Multi-site modified enkephalin and neurotensin (8-13) coupled cyclic hybrid peptide and its synthesis method and application

Abstract

The invention provides a multi-site modified enkephalin and neurotensin (8-13) coupled cycled hybrid peptide, a compounding method and an application thereof and relates to a cycled hybrid peptide, the compounding method and the application thereof. The invention aims to solve the problems of inferior anti-enzymolysis capacity and non-ideal anti-neuropathic pain effect of present opioids. The hybrid peptide is a hybrid peptide 1, a hybrid peptide 2, a hybrid peptide 3 or a hybrid peptide 4. The preparation method comprises the following steps: 1) pre-treating 'Fmoc' protected Wang resin; 2) removing 'Fmoc' protecting groups; 3) triggering condensation reaction of amino acid; 4) prolonging peptide chain; 5) forming a disulfide bond; 6) cutting peptide chain from the resin; 7) desalting andpurifying crude peptide. According to the invention, the biological stability of hybrid peptide can be enhanced and an anti-neuropathic pain effect can be endowed, through multi-site unnatural amino acid substitution and cyclizing modification. The hybrid peptide provided by the invention can be used for preparing drugs for relieving neuropathic pain.

Description

technical field

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

[0002] Opioids and their extracts have been used in analgesic therapy for centuries. 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. It is well known that opioid peptides play an important role in the transmission and modulation of pain information. As early as 1974, scholars extracted two pentapeptides with morphine-like opioid activity from pig brains, namely Met-enkephalin and Leu-enkephalin , both collectively referred to as enkephalins (enkephalins). T...

Examples

specific Embodiment approach 1

[0057] Specific embodiment 1: In this embodiment, the multi-site modified enkephalin and neurotensin (8-13) coupled cyclic hybrid peptides are hybrid peptide 1, hybrid peptide 2, and hybrid peptide 3 or hybrid peptide 4;

[0058] Wherein the amino acid sequence of hybrid peptide 1 is:

[0059] Dmt-Gly-c(Cys-NMePhe-Met-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0060] The amino acid sequence of hybrid peptide 2 is:

[0061] Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0062] The amino acid sequence of hybrid peptide 3 is:

[0063] Dmt-Gly-c(Cys-NMePhe-Met-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0064] The amino acid sequence of hybrid peptide 4 is:

[0065] Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu.

[0066] In this embodiment, the amino acid sequence "Tyr-Gly-Gly-Phe-Met / Leu" of methionine / leucine enkephalin is modified and cyclized to obtain "Dmt-Gly-c(Cys- NMePhe-Met-Cys)" and "Dmt-Gly-c (Cys-NMePhe-Leu-Cys)" amino acid residue seq...

specific Embodiment approach 2

[0068] Specific embodiment two: the synthetic method of the cyclized hybrid peptide of the multi-site modified enkephalin and neurotensin (8-13) coupling of the present embodiment, comprises the following steps:

[0069] 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;

[0070] 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 concentr...

specific Embodiment approach 3

[0078] Embodiment 3: This embodiment differs from Embodiment 2 in that the molar weight of the amino acid protected by the "Fmoc" group in step 3 is 2.5-3 times the molar weight 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] Opioids and their extracts have been used in analgesic therapy for centuries. 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. It is well known that opioid peptides play an important role in the transmission and modulation of pain information. As early as 1974, scholars extracted two pentapeptides with morphine-like opioid activity from pig brains, namely Met-enkephalin and Leu-enkephalin , both collectively referred to as enkephalins (enkephalins). T...

Examples

specific Embodiment approach 1

[0057] Specific embodiment 1: In this embodiment, the multi-site modified enkephalin and neurotensin (8-13) coupled cyclic hybrid peptides are hybrid peptide 1, hybrid peptide 2, and hybrid peptide 3 or hybrid peptide 4;

[0058] Wherein the amino acid sequence of hybrid peptide 1 is:

[0059] Dmt-Gly-c(Cys-NMePhe-Met-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0060] The amino acid sequence of hybrid peptide 2 is:

[0061] Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0062] The amino acid sequence of hybrid peptide 3 is:

[0063] Dmt-Gly-c(Cys-NMePhe-Met-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;

[0064] The amino acid sequence of hybrid peptide 4 is:

[0065] Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu.

[0066] In this embodiment, the amino acid sequence "Tyr-Gly-Gly-Phe-Met / Leu" of methionine / leucine enkephalin is modified and cyclized to obtain "Dmt-Gly-c(Cys- NMePhe-Met-Cys)" and "Dmt-Gly-c (Cys-NMePhe-Leu-Cys)" amino acid residue seq...

specific Embodiment approach 2

[0068] Specific embodiment two: the synthetic method of the cyclized hybrid peptide of the multi-site modified enkephalin and neurotensin (8-13) coupling of the present embodiment, comprises the following steps:

[0069] 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;

[0070] 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 concentr...

specific Embodiment approach 3

[0078] Embodiment 3: This embodiment differs from Embodiment 2 in that the molar weight of the amino acid protected by the "Fmoc" group in step 3 is 2.5-3 times the molar weight of the Fmoc-Leu-Wang resin. Others are the same as in the second embodiment.

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