CN102206285B - Chimeric peptide based on endomorphins 2 and neuropeptides FF, and synthesis and application thereof - Google Patents
Chimeric peptide based on endomorphins 2 and neuropeptides FF, and synthesis and application thereof Download PDFInfo
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Abstract
The invention provides chimeric peptide based on endomorphins 2 and neuropeptides FF, wherein the chimeric peptide is the novel chimeric peptide EN-9 which is constructed by using the characteristic that both a C end of the endomorphins 2 and an N end of the neuropeptides FF have phenylalanine residues, on the basis of reserving an N end structure of the endomorphins 2 and a C end structure of the neuropeptides FF. Through in-vivo pharmacological study, it is found that the EN-9 shows stronger and more persistent analgesic activity than the endomorphins 2, and has the advantages of no enduring action, low addiction and the like. In addition, hypodermic compound EN-9 can play obvious analgesic action, can overcome the defect that the endomorphins have no analgesic activity through injecting in peripheral regions, therefore the chimeric peptide has a potential using value of being used for clinical pain treatment.
Description
Technical field
The invention belongs to the biochemical technology field, relate to a kind of based on interior morphine peptide 2 and neuropeptide FF and the chimeric peptide and the synthetic method thereof that make up; The present invention also relates to the application of this chimeric peptide in the preparation analgesic simultaneously.
Background technology
Clinically, the opium medicine of morphine class is widely used in treatment and alleviates various pain by people, but because the anodyne of opiates can cause serious untoward reaction and opioid dependence effect, and greatly limit its application in clinical treatment.Existing Mammals maincenter and the periphery of studies show that all extensively exists opiate receptor.1997, morphine peptide (endomorphins, EMs), i.e. endomorphin 1 (EM-1, Tyr-Pro-Trp-Phe-NH in finding
2) and 2(EM-2, Tyr-Pro-Phe-Phe-NH
2) be the agonist of the endogenic efficient selective of mu opioid receptor (MOR), they are to the K of mu opioid receptor
iBe respectively 360 pM and 690 pM (
Nature. 1997,386:499;
Ann. N. Y. Acad. Sci.1999; 897:136).Their performance is similar to morphine, can participate in the adjusting of different physiological roles in the body, and especially the pain sensation is regulated, therefore be regarded as potential alternative morphine profound analgesic (
Jpn. J. Pharmacol.1998,78:337;
Brain Res.2000,881:1;
J. Pharmacol. Exp. Ther.1999,291:12;
Pharmacol. Rev.2007,59:88 etc.).The efficient selective agonist that interior morphine peptide is μ-opiate receptor can mediate obvious analgesic activities.Existing research discovery maincenter injection EM-1 and EM-2 show antipodal result in the CPP experiment, namely cause respectively the preference of environment and detest (
J. Pharmacol. Exp. Ther.2004,309:816;
Neurosci. Lett.2004,365:157 etc.).Therefore EM-2 has lower drug addiction than EM-1.
1985, neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH
2) be isolated and identified from the ox brain as a kind of anti-opioid, and find it have the effect of anti-morphine analgesia (
Proc. Nat. Acad. Sci. U.S.A.1985,82:7757).Studies show that subsequently, the neuropeptide FF system relates to two kinds of different acceptor precursor (Pro-NPFF
AAnd NPFF
BAcceptor) and two kinds of isoacceptor (NPFF not
1And NPFF
2Acceptor) (
FEBS Lett.1997,409:426;
Mol. Pharmacol.1999,55:804;
Nat. Cell Biol.2000,2:703;
J. Biol. Chem.2001,276:36961;
J. Biol. Chem.2000,275:25965;
J. Biol. Chem.2000,275:39324 etc.).A large amount of results of study show, NPFF and relevant Toplink mediation various biological thereof are active, namely movable in the pain sensation, cardiac vascular activity, body temperature, gi tract, ingest, the aspects such as internal secretion, opiate tolerance and dependence have certain regulating effect, particularly on the caused analgesia of opioid, tolerance and habituation all have important impact (
Prog. Neurobiol.1996,48:461;
Eur. J. Pharmacol.1998,345 (1): 1;
Curr. Top. Med. Chem.2005,5:341 etc.).
As everyone knows, endogenic neuropeptide can both mediate one or more main physiology and pharmacological activities in body.But neuropeptide often is accompanied by and has produced its less important activity, and these less important activity just becomes its side effect in its main activity of performance.Such as, opioid peptides can be accompanied by the appearance of the side effects such as tolerance, habituation and respiration inhibition in the lenitive while.In order to solve this contradiction, traditional research strategy is that parent take neuropeptide is as chemical template, carry out the chemically modified of system and filter out the neuropeptide analogue that receptor-selective is higher and have preferred conformation, keep than high biological activity and reduce the desired result of its side effect thereby reach.Also developed in recent years some New Policies, based on endogenous neuropeptide design and made up a series of T1249s and it is studied, in the hope of make chimeric peptide can performance main active in, reduce to greatest extent the appearance of its side effect.Especially, the people such as Foran based on opioid peptides EM-2 and tachykinin Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) and successfully design construction the brand-new T1249 ESP7(Tyr-Pro-Phe-Phe-Gly-Leu-Met-NH of one class
2), and find that spinal levels injection ESP7 can produce the analgesic activity that opiate receptor relies on, but do not produce analgesia tolerance phenomenon side effect (
Proc. Nat. Acad. Sci. U.S.A.2000,97:7621).
In recent years pharmacological activity identifies and shows that NPFF not only has the function of anti-opium also have former opioid activity, thus be classified as the important opium of a class regulate peptide (
Curr. Top. Med. Chem.2005,5:341).NPFF self, namely affects not quite in the biologic activity of physiological dose level on body without impact the Basic Pain Threshold value.But NPFF regulates one of peptide as opium, plays an important role in the endogenous opiate of keeping body and anti-opiate system equilibrium process, has participated in the adjusting of analgesia tolerance, and can weaken the caused condition Place Preference of the opioids effect such as morphine (
Curr. Top. Med. Chem.2005,5:341;
Peptides. 2006,27:964;
Peptides. 2007,28:2235;
Peptides. 2010,31:1374 etc.).Therefore, the pharmacological characteristics of NPFF makes it be suitable as the chemical template molecule and develops into the brand-new opiates chimeric peptide of a class.
Summary of the invention
The objective of the invention is in clinical application, to have the side effects such as opioid dependence and habituation for opium kind analgesics things such as morphines, a kind of chimeric peptide based on interior morphine peptide 2 and neuropeptide FF is provided.
Another object of the present invention provides a kind of method of synthesizing based on the chimeric peptide of interior morphine peptide 2 and neuropeptide FF.
A further object of the invention just provides the application of this chimeric peptide in the preparation analgesic.
(1)
Chimeric peptide based on interior morphine peptide 2 and neuropeptide FF
The present invention is based on the chimeric peptide of interior morphine peptide 2 and neuropeptide FF, make up based on interior morphine peptide 2 and neuropeptide FF, its N-terminal and C-terminal are respectively the structure of EM-2 and neuropeptide FF (3-8), and its structural formula is as follows:
。
(2)
Synthetic based on the chimeric peptide of interior morphine peptide 2 and neuropeptide FF
The present invention is based on chimeric peptide synthetic of interior morphine peptide 2 and neuropeptide FF, comprise following processing step:
(1) resin pre-treatment: the Rink-Amide-MBHA resin is stirred 30-40 min in methylene dichloride, make decompressing and extracting solvent after the abundant swelling of resin;
(2) remove the Fmoc radical protection: make swelling, drain resin behind the solvent in the hexahydropyridine DMF of volumetric concentration 20-22% solution, drain after stirring 3-5min, repeat 2-3 time; Join again the hexahydropyridine DMF solution of volumetric concentration 20-22%, stir 10-15min, the Fmoc group is removed fully, then drain solvent; At last with DMF washing Ex-all hexahydropyridine;
(3) condensation: successively with 2-4eq's
N-α-Fmoc blocking group amino acid,
N-hydroxy benzo triazole (HOBt), O-benzotriazole-
N, N, N', N'-tetramethyl-urea-hexafluorophosphate (HBTU) is dissolved among the DMF fully, adds the rear mixing mixing solutions of diisopropylethylamine (DIEA) of 4-8eq again; Then under argon shield, make the resin stirring reaction 50-60min in above-mentioned mixing solutions that removes the Fmoc radical protection, drain solvent; Remove unreacted with the DMF repeated washing
N-α-Fmoc blocking group amino acid, HOBt, HBTU;
(4) prolongation of peptide chain: repeating step (2), (3), successively will with
N-α-Fmoc blocking group amino acid is condensed on the resin one by one, until finish the condensation of all amino-acid residues, obtains peptide resin;
(5) cutting of peptide chain from the resin: after the peptide chain condensation is all finished, according to the method for step (2) last amino acid whose Fmoc group of peptide resin is removed fully; Replace washing resin with DCM, MeOH, fully drain solvent after, add the cutting agent of 15-20ml by every gram peptide resin, under room temperature cleavage reaction 2-3 hour; Filter, decompression is spin-dried for filtrate not being higher than under 37 ℃ the condition fully, then separates out precipitation with the ether that is not higher than-10 ℃; Leave standstill the rear ether of removing first supernatant, remove the ether phase with separating funnel after water fully dissolves again, merge water, lyophilize gets white thick peptide pressed powder; Described cutting agent is to mix the solvent that forms by trifluoroacetic acid (TFA), tri isopropyl silane (TIS), water with the volume ratio of 95:2.5:2.5;
(6) desalination and the purifying of thick peptide: take the acetic acid solution of volumetric concentration 15-20% as moving phase, thick peptide is crossed the desalination of Sephadex G25 cross-linked dextran gel column, utilize Ultraviolet Detector to collect the main peak postlyophilization, get the peptide compounds of desalination; Recycle reverse performance liquid chromatographic column and carry out separation and purification, collect main peak, obtain the pure peptide pressed powder of white after the lyophilize.
The product of aforesaid method preparation detects through mass spectrum and stratographic analysis, and is consistent with the compound structure of design.Show the chimeric peptide that successfully synthesizes based on interior morphine peptide 2 and neuropeptide FF, sample purity is more than 98% behind the purifying.
(3) based on the analgesic experiment of the chimeric peptide of interior morphine peptide 2 and neuropeptide FF
The present invention is based on the chimeric peptide of interior morphine peptide 2 and neuropeptide FF, be based on the chimeric brand-new compd E N-9 that forms of interior morphine peptide 2 and neuropeptide FF two class endogenous neuropeptides, it has kept the bioactive key structure of these two kinds of neuropeptides of decision zone simultaneously, therefore, when having kept the opium analgesic activity, can effectively reduce the side effects such as opiate tolerance and habituation.Analgesia and the tolerance effect of the EN-9 of chimeric peptide of the present invention are described below by pharmacological evaluation.
1, the analgesic experiment of EN-9
Compd E N-9 of the present invention and interior morphine peptide EM-2, after the injection of tricorn maincenter, the activity of utilizing the experiment of mouse photo-thermal whipping to carry out pain sensation regulating effect in the body detects and compares.
(1) mouse tricorn pipe laying
Stereotaxic apparatus is gulf, river I type.Kunming is male mice, body weight 20 ± 2 g.After vetanarcol anesthesia (i.p., 80mg/kg Mouse Weight), place on the stereotaxic apparatus.With mouse head field of operation iodophor disinfection, cut off hair, cut scalp along sagittal suture, expose skull, find the bregma position.From bregma 3 mm backward, to left/right 1 mm, be the top position of tricorn.Self-control stainless steel tube (a section of 24-gauge, the above connects a bit of PE-10 pipe) inserts 3 mm downwards by above-mentioned position, is tricorn.One section stainless steel string (28-gauge) is inserted in the above-mentioned steel pipe, to prevent that cerebrospinal fluid is excessive or to infect.With the fixing steel pipe of medical courses in general dental base acrylic resin powder, after solidifying, sew up a wound.After the operation, mouse recovered 4 days, the 5th day beginning subsequent experimental.The instruments that relates in the experiment, stainless steel tube etc. are all sterilized.Medicine dissolution-20 ℃ of preservations, thaws before the use in physiological saline.
(2) pain sensation test experience
Use above-mentioned tricorn pipe laying mouse, utilize photo-thermal whipping instrument to come detection of drugs on the impact of pain sensation effect.Envrionment temperature is controlled at 20 ± 1 ℃, but laboratory animal ad lib, drinking-water.Mouse tricorn per injection 4 μ l medicines.Measure first basic TFL (3-5 second) before the administration, too responsive or blunt mouse discards need not.The record administration after the 5th, 10,15,20,25,30,40,50,60 minutes TFLs.For preventing scalding, the whipping time surpasses 10 seconds and all calculated with 10 seconds.
(3) MPE and the AUC value of calculating medicine
Pain sensation regulating effect is utilized MPE(maximum possible effect) value estimates, MPE(%)=[(threshold of pain-Basic Pain Threshold after the administration)/(10 seconds-Basic Pain Threshold)].Because medicine analgesic is mainly 0-30 min action time, therefore the MPE data of every mouse are converted into area under curve AUC(area under the curve) value (0-30 min), estimate medicine to the regulating effect of the mouse pain sensation.The AUC data that obtain mean+/-standard error (Means ± S.E.M.) expression, the group difference that different pharmaceutical concentration is processed carries out data statistics and analysis with one-way analysis of variance (the Dunnett check of one-way ANOVA),
*P＜0.01,
* *There is significant difference between the AUC value of P＜0.001 expression drug treating group and the physiological saline group.Experimental result as depicted in figs. 1 and 2.
The physiological saline group is the blank solvent control group, and the drug level of EN-9 is respectively 5,10,20 and 30 nmol, and the drug level of EM-2 is respectively 1,5,10,20 and 30 nmol.Every treated animal number is 8-11.Experimental result shows that mouse intracerebroventricular injection new compound EN-9 can rely on ground generation analgesic activity by dosage.After injection the 10th minute of the analgesic activity of EN-9 reaches maximum effect.After administration front 15 minutes, EN-9 has kept stronger analgesic activities, and the Duration Ratio EM-2's of its analgesic activity is longer.As shown in Figure 2, the data of each group are converted into area under curve AUC value (0-30 min), compare intracerebroventricular injection EM-2(5 ~ 30 nmol with blank solvent contrast physiological saline group) and EN-9(10 ~ 30 nmol) TFL of mouse all increased significantly.Compare with the EM-2 with dosage, intracerebroventricular injection EN-9 shows stronger, more lasting analgesic activities.
2, the analgesia of EN-9 tolerance experiment
Further compd E N-9 more of the present invention and EM-2 are in the biological activity of analgesia aspect the tolerance.By continuous six days injectable drugs of mouse tricorn, them have been detected to the tolerance phenomenon of analgesic activity with the photo-thermal tail-flick method.
(1) pain sensation of mouse tolerance experimental technique
Kunming is male mice, recovers 5 days behind the tricorn pipe laying, injects continuously high dose medicament 6 days, and inject once every day, utilizes photo-thermal whipping instrument to come the continuous injection of detection of drugs on the impact of pain sensation effect.Envrionment temperature is controlled at 22 ± 1 ℃, but laboratory animal ad lib, drinking-water.Mouse tricorn per injection 4 μ l medicines.Experiment was carried out between 9 o'clock to 11 o'clock every day.Measure basic TFL (3-5 second) before the first day drug injection, too responsive or blunt mouse discards need not.The the 5th, 10,15,20,25 and 30 minute TFL after the record administration in the experiment.For preventing scalding, the whipping time is no more than 10 seconds.
(2) tolerance experimental data statistics
Experimental data is with MPE(maximum possible effect) represent, MPE(%)=100 * [(threshold of pain-Basic Pain Threshold after the administration)/(10 seconds-Basic Pain Threshold)].The MPE data of every mouse are converted into area under curve AUC value, estimate medicine to the regulating effect of the mouse pain sensation.The AUC data that obtain mean+/-standard error (Means ± S.E.M.) expression, the difference of mouse analgesia effect in six days is added up with one-way analysis of variance (the Tukey HSD check of one-way ANOVA),
*P＜0.01,
* *P＜0.001 expression AUC value has been compared significant difference with first day EM-2 analgesic activity.Experimental result as shown in Figure 3.
The physiological saline group is the blank solvent control group, and the drug level of EN-9 is 30 and 60 nmol, and the drug level of EM-2 is 60 nmol.Every treated animal number is 8-15, and every group data are converted into area under curve AUC value (0-30 min).After experimental data showed the tricorn of mouse injection in continuous six days blank solvent contrast physiological saline, mouse did not produce analgesic activities.Compare first day intracerebroventricular injection EM-2(60 nmol with blank solvent contrast physiological saline group) and EN-9(30 and 60 nmol) TFL of mouse all increased significantly.But the mouse tricorn is six days EM-2 of injection continuously, can begin its AUC value the 4th day of injection and just reduce significantly, and namely analgesic activity the tolerance phenomenon occurred from the 4th day.Compare with first day, the 4th day otherness highly significant (P＜0.01), the otherness of the 5th day and the 6th day is (P＜0.001) extremely significantly.And 30 and the continuously injection during six days of the EN-9 tricorn of 60nmol, the AUC value of its analgesic activities is all without considerable change (P〉0.05), keeps preferably without the analgesic activities that tolerates in six days of continuous injection always.
3, the drug habit of EN-9 experiment
Condition Place Preference (CPP) is tested, and is widely used in the award character of detection of drugs, is a kind of reactive model of strengthening.Through Improvement and perfection constantly, this experiment has been successfully used to estimate the effect of all kinds of psychotropics, and the result who detects is with classical drug habit experimental model---the result of study of self administration experiment is consistent, thereby this method admitted widely, and becomes one of method of estimating Drug psychological dependence begetting power and mechanism of action.
Consider that endomorphin 1 and 2 shows antipodal result in the CPP experiment, namely cause respectively preference and the detest of environment, so select the CPP experiment as the research model of research compd E N-9 drug habit of the present invention.The principle of this experiment is mainly Pavlovian conditioning, and when certain main reinforcing stimulus was related with environmental facies, this environment had just obtained strengthening effect.Its major advantage is to measure the award effect of medicine, and can measure the detest effect of medicine, and generally is used for estimating the award effect of the different system that comprises opium.The time of in this experiment mouse being stayed in the grid of administration is as the indirect measurement of drug craving.
(1) experimental model device of non-deviation CPP
Three grid altogether.About two large grid (20 * 20 * 20 cm) by middle little (5 * 20 * 20 cm) separately.The wicket that open one 5 * 5 two large grid bottoms is come in and gone out for mouse, and wicket can be closed, and when mouse was come in and gone out large grid, telescopic joint two wickets with " Π " shape prevented that mouse from staying in the grid of centre.Modify as follows grid, cause the difference of vision and sense of touch: chequered with black and white striped (black 2 mm, white 18 mm) is sticked at the wall of a large grid and top, and the bottom is coarse wire netting floor; White background black splotch (diameter 10 mm, interval 10 mm) is sticked at the wall of another large grid and top, and the bottom is smooth synthetic glass floor.
(2) specific operation process of CPP experiment.
First day: survey basic value.Had a rest at least 3 days, and selected behavior, figure and active normal tricorn pipe laying mouse.The CPP device cleans up, and wicket is opened so that mouse can move freely between two grid.Mouse is put into the CPP box, survey the time that it stops in two grid.Mensuration total time is 15min.Deletion surpasses 60% mouse residence time in certain grid after measuring.The mouse numbering of picking out.
Second to four day: training.The wicket of CPP box is closed, so that mouse can not leave residing grid.In morning (9:00-12:00), then mouse intracerebroventricular injection physiological saline place the spot grid to train 15min immediately.In afternoon (15:00-18:00), mouse intracerebroventricular injection medicine (saline control, morphine, EM-2 or EN-9) then places the striped grid to train 15min immediately.The time that keeps mouse to accept injection was fixed in every day, trained continuously three days.
The 5th day: estimate the CPP performance.The same with first day, measure after the administration residence time of mouse in each grid.
(3) interpretation and statistics
The CPP experimental result represents with CPP score (CPP score).The CPP score is defined as the 5th day mouse and deducts the residence time of first day mouse in the related grid of medicine the residence time in the related grid of medicine (being the striped grid of this experiment).Experimental data represents with mean+/-standard error, and analyzes and add up with single factor variance method (the Tukey HSD check of one-way ANOVA),
*P＜0.05,
*P＜0.01,
* *The CPP score of P＜0.001 expression drug treating group is compared with the saline control group, mouse condition Place Preference or detest variant.Experimental result as shown in Figure 4.
The physiological saline group is the blank solvent control group, and the drug level of morphine is 6 nmol, and the drug level of EM-2 is 15 and 30 nmol, and the drug level of EN-9 is respectively 15,30 and 60 nmol.Every group of number of mice is 10-12.After experimental result was found the intracerebroventricular injection blank solvent contrast physiological saline of mouse, mouse neither produced the condition Place Preference and does not also occur detesting.Compare with the physiological saline control group, intracerebroventricular injection 6 nmol morphines can produce significant condition Place Preference.And the EM-2 of intracerebroventricular injection 15 and 30 nmol, mouse can show the condition position to be detested, and its condition position detest effect is remarkable when 30 nmol high density.Yet the EN-9 of 15 nmol neither produces the condition Place Preference and does not also occur detesting.Compare with the physiological saline group, the EN-9 of 30 nmol shows slight condition position and detests (P〉0.05), and the EN-9(60nmol of higher dosage) just show highly significant condition position and detest activity (P＜0.01).These results show that compd E N-9 of the present invention compares with morphine, have low addicted advantage, and it have lower condition position detest effect than EM-2.
4, EN-9 when the periphery horizontal injection to the adjusting of the pain sensation
(1) pain sensation test experience
Kunming is male mice, body weight 20 ± 2 g.Utilize photo-thermal whipping instrument to come detection of drugs on the impact of pain sensation effect.Envrionment temperature is controlled at 22 ± 1 ℃, but laboratory animal ad lib, drinking-water.The volume of mouse subcutaneous injection medicine is 100 μ l.EN-9 and EM-2 all use aseptic physiological saline solution ,-20 ℃ of preservations, thaw before the use.Measure first basic TFL (3-5 second) before the administration, too responsive or blunt mouse discards need not.The 5th, 10,15,20,30,40,50,60 minutes TFLs after the record administration.For preventing scalding, the whipping time surpasses 10 seconds and all calculated with 10 seconds.
(2) the MPE value of calculating medicine
Pain sensation regulating effect is utilized MPE(maximum possible effect) value estimates, MPE(%)=100 * [(threshold of pain-Basic Pain Threshold after the administration)/(10 seconds-Basic Pain Threshold)].Data mean+/-standard error (Means ± S.E.M.) expression, the MPE value that each time point of different pharmaceutical treatment group is measured and the difference between the blank solvent control group are analyzed and are added up with single factor variance (the Bonferroni check of one-way ANOVA)
*P＜0.01,
* *The MPE value of P＜0.001 expression drug treating group is compared significant difference with the MPE value of physiological saline group same time point.Experimental result as shown in Figure 5.
The physiological saline group is the blank solvent control group, and the drug level of EN-9 and EM-2 treatment group is 30 mg/kg.Every treated animal number is 7-8.Experimental result shows that mouse subcutaneous injection blank solvent contrast physiological saline does not affect its Basic Pain Threshold value substantially.And also basic analgesia regulating effect of the EM-2 of subcutaneous injection 30 mg/kg.But compare with blank solvent contrast physiological saline group, mouse subcutaneous injection new compound EN-9 of the present invention can cause analgesic activity in time-dependent ground.After injection the 10th minute of the analgesic activity of EN-9 reaches maximum effect (the MPE values of about 65 %), and its analgesic activity can continue 50 minutes.Thereby show that new compound EN-9 can overcome interior morphine peptide in the weakness of periphery horizontal injection without analgesic activities.
The result of study of a series of pharmacological experiments such as pain sensation adjusting, tolerance and condition Place Preference in the body shows: it is active that the chimeric peptide EN-9 that the present invention is based on interior morphine peptide 2 and neuropeptide FF shows the central analgesia stronger, more lasting than interior morphine peptide 2, and have without advantages such as tolerance effect, low habituation.In addition, subcutaneous EN-9 can cause significant analgesic activity, can overcome interior morphine peptide in the defective of peripheral injection without analgesic activities, has the potential clinical value for the treatment of pain.
Description of drawings
Fig. 1 mouse intracerebroventricular injection EN-9 produces the Time-activity-curve of dose-dependently analgesic activity.
The comparison of Fig. 2 mouse intracerebroventricular injection EN-9 and analgesic activity that EM-2 produces.
The variation of continuous six days injections EN-9 and the caused analgesic activities of EM-2 in Fig. 3 mouse tricorn.
The mouse condition position that Fig. 4 mouse intracerebroventricular injection EN-9 and EM-2 induce is detested.
The comparison of Fig. 5 mouse subcutaneous injection EN-9 and analgesic activity that EM-2 produces.
Embodiment
Be described further below by the synthetic method of specific embodiment to chimeric peptide of the present invention.
The I material
Instrument: high performance liquid chromatograph (HPLC) is the Delta 600 of Waters company; Analytical column: DELTA PAK 5 μ C18 300 3.9 * 150mm; Preparative column: DELTA PAK 15 μ C18 300 7.8 * 300mm.Mass spectrograph is PE Biosystems, Mariner System 5074.The manual solid-phase Peptide synthesizer, by making (the 14th page of Fig. 4 in the principle of design of synthesizer " the Fmoc solid phase peptide synthesis " that specifically compile with reference to Chen WC and White PD by the glazier after this lab design, and make on its basis part and improve, the mechanical stirring mode is substituted drum nitrogen method, thereby reach the well-mixed purpose of reaction soln).Reagent: resin is Rink-Amide-MBHA-Resin (1% DVB, 200 ~ 400mesh, substitution value S=0.40 mmol/g resin), available from Tianjin Nankai with become company.
NThe amino acid (Fmoc-Aa) of-α-Fmoc protection,
N-hydroxy benzo triazole (HOBt), O-benzotriazole-
N, N, N', N'-tetramethyl-urea-hexafluorophosphate (HBTU), diisopropylethylamine (DIEA) and tri isopropyl silane (TIS) are available from the biochemical (Shanghai) Co., Ltd. of gill.Triketohydrindene hydrate is Shanghai reagent three factory's products.Methylene dichloride (DCM),
N, N-dimethyl formamide (DMF), hexahydropyridine (piperidines), methyl alcohol (MeOH) and pyridine are all available from Tianjin the second chemical reagent work, and trifluoroacetic acid (TFA), phenol and pyridine are Tianjin reagent one factory's product; Before using, all processes through overweight steaming above organic reagent.
Thick peptide is synthetic
Adopt the solid-phase polypeptide synthesis method of Fmoc protection strategy.Peptide elongation adopts the peptide method (step by step) that connects one by one.Concrete operation step is as follows:
(1) resin pre-treatment: 900 mg Rink-Amide-MBHA resins are added in the synthesizer, stir 30 min after adding the DCM of 12 ml, make decompressing and extracting solvent after the abundant swelling of resin.
(2) remove the Fmoc radical protection: in swelling, drain in the resin of solvent, add the hexahydropyridine DMF solution of the volumetric concentration 20% of 12ml, drain after stirring 5min, repeat 2 times.The hexahydropyridine DMF solution that adds again the volumetric concentration 20% of 12ml is drained behind the stirring 15min.The DMF that adds at last 12ml drains behind the stirring 3min, repeats 4 times, gets the peptide resin sample; Carry out the indenes inspection according to step (3) operation, it is complete that solution, resin are blue expression deprotection.
(3) indenes inspection: building-up process checks to determine with ninhydrin reagent whether the condensation in each step and deprotection be complete.Be used for the indenes inspection three kinds of reagent join method: reagent
80g phenol/20ml ethanol; Reagent
2.0ml 0.001M potassium cyanide (water)/98ml pyridine; Reagent
5g triketohydrindene hydrate/100ml ethanol.Add in the following order: peptide resin sample+0.1ml reagent
+ 0.2ml reagent
+ 0.1ml reagent
, observe behind the boiling water bath 3-10min.
(4) condensation: in small beaker, successively 2.5eq Fmoc-Aa, HOBt, HBTU are dissolved fully with DMF; mix fully after adding again 5eq DIEA; the mixing solutions of 12ml joined be equipped with in the synthesizer that removes the Fmoc resin, stirring reaction 60min drains solvent under argon shield.The DMF that adds 12ml drains behind the stirring 3min, repeats 3 times, gets the peptide resin sample; Carry out indenes inspection according to (3), indenes inspection solution is that yellowish, resin is that colourless expression condensation is complete.
(5) prolongation of peptide chain: repeating step (2), (4), according to the chimeric peptide structure by the order of C-end to the N-end, successively will with
N-α-Fmoc blocking group amino acid is condensed on the resin one by one, after per step, reaction was finished, filters reagent in the removal reactor through suction function.Until finish the condensation of all amino-acid residues.
After the peptide chain condensation is all finished, according to operation in the above-mentioned steps (2) last amino acid whose Fmoc group of peptide resin is removed fully.Then according to DCM 3 min * 2 times, MeOH 3 min * 1 time; DCM 3 min * 1 time, resin is alternately washed in the operation of MeOH 3 min * 2 times.Remove splash bar, with synthesizer sealing (plug), thoroughly drain (at least 2 hours).The peptide chain resin of drying is placed reactor, and the cutting agent of adding 18ml (volume ratio is TFA:TIS: water=95:2.5:2.5), cleavage reaction 2.5 hours (stirred once, stirred 1 minute at every turn in per 15 minutes) under room temperature.Filter, decompression is spin-dried for filtrate not being higher than under 37 ℃ the condition fully, then separates out precipitation with the ether that is not higher than-10 ℃, and vibration makes thick peptide fully separate out with the form of white precipitate.With the sucking-off of supernatant ether, after adding water and fully dissolving, utilize separating funnel that ether is separated from aqueous phase and remove after leaving standstill, merge water and get white thick peptide pressed powder 383.7 mg by lyophilize, productive rate is 86.8 %.
Desalination and the purifying of thick peptide.
Take by weighing 90mg left and right sides crude product, be dissolved in 20% acetic acid solution, solution is crossed Sephadex G25 cross-linked dextran gel column, and (2.0 * 25cm), moving phase is volumetric concentration 20% acetic acid solution.Utilize Ultraviolet Detector to collect the main peak postlyophilization, get white powder 323.1 mg after the desalting treatment.Use again reverse high performance liquid chromatography (HPLC) C
18Post (XBridge TM BEH 130 C
18, 19 mm * 250mm) separation and purification is carried out in the peptide compounds of gained desalination collect the sample main peak after separating, and the lyophilize gets white pure peptide pressed powder 209.8 mg.EN-9 sample purity behind the purifying is more than 98%.Mass spectrum and stratographic analysis detected result are as shown in table 2.
The mass spectrum of table 1 EN-9 and stratographic analysis detected result
Annotate:
Method 1: gradient elution: 10 –, 100% acetonitrile/water (0.05% TFA) (finishing in 30 minutes), flow velocity is: 1 mL/min, detecting wavelength is 220 nm, analyzes chromatographic column to be: Delta Pak C
18, 5 μ m, 150 * 3.9 mm;
Method 2: gradient elution: 10 –, 80% acetonitrile/water (0.05% TFA) (finishing in 30 minutes), flow velocity is: 1 mL/min, detecting wavelength is 220 nm, analyzes chromatographic column to be: Delta Pak C
18, 5 μ m, 150 * 3.9 mm.
From mass spectrum interpretation of result explanation, the peptide compounds EN-9 that is synthesized is consistent with the compound structure of design.The chromatogram detected result shows that the peptide compounds EN-9 that is synthesized has different retention time from interior morphine peptide 2 and neuropeptide FF, and is consistent with expected result.
Claims (4)
1. based on the chimeric peptide of interior morphine peptide 2 and neuropeptide FF, its structural formula:
。
2. as claimed in claim 1 based on the preparation method of the chimeric peptide of interior morphine peptide 2 and neuropeptide FF, comprise following processing step:
(1) resin pre-treatment: the Rink-Amide-MBHA resin is stirred 30-40 min in methylene dichloride, make decompressing and extracting solvent after the abundant swelling of resin;
(2) remove the Fmoc radical protection: make swelling, drain resin behind the solvent in the hexahydropyridine DMF of volumetric concentration 20-22% solution, drain after stirring 3-5min, repeat 2-3 time; Join again the hexahydropyridine DMF solution of volumetric concentration 20-22%, stir 10-15min, the Fmoc group is removed fully, then drain solvent; At last with DMF washing Ex-all hexahydropyridine;
(3) condensation: successively with 2-4eq's
N-α-Fmoc blocking group amino acid,
N-hydroxy benzo triazole, O-benzotriazole-
N, N, N', N'-tetramethyl-urea-hexafluorophosphate is dissolved among the DMF fully, adds mixing mixing solutions behind the diisopropylethylamine of 4-8eq again; Then under argon shield, make the resin stirring reaction 50-60min in above-mentioned mixing solutions that removes the Fmoc radical protection, drain solvent; Remove unreacted with the DMF repeated washing
N-α-Fmoc blocking group amino acid,
N-hydroxy benzo triazole, O-benzotriazole-
N, N, N', N'-tetramethyl-urea-hexafluorophosphate;
(4) prolongation of peptide chain: repeating step (2), (3), successively will with
NThe amino acid of-α-Fmoc blocking group is condensed on the resin one by one, until finish the condensation of all amino-acid residues, obtains peptide resin;
(5) cutting of peptide chain from the resin: after the peptide chain condensation is all finished, according to the method for step (2) last amino acid whose Fmoc group of peptide resin is removed fully; Replace washing resin with DCM, MeOH, fully drain solvent after, add the cutting agent of 15-20ml by every gram peptide resin, under room temperature cleavage reaction 2-3 hour; Filter, decompression is spin-dried for filtrate not being higher than under 37 ℃ the condition fully, then separates out precipitation with the ether that is not higher than-10 ℃; Leave standstill the rear ether of removing first supernatant, remove the ether phase with separating funnel after water fully dissolves again, merge water, lyophilize gets white thick peptide pressed powder;
(6) desalination and the purifying of thick peptide: take the acetic acid solution of volumetric concentration 15-20% as moving phase, thick peptide is crossed the desalination of Sephadex G25 cross-linked dextran gel column, utilize Ultraviolet Detector to collect the main peak postlyophilization, get the peptide compounds of desalination; Recycle reverse performance liquid chromatographic column and carry out separation and purification, collect main peak, obtain the pure peptide pressed powder of white after the lyophilize.
3. as claimed in claim 2 based on the preparation method of the chimeric peptide of interior morphine peptide 2 and neuropeptide FF, it is characterized in that: the described cutting agent of step (5) is to mix the solvent that forms by trifluoroacetic acid, tri isopropyl silane, water with the volume ratio of 95:2.5:2.5.
4. as claimed in claim 1 based on the application at the preparation anodyne of the chimeric peptide of interior morphine peptide 2 and neuropeptide FF.
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| CN104710508B (en) * | 2015-03-20 | 2017-11-28 | 兰州大学 | Peptide and its synthetic method and application are hybridized based on interior morphine peptide 2 and NPFF receptor antagonists RF9 branch type |
| CN110194785B (en) * | 2016-04-21 | 2023-02-24 | 上海天慈生命科学发展有限公司 | Multi-target peptide molecule of opioid and neuropeptide FF receptor and preparation and application thereof |
| CN109021117B (en) * | 2018-08-20 | 2020-05-08 | 兰州大学 | Opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue, and preparation method and application thereof |
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