CN107964047B - Chimeric peptide and its synthetic method based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13) and application - Google Patents
Chimeric peptide and its synthetic method based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13) and application Download PDFInfo
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Abstract
Chimeric peptide and its synthetic method based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13) and application are related to a kind of chimeric peptide and its synthetic method and application.Be to solve existing Tyr-Pro-Trp-Phe-NH2 analgesia duration it is shorter, peripherally administered analgesic activities are relatively low, and with analgesia tolerance and gastrointestinal side effect the problem of.The amino acid sequence of the chimeric peptide is Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu.Method：One, the Wang resins pretreatment of " Fmoc " protection；Two, " Fmoc " blocking group is removed；Three, amino acid condensation reacts；Four, the extension of peptide chain；Five, peptide chain is from the cutting on resin；Six, the desalination and purifying of thick peptide.Chimeric peptide analgesia duration of the present invention is long, and has the advantages that no analgesia tolerance is low with to gastrointestinal side effect.Chimeric peptide of the present invention is used to prepare polypeptide analgesic.
Description
Technical field
The present invention relates to a kind of chimeric peptide and its synthetic method and applications.
Background technology
Neurogenic pain is defined as by central or peripheral nervous system by pain research federation of the world (IASP)
Pain syndrome caused by feeling pathway injury or dysfunction, with spontaneous pain, hyperalgia, allodynia or lures tactile
It is sore to be characterized.The neuropathic pain duration is long, restores slower, and Theory of Pain Mechanism is also imperfectly understood, and clinically lacks full
The therapy of meaning drastically influences the quality of life of the mankind.Research of the neuropeptide in terms of nociception and modulation is always
The hot spot of Neuroscience Research.It has now been found that and plays important work in the transmission of pain sensation information and modulated process there are many neuropeptide
With wherein being exactly opioid peptides known to the most.
Interior morphine peptide is the endogenic μ-opiate receptors ligands being widely present in the mammalian body, is divided into Tyr-Pro-Trp-Phe-NH2
(Tyr-Pro-Trp-Phe-NH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2).All known
In opioids, interior morphine peptide has high compatibility and selectivity to μ-opiate receptor.Interior morphine peptide passes through even with G-protein
The μ of connection-opiate receptor is combined, and is participated in the pain sensation, angiocarpy, breathing, stomach and intestine, movement, behavior, endocrine and many work(such as immune
The adjusting of energy, but its main function is potent analgesic activities, is especially also shown in neuropathic pain animal model bright
Aobvious analgesic activities, it is all more more effective than most of opioid peptides.In addition, interior morphine peptide has excellent acology characteristic, can generate
Activity is better than morphine and the less analgesic activity of side effect, such as awards the separation of behavior and analgesic activity, and the formation of tolerance is slower,
Analgesic dose is significantly lower than the dosage etc. for causing respiration inhibition and Cardiovascular, makes it have potential clinical value.
However, interior morphine peptide has analgesia time short, only persistently 20min effects, enzymolysis stability is poor, is not easy to lack through blood-brain barrier etc.
Point, to limit its application as clinical analgesic.In recent years, scholars are dedicated to research based on interior morphine peptide structure
Novel, efficient, less toxic side effect polypeptide analgesic.Have the internal morphine peptide of relevant report carry out glycosylation and it is lipidization
Modification, can pass through the peripherally administered analgesic effect that maincenter is generated into central nervous system.
Invention content
The present invention is to solve the analgesia duration of existing Tyr-Pro-Trp-Phe-NH2 is shorter, peripherally administered analgesic activities compared with
It is low, and the problem of with analgesia tolerance and gastrointestinal side effect, provide based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
Chimeric peptide and its synthetic method and application.
The present invention is based on the amino acid sequence of Tyr-Pro-Trp-Phe-NH2 and the chimeric peptide of neurotensin (8-13) is as follows：
Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu。
Wherein sequence " Tyr-Pro-Trp-Phe " is four amino acid residue sequences of Tyr-Pro-Trp-Phe-NH2, and " Gly-Gly " is
The intermediate connexon of chimeric peptide, " Arg-Arg-Pro-Tyr-Ile-Leu " are the 8-13 amino acids residues of neurotensin,
That is six amino acid residue sequences of neurotensin (8-13).
The present invention is based on the synthetic methods of Tyr-Pro-Trp-Phe-NH2 and the chimeric peptide of neurotensin (8-13), including following step
Suddenly：
One, the Wang resins pretreatment of " Fmoc " protection：The air-tightness for checking solid phase synthetic instrument, will be with there are one amino acid
The Fmoc-Leu-Wang resins of residue are put into synthesizer, and add methylene chloride 30~40min of stirring, and resin is made fully to impregnate swelling
Afterwards, decompression filters solvent；Wherein there are one the bodies of the quality and dichloromethane of the Fmoc-Leu-Wang resins of amino acid residue for band
Product is than being 1g：(7~12) mL；
Two, " Fmoc " blocking group is removed：Swellable resins after draining are 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 makes " Fmoc " blocking group fully remove, drains solvent later, finally washs cleared deprotection solution with DMF, obtains
Remove the resin of " Fmoc " blocking group；The volume ratio of the wherein quality of resin and the deprotection solution being added for the first time is 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 triazoles, O- benzene of " Fmoc " radical protection
And triazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, mixing must mix after adding diisopropylethylamine
Solution is closed, then mixed solution is added in the resin that step 2 removes " 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 with DMF to remove not instead after the reaction was complete
The residual liquid answered；
Four, the extension of peptide chain：Step 2 and three is repeated, according to the sequence of chimeric peptide amino acid sequence, from the ends C- of polypeptide
The amino acid of " Fmoc " radical protection is condensed on resin one by one successively to the ends N-, until all amino acid residues have been condensed
At obtaining peptide resin；
Five, peptide chain is from the cutting on resin：" Fmoc " group of most latter linked amino acid on peptide resin is removed completely,
Then dichloromethane and methanol alternately washing peptide resin are used, after fully draining solvent, cutting reagent is added into peptide resin, in room
3~5h of the lower 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 fully dissolves precipitation, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white
The thick peptide of solid powder；
Six, 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 desalinations are freeze-dried after collecting main peak using Ultraviolet Detector, obtain 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 Fmoc-Arg (pbf)-Wang resin moles in step 3
2.5-3 times.
The mole of N- hydroxy benzo triazoles is the 2.5-3 of Fmoc-Arg (pbf)-Wang resin moles in step 3
Times.
The mole of O- benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphates is Fmoc-Arg in step 3
(pbf) 2.5-3 times of-Wang resins mole.
The mole of diisopropylethylamine is 5-6 times of Fmoc-Leu-Wang resin moles in step 3.
The cutting reagent of 10-25mL is added in every gram of peptide resin in step 5.
Cutting reagent described in step 5 is by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95：2.5：2.5 mixed
It closes.
It is by the specific method that " Fmoc " group of most latter linked amino acid on peptide resin removes completely in step 5：
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
A concentration of 20%~25% piperidines/DMF is deprotected solution, stirs 15~20min, " Fmoc " blocking group is made fully to remove,
Solvent is drained later, is finally washed cleared deprotection solution with DMF, is removed the resin of " Fmoc " blocking group.
Above-mentioned chimeric peptide the answering in preparing polypeptide analgesic based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
With.
Beneficial effects of the present invention：
It is by by the four of Tyr-Pro-Trp-Phe-NH2 the present invention is based on the chimeric peptide of Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
A amino acid residue sequence " Tyr-Pro-Trp-Phe " passes through intermediate connexon " Gly-Gly " and neurotensin (8-13)
Six amino acid residue sequences " Arg-Arg-Pro-Tyr-Ile-Leu " are mutually coupled, and are detected by mass spectrum and chromatography, and set
The structure of meter is consistent, shows to successfully synthesize chimeric peptide EMNT.
The intermediate connexon that is added of the chimeric peptide can be improved into the flexibility of chimeric peptide, enhance the bioactivity of chimeric peptide.
The Tyr-Pro-Trp-Phe-NH2 part of chimeric peptide is easier to be combined with μ-opiate receptor, retains the opioid activity of the part.Neurotensin
8-13 amino acids, i.e. neurotensin (8-13) is the minimum active fragment of neurotensin, the nerve drop of chimeric peptide
Pressure plain (8-13) partly can activate opiate system while generating analgesic effect, cause the release of endogenous opiatepeptide, into
And generate synergic antalgic effect with the Tyr-Pro-Trp-Phe-NH2 part of chimeric peptide.In addition, there are two positively charged arginine for chimeric peptide tool
Residue, arginic guanidino group can form the hydrogen bond of divalent with the anionic group of cell surface, enhance the biology of polypeptide
Membrane permeability improves transport capacity of the drug to central nervous system, and then by peripherally administered such as the permeability of blood-brain barrier
The efficient analgesic activities that generation maincenter mediates.Moreover, while being entrenched in performance analgesic activity, due to neurotensin (8-13)
Partial participation, opium adverse side effect can be also substantially reduced.
The analgesia duration of chimeric peptide of the present invention is longer.It is real by carrying out analgesia in mouse intracerebroventricular injection chimeric peptide
It tests, as a result proves that 50min measurement upon administration, analgesia %MPE remain to reach the left sides 10%-20% under 1-10nmol dosage
The right side shows that chimeric peptide has efficient, duration length central analgesia activity.In addition, passing through 10 μm of ol/kg's of hypodermic injection
Chimeric peptide, 60min is measured as 17.07% to analgesia %MPE upon administration, and 16.8% is measured as in 90min, shows that chimeric peptide is logical
90min or more can be reached by spending the peripherally administered duration.The result proves to be based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-
13) structure and the chimeric peptide that designs greatly enhance analgesic effect and the action time of drug, i.e. chimeric peptide has efficiently
The feature of central analgesia activity.
Therefore, it is a kind of efficient, low secondary work the present invention is based on the chimeric peptide of Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
Polypeptide analgesic has very high clinical value.
Description of the drawings
Fig. 1 is analgesic effect-time graph of intracerebroventricular injection chimeric peptide in embodiment 1；
Fig. 2 is analgesic effect-time graph that chimeric peptide is subcutaneously injected in embodiment 1；
Fig. 3 is the analgesia tolerance dose curve of intracerebroventricular injection Tyr-Pro-Trp-Phe-NH2 in embodiment 1；
Fig. 4 is the analgesia tolerance dose curve of intracerebroventricular injection chimeric peptide in embodiment 1；
Fig. 5 is the effect that intracerebroventricular injection Tyr-Pro-Trp-Phe-NH2 and chimeric peptide transport colon in embodiment 1；
Fig. 6 is the effect that intracerebroventricular injection Tyr-Pro-Trp-Phe-NH2 and chimeric peptide promote gastrointestinal tract charcoal meal in embodiment 1.
Specific implementation mode
Technical solution of the present invention is not limited to act specific implementation mode set forth below, further includes between each specific implementation mode
Arbitrary combination.
Specific implementation mode one：Present embodiment is based on the ammonia of the chimeric peptide of Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
Base acid sequence is as follows：
Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu。
Present embodiment remains the bioactivity of two neural peptide fragments, when the analgesia for solving Tyr-Pro-Trp-Phe-NH2 continues
Between it is shorter, peripherally administered analgesic activities are relatively low, and with analgesia tolerance and gastrointestinal side effect the problem of.By various in vitro
In body biological experiment, pharmacological activity identification is carried out to the chimeric peptide of present embodiment.The result shows that present embodiment is embedding
Peptide is closed to μ-opiate receptor with higher compatibility and to the opioid activity of isolated preparation, especially to the activity height of GPI samples
In Tyr-Pro-Trp-Phe-NH2.In addition, chimeric peptide has maincenter and peripherally administered efficient analgesic activities, analgesia duration is long, and has
Whether there is or not analgesia tolerance and the advantage low to gastrointestinal side effect, effectively overcome opioid analgesic drug generally existing tolerance and
The side-effect problems such as constipation.Therefore, the chimeric peptide of present embodiment has potential in terms of preparing clinical polypeptide analgesic
Application value.
Specific implementation mode two：Present embodiment is based on the conjunction of the chimeric peptide of Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
At method, include the following steps：
One, the Wang resins pretreatment of " Fmoc " protection：The air-tightness for checking solid phase synthetic instrument, will be with there are one amino acid
The Fmoc-Leu-Wang resins of residue are put into synthesizer, and add methylene chloride 30~40min of stirring, and resin is made fully to impregnate swelling
Afterwards, decompression filters solvent；Wherein there are one the bodies of the quality and dichloromethane of the Fmoc-Leu-Wang resins of amino acid residue for band
Product is than being 1g：(7~12) mL；
Two, " Fmoc " blocking group is removed：Swellable resins after draining are 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 makes " Fmoc " blocking group fully remove, drains solvent later, finally washs cleared deprotection solution with DMF, obtains
Remove the resin of " Fmoc " blocking group；The volume ratio of the wherein quality of resin and the deprotection solution being added for the first time is 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 triazoles, O- benzene of " Fmoc " radical protection
And triazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, mixing must mix after adding diisopropylethylamine
Solution is closed, then mixed solution is added in the resin that step 2 removes " 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 with DMF to remove not instead after the reaction was complete
The residual liquid answered；
Four, the extension of peptide chain：Step 2 and three is repeated, according to the sequence of chimeric peptide amino acid sequence, from the ends C- of polypeptide
The amino acid of " Fmoc " radical protection is condensed on resin one by one successively to the ends N-, until all amino acid residues have been condensed
At obtaining peptide resin；
Five, peptide chain is from the cutting on resin：" Fmoc " group of most latter linked amino acid on peptide resin is removed completely,
Then dichloromethane and methanol alternately washing peptide resin are used, after fully draining solvent, cutting reagent is added into peptide resin, in room
3~5h of the lower 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 fully dissolves precipitation, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white
The thick peptide of solid powder；
Six, 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 desalinations are freeze-dried after collecting main peak using Ultraviolet Detector, obtain 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 implementation mode three：Present embodiment is unlike specific implementation mode two：" Fmoc " group in step 3
The mole of the amino acid of protection is 2.5-3 times of Fmoc-Arg (pbf)-Wang resin moles.Other and specific embodiment party
Formula two is identical.
Specific implementation mode four：Present embodiment is unlike specific implementation mode two or three：N- hydroxyls in step 3
The mole of benzotriazole is 2.5-3 times of Fmoc-Arg (pbf)-Wang resin moles.Other and specific implementation mode
Two or three is identical.
Specific implementation mode five：Unlike one of present embodiment and specific implementation mode two to four：O- in step 3
The mole of benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphate is Fmoc-Arg (pbf)-Wang resins mole
2.5-3 times of amount.It is other identical as one of specific implementation mode two to four.
Specific implementation mode six：Unlike one of present embodiment and specific implementation mode two to five：Two in step 3
The mole of wopropyl ethyl amine is 5-6 times of Fmoc-Leu-Wang resin moles.It is other with specific implementation mode two to five it
One is identical.
Specific implementation mode seven：Unlike one of present embodiment and specific implementation mode two to six：It is every in step 5
The cutting reagent of 10-25mL is added in gram peptide resin.It is other identical as one of specific implementation mode two to six.
Specific implementation mode eight：Unlike one of present embodiment and specific implementation mode two to seven：Institute in step 5
Cutting reagent is stated by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95：2.5：2.5 mixing.It is other with it is specific
One of embodiment two to seven is identical.
Specific implementation mode nine：Unlike one of present embodiment and specific implementation mode two to eight：It will in step 5
The specific method that " Fmoc " group of most latter linked amino acid removes completely on peptide resin is：
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
A concentration of 20%~25% piperidines/DMF is deprotected solution, stirs 15~20min, " Fmoc " blocking group is made fully to remove,
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 implementation mode two to eight is identical.
Specific implementation mode ten：Present embodiment is based on Tyr-Pro-Trp-Phe-NH2 and the chimeric peptide of neurotensin (8-13) is being made
Application in standby polypeptide analgesic.
Elaborate below to the embodiment of the present invention, following embodiment under based on the technical solution 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 present embodiment is based on the synthetic method of the chimeric peptide of Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13), including following step
Suddenly：
One, the Wang resins pretreatment of " Fmoc " protection：The air-tightness for checking solid phase synthetic instrument, by 1g bands, there are one amino
The Fmoc-Leu-Wang resins of sour residue are put into synthesizer, add 10mL dichloromethane to stir 30min, so that resin is fully impregnated molten
After swollen, decompression filters solvent.
Two, " Fmoc " blocking group is removed：Swellable resins after draining are washed into 3min with DMF, drains, is repeated 3 times.?
Piperidines/DMF that 10mL concentration expressed in percentage by volumes are 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 volumes are 20% and is deprotected solution, stir 15min, " Fmoc " blocking group is made to fill
Divide removing, drains solvent later, finally wash cleared deprotecting regent with 10mL DMF, removed " Fmoc " blocking group
Resin.
Three, amino acid condensation reacts：It will be equivalent to Fmoc-Arg (pbf)-Wang 2.5-3 times of mole of resin successively
The amino acid, N- hydroxy benzo triazoles, O- benzotriazole-N, N, N', N'- tetramethylurea-hexafluoro of " Fmoc " radical protection
Phosphate is dissolved in 10mL DMF, after adding the diisopropylethylamine for being equivalent to 5-6 times of mole of Fmoc-Leu-Wang resins
Mixing obtains mixed solution, is then added to mixed solution in the resin of the deprotection, is stirred to react under protection of argon gas
60min.The degree that condensation reaction is completed is detected by indenes check reagent, is washed repeatedly with DMF to remove unreacted after the reaction was complete
Residual liquid.
Four, the extension of peptide chain：Step 2 and three is repeated, according to the sequence of chimeric peptide amino acid sequence, from the ends C- of polypeptide
The amino acid of " Fmoc " radical protection is condensed on resin one by one successively to the ends N-, until all amino acid residues have been condensed
At obtaining peptide resin.
Five, peptide chain is from the cutting on resin：According to the method for step 2 by most latter linked amino acid on peptide resin
" Fmoc " group removes completely.Resin is alternately washed with dichloromethane, methanol, after fully draining solvent, is added by every gram of peptide resin
Enter the cutting reagent of 18 mL, cutting reagent is by trifluoroacetic acid, diisopropylsilyl, water with 95：2.5：2.5 volume ratio mixing
It forms, at room temperature 3 h of cleavage reaction.It collects cutting reagent and depressurizes and be spin-dried for, be precipitated and precipitated with ice-cold ether, stood in a moment
Ether supernatant is removed, then precipitation is fully dissolved with water and a small amount of acetic acid, pours into separatory funnel, and ethyl acetate extraction is added, is received
Collect water phase, it is freeze-dried, obtain the thick peptide of white solid powder.
Six, 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 desalinations are freeze-dried after collecting main peak using Ultraviolet Detector, obtain desalination
Polypeptide compound.It is isolated and purified again by Reversed Phase High Performance, collects main peak, white is obtained after freeze-drying
The pure peptide of solid powder, i.e. chimeric peptide.
The ultimate yield of chimeric peptide prepared by above example is 41%, mass spectrum and chromatography testing result such as 1 institute of table
Show.
The mass spectrum and chromatography testing result of 1. chimeric peptide EMNT of table
Table 1 the result shows that, the detected value for the chimeric peptide that the present invention obtains is consistent with theoretical value.
The biological activity test of chimeric peptide manufactured in the present embodiment based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
It is as follows：
1, the affine experiment of the ligand-receptor of isotope labelling
Opiate receptor is obtained using rat brain membrane albumen is prepared, experiment is divided into total binding pipe, competitive binding pipe and non-specific
Property combine pipe, sequentially add radioligand, naloxone (Naloxone, Nx)/on-radiation drug, Tris- in each reaction tube
HCl (pH=7.4) and brain membrane proteins.Wherein, be only added in total binding pipe [3H] DAMGO (0.5nM) or [3H]DPDPE(1nM)
And the analog of 10 μM of Nx/ various concentrations is separately added in non-specific binding pipe and competitive binding pipe in brain membrane proteins respectively
And brain membrane proteins, Tris-HCl buffer solutions (pH=7.4) are finally supplemented to 0.5 mL of total volume.Oscillator mixing reaction solution, 37
DEG C shaking table reacts 1 h, takes out centrifuge tube, reaction solution is through ZT-II type cell harvesters, GF/C type glass fibre element film mistakes
Filter.Filter membrane is taken out after toasting 30 min of glass fibre element film under 80 DEG C of oil baths, is put into 24 orifice plates, 0.7 mL flickers are added per hole
Liquid, capping film capping.β liquid scintillation counters record radioactive intensity (CPM) overnight.It is given birth to using Chengdu Tai Meng BL-420F
Object records inhibiting rate of the system-computed analog to specific binding, and using the negative logarithm of concentration as abscissa, inhibiting rate is vertical sits
Mark, linear regression acquire IC50Value, according to formula Ki=IC50/ (1+ [L]/KD) calculates KiValue.Each experimental data difference is parallel
3 groups of experiments are done to average.
Experimental result is shown in Table 2.The data of table 2 show that chimeric peptide is lower than Tyr-Pro-Trp-Phe-NH2 to the compatibility of μ-opiate receptor
About 2.5 times or so, to the compatibility K of δ-opiate receptori(δ)>10000nM shows that chimeric peptide still has μ-opiate receptor
Higher compatibility feature, this may have the connexon of the addition among chimeric peptide related." Gly-Gly " increases chimeric peptide
Flexibility makes it easier for being combined with μ-opiate receptor, and the affine activity of the opium for therefore remaining Tyr-Pro-Trp-Phe-NH2 part.
The opiate receptor compatibility of 2. chimeric peptide EMNT of table
| Polypeptide | Ki(μ)[nM] | Ki(δ)[nM] | Ki(δ)/Ki(μ) |
| Tyr-Pro-Trp-Phe-NH2 | 4.55±0.16 | 5093±660 | 1119 |
| EMNT | 11.2±1.6 | >10000 | >893 |
2, in vitro biological sample opioid activity measures
Guinea pig ileum longitudinal muscle tests (GPI experiments)：Cavy 250-300g, male and female are unlimited, fasting 12h before testing, drinking-water
It is unlimited.Stick hits occipitalia execution, cuts open the belly and takes out two sections of the ileum 6cm at nearly caecum end, and immersion is continually fed into 95%O2And 5%CO2Mixing
Kreb ' s liquid (the g/L of gas：NaCl 6.9, CaCl20.28, KCl 0.35, KH2PO40.16, MgSO4·7 H2O 0.30,
NaHCO32.1, diphenhydramine 5 × 10-5, choline chloride 2.8 × 10-3, glucose 1.98) in, moistening rear enclosure is in a glass bar
On, a vertical shape blood vessel along ileum wall detaches longitudinal muscle with ring muscle, is then tied up one end of longitudinal muscle with No. zero silk thread
On the small hook of glass electrode, the other end is tied on JZ-1 type muscle tension sensors, is immediately placed in and is filled persistently after preparing
It is passed through gas (95%O2And 5%CO2) Kreb ' s liquid, 37 ± 0.2 DEG C of constant temperature glass bath pipe in, preload 500mg.Before
30min changes one time of nutrition liquid per 5min, is changed the liquid once later per 15min, starts to test after balancing 2h.Electro photoluminescence is given, is stimulated
Parameter：The wide 0.3-0.5ms folk prescriptions wave of wave, frequency 6 times/min, load voltage 50V.The basic contraction intensity of record, then give appropriate
Morphine identifies its activity；Then drug 3-4 to be measured different concentration are selected, bath pipe is added, it is measured and inhibits sample electro photoluminescence
Cause the percentage shunk, inhibition level should be between 20%-80%.It after each drug effect, is rinsed 5 times, is put down with nutrient solution
Weigh 15min.Height × 100% is shunk before inhibiting percentage=(shrinking height-reaction after-contraction height before reaction)/reaction.So
Docs-effect semilog graphing method is used afterwards, to inhibit percentage as ordinate, using the logarithm of the concentration of drug to be measured as abscissa
Mapping acquires and inhibits the 50% drug concentration (IC for shrinking height50)。
Mouse vas deferens test (MVD experiments)：Weight 30-35g male Kunming white mouses, cervical dislocation put to death, cut open the belly
Both sides vas deferens is taken out, immersion is continually fed into 95%O2And 5%CO2Kreb ' s liquid (the g/L of gaseous mixture：NaCl 6.9, CaCl2
0.28, KCl 0.35, KH2PO40.16, NaHCO32.1, glucose 1.98) in, remove fat and blood vessel attached to it.
The one end for cutting off vas deferens becomes a hollow pipe with the small cotton ball lightly sperm from end into open end blow-off pipe,
Then one end being tied on the small hook of glass electrode with No. zero silk thread, the other end is tied on JZ-1 type muscle tension sensors,
It is immediately placed in fill after preparing and is continually fed into gas (95%O2And 5%CO2) Kreb ' s liquid, 36.5 ± 0.5 DEG C of constant temperature glass
Glass is bathed in pipe, preload 100mg.Preceding 30min changes one time of nutrition liquid per 5min, is changed the liquid once later per 10min, is opened after balancing 2h
Begin to test.Give electro photoluminescence, stimulation parameter：The wide 2ms of wave, frequency 6 times/min, load voltage 50V.The basic contraction intensity of record,
Appropriate morphine is given again identifies its activity；That then gives various dose respectively waits for reagent object, records contraction intensity.Each drug
It after effect, is rinsed 5 times with nutrient solution, balances 15min.IC50Assay method is tested with GPI.
Experimental result is shown in Table 3.The data of table 3 show, the ligand-of the result and isotope labelling of the experiment of GPI and MVD samples
The trend for experimental result that receptor is affine is consistent.μ-opiate receptor is mainly contained in GPI samples, mainly contained in MVD samples μ-and
δ-opiate receptor.In GPI experiments, the opium alkaloids activity of chimeric peptide is suitable with Tyr-Pro-Trp-Phe-NH2, IC50Value is than interior morphine
Peptide -1 is slightly lower.In MVD experiments, the IC of chimeric peptide50Value is then slightly below Tyr-Pro-Trp-Phe-NH2.Should the result shows that, chimeric peptide is in vitro
Biological sample has higher opium agonist activity, is especially higher than Tyr-Pro-Trp-Phe-NH2 to the agonist activity of GPI samples.
The GPI/MVD sample opioid activities of 3. chimeric peptide of table
3, maincenter and periphery analgesic experiment
Choose Kunming system male mice, 18-22g, environment temperature：20 DEG C, bath temperature：50±0.5℃.It is first surveyed before administration
The 1/3-1/2 of mouse rat-tail is immersed water-bath by the Basic Pain Threshold (control latency, CL) for determining mouse, and record rat-tail is from rigid
Water-bath is immersed to the time shunk.Excessively sensitive (<3s) or it is blunt (>5s) mouse is discarded, deadline 10s,
To prevent mouse from scalding.5,10,15,20,25,30,40and 50min respectively survey a flick latency after telocoele administration
(test latency, TL), the 5th, 10,15,20,25,30,45,60and 90min respectively surveys a TL after subcutaneous administration, and 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).
Maincenter and periphery analgesic experiment result are as illustrated in fig. 1 and 2, and maincenter intracerebroventricular injection 0.3-10nmol dosage is fitted into
Peptide generate concentration-dependant efficient analgesic effect (in Fig. 1 ■ indicate injection 10nmol dosage chimeric peptide, ● indicate injection
The chimeric peptide of 3nmol dosage, ▲ indicate to inject the chimeric peptide of 1nmol dosage, ▼ indicates the chimeric peptide of injection 0.3nmol dosage,
◆ indicate physiological saline；■ indicates that 10 μm of ol/kg chimeric peptides are subcutaneously injected in Fig. 2, ● indicate that physiological saline is subcutaneously injected).?
Maximum analgesic effect is generated after injecting drug 5min to 10min, and analgesia %MPE can reach about 80% or so in the meantime.
Moreover, the analgesia duration of chimeric peptide is longer, 50min is measured upon administration, analgesia %MPE under 1-10nmol dosage still
10%-20% or so can be reached, show that chimeric peptide has efficient central analgesia activity.In addition, by the way that 10 μm of ol/ are subcutaneously injected
The chimeric peptide of kg generates most as a result, it has been found that chimeric peptide has efficient peripherally administered analgesic activities after injecting drug 10min
Big analgesic activity, and analgesia duration is longer, 60min is measured as 17.07% to analgesia %MPE upon administration, in 90min
It is measured as 16.8%, shows that chimeric peptide can reach 90min or more by the peripherally administered duration.The result is proved based on interior
When morphine peptide -1 and neurotensin (8-13) structure and the chimeric peptide that designs greatly enhance analgesic effect and the effect of drug
Between, i.e., chimeric peptide has the feature of efficient central analgesia activity.
4, Acute brain block is tested
Choose Kunming system male mice, 18-22g, environment temperature：20℃.Mouse telocoele is buried using PE-10 intubations
Pipe is administered for 3 days after operation.According to the analgesic activities of chimeric peptide, drug dose is determined, measure acute analgesia tolerance.Mouse shifts to an earlier date 1
Hour intracerebroventricular injection drug or physiological saline 1 time, inject the drug of various dose, measure pain threshold variation respectively later,
And then analgesia tolerance assessment is carried out to Tyr-Pro-Trp-Phe-NH2 and chimeric peptide.
(■ indicates physiological saline+interior to the analgesia tolerance result of Tyr-Pro-Trp-Phe-NH2 and chimeric peptide in Fig. 3 as shown in Figures 3 and 4
Deltorphin delta -1, ● indicate Tyr-Pro-Trp-Phe-NH2+Tyr-Pro-Trp-Phe-NH2 of 10nmol dosage；■ indicates physiological saline+chimeric peptide in Fig. 4, ●
Indicate chimeric peptide+chimeric peptide of 10nmol dosage), by shifting to an earlier date 1 hour to mouse intracerebroventricular injection physiological saline, rear side brain
The Tyr-Pro-Trp-Phe-NH2 and chimeric peptide of room injection 0.3-10nmol dosage generate significant analgesic activity, and are in concentration-dependant,
It is similar to the result of Fig. 1, show that injecting normal saline in advance does not influence the performance of Tyr-Pro-Trp-Phe-NH2 and chimeric peptide analgesic activity.So
And the 1 hour in advance Tyr-Pro-Trp-Phe-NH2 to mouse intracerebroventricular injection 10nmol dosage, inject the interior morphine of various dose again later
Peptide -1, analgesic dose curve obviously move to right, and analgesic effect significantly reduces, that is, produces analgesia tolerance.But mouse is shifted to an earlier date
The chimeric peptide of 1 hour intracerebroventricular injection 10nmol dosage, then inject the chimeric peptide of various dose, analgesic dose curve in advance
The dose curve of injecting normal saline almost overlaps, and there is no generate significantly to move to right.It should be the result shows that injecting chimeric peptide in advance
To the no significant impact of performance of the analgesic activity of second of injection EMNT, it was demonstrated that chimeric peptide has the height without tolerance side effect
Imitate analgesic activities.
5, colon transport experiment
Male kunming mouse, weight 25-30g, fasting 1h, light anaesthesia with diethyl ether, by the glass of d=3mm before experiment starts
Glass pearl plug is at terminal colon (away from anus 2cm), and mouse freely activity measures basis row's pearl time, and row's pearl time is more than 30min
Mouse reject.Under the same terms, intracerebroventricular injection various concentration drug, 5 μ L of volume injected.When starting the row of measurement pearl after 5min
Between, transport calculation formula：%MPE=(m- basis row's pearl time when administration heel row pearl)/basis row pearl time × 100.
Experimental result as shown in Figure 5 (in Fig. 5 ■ indicate Tyr-Pro-Trp-Phe-NH2,Indicate chimeric peptide), intracerebroventricular injection 0.5-
The enhancing mouse Colon row pearl that the Tyr-Pro-Trp-Phe-NH2 and chimeric peptide of 5nmol dosage generate concentration-dependant is potency, and concentration is higher,
It is longer to arrange the pearl time.But chimeric peptide is significantly lower than Tyr-Pro-Trp-Phe-NH2 to the effect of colon row's pearl transhipment.The heel row pearl time is administered
It is longer, show that the inhibiting effect that drug transports mouse Colon section is stronger.It should be the result shows that Central injection chimeric peptide be substantially reduced
The effect of the intestinal transport of mouse Colon section.
6, gastrointestinal tract charcoal meal Promoting Experiment
Male kunming mouse, weight 25-30g, fasting 20h start to test later, intracerebroventricular injection various concentration drug,
5 μ L of volume injected.The charcoal meal (5% Arabic gum, 10% activated carbon aqueous solution) of Mouse oral 0.2mL is given later.After 30min
Mouse is put to death, small intestine is removed.Measure small intestine overall length (pylorus knot to cap end) and charcoal meal promoted in small intestine away from
From.The ratio percentage table for the distance and entire small intestinal length that the gastrointestinal transit activity of drug is promoted with charcoal meal in small intestine
Show.
Experimental result as shown in Figure 6 (in Fig. 6 ■ indicate Tyr-Pro-Trp-Phe-NH2,Indicate chimeric peptide), intracerebroventricular injection 0.5-
The Tyr-Pro-Trp-Phe-NH2 and chimeric peptide of 5nmol dosage generate the inhibition gastrointestinal tract charcoal meal progradation of concentration-dependant, and concentration is higher,
Inhibiting effect is stronger, but the inhibiting effect that chimeric peptide promotes gastrointestinal tract charcoal meal is significantly lower than Tyr-Pro-Trp-Phe-NH2.After administration, charcoal
Meal is lower in gastrointestinal transit activity, shows that the inhibiting effect that drug promotes mouse GI tract charcoal meal is stronger.This shows maincenter
Injection chimeric peptide reduces the meal progradation of gastrointestinal tract carbon, that is, has the advantages of reduction gastrointestinal side effect.
In conclusion the present invention is by by four amino acid residue sequences " Tyr-Pro-Trp-Phe " of Tyr-Pro-Trp-Phe-NH2
Pass through six amino acid residue sequence " Arg-Arg-Pro- of intermediate connexon " Gly-Gly " and neurotensin (8-13)
Tyr-Ile-Leu " is mutually coupled, a kind of chimeric peptide being based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13) of structure.The purpose is to
In order to retain the bioactivity of two neural peptide fragments, the analgesia duration for solving Tyr-Pro-Trp-Phe-NH2 is shorter, peripherally administered
Analgesic activities are relatively low, and have the problem of analgesia tolerance and gastrointestinal side effect.It is right by various in vitro in body biological experiment
The chimeric peptide that the present invention synthesizes carries out pharmacological activity identification.The result shows that chimeric peptide of the invention has μ-opiate receptor
Higher compatibility, and to the higher opium agonist activity of in vitro biological sample, interior morphine especially is higher than to the activity of GPI samples
Peptide -1.By maincenter telocoele and periphery subcutaneous administration, chimeric peptide all has efficient analgesic activities.In addition, Central injection is embedding
Peptide is closed with no analgesia tolerance characteristic, and its gastrointestinal side effect is substantially reduced.Therefore, chimeric peptide of the invention is preparing height
It is had potential application in terms of effect, the polypeptide analgesic without analgesia tolerance and reduction gastrointestinal side effect.
Claims (9)
1. the chimeric peptide based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13), it is characterised in that the amino acid sequence of the chimeric peptide
It is as follows：
Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu。
2. the synthetic method of the chimeric peptide as described in claim 1 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that this approach includes the following steps：
One, the Wang resins pretreatment of " Fmoc " protection：The air-tightness for checking solid phase synthetic instrument, will be with there are one amino acid residues
Fmoc-Leu-Wang resins be put into synthesizer, add methylene chloride 30~40min of stirring, make resin fully impregnate swelling after, subtract
Pressure filters solvent；Wherein there are one the volume ratios of the quality and dichloromethane of the Fmoc-Leu-Wang resins of amino acid residue for band
For 1g：(7~12) mL；
Two, " Fmoc " blocking group is removed：Swellable resins after draining are washed into 3~5min with DMF, are drained, are repeated 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 makes " Fmoc " blocking group fully remove, drains solvent later, finally washs cleared deprotection solution with DMF, obtains
Remove the resin of " Fmoc " blocking group；The volume ratio of the wherein quality of resin and the deprotection solution being added for the first time is 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 triazoles, O- benzos three of " Fmoc " radical protection
Nitrogen azoles-N, N, N', N'- tetramethylurea-hexafluorophosphate is dissolved in DMF, add mixing after diisopropylethylamine must mix it is molten
Then mixed solution is added in the resin that step 2 removes " 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 washings after the reaction was complete
Residual liquid；
Four, the extension of peptide chain：Step 2 and three is repeated, according to the sequence of chimeric peptide amino acid sequence, from the ends C- of polypeptide to N-
The amino acid of " Fmoc " radical protection is condensed on resin by end one by one successively, until the condensation of all amino acid residues is completed, is obtained
To peptide resin；
Five, peptide chain is from the cutting on resin：" Fmoc " group of most latter linked amino acid on peptide resin is removed completely, then
Peptide resin is alternately washed with dichloromethane and methanol, after fully 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
Fully dissolving precipitation, pours into separatory funnel, and ethyl acetate extraction is added, and collects water phase, freeze-dried, obtains white solid
The thick peptide of powder；
Six, 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 desalinations are freeze-dried after collecting main peak using Ultraviolet Detector, obtain 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 synthetic method of the chimeric peptide according to claim 2 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that the mole of the amino acid of " Fmoc " radical protection is Fmoc-Arg (pbf)-Wang resins mole in step 3
2.5-3 times of amount.
4. the synthesis side of the chimeric peptide according to claim 2 or 3 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13)
Method, it is characterised in that the mole of N- hydroxy benzo triazoles is Fmoc-Arg (pbf)-Wang resin moles in step 3
2.5-3 again.
5. the synthetic method of the chimeric peptide according to claim 4 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that the mole of O- benzotriazole-N, N, N', N'- tetramethylurea-hexafluorophosphates is Fmoc- in step 3
2.5-3 times of Arg (pbf)-Wang resin moles.
6. the synthetic method of the chimeric peptide according to claim 5 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that the mole of diisopropylethylamine is 5-6 times of Fmoc-Leu-Wang resin moles in step 3.
7. the synthetic method of the chimeric peptide according to claim 6 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that the cutting reagent of 10-25mL is added in every gram of peptide resin in step 5.
8. the synthetic method of the chimeric peptide according to claim 7 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that cutting reagent described in step 5 by trifluoroacetic acid, diisopropylsilyl and water according to volume ratio 95：2.5：2.5
It mixes.
9. the synthetic method of the chimeric peptide according to claim 8 based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13),
It is characterized in that being by the specific method that " Fmoc " group of most latter linked amino acid on peptide resin removes completely in step 5：
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, is drained 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, " Fmoc " blocking group is made fully to remove, it
After drain solvent, finally wash cleared deprotection solution with DMF, removed the resin of " Fmoc " blocking group.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1192246A (en) * | 1995-06-07 | 1998-09-02 | 细胞治疗公司 | Cell line producing analgesic compounds for treating pain |
| WO2008052043A9 (en) * | 2006-10-24 | 2008-07-31 | Cogenesys Inc | Opioid receptor agonist fusion proteins |
| CN103665170A (en) * | 2008-12-05 | 2014-03-26 | 安吉奥开米公司 | Peptide therapeutic conjugates and uses thereof |
| CN104877005A (en) * | 2015-04-16 | 2015-09-02 | 兰州大学 | Non-natural amino acid modified endomorphin-1 analogue as well as synthesis method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1192246A (en) * | 1995-06-07 | 1998-09-02 | 细胞治疗公司 | Cell line producing analgesic compounds for treating pain |
| WO2008052043A9 (en) * | 2006-10-24 | 2008-07-31 | Cogenesys Inc | Opioid receptor agonist fusion proteins |
| CN103665170A (en) * | 2008-12-05 | 2014-03-26 | 安吉奥开米公司 | Peptide therapeutic conjugates and uses thereof |
| CN104877005A (en) * | 2015-04-16 | 2015-09-02 | 兰州大学 | Non-natural amino acid modified endomorphin-1 analogue as well as synthesis method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| ***及神经降压素对大鼠尾壳核神经元μ、δ、κ阿片受体表达的影响;刘青海;《中国优秀硕士学位论文全文数据库》;20120315(第3期);前言部分，第37页第3段，第41页第4段 * |
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