CN102675417A - Monocyte migration inhibition factor analogue, synthesis thereof and application thereof - Google Patents
Monocyte migration inhibition factor analogue, synthesis thereof and application thereof Download PDFInfo
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Abstract
The invention discloses a novel monocyte migration inhibition factor active fragment analogue and belongs to the field of biological medicines. Cationic amino acid Arg is introduced in an N secion of a bioactive peptide section Cys-Asn-Ser of a monocyte migration inhibition factor, or an obtained peptide sequence Arg-Cys-Asn-Ser or D-Cys-Asn-Ser is obtained after D-Cys substitutes for Cys, and penetrating capability of cell membranes of the analogue and enzymolysis stability of small molecule peptides in bodies are both improved. A model of the focal cerebral ischemia-reperfusion injury in rats of a suture-occluded method shows that the monocyte migration inhibition factor (MLIF) analogue can improve brain infarct volume caused by rat cerebral ischemia, improves neurological functions of rats and has a good improvement effect on cerebral ischemia-reperfusion injury. Therefore, the novel monocyte migration inhibition factor analogue has good application prospects in clinic drug preparation.
Description
Technical field
The invention belongs to the biological medicine technology field, relate to a series of new monocyte migration inhibition factor analogues and compound method thereof; The present invention also relates to the application of this monocyte migration inhibition factor analogue simultaneously---the application in preparation control ischemic cerebrovascular medicine.
Background technology
Monocyte migration inhibition factor (MLIF), a kind of polypeptide of anti-inflammatory response is the pentapeptide of from the entamoeba histolytica of sterile culture, finding, its structure is Met-Gln-Cys-Asn-Ser.Vitro culture and in vivo tests prove that all this small molecules Toplink suppresses moving of person monocytic cell, polymorphonuclear leukocyte.Entamoeba histolytica influences the secretion of cytokine through producing this anti-inflammatory peptides, and host immune is escaped in the generation of inflammation-inhibiting reaction.Bibliographical information; This pentapeptide molecule not only can directly act on inflammatory cell; Also can influence the process of inflammatory reaction through the secretion of disturbing inflammatory cytokine; Secretion and corresponding receptor expression to inflammatory cytokine such as TNF-α, IL-1 β have the obvious suppression effect, can also suppress the expression of inflammation chemokines such as MIP-1 α, IP-1 β simultaneously.
Morales-Mart ' nez etc. are in order to study the active site of this small-molecular peptides; Met-Gln-Cys-Asn-Ser being cut apart, be divided into three small-molecular peptides, is respectively Met-Gln-Cys, Gln-Cys-Asn and Cys-Asn-Ser; Or the amino acid in the peptide chain replaced; Study its anti-inflammatory activity respectively, discover the anti-inflammatory activity group that its terminal tripeptides Cys-Asn-Ser is this small-molecular peptides, its anti-inflammatory activity and Met-Gln-Cys-Asn-Ser are suitable.
Yang Peng far waits the people to find that MLIF and reactive group Cys-Asn-Ser thereof can significantly reduce the cerebral infarction volume that the mouse brain ischemia-reperfusion causes, and can obviously improve the neurobehavioral behind the mouse ischemic.The cerebral protection of this polypeptide small molecule is stronger, and because molecular weight is little, is easy to see through hemato encephalic barrier, has significant brain permeability advantage.
Ischemic brain injury is the pathophysiological process of a complicacy; Thought once that ischemic brain injury was owing to have no progeny in the cerebral blood flow; The brain cell energy is under-supply and then trigger apoptosis amount, discovers afterwards that cerebral blood flow interrupted making brain cell produce the damage cascade reaction with pouring into again.The lipid peroxidation that inflammatory reaction that wherein causes behind the cerebral ischemia re-pouring and radical cause plays a part can not be ignored in the pathogenic process of ischemic brain injury.Behind the ischemia-reperfusion, the acute phase inflammatory reaction can increase the weight of the cerebral tissue oedema, mediates insecondary brain tissue impairment.In inflammatory reaction; The expression of the local inflammation factor, chemokine and adhesion molecule is a cerebral ischemic injury to the basis that inflammatory injury changes after the cerebral ischemia, and the expression that therefore suppresses infiltration and inflammatory cytokine, the adhesion molecule etc. of inflammatory cell is expected to become the effective measure of control cerebral ischemia diseases.In addition, the generation of radical increases during ischemia-reperfusion, and peroxidatic reaction of lipid strengthens, and the free radical scavenging function reduction sustains damage neurocyte.The interior enzymolysis stability of the body of polypeptide drug also is one simultaneously influences one of active important factor of medicine itself all the time, therefore, also is one of direction of peptide medicament transformation in recent years from increasing medicine resistance to enzymolysis ability.
Summary of the invention
One of the object of the invention provides the monocyte migration inhibition factor analogue of one type of brand new;
Two of the object of the invention provides the application of above-mentioned monocyte migration inhibition factor analogue in preparation clinical treatment medicine;
Three of the object of the invention provides a kind of compound method of above-mentioned monocyte migration inhibition factor analogue.
(1) MLIF analogue
Monocyte migration inhibition factor analogue of the present invention (MLIF analogue) is to introduce amino acid Arg or replace peptide sequence Arg-Cys-Asn-Ser or the D-Cys-Asn-Ser that obtains behind the Cys with D-Cys at the N of the active peptide segment Cys-Asn-Ser of monocyte migration inhibition factor end, with penetrativity and the raising small-molecular peptides enzymolysis stability in vivo that increases its cytolemma.
Micromolecular active peptide segment Cys-Asn-Ser of MLIF and MLIF analogue D-Cys-Asn-Ser, Arg-Cys-Asn-Ser, structure following:
Active peptide segment Cys-Asn-Ser
Arg-Cys-Asn-Ser
D-Cys-Asn-Ser
(2) the MLIF analogue is synthetic
The compound method of monocyte migration inhibition factor analogue of the present invention comprises following process step:
(1) activation of resin and pre-treatment
The wang resin is dipped in the methylene dichloride, stirs and make the abundant swelling of resin, drain solvent;
(2) amino acid whose coupling
The coupling of a, Ser: will pass through pretreated resin and wash with DMF, drying joins the mixing solutions of hexahydropyridine and DMF, stirs it is fully reacted; After reaction is accomplished,, obtain removing the resin of Fmoc protection with the DMF washing; The Ser of Fmoc protection is dissolved among the DMF, and stirring is fully dissolved it, adds excessive HOBT and HBTU; Add DIEA after the stirring and dissolving again, then with the mixed with resin that removes the Fmoc protection, stirring reaction 1 ~ 2h under argon shield, the room temperature; Drain, the DMF washing obtains Fmoc-Ser-Wang;
The coupling of b, Asn: Fmoc-Ser-Wang is added the mixing solutions of hexahydropyridine and DMF, stir it is fully reacted; After reaction is accomplished,, obtain Ser-Wang with the DMF washing; The Asn of Fmoc protection is dissolved among the DMF, and stirring is fully dissolved it, adds HOBT and HBTU; Add DIEA after the stirring and dissolving again, mix with Ser-Wang then, stirring reaction 1 ~ 2h under argon shield, the room temperature; Drain, wash, obtain Fmoc-Asn-Ser-Wang with DMF;
The coupling of c, Cys or D-Cys: Fmoc-Asn-Ser-Wang is joined the mixing solutions of hexahydropyridine and DMF, stir it is fully reacted; After reaction is accomplished,, obtain Asn-Ser-Wang with the DMF washing; The Cys or the D-Cys of Fmoc protection are dissolved among the DMF, and stirring is fully dissolved it, adds HOBT and HBTU; Add DIEA after the stirring and dissolving again; Mix with Asn-Ser-Wang then, stirring reaction 1 ~ 2h drains under argon shield, the room temperature; The DMF washing obtains Fmoc-Cys-Asn-Ser-Wang or Fmoc-D-Cys-Asn-Ser-Wang;
The coupling of d, Arg: Fmoc-Cys-Asn-Ser-Wang is joined hexahydropyridine and DMF mixing solutions, and stirring is fully reacted it; After reaction is accomplished,, obtain Cys-Asn-Ser-Wang with the DMF washing; The Arg of Fmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds HOBT and HBTU, adds DIEA after the stirring and dissolving again; Mix with Cys-Asn-Ser-Wang then; Stirring reaction 1 ~ 2h under argon shield, the room temperature drains, washs with DMF, obtains Fmoc-Arg-Cys-Asn-Ser-Wang;
The add-on of said HOBT is 1 ~ 10 times (preferred 2 ~ 4 times) of corresponding weight resin; The add-on of HBTU is 1 ~ 10 times (preferred 2 ~ 4 times) of corresponding weight resin; The add-on of DIEA is 1 ~ 10 times (preferred 2 ~ 4 times) of corresponding weight resin;
E, take off Fmoc protection: Fmoc-Arg-Cys-Asn-Ser-Wang or Fmoc-D-Cys-Asn-Ser-Wang are added in the mixing solutions of hexahydropyridine and DMF, stirring is fully reacted it; After reaction is accomplished,, obtain Arg-Cys-Asn-Ser-Wang or D-Cys-Asn-Ser-Wang with the DMF washing;
In above-mentioned each step, in said hexahydropyridine and the DMF mixing solutions, the volume ratio of hexahydropyridine and DMF is 1:20 ~ 1:0.5 (preferred 1:1 ~ 1:10).
Stirring reaction in said each step is: stirred 3 ~ 5 minutes, and drained; Repeat to stir 2 ~ 3 times.DMF washing in said each step is: washing 2 ~ 3min, drain; Repeat 3 ~ 4 times.
(3) polypeptide cutting
Arg-Cys-Asn-Ser-Wang or D-Cys-Asn-Ser-Wang are alternately washed with DCM and MeOH respectively, drain the back and add in the solid phase synthetic instrument room temperature reaction 2.5 ~ 3.5h with cutting reagent; After reaction finishes; Reaction solution filters through core, and the TFA washing concentrates in underpressure distillation below 40 ℃; Add freezing ether sedimentation polypeptide in the liquid concentrator; Then with polypeptide with the dissolving of the glacial acetic acid aqueous solution of volume(tric)fraction 0 ~ 50%, in-20 ~-80 ℃ solidify, freeze-drying, thick peptide Arg-Cys-Asn-Ser or D-Cys-Asn-Ser;
Said cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water; Wherein the percent by volume of each component in system is respectively: trifluoroacetic acid: 65 ~ 96.5%, and phenol: 1 ~ 10%, thioanisole: 1 ~ 10%, 1: 0.5 ~ 2.5%, water: 1 ~ 10%; The consumption of said cutting reagent is 5 ~ 20mL/g resin.
(4) purifying of polypeptide
A, thick peptide desalination: thick peptide Arg-Cys-Asn-Ser or D-Cys-Asn-Ser are dissolved in the glacial acetic acid aqueous solution of volume(tric)fraction 0.1 ~ 50% fully, add in the gel column wash-out; Monitor with UV-detector; Collection has the component of absorption, and the dilution postlyophilization obtains the desalination peptide;
B, performance liquid purifying: the desalination peptide is fully dissolved with deionized water, filtering with microporous membrane, high performance liquid chromatograph on the filtrating, each sample introduction 3 ~ 4 mL collect the effluent of main absorption peak, and freeze-drying obtains target peptide;
Performance liquid purification condition: applied sample amount: 40 ~ 80 mg/ time; Mobile phase A: 0.1%TFA/ water; Mobile phase B: 0.1%TFA/ acetonitrile; Linear gradient is 0～30 min, Mobile phase B 5%～30%; Chromatographic column: semipreparative column Waters Delta-PakC18 column (19 * 250 mm); Detect wavelength: 220 nm; Flow velocity: 8 mL/min.
Identify that through mass spectrum target peptide---monocyte migration inhibition factor analogue synthesizes successfully.
Synthetic route is following:
(three
) the MLIF analogue is to the focal brain ischemia-reperfusion injury in rats protection test
1, line bolt method is set up the focal brain ischemia-reperfusion injury in rats model
(1) preparation of line bolt and pre-treatment: (its line body diameter is 0.26 mm to adopt commercially available line bolt; Ball section diameter is 0.36 ± 0.02 mm); With the line bolt with 75% alcohol disinfecting, with the blueness of waterproof or black marking pen in 18,20,22 mm places marked respectively, as the reference of depth of penetration apart from the pommel; After drying about 2 cm of line bolt leading portion are immersed in the left-handed poly-lysine; Take out behind about 1 min, place 60 ℃ of baking ovens to dry then 1 ~ 2 hour, place the SPSS of 1% heparin sodium subsequent use on the line bolt before the experiment beginning.
(2) model preparation: improve preparation rats with left middle cerebral artery occlusion re-perfusion model slightly according to the method for Longa.The fasting in preceding 12 hours of male SD rat art is freely drunk water.Adopt 10% Chloral Hydrate (350 mg/kg) abdominal injection to anaesthetize before the art; Lie on the back after the anesthesia and be fixed on the operating table; Behind Iodophor and the alcohol disinfecting, row is about the otch of 3 cm, successively chorista in the neck center; It is soft that this process action is wanted, avoid excessive tractive neural, stimulate tracheae and cause that rats breathing road secretory product increases.Separate left carotid (common carotid artery, CCA), external carotid artery (external carotid artery, ECA), internal carotid artery (internal carotid artery; ICA), permanent ligation CCA proximal part, ECA root, the temporary transient folder of arteriole folder closes the distal end of ICA; Cut a kerf in ECA and ICA bifurcated below rapidly, insert nylon wire through this otch, depth of penetration is about 20 mm; Till slight resistance sense is arranged; This moment, the line bolt was gone into cranium to arteria cerebri anterior (anterior cerebral artery, initial part ACA), thereby inaccessible arteria cerebri media (middle cerebral artery through the CCA crotch through ICA; MCA) initial part has caused the infarct of MCA blood supply relevant range.Ligation CCA distal end with fixing nylon wire with prevent hemorrhage, layer-by-layer suture skin, the nylon wire ends exposed is external.Extract about 1 cm of institute's the end of a thread that stays behind 90 min gently out, keep the blood supply that damages side internal carotid artery, arteria cerebri media by opposing carotid system, basal arteries jointly through Wiilis ring and external carotid artery, realize that arteria cerebri media pours into again, modeling is accomplished.Sham operated rats ligation CCA and ECA.At anestheticing period, keep the body temperature of rat with electric blanket and the heating of 60 W incandescent light.Postoperative also needs intraperitoneal injection of saline to replenish body fluid.Sham operated rats except that plug wire not, the same experimental group of all the other steps.Postoperative is put into the single cage of mouse cage and is raised, and it is that master's neurological deficit symptom is the successful sign of model that the clear-headed back of rat occurs with the left fore hemiplegia.
2, animal divides into groups and administration
Male SD rat is divided into six groups at random, 3 of sham operated rats, other organize 8 every group; Administration: I group: sham operated rats (injection equal-volume saline water); II group: model group (injection equal-volume saline water); III group: nimodipine group (cerebral ischemia re-pouring is at once with the dosage tail vein injection administration of 0.4 mg/kg); IV group: Cys-Asn-Ser group (preceding 30 min of cerebral ischemia, perfusion is poured into 12 h at once and again and distinguished the tail vein injection administration with the dosage of 1 mg/kg again); V group: D-Cys-Asn-Ser (medication is with the IV group); VI group: Arg-Cys-Asn-Ser (medication is with the IV group).
3, neural function scoring
The neural function scoring is evaluated when cerebral ischemic reperfusion in rats 24 h with reference to 5 grades of point systems of Zea Longa, 0 minute and the person's of remaining unconscious rejecting.
Standards of grading:
0 minute, impassivity functional impairment symptom;
1 minute, slight neurologic impairment can not full extension left side fore paw;
2 minutes, the focal neurologic impairment of moderate was turn-taked to the left;
3 minutes, the focal neurologic impairment of severe was toppled over to the left;
4 minutes, can not spontaneously walk, level of consciousness descends.Scoring is divided into the modeling success at 1-3, and dead and non-compliant animal is rejected.
Experimental result: as shown in Figure 1.Nimodipine group, Cys-Asn-Ser group, D-Cys-Asn-Ser group, Arg-Cys-Asn-Ser group all can be improved rat nerves afunction symptom to a certain extent, and wherein two novel analogs among the present invention have all shown nimodipine group, the more significantly improvement effect of comparing of Cys-Asn-Ser group.
4, cerebral infarction stereometry
After the cerebral ischemic reperfusion in rats 24 h scoring, with excessive 10% chloral hydrate anesthesia rat, broken end is got brain; Remove olfactory bulb, cerebellum and low brain stem, the method for reference is cut to 6, every thick about 2 mm with remainder is crown on ice pan; Rapidly the brain sheet is put in the 2%TTC solution then; Lucifuge is hatched 20 min for 37 ℃, whenever stirs once at a distance from 5 min therebetween.TTC is a kind of water-soluble salt, it can with dehydrogenase reaction in the viable cell plastosome, generate the scarlet lipid-soluble substance, non-viable non-apoptotic cell is because mitochondrial dehydrogenase inactivation and not developing the color.After TTC dyeing, normal cerebral tissue is rose, and blocking tissue is white in color.Hatch the brain sheet to be put to keep in Dark Place in 10% Superlysoform after finishing and spend the night; Digital camera is taken record; Again with stained brain picture input computingmachine; Utilize image processing software Image-Pro Plus 6.0, the area that calculates each brain sheet damage zone of each sample respectively is designated as A, carries out the calculating of Infarction volume with following formula:
V=[2×(A1+A2+A3+A4+A5+A6)]?mm
3
Cerebral ischemia re-pouring injured protection experimental result such as Fig. 2, shown in Figure 3, two novel MLIF analogue D-Cys-Asn-Ser, Arg-Cys-Asn-Ser have all shown the better improvement effect of comparing of female peptide Cys-Asn-Ser.
Above-mentioned experimental result shows that medication therapy groups can be improved the pathological symptom of MCAO rat in various degree: reduce the neural function scoring; Reduce the volume of cerebral infarction; The brain nervous cell form is made moderate progress, and oedema alleviates.Visible by experimental result, Cys-Asn-Ser compares with small molecule active peptide section, and the activity of MLIF analogue D-Cys-Asn-Ser, Arg-Cys-Asn-Ser all increases.For the treatment ischemic cerebrovascular better curative effect is arranged, therefore, have important effect and application prospect aspect the preparation control ischemic cerebrovascular medicine.
Description of drawings
Fig. 1 is MLIF analogue of the present invention and control group neural function appraisal result figure;
Fig. 2 is a MCAO rat brain slice TTC dyeing photo comparison diagram;
Fig. 3 respectively organizes MCAO rat cerebral infarction volume figure as a result;
Fig. 4 is the mass spectrum of Cys-Asn-Ser;
Fig. 5 is the mass spectrum of Arg-Cys-Asn-Ser;
Fig. 6 is the mass spectrum of D-Cys-Asn-Ser.
Embodiment
Below through specific embodiment to MLIF analogue of the present invention and synthetic being described further thereof.
(1) activation of resin and pre-treatment
Accurately take by weighing 1.0g wang resin, add in the polypeptide solid phase synthetic instrument, add DCM, be advisable, stir 30 min, make the abundant swelling of resin, remove whisking appliance then, drain DCM (30 ~ 40 min) to complete submergence resin along tube wall.
(2) amino acid whose coupling
The coupling of a, Ser: will pass through pretreated resin with DMF washing three times, each 3 min (3 min * 3 time) drain the solvent in the reactor drum with vacuum pump; Hexahydropyridine/DMF the solution that adds volume(tric)fraction 20%; Stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success; Obtain removing the resin of Fmoc protection; The Ser of 563.6mgFmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds 198.7mgHOBT and 557.4mg HBTU, adds 485.9 μ l DIEA after the stirring and dissolving again; Then with the above-mentioned mixed with resin that removes the Fmoc protection; Stirring reaction 1h drains under argon shield, the room temperature, and DMF washs (3 min * 3); With the ninhydrin calibrating, if resin is blue, explain that coupling is incomplete, repeat above step; If resin is colourless, Ser is coupled successfully, obtains Fmoc-Ser-Wang;
The coupling of b, Asn: Fmoc-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains Ser-Wang; The Asn of 877.0mgFmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds 198.7mg HOBT and 557.4mg HBTU, adds 485.9 μ l DIEA after the stirring and dissolving again; Mix with Ser-Wang then; Stirring reaction 1h drains under argon shield, the room temperature, and DMF washs (3 min * 3); With the ninhydrin calibrating, if resin is blue, explain that coupling is incomplete, repeat above step; If resin is colourless, Asn is coupled successfully, obtains Fmoc-Asn-Ser-Wang;
The coupling of c, Cys: Fmoc-Asn-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains Asn-Ser-Wang; The Cys of 861.0mgFmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds 198.7mg HOBT and 557.4mg HBTU, adds 485.9 μ l DIEA after the stirring and dissolving again; Mix with Asn-Ser-Wang then; Stirring reaction 1 ~ 1.2h under argon shield, the room temperature drains, washs with DMF, obtains Fmoc-Cys-Asn-Ser-Wang;
The coupling of d, Arg: Fmoc-Cys-Asn-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains Cys-Asn-Ser-Wang; The Arg of 953.7mgFmoc protection is dissolved among the DMF; Stirring is fully dissolved it; Add 198.7mg HOBT and 557.4mg HBTU, add 485.9 μ l DIEA after the stirring and dissolving again, mix with Cys-Asn-Ser-Wang then; Stirring reaction 1h under argon shield, the room temperature drains, DMF washs (3 min * 3); With the ninhydrin calibrating, if resin is blue, explain that coupling is incomplete, repeat above step; If resin is colourless, Arg is coupled successfully, with the DMF washing, obtains Fmoc-Arg-Cys-Asn-Ser-Wang;
E, take off Fmoc protection: Fmoc-Arg-Cys-Asn-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains Arg-Cys-Asn-Ser-Wang.
In above-mentioned each step, the volume ratio of hexahydropyridine and DMF is 1:5.
(3) polypeptide cutting
Arg-Cys-Asn-Ser-Wang is replaced washing resin three times with DCM and MeOH, and each 3 min thoroughly drain.The cutting reagent adding for preparing is equipped with in the solid phase synthetic instrument of peptide resin, and per 20 min stir 1 min, coreaction 3 h.After reaction finished, reaction solution filtered through core, with an amount of TFA washing.35 ℃ of underpressure distillation to volumes reaction solution and filtrating all are collected in the flask, till can not reduce again.Add an amount of freezing ether sedimentation polypeptide in the liquid concentrator, treat abandoning supernatant behind sedimentation 2 ~ 3 h, add freezing ether and wash once more, sedimentation is spent the night.The next day abandoning supernatant, collecting precipitation.Deposition divides to install in the small beaker with the dissolving of the glacial acetic acid water mixed solution of volume(tric)fraction 20%, and it is freezing to put cryogenic refrigerator, solidifies that freeze-drying gets thick peptide in the rearmounted Freeze Drying Equipment.
Said cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 82.5:5:5:2.5:5; The consumption 10mL/g resin of cutting reagent.
(4) purifying of polypeptide
A, thick peptide desalination: with the glacial acetic acid water mixed solution balance Sephadex G-10 gel column of volume(tric)fraction 20%, the gel column lower end is connected with UV-detector, appearance in the gradation.Take by weighing about about 40 mg of thick peptide, concrete amount is decided according to the solubleness of polypeptide, is dissolved in 2 ml, 20% glacial acetic acid-water mixed solution, adds gel column at every turn, and wash-out with the UV-detector monitoring, is collected the component that absorption is arranged.Refined liquid dilution postlyophilization with collecting gained obtains the desalination peptide.
B, performance liquid purifying:
RP-HPLC purification condition: applied sample amount: 40 mg/ time; Mobile phase A: 0.1%TFA/ water; Mobile phase B: 0.1%TFA/ acetonitrile; Linear gradient is 0～30 min, Mobile phase B 5%～30%; Chromatographic column: semipreparative column Waters Delta-PakC18 column (19 * 250 mm); Detect wavelength: 220 nm; Flow velocity: 8 mL/min.
Purge process: take by weighing desalination peptide 40 mg, add deionized water dissolving,, can add the acetonitrile hydrotropy if solubleness is bad; With 0.45 μ m filtering with microporous membrane, preparative high performance liquid chromatography appearance on the filtrating, each sample introduction 3 ~ 4 mL collect the effluent of main absorption peak; Packing, lyophilized powder is collected in freeze-drying; Identify the medicine peak with mass spectrum, collect target peptide, 105.5mg weighs.Productive rate 45.0%.
The mass spectrum of product is seen Fig. 5.
(1) activation of resin and pre-treatment
With embodiment 1.
(2) amino acid whose coupling
The coupling of a, Ser: with embodiment 1.
The coupling of b, Asn: with embodiment 1.
The coupling of c, D-Cys: Fmoc-Asn-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains Asn-Ser-Wang; The 861.0mgD-Cys of Fmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds 198.7mg HOBT and 557.4mg HBTU, adds 485.9 μ l DIEA after the stirring and dissolving again; Mix with Asn-Ser-Wang then; Stirring reaction 1 ~ 1.2h under argon shield, the room temperature drains, washs with DMF, obtains Fmoc-D-Cys-Asn-Ser-Wang;
D, take off Fmoc protection: Fmoc-D-Cys-Asn-Ser-Wang is added the hexahydropyridine/DMF solution of volume(tric)fraction 20%, stir (5 min * 2,15 min * 1), drain.After reaction is accomplished, with DMF washing 3 ~ 4 times, each 3 min.Whether ninhydrin calibrating Fmoc protection base is sloughed: if colourless, Fmoc protection base does not remove, and repeats above-mentioned steps; If blue, Fmoc protection base removes success, obtains D-Cys-Asn-Ser-Wang;
In above-mentioned each step, the volume ratio of hexahydropyridine and DMF is 1:5.
(3) polypeptide cutting
D-Cys-Asn-Ser-Wang is replaced washing resin three times with DCM and MeOH, and each 3 min thoroughly drain.The cutting reagent adding for preparing is equipped with in the solid phase synthetic instrument of peptide resin, and per 20 min stir 1 min, coreaction 3 h.After reaction finished, reaction solution filtered through core, with an amount of TFA washing.35 ℃ of underpressure distillation to volumes reaction solution and filtrating all are collected in the flask, till can not reduce again.Add an amount of freezing ether sedimentation polypeptide in the liquid concentrator, treat abandoning supernatant behind sedimentation 2 ~ 3 h, add freezing ether and wash once more, sedimentation is spent the night.The next day abandoning supernatant, collecting precipitation.Deposition divides to install in the small beaker with the dissolving of the glacial acetic acid water mixed solution of volume(tric)fraction 20%, and it is freezing to put cryogenic refrigerator, solidifies that freeze-drying gets thick peptide in the rearmounted Freeze Drying Equipment.
Said cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 82.5:5:5:2.5:5; The consumption of cutting reagent is the 10mL/g resin.
(4) purifying of polypeptide
A, thick peptide desalination: with the glacial acetic acid water mixed solution balance Sephadex G-10 gel column of volume(tric)fraction 20%, the gel column lower end is connected with UV-detector, appearance in the gradation.Take by weighing about about 40 mg of thick peptide, concrete amount is decided according to the solubleness of polypeptide, is dissolved in 2 ml, 20% glacial acetic acid-water mixed solution, adds gel column at every turn, and wash-out with the UV-detector monitoring, is collected the component that absorption is arranged.Refined liquid dilution postlyophilization with collecting gained obtains the desalination peptide.
B, performance liquid purifying:
RP-HPLC purification condition: applied sample amount: 40 mg/ time; Mobile phase A: 0.1%TFA/ water; Mobile phase B: 0.1%TFA/ acetonitrile; Linear gradient is 0～30 min, Mobile phase B 5%～30%; Chromatographic column: semipreparative column Waters Delta-PakC18 column (19 * 250 mm); Detect wavelength: 220 nm; Flow velocity: 8 mL/min.
Purge process: take by weighing desalination peptide 40 mg, add deionized water dissolving,, can add the acetonitrile hydrotropy if solubleness is bad; With 0.45 μ m filtering with microporous membrane, preparative high performance liquid chromatography appearance on the filtrating, each sample introduction 3 ~ 4 mL collect the effluent of main absorption peak; Packing, lyophilized powder is collected in freeze-drying; Identify the medicine peak with mass spectrum, collect target peptide, 102.0mg weighs.Productive rate 64.6%
The mass spectrum of product is seen Fig. 6.
(1) activation of resin and pre-treatment: with embodiment 1.
(2) amino acid whose coupling:
The consumption of the Ser of the coupling of a, Ser: Fmoc protection is 563.6mg, and the add-on of HOBT is 33.1mg, and the add-on of HBTU is 92.9mg, and the add-on of DIEA is 40.5 μ l, and other is identical with embodiment 1.
The consumption of the Asn of the coupling of b, Asn: Fmoc protection is 877.0mg, and the add-on of HOBT is 33.1mg, and the add-on of HBTU is 92.9mg, and the add-on of DIEA is 40.5 μ l, and other are identical with embodiment 1.
The add-on of the Cys of the coupling of c, Cys: Fmoc protection is 861.0mg, and the add-on of HOBT is 33.1mg, and the add-on of HBTU is 92.9mg, and the add-on of DIEA is 40.5 μ l, and other are identical with embodiment 1.
The add-on of the Arg of the coupling of d, Arg: Fmoc protection is 953.7mg, and the add-on of HOBT is 33.1mg, and the add-on of HBTU is 92.9mg, and the add-on of DIEA is 40.5 μ l, and other are identical with embodiment 1.
E, take off Fmoc protection: with embodiment 1.
In above-mentioned each step, in hexahydropyridine/DMF solution, the volume ratio of hexahydropyridine and DMF is 1:20.
(3) polypeptide cutting
Cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 96.5:1:1:0.5:1; The consumption of cutting reagent is the 5mL/g resin, and other and embodiment 1 are together.
(4) purifying of polypeptide
A, thick peptide desalination: in the glacial acetic acid water mixed solution, the volume(tric)fraction 50% of glacial acetic acid, other is identical with embodiment 1.
B, performance liquid purifying: purification condition and purge process are with embodiment 1.The weight of the target peptide of collecting is 33.4mg, and productive rate is 28.5%.
Embodiment 4, Arg-Cys-Asn-Ser's is synthetic
(1) activation of resin and pre-treatment: with embodiment 1.
(2) amino acid whose coupling:
The consumption of the Ser of the coupling of a, Ser: Fmoc protection is 563.6mg, and the add-on of HOBT is 662.5mg, and the add-on of HBTU is 1858.1mg, and the add-on of DIEA is 809.8 μ l, and other is identical with embodiment 1.
The consumption of the Asn of the coupling of b, Asn: Fmoc protection is 877.0mg, and the add-on of HOBT is 662.5mg, and the add-on of HBTU is 1858.1mg, and the add-on of DIEA is 809.8 μ l, and other are identical with embodiment 1.
The add-on of the Cys of the coupling of c, Cys: Fmoc protection is 861.0mg, and the add-on of HOBT is 662.5mg, and the add-on of HBTU is 1858.1mg, and the add-on of DIEA is 809.8 μ l, and other are identical with embodiment 1.
The add-on of the Arg of the coupling of d, Arg: Fmoc protection is 953.7mg, and the add-on of HOBT is 662.5mg, and the add-on of HBTU is 1858.1mg, and the add-on of DIEA is 809.8 μ l, and other are identical with embodiment 1.
E, take off Fmoc protection: with embodiment 1.
In above-mentioned each step, in hexahydropyridine/DMF solution, the volume ratio of hexahydropyridine and DMF is 1:10.
(3) polypeptide cutting
Cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 65:10:10:5:10; The consumption of cutting reagent is the 15mL/g resin, and other and embodiment 1 are together.
(4) purifying of polypeptide
A, thick peptide desalination: in the glacial acetic acid water mixed solution, the volume(tric)fraction 50% of glacial acetic acid, other is identical with embodiment 1.
B, performance liquid purifying: purification condition and purge process are with embodiment 1.The weight of the target peptide of collecting is 181.2mg, and productive rate is 77.3%.
Embodiment 5, D-Cys-Asn-Ser's is synthetic
(1) activation of resin and pre-treatment: with embodiment 1.
(2) amino acid whose coupling
The coupling of a, Ser: with embodiment 1.
The coupling of b, Asn: with embodiment 1.
The add-on of the D-Cys of the coupling of c, D-Cys: Fmoc protection is 861.0mg, and the add-on of HOBT is 33.1mg, and the add-on of HBTU is 92.9mg, and the add-on of DIEA is 40.5 μ l,, other are identical with embodiment 2.
In above-mentioned each step, in hexahydropyridine/DMF solution, the volume ratio of hexahydropyridine and DMF is 1:1.
(3) polypeptide cutting
Cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 96.5:1:1:0.5:1; The consumption of cutting reagent is the 5mL/g resin, and other and embodiment 2 are together.
(4) purifying of polypeptide
A, thick peptide desalination: in the glacial acetic acid water mixed solution, the volume(tric)fraction 50% of glacial acetic acid, other is identical with embodiment 2.
B, performance liquid purifying: purification condition and purge process are with embodiment 2.The weight of the target peptide of collecting is 24.7mg, and productive rate is 31.3%.
Embodiment 6, D-Cys-Asn-Ser's is synthetic
(1) activation of resin and pre-treatment: with embodiment 1.
(2) amino acid whose coupling
The coupling of a, Ser: with embodiment 2.
The coupling of b, Asn: with embodiment 2.
The add-on of the D-Cys of the coupling of c, D-Cys: Fmoc protection is 861.0mg, and the add-on of HOBT is 662.5mg, and the add-on of HBTU is 1858.1mg, and the add-on of DIEA is 809.8 μ l, and other are identical with embodiment 2.
In the hexahydropyridine/DMF solution in above-mentioned each step, the volume ratio of hexahydropyridine and DMF is 1:0.5.
(3) polypeptide cutting
Cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water, and its volume ratio is 65:10:10:5:10; The consumption of cutting reagent is the 20mL/g resin, and other and embodiment 2 are together.
(4) purifying of polypeptide
A, thick peptide desalination: in the glacial acetic acid water mixed solution, the volume(tric)fraction 50% of glacial acetic acid, other is identical with embodiment 2.
B, performance liquid purifying: purification condition and purge process are with embodiment 2.The weight of the target peptide of collecting is 126.0mg, and productive rate is 79.8%.
Claims (10)
1. monocyte migration inhibition factor analogue is characterized in that: introduce amino acid Arg or replace peptide sequence Arg-Cys-Asn-Ser or the D-Cys-Asn-Ser that obtains behind the Cys with D-Cys at the N end of the active peptide segment Cys-Asn-Ser of monocyte migration inhibition factor.
2. the application of monocyte migration inhibition factor analogue in preparation anti-inflammatory, anti-oxidant, control ischemic cerebrovascular medicine according to claim 1.
3. the compound method of monocyte migration inhibition factor analogue according to claim 1 comprises following process step:
(1) activation of resin and pre-treatment:
The wang resin is dipped in the methylene dichloride, stirs and make the abundant swelling of resin, drain solvent;
(2) amino acid whose coupling
The coupling of a, Ser: will pass through pretreated resin and wash with DMF, drying joins the mixing solutions of hexahydropyridine and DMF, stirs it is fully reacted; After reaction is accomplished,, obtain removing the resin of Fmoc protection with the DMF washing; The Ser of Fmoc protection is dissolved among the DMF, and stirring is fully dissolved it, adds excessive HOBT and HBTU; Add DIEA after the stirring and dissolving again, then with the mixed with resin that removes the Fmoc protection, stirring reaction 1 ~ 2h under argon shield, the room temperature; Drain, the DMF washing obtains Fmoc-Ser-Wang;
The coupling of b, Asn: Fmoc-Ser-Wang is added the mixing solutions of hexahydropyridine and DMF, stir it is fully reacted; After reaction is accomplished,, obtain Ser-Wang with the DMF washing; The Asn of Fmoc protection is dissolved among the DMF, and stirring is fully dissolved it, adds HOBT and HBTU; Add DIEA after the stirring and dissolving again, mix with Ser-Wang then, stirring reaction 1 ~ 2h under argon shield, the room temperature; Drain, wash, obtain Fmoc-Asn-Ser-Wang with DMF;
The coupling of c, Cys or D-Cys: Fmoc-Asn-Ser-Wang is joined the mixing solutions of hexahydropyridine and DMF, stir it is fully reacted; After reaction is accomplished,, obtain Asn-Ser-Wang with the DMF washing; The Cys or the D-Cys of Fmoc protection are dissolved among the DMF, and stirring is fully dissolved it, adds HOBT and HBTU; Add DIEA after the stirring and dissolving again; Mix with Asn-Ser-Wang then, stirring reaction 1 ~ 2h drains under argon shield, the room temperature; The DMF washing obtains Fmoc-Cys-Asn-Ser-Wang or Fmoc-D-Cys-Asn-Ser-Wang;
The coupling of d, Arg: Fmoc-Cys-Asn-Ser-Wang is joined hexahydropyridine and DMF mixing solutions, and stirring is fully reacted it; After reaction is accomplished,, obtain Cys-Asn-Ser-Wang with the DMF washing; The Arg of Fmoc protection is dissolved among the DMF; Stirring is fully dissolved it, adds HOBT and HBTU, adds DIEA after the stirring and dissolving again; Mix with Cys-Asn-Ser-Wang then; Stirring reaction 1 ~ 2h under argon shield, the room temperature drains, washs with DMF, obtains Fmoc-Arg-Cys-Asn-Ser-Wang;
E, take off Fmoc protection: Fmoc-Arg-Cys-Asn-Ser-Wang or Fmoc-D-Cys-Asn-Ser-Wang are added in the mixing solutions of hexahydropyridine and DMF, stirring is fully reacted it; After reaction is accomplished,, obtain Arg-Cys-Asn-Ser-Wang or D-Cys-Asn-Ser-Wang with the DMF washing;
(3) polypeptide cutting
Arg-Cys-Asn-Ser-Wang or D-Cys-Asn-Ser-Wang are alternately washed with DCM and MeOH respectively, drain the back and add in the solid phase synthetic instrument room temperature reaction 2.5 ~ 3.5h with cutting reagent; After reaction finishes; Reaction solution filters through core, and the TFA washing concentrates in underpressure distillation below 40 ℃; Add freezing ether sedimentation polypeptide in the liquid concentrator; Then with polypeptide with the dissolving of the glacial acetic acid aqueous solution of volume(tric)fraction 0 ~ 50%, in-20 ~-80 ℃ solidify, freeze-drying, thick peptide Arg-Cys-Asn-Ser or D-Cys-Asn-Ser;
(4) purifying of polypeptide
A, thick peptide desalination: thick peptide Arg-Cys-Asn-Ser or D-Cys-Asn-Ser are dissolved in the glacial acetic acid aqueous solution of volume(tric)fraction 0.1 ~ 50% fully, add in the gel column wash-out; Monitor with UV-detector; Collection has the component of absorption, and the dilution postlyophilization obtains the desalination peptide;
B, performance liquid purifying: the desalination peptide is fully dissolved with deionized water, filtering with microporous membrane, high performance liquid chromatograph on the filtrating, each sample introduction 3 ~ 4 mL collect the effluent of main absorption peak, and freeze-drying obtains target peptide.
4. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: in above-mentioned each step, in said hexahydropyridine and the DMF mixing solutions, the volume ratio of hexahydropyridine and DMF is 1:20 ~ 1:0.5.
5. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: the stirring reaction in said each step is: stirred 3 ~ 5 minutes, and drained; Repeat to stir 2 ~ 3 times.
6. like the compound method of the said monocyte migration inhibition factor of claim 1 analogue, it is characterized in that: in the said step (2), the DMF washing is: washing 2 ~ 3min, drain; Repeat 3 ~ 4 times.
7. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: in the step (2), the add-on of said HOBT is 1 ~ 10 times of corresponding weight resin.
8. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: in the step (3), the add-on of said HBTU is 1 ~ 10 times of corresponding weight resin.
9. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: in the step (3), the add-on of said DIEA is 1 ~ 10 times of corresponding weight resin.
10. the compound method of monocyte migration inhibition factor analogue according to claim 1, it is characterized in that: in the step (3), said cutting reagent is made up of trifluoroacetic acid, phenol, thioanisole, 1, water; Wherein the percent by volume of each component in system is respectively: trifluoroacetic acid: 65 ~ 96.5%, and phenol: 1 ~ 10%, thioanisole: 1 ~ 10%, 1: 0.5 ~ 2.5%, water: 1 ~ 10%; The consumption of said cutting reagent is 5 ~ 20mL/g resin.
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| CN103191407A (en) * | 2013-04-15 | 2013-07-10 | 中国人民解放军第二军医大学 | Novel use of pentapeptide and metabolite thereof in preparation of anti-dementia product |
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