Dynorphin A (1-8) and neurotensin (8-13) coupled cyclic hybrid peptide, as well as synthesizing method and application thereof

Abstract

The invention provides a dynorphin A (1-8) and neurotensin (8-13) coupled cyclic hybrid peptide, as well as a synthesizing method and an application thereof, relates to cyclic hybrid peptides, as wellas a synthesizing method and an application thereof, and aims at solving the problems of relatively poor biological stability and poor neuropathic pain relieving effect of existing opioid medicines.The hybrid peptide has an amino acid sequence of Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu. The method comprises the following steps: 1, pretreating 'Fmoc' protected Wang resin; 2, removing the 'Fmoc' protective group; 3, performing an amino acid condensation reaction; 4, prolonging a peptide chain; 5, forming a disulfide bond; 6, cutting off the peptide chain from resin; and 7, desalting and purifying crude peptide. The biological stability of hybrid peptide can be enhanced through multi-site unnatural amino acid substitution and cyclic modification, and the cyclic hybrid peptide has an effect of relieving neuropathic pains. The hybrid peptide is used for preparing neuropathic pain relieving medicines.

Description

technical field

[0001] The invention relates to a cyclized hybrid peptide and its synthesis method and application. Background technique

[0002] It is well known that opioids and their extracts have been used in analgesic therapy. Morphine, isolated from opioids, is one of the most widely used analgesic drugs in clinical practice. However, morphine and other drugs have many defects in safe clinical use, such as inhibiting breathing, causing blood pressure drop and bradycardia, constipation, easy to cause mental dependence, addiction and analgesic tolerance. Furthermore, morphine is less effective in the treatment of chronic pain such as neuropathic pain. Studies have shown that opioid peptides play an important role in the transmission and modulation of pain information. Following the discovery of enkephalin and endorphin, in 1979, researchers discovered an endogenous seventeen peptide with opioid activity from pig pituitary gland, and named it dynorphin. Dynorphin (dyn...

Examples

specific Embodiment approach 1

[0046] Specific embodiment 1: The amino acid sequence of the cyclized hybrid peptide coupled with dynorphin A (1-8) and neurotensin (8-13) in this embodiment is as follows:

[0047] Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu.

[0048] In this embodiment, the amino acid sequence "Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile" of dynorphin A (1-8) is obtained through multi-site modification and Cys disulfide bond cyclization to obtain "Dmt- Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)" amino acid residue sequence, the amino acid sequence "Arg-Arg-Pro-Tyr-Ile-Leu" of neurotensin (8-13) The "NMeArg-Lys-Pro-Trp-Tle-Leu" amino acid residue sequence was obtained through multi-site modification, and the two parts of the amino acid residue sequence were coupled using the intermediate linker "Gly" to construct a highly efficient hybrid peptide .

[0049] Multi-site non-natural amino acid substitution can enhance the anti-enzymatic ability of polypeptides, and cyclization mod...

specific Embodiment approach 2

[0050] Specific embodiment two: The synthesis method of the cyclization hybrid peptide coupled with dynorphin A (1-8) and neurotensin (8-13) in this embodiment comprises the following steps:

[0051] 1. Pretreatment of Wang resin protected by "Fmoc": check the air tightness of the solid phase synthesizer, put the Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add dichloromethane and stir for 30-40min, After the resin is fully soaked and swollen, filter the solvent under reduced pressure; the mass ratio of the Fmoc-Leu-Wang resin with one amino acid residue to the volume ratio of dichloromethane is 1g: (7-12)mL;

[0052] 2. Remove the "Fmoc" protecting group: wash the swelled resin with DMF for 3 to 5 minutes, dry it, repeat 3 to 5 times, and then add 20% to 25% vol. Piperidine / DMF deprotection solution, stirred for 5-10 minutes, drained, repeated 2-3 times, then added piperidine / DMF deprotection solution with volume percentage concentration of 20%-25%, s...

specific Embodiment approach 3

[0060] Embodiment 3: The difference between this embodiment and Embodiment 2 is that the molar weight of N-hydroxybenzotriazole in step 3 is 2.5-3 times that of the Fmoc-Leu-Wang resin. Others are the same as in the second embodiment.

technical field

[0001] The invention relates to a cyclized hybrid peptide and its synthesis method and application. Background technique

[0002] It is well known that opioids and their extracts have been used in analgesic therapy. Morphine, isolated from opioids, is one of the most widely used analgesic drugs in clinical practice. However, morphine and other drugs have many defects in safe clinical use, such as inhibiting breathing, causing blood pressure drop and bradycardia, constipation, easy to cause mental dependence, addiction and analgesic tolerance. Furthermore, morphine is less effective in the treatment of chronic pain such as neuropathic pain. Studies have shown that opioid peptides play an important role in the transmission and modulation of pain information. Following the discovery of enkephalin and endorphin, in 1979, researchers discovered an endogenous seventeen peptide with opioid activity from pig pituitary gland, and named it dynorphin. Dynorphin (dyn...

Examples

specific Embodiment approach 1

[0046] Specific embodiment 1: The amino acid sequence of the cyclized hybrid peptide coupled with dynorphin A (1-8) and neurotensin (8-13) in this embodiment is as follows:

[0047] Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu.

[0048] In this embodiment, the amino acid sequence "Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile" of dynorphin A (1-8) is obtained through multi-site modification and Cys disulfide bond cyclization to obtain "Dmt- Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)" amino acid residue sequence, the amino acid sequence "Arg-Arg-Pro-Tyr-Ile-Leu" of neurotensin (8-13) The "NMeArg-Lys-Pro-Trp-Tle-Leu" amino acid residue sequence was obtained through multi-site modification, and the two parts of the amino acid residue sequence were coupled using the intermediate linker "Gly" to construct a highly efficient hybrid peptide .

[0049] Multi-site non-natural amino acid substitution can enhance the anti-enzymatic ability of polypeptides, and cyclization mod...

specific Embodiment approach 2

[0050] Specific embodiment two: The synthesis method of the cyclization hybrid peptide coupled with dynorphin A (1-8) and neurotensin (8-13) in this embodiment comprises the following steps:

[0051] 1. Pretreatment of Wang resin protected by "Fmoc": check the air tightness of the solid phase synthesizer, put the Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add dichloromethane and stir for 30-40min, After the resin is fully soaked and swollen, filter the solvent under reduced pressure; the mass ratio of the Fmoc-Leu-Wang resin with one amino acid residue to the volume ratio of dichloromethane is 1g: (7-12)mL;

[0052] 2. Remove the "Fmoc" protecting group: wash the swelled resin with DMF for 3 to 5 minutes, dry it, repeat 3 to 5 times, and then add 20% to 25% vol. Piperidine / DMF deprotection solution, stirred for 5-10 minutes, drained, repeated 2-3 times, then added piperidine / DMF deprotection solution with volume percentage concentration of 20%-25%, s...

specific Embodiment approach 3

[0060] Embodiment 3: The difference between this embodiment and Embodiment 2 is that the molar weight of N-hydroxybenzotriazole in step 3 is 2.5-3 times that of the Fmoc-Leu-Wang resin. Others are the same as in the second embodiment.

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