Novel phase separation peptide for intracellular protein delivery as well as synthesis method and application of novel phase separation peptide

Abstract

The invention discloses a novel phase separation peptide for intracellular protein delivery and a synthesis method and application thereof. The structural formula of the novel phase separation peptide is shown in the specification. According to the novel phase separation peptide, coacervate microdroplets are formed through liquid-liquid separation (LLPS) self-assembly, meanwhile, protein can be rapidly collected, and after a coacervate enters cells by bypassing a classical endocytosis way, glutathione (GSH) in the cells triggers dissociation to release the protein.

Description

A novel phase-separated peptide for intracellular protein delivery, its synthesis method, and its applications. Technical Field

[0001] This invention relates to a novel phase-separated peptide for intracellular protein delivery, its synthesis method, and its application, belonging to the field of protein synthesis technology. Background Technology

[0002] Synthetic proteins with cell-penetrating capabilities are widely used in studies of intracellular protein-protein interactions, protein localization, and structure-function relationships. Intracellular delivery of proteins related to disease development, regulation of cellular physiological states, and responses to extracellular stimuli holds potential value for basic research and disease treatment. However, due to the physical properties of these proteins and the barrier effect of the cell membrane, they are difficult to penetrate the cell membrane and enter the cell interior, limiting their applications to most extracellular targets or immobilized tissues. Therefore, developing more efficient protein delivery strategies will significantly expand the potential applications of protein reagents and therapeutic agents.

[0003] Over the past few decades, numerous different methods have been developed for protein delivery into cells, including physical transmembrane methods such as microinjection or electroporation, and delivery systems utilizing viral vectors, nanoparticles, pressurized proteins, and lipid-mediated delivery systems. Among these, phase-separated peptides drive and regulate the formation of dynamic, membrane-free organelles or aggregates through liquid-liquid phase separation. This results in highly concentrated liquid microdomains within the cell. This process occurs spontaneously, reversibly, and in a concentration-dependent manner, independent of classical membrane structures or active energy consumption, and maintains cellular homeostasis under specific concentration and environmental conditions without disrupting normal cell viability, thus introducing proteins into living cells.

[0004] Phase-separating peptides (PSPs) are a class of biomaterials capable of self-assembling into coacervate microdroplets (CMs) via liquid-liquid phase separation (LLPS). Their primary function is as efficient and programmable intracellular macromolecule delivery carriers. The core sequence of these peptides is typically designed based on the modular structure of cephalopod beak proteins (such as HBpep), with the basic repeating unit being the pentapeptide sequence GHGXY (glycine-histidine-glycine-X-tyrosine), where the amino acid at the X site can be systematically replaced as needed. By introducing lysine residues into the peptide chain and attaching a disulfide self-cleaving group, the resulting coacervate microdroplets further acquire dual pH and redox responsiveness, exhibiting liquid-liquid phase separation behavior and thus facilitating transmembrane crossing. Furthermore, this design makes them reduction-responsive, allowing them to dissociate and release their cargo upon triggering intracellular glutathione (GSH). During the formation of microdroplets from phase-separated peptides, a variety of macromolecules can be rapidly recruited, including small peptides, enzymes up to 430 kDa, and messenger RNA (mRNA). The protein-loaded condensates bypass the classical endocytic pathway and enter the cell, where they undergo glutathione-mediated payload release in the cytoplasm, releasing the biologically active protein into the cell.

[0005] The article (Nat. Chem. 2022, 14, 274-283) reports an intracellular macromolecule delivery strategy based on liquid-liquid phase separation (LLPS), the core of which is the design of histidine-enriched beak protein-derived peptides (HBpep). This strategy involves inserting a lysine residue into the HBpep sequence and further linking it with a self-cleaving module containing disulfide bonds (such as NHS-SS-Ac or NHS-SS-Ph) to construct peptides (HBpep-SA / HBpep-SP) with dual pH and redox responses. These modified peptides can self-assemble into condensed layer droplets of approximately 1 μm in size via LLPS at near-physiological pH (approximately 6.5), and can efficiently encapsulate various macromolecules such as proteins, peptides, and mRNA during droplet formation. Microdroplets enter cells via a non-classical memory pathway and utilize high intracellular glutathione (GSH) concentrations to trigger disulfide bond reduction and self-fragmentation, causing the condensate layer to disintegrate and releasing the carried cargo, thus achieving delivery. However, this strategy relies on chemical modification to introduce self-fragmentation units, requiring multiple organic reactions, resulting in relatively complex synthesis steps, high production costs, and the potential generation of byproducts from the NHS esters used in the modification process, affecting peptide purity and biocompatibility. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a novel phase-separated peptide, its synthesis method, and its applications. The desired peptide can be obtained via the SPPS method without organic chemical modification, resulting in a relatively simple synthesis process and low production costs. This novel phase-separated peptide self-assembles into condensate droplets through liquid-liquid phase separation (LLPS) and can rapidly recruit proteins. After the condensate bypasses the classic endocytosis pathway and enters the cell, intracellular glutathione (GSH) triggers dissociation, releasing the protein.

[0007] The novel phase-separated peptide of this invention has the following structure:

[0008]

[0009] The present invention discloses a novel phase-separated peptide synthesis method. First, linear sequence peptide fragments (1) and (2) are synthesized using the N-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis method. Then, the amino protecting group ivDde of the lysine side chain of peptide fragment (1) is removed and coupled with [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid and benzoic acid. Finally, a novel phase-separated peptide molecule is obtained by forming a disulfide bond between the two fragments.

[0010] The polypeptide fragment (1) is H2N-GHGRYGHGRYGHGPYK(ivDde)-CONHNH2; the polypeptide fragment (2) is HS-CH2-CO-HN-GHGPYGHGYYW-COOH.

[0011] The synthesis route is shown below:

[0012]

[0013] This invention discloses a novel method for synthesizing phase-separated peptides, employing solid-phase peptide synthesis (SPPS), comprising the following steps:

[0014] Step 1: Synthesize the linear sequence of the polypeptide fragment (1) using the Fmoc solid-phase polypeptide synthesis method.

[0015] 1a. Take 0.1 mmol of 2-Cl-Trt-Cl resin (degree of substitution is 0.32 mmol / g), add 10 mL of N,N-dimethylformamide (DMF), and let the resin swell for 30 minutes. Use a diaphragm pump as the power source to dry the swollen product to obtain the swollen resin.

[0016] 1b. Add 4 mL of DMF solution containing 5% (volume fraction) hydrazine hydrate to the resin obtained in 1a to induce acylation. Place the resin in a shaker at room temperature and shake for 30 minutes. Then wash the resin three times each with DMF, dichloromethane (DCM), and DMF in sequence. Add 4 mL of DMF solution containing 5% hydrazine hydrate again and react for 30 minutes. After washing with the same method, add 4 mL of DMF solution containing 5% (volume fraction) methanol and shake for another 10 minutes to block the unreacted active groups on the resin. After thorough washing, dry the resin to obtain the polypeptide hydrazine resin, which can be used for subsequent solid-phase synthesis.

[0017] 1c. According to the linear amino acid sequence of the polypeptide fragment (1), a certain amount of K (ivDde), HATU and HOAT with side chains protected by the ivDde protecting group were weighed according to the standard Fmoc solid phase polypeptide synthesis (SPPS) method with the following molar ratio: resin (Resin): amino acid (aa): 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU): N-hydroxy-7-azabenzotriazole (HOAT): N,N-diisopropylethylamine (DIEA) = 1:5:5:5:10. A certain volume of DIEA was added to DMF in an EP tube to dissolve it, mixed well, and added to the synthesis tube. The mixture was incubated twice in a shaker at room temperature for 30 min each time. After the first amino acid was added, the remaining amino acids were synthesized in the order YPGHGYRGHGYRGHG using an automated microwave peptide synthesizer (all unfolded peptides in this invention were synthesized using a Liberty Blue™ automated microwave peptide synthesizer from CEM Corporation, USA, with a synthesis scale of 0.1 mmol resin. Before use, the resin was swollen in 20 mL DMF for 20 minutes and washed with DMF. Except for His, which was assembled using the standard double coupling method at 50 °C, the other amino acids were standard double-coupled at 90 °C. Each standard coupling cycle included deprotection of Fmoc using 20% ​​piperidine and 0.1 M Oxyma in DMF, and amino acid coupling using 5 times the excess of 0.2 M fmoc-protected amino acids in DMF, 0.5 M DIC in DMF, and 1.0 M Oxyma in DMF. After each reaction, the resin was washed three times with DMF).

[0018]

[0019] Step 2: Synthesize polypeptide fragments (1)

[0020] 2a. Take the peptide obtained in step 1 out of the fully automated microwave peptide synthesizer, clean it with DMF, and remove ivDde. Add 10 ml of 5% hydrazine hydrate to the resin and shake it in a shaker at room temperature for 10 min. Then clean it with DMF. Repeat this operation 5-6 times.

[0021] 2b. Link [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (AEEA) to the lysine side. Weigh out a fixed amount of AEEA, HATU, and HOAT according to the molar ratio of Resin:AEEA:HATU:HOAT:DIEA = 1:5:5:5:10, and take a certain volume of DIEA. Dissolve it in DMF in an EP tube, mix well, and add it to the synthesis tube. Inoculate twice at room temperature on a shaker, 30 min each time. Remove Fmoc by adding 20% ​​piperidine (100 mL piperidine dissolved in 400 mL DMF) and desorb at a high temperature shaker (75 °C) for 5 min. Wash the resin. Weigh out measured amounts of benzoic acid, HATU, and HOAT according to the molar ratio of Resin:benzoic acid:HATU:HOAT:DIEA = 1:2:2:2:4, and take a certain volume of DIEA. Dissolve the DIEA in DMF in an EP tube, mix well, and add it to the synthesis tube. Inject the mixture twice at room temperature on a shaker, 30 min each time. After injection, clean the resin for the final rinse with DCM, and then dry the resin.

[0022] 2c. The peptide synthesized in 2b is subjected to a global deprotection and cleavage step to remove all side-chain protecting groups and cleave the peptide from the resin. Using a lysis buffer containing TFA (10 mL TFA lysis mixture (524 mg phenol, 500 μL anisole, 500 μL water, 500 μL tips, 250 μL 1,2-ethylene glycol, and 8 mL trifluoroacetic acid)), the crude peptide is cleaved from the hydrazine resin at room temperature for 3 h. The resin and lysis buffer are separated. Under positive pressure nitrogen, the lysis buffer is blown down to a volume below 5 mL. Excess cold diethyl ether (0℃-10℃) is added to the lysis buffer, followed by centrifugation to collect the precipitate. This precipitation is repeated 2-3 times. The crude peptide is then air-dried. Purification is performed using semi-preparative high-performance liquid chromatography (HPLC). The purified solution is collected and lyophilized.

[0023] 2d. The lyophilized peptide (5 mg) obtained in 2c and NaNO2 (13.8 mg) were dissolved in 1 mL of Gn-HCl at pH 2-3. 1 mL of guanidine hydrochloride at pH 2.3 was added, and the mixture was pre-cooled in an ice-salt bath for 5 min. Then, 34.36 μL of NaNO2 was added, and the system pH was adjusted to 2-3. The reaction was allowed to proceed for 10 min. Then, 4.619 mg of MPAA (40 eq) was dissolved in 40 μL of 1M NaOH solution and added to the system. The pH of the system was adjusted to 4.8, and the reaction was allowed to proceed at room temperature for 10 min. 1.61 mg of MEA (7 eq) was weighed, dissolved in 300 μL of guanidine hydrochloride at pH 7.0, and the pH was adjusted to 6.8. After reacting for 3 h, dithiothreitol (DTT) (500 mM stock solution) was added to monitor the reaction. After monitoring, the solution was purified by semi-preparative high-performance liquid chromatography. The purified solution was collected and lyophilized to obtain the peptide fragment (1).

[0024]

[0025] Step 3: Synthesize the linear sequence of polypeptide fragment (2) using the Fmoc solid-phase polypeptide synthesis method.

[0026] 3a. Weigh 0.1 mmol of Wang resin (degree of substitution 0.42 mmol / g), add 10 mL of DMF, and allow the resin to swell for 30 minutes. Use a diaphragm pump as the power source to dry the swollen product to obtain the swollen resin.

[0027] 3b. Weigh out a fixed amount of tryptophan (W), Oxyma, and DMAP according to the molar ratio of Resin:aa:DIC:Oxyma:DMAP = 1:8:8:10:1, and take a certain volume of DIC. Place the swollen resin into a synthesis tube, and wash the resin three times with DMF, DCM, and DMF in sequence. Dissolve the weighed W, DIC, Oxyma, and DMAP in DMF in an EP tube, mix well, and add to the synthesis tube. React overnight on a shaker at room temperature. After adding the first amino acid, wash the resin three times with DMF, DCM, and DMF in sequence. Then, block twice with acetic anhydride:DIEA:DMF = 1:1:8, 10 min each time (blocking unreacted OH on the resin). After blocking, wash the resin using the above method. Then add 20% piperidine (100 mL piperidine dissolved in 400 mL DMF) and de-Fmoc on a high-temperature shaker (75 °C). After 5 minutes, clean the resin. Then, weigh out a fixed amount of tyrosine (Y), HATU, and HOAT according to the molar ratio of Resin:aa:HATU:HOAT:DIEA = 1:5:5:5:10, and take a certain volume of DIEA. Dissolve the DIEA in DMF in an EP tube, mix well, and add it to the synthesis tube. Incubate twice at room temperature on a shaker for 30 minutes each time. Remove Fmoc and repeat the steps to inoculate the third amino acid Y. After coupling, clean the resin three times with DMF, DCM, and DMF in sequence. After inoculating the three amino acids W, Y, and Y, synthesize the remaining amino acids in the order of GHGYPGHG 2-[(triphenylmethyl)thio]acetic acid using a fully automated microwave peptide synthesizer. Remove the completed peptide from the fully automated microwave peptide synthesizer.

[0028]

[0029] Step 4: Synthesize polypeptide fragments (2)

[0030] 4a. Weigh out a certain amount of mercaptoacetic acid, HATU and HOAT according to the molar ratio of Resin:thioglycolic acid:HATU:HOAT:DIEA = 1:5:5:5:10, and take a certain volume of DIEA. Dissolve it in DMF in an EP tube, mix well, and add it to the synthesis tube. Incubate in a shaker at room temperature for 30 minutes. After incubation, clean the tube with DCM and dry it. A global deprotection and lysis step was performed to remove all side-chain protecting groups and lyse the peptide from the resin. The crude peptide was lysed from the hydrazine resin by shaking in a shaker at room temperature for 3 hours using a lysis buffer containing TFA (10 mL TFA lysis mixture (524 mg phenol, 500 μL anisole, 500 μL water, 500 μL tips, 250 μL 1,2-ethylene glycol and 8 mL trifluoroacetic acid)). The resin and lysis buffer were separated, and the lysis buffer was blown down to a volume of less than 5 mL under positive pressure nitrogen. After adding excess cold diethyl ether to the lysis buffer, the mixture was centrifuged and the precipitate was collected. The precipitation was repeated 2-3 times, and the crude peptide was allowed to air dry.

[0031] 4b. Dissolve the crude peptide obtained in 4a in guanidine hydrochloride at pH 7.0 (2-3 mL). Weigh DTNB (2 eq) and dissolve it in 4 mL of guanidine hydrochloride at pH 7.0. Add the crude peptide solution dropwise to the DTNB solution, adjust the pH to 7.0, and react at room temperature for 1 h. After the reaction is complete, purify the peptide using semi-preparative high-performance liquid chromatography (HPLC). Collect the purified solution and freeze-dry it to obtain the polypeptide fragment (2).

[0032]

[0033] Step 5: Synthesize novel phase-separated peptides

[0034] The polypeptide fragment (1) obtained in step 2 and the polypeptide fragment (2) obtained in step 4 were dissolved in guanidine hydrochloride, the pH was adjusted to 7.0, and after reacting for 1 h, they were purified by semi-preparative high performance liquid chromatography. The purified solution was collected and lyophilized to obtain a novel phase-separated peptide linked by disulfide bonds.

[0035]

[0036] The present invention relates to the application of novel phase-separated peptides as delivery carriers in intracellular protein delivery processes.

[0037] The novel phase-separated peptides of this invention self-assemble into condensed layer droplets through liquid-liquid phase separation (LLPS). During droplet formation, proteins can be rapidly recruited. After the condensates bypass the classic endocytosis pathway and enter the cell, intracellular glutathione (GSH) triggers dissociation and release of proteins.

[0038] The beneficial effects of this invention are reflected in:

[0039] This invention relates to novel phase-separated peptides for intracellular protein delivery, characterized by high preparation yield, high synthetic purity, simple operation, and large-scale production capability. As natural amino acid building blocks, they exhibit low toxicity, simple synthesis steps, and low synthesis cost. A key feature is their ability to form dynamic, membrane-free biomolecular condensates through liquid-liquid phase separation. As advanced protein delivery carriers, tissue engineering scaffolds, and other biomedical materials, they create biomimetic microenvironments that promote cell growth. Attached Figure Description

[0040] Figure 1 is a high-performance liquid chromatogram of the hydrazine-terminated polypeptide fragment (1). As can be seen from Figure 1, the elution time of the hydrazine-terminated polypeptide fragment (1) is as follows.

[0041] Figure 2 is the mass spectrum of the hydrazine-terminated polypeptide fragment (1). As can be seen from Figure 2, the molecular weight of the hydrazine-terminated polypeptide fragment (1) is consistent with the theoretical molecular weight.

[0042] Figure 3 is a high-performance liquid chromatogram of the MEA terminus of polypeptide fragment (1). As can be seen from Figure 3, the elution time of the MEA terminus of polypeptide fragment (1) is as follows.

[0043] Figure 4 is the mass spectrum of the MEA terminus of polypeptide fragment (1). As can be seen from Figure 4, the molecular weight of the MEA-terminated polypeptide fragment (1) is consistent with the theoretical molecular weight.

[0044] Figure 5 is a high-performance liquid chromatogram of the thioglycolic acid terminus of polypeptide fragment (2). As can be seen from Figure 5, the elution time of the thioglycolic acid terminus of polypeptide fragment (2) is shown.

[0045] Figure 6 is the mass spectrum of the thioglycolic acid terminus of polypeptide fragment (2). As can be seen from Figure 6, the molecular weight of the thioglycolic acid terminus of polypeptide fragment (2) is consistent with the theoretical molecular weight.

[0046] Figure 7 is a high-performance liquid chromatogram of the DTNB-terminated peptide fragment (2). As can be seen from Figure 7, the elution time of the DTNB-terminated peptide fragment (2) is as follows.

[0047] Figure 8 is the mass spectrum of the DTNB terminus of polypeptide fragment (2). As can be seen from Figure 8, the molecular weight of the DTNB terminus of polypeptide fragment (2) is consistent with the theoretical molecular weight.

[0048] Figure 9. High-performance liquid chromatography (HPLC) chromatogram of the novel phase-separated peptide. Figure 9 shows the elution times of the novel phase-separated peptide.

[0049] Figure 10. Mass spectrum of the novel phase-separated peptide. As can be seen from Figure 10, the molecular weight of the novel phase-separated peptide is consistent with the theoretical molecular weight.

[0050] Figure 11. Confocal fluorescence microscopy image of EGFP delivery using the novel phase-separated peptide. As shown in Figure 11, EGFP was successfully delivered into the cell and released after 4 hours. Detailed Implementation

[0051] To facilitate understanding of the present invention, the implementation process of the present invention will be further described below with reference to specific embodiments. These descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims.

[0052] Example 1:

[0053] 1a. Take 0.1 mmol of 2-Cl-Trt-Cl resin (degree of substitution is 0.32 mmol / g), add 10 mL of N,N-dimethylformamide (DMF), and let the resin swell for 30 minutes. Use a diaphragm pump as the power source to dry the swollen product to obtain the swollen resin.

[0054] 1b. Add 4 mL of DMF solution containing 5% (volume fraction) hydrazine hydrate to the resin obtained in 1a to induce acylation. Place the resin in a shaker at room temperature and shake for 30 minutes. Then wash the resin three times each with DMF, DCM and DMF respectively. Add 4 mL of DMF solution containing 5% hydrazine hydrate again and react for 30 minutes. After washing with the same method, add 4 mL of DMF solution containing 5% (volume fraction) methanol and shake for another 10 minutes to block the unreacted active groups on the resin. After thorough washing, dry the resin to obtain the polypeptide hydrazine resin for solid-phase synthesis.

[0055] 1c. According to the linear amino acid sequence of polypeptide fragment (1), a certain amount of K (ivDde), HATU, and HOAT with side chains protected by the ivDde protecting group were weighed according to the standard Fmoc solid-phase polypeptide synthesis (SPPS) method in the molar ratio of Resin:aa:2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU): N-hydroxy-7-azabenzotriazole (HOAT): N,N-diisopropylethylamine (DIEA) = 1:5:5:5:10. A certain volume of DIEA was dissolved in DMF in an EP tube, mixed well, and added to the synthesis tube. The mixture was added twice in a shaker at room temperature for 30 min each time. After adding the first amino acid, the remaining amino acids were synthesized in the order YPGHGYRGHGYRGHG using an automated microwave polypeptide synthesizer.

[0056] Example 2:

[0057] Take the peptide obtained in Example 1 out of the fully automated microwave peptide synthesizer, clean it with DMF, and remove ivDde. Add 10 ml of 5% hydrazine hydrate to the resin and shake it at room temperature for 10 min. Then clean it with DMF. Repeat this operation 5-6 times.

[0058] [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (AEEA) was linked to the lysine side. A measured amount of AEEA, HATU, and HOAT were weighed according to the molar ratio of Resin:AEEA:HATU:HOAT:DIEA = 1:5:5:5:10. A certain volume of DIEA was also taken, dissolved in DMF in an EP tube, mixed well, and added to the synthesis tube. The mixture was incubated twice on a shaker at room temperature, 30 min each time. Fmoc was removed by adding 20% ​​piperidine (100 mL piperidine dissolved in 400 mL DMF), and the mixture was incubated on a shaker at 75°C for 5 min. The resin was then washed. Weigh out measured amounts of benzoic acid, HATU, and HOAT according to the molar ratio of Resin:benzoic acid:HATU:HOAT:DIEA = 1:2:2:2:4, and take a certain volume of DIEA. Dissolve the DIEA in DMF in an EP tube, mix well, and add it to the synthesis tube. Inject the mixture twice at room temperature on a shaker, 30 minutes each time. After injection, clean the resin for the final rinse with DCM, and then dry the resin.

[0059] The synthesized peptide underwent a global deprotection and cleavage process to remove all side-chain protecting groups and cleave the peptide from the resin. The crude peptide was cleaved from the hydrazine resin by shaking with a lysis buffer containing TFA in a shaker at room temperature for 3 hours. The resin and lysis buffer were separated, and the lysis buffer was blown down to a volume of less than 5 ml under positive nitrogen pressure. Excess cold diethyl ether was added to the lysis buffer, followed by centrifugation to collect the precipitate. This process was repeated, with the crude peptide passed through cold diethyl ether three times before air-drying. The peptide was purified using semi-preparative high-performance liquid chromatography (HPLC), and the purified solution was collected and lyophilized.

[0060] Weigh 5 mg of the lyophilized peptide, and 13.8 mg of NaNO2. Dissolve the peptide in 1 mL of pH 2-3 Gn-HCl. Add approximately 1 mL of guanidine hydrochloride (pH 2.3) to the peptide. Pre-cool in an ice-salt bath for 5 min. Add 34.36 μL of NaNO2 and measure the pH to between 2 and 3. Activate for 20 min. Dissolve 4.619 mg of MPAA (40 eq) in 40 μL of 1M NaOH solution. Add the dissolved MPAA to the peptide and adjust the pH to 4.8. React at room temperature for 10 min. Weigh MEA and dissolve it in 300 μL of pH 7.0 guanidine hydrochloride. Add the dissolved MEA to the thioester intermediate and adjust the pH to 6.8. React for 3 h. Add DTT (500 mM stock solution) for monitoring. Then, purify the peptide using semi-preparative high-performance liquid chromatography. Collect the purified solution and lyophilize to obtain the peptide fragment (1).

[0061] Example 3:

[0062] Weigh 0.1 mmol of Wang resin (degree of substitution 0.42 mmol / g), add 10 mL of DMF, and allow the resin to swell for 30 minutes. Use a diaphragm pump as the power source to dry the swollen product to obtain the swollen resin.

[0063] Weigh out a fixed amount of tryptophan (W), Oxyma, and DMAP according to the molar ratio of Resin:aa:DIC:Oxyma:DMAP = 1:8:8:10:1, and take a certain volume of DIC. Place the swollen resin into a synthesis tube, and wash the resin three times sequentially with DMF, DCM, and DMF. Dissolve the weighed W, DIC, Oxyma, and DMAP in DMF in an EP tube, mix well, and add to the synthesis tube. Place in a shaker at room temperature overnight. After adding the first amino acid, wash the resin three times sequentially with DMF, DCM, and DMF. Then, block twice with acetic anhydride:DIEA:DMF = 1:1:8, 10 min each time. After blocking, wash the resin using the above method. Remove Fmoc by adding 20% ​​piperidine and de-de-oxidizing on a high-temperature shaker (75℃) for 5 min, and then wash the resin. Weigh out a fixed amount of tyrosine (Y), HATU, and HOAT according to the molar ratio of Resin:aa:HATU:HOAT:DIEA = 1:5:5:5:10, and dissolve a certain volume of DIEA in DMF in an EP tube. Mix well and add to the synthesis tube. Incubate twice at room temperature on a shaker for 30 minutes each time. Remove Fmoc. Repeat the steps to add the third amino acid Y. After coupling, wash the resin three times with DMF, DCM, and DMF in sequence. After adding the three amino acids W, Y, and Y, synthesize the remaining amino acids in the order of GHGYPGHG 2-[(triphenylmethyl)thio]acetic acid using a fully automated microwave peptide synthesizer.

[0064] Example 4:

[0065] Weigh out quantitative amounts of thioglycolic acid, HATU, and HOAT according to the molar ratio of Resin:thioglycolic acid:HATU:HOAT:DIEA = 1:5:5:5:10, and take a certain volume of DIEA. Dissolve the DIEA in DMF in an EP tube, mix well, and add it to the synthesis tube. Incubate on a shaker at room temperature for 30 min. After incubation, clean with DCM and dry. Perform global deprotection and lysis steps to remove all side-chain protecting groups and lyse the peptide from the resin. Use lysis buffer containing TFA and shake on a shaker at room temperature for 3 h to lyse the crude peptide from the hydrazine resin. Separate the resin from the lysis buffer. Under positive pressure nitrogen, blow the lysis buffer to a volume of less than 5 ml. Add excess cold diethyl ether to the lysis buffer, centrifuge, and retain the precipitate. Repeat this operation. Pass the crude peptide through cold diethyl ether three times and then air dry.

[0066] The crude peptide was dissolved in guanidine hydrochloride at pH 7.0 (2-3 mL). DTNB (2 eq) was weighed and dissolved in 4 mL of guanidine hydrochloride at pH 7.0. The crude peptide solution was then added dropwise to the DTNB solution to adjust the pH to 7.0. The reaction was carried out at room temperature for 1 h. After the reaction was complete, the peptide was purified using semi-preparative high-performance liquid chromatography (HPLC). The purified solution was collected and lyophilized. Peptide fragment (2) was obtained.

[0067] Example 5:

[0068] Peptide fragment (1) and peptide fragment (2) were dissolved in guanidine hydrochloride, pH was adjusted to 7.0, and after reacting for 1 h, they were purified by semi-preparative high performance liquid chromatography. The purified solution was collected and lyophilized to obtain a novel phase-separated peptide linked by disulfide bonds.

[0069] Example 6:

[0070] HeLa cells were cultured under standard conditions (37°C, 5% CO2) in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 µg / mL streptomycin. For passage, cells were first detached by trypsin treatment, then collected by centrifugation (300×g, 5 min), and the cell pellet was resuspended in fresh medium for passage or experiments. Cells were resuspended in 1.5 mL of complete medium and transferred to a 35 cm³ culture medium. 2In culture dishes. When cell confluence reached approximately 80%, the medium was replaced with 900 µL of Opti-MEM, followed by the addition of 100 µL of freshly prepared CMS (0.3 mM novel phase-separating peptide, 0.1 mg / mL EGFP). This CMS solution was prepared by adding 10 µL of a novel phase-separating peptide stock solution (10 µL, prepared as a 3 mM stock solution by dissolving the novel phase-separating peptide in PBS buffer at pH 7.0) to a buffer containing 90 µL of EGFP (prepared as a 0.1 mg / mL stock solution by dissolving EGFP in PBS buffer at pH 7.0). The treated cells were imaged using a fluorescence microscope (AxioObserver.ZI, Zeiss) after 4 hours.

[0071] In summary, this invention provides a novel phase-separated peptide capable of intracellular protein delivery.

[0072] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, nor does the order of the various embodiments limit the present invention in any way. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A novel phase-separated peptide for intracellular protein delivery, characterized in that... Its structure is as follows: 。 2. The method for synthesizing the novel phase-separated peptide according to claim 1, characterized in that: First, linear sequence peptide fragments (1) and (2) were synthesized using the Fmoc solid-phase peptide synthesis method. Then, the lysine side chain amino protecting group ivDde of peptide fragment (1) was removed, and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid and benzoic acid were coupled. Finally, a novel phase-separated peptide molecule was obtained by forming a disulfide bond between the two fragments. The peptide fragment (1) is H2N-GHGRYGHGRYGHGPYK(ivDde)-CONHNH2; the peptide fragment (2) is HS-CH2-CO-HN-GHGPYGHGYYW-COOH. The synthetic route is shown below: 。 3. The synthesis method according to claim 2, characterized in that... The steps include: Step 1: Synthesize polypeptide fragments (1) linear sequence 1a using the Fmoc solid-phase polypeptide synthesis method. Take 2-Cl-Trt-Cl resin and add it to N,N-dimethylformamide to swell the resin. Use a diaphragm pump as a power source to dry the swollen product and obtain the swollen resin. 1b. Add a DMF solution containing 5% hydrazine hydrate to the resin obtained in 1a to induce acylation. Then wash with DMF, DCM and DMF in sequence. Add a DMF solution containing 5% hydrazine hydrate again to react. After washing with the same method, add a DMF solution containing 5% methanol and continue shaking for 10 minutes to block the unreacted active groups on the resin. After thorough washing, dry to obtain the polypeptide hydrazine resin for subsequent solid-phase synthesis. 1c. According to the linear amino acid sequence of the polypeptide fragment (1), weigh K (ivDde), HATU and HOAT with the ivDde protecting group on the side chain according to the standard Fmoc solid-phase polypeptide synthesis method. Take a certain volume of DIEA, add DMF to the EP tube to dissolve, mix well, add to the synthesis tube, and react in a shaker at room temperature. After adding the first amino acid, the remaining amino acids are synthesized in the order YPGHGYRGHGYRGHG using an automatic microwave polypeptide synthesizer. Step 2: Synthesize peptide fragments (1) 2a. Take the peptide obtained in step 1 from the fully automated microwave peptide synthesizer, clean it with DMF, remove ivDde, add 5% hydrazine hydrate to the resin and shake at room temperature, then clean with DMF; 2b. Next, weigh AEEA, HATU, HOAT and DIEA, add DMF to the EP tube to dissolve, mix well, add to the synthesis tube, remove Fmoc after reaction, and clean the resin; then weigh benzoic acid, HATU, HOAT and DIEA, add DMF to the EP tube to dissolve, mix well, add to the synthesis tube, remove Fmoc after reaction, and clean the resin; then weigh benzoic acid, HATU, HOAT and DIEA, add DMF to the EP tube to dissolve, mix well, add to the synthesis tube 1. React in a tube, wash the resin and dry it; 2c. Perform global deprotection and cleavage steps on the peptide synthesized in 2b to remove all side chain protecting groups and cleave the peptide from the resin; use lysis buffer containing TFA to shake in a shaker at room temperature for 3 hours to cleave the crude peptide from the hydrazine resin, separate the resin from the lysis buffer, blow the lysis buffer to a volume of less than 5 ml under positive pressure nitrogen, add excess cold diethyl ether to the lysis buffer, centrifuge and retain the precipitate, repeat the precipitation 2-3 times, and let the crude peptide air dry naturally; purify using semi-preparative high performance liquid chromatography, collect the purified solution and freeze dry; 2d. The lyophilized peptide obtained in 2c and NaNO2 were dissolved in Gn-HCl at pH 2-3, guanidine hydrochloride at pH 2.3 was added, and the mixture was pre-cooled in an ice-salt bath. Then NaNO2 was added, and the pH of the system was adjusted to 2-3. The reaction was carried out for 10 min. Then MPAA was dissolved in NaOH solution and added to the system. The pH of the system was adjusted to 4.8, and the reaction was carried out at room temperature for 10 min. MEA was weighed and dissolved in guanidine hydrochloride at pH 7.

0. The pH was adjusted to 6.8, and the reaction was carried out for 3 h. Dithiothreitol was added to monitor the reaction. After monitoring, the peptide was purified by semi-preparative high performance liquid chromatography. The purified solution was collected and lyophilized to obtain the peptide fragment (1). Step 3: Synthesize the polypeptide fragment (2) linear sequence using the Fmoc solid-phase polypeptide synthesis method. 3a. Weigh the Wang resin and add it to DMF to swell. Use a diaphragm pump as the power source to dry the swollen product and obtain the swollen resin. 3b. Put the swollen resin into the synthesis tube. Dissolve tryptophan, DIC, Oxyma and DMAP in DMF in the EP tube, mix well, and add to the synthesis tube. React overnight on a shaker at room temperature. After adding the first amino acid, wash the resin and seal the unreacted OH on the resin. Then add piperidine and remove Fmoc on a shaker at high temperature. Then weigh tyrosine, HATU, HOAT and DIEA, dissolve them in DMF in the EP tube, mix well, add to the synthesis tube, and add twice on a shaker at room temperature to remove Fmoc. Repeat the above steps to add the third amino acid Y. After coupling, wash the resin. After adding the three amino acids W, Y and Y, the remaining amino acids are added according to GHGYPGHG. 2-[(triphenylmethyl)thio]acetic acid was synthesized using a fully automated microwave peptide synthesizer. The completed peptide was then removed from the fully automated microwave peptide synthesizer. Step 4: Synthesize polypeptide fragment (2) 4a. Weigh mercaptoacetic acid, HATU, HOAT and DIEA, add DMF to an EP tube to dissolve, mix well, add to a synthesis tube, and react in a shaker at room temperature; use a lysis buffer containing TFA to shake the reaction in a shaker at room temperature to lyse the crude peptide from the hydrazine resin; separate the resin from the lysis buffer, and under positive pressure nitrogen, blow the lysis buffer to a volume of less than 5 ml; add excess cold ether to the lysis buffer, centrifuge and retain the precipitate, repeat the precipitation 2-3 times, and let the crude peptide air dry naturally; 4b. Dissolve the crude peptide obtained in 4a in guanidine hydrochloride at pH=7.0, weigh DTNB and dissolve it in guanidine hydrochloride at pH=7.0, add the crude peptide solution dropwise to the DTNB solution, adjust the pH to 7.0, and react at room temperature for 1 h; after the reaction is completed, purify using semi-preparative high performance liquid chromatography, collect the purified solution and freeze dry to obtain polypeptide fragment (2). Step 5: Synthesize novel phase-separated peptides. Dissolve the polypeptide fragment (1) obtained in step 2 and the polypeptide fragment (2) obtained in step 4 in guanidine hydrochloride, adjust the pH to 7.0, react for 1 h, and then purify using semi-preparative high performance liquid chromatography. Collect the purified solution and freeze dry to obtain novel phase-separated peptides linked by disulfide bonds. 。 4. The application of the novel phase-separated peptide of claim 1 as a delivery carrier in intracellular protein delivery.

5. The application according to claim 4, characterized in that: The novel phase-separated peptides self-assemble into condensed layer droplets through liquid-liquid phase separation. During droplet formation, proteins can be rapidly recruited. After the condensates bypass the classic endocytosis pathway and enter the cell, intracellular glutathione (GSH) triggers dissociation and release of proteins.

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