Stabilized cell-penetrating peptide with hydrophobic side chain and preparation method and use of stabilized cell-penetrating peptide

A membrane-penetrating peptide and hydrophobic technology, applied in the field of biomedicine, can solve the problems of low cell penetrating ability and poor metabolic stability of cell-penetrating peptides, and achieve strong cell penetrating ability, improve metabolic stability, and fast penetration. The effect of penetration speed

Active Publication Date: 2021-01-22
SOUTHWEST JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention solves the technical problems of poor metabolic stability and low cell penetration ability of existing cell penetrating peptides

Method used

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  • Stabilized cell-penetrating peptide with hydrophobic side chain and preparation method and use of stabilized cell-penetrating peptide
  • Stabilized cell-penetrating peptide with hydrophobic side chain and preparation method and use of stabilized cell-penetrating peptide
  • Stabilized cell-penetrating peptide with hydrophobic side chain and preparation method and use of stabilized cell-penetrating peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Since each ascending turn of the α-helix contains 3.6 amino acid residues, the corresponding cyclization positions need to be i+4, i+7. In order to explore the effect of hydrophobicity on the penetrating ability of CPPs, based on the sequences of R8 and Tat derivatives, small molecules with different hydrophilicity and hydrophobicity were used to cyclize the polypeptide, using weakly hydrophobic perfluorobenzene and 1,3 -Bis(bromomethyl)benzene is cyclized at the i+4 position, and decafluorobiphenyl and 4,4'-bis(bromomethyl)biphenyl with strong hydrophobicity are cyclized at the i+7 position. At the same time, in order to understand the effect of amino acid conformation on the penetration ability of CPPs, the same chirality (L) sequence and different chirality (L, D) alternating cyclic peptides were designed. Constructed 12 test examples of stabilized cell-penetrating cyclic peptides with hydrophobic side chains, named sR8-1, sR8-2, sR8-3, sR8-4, sR8-5, sR8-6 and sTat- ...

Embodiment 2

[0050] Take the synthetic route of R8, sR8-1, sR8-2, sR8-3, sR8-4, sR8-5, sR8-6 as an example, the synthetic route is as attached in the manual figure 1 As shown, the synthesis methods of Tat, sTat-1, sTat-2, sTat-3, sTat-4, sTat-5, and sTat-6 are the same.

[0051] Concrete preparation process is as follows:

[0052] 1. Cyclization method

[0053] Decafluorobiphenyl and 4,4'-bis(bromomethyl)biphenyl cyclization method: Swell 200mg (RCRRRRRRCR, rCRrRrRrCR, GRKCRRQRRRC and GrKCRrQrRrC) resin with DCM for 10 minutes, remove DCM, add the mixed solution to the reaction tube (2%TFA / 3%TIS / 95%DCM, v / v / v) selectively remove the Cys(Trt) protecting group, each time for 2 minutes, until the yellow color in the solution disappears, wash with DCM and DMF alternately for several times. Dissolve 1.2eq. of decafluorobiphenyl or 4,4'-bis(bromomethyl)biphenyl and 2.4eq. of DIPEA in DMF, mix well and add to the reaction tube to react for 4 hours.

[0054] Perfluorobenzene and 1,3-bis(bromometh...

Embodiment 3

[0062] The hydrophilicity and hydrophobicity of the synthesized CPPs were compared using the retention time of reversed-phase HPLC. The lyophilized peptide was dissolved in aqueous solution and detected by reversed-phase HPLC (Agilent Poroshell 120 EC-C18: 4.6×150mm, 4μm, flow rate 1.0mL / min) The peak time of each polypeptide can be used to judge the hydrophobicity of the polypeptide. The later the peak time, the stronger the hydrophobicity of the polypeptide. Experimental conditions: a gradient system in which solvent B (solvent A: 1‰ (v / v) TFA in water, solvent B: ACN) rises from 10% to 80% at a flow rate of 1.0ml / min in 30 minutes, detects 220nm and UV signal at 254nm.

[0063] The retention times of 14 CPPs including linear peptides detected on reversed-phase HPLC are shown in Table 2 and Table 3. The results show that the retention times of cyclized CPPs are increased compared with the corresponding linear CPPs, proving that cyclized CPPs The hydrophobicity has been impr...

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Abstract

The present invention discloses a stabilized cell-penetrating peptide with a hydrophobic side chain and a preparation method and use of the stabilized cell-penetrating peptide. The stabilized cell-penetrating peptide comprises a cell-penetrating peptidyl peptide and the hydrophobic side chain, and the cell-penetrating peptidyl peptide is a cell-penetrating peptide rich in arginine; and the hydrophobic side chain contains hydrophobic small molecules, the stabilized cell-penetrating peptide is a cyclic peptide with a cyclized side chain and obtained by introducing two natural cysteines into a sequence of the cell-penetrating peptidyl peptide and crosslinking the cell-penetrating peptidyl peptide with the hydrophobic small molecules by an alkylation/arylation reaction of the cysteines. The preparation method of the stabilized cell-penetrating peptide is simple and effective, besides, metabolic stability and cell-penetrating capacity of the cell-penetrating peptide are remarkably improvedby adjusting conformation of the amino acids and hydrophilicity and hydrophobicity of the side chain, deep penetration of in-vitro cell spheres and living tumor tissues by the cell-penetrating peptideis realized, and the cell-penetrating peptide has a good application prospect in the field of medicine delivery.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a stabilized cell-penetrating peptide with a hydrophobic side chain, a preparation method and application thereof. Background technique [0002] The entry of drug molecules into cells to play a role will be hindered by various physiological barriers. For those drugs with small molecular weight and strong hydrophobicity, they can enter the cell interior by passive diffusion. However, for those macromolecular drugs with larger molecular weight and more hydrophilic, it is an urgent problem to overcome the cell membrane barrier and exert their biological activity. For this problem, researchers have developed a variety of strategies such as electroporation, microinjection, construction of viral vector systems, and liposome-based vector systems to deliver macromolecular drugs across the membrane. However, these methods usually have problems such as high biological toxicity, low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K49/00A61K45/00C07K7/06A61P35/00
CPCA61K47/64A61K45/00A61K49/0056C07K7/06A61P35/00Y02P20/55
Inventor 田原史梦真
Owner SOUTHWEST JIAOTONG UNIV
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