CN112830975B - Pro-apoptotic bicyclic polypeptide with stable α-helical conformation, preparation method and application - Google Patents

Abstract

The invention discloses a pro-apoptotic bicyclic polypeptide with stable alpha-helix conformation, and a preparation method and application thereof, wherein the pro-apoptotic bicyclic polypeptide comprises a pro-apoptotic functional peptide segment, a tumor targeting functional peptide segment, a corner amino acid residue and a thioether-containing molecular skeleton, the pro-apoptotic peptide segment is an amphiphilic sequence rich in lysine and leucine, the corner amino acid is proline, and the thioether-containing molecular skeleton adds two molecules of mercaptopropionic acid to an alkynyl-containing unnatural amino acid side chain through thiol-alkynyl click chemistry and is obtained through macrolactamization. The preparation method of the polypeptide is simple and effective, and the cyclization efficiency can be obviously improved by carrying out double cyclization at the ith and i +7 amino acid residue positions of the apoptosis-promoting peptide functional peptide segment.

Description

Pro-apoptotic bicyclic polypeptide with stable α-helix conformation, preparation method and application

technical field

The invention belongs to the field of biomedicine, and in particular relates to a pro-apoptotic bicyclic polypeptide with a stable α-helix conformation, a preparation method and application thereof.

Background technique

The pro-apoptotic peptide KLA (KLAKLAKKLAKLAK) is an α-helical amphipathic polypeptide with antibacterial activity, which can target mitochondria and damage mitochondrial membranes, resulting in the release of cytochrome C and inducing apoptosis. It has been widely reported that it can be used in malignant Tumor treatment. However, linear KLA polypeptides have problems such as poor stability, poor cell penetration, and poor cell activity. At present, it is reported in the literature that the pro-apoptotic peptide KLA is delivered into cells mainly by coupling ligands and using receptor-mediated endocytosis. For example, by further modifying the cyclic NGR polypeptide ligand in the pro-apoptotic peptide KLA sequence, the AHNP polypeptide ligand or by folic acid. Therefore, the pro-apoptotic peptide KLA can effectively target the mitochondria of cells and destroy the mitochondrial membrane, thereby causing the release of cytochrome C, activating the apoptosis pathway of cells, and finally being used for the treatment of malignant tumors. Therefore, pro-apoptotic peptide drugs have gradually become research hotspot.

In the presence of the advantages of peptide drugs over small molecule drugs, traditional peptide drugs still have various problems, such as poor stability of linear peptides, poor cell penetration, functional peptides without targeting, and Targeted polypeptides have disadvantages such as no functionality, and can only target receptors in cells or on the surface of cells alone. Therefore, it is still a huge challenge to modify and modify the shortcomings of peptide drugs.

Contents of the invention

The invention solves the technical problems of poor metabolic stability, low cell penetration ability and weak targeting of traditional polypeptides.

The applicant developed a variety of new peptide stabilization systems in the previous research, and found that chemical cyclization can effectively improve the stability of the peptide, enhance the cell penetration ability of the peptide, improve the activity of the peptide, and chemically modify different side chains. It can also significantly affect the conformation of the polypeptide, amphiphilicity and other biophysical properties, but this type of monocyclic polypeptide lacks tumor targeting. Based on the above research results, the applicant further constructed a sulfide-containing ether by photo-induced thiol-alkyne click chemistry The α-helical conformation of the side chain with dual targeting capabilities stabilizes the bicyclic polypeptide.

According to the first aspect of the present invention, a pro-apoptotic bicyclic polypeptide with a stable α-helical conformation is provided, the pro-apoptotic bicyclic polypeptide has a stable α-helical secondary structure, which includes a pro-apoptotic functional peptide, tumor Targeting functional peptides, corner amino acid residues and thioether-containing molecular skeletons, the pro-apoptosis functional peptides and tumor targeting functional peptides are connected through the corner amino acid residues and the linking molecules are used to form a thioether-containing molecular skeleton double ring structure.

Preferably, the linking molecule is any one of mercaptopropionic acid, mercaptoacetic acid or mercaptobutyric acid.

Preferably, the turn amino acid residue is any one of proline or glycine, and proline and glycine are most protein turn amino acids, and this turn function can provide a gap between the pro-apoptotic peptide and the targeting peptide. flexibility.

Preferably, _the tumor-targeting functional peptide is the RGD sequence targeting _αvβ3 integrin, the somatostatin receptor octreotide fCYwKTCT sequence, the lanreotide sequence D-2-Nal-CYwKVCT sequence and the Any one of the polypeptide sequences YCDGFYACYMDV sequences of the Her2 receptor.

Preferably, the thioether-containing molecular skeleton is located at the i-th amino acid position in the proapoptotic functional peptide sequence, and the cyclization positions of the bicyclic structure respectively start with the amino acid residues of the thioether-containing side chain, and The amino acid residue at position i+4, i+7 or i+11 in the C-terminal direction of the polypeptide forms a loop and forms a ring with the amino acid residue at the N-terminal of the polypeptide to form a bicyclic molecular skeleton.

Cyclization at the positions of i and i+4, i and i+7, i and i+11 can significantly improve the efficiency of cyclization, because in the polypeptide sequence of α-helical structure, i and i+4, i The amino acid side chains at positions +7, i+11 are on the same side of the helix.

Preferably, the structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 1:

Alternatively, the structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 2:

According to another aspect of the present invention, a method for preparing a pro-apoptotic bicyclic polypeptide with a stable α-helical conformation is provided, and the specific preparation process is as follows:

S1. Synthesize the linear polypeptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc)K-Resin using Fmoc solid-phase peptide synthesis technology, wherein X is an unnatural amino acid with an alkynyl side chain;

S2. Remove the Fmoc group from the tryptophan at the end of the linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc)K-Resin to obtain a linear polypeptide;

S3. Formation of a thioether-containing side chain molecular skeleton precursor using photoinitiated mercapto-alkyne click chemistry reaction: Add 2 equivalents of photoinitiator 2,2-dimethylolpropionic acid to 1 equivalent of linear polypeptide product obtained in step S2 DMPA, 4 equivalents of mercaptopropionic acid, reacted under 365nm ultraviolet light for 2h;

S4. Using the reaction product of step S3 to carry out macrocyclic lactamization to construct a bicyclic skeleton containing a thioether structure;

S5. The cyclized polypeptide is cut off from the resin, purified by high performance liquid chromatography, and then freeze-dried to obtain the target bicyclic polypeptide.

Preferably, in step S5, the polypeptide is cleaved from the resin with a cleavage solution composed of 2.5% H ₂ O, 2.5% TIS and 95% TFA.

According to another aspect of the present invention, application of the above-mentioned pro-apoptotic bicyclic polypeptide with stable α-helical conformation in tumor treatment is provided.

Generally speaking, compared with the prior art, the above technical solution conceived by the present invention mainly has the following beneficial effects:

(1) Creatively construct a bicyclic polypeptide backbone with a thioether structure with dual effects of therapeutic and targeting properties, and at the same time, the bicyclic polypeptide has a stable α-helical secondary conformation; The pro-apoptotic peptide that breaks down the mitochondria to promote cell apoptosis and the RGD peptide that targets the overexpressed αvβ3 integrin in tumor cells have achieved dual targeting functions in the same polypeptide backbone; at the same time, using mercapto-alkyne click chemistry The formation of a thioether-containing bicyclic molecular skeleton has low non-specific toxicity and can further increase the stability and cell penetration ability of the polypeptide.

(2) Creatively use the light reaction to synthesize a bicyclic polypeptide with a molecular skeleton containing a thioether bond, and pass the i-th and i+4 or i-th and i+7 or i-th and i+11 amino acids in the pro-apoptotic peptide segment The double cyclization at the residue position can significantly improve the cyclization efficiency, and the cyclization efficiency can reach 80%.

(3) The constructed bicyclic polypeptide has a higher degree of α-helix than the linear polypeptide, has good α-helix stability, and has a low hemolysis rate, has good blood stability and low non-specific cytotoxicity, and compared with Linear polypeptides and bicyclic polypeptides show more obvious mitochondrial killing ability on tumor cells with high integrin expression, but less killing ability on normal cells, and have good application prospects for tumor treatment.

Description of drawings

Fig. 1 is a synthetic route diagram of a pro-apoptotic bicyclic polypeptide with a stable α-helical conformation.

Figure 2 is a liquid phase diagram of the purified linear polypeptide.

Fig. 3 is a liquid phase diagram of the purified bicyclic polypeptide Cyclic.

Figure 4 is a liquid phase diagram of Cyclic-1 after separation from the two isomers.

Figure 5 is a liquid phase diagram of Cyclic-2 after separation from the two isomers.

Figure 6 is the mass spectrum of the purified linear polypeptide.

Figure 7 is the mass spectrum of Cyclic-1 after separation from the two isomers.

Figure 8 is the mass spectrum of Cyclic-2 after separation from the two isomers.

Figure 9 is the circular dichroism chromatograms of linear polypeptides and bicyclic polypeptides Cyclic-1 and Cyclic-2.

Fig. 10 is a statistical graph of the hemolysis rate of linear polypeptide and bicyclic polypeptide Cyclic at different concentrations.

Fig. 11 is a graph showing the cytotoxicity of linear polypeptides and bicyclic polypeptides cultured with different cells.

Fig. 12 is a fluorescence micrograph of linear polypeptide and bicyclic polypeptide co-incubated with 4T1.

Fig. 13 is a fluorescence micrograph of linear polypeptide and bicyclic polypeptide co-incubated with MCF-7.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

Example 1

This example provides a pro-apoptotic bicyclic polypeptide with a stable α-helical conformation, which includes a pro-apoptotic peptide functional peptide, targeting α _v β ₃ integrin RGD, a corner amino acid proline, and a thioether-containing molecular skeleton, which promotes The sequence of the functional peptide of the apoptotic peptide is KLAKLXKKLAKLKK, which is a linear sequence rich in lysine, and X is an unnatural amino acid with an alkynyl side chain, and the functional peptide of the pro-apoptotic peptide is connected to RGD through proline In order to reduce the mutual interference between the pro-apoptotic peptide and RGD, at the same time, it is convenient to turn into a loop and improve the flexibility of the junction. At the same time, the linker molecule mercaptopropionic acid is used to construct a thioether molecule on the linear peptide through a thiol-alkyne click chemical reaction. The double-ring structure of the backbone.

The amino acids in the sequence of the pro-apoptotic peptide functional peptide are all L-type amino acids, and have an α-helical conformation, which can exist more stably in vivo.

The unnatural amino acid X with an alkynyl side chain is used to replace the position of the sixth amino acid alanine A on the pro-apoptotic peptide KLAKLAKKLAKLAK, because in the sequence of the pro-apoptotic peptide KLA, mainly composed of lysine K , leucine L, and alanine A sequence composed of three amino acids, wherein lysine is a positively charged amino acid, and leucine as a hydrophobic amino acid has a significant impact on the amphipathicity of the polypeptide sequence. The sexual structure has a significant impact on the ability of the peptide to target mitochondria, so the unnatural amino acid X with an alkynyl side chain replaced alanine as the ring-forming site, and correspondingly replaced the 13th in the pro-apoptotic peptide KLAKLAKKLAKLAK with lysine The position of the amino acid alanine A.

The thioether molecular skeleton is located at the i-th amino acid position in the pro-apoptosis functional peptide sequence, and the cyclization position of the double ring structure starts from the amino acid residue of the thioether side chain and is aligned with the C-terminal direction of the polypeptide. The side chain of the i+7 amino acid residue forms a loop and forms a loop with the N-terminal amino acid residue of the polypeptide to form a bicyclic molecular skeleton.

The structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 1:

Alternatively, the structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 2:

In another optional embodiment, the linking molecule used for ring formation may also be any one of mercaptoacetic acid or mercaptobutyric acid.

In another optional embodiment, the turn amino acid can also be glycine.

In another optional embodiment, the tumor-targeting peptide can also be targeting the somatostatin receptor octreotide fCYwKTCT, the sequence of lanreotide D-2-Nal-CYwKVCT and the polypeptide sequence YCDGFYACYMDV targeting the Her2 receptor any of the.

In another optional embodiment, the cyclization position of the bicyclic structure starts with the amino acid residue of the sulfide-containing side chain, and is connected to the side of the amino acid residue at position i+4 or position i+11 in the C-terminal direction of the polypeptide. The chain forms a ring and forms a ring with the N-terminal amino acid residue of the polypeptide to form a bicyclic molecular backbone.

Example 2

This example provides a method for preparing a pro-apoptotic bicyclic polypeptide with a stable α-helical conformation, as shown in Figure 1 of the description, and the specific preparation process is as follows:

S1. Synthesize the linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc)K-Resin containing the allyloxycarbonyl Alloc side chain protection group by Fmoc solid-phase synthesis method, wherein the Alloc side chain protection group is located at the lysine on the right side of the sequence On the above, X is an unnatural amino acid with an alkyne group, and tryptophan is connected to the end of RGD. Tryptophan has an absorption peak at 280nm, and the concentration of the polypeptide can be determined by a microplate reader. The linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc) The specific structure of K-Resin is as follows:

The specific synthesis process is as follows:

First weigh 400mg of Rink resin, swell it with N,N-dimethylformamide DMF for 20min, and use deprotection solution (50% morpholine, 50% DMF, (V:V)) to remove the Fmoc protecting group on the resin , then washed three times with DMF, then washed three times with dichloromethane DCM, and then washed three times with DMF, then added the amino acid raw material dissolved in DMF and the HATU mixture, each amino acid was connected for two hours, and each amino acid was followed by the same steps React twice to increase conversion efficiency.

Polypeptides are synthesized by Fmoc solid-phase synthesis method, and Fmoc is used as the protecting group of amino acid α-amino group. It is stable under acidic conditions and is not affected by reagents such as TFA. It can be deprotected by mild alkali treatment, so the side chain can be used easily. The Boc protecting group removed by acid is used for protection, and the Fmoc method has mild reaction conditions, few side reactions, and high yield, and the Fmoc group itself has characteristic ultraviolet absorption, which is easy to monitor and control the progress of the reaction.

S2. Remove the Fmoc group from the tryptophan at the end of the linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK (Alloc) K-Resin;

The specific process is as follows:

Add 0.1 equivalent of tetrakis(triphenylphosphine) palladium and 4 equivalents of 1,3-dimethylbarbituric acid to 1 equivalent of linear peptide sequence containing allyloxycarbonyl Alloc side chain protection group, and use dichloromethane as Solvent, react twice, two hours each time. After the reaction, use DMF-dissolved sodium diethyldithiocarbamate (copper reagent) to wash off excess tetrakis(triphenylphosphine) palladium (Pd(PPh3)4) attached to the resin to obtain NH ₂ -WRGDfVPKLAKLXKKLAKLKK-Resin ;

S3. Using light to initiate a mercaptoalkynyl click chemical reaction to form a thioether-containing side chain molecular skeleton precursor;

The specific process is as follows:

In 1 equivalent of resin NH ₂ -WRGDfVPKLAKLXKKLAKLKK-Resin with polypeptide dissolved in N-methylpyrrolidone NMP, add 2 equivalents of photoinitiator 2,2-dimethylolpropionic acid DMPA, 4 equivalents of mercaptopropionic acid, in Addition reaction of mercaptopropionic acid and alkyne occurred under 365nm ultraviolet light irradiation for 2h.

S4. Using the reaction product of the previous step to carry out macrolactamization to construct a bicyclic skeleton containing a thioether structure;

The specific process is as follows:

Add 2 equivalents of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate PyBop, 2 equivalents of 1-hydroxybenzotriazole HOBT, 2 equivalents of N-methyl Morpholine NMM, and placed on a shaker for 12 hours.

S5. The cyclized polypeptide is excised using a shear solution prepared by mixing 2.5% H ₂ O, 2.5% TIS, and 95% TFA, purified by high performance liquid chromatography (HPLC), and then freeze-dried to obtain the target bicyclic polypeptide.

Wherein, the preparation parameter of high performance liquid chromatography is:

Undegraded polypeptides were analyzed by reverse phase HPLC (Agilent ZORBAX SB-Aq: 4.6×250 mm, 5 μm, flow rate 1.0 mL/min, 220 nm). The analysis conditions were gradient elution of solvent B from 10% to 90% within 40 minutes (solvent A: water containing 1‰ TFA (v/v), solvent B: acetonitrile).

The liquid phase chromatogram combined with linear polypeptide and cyclic peptide is shown in Figure 2-3 of the specification. It can be seen that the retention time of the purified linear polypeptide is 15.067min, while the retention time of the double-cyclized polypeptide and linear polypeptide increases, which proves that the double-cyclized The hydrophobicity of the polypeptide has been improved, and according to Figure 3, it can be seen that the retention time of the double-cyclized polypeptide is concentrated in two similar double peaks, corresponding to 18.373min and 18.767min respectively, which proves that two types of isomers can be obtained by separation and purification .

Further separation and purification by high performance liquid chromatography, and two types of bicyclic isomers Cyclic-1 and Cyclic-2 were obtained according to the sequence of peak eluting time, and the obtained liquid phase spectra were shown in 4-5, corresponding to the following structural formulas respectively:

In order to further verify the synthesis of the expected product, two different bicyclic isomers were detected by mass spectrometry. The mass spectrometry manufacturer is Waters, the model is Q-TOF, the mobile phase is methanol and water, and the analysis conditions are within 5 minutes. 10% to 90%;

Figures 6-8 are the mass spectrograms of purified linear peptides and cyclic peptides Cyclic-1 and Cyclic-2, respectively, where [M+5] ⁺ represents the molecular weight with five positive charges, and [M+4] ⁺ represents the molecular weight with The molecular weight of four positive charges, [M+3] ⁺ means the molecular weight of three positive charges, from the analysis of the mass spectrum, it can be seen that the bicyclic polypeptide with the correct molecular weight has been successfully obtained.

Cyclization can effectively constrain the conformation of polypeptides, and the conformation of polypeptides is considered to have an important impact on its penetration ability. Polypeptides can form various secondary structures such as α-helices and β-sheets. Here, circular dichroism is used to investigate linear polypeptides and bicyclic For the secondary structure of peptides, linear peptides and bicyclic peptides were dissolved in 30 mM sodium dodecyl sulfate (SDS). Circular dichroism data were collected by Jasco J-810 at room temperature, and the detection conditions were as follows: the step resolution was 0.5nm, the speed was 20nm/s, the accumulation was 10 times, the response time was 1s, the bandwidth was 1nm, and the path length was 10mm. Circular dichroism spectra were converted to mean residue molar ellipticities.

The obtained circular dichroism chromatogram is shown in Figure 9. It can be seen that the bicyclic polypeptide has a positive peak at around 190 and two negative peaks at 208nm and 222nm. This is an obvious characteristic peak of the α-helical structure. Compared with the linear polypeptide, the peak width at 222nm is significantly enhanced and broadened, which shows that the helical degree of the constructed bicyclic polypeptide is higher than that of the linear polypeptide, and further proves that the bicyclic polypeptide was successfully obtained, because the α -The helix content is significantly increased, and two negative peaks characteristic of α-helices appear at 208nm and 222nm.

Different concentrations of linear peptides and bicyclic peptides were incubated with fresh rabbit blood for 1 hour to examine their hemolytic properties. As shown in Figure 10, it can be found that both bicyclic peptides and linear peptides have low hemolytic properties and have good biological safety. .

In order to explore the selective inhibitory effect of bicyclic peptides on tumor cells, 0 μM, 5 μM, 10 μM, 20 μM, 40 μM linear polypeptides and cells with high expression of bicyclic polypeptides and α _v β ₃ integrin were used respectively. Mouse melanoma cell B16, mouse mammary gland Cancer cell 4T1 and cells with low expression of α _v β ₃ integrin, human breast cancer cell MCF-7, and normal liver cell L02 with low expression of α _v β ₃ integrin were co-incubated for 24 hours, then stained with AB dye, and detected by microplate reader OD value was used to detect cell viability. As shown in Figure 11, it can be seen that for normal cell lines, both bicyclic peptides and linear peptides exhibit lower non-specific cytotoxicity, while for tumor cell lines, bicyclic peptides have a better effect on cell killing than linear peptides , and has a better effect on cells with high integrin expression, thereby verifying the selective pro-apoptotic effect of the bicyclic polypeptide on tumor cells.

In order to verify the role of Bicyclic Peptide in targeting mitochondria, the mitochondrial membrane potential of cells was characterized by JC-1 dye for mitochondrial staining. 4T1 cells and MCF-7 cells were seeded in a confocal dish at a density of 1.2×10 ⁵ cells/well, added 10% fetal calf serum medium and cultured at 37°C, 5% CO ₂ at a constant temperature and atmosphere for 24 hours. Then add the polypeptide with a final concentration of 15 μM and incubate for 24 hours, then add the newly prepared JC-1 staining solution for staining for 10 minutes, and then wash with PBS buffer for 3 times. Put the sample under an inverted fluorescence microscope to observe and take pictures as shown in Figure 12-Figure 13. Linear polypeptides are used as controls. It can be seen that bicyclic polypeptides have a significant ability to affect mitochondrial membrane potential compared with linear polypeptides, and compared with The effect of cell lines with high integrin expression is more obvious, so it can be proved that the bicyclic polypeptide can target cell surface integrin receptors and intracellular mitochondria, selectively disturb the mitochondrial membrane potential of tumor cells with high integrin expression, and has a good application prospect in tumor therapy .

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (4)

1. A pro-apoptotic bicyclic polypeptide with stable α-helical conformation, characterized in that the pro-apoptotic bicyclic polypeptide has a stable α-helical secondary structure, which includes pro-apoptotic functional peptides and tumor targeting functional peptides segments, corner amino acid residues and a thioether-containing molecular skeleton, the pro-apoptosis functional peptide and the tumor-targeting functional peptide are connected through a turn-angle amino acid residue and a bicyclic structure containing a thioether molecular skeleton is formed by using linking molecules; The structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 1:

Alternatively, the structure of the pro-apoptotic bicyclic polypeptide is shown in Formula 2:

Or, the linking molecule in formula 1 or formula 2 is any one of thioglycolic acid or mercaptobutyric acid. 2. The preparation method of a kind of α-helical conformation stable proapoptotic bicyclic polypeptide as claimed in claim 1, the specific preparation process is as follows: S1. Synthesize the linear polypeptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc)K-Resin using Fmoc solid-phase peptide synthesis technology, wherein X is an unnatural amino acid with an alkynyl side chain; S2. Remove the Fmoc group from the tryptophan at the end of the linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK(Alloc)K-Resin to obtain a linear polypeptide; S3. Formation of a thioether-containing side chain molecular skeleton precursor using photoinitiated mercapto-alkyne click chemistry reaction: Add 2 equivalents of photoinitiator 2,2-dimethylolpropionic acid to 1 equivalent of linear polypeptide product obtained in step S2 DMPA, 4 equivalents of mercaptopropionic acid, reacted under 365nm ultraviolet light for 2h; S4. Using the reaction product of step S3 to carry out macrocyclic lactamization to construct a bicyclic skeleton containing a thioether structure; S5. The cyclized polypeptide is cut off from the resin, purified by high performance liquid chromatography, and then freeze-dried to obtain the target bicyclic polypeptide. 3. The preparation method of a pro-apoptotic bicyclic polypeptide with stable α-helical conformation as claimed in claim 2, characterized in that, in step S5, a mixture of 2.5% H ₂ O, 2.5% TIS and 95% TFA is used. The cleavage solution cleaves the peptide from the resin. 4. The application of a pro-apoptotic bicyclic polypeptide with stable α-helical conformation as claimed in claim 1 in the preparation of tumor therapeutic drugs.

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