CN109232748B - The cyclisation hybrid peptide and its synthetic method and application that the enkephalins of multidigit point modification is mutually coupled with neurotensin (8-13) - Google Patents

The cyclisation hybrid peptide and its synthetic method and application that the enkephalins of multidigit point modification is mutually coupled with neurotensin (8-13) Download PDF

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CN109232748B
CN109232748B CN201811126681.0A CN201811126681A CN109232748B CN 109232748 B CN109232748 B CN 109232748B CN 201811126681 A CN201811126681 A CN 201811126681A CN 109232748 B CN109232748 B CN 109232748B
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peptide
resin
fmoc
amino acid
hybrid peptide
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CN201811126681.0A
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CN109232748A (en
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王长林
张雨哲
袁碧玉
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哈尔滨工业大学
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Abstract

The cyclisation hybrid peptide that is mutually coupled with neurotensin (8-13) of enkephalins and its synthetic method of the modification of multidigit point and application are related to a kind of cyclisation hybrid peptide and its synthetic method and application.Be to solve existing opioid drug resistance to enzymolysis ability it is poor, the ineffective problem of anti-neuropathic pain.The hybrid peptide is hybrid peptide 1, hybrid peptide 2, hybrid peptide 3 or hybrid peptide 4.Method: one, the Wang resin pretreatment of " Fmoc " protection;Two, " Fmoc " blocking group is removed;Three, amino acid condensation reacts;Four, the extension of peptide chain;Five, the formation of disulfide bond;Six, peptide chain is from the cutting on resin;Seven, the desalination and purifying of thick peptide.The present invention, which replaces and be cyclized modification by multidigit point unnatural amino acid, can also enhance the biological stability of hybrid peptide, and have the function of anti-neuropathic pain.Hybrid peptide of the invention is used to prepare anti-neuropathic pain drug.

Description

The cyclisation heterozygosis that the enkephalins of multidigit point modification is mutually coupled with neurotensin (8-13) Peptide and its synthetic method and application

Technical field

The present invention relates to a kind of cyclisation hybrid peptide and its synthetic method and applications.

Background technique

For centuries, opium and its extract are used for always analgesia therapy.The morphine isolated from opium is exactly One of widest analgesic of present clinical application.However the defect that the drugs such as morphine are used in safety clinical is also very much, such as Inhibit breathing, cause blood pressure reduction and areocardia, constipation, Yi Yinqi psychological dependence, habituation and analgesia tolerance etc..In addition, Coffee is bad to the therapeutic effect of chronic ache such as neurogenic pain.It is well known that opioid peptides pain sensation information transmitting and It plays an important role in modulated process.Early in 1974, scholars have just extracted two from pig brain had morphine sample opium Active pentapeptide, i.e. Methionine enkephalin (Met-enkephalin) and leucine enkephalin (Leu-enkephalin), two Person is referred to as enkephalins (enkephalins).They are to δ-opiate receptor compatibility with higher and selectivity, it is considered to be δ-opiate receptor endogenic ligand.Two kinds of enkephalins by reduced in conjunction with its receptor in nerve cell cAMP level and calcium from The conduction of son inhibits the neurotransmission of pain sensation regulating system, and then plays analgesic activity.Under physiological concentration, methionine brain Deltorphin delta and leucine enkephalin are not easy to generate body psychic dependence, and drug resistance formation is slower.However enkephalins Analgesic activities are relatively weak, hence it is evident that lower than the analgesic effect of the mediations such as μ-opioid receptor agonist such as morphine.In addition, enkephalins Biological stability is poor, and analgesia duration is shorter, and blood-brain barrier Penetration ration is lower, to limit it as clinical antalgesic The development of object.

American Physiological man in 1973 has found the active nerve peptide being made of 13 amino acid from ox hypothalamus, i.e., refreshing Through hypotensor medicine (neurotensin, NT).In central nervous system, neurotensin is as neurotransmitter or quenched and play Effect, effect include cooling, analgesia, adjust dopaminergic transmitting and the release of Stimulation of Pituitary Gland anterior lobe normone.Neurotensin exists Inhibit have important relationship with opioid peptides in pain sensation transmittance process.Neurotensin nerve endings and neurotensin receptor exist Be distributed widely in central nervous system with the related region of easing pain, and with endogenous opiatepeptide nerve endings and opiate receptor It is distributed companion lines.Some researches show that the analgesic effect and activation opiate system that Central injection neurotensin generates cause endogenous The release of property opioid peptides is related.Furthermore, it has already been proven that the 8-13 amino acids " Arg-Arg-Pro-Tyr- of neurotensin Ile-Leu " is the active minimal segment of neurotensin, and there are two positively charged arginine in amino acid sequence.Arginine Guanidino group can form the hydrogen bond of divalent with the anionic group of cell surface, enhancing polypeptide to the permeability of biomembrane, Such as blood-brain barrier.

Summary of the invention

The present invention is to solve the resistance to enzymolysis ability of existing opioid drug is poor, anti-neuropathic pain is ineffective to be asked Topic provides cyclisation hybrid peptide and its synthetic method and answer that the enkephalins that multidigit point is modified mutually is coupled with neurotensin (8-13) With.

The enkephalins of multidigit point modification of the present invention and the cyclisation hybrid peptide that neurotensin (8-13) is mutually coupled are hybrid peptide 1, hybrid peptide 2, hybrid peptide 3 or hybrid peptide 4;

The wherein amino acid sequence of hybrid peptide 1 are as follows:

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

The amino acid sequence of hybrid peptide 2 are as follows:

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

The amino acid sequence of hybrid peptide 3 are as follows:

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

The amino acid sequence of hybrid peptide 4 are as follows:

Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu。

Wherein sequence " Dmt-Gly-c (Cys-NMePhe-Met-Cys) " and " Dmt-Gly-c (Cys-NMePhe-Leu- Cys) " be respectively multidigit point modification cyclisation Methionine enkephalin and leucine enkephalin residue sequence, " Gly " and " D-Ala " is the intermediate connexon of hybrid peptide, and " NMeArg-Lys-Pro-Trp-Tle-Leu " is the nerve drop of multidigit point modification Press the residue sequence of plain (8-13).(Dmt represents 2,6- dimethyl Tyr, and NMePhe represents N- methyl Phe, and NMeArg represents N- first Base Arg, Tle represent tert-Leu)

The synthetic method for the cyclisation hybrid peptide that the enkephalins of above-mentioned multidigit point modification is mutually coupled with neurotensin (8-13), The following steps are included:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, an amino acid will be had The Fmoc-Leu-Wang resin of residue is put into synthesizer, and add methylene chloride 30~40min of stirring, and resin is made sufficiently to impregnate swelling Afterwards, decompression filters solvent;The wherein body of the quality of the Fmoc-Leu-Wang resin with an amino acid residue and methylene chloride Product is than being 1g:(7~12) mL;

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3~5min with DMF, are drained, repetition 3~ 5 times, piperidines/DMF that concentration expressed in percentage by volume is 20%~25% is then added in resin and is deprotected solution, stirs 5~10min After drain, repeat 2~3 times, add concentration expressed in percentage by volume be 20%~25% piperidines/DMF be deprotected solution, stirring 15~ 20min removes " Fmoc " blocking group sufficiently, drains solvent later, finally washs cleared deprotection solution with DMF, obtains Remove the resin of " Fmoc " blocking group;Wherein the quality of resin and the volume ratio for the deprotection solution being added for the first time are 1g: (8~12) mL;The volume ratio for the deprotection solution that the quality of resin is added with second is 1g:(10~14) mL;

Three, amino acid condensation reacts: successively by the amino acid, N- hydroxy benzo triazole, O- benzene of " Fmoc " radical protection And triazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, mixes mixed after adding diisopropylethylamine Solution is closed, then mixed solution is added in the resin of step 2 removing " Fmoc " blocking group, is stirred under protection of argon gas React 60~72min;

The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove not instead The residual liquid answered;

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence of heterozygosis peptide amino acid sequence, from the end C- of polypeptide Successively the amino acid of " Fmoc " radical protection is condensed on resin one by one to the end N-, until all amino acid residues have been condensed At obtaining peptide resin;The wherein amino acid sequence of hybrid peptide 1 are as follows: Dmt-Gly-c (Cys-NMePhe-Met-Cys)-Gly- NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 2 are as follows: Dmt-Gly-c (Cys-NMePhe-Leu- Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 3 are as follows: Dmt-Gly-c (Cys- NMePhe-Met-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 4 are as follows: Dmt- Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;

Five, the formation of disulfide bond: I is weighed2It is dissolved in anhydrous methanol and DMF mixed liquor, is made into I2Solution, and by I2Solution It is added in peptide resin, is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, drain reaction solution;Wherein mixed solution The volume ratio of middle anhydrous methanol and DMF are 4:1, I2Quality and mixed solution volume ratio be 2.6g:100mL, I2Solution and peptide The volume ratio of resin is 100mL:1g;

Six, peptide chain is from the cutting on resin: " Fmoc " group of amino acid most latter linked on peptide resin is removed completely, Then peptide resin is alternately washed with methylene chloride and methanol, after sufficiently draining solvent, cutting reagent is added into peptide resin, in room Lower 3~the 5h of cleavage reaction of temperature;

Collect cutting reagent and depressurize and be spin-dried for, be precipitated and precipitated with ice-cold ether, remove ether supernatant after standing, then with water and Acetic acid sufficiently dissolves precipitating, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white The thick peptide of solid powder;

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 10%-20% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound, then isolated and purified by Reversed Phase High Performance, main peak is collected, white is obtained after freeze-drying The pure peptide of solid powder.

The mole of the amino acid of " Fmoc " radical protection is the 2.5- of Fmoc-Leu-Wang resin mole in step 3 3 times.

The mole of N- hydroxy benzo triazole is 2.5-3 times of Fmoc-Leu-Wang resin mole in step 3.

The mole of O- benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is Fmoc-Arg in step 3 (pbf) 2.5-3 times of-Wang resin mole.

The mole of diisopropylethylamine is 5-6 times of Fmoc-Leu-Wang resin mole in step 3.

The cutting reagent of 10-25mL is added in every gram of peptide resin in step 6.

Cutting reagent described in step 6 is mixed by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95:2.5:2.5 It closes.

" Fmoc " group of amino acid most latter linked on peptide resin is removed completely in step 6 method particularly includes:

Peptide resin is washed into 3~5min with DMF, is drained, repeats 3~5 times, concentration expressed in percentage by volume is then added in resin It is deprotected solution for 20%~25% piperidines/DMF, is drained after stirring 5~10min, is repeated 2~3 times, add volume basis The piperidines that concentration is 20%~25%/DMF is deprotected solution, stirs 15~20min, removes " Fmoc " blocking group sufficiently, Solvent is drained later, is finally washed cleared deprotection solution with DMF, is removed the resin of " Fmoc " blocking group.

The cyclisation hybrid peptide that the enkephalins of above-mentioned multidigit point modification is mutually coupled with neurotensin (8-13) is preparing polypeptide Application in analgesic.

The cyclisation hybrid peptide that the enkephalins of above-mentioned multidigit point modification is mutually coupled with neurotensin (8-13) is preparing anti-mind Through the application in property pain medication.

Beneficial effects of the present invention:

The cyclisation hybrid peptide that the enkephalins of multidigit point modification of the present invention is mutually coupled with neurotensin (8-13), is by first sulphur Propylhomoserin/leucine enkephalin amino acid sequence " Tyr-Gly-Gly-Phe-Met/Leu " is modified and is cyclized by multidigit point To " Dmt-Gly-c (Cys-NMePhe-Met-Cys) " and " Dmt-Gly-c (Cys-NMePhe-Leu-Cys) " amino acid residue Sequence modifies the amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13) by multidigit point To " NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence, using intermediate connexon " Gly " or " D-Ala " by two Partial amino-acid residue sequence is mutually coupled, and constructs a kind of new and effective hybrid peptide.

Wherein multidigit point modification is by methionine/leucine enkephalin amino acid sequence " Tyr-Gly-Gly-Phe- 1 Tyr of Met/Leu " is substituted with Dmt, and 3 Gly are substituted with Cys, and 4 Phe are substituted with NMePhe and 6 increase Cys. Then it is cyclized, is specifically cyclized 3 and 6 Cys by disulfide bond.

The resistance to enzymolysis ability of polypeptide can be enhanced by the replacement of multidigit point unnatural amino acid, and be cyclized modification can also mention The biological stability of high polypeptide drugs, various hydrolases are not easily decomposed the peptide bond of cyclized polypeptide, further enhance the anti-of polypeptide Enzymatic hydrolysis ability, and then enhance transport capacity of the drug to cental system, improve the utilization rate and efficiency of drug.

As long as polypeptide is modified by site unnatural amino acid, then various hydrolases non-natural amino not easy to identify Acid, not degradable polypeptide, and then enhance the resistance to enzymolysis ability of polypeptide.The modification of multidigit point, the resistance to enzymolysis ability of polypeptide is with regard to stronger.It is more Site modification and cyclisation synergistic effect, significantly enhance the enzymatic hydrolysis stability of polypeptide.Experiment shows the small of hybrid peptide 1,2,3 and 4 Mouse brain plasma membrane half-life period respectively reach 297.1 ± 22.0min, 302.6 ± 28.7min, 447.1 ± 28.2min, 412.0 ± The serum half-life of 31.4min, hybrid peptide 1,2,3 and 4 respectively reach 267.0 ± 20.1min, 258.5 ± 21.4min, 339.7 ±21.7min、311.2±31.9min。

It is small by being added among the modification sequence of the methionine/leucine enkephalin and neurotensin (8-13) of hybrid peptide The flexible conformation of hybrid peptide can be improved in molecule Amino acid linker " Gly " or " D-Ala ", retains methionine/leucine brain The bioactivity of deltorphin delta and neurotensin (8-13) two parts modification sequence.Connexon " D-Ala " is because introduce D type amino Acid will increase the resistance to enzymolysis ability of hybrid peptide.Also can moreover, modification is replaced and be cyclized by multidigit point unnatural amino acid Enhance the biological stability of hybrid peptide.Neurotensin (8-13) is the minimum active fragment of neurotensin, the mind of hybrid peptide Through hypotensor medicine (8-13) partially endogenic opioid peptides can be caused to discharge, analgesia is participated in and make by activating Opioid Receptor System Performance, and then efficient analgesic activities are generated with the enkephalins part of hybrid peptide.Moreover, the hybrid peptide tool there are two band just The amino acid of electricity, can form the hydrogen bond of divalent with the anionic group of cell surface, and enhancing hybrid peptide wears blood-brain barrier Saturating ability, and then improve the maincenter utilization rate of drug.In addition, the neurotensin (8-13) in hybrid peptide partially can reduce Ah The adverse side effects such as the analgesia tolerance and angiocarpy of opiates.

In addition, hybrid peptide 3 and 4 of the invention also has efficient analgesic activities to neuropathology pain.Experiment shows The maximum analgesic effect of hybrid peptide 3 and 4 generates after injecting drug 10min, and analgesia duration is longer, it is sustainable at least 40min。

Hybrid peptide 1-4 of the invention enzymatic hydrolysis stability with higher.By central administration, hybrid peptide 1-4 is in hot whipping All there are efficient analgesic activities in experiment.Moreover, hybrid peptide 3 and 4 has peripherally administered efficient analgesic effect, and significant Anti- neuropathic pain activity.In addition, the medicine analgesic tolerance side effect of hybrid peptide 3 and 4 significantly reduces, especially hybrid peptide 3 has Without analgesia tolerance characteristic, and it is significantly reduced the side effect of cardiovascular system.

Therefore, hybrid peptide of the invention is in the polypeptide analgesic for preparing efficient, low analgesia tolerance and cardiovascular side effects Aspect has potential application and scientific meaning.

Detailed description of the invention

Fig. 1 is analgesic effect-time graph of intracerebroventricular injection hybrid peptide 1;Wherein ■ indicates hybrid peptide 1,10nmol, ● Indicate hybrid peptide 1,3nmol, ▲ indicate that hybrid peptide 1,1nmol, ▼ indicate hybrid peptide 1,0.3nmol, ◆ indicate physiological saline;

Fig. 2 is analgesic effect-time graph of intracerebroventricular injection hybrid peptide 2;Wherein ■ indicates hybrid peptide 2,10nmol, ● Indicate hybrid peptide 2,3nmol, ▲ indicate that hybrid peptide 2,1nmol, ▼ indicate hybrid peptide 2,0.3nmol, ◆ indicate physiological saline;

Fig. 3 is analgesic effect-time graph of intracerebroventricular injection hybrid peptide 3;Wherein ■ indicates hybrid peptide 3,10nmol, ● Indicate hybrid peptide 3,3nmol, ▲ indicate that hybrid peptide 3,1nmol, ▼ indicate hybrid peptide 3,0.3nmol, ◆ indicate physiological saline;

Fig. 4 is analgesic effect-time graph of intracerebroventricular injection hybrid peptide 4;Wherein ■ indicates hybrid peptide 4,10nmol, ● Indicate hybrid peptide 4,3nmol, ▲ indicate that hybrid peptide 4,1nmol, ▼ indicate hybrid peptide 4,0.3nmol, ◆ indicate physiological saline;

Fig. 5 is the periphery analgesic effect that hybrid peptide 3 and 4 is subcutaneously injected;Wherein ■ indicates hybrid peptide 3 (10 μm of ol/kg, skin Under), ● indicate hybrid peptide 4 (10 μm of ol/kg, subcutaneous), ▲ indicate physiological saline (subcutaneous);

Fig. 6 is the analgesia tolerance dose curve of intracerebroventricular injection morphine;Wherein ■ indicates physiological saline+morphine, ● it indicates Morphine (10nmol)+morphine;

Fig. 7 is the analgesia tolerance dose curve of intracerebroventricular injection hybrid peptide 3;Wherein ■ indicates physiological saline+hybrid peptide 3, ● indicate hybrid peptide 3 (10nmol)+hybrid peptide 3;

Fig. 8 is the analgesia tolerance dose curve of intracerebroventricular injection hybrid peptide 4;Wherein ■ indicates physiological saline+hybrid peptide 4, ● indicate hybrid peptide 4 (10nmol)+hybrid peptide 4;

Fig. 9 is the anti-neuropathic pain activity of intracerebroventricular injection hybrid peptide 3;Wherein ■ indicates hybrid peptide 3,10nmol, ● Indicate hybrid peptide 3,3nmol, ▲ indicate that hybrid peptide 3,1nmol, ▼ indicate physiological saline;

Figure 10 is the anti-neuropathic pain activity of intracerebroventricular injection hybrid peptide 4;Wherein ■ indicates hybrid peptide 4,10nmol, ● Indicate hybrid peptide 4,3nmol, ▲ indicate that hybrid peptide 4,1nmol, ▼ indicate physiological saline;

Figure 11 is the effect of intravenous morphine and hybrid peptide 3 and 4 pair rat system angiosthenia;

Figure 12 is the effect of intravenous morphine and hybrid peptide 3 and 4 pair rat heart rate.

Specific embodiment

The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment Any combination.

Specific embodiment 1: what the enkephalins of present embodiment multidigit point modification was mutually coupled with neurotensin (8-13) Cyclisation hybrid peptide is hybrid peptide 1, hybrid peptide 2, hybrid peptide 3 or hybrid peptide 4;

The wherein amino acid sequence of hybrid peptide 1 are as follows:

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

The amino acid sequence of hybrid peptide 2 are as follows:

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

The amino acid sequence of hybrid peptide 3 are as follows:

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

The amino acid sequence of hybrid peptide 4 are as follows:

Dmt-Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu。

Present embodiment is by methionine/leucine enkephalin amino acid sequence " Tyr-Gly-Gly-Phe-Met/ Leu " obtains " Dmt-Gly-c (Cys-NMePhe-Met-Cys) " and " Dmt-Gly-c (Cys- with cyclisation by the modification of multidigit point NMePhe-Leu-Cys) " amino acid residue sequence, by the amino acid sequence " Arg-Arg-Pro- of neurotensin (8-13) Tyr-Ile-Leu " modifies to obtain " NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence by multidigit point, utilizes Two parts amino acid residue sequence is mutually coupled by intermediate connexon " Gly " or " D-Ala ", constructs a kind of efficient hybrid peptide.

The resistance to enzymolysis ability of polypeptide can be enhanced by the replacement of multidigit point unnatural amino acid, and be cyclized modification can also mention The biological stability of high polypeptide drugs, various hydrolases are not easily decomposed the peptide bond of cyclized polypeptide, further enhance the anti-of polypeptide Enzymatic hydrolysis ability, and then enhance transport capacity of the drug to cental system, improve the utilization rate and efficiency of drug.

Specific embodiment 2: what the enkephalins of present embodiment multidigit point modification was mutually coupled with neurotensin (8-13) It is cyclized the synthetic method of hybrid peptide, comprising the following steps:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, an amino acid will be had The Fmoc-Leu-Wang resin of residue is put into synthesizer, and add methylene chloride 30~40min of stirring, and resin is made sufficiently to impregnate swelling Afterwards, decompression filters solvent;The wherein body of the quality of the Fmoc-Leu-Wang resin with an amino acid residue and methylene chloride Product is than being 1g:(7~12) mL;

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3~5min with DMF, are drained, repetition 3~ 5 times, piperidines/DMF that concentration expressed in percentage by volume is 20%~25% is then added in resin and is deprotected solution, stirs 5~10min After drain, repeat 2~3 times, add concentration expressed in percentage by volume be 20%~25% piperidines/DMF be deprotected solution, stirring 15~ 20min removes " Fmoc " blocking group sufficiently, drains solvent later, finally washs cleared deprotection solution with DMF, obtains Remove the resin of " Fmoc " blocking group;Wherein the quality of resin and the volume ratio for the deprotection solution being added for the first time are 1g: (8~12) mL;The volume ratio for the deprotection solution that the quality of resin is added with second is 1g:(10~14) mL;

Three, amino acid condensation reacts: successively by the amino acid, N- hydroxy benzo triazole, O- benzene of " Fmoc " radical protection And triazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, mixes mixed after adding diisopropylethylamine Solution is closed, then mixed solution is added in the resin of step 2 removing " Fmoc " blocking group, is stirred under protection of argon gas React 60~72min;

The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove not instead The residual liquid answered;

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence of heterozygosis peptide amino acid sequence, from the end C- of polypeptide Successively the amino acid of " Fmoc " radical protection is condensed on resin one by one to the end N-, until all amino acid residues have been condensed At obtaining peptide resin;The wherein amino acid sequence of hybrid peptide 1 are as follows: Dmt-Gly-c (Cys-NMePhe-Met-Cys)-Gly- NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 2 are as follows: Dmt-Gly-c (Cys-NMePhe-Leu- Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 3 are as follows: Dmt-Gly-c (Cys- NMePhe-Met-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;The amino acid sequence of hybrid peptide 4 are as follows: Dmt- Gly-c(Cys-NMePhe-Leu-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;

Five, the formation of disulfide bond: I is weighed2It is dissolved in anhydrous methanol and DMF mixed liquor, is made into I2Solution, and by I2Solution It is added in peptide resin, is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, drain reaction solution;Wherein mixed solution The volume ratio of middle anhydrous methanol and DMF are 4:1, I2Quality and mixed solution volume ratio be 2.6g:100mL, I2Solution and peptide The volume ratio of resin is 100mL:1g;

Six, peptide chain is from the cutting on resin: " Fmoc " group of amino acid most latter linked on peptide resin is removed completely, Then peptide resin is alternately washed with methylene chloride and methanol, after sufficiently draining solvent, cutting reagent is added into peptide resin, in room Lower 3~the 5h of cleavage reaction of temperature;

Collect cutting reagent and depressurize and be spin-dried for, be precipitated and precipitated with ice-cold ether, remove ether supernatant after standing, then with water and Acetic acid sufficiently dissolves precipitating, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white The thick peptide of solid powder;

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 10%-20% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound, then isolated and purified by Reversed Phase High Performance, main peak is collected, white is obtained after freeze-drying The pure peptide of solid powder.

Specific embodiment 3: present embodiment is unlike specific embodiment two: " Fmoc " group in step 3 The mole of the amino acid of protection is 2.5-3 times of Fmoc-Leu-Wang resin mole.Other and specific embodiment two-phase Together.

Specific embodiment 4: present embodiment is unlike specific embodiment two or three: N- hydroxyl in step 3 The mole of benzotriazole is 2.5-3 times of Fmoc-Leu-Wang resin mole.Other and specific embodiment two or three It is identical.

Specific embodiment 5: unlike one of present embodiment and specific embodiment two to four: O- in step 3 The mole of benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is Fmoc-Arg (pbf)-Wang resin mole 2.5-3 times of amount.It is other identical as one of specific embodiment two to four.

Specific embodiment 6: unlike one of present embodiment and specific embodiment two to five: two in step 3 The mole of wopropyl ethyl amine is 5-6 times of Fmoc-Leu-Wang resin mole.It is other with specific embodiment two to five it One is identical.

Specific embodiment 7: unlike one of present embodiment and specific embodiment two to six: every in step 6 The cutting reagent of 10-25mL is added in gram peptide resin.It is other identical as one of specific embodiment two to six.

Specific embodiment 8: present embodiment is unlike specific embodiment seven: cutting examination described in step 6 Agent is mixed by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95:2.5:2.5.Other and specific embodiment Seven is identical.

Specific embodiment 9: unlike one of present embodiment and specific embodiment two to eight: will in step 6 " Fmoc " group of most latter linked amino acid removes completely on peptide resin method particularly includes:

Peptide resin is washed into 3~5min with DMF, is drained, repeats 3~5 times, concentration expressed in percentage by volume is then added in resin It is deprotected solution for 20%~25% piperidines/DMF, is drained after stirring 5~10min, is repeated 2~3 times, add volume basis The piperidines that concentration is 20%~25%/DMF is deprotected solution, stirs 15~20min, removes " Fmoc " blocking group sufficiently, Solvent is drained later, is finally washed cleared deprotection solution with DMF, is removed the resin of " Fmoc " blocking group.It is other with One of specific embodiment two to eight is identical.

Specific embodiment 10: what the enkephalins of present embodiment multidigit point modification was mutually coupled with neurotensin (8-13) Cyclisation hybrid peptide is preparing the application in polypeptide analgesic.

Specific embodiment 11: the enkephalins of present embodiment multidigit point modification is mutually coupled with neurotensin (8-13) Cyclisation hybrid peptide preparing the application in anti-neuropathic pain drug.

Elaborate below to the embodiment of the present invention, following embodiment under the premise of the technical scheme of the present invention into Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities Apply example.

Embodiment 1:

The synthesis for the cyclisation hybrid peptide 1 that the enkephalins of the present embodiment multidigit point modification is mutually coupled with neurotensin (8-13) Method, comprising the following steps:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, 1g is had into an amino The Fmoc-Leu-Wang resin of sour residue is put into synthesizer, adds 10mL methylene chloride to stir 30min, impregnates resin sufficiently molten After swollen, decompression filters solvent.

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3min with DMF, drains, is repeated 3 times.? Piperidines/DMF that 10mL concentration expressed in percentage by volume is 20% is added in resin and is deprotected solution, drains, is repeated 2 times after stirring 5min, It adds piperidines/DMF that 12mL concentration expressed in percentage by volume is 20% and is deprotected solution, stir 15min, fill " Fmoc " blocking group Divide removing, drains solvent later, finally wash cleared deprotecting regent with 10mL DMF, removed " Fmoc " blocking group Resin.

Three, amino acid condensation reacts: successively will be equivalent to " Fmoc " of 2.5-3 times of mole of Fmoc-Leu-Wang resin Amino acid, N- hydroxy benzo triazole, O- benzotriazole-N, N, N', the N'- tetramethylurea-hexafluorophosphate of radical protection It is dissolved in 10mL DMF, is mixed after adding the diisopropylethylamine for being equivalent to 5-6 times of mole of Fmoc-Leu-Wang resin Then mixed solution is added in the resin of the deprotection, is stirred to react 60min under protection of argon gas by mixed solution. The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove unreacted residual Liquid.

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence Dmt-Gly-Cys- of 1 amino acid sequence of hybrid peptide NMePhe-Met-Cys-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu, from the end C- of polypeptide to the end N- successively by " Fmoc " The amino acid of radical protection is condensed on resin one by one, until the condensation of all amino acid residues is completed, obtains peptide resin.

Five, the formation of disulfide bond: the I of 2.6g is weighed2It is dissolved in the DMF mixed liquor of 80mL anhydrous methanol and 20mL, is made into The I of 0.1mol/L2Solution, and add it in peptide resin, it is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, Drain reaction solution.

Six, peptide chain is from the cutting on resin: according to the method for step 2 by amino acid most latter linked on peptide resin " Fmoc " group removes completely.Resin is alternately washed with methylene chloride, methanol, after sufficiently draining solvent, is added by every gram of peptide resin Enter the cutting reagent of 18mL, cutting reagent by trifluoroacetic acid, diisopropylsilyl, water with the volume ratio mixing of 95:2.5:2.5 and At cleavage reaction 3h at room temperature.It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, standing is gone in a moment Precipitating is sufficiently dissolved except ether supernatant, then with water and a small amount of acetic acid, pours into separatory funnel, and ethyl acetate extraction is added, is collected Water phase, it is freeze-dried, obtain the thick peptide of white solid powder.

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 16% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound.It is isolated and purified again by Reversed Phase High Performance, collects main peak, white is obtained after freeze-drying Solid end product hybrid peptide 1.

Embodiment 2:

The synthesis for the cyclisation hybrid peptide 1 that the enkephalins of the present embodiment multidigit point modification is mutually coupled with neurotensin (8-13) Method, comprising the following steps:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, 1g is had into an amino The Fmoc-Leu-Wang resin of sour residue is put into synthesizer, adds 10mL methylene chloride to stir 30min, impregnates resin sufficiently molten After swollen, decompression filters solvent.

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3min with DMF, drains, is repeated 3 times.? Piperidines/DMF that 10mL concentration expressed in percentage by volume is 20% is added in resin and is deprotected solution, drains, is repeated 2 times after stirring 5min, It adds piperidines/DMF that 12mL concentration expressed in percentage by volume is 20% and is deprotected solution, stir 15min, fill " Fmoc " blocking group Divide removing, drains solvent later, finally wash cleared deprotecting regent with 10mL DMF, removed " Fmoc " blocking group Resin.

Three, amino acid condensation reacts: successively will be equivalent to " Fmoc " of 2.5-3 times of mole of Fmoc-Leu-Wang resin Amino acid, N- hydroxy benzo triazole, O- benzotriazole-N, N, N', the N'- tetramethylurea-hexafluorophosphate of radical protection It is dissolved in 10mL DMF, is mixed after adding the diisopropylethylamine for being equivalent to 5-6 times of mole of Fmoc-Leu-Wang resin Then mixed solution is added in the resin of the deprotection, is stirred to react 60min under protection of argon gas by mixed solution. The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove unreacted residual Liquid.

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence Dmt-Gly-Cys- of 1 amino acid sequence of hybrid peptide NMePhe-Leu-Cys-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu, from the end C- of polypeptide to the end N- successively by " Fmoc " The amino acid of radical protection is condensed on resin one by one, until the condensation of all amino acid residues is completed, obtains peptide resin.

Five, the formation of disulfide bond: the I of 2.6g is weighed2It is dissolved in the DMF mixed liquor of 80mL anhydrous methanol and 20mL, is made into The I of 0.1mol/L2Solution, and add it in peptide resin, it is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, Drain reaction solution.

Six, peptide chain is from the cutting on resin: according to the method for step 2 by amino acid most latter linked on peptide resin " Fmoc " group removes completely.Resin is alternately washed with methylene chloride, methanol, after sufficiently draining solvent, is added by every gram of peptide resin Enter the cutting reagent of 18mL, cutting reagent by trifluoroacetic acid, diisopropylsilyl, water with the volume ratio mixing of 95:2.5:2.5 and At cleavage reaction 3h at room temperature.It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, standing is gone in a moment Precipitating is sufficiently dissolved except ether supernatant, then with water and a small amount of acetic acid, pours into separatory funnel, and ethyl acetate extraction is added, is collected Water phase, it is freeze-dried, obtain the thick peptide of white solid powder.

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 16% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound.It is isolated and purified again by Reversed Phase High Performance, collects main peak, white is obtained after freeze-drying Solid end product hybrid peptide 2.

Embodiment 3:

The synthesis for the cyclisation hybrid peptide 1 that the enkephalins of the present embodiment multidigit point modification is mutually coupled with neurotensin (8-13) Method, comprising the following steps:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, 1g is had into an amino The Fmoc-Leu-Wang resin of sour residue is put into synthesizer, adds 10mL methylene chloride to stir 30min, impregnates resin sufficiently molten After swollen, decompression filters solvent.

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3min with DMF, drains, is repeated 3 times.? Piperidines/DMF that 10mL concentration expressed in percentage by volume is 20% is added in resin and is deprotected solution, drains, is repeated 2 times after stirring 5min, It adds piperidines/DMF that 12mL concentration expressed in percentage by volume is 20% and is deprotected solution, stir 15min, fill " Fmoc " blocking group Divide removing, drains solvent later, finally wash cleared deprotecting regent with 10mL DMF, removed " Fmoc " blocking group Resin.

Three, amino acid condensation reacts: successively will be equivalent to " Fmoc " of 2.5-3 times of mole of Fmoc-Leu-Wang resin Amino acid, N- hydroxy benzo triazole, O- benzotriazole-N, N, N', the N'- tetramethylurea-hexafluorophosphate of radical protection It is dissolved in 10mL DMF, is mixed after adding the diisopropylethylamine for being equivalent to 5-6 times of mole of Fmoc-Leu-Wang resin Then mixed solution is added in the resin of the deprotection, is stirred to react 60min under protection of argon gas by mixed solution. The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove unreacted residual Liquid.

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence Dmt-Gly-Cys- of 1 amino acid sequence of hybrid peptide NMePhe-Met-Cys-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu successively will from the end C- of polypeptide to the end N- The amino acid of " Fmoc " radical protection is condensed on resin one by one, until the condensation of all amino acid residues is completed, obtains peptide resin.

Five, the formation of disulfide bond: the I of 2.6g is weighed2It is dissolved in the DMF mixed liquor of 80mL anhydrous methanol and 20mL, is made into The I of 0.1mol/L2Solution, and add it in peptide resin, it is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, Drain reaction solution.

Six, peptide chain is from the cutting on resin: according to the method for step 2 by amino acid most latter linked on peptide resin " Fmoc " group removes completely.Resin is alternately washed with methylene chloride, methanol, after sufficiently draining solvent, is added by every gram of peptide resin Enter the cutting reagent of 18mL, cutting reagent by trifluoroacetic acid, diisopropylsilyl, water with the volume ratio mixing of 95:2.5:2.5 and At cleavage reaction 3h at room temperature.It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, standing is gone in a moment Precipitating is sufficiently dissolved except ether supernatant, then with water and a small amount of acetic acid, pours into separatory funnel, and ethyl acetate extraction is added, is collected Water phase, it is freeze-dried, obtain the thick peptide of white solid powder.

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 16% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound.It is isolated and purified again by Reversed Phase High Performance, collects main peak, white is obtained after freeze-drying Solid end product hybrid peptide 3.

Embodiment 4:

The synthesis for the cyclisation hybrid peptide 1 that the enkephalins of the present embodiment multidigit point modification is mutually coupled with neurotensin (8-13) Method, comprising the following steps:

One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, 1g is had into an amino The Fmoc-Leu-Wang resin of sour residue is put into synthesizer, adds 10mL methylene chloride to stir 30min, impregnates resin sufficiently molten After swollen, decompression filters solvent.

Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3min with DMF, drains, is repeated 3 times.? Piperidines/DMF that 10mL concentration expressed in percentage by volume is 20% is added in resin and is deprotected solution, drains, is repeated 2 times after stirring 5min, It adds piperidines/DMF that 12mL concentration expressed in percentage by volume is 20% and is deprotected solution, stir 15min, fill " Fmoc " blocking group Divide removing, drains solvent later, finally wash cleared deprotecting regent with 10mL DMF, removed " Fmoc " blocking group Resin.

Three, amino acid condensation reacts: successively will be equivalent to " Fmoc " of 2.5-3 times of mole of Fmoc-Leu-Wang resin Amino acid, N- hydroxy benzo triazole, O- benzotriazole-N, N, N', the N'- tetramethylurea-hexafluorophosphate of radical protection It is dissolved in 10mL DMF, is mixed after adding the diisopropylethylamine for being equivalent to 5-6 times of mole of Fmoc-Leu-Wang resin Then mixed solution is added in the resin of the deprotection, is stirred to react 60min under protection of argon gas by mixed solution. The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly after fully reacting with DMF to remove unreacted residual Liquid.

Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence Dmt-Gly-Cys- of 1 amino acid sequence of hybrid peptide NMePhe-Leu-Cys-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu successively will from the end C- of polypeptide to the end N- The amino acid of " Fmoc " radical protection is condensed on resin one by one, until the condensation of all amino acid residues is completed, obtains peptide resin.

Five, the formation of disulfide bond: the I of 2.6g is weighed2It is dissolved in the DMF mixed liquor of 85mL anhydrous methanol and 10mL, is made into The I of 0.1mol/L2Solution, and add it in peptide resin, it is passed through argon gas protection, is protected from light and is stirred to react 3~5 hours cyclization, Drain reaction solution.

Six, peptide chain is from the cutting on resin: according to the method for step 2 by amino acid most latter linked on peptide resin " Fmoc " group removes completely.Resin is alternately washed with methylene chloride, methanol, after sufficiently draining solvent, is added by every gram of peptide resin Enter the cutting reagent of 18mL, cutting reagent by trifluoroacetic acid, diisopropylsilyl, water with the volume ratio mixing of 95:2.5:2.5 and At cleavage reaction 3h at room temperature.It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, standing is gone in a moment Precipitating is sufficiently dissolved except ether supernatant, then with water and a small amount of acetic acid, pours into separatory funnel, and ethyl acetate extraction is added, is collected Water phase, it is freeze-dried, obtain the thick peptide of white solid powder.

Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 16% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound.It is isolated and purified again by Reversed Phase High Performance, collects main peak, white is obtained after freeze-drying Solid end product hybrid peptide 4.

The ultimate yield of the hybrid peptide of above embodiments preparation reaches 25% or more, mass spectrum and chromatography testing result As shown in table 1.

The mass spectrum and chromatography testing result of 1. hybrid peptide 1-4 of table

Table 1 the result shows that, the detected value for the hybrid peptide 1-4 that the present invention obtains is consistent with theoretical value.

The cyclisation heterozygosis that the enkephalins of the multidigit point modification of the present embodiment 1-4 preparation is mutually coupled with neurotensin (8-13) The biological activity test of peptide is as follows:

1, unorganized ferment Numerical solution is tested

100% mice serum and 15% brain membrane sample are prepared, 10 μ L polypeptide mother liquors (10 are taken-2M), it is added to 190 μ L's In 100% mice serum or 15% brain plasma membrane, oscillation is mixed immediately, then takes out 20 μ L mixed liquors rapidly, is added in centrifuge tube Timing is 0min, and remaining person continues to be incubated at 37 DEG C, and respectively in 5min, 10min, 15min, 30min, 60min, 120min, 240min takes out 20 μ L.The termination of enzymolysis process: the oscillation of 90 μ L acetonitriles is added in the sample of taking-up and mixes, sample is placed on ice 5min is placed, then with the ice-cold acetic acid dilution of 90 μ L 0.5% to ensure that enzymolysis process stops.13,000g centrifugation 15min, are collected Supernatant, -80 DEG C freeze, until RP-HPLC is analyzed.

Experimental result is shown in Table 2.Table 2 the results show that the mouse brain plasma membrane long half time of hybrid peptide 1 in serum half-life, The mouse brain plasma membrane and serum half-life and hybrid peptide 1 of hybrid peptide 2 are suitable.And the brain plasma membrane and serum half-life of hybrid peptide 3 and 4 It is all considerably longer than hybrid peptide 1 and 2, shows space connexon " D-Ala " because introducing D type amino acid, enhances hybrid peptide Resistance to enzymolysis ability.Wherein, half-life period longest of the hybrid peptide 3 in two kinds of samples, i.e. enzymatic hydrolysis stability highest.It should be the result shows that logical Excessive site unnatural amino acid modification, the introducing and polypeptide cyclisation of space connexon " D-Ala " can significantly improve heterozygosis The enzymatic hydrolysis stability of peptide 3 and 4.

The brain plasma membrane and serum half-life of 2. hybrid peptide 1-4 of table

2, hot whipping analgesic experiment

It tests and uses Kunming system male mice, 18-22g, environment temperature: 20 DEG C, bath temperature: 50 ± 0.5 DEG C.Before administration The 1/3-1/2 of mouse rat-tail is immersed water-bath, records rat-tail by the Basic Pain Threshold (control latency, CL) for first measuring mouse Water-bath is immersed to the time shunk from rigid.Too sensitive (<3s) or blunt (>5s) mouse is discarded, deadline 10s, to prevent mouse from scalding.It is latent respectively to survey a whipping by 5,10,15,20,25,30,40and 50min after telocoele administration Phase (test latency, TL), the 5th, 10,15,20,25,30,45,60and 90min respectively surveys a TL after subcutaneous administration, 0.9% physiological saline is as blank control.As a result with maximum possible effect percentage (maximum possible effect, % MPE it) indicates: %MPE=100 × (TL-CL)/(10-CL).

The hot whipping analgesic experiment result of the mouse of central administration is as shown in Fig. 1,2,3 and 4, intracerebroventricular injection 0.3-10nmol The hybrid peptide 1-4 of dosage all has significant analgesic activities, and is in concentration-dependant, and maximum analgesic effect is in injection drug It is generated after 10min.Wherein, the hybrid peptide 3 and 4 of Central injection 10nmol high dose, maximum analgesia %MPE value are respectively 92.46% and 87.91%.In addition, hybrid peptide 1-4 have longer analgesia duration, sustainable at least 50min or more, this Show that hybrid peptide has efficient central analgesia activity.In addition, peripherally administered analgesic experiment result is as shown in figure 5, pass through skin The hybrid peptide 3 and 4 of 10 μm of ol/kg dosage of lower injection, as a result, it has been found that hybrid peptide 3 and 4 still has efficient analgesic activities.Heterozygosis The maximum analgesic effect of peptide 3 and 4 generates after injecting drug 15min, and maximum analgesia %MPE value is respectively 85.16% He 81.78%.The analgesia %MPE value that 90min measures hybrid peptide 3 and 4 upon administration remains to reach 31.68% and 28.26%, shows The analgesia duration of peripheral injection hybrid peptide 3 and 4 can reach 90min or more, i.e., the analgesia duration of hybrid peptide 3 and 4 compared with It is long.The novel cyclized hybrid peptide that the above results show that enkephalins and the neurotensin (8-13) of multidigit point modification are mutually coupled is significant Analgesic activities and the duration of drug are enhanced, has the characteristics of efficient analgesic activities.

3, Acute brain block is tested

Kunming system male mice, 18-22g, environment temperature: 20 DEG C are chosen in experiment.It is intubated using PE-10 to mouse telocoele Pipe laying is administered for 3 days after operation.According to the analgesic activities of hybrid peptide, drug dose is determined, measure acute analgesia tolerance.Mouse mentions Preceding 1 hour intracerebroventricular injection drug or physiological saline 1 time, inject the drug of various dose respectively later, and measurement pain threshold becomes Change, and then analgesia tolerance assessment is carried out to hybrid peptide, by morphine as positive control.

Central injection morphine, hybrid peptide 3 and 4 analgesia tolerance result as shown in Fig. 6,7 and 8,1 hour in advance to mouse side Intracerebroventricular physiological saline, the morphine, hybrid peptide 3 and 4 of intracerebroventricular injection 0.3-10nmol various dose generates obviously later Analgesic effect, and be in concentration-dependant feature, show that shifting to an earlier date injecting normal saline to mouse has no effect on morphine, hybrid peptide 3 With the activity of 4 equal analgesics.However, being injected again later by the morphine for shifting to an earlier date 1 hour intracerebroventricular injection 10nmol dosage The corresponding drug morphine of 0.3-10nmol various dose, medicine analgesic dose curve obviously move to right, and central analgesia activity is aobvious Writing reduces, i.e., morphine produces apparent medicine analgesic tolerance.But 1 hour in advance intracerebroventricular injection 10nmol dosage is miscellaneous Peptide 3 is closed, then injects the hybrid peptide 3 of 0.3-10nmol various dose, the town of analgesic dose curve and injecting normal saline in advance Pain dose curve is almost overlapped, and there is no generate significantly to move to left or move to right.It should be the result shows that injection hybrid peptide 3 be to the in advance The analgesic activities of secondary acute injection hybrid peptide 3 have little effect, and show that hybrid peptide 3 has the analgesia without tolerance side effect special Property.In addition, the hybrid peptide 4 of 1 hour in advance intracerebroventricular injection 10nmol dosage, then inject the heterozygosis of 0.3-10nmol various dose Peptide 4, medicine analgesic dose curve move to right that degree is smaller compared with morphine, show that the analgesia tolerance of hybrid peptide 4 is substantially reduced. The above result shows that the medicine analgesic tolerance side effect of hybrid peptide 3 and 4 significantly reduces, especially hybrid peptide 3 has the height without tolerance Imitate analgesic properties.

4, anti-neuropathic pain experiment

Animal models of neuropathic pain is established by the method stabilization that mouse sciatic nerve branching selection damages.Experiment Selection Kunming system male mice, 20-25g, wherein two for tightly pricking and cutting off three branch of sciatic nerve end of mouse side, i.e., Nervus tibialis and nervus peroneus communis branch retain nervus suralis branch.The postoperative 24 hours pain for starting duration occur, duration Up to 7 weeks or more, i.e., induction of stable neuropathic pain animal model.The mechanical threshold of pain, research are measured by Von Frey fiber The anti-neuropathic pain activity of Central injection drug.

The anti-neuropathic pain exercising result of Central injection hybrid peptide 3 and 4 is as shown in Figures 9 and 10, intracerebroventricular injection 1- The hybrid peptide 3 and 4 of 10nmol is dose-dependent to cause the paw withdrawal threshold of reaction to increase, it was demonstrated that hybrid peptide 3 and 4 has apparent anti-mind Through property pain activity.The maximum analgesic effect of hybrid peptide 3 and 4 generates after injecting drug 10min, and analgesia duration compared with Long, sustainable at least 40min shows that hybrid peptide 3 and 4 pair neuropathology pain has efficient analgesic activities.

5, rat in vivo Blood Pressure Experiment

Using Wistar rat, 200-300g, with 20% Anaesthesia with Ethyl Carbamate (l.2g/kg, i.p.), in order to protect It is good to hold narcosis, it may be necessary to temporarily add anaesthetic.The tracheae of incision rat, removes mucus in managing, and animal can be certainly Main breathing.PE-50 conduit is inserted into vena jugularis externa in case intravenously administrable, PE-50 conduit is inserted into neck main artery, and presses with YP-100 Force snesor is connected, the variation of blood pressure after record administration.System arterial pressure and heart rate data mainly pass through Chengdu Tai Meng BL-420F Biological recorder system handling averagely obtains.100 μ L of medical intravenous per injection is injected in 10~15 seconds and is completed.

The experimental result that morphine, hybrid peptide 3 and 4 pair Rat Cardiovascular act on is as shown in FIG. 11 and 12 (in Figure 11 and 12Indicate morphine,Indicate hybrid peptide 3,Indicate hybrid peptide 4), intravenous injection 1-10nmol dosage Coffee, hybrid peptide 3 and 4 significantly reduce rat system arterial pressure, and are in dose dependent.However hybrid peptide 3 under all concentration The effect of arterial pressure is reduced significantly lower than the morphine under corresponding concentration with 4.In addition, intravenous injection 1-10nmol dosage The dose-dependent heart rate for reducing rat of coffee, hybrid peptide 3 and 4.It is similar with system arterial pressure, hybrid peptide 3 and 4 pair under all concentration The effect that rat heart rate reduces is significantly lower than the morphine under corresponding concentration.Should the result shows that, hybrid peptide 3 and 4 pair is in body-centered blood vessel The function and effect of system are significantly lower than morphine, that is, have the advantages that lower cardiovascular side effects.

In conclusion the cyclisation heterozygosis that the enkephalins of multidigit point modification of the present invention is mutually coupled with neurotensin (8-13) Peptide is to repair methionine/leucine enkephalin amino acid sequence " Tyr-Gly-Gly-Phe-Met/Leu " by multidigit point Decorations obtain " Dmt-Gly-c (Cys-NMePhe-Met-Cys) " and " Dmt-Gly-c (Cys-NMePhe-Leu-Cys) " with cyclisation Amino acid residue sequence passes through the amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13) more Site is modified to obtain " NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence, using intermediate connexon " Gly " or Two parts amino acid residue sequence is mutually coupled by " D-Ala ", constructs a kind of new and effective hybrid peptide.By the first sulphur ammonia of hybrid peptide Be added among the modification sequence of acid/leucine enkephalin and neurotensin (8-13) small molecule Amino acid linker " Gly " or " D-Ala " can be improved the flexible conformation of hybrid peptide, retain methionine/leucine enkephalin and neurotensin (8-13) The bioactivity of two parts modification sequence.Modified by multidigit point unnatural amino acid, the introducing of space connexon " D-Ala " with And polypeptide cyclisation can significantly increase the resistance to enzymolysis ability of hybrid peptide.Moreover, part neurotensin (8-13) in hybrid peptide It can reduce the adverse side effects such as analgesia tolerance and the angiocarpy of opioid drug, solve the resistance to enzymolysis energy of existing opioid drug Power is poor, and analgesic activities are lower and the duration is shorter, and anti-neuropathic pain is ineffective, and has analgesia tolerance and angiocarpy The problem of system side effect.

Tested by vitro biological stability, analgesia with Acute brain block test, anti-neurogenic pain activity experiment with And in body Blood Pressure Experiment, pharmacological activity identification is carried out to the hybrid peptide that the present invention synthesizes.The result shows that hybrid peptide of the invention Resistance to enzymolysis ability with higher.By central administration, hybrid peptide 1-4 in hot tail-flick test there is efficient analgesia to live Property.Moreover, hybrid peptide 3 and 4 has peripherally administered efficient analgesic effect, and significant anti-neuropathic pain activity.In addition, miscellaneous The medicine analgesic tolerance side effect for closing peptide 3 and 4 significantly reduces, and especially hybrid peptide 3 is with no analgesia tolerance characteristic, and it is to painstaking effort The side effect of guard system is significantly reduced.Therefore, hybrid peptide of the invention is preparing efficient, low analgesia tolerance and cardiovascular secondary work It is had potential application in terms of polypeptide analgesic.

Claims (8)

1. the cyclisation hybrid peptide that the enkephalins of multidigit point modification is mutually coupled with neurotensin (8-13), it is characterised in that the heterozygosis The amino acid sequence of peptide are as follows:
Dmt-Gly-c(Cys-NMePhe-Met-Cys)-D-Ala-NMeArg-Lys-Pro-Trp-Tle-Leu;
Wherein Dmt represents 2,6- dimethyl Tyr, and NMePhe represents N- methyl Phe, and NMeArg represents N- methyl Arg, and Tle is represented tert-Leu。
2. the cyclisation hybrid peptide that the enkephalins of multidigit point modification as described in claim 1 is mutually coupled with neurotensin (8-13) Synthetic method, it is characterised in that method includes the following steps:
One, it the Wang resin pretreatment of " Fmoc " protection: checks the air-tightness of solid phase synthetic instrument, an amino acid residue will be had Fmoc-Leu-Wang resin be put into synthesizer, add methylene chloride 30 ~ 40 min of stirring, make resin sufficiently impregnate swelling after, subtract Pressure filters solvent;The wherein volume ratio of the quality of the Fmoc-Leu-Wang resin with an amino acid residue and methylene chloride For 1g:(7 ~ 12) mL;
Two, it removes " Fmoc " blocking group: the swellable resins after draining being washed into 3 ~ 5 min with DMF, are drained, repeat 3 ~ 5 times, Then piperidines/DMF that concentration expressed in percentage by volume is 20% ~ 25% is added in resin and is deprotected solution, is drained after stirring 5 ~ 10 min, It repeats 2 ~ 3 times, adds piperidines/DMF that concentration expressed in percentage by volume is 20% ~ 25% and be deprotected solution, stir 15 ~ 20 min, make " Fmoc " blocking group sufficiently removes, and drains solvent later, finally washs cleared deprotection solution with DMF, is removed The resin of " Fmoc " blocking group;Wherein the quality of resin and for the first time be added deprotection solution volume ratio be 1g:(8 ~ 12) mL;The volume ratio for the deprotection solution that the quality of resin is added with second is 1g:(10 ~ 14) mL;
Three, amino acid condensation reacts: successively by the amino acid, N- hydroxy benzo triazole, O- benzo of " Fmoc " radical protection Triazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, mixes mixed after adding diisopropylethylamine Solution is closed, then mixed solution is added in the resin of step 2 removing " Fmoc " blocking group, is stirred under protection of argon gas React 60 ~ 72 min;
The degree that condensation reaction is completed is detected by indenes check reagent, it is unreacted to remove with DMF repeated washing after fully reacting Residual liquid;
Four, the extension of peptide chain: step 2 and three is repeated, according to the sequence of heterozygosis peptide amino acid sequence, from the end C- of polypeptide to N- The amino acid of " Fmoc " radical protection is successively condensed on resin by end one by one, until the condensation of all amino acid residues is completed, is obtained To peptide resin;The wherein amino acid sequence of hybrid peptide are as follows: Dmt-Gly-c (Cys-NMePhe-Met-Cys)-D-Ala-NMeArg- Lys-Pro-Trp-Tle-Leu;
Five, the formation of disulfide bond: I is weighed2It is dissolved in anhydrous methanol and DMF mixed liquor, is made into I2Solution, and by I2Solution is added Into peptide resin, it is passed through argon gas protection, is protected from light and is stirred to react 3 ~ 5 hours cyclization, drain reaction solution;It is wherein anhydrous in mixed solution The volume ratio of methanol and DMF are 4:1, I2Quality and mixed solution volume ratio be 2.6 g:100 mL, I2Solution and peptide resin Volume ratio be 100 mL:1g;
Six, peptide chain is from the cutting on resin: " Fmoc " group of amino acid most latter linked on peptide resin being removed completely, then Peptide resin is alternately washed with methylene chloride and methanol, after sufficiently draining solvent, cutting reagent is added into peptide resin, at room temperature 3 ~ 5 h of cleavage reaction;
It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, remove ether supernatant after standing, then with water and acetic acid Sufficiently dissolution precipitating, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white solid The thick peptide of powder;
Seven, the desalination and purifying of thick peptide: using the acetic acid solution of volumetric concentration 10%-20% as mobile phase, thick peptide is passed through Sephadex G25 cross-linked dextran gel column desalination is freeze-dried after collecting main peak using Ultraviolet Detector, obtains desalination Polypeptide compound, then isolated and purified by Reversed Phase High Performance, main peak is collected, white is obtained after freeze-drying The pure peptide of solid powder.
3. the cyclisation heterozygosis that the enkephalins of multidigit point modification according to claim 2 is mutually coupled with neurotensin (8-13) The synthetic method of peptide, it is characterised in that: the mole of the amino acid of " Fmoc " radical protection is Fmoc-Leu- in step 3 2.5-3 times of Wang resin mole.
4. the cyclisation that the enkephalins of multidigit point modification according to claim 2 or 3 is mutually coupled with neurotensin (8-13) The synthetic method of hybrid peptide, it is characterised in that: the mole of N- hydroxy benzo triazole is Fmoc-Leu-Wang tree in step 3 2.5-3 times of rouge mole.
5. the cyclisation heterozygosis that the enkephalins of multidigit point modification according to claim 4 is mutually coupled with neurotensin (8-13) The synthetic method of peptide, it is characterised in that: the mole of diisopropylethylamine is Fmoc-Leu-Wang resin mole in step 3 5-6 times.
6. the cyclisation heterozygosis that the enkephalins of multidigit point modification according to claim 5 is mutually coupled with neurotensin (8-13) The synthetic method of peptide, it is characterised in that: the cutting reagent of 10-25 mL is added in every gram of peptide resin in step 6.
7. the cyclisation heterozygosis that the enkephalins of multidigit point modification according to claim 6 is mutually coupled with neurotensin (8-13) The synthetic method of peptide, it is characterised in that: cutting reagent described in step 6 is by trifluoroacetic acid, diisopropylsilyl and water according to body Product is mixed than 95:2.5:2.5.
8. the cyclisation heterozygosis that the enkephalins of multidigit point modification according to claim 7 is mutually coupled with neurotensin (8-13) The synthetic method of peptide, it is characterised in that: in step 6 that " Fmoc " group of amino acid most latter linked on peptide resin is completely de- It removes method particularly includes:
Peptide resin is washed into 3 ~ 5 min with DMF, is drained, is repeated 3 ~ 5 times, it is 20% that concentration expressed in percentage by volume is then added in resin ~ 25% piperidines/DMF is deprotected solution, drains after stirring 5 ~ 10 min, repeats 2 ~ 3 times, and adding concentration expressed in percentage by volume is 20% ~ 25% piperidines/DMF is deprotected solution, stirs 15 ~ 20 min, removes " Fmoc " blocking group sufficiently, drain solvent later, Cleared deprotection solution finally is washed with DMF, is removed the resin of " Fmoc " blocking group.
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CN107964047A (en) * 2017-12-18 2018-04-27 哈尔滨工业大学 Chimeric peptide based on endomorphin-1 and neurotensin (8-13) and synthesis method and application thereof

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