CN109232747B - The cyclisation hybrid peptide and its synthetic method and application that dynorphin A (1-8) is mutually coupled with neurotensin (8-13) - Google Patents
The cyclisation hybrid peptide and its synthetic method and application that dynorphin A (1-8) is mutually coupled with neurotensin (8-13) Download PDFInfo
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Abstract
The cyclisation hybrid peptide and its synthetic method and application that dynorphin A (1-8) is mutually coupled with neurotensin (8-13) are related to a kind of cyclisation hybrid peptide and its synthetic method and application.Be to solve existing opioid drug biological stability it is poor, the ineffective problem of anti-neuropathic pain.The amino acid sequence of the hybrid peptide is Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-Leu.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
Technical field
The present invention relates to a kind of cyclisation hybrid peptide and its synthetic method and applications.
Background technique
It is well known that opium and its extract are used for always analgesia therapy.The morphine isolated from opium is exactly present
One of widest analgesic of clinical application.However the defect that the drugs such as morphine are used in safety clinical is also very much, such as inhibits
Breathe, cause blood pressure reduction and areocardia, constipation, Yi Yinqi psychological dependence, habituation and analgesia tolerance etc..In addition, morphine pair
The therapeutic effect of chronic ache such as neurogenic pain is bad.Studies have shown that transmitting and modulation of the opioid peptides in pain sensation information
It plays an important role in the process.After enkephalins and endorphin discovery, 1979, researcher had found from the hypophysis of pig again
A kind of ten heptapeptide of endogenous with opioid activity, and it is named as dynorphin.Dynorphin (dynorphin) is to κ-opiate receptor
It is κ-receptor endogenic ligand with high compatibility and selectivity, is belonged to enkephalins, endorphin and interior morphine peptide
In endogenous opiatepeptide family.Prodynorphin is the precursor of dynorphin, is eventually converted into dynorphin A (dynorphin in vivo
A, Dyn A) and dynorphin B (dynorphin B, Dyn B).Endogenic dynorphin A is there are many moiety structures, wherein dynorphin
A (1-8) is dynorphin minimum active fragment.Research about dynorphin in pain field is more, and dynorphin is as a kind of analgesia
Medium is widely used in evaluating analgesic effect.However the analgesic activities of dynorphin are relatively weak, hence it is evident that swash lower than μ-opiate receptor
The analgesic effect of the mediations such as dynamic agent such as morphine.In addition, the biological stability of dynorphin is poor, analgesia duration is shorter, blood brain
Barrier Penetration ration is lower, to limit its development as clinical analgesic.
The active nerve peptide being made of 13 amino acid is had found from the hypothalamus of ox early in American scholar in 1973,
That is neurotensin (neurotensin, NT).In central nervous system, neurotensin as neurotransmitter or it is quenched and
It plays a role, effect includes cooling, analgesia, adjusts dopaminergic transmitting and the release of Stimulation of Pituitary Gland anterior lobe normone.Neurotensin
Element has important relationship with opioid peptides in inhibiting pain sensation transmittance process.Neurotensin nerve endings and neurotensin by
Body be distributed widely in central nervous system with the related region of easing pain, and with endogenous opiatepeptide nerve endings and opium
Receptor is distributed companion lines.Some researches show that the analgesic effect and activation opiate system that Central injection neurotensin generates cause
The release of endogenous opiatepeptide is related.Furthermore, it has already been proven that the 8-13 amino acids " Arg-Arg-Pro- of neurotensin
Tyr-Ile-Leu " is the active minimal segment of neurotensin, and there are two positively charged arginine in amino acid sequence.Essence
The guanidino group of propylhomoserin can form the hydrogen bond of divalent with the anionic group of cell surface, enhance polypeptide to the penetrating of biomembrane
Property, such as blood-brain barrier.The analog sequence NMeArg-Lys-Pro- that neurotensin (8-13) is modified by site
Trp-Tle-Leu has significant peripherally administered analgesic activities.
Summary of the invention
The present invention is to solve the biological stability of existing opioid drug is poor, anti-neuropathic pain is ineffective to be asked
Topic, provides the cyclisation hybrid peptide and its synthetic method and application that dynorphin A (1-8) is mutually coupled with neurotensin (8-13).
The amino acid sequence for the cyclisation hybrid peptide that dynorphin A (1-8) of the present invention is mutually coupled with neurotensin (8-13) is such as
Under:
Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-
Leu。
Wherein sequence " Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys) " is the cyclisation of multidigit point modification
Dynorphin A (1-8) residue sequence, " Gly " be hybrid peptide intermediate connexon, " NMeArg-Lys-Pro-Trp-Tle-
Leu " is the residue sequence of the neurotensin (8-13) of multidigit point modification.(Dmt represents 2,6- dimethyl Tyr, and NMePhe is represented
N- methyl Phe, NMeArg represent N- methyl Arg, Tle and represent tert-Leu)
The synthetic method for the cyclisation hybrid peptide that above-mentioned dynorphin A (1-8) is mutually coupled with neurotensin (8-13), including with
Lower step:
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 are as follows: Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-
Cys)-Gly-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 dynorphin A (1-8) of above-mentioned multidigit point modification is mutually coupled with neurotensin (8-13) is being made
Application in standby polypeptide analgesic.
The cyclisation hybrid peptide that the dynorphin A (1-8) of above-mentioned multidigit point modification is mutually coupled with neurotensin (8-13) is being made
Application in standby anti-neuropathic pain drug.
Beneficial effects of the present invention:
The cyclisation hybrid peptide that the dynorphin A (1-8) of multidigit point of the present invention modification is mutually coupled with neurotensin (8-13) is
By the amino acid sequence " Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile " of dynorphin A (1-8) by multidigit point modification with
Cys disulfide bond is cyclized to obtain " Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys) " amino acid residue sequence, will
The amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13) is modified to obtain by multidigit point
" NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence, using intermediate connexon " Gly " by two parts amino acid
Residue sequence is mutually coupled, and constructs a kind of new and effective hybrid peptide.
Wherein multidigit point modification is by the amino acid sequence " Tyr-Gly-Gly-Phe-Leu-Arg- of dynorphin A (1-8)
1 Tyr of Arg-Ile " is substituted with Dmt, and 3 Gly are substituted with Cys, and 4 Phe are substituted with NMePhe and 9 increase Cys.
1 Arg of the amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13) is substituted with NMeArg, 2
Position Arg is substituted with Lys, and 4 Tyr are substituted with Trp, and 5 Ile are substituted with Tle.
Then it is cyclized, specifically is cyclized to obtain " Dmt-Gly-c (Cys-NMePhe- by 3 and 6 Cys disulfide bond
Leu-Arg-Arg-Ile-Cys) " amino acid residue sequence.
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 mouse brain plasma membrane of hybrid peptide
Half-life period reaches 396.2 ± 28.0min, and the serum half-life of hybrid peptide reaches 308.0 ± 29.5min.
In addition, one small by being added among the dynorphin A (1-8) of hybrid peptide and the modification sequence of neurotensin (8-13)
The flexible conformation of hybrid peptide can be improved in molecule Amino acid linker " Gly ", retains dynorphin A (1-8) and neurotensin
The bioactivity of (8-13) two parts modification sequence.Neurotensin (8-13) is the minimum active fragment of neurotensin, miscellaneous
The neurotensin (8-13) for closing peptide partially can cause endogenic opioid peptides to discharge, ginseng by activating Opioid Receptor System
With the performance of analgesic activity, and then efficient synergic antalgic is partially generated with the dynorphin A of hybrid peptide (1-8) and is acted on.Moreover, should
Hybrid peptide has multiple positively charged amino acid residues, can enhance hybrid peptide to the penetration capacity of blood-brain barrier, and then improve
The maincenter utilization rate of drug.In addition, the neurotensin (8-13) in hybrid peptide partially can reduce the analgesia of opioid drug
The adverse side effects such as tolerance, gastrointestinal tract and angiocarpy.
By digesting stability experiment, analgesia is tested with Acute brain block, anti-neurogenic pain activity experiment, Ji Ti
Gastrointestinal tract and Blood Pressure Experiment carry out pharmacological activity identification to the hybrid peptide that the present invention synthesizes.The result shows that heterozygosis of the invention
Peptide enzymatic hydrolysis stability with higher.By central administration, there are apparent analgesic activities in hot tail-flick test.Moreover, miscellaneous
Closing peptide has peripherally administered efficient analgesic effect, and significant anti-neuropathic pain activity, and maximum analgesic effect is in side brain
It is generated after room injection drug 10min, the maximum paw withdrawal threshold of reaction is 1.134g, moreover, when its anti-neuropathic pain effect lasts
Between longer, sustainable at least 40min or more.In addition, hybrid peptide has the analgesic properties without tolerance side effect, and it is to gastrointestinal tract
Side effect with cardiovascular system is significantly reduced.
Therefore, hybrid peptide of the invention is preparing the efficient, polypeptide without analgesia tolerance, low gastrointestinal tract and cardiovascular side effects
It is had potential application in terms of analgesic.
Detailed description of the invention
Fig. 1 is analgesic effect-time graph of intracerebroventricular injection hybrid peptide DANT；Wherein ■ indicate hybrid peptide DANT,
10nmol, ● indicate hybrid peptide, 3nmol, ▲ indicate that hybrid peptide DANT, 1nmol, ▼ indicate hybrid peptide DANT, 0.3nmol, ◆
Indicate physiological saline；
Fig. 2 is the periphery analgesic effect that hybrid peptide DANT is subcutaneously injected；Wherein ■ indicate hybrid peptide DANT (10 μm of ol/kg,
Subcutaneously), ● indicate physiological saline (subcutaneous)；
Fig. 3 is the analgesia tolerance dose curve of intracerebroventricular injection morphine；Wherein ■ indicates physiological saline+morphine, ● it indicates
Morphine (10nmol)+morphine；
Fig. 4 is the analgesia tolerance dose curve of intracerebroventricular injection hybrid peptide DANT；Wherein ■ indicates physiological saline+hybrid peptide
DANT, ● indicate hybrid peptide DANT (10nmol)+hybrid peptide DANT；
The anti-neuropathic pain activity that Fig. 5 is intracerebroventricular injection hybrid peptide DANT；Wherein ■ indicate hybrid peptide DANT,
10nmol, ● indicate hybrid peptide DANT, 3nmol, ▲ indicate that hybrid peptide DANT, 1nmol, ▼ indicate physiological saline；
Fig. 6 is the effect that intracerebroventricular injection hybrid peptide DANT promotes gastrointestinal tract charcoal meal；
Fig. 7 is the effect of intravenous morphine and hybrid peptide DANT to rat system angiosthenia；
Fig. 8 is the effect of intravenous morphine and hybrid peptide DANT to 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: present embodiment dynorphin A (1-8) and the cyclisation that neurotensin (8-13) is mutually coupled are miscellaneous
The amino acid sequence for closing peptide is as follows:
Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-NMeArg-Lys-Pro-Trp-Tle-
Leu。
Present embodiment is by the amino acid sequence " Tyr-Gly-Gly-Phe-Leu-Arg-Arg- of dynorphin A (1-8)
Ile " is cyclized to obtain " Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile- by the modification of multidigit point with Cys disulfide bond
Cys) " amino acid residue sequence is logical by the amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13)
Excessive site is modified to obtain " NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence, utilizes intermediate connexon
Two parts amino acid residue sequence is mutually coupled by " Gly ", 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: present embodiment dynorphin A (1-8) and the cyclisation that neurotensin (8-13) is mutually coupled are miscellaneous
Close the synthetic method of 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 are as follows: Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-
Cys)-Gly-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: N- hydroxy benzo in step 3
The mole of triazole is 2.5-3 times of Fmoc-Leu-Wang resin mole.It is other to be identical with embodiment two.
Specific embodiment 4: present embodiment is unlike specific embodiment two or three: in step 3 " Fmoc "
The mole of the amino acid of radical protection is 2.5-3 times of Fmoc-Leu-Wang resin mole.Other and specific embodiment
Two or three 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: the dynorphin A (1-8) and neurotensin (8-13) phase of the modification of present embodiment multidigit point
The cyclisation hybrid peptide of coupling is preparing the application in polypeptide analgesic.
Specific embodiment 11: the dynorphin A (1-8) and neurotensin (8-13) of present embodiment multidigit point modification
Mutually the cyclisation hybrid peptide of coupling is 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 cyclisation hybrid peptide that the dynorphin A (1-8) of the present embodiment multidigit point modification is mutually coupled with neurotensin (8-13)
The synthetic method of DANT, 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- of hybrid peptide DANT amino acid sequence
Cys-NMePhe-Leu-Arg-Arg-Ile-Cys-Gly-NMeArg-Lys-Pro-Trp-Tl e-Leu, from the end C- of polypeptide to
The amino acid of " Fmoc " radical protection is successively condensed on resin by the end N- one by one, until the condensation of all amino acid residues is completed,
Obtain 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 DANT, whole yield are 27%, and mass spectrum and chromatography testing result are as shown in table 1.
The mass spectrum and chromatography testing result of 1. hybrid peptide DANT of table
Table 1 the result shows that, the detected value for the hybrid peptide DANT that the present invention obtains is consistent with theoretical value.
The dynorphin A (1-8) of multidigit point modification manufactured in the present embodiment is mutually coupled novel with neurotensin (8-13)
The biological activity test for being cyclized hybrid peptide is as follows:
1, stability experiment is digested
100% mice serum and 15% brain membrane sample are prepared by ex vivo approach, takes 10 μ L polypeptide mother liquors (10-2M), add
Enter into 100% mice serum or 15% brain plasma membrane of 190 μ L, oscillation mixes immediately, then takes out 20 μ L mixed liquors rapidly, adds
Entering 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 take out 20 μ L.The termination of enzymolysis process: it is mixed that the oscillation of 90 μ L acetonitriles is added in the sample of taking-up
Even, sample is placed in and places 5min on ice, then with the ice-cold acetic acid dilution of 90 μ L0.5% to ensure that enzymolysis process stops.13,000g
It is centrifuged 15min, collects supernatant, -80 DEG C freeze, until RP-HPLC is analyzed.
Experimental result is shown in Table 2.Table 2 the results show that dynorphin A (1-8) mouse brain plasma membrane and serum half-life than mind
It is slightly higher through hypotensor medicine (8-13), however the mouse brain plasma membrane and serum half-life of dynorphin A (1-8) and neurotensin (8-13)
It is shorter, it is no more than 20min.But the brain plasma membrane and serum half-life of hybrid peptide DANT are all considerably longer than dynorphin A (1-8)
Thirtyfold or so is arrived with neurotensin (8-13), high about 20.It should be the result shows that being modified by multidigit point unnatural amino acid
And polypeptide cyclisation, the enzymatic hydrolysis stability of hybrid peptide DANT can be significantly improved.
2. dynorphin A of table (1-8), the brain plasma membrane and serum half-life of neurotensin (8-13) and hybrid peptide DANT
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 Figure 1, intracerebroventricular injection 0.3-10nmol difference agent
The hybrid peptide DANT of amount shows the significant analgesic activities of concentration-dependant.Maximum analgesic effect is in intracerebroventricular injection drug
It is generated after 10min, maximum analgesia %MPE value is 90.11%.After injecting drug 15min, analgesia %MPE value remains to reach
87.07%, show that the duration of the higher analgesic activities of hybrid peptide DANT generation is longer.It is detected in 50min, hybrid peptide DANT's
Analgesia %MPE value 24.13%, i.e. analgesia duration at least 50min or more, this shows that hybrid peptide DANT has efficient maincenter
Analgesic activities.In addition, peripherally administered analgesic experiment result is as shown in Fig. 2, pass through 10 μm of ol/kg fixed dosages of subcutaneous injection
Hybrid peptide DANT, as a result, it has been found that DANT still has high-efficiency continuous analgesic activities.Maximum analgesic effect is in injection drug 15min
After generate, analgesia %MPE value be 84.24%.The analgesia %MPE value that 90min measures DANT upon administration remains to reach
27.03%, show that the analgesia duration of peripheral injection hybrid peptide DANT can reach 90min or more, i.e. peripheral injection hybrid peptide
The analgesia duration of DANT is longer.The above results show the dynorphin A (1-8) and neurotensin (8-13) of multidigit point modification
Mutually the novel cyclized hybrid peptide of coupling significantly enhances analgesic activities and the duration of drug, i.e. hybrid peptide has efficient town
The feature of pain activity.
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.
The analgesia tolerance result of Central injection morphine and hybrid peptide DANT are as shown in Figures 3 and 4, and 1 hour in advance to mouse side
Intracerebroventricular physiological saline, the morphine of intracerebroventricular injection 0.3-10nmol various dose and hybrid peptide DANT are generated significantly 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 and hybrid peptide
The active performance of the analgesics such as DANT.However, by the morphine for shifting to an earlier date 1 hour intracerebroventricular injection 10nmol dosage, Zhi Houzai
The corresponding drug morphine of 0.3-10nmol various dose is injected, medicine analgesic dose curve obviously moves to right, and central analgesia is living
Property significantly reduce, i.e., morphine produces the tolerance of apparent medicine analgesic.But 1 hour in advance intracerebroventricular injection 10nmol dosage
Hybrid peptide DANT, then inject the hybrid peptide 3 of 0.3-10nmol various dose, analgesic dose curve and inject physiology salt in advance
The analgesic dose curve of water is almost overlapped, and there is no generate significantly to move to left or move to right.It should be the result shows that injecting heterozygosis in advance
Peptide D ANT has little effect the analgesic activities of second of acute injection DANT, shows that DANT has the town without tolerance side effect
Pain characteristic.Should the result shows that, efficient analgesic properties of the hybrid peptide DANT with no drug resistance side effect.
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 DANT is as shown in figure 5, intracerebroventricular injection 1-10nmol
The DANT of various dose dose-dependent can cause the paw withdrawal threshold of reaction to increase, it was demonstrated that hybrid peptide DANT is to neuropathic pain
With significant analgesic activities.Its maximum analgesic effect generates after intracerebroventricular injection drug 10min, the maximum paw withdrawal threshold of reaction
For 1.134g.Moreover, the anti-neuropathic pain acting duration of DANT is longer, sustainable at least 40min or more shows heterozygosis
Peptide D ANT has efficient analgesic activities to neuropathology pain.
5, gastrointestinal tract charcoal meal Promoting Experiment
Experiment starts to test later using male kunming mouse, weight 25-30g, fasting 20h, and intracerebroventricular injection is different
Acute drug, 5 μ L of volume injected.(5% Arabic gum, 10% active carbon aqueous solution) is eaten to the charcoal of Mouse oral 0.2mL later.
30min puts to death mouse later, and small intestine is removed.The overall length (pylorus knot to cap end) and charcoal for measuring small intestine are eaten in small intestine
The distance of middle propulsion.The ratio of distance and entire small intestinal length that the gastrointestinal transit activity of drug is promoted in small intestine with charcoal meal
Percentage indicates.
Gastrointestinal tract charcoal meal Promoting Experiment experimental result is as shown in Figure 6 (in Fig. 6Indicate morphine,Indicate miscellaneous
Close Peptide D ANT), after telocoele administration, charcoal meal is lower in gastrointestinal transit activity, then drug promotes mouse GI tract charcoal meal
Inhibiting effect is stronger.Pass through the morphine and hybrid peptide DANT of intracerebroventricular injection 0.5-5nmol various dose, generation concentration dependant
The inhibition gastrointestinal tract charcoal meal progradation of type, and drug concentration is higher, and inhibiting effect is stronger.But opium under all concentration
The inhibitory effect that alkaloid morphine promotes gastrointestinal tract charcoal meal is significantly higher than the hybrid peptide DANT under corresponding concentration, this shows maincenter
Injection hybrid peptide DANT reduces gastrointestinal tract carbon meal progradation effect, i.e., excellent with lower gastrointestinal system side effect
Point.
6, rat in vivo Blood Pressure Experiment
Experiment uses Wistar rat, and 200-300g is with 20% Anaesthesia with Ethyl Carbamate (l.2g/kg, i.p.)
Holding narcosis is good, it may be necessary to temporarily add anaesthetic.The tracheae of rat is cut, mucus in managing, animal are removed
It can autonomous respiration.PE-50 conduit is inserted into vena jugularis externa in case intravenously administrable, PE-50 conduit are inserted into neck main artery, and and YP-
100 pressure sensors are 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 and hybrid peptide DANT act on Rat Cardiovascular is as shown in FIG. 7 and 8 (in Fig. 7 and 8
Indicate morphine,Indicate hybrid peptide DANT), it is injected intravenously the morphine and the equal energy of hybrid peptide DANT of 1-10nmol various dose
Rat system arterial pressure is enough significantly reduced, and is in dose dependent feature.However hybrid peptide DANT is reduced under all concentration
The effect of arterial pressure is significantly lower than the morphine under corresponding concentration.In addition, the morphine and heterozygosis of intravenous injection 1-10nmol dosage
The dose-dependent heart rate for reducing rat of Peptide D ANT.Similar with system arterial pressure, hybrid peptide DANT reduces artery under all concentration
The effect of blood pressure is significantly lower than the morphine under corresponding concentration.Should the result shows that, hybrid peptide DANT is to the work in body-centered vascular system
It is significantly lower than morphine with effect, that is, has the advantages that lower cardiovascular side effects.
In conclusion the dynorphin A (1-8) of multidigit point of the present invention modification be mutually coupled with neurotensin (8-13) it is novel
It is cyclized hybrid peptide, is to pass through the amino acid sequence " Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile " of dynorphin A (1-8)
The modification of multidigit point is cyclized to obtain " Dmt-Gly-c (Cys-NMePhe-Leu-Arg-Arg-Ile-Cys) " amino acid with disulfide bond residual
Basic sequence modifies the amino acid sequence " Arg-Arg-Pro-Tyr-Ile-Leu " of neurotensin (8-13) by multidigit point
" NMeArg-Lys-Pro-Trp-Tle-Leu " amino acid residue sequence is obtained, using intermediate connexon " Gly " by two parts ammonia
Base acid residue sequence is mutually coupled, and constructs a kind of new and effective hybrid peptide.Pass through the replacement of multidigit point unnatural amino acid and ring
The biological stability of hybrid peptide can be significantly increased by changing modification.By the dynorphin A (1-8) of hybrid peptide and neurotensin (8-13)
Modification sequence among be added a small molecule Amino acid linker " Gly ", the flexible conformation of hybrid peptide can be improved, remain
The bioactivity of dynorphin A (1-8) and neurotensin (8-13) two parts sequence, the biology for solving existing opioid drug are steady
Qualitative poor, analgesic activities are lower and the duration is shorter, and anti-neuropathic pain is ineffective, and have analgesia tolerance, stomach and intestine
The problem of road and cardiovascular side effect.
By digesting stability experiment, analgesia is tested with Acute brain block, anti-neurogenic pain activity experiment, Ji Ti
Gastrointestinal tract and Blood Pressure Experiment carry out pharmacological activity identification to the hybrid peptide that the present invention synthesizes.The result shows that heterozygosis of the invention
Peptide enzymatic hydrolysis stability with higher.By central administration, there are apparent analgesic activities in hot tail-flick test.Moreover, miscellaneous
Closing peptide has peripherally administered efficient analgesic effect, and significant anti-neuropathic pain activity.In addition, hybrid peptide is with no tolerance
The analgesic properties of side effect, and it is significantly reduced the side effect of gastrointestinal tract and cardiovascular system.Therefore, heterozygosis of the invention
Peptide preparation efficiently, without the polypeptide analgesic of analgesia tolerance, low gastrointestinal tract and cardiovascular side effects in terms of have and potentially answer
With value.
Sequence table
<110>Harbin Institute of Technology
<120>the cyclisation hybrid peptide and its synthetic method and answer that dynorphin A (1-8) is mutually coupled with neurotensin (8-13)
With
<160>1
<210> 1
<211> 16
<212> PRT
<213>artificial sequence
<220>
<223>it is cyclized hybrid peptide
<400> 1
Dmt Gly Cys MePhe Leu Arg Arg Ile Cys Gly MeArg Lys Pro Trp Tle Leu
Claims (9)
1. the cyclisation hybrid peptide that dynorphin A (1-8) is mutually coupled with neurotensin (8-13), it is characterised in that the ammonia of the hybrid peptide
Base acid sequence is as follows:
Dmt-Gly-c(Cys-NMePhe-Leu-Arg-Arg-Ile-Cys)-Gly-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, Tle and represents tert-
Leu。
2. the synthesis for the cyclisation hybrid peptide that dynorphin A (1-8) as described in claim 1 is mutually coupled with neurotensin (8-13)
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 30~40min 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 3~5min 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 taken out after stirring 5~10min
It is dry, it repeats 2~3 times, adds piperidines/DMF that concentration expressed in percentage by volume is 20%~25% and 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- benzo three of " Fmoc " radical protection
Nitrogen azoles-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, add after diisopropylethylamine mix mixing is molten
Then mixed solution is added in the resin of step 2 removing " Fmoc " blocking group, is stirred to react under protection of argon gas by liquid
60~72min；
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-Leu-Arg-Arg-Ile-Cys)-
Gly-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；Wherein nothing in mixed solution
The volume ratio of water methanol and DMF are 4:1, I2Quality and mixed solution volume ratio be 2.6g:100mL, I2Solution and peptide resin
Volume ratio be 100mL: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
3~5h 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 conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that the mole of N- hydroxy benzo triazole is Fmoc-Leu-Wang resin mole in step 3
2.5-3 again.
4. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that the mole of the amino acid of " Fmoc " radical protection is that Fmoc-Leu-Wang resin rubs in step 3
2.5-3 times of that amount.
5. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that the mole of O- benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is in step 3
2.5-3 times of Fmoc-Arg (pbf)-Wang resin mole.
6. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that the mole of diisopropylethylamine is the 5-6 of Fmoc-Leu-Wang resin mole in step 3
Times.
7. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that the cutting reagent of 10-25mL is added in every gram of peptide resin in step 6.
8. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that cutting reagent described in step 6 is by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95:
2.5:2.5 mixing.
9. the conjunction for the cyclisation hybrid peptide that dynorphin A (1-8) according to claim 2 is mutually coupled with neurotensin (8-13)
At method, it is characterised in that remove " Fmoc " group of amino acid most latter linked on peptide resin completely in step 6 specific
Method are as follows:
Peptide resin is washed into 3~5min with DMF, is drained, is repeated 3~5 times, concentration expressed in percentage by volume is then added in resin is
20%~25% piperidines/DMF is deprotected solution, drains after stirring 5~10min, repeats 2~3 times, it is dense to add volume basis
Degree is deprotected solution for 20%~25% piperidines/DMF, stirs 15~20min, removes " Fmoc " blocking group sufficiently, it
After drain solvent, finally wash cleared deprotection solution with DMF, removed the resin of " Fmoc " blocking group.
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