] The Fmoc-YK is co-assembled with SO 4 2- The system used is the H 2 O system.

[0047] The Fmoc-YK is co-assembled with SO 4 2- For the co-assembly, the pH range of the co-assembly of Fmoc-YK and SO 4 2- is within 0 to 4.0, and the preferred pH range is 2.0 to 3.0. The molar ratio of Fmoc-YK to SO 4 2- is <5:2, and the preferred molar ratio is 5:8 to 5:4, where 5:5 is the optimal molar ratio. The volume ratio of Fmoc-YK to SO 4 2- is >5:2, and the preferred volume ratio is 10:1 to 5:2, where 5:1 is the optimal volume ratio. The static assembly temperature is 4 to 80 °C, and the preferred static assembly temperature is 25 to 37 °C. The static assembly time is 10 to 60 min, and 60 min is the optimal static assembly time.

[0048] The pH-responsive micron rolls and micron bands are in-situ alternately transformed. OH - includes but is not limited to NaOH, KOH, NaHCO 3 , where NaOH is the best. H + includes but is not limited to H 2 SO 4 , HCl, CH 3 COOH, HNO3 , where H 2 SO 4 is optimal. Fmoc-YK / SO 4 2- The micron rolls are stable at pH < 4.0, with the optimal range being pH 2.0 - 3.0. The micron belts are stable at 4.0 < pH < 10.3, with the optimal range being pH 6.0 - 8.0. The assembly time from micron rolls to micron belts is 1 - 2 h, with 2 h being the optimal assembly time. The assembly time from micron belts to micron rolls is 5 - 20 min, with 20 min being the optimal assembly time.

[0049] Chemical reagents: Sodium sulfate (analytical grade) was purchased from Beijing Chemical Industry Company; the acids and bases used to adjust pH were purchased from Shanghai Reagent.

[0050] Testing instruments and conditions: Cold field emission scanning electron microscope (SEM): 5 μL of the sample was pipetted onto a clean silicon wafer, and the moisture on the sample surface was removed by drying with a vacuum pump. The obtained sample was adhered to the scanning stage with a conductive adhesive, and a layer of platinum (20 mA, sputtered for 40 s) was needed to be coated on the surface to increase conductivity. The microscopic morphology of the sample surface was photographed under the conditions of a voltage of 10 kV and a current of 10 μA. Transmission electron microscope (TEM): 5 μL of the sample was dropped onto a carbon-coated copper grid (300 mesh), and after standing for a while, the excess sample was sucked away from one end of the copper grid with a filter paper. The moisture on the sample surface was removed by drying with a vacuum pump, and then TEM images were obtained under an accelerating voltage of 100 - 120 kV. Microscope for photographing in-situ morphology transformation video: 20 μL of the micron roll suspension was placed on a glass slide. While pressing the shooting button, a small amount of NaOH was dropped into the micron roll suspension, and the whole process from the disappearance of the micron roll precipitate until it became transparent was photographed; keeping the position unchanged, a small amount of H 2 SO 4 , and the whole process of the precipitation of white micron roll precipitate in the transparent solution was photographed. Circular dichroism (CD) spectrum: 50 - 100 μL of the sample was pipetted into a quartz cell with an optical path of 0.2 mm, and the CD spectral information in the corresponding wavelength range was detected using a circular dichroism spectrometer at room temperature. The scanning speed was 200 nm / min, and the bandwidth was 1.0 nm.

[0051] Example 1:

[0052] 5.32 mg of Fmoc-YK was dissolved in 1 mL of ddH 2 O, and an Fmoc-YK solution with a concentration of 10 mM was obtained by ultrasonic assistance for dissolution;

[0053] 200 μL of a sodium sulfate solution with a concentration of 50 mM was mixed with the above-obtained Fmoc-YK solution, and left to stand at room temperature for 1 h to obtain Fmoc-YK / SO 42- Micron roll.

[0054] Example 2: Fmoc-YK / SO obtained in Example 1 4 2- Morphology characterization of the micron roll.

[0055] Fmoc-YK / SO prepared in Example 1 of the present invention 4 2- The SEM characterization results of the micron roll are as Figure 1 shown in a, and the TEM characterization results are as Figure 1 shown in b. From the morphology characterization, it can be seen that the Fmoc-YK / SO obtained in the present invention 4 2- micron roll is a hollow tubular structure with an outer diameter of about 1 μm formed by the inward curling of 2D nanosheets.

[0056] Example 3: Fmoc-YK / SO obtained in Example 1 4 2- Growth process of the micron roll.

[0057] Use SEM to observe its growth process. The results are as Figure 2 shown. For Fmoc-YK / SO 4 2- nanosheets precipitate after 1 min, and the nanosheets have a certain curling curvature. The reason for this phenomenon may be that SO 4 2- causes the surface tension on both sides of the nanosheets to be asymmetric; as the aging time extends to 3 min, the curling effect is amplified while the 2D nanosheets are growing, and they spontaneously curl into a semi-tubular or even tubular structure; subsequently, the nanosheets use the curled tubular structure as a template and continue to wind and grow along the tube wall, while extending in the direction of the tube diameter, and finally form multi-walled micron rolls after 16 min.

[0058] Example 4: Fmoc-YK / SO obtained in Example 1 4 2- pH responsiveness of the in-situ alternating transformation from chiral micron rolls to micron belts.

[0059] Add an appropriate amount of NaOH to the chiral micron rolls prepared in Example 1, mix immediately, and adjust the pH of the system to 7.0. At this time, the solution micron roll precipitate disappears rapidly within 18 s. After standing and assembling for 2 h, a micron belt hydrogel is obtained. Add an appropriate amount of H 4 2- to the micron belt hydrogel. 2 SO 4, immediately mix well, adjust the pH of the system to 2.8. At this time, the hydrogel collapses within 2 s, and after standing and assembling for 20 min, a microtube precipitate is obtained again. With the alternating addition of NaOH and H 2 SO 4 , the morphological transformation from microtubes to microbelts and then back to microtubes can be cycled in situ more than 3 times, and its structure and assembly mechanism are as Figure 5 shown.

[0060] Example 5: Morphological characterization of the in-situ alternating transformation from microtubes to microbelts obtained in Example 4.

[0061] The SEM characterization results of the in-situ alternating transformation from microtubes to microbelts prepared in Example 4 of the present invention are as shown in a of Figure 3 , and the video screenshot taken by the microscope is as shown in b of Figure 3 . The results show that after Fmoc-YK and SO 4 2- co-assemble to form microtubes, the protonation of the terminal carboxyl group plays a very important role in maintaining the stability of the microtubes. When the system is under the condition of 4.0 < pH < 10.3, while the amino group remains protonated, the carboxyl group will be deprotonated, and Fmoc-YK exists in the form of zwitterions and is in a charge balance state. At this time, Fmoc-YK will not co-assemble with SO 4 2- , and the microtubes will disappear instantly and turn into a clear and transparent solution within 18 s. After standing at room temperature for 2 h, the clear and transparent solution will turn into a translucent hydrogel. The SEM results show that the hydrogel network is composed of microbelts. When an appropriate amount of H 2 SO 4 is added dropwise to the microbelt hydrogel network to adjust the pH to 2.8, the carboxyl group of Fmoc-YK is reprotonated at this time. After adding H 2 SO 4 for 2 s, the hydrogel network is instantly destroyed and completely turns into a microtube precipitate after 5 min. With the alternating addition of NaOH and H 2 SO 4 , the cyclic transformation of the microtube-to-microbelt structure can be carried out in situ more than 3 times.

[0062] Example 6: CD characterization of the microtubes and microbelts obtained in Example 4.

[0063] The CD characterization results of the microtubes and microbelts obtained in Example 4 of the present invention show that the CD spectra of the microtubes and microbelts are almost mirror images of each other, indicating that the two assemblies have opposite supramolecular chirality. It can be seen from this that under pH control, while the morphology of the Fmoc-YK / SO 4 2- system is reversibly switched, the supramolecular chirality is also reversed.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a peptide-based crystal with in situ adjustable pH response, characterized in that: Comprising: Mix the Fmoc-YK solution and the sulfate solution evenly, and let it stand for assembly to form micro-rolls under the condition that the pH is 0-4.0, thus obtaining the product. Among them, the structural formula of the Fmoc-YK is as follows:

2. The method for preparing a peptide-based crystal with in-situ adjustable pH response according to claim 1, characterized in that: The sulfate is selected from at least one of sodium sulfate, magnesium sulfate, ferrous sulfate, potassium sulfate, and ammonium sulfate.

3. The method for preparing a peptide-based crystal with in-situ adjustable pH response according to claim 1, characterized in that: The pH value is 2.0-3.

0.

4. The method for preparing a peptide-based crystal with in-situ adjustable pH response according to claim 1, characterized in that: The molar ratio of the Fmoc-YK to the sulfate is <5:2, or 5:8-5:4, or 5:

5.

5. The method for preparing a peptide-based crystal with in-situ adjustable pH response according to claim 1, characterized in that: The temperature for standing assembly is 4-80 °C, or 25-37 °C.

6. The method for preparing a peptide-based crystal with in-situ adjustable pH response according to claim 1, characterized in that: The time for standing assembly is 10-60 min, or 60 min.

7. The peptide-based crystal prepared by the method according to any one of claims 1-6.

8. A method for in-situ alternating transformation of peptide-based crystals according to claim 7, characterized in that: Comprising: By controlling the embedding and extraction of sulfate in the peptide-based crystal through pH, the morphological transformation from micro-rolls to micro-belts, or from micro-belts to micro-rolls is achieved. Among them, when an alkaline substance is added to the micro-rolls and 4.0 < pH < 10.3, micro-belts are formed. When an acidic substance is added to the micro-belts and pH ≤ 4.0, micro-rolls are formed.

9. The in-situ alternating transformation method according to claim 8, characterized in that: When the pH is 6.0-8.0, micro-belts are formed. Or, when the pH is 2.0-3.0, micro-rolls are formed. Or, the alkaline substance is selected from at least one of NaOH, KOH, and NaHCO3. Or, the acidic substance is selected from at least one of H2SO4, HCl, CH3COOH, and HNO3.

10. The in-situ alternating transformation method according to claim 8, characterized in that: The assembly time for the micro-rolls to form micro-belts is 1-2 h, or 2 h. Or, the assembly time for the micro-belts to form micro-rolls is 5-20 min, or 20 min.

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