CN110627875A

CN110627875A - Mitochondrion targeting membrane-penetrating cyclopeptide and preparation method and application thereof

Info

Publication number: CN110627875A
Application number: CN201910991319.8A
Authority: CN
Inventors: 孟祥明; 杨倩倩; 方葛敏; 朱满洲
Original assignee: Hefei Xiuhe Biotechnology Co Ltd
Current assignee: Hefei Xiuhe Biotechnology Co Ltd
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2019-12-31

Abstract

The invention discloses a mitochondrion targeting transmembrane cyclopeptide and a preparation method and application thereof, wherein the structure of the mitochondrion targeting peptide is as follows:the mitochondrion targeting transmembrane cyclopeptide can specifically target cell mitochondrion, and confocal inverted fluorescence microscopic imaging experiments show that the structure of the invention has good transmembrane property on Hela cells and is comparable to commercial cellsThe co-localization coefficient Pr of the mitochondrial dye is 0.74, and the structure can be targeted and localized to mitochondria efficiently.

Description

Mitochondrion targeting membrane-penetrating cyclopeptide and preparation method and application thereof

Technical Field

The invention relates to chemical synthesis of a mitochondrion-targeted transmembrane cyclopeptide and application of the mitochondrion-targeted transmembrane cyclopeptide in the aspect of active substance delivery.

Background

The main function of mitochondria is to efficiently convert the energy stored in organic matter into direct energy for cell life activities, which is needed for cell life activities. Mitochondria have been reputed as a "power plant" as the primary site of animal cell respiration. Mitochondria play a crucial role in maintaining normal cellular activities. Mitochondria are involved in many links of the cell's life process, including cell physiology, reactive oxygen species production, calcium homeostasis, and apoptosis. Mitochondrial dysfunction is closely associated with the development of various diseases, such as: brain necrosis, cardiomyopathy, tumor, infertility, Parkinson's syndrome, etc. The permselectivity of cell membranes and the high negative membrane potential of the mitochondrial bilayer membrane act as a large barrier for macromolecular bioactive substances to enter mitochondria. The inability of targeted delivery of bioactive substances to mitochondria makes the treatment of mitochondrial-related diseases difficult to combat. With the development of molecular tools targeting mitochondria, selective bioimaging of mitochondria is achieved. Makes it possible to functionally intervene and treat mitochondrial-related diseases.

Over the past few decades, there has been a constant search for the targeted delivery of various bioactive substances to mitochondria to achieve the regulation and treatment of diseases associated with mitochondria. Through the step-by-step effort of people. Currently, the most common method of targeted drug delivery to mitochondria is to covalently attach a lipophilic cation (such as alkyltriphenylphosphino) to the drug group of interest. Nanoparticles, as well as natural and synthetic mitochondrial targeting peptide carriers, have also been used for the delivery of mitochondrial small molecules. Different kinds of active biomolecules require different kinds and structures of carriers to achieve the purpose of targeted delivery. The development of the mitochondrion targeting technology opens a new window for researching the mitochondrion physiology and dysfunction and the mutual connection between the mitochondrion and other subcellular organelles, and provides a new idea for treating various mitochondrion related diseases. It is worth noting that the mitochondrial targeting peptide as a delivery scaffold has unique advantages, such as easy synthesis, tunability, biocompatibility and high uptake in cells and in vivo. The simple solid-phase synthesis method can realize the synthesis of different sequences, and various side chain groups can meet the modification of different types of substances. Due to its simple and modular synthesis and multiple structural sites available for modification, these peptides are well suited for the delivery of different types of cargo.

Cationic peptides consisting of the unnatural amino acids cyclohexylalanine and D-arginine, developed by Kelley and his colleagues, showed better mitochondrial targeting ability. Bioactive molecules such as antitumor drugs (Dox), siRNA, plasmid DNA and the like are successfully transported to mitochondria by applying the structure, and the specificity of the bioactive molecules can be improved by modifying a peptide sequence or introducing an artificially synthesized amino acid side chain. However, even minimal sequence changes can have a dramatic effect on the membrane-penetrating transport activity of the peptide. Therefore, optimizing and providing diverse mitochondrial targeting peptides is of great importance for the delivery of bioactive molecules and the treatment of mitochondrial diseases.

Disclosure of Invention

The invention aims at the defect that a mitochondrion targeting peptide is not easy to modify and aims at providing a mitochondrion targeting transmembrane cyclopeptide and a preparation method and application thereof. The invention optimizes the reported mitochondrion membrane penetrating peptide to form a mitochondrion targeting peptide with more stable structure and high modifiability. In addition, the invention also carries out cell imaging experiment on the screened mitochondrial penetrating peptide. The effect of the mitochondria on penetrating the cell membrane of the peptide was evaluated in comparison with commercial mitochondrial dyes.

The mitochondrion targeting transmembrane cyclopeptide is abbreviated as CMPP-1, and the general structural formula of the mitochondrion targeting transmembrane cyclopeptide is shown as the following formula I:

the preparation method of the mitochondrion targeting transmembrane cyclopeptide comprises the following steps:

step 1: synthesis of hydrazide resins

Adding 200mg of 2-Cl-Trt-Cl resin into 2mL of DMF for swelling, dropwise adding a mixed solution of 266uL DIEA (N, N-diisopropylethylamine), 80uL hydrazine hydrate and 800uL DMF under the conditions of ice bath and stirring, stirring for 20min under ice bath, transferring the system to room temperature, stirring for reacting for 70min, and adding 40uL methanol to finish the reaction; transfer the resulting hydrazide-Trt (2-Cl) resin to a sand core funnel, wash thoroughly with DMF, MeOH, ether sequentially for 3-5 times, and dry under vacuum for 1 h.

Step 2: synthesis method of CMPP-1

Using Fmoc solid phase synthesis method to synthesize hydrazide-Trt (2-Cl) resin obtained in the step 1, wherein the sequence of the hydrazide-Trt (2-Cl) resin is Boc-C (Trt) -K (alloc) -r (Pbf) -Cha-r (Pbf) hydrazide polypeptide; then removing protective group of Alloc side chain on lysine (K), condensing naked amino group on lysine side chain with 5(6) carboxyfluorescein to obtain sequence Boc-C (Trt) -K (FAM) -r (Pbf) -Cha-r (Pbf), then cracking polypeptide sequence from resin, purifying to obtain pure H-C-K (FAM) -r-Cha-r-NH₂And finally, performing head-to-tail cyclization on the obtained polypeptide by adopting a natural connection method to obtain a CMPP-1 product.

According to the mitochondrion targeted membrane-penetrating cyclopeptide, the head and the tail of a polypeptide sequence are cyclized by a natural connection method, and a fluorophore is connected to a lysine side chain amino group of the cyclopeptide, so that cell fluorescence visualization is realized.

The mitochondrion targeting transmembrane cyclopeptide is used as a mitochondrion targeting peptide carrier to deliver different kinds of bioactive substances to the mitochondrion in a targeted manner. The bioactive substances include anticancer drugs, inhibitors, negatively charged dyes, etc. The anticancer drugs include adriamycin, chlorambucil, cisplatin, etc. The inhibitor comprises methotrexate, clavulanic acid, sulbactam, etc. Negatively charged dyes include bromocresol green, and dyes containing a-COOH group.

Compared with the prior art, the invention has the beneficial effects that:

the mitochondrion targeting membrane-penetrating cyclopeptide provided by the invention can specifically target cell mitochondrion, and confocal inverted fluorescence microscopy imaging experiments show that the structure has good membrane-penetrating property on Hela cells, and the co-localization coefficient Pr of the structure and commercial mitochondrion dye is 0.74, so that the structure can efficiently target and localize to the mitochondrion. Since the ability of a polypeptide to penetrate cell membranes is susceptible to structural changes, and with minor structural changes, its ability to penetrate cell membranes can vary greatly. Compared with the published H-Cha-r-NH 2 linear mitochondrion targeting peptide, the mitochondrion targeting membrane penetrating peptide provided by the invention can stably position mitochondrion, provides a structural site which is easy to modify, modifies amino acids with different structures on the mitochondrion targeting membrane penetrating peptide, and still has better membrane penetrating capability. Provides a delivery tool with potential for targeted delivery of active substances with different structures, such as small molecules, polypeptides and proteins.

Drawings

FIG. 1 shows the results of confocal imaging of CMPP-1, CMPP-2 and CMPP-3 in Hela cells, and it can be seen from FIG. 1 that the mitochondrial targeting peptide CMPP-1 provided by the publication can be effectively localized to mitochondria and can stably deliver amino acid sequences with different structures.

FIG. 2 is a screen work on mitochondrial targeting peptides from reported H-Cha-r-NH₂The targeting peptide starts from changing the sequence of the polypeptide and the conformation of arginine, and performs head-to-tail cyclization on the polypeptide. The polypeptide structure CMPP-1 which can be effectively positioned to mitochondria is screened out, and as can be seen from figure 2, the capability of the polypeptide to penetrate cell membranes is easily affected by structural change, and the capability of the polypeptide to penetrate the cell membranes has great difference due to small structural change.

Detailed Description

Example 1: synthesis of hydrazide resins

Swelling 2-Cl-Trt-Cl resin (200mg) with DMF (2ml), dropwise adding a mixture of DIEA (266ul), hydrazine hydrate (80ul) and DMF (800ul) under stirring in an ice bath, stirring in the ice bath for 20min, reacting the system for 70min under stirring at normal temperature, and adding 40ul methanol to finish the reaction. Transfer the resulting hydrazide-Trt (2-Cl) resin to a sand core funnel, wash thoroughly with DMF, MeOH, ether in that order for 3-5 times, and vacuum dry the resin for 1 h.

Example 2: fmoc method for solid phase synthesis of hydrazide polypeptide

The synthesis sequence is as follows: Boc-C (Trt) -K (alloc) -r (Pbf) -Cha-r (P)bf)-Cha-r(Pbf)-NH-NH₂

1. 50mg (20. mu. mol) of hydrazide-Trt (2-Cl) resin was added to the solid phase reactor and swollen with DMF for 15 min.

2. Oxyma, DIC and a DMF mixed solution of Fmoc-D-r (pdf) -OH (Oxyma (12.6mg,4.5 equiv.): DIC (14ul,4.5 equiv.): Fmoc-D-r (pdf) -OH (58.4mg, 4.5 equiv.)) were prepared and added to a reactor, and reacted at 55 ℃ for 40min, and after completion of the reaction, the resulting resin was washed with DMF 3 to 5 times.

3. Adding a proper amount of blocking reagent (acetic anhydride: 2, 6-lutidine: DMF: 5: 6: 89) into the reaction system for reaction for 3min, blocking unreacted amino acid, and washing the obtained resin with DMF for 3-5 times;

4. and (3) adding the resin obtained in the step (3) into a DMF (dimethyl formamide) solution containing 20% of piperidine for treatment twice, wherein the first treatment time is 7min, the second treatment time is 8min, the volumes of the DMF solutions containing 20% of piperidine used in the two treatments are the same, so as to remove the Fmoc protecting group on the amino group, and then washing the resin 3-5 times by using DMF.

5. The freshly prepared DMF mixed solution of Oxyma, DIC and Fmoc-L-Cha-OH (Oxyma (12.6mg,4.5 equiv.) DIC (14ul,4.5 equiv.) Fmoc-L-Cha-OH (35.4mg,4.5 equiv.) dissolved in 400ml DMF was then added and reacted at 55 ℃ for 40 min;

repeating the above operations 3, 4 and 5 in sequence until the last amino acid Boc-Cys (Trt) -OH finishes condensation, and obtaining the sequence Boc-C (Trt) -K (alloc) -r (Pbf) -Cha-r (Pbf) -NH₂The hydrazide polypeptide of (1).

Example 3: deprotection of the Alloc protecting group of the resin polypeptide

Weighing 24mg of Pd (PPh3)4(20.8umol) and 60ul of N-methylaniline (554.4umol) to dissolve in 1.2mL of THF, mixing uniformly, sucking into the resin peptide obtained in example 2, and reacting for 2h at normal temperature and in a dark place; after the reaction was completed, 0.5% NaS was prepared from DMF (3 mL. times.3) and DMF in this order₂CN(C₂H₅)₂The resin was washed (3 mL. times.6), DCM (3 mL. times.3), and DMF (3 mL. times.3) until the dark brown disappeared.

Example 4: 5(6) carboxyfluorescein conjugated with lysine side chain amino group

Weighing 37.5mg5(6) carboxyl fluorescein, 52mg PyBoP and 22ul NMM, dissolving in 400ul DMF, mixing well, sucking into the resin peptide obtained in example 3, and reacting for 16h at normal temperature and in dark place; after the reaction is finished, the resin is washed 3-5 times by DMF, the DMF solution containing 20% of piperidine is added for treatment twice, the treatment time of the two times is 10min, the volume of the DMF solution containing 20% of piperidine used for the treatment twice is the same, and the resin is washed 3-5 times by DMF again.

Example 5: cleavage of polypeptides

The resin obtained in example 4 was washed with a large amount of DMF and DCM, treated with an additional acidic cleavage reagent (85% TFA: 5% phenol: 5% water: 2.5% TIPS)2mL for 2h, concentrated with the acidic cleavage reagent containing the target polypeptide, precipitated with 20 equivalents of glacial ethyl ether, and centrifuged to obtain a powdery initial peptide, which was analyzed by HPLC to determine whether it was the target product, and then purified by HPLC to obtain the target peptide (mass spectrometry), and vacuum-lyophilized to obtain the target polypeptide hydrazide with high purity.

Example 6: end-to-end cyclization of hydrazide polypeptides

1. Weighing polypeptide hydrazide H-Cys-Lys (FAM) -r-Cha-r-NH₂(2.4mg) was dissolved in 1ml of a mixed solution of PBS (6.0M guanidine hydrochloride, 0.2M phosphate, pH 2.3) and DMF (4:1), followed by addition of magnetons and stirring at-10 ℃ for 10min in an ice salt bath.

2. Taking 7mg NaNO₂Dissolved in 1ml of water and added dropwise with stirring in an ice salt bath (-10 ℃) 170uLNaNO₂(10 equivalents) reacting the aqueous solution for 20 minutes at low temperature; after the reaction was completed, 20uL of the reaction solution was subjected to HPLC detection.

3. After the oxidation was complete, 27.8mg of MESNa (100 equivalents) was added dropwise to a guanidine hydrochloride solution (6.0M guanidine hydrochloride, 0.2M phosphate, pH 7.0), the ice salt bath was removed, and the acidity of the reaction solution was carefully adjusted to neutral pH 7 with NaOH (2.0M) solution. (acidity was measured with a micro glass pH meter).

4. After reacting for 2 hours, 20uL of reaction solution is taken for HPLC detection, a new peak is separated, and the mass spectrum is determined as a cyclization product.

5. HPLC purification of the target peptide (mass spectrometric determination), vacuum freeze-drying of the target cyclic polypeptide in high purity.

Example 7: fmoc method for solid phase synthesis of linear polypeptide

1. 15mg (5umol) of Rinker Amide resin is added into a solid phase reactor and swelled with DMF for 15min, and is added with a proper amount of 20% piperidine DMF solution for treatment twice, the first treatment time is 7min, the second treatment time is 8min, and the volumes of 20% piperidine DMF solutions used in the two treatments are the same.

2. Oxyma, DIC and a DMF mixture of Fmoc-D-Arg (pdf) -OH (Oxyma (3.2mg,4.5 equiv.): DIC (3.5,4.5 equiv.): Fmoc-D-Arg (pdf) -OH (14.6mg,4.5 equiv.) were dissolved in 400ml DM and the mixture was taken up into a resin and reacted at 55 ℃ for 40min, after which the resulting resin was washed 3-5 times with DMF.

3. The resin was soaked with the appropriate amount of blocking reagent (acetic anhydride: 2, 6-lutidine: DMF: 5: 6: 89) for 3min, the unreacted amino acids were blocked, and the resin was washed again with DMF 3-5 times.

4. Adding appropriate amount of 20% piperidine in DMF solution for two times, the first time is 7min, the second time is 8min, the volume of the 20% piperidine-containing DMF solution used in the two times is the same, removing the Fmoc protecting group on the amino group, and washing the resin with DMF for 3-5 times.

5. Add the DMF mixture of now prepared Oxyma, DIC and Fmoc-L-Cha-OH (Oxyma (3.2mg,4.5 equiv.) DIC (3.5ul,4.5 equiv.) Fmoc-L-Cha-OH (8.9mg,4.5 equiv.) dissolved in 400ml DMF); the reaction was carried out at 55 ℃ for 40 min.

Repeating the operations 3, 4 and 5 in sequence until the last amino acid Boc-Cys (Trt) -OH finishes condensation.

6. According to the method for removing Alloc from the lysine side chain of the hydrazide polypeptide, removing the Alloc protecting group of the lysine side chain of the linear peptide, connecting 5(6) carboxyfluorescein to the amino group of the lysine side chain, cracking to obtain the linear fluorescent polypeptide, and analyzing and purifying by HPLC to obtain the target yellow powder.

Example 8: synthesis of mitochondrial targeting peptides of different structures

1. The cyclic peptides in the following table were synthesized according to the methods of examples 1-6.

Name (R)	Sequence of
		1	Cyclo[C-K(FAM)-R-Cha-R-Cha-R]
2	Cyclo[C-K(FAM)-Cha-R-Cha-R-Cha-R]
		3	Cyclo[C-K(FAM)-R-Cha-R-Cha-R-Cha-R]
4	Cyclo[C-K(FAM)-Cha-r-Cha-r-Cha-r]
		5	Cyclo[C-K(FAM)-r-Cha-r-Cha-r-Cha-r]
6	Cyclo[C-K(FAM)-K-Cha-R-K-Cha-R-K-Cha-R]
		7	Cyclo[C-K(FAM)-K-Cha-R-K-Cha-R-K-Cha-R-K-Cha-R]
8	Cyclo[C-K(FAM)-R-Cha-r-Cha-r]
		9	Cyclo[C-K(FAM)-r-Cha-R-Cha-r]
10	Cyclo[C-K(FAM)-r-Cha-r-Cha-R]
		11	Cyclo[C-K(FAM)-R-Cha-R-Cha-r]
12	Cyclo[C-K(FAM)-R-Cha-r-Cha-R]
		13	Cyclo[C-K(FAM)-r-Cha-R-Cha-R]

2. Following the procedure of example 7, linear peptides in the following table were synthesized:

name (R)	Sequence of
		14	H-C-K(FAM)-R-Cha-R-Cha-R-NH₂
15	H-C-K(FAM)-Cha-R-Cha-R-Cha-R-NH₂
		16	H-C-K(FAM)-R-Cha-R-Cha-R-Cha-R-NH₂
17	H-C-K(FAM)-r-Cha-r-Cha-r-NH₂
		18	H-C-K(FAM)-Cha-r-Cha-r-Cha-r-NH₂
19	H-C-K(FAM)-r-Cha-r-Cha-r-Cha-r-NH₂
		20	H-C-K(FAM)-K-Cha-R-K-Cha-R-K-Cha-R-NH₂
21	H-C-K(FAM)-K-Cha-R-K-Cha-R-K-Cha-R-K-Cha-R-NH₂

The polypeptide sequence synthesized in example 8 is the reported mitochondrial targeting peptide H-Cha-r-NH₂On the basis of the sequence, a structure with better mitochondrial localization effect is expected to be obtained through structural change, and firstly, the sequences 14, 15, 16, 17, 18, 19, 1, 2, 3 and CMPP-1, 4 and 5 are designed. The influence of structure on mitochondrial localization was explored by varying the length and head-to-tail ligation of the sequences. Through confocal imaging (FIGS. 1 and 2), we obtained a structure CMPP-1 with better localization effect, and then we explored the effect of arginine conformation on different positions on localization effect. Sequences 8, 9, 10, 11, 12, 13 were synthesized. The best mitochondrial localization of arginine form D was shown by confocal imaging (figure 2). Finally, the inventors explored whether head-to-tail cyclization of different sequences can improve the cell membrane penetration capacity of the sequences, and designed the sequences 20, 21, 6 and 7. Confocal imaging showed (figure 2) that the increase in cell membrane penetration was different for polypeptides of different sequences, cyclized head to tail.

Example 9: delivery of different types of small peptides by using mitochondrion-targeted transmembrane cyclopeptide as scaffold

1. Synthesis of the sequence Cyclo [ C-K (FAM-^βAGWIYA)-r-Cha-r-Cha-r]The general formula of the structure is shown as formula II:

the synthesis steps are as follows:

in the 2-Cl-Trt-Cl resin synthesis sequence Boc-C (Trt) -K (Alloc) -r (Pbf) -Cha-r (Pbf), after the lysine side chain protecting group Alloc is removed, on the lysine side chain amino group, the sequence Boc-C (Trt) -K (Alloc) -r (Pbf) is completed according to Fmoc solid phase synthesis method^βCondensation of A-G-W-I-Y-A, 5(6) carboxyfluorescein binding to beta alanine; after the reaction is finished, the polypeptide is cracked from the resin, and the synthesis of the cyclic polypeptide is realized according to a head-to-tail cyclization method of the hydrazide polypeptide.

2. Synthesizing a sequence Cyclo [ C-K (FAM-F-Y-F-Y-F-Y) -r-Cha-r ], wherein the structural general formula is shown as formula III:

the synthesis steps are as follows:

synthesizing a sequence Boc-C (Trt) -K (Alloc) -r (Pbf) -Cha-r (Pbf) on a 2-Cl-Trt-Cl resin, after the lysine side chain protecting group Alloc is removed, completing condensation of a sequence F-Y-F-Y-F-Y on the amino group of a lysine side chain according to Fmoc solid phase synthesis, and 5(6) combining carboxyfluorescein with phenylalanine; after the reaction is finished, the polypeptide is cracked from the resin, and the synthesis of the cyclic polypeptide is realized according to a head-to-tail cyclization method of the hydrazide polypeptide.

Example 10: mitochondrial targeting peptide cellular imaging assay

The prepared mitochondrial targeting peptide solid powder was dissolved in DMSO to prepare a 1mM test stock solution, Hela cells were cultured in a culture solution (DMEM: FBS: AK ═ 9:1:0.1), and the Hela cells were divided into 1ml laser confocal dishes one day before cell imaging test, and the Hela cells and a 10. mu.M mitochondrial targeting peptide CMPP-1 solution were imaged at 37 ℃ in 5% CO₂The cells of (1) were incubated in a cell incubator with 20. mu.M CMPP-2 and CMPP-3 at 37 ℃ and 5% CO₂Was incubated for 2 hours in the cell incubator of (1). Then 0.5. mu.M commercial mitochondrial stain MitoTracker Red FM solution is added into the culture dish for further incubation for 15 minutesAnd washed 3 times with neutral PBS buffer. Adding 1mL of culture medium for two-photon fluorescence confocal imaging, setting a green channel tracker1, wherein the excitation wavelength is 488nm, the emission wavelength is 510-560nm, and the channel is used for receiving the fluorescence emitted by the mitochondrion targeting peptide. A Red channel tracker2 was set at an excitation wavelength of 559nm and an emission wavelength of 580-620nm, and this channel was used to receive fluorescence emitted by the commercial mitochondrial stain Mitotracker Red FM.

The results of the experiment are shown in FIG. 1. CMPP-1 has higher mitochondrion positioning capability, the co-positioning coefficient with commercial mitochondrion positioning dye is 0.74, and CMPP-2 modifies the structure of CMPP-1^βThe co-localization coefficient of the CMPP-2 and the commercial mitochondrial localization dye is 0.76, the CMPP-3 is the modified F-Y-F-Y-F-Y sequence on the basis of the CMPP-1 structure, and the co-localization coefficient of the CMPP-3 and the commercial mitochondrial localization dye is 0.45. As can be seen from the figure I, the CMPP-1 provided by the invention can be effectively positioned to mitochondria, and amino acids with different structures are modified on the basis of the structure of the CMPP-1, so that the CMPP-1 still has the capability of positioning the mitochondria. These results indicate that the CMPP-1 structure provided by the invention has the advantages of easy modification and stable positioning to mitochondria.

Claims

1. A mitochondrial-targeted membrane-penetrating cyclopeptide, characterized by:

2. the method for preparing the mitochondrion-targeted transmembrane cyclopeptide according to claim 1, which comprises the following steps:

step 1: synthesis of hydrazide resins

Adding 200mg of 2-Cl-Trt-Cl resin into DMF for swelling, dropwise adding a mixed solution of 266uL DIEA, 80uL hydrazine hydrate and DMF under the conditions of ice bath and stirring, stirring for 20min under ice bath, transferring the system to normal temperature, stirring for reacting for 70min, and adding 40uL methanol to finish the reaction; transferring the obtained hydrazide-Trt (2-Cl) resin to a sand core funnel, fully washing with DMF, MeOH and diethyl ether in sequence, and drying in vacuum;

step 2: synthesis method of CMPP-1

Synthesizing the hydrazide-Trt (2-Cl) resin obtained in the step 1 into a hydrazide polypeptide with the sequence of Boc-C (Trt) -K (alloc) -r (Pbf) -Cha-r (Pbf) by using Fmoc solid phase synthesis; then removing protective group of Alloc side chain on lysine (K), condensing naked amino group on lysine side chain with 5(6) carboxyfluorescein to obtain sequence Boc-C (Trt) -K (FAM) -r (Pbf) -Cha-r (Pbf), then cracking polypeptide sequence from resin, purifying to obtain pure H-C-K (FAM) -r-Cha-r-NH₂And finally, performing head-to-tail cyclization on the obtained polypeptide by adopting a natural connection method to obtain a CMPP-1 target product.

3. Use of the mitochondrially targeted transmembrane cyclopeptide according to claim 1, which comprises:

it is used as a mitochondrion targeting peptide carrier to target and deliver different kinds of bioactive substances to mitochondrion.

4. Use according to claim 3, characterized in that:

the bioactive substances include anticancer drugs, inhibitors, negatively charged dyes, etc.