CN101289499B - Cyclic peptide with -Ile-Sta-Sta-Pro- residue segment and used as immunity inhibitor and synthetic process thereof - Google Patents
Cyclic peptide with -Ile-Sta-Sta-Pro- residue segment and used as immunity inhibitor and synthetic process thereof Download PDFInfo
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- CN101289499B CN101289499B CN2008100610327A CN200810061032A CN101289499B CN 101289499 B CN101289499 B CN 101289499B CN 2008100610327 A CN2008100610327 A CN 2008100610327A CN 200810061032 A CN200810061032 A CN 200810061032A CN 101289499 B CN101289499 B CN 101289499B
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- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 108010011270 yunnanin F Proteins 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention relates to a cyclic peptide which is provided with a residue fragment of -Ile-Sta-Pro- and is used as immunosuppressive agents, and the synthesis process of the cyclic peptide (Ile-Ile-Sta-Pro-Tyr-Val-Pro-Leu) consists of 1) removing amino-protecting groups, 2) transpeptidase reaction, 3) the cutting of peptide chain, 4) cyclization of peptide chain. The cyclic peptide compounds which are provided with the residue fragment of -Ile-Sta-Pro- and used as immunosuppressive agents and have a high yield and a high purity can be obtained, and the immunosuppressive activity of the cyclic peptide compounds is higher. Through the experiments of the retarding effect of A1HS1 towards the delayed hypersensitivity of mice, the influence of phagocytic function of macrophages, and the retarding effect of the proliferation of lymphocytes, the A1HS1 shows a stronger immunosuppressive effect than HS-1, which reaches or surpasses the immunosuppressive of the positive contrast (cyclosporine).
Description
Technical field
The present invention relates to peptide compounds, specifically is the cyclic peptide and the synthesis technique thereof of the having of a kind of useful as immunosuppressants-Ile-Sta-Pro-residue segment.
Background technology
Hymenistatin-1 (HS-1) separates a kind of ring octapeptide (the Pettit G.R. that obtains by investigators such as Pettit from the hymeniacidon sponge of the Pacific Ocean, Clewlow P.W., Dufresne C., et al.Isolation and structure of the cyclic peptide hymenistatin I.Can.J.Chem.1990,68:708-711.).The peptide chain-ordering is: cyclo (Pro-Pro-Tyr-Val-Pro-Leu-Ile-Ile).Convenient and accurate in order to discuss, give amino-acid residue with numbering, promptly HS-1 is expressed as (Ile
1-Ile
2-Pro
3-Pro
4-Tyr
5-Val
6-Pro
7-Leu
8).
Have and report that HS-1 in the research of the P388 of mouse lymphocytoblast aleukemic leukemia, has cytostatic effect, but the result is not fully confirmed in follow-up research.
Investigators such as Siemion have disclosed HS-1 and have had the immunosuppressive action that some suppresses body fluid and cell immune response.Document (Poojary B, Belagali SL.Synthetic studies on cyclicoctapeptides:Yunnanin F and Hymenistatin.Eur.J.Med.Chem.2005,40 (4): synthetic HS-1 demonstration place is to the restraining effect of bacterial growth 407-412.), but the restraining effect to fungi is not strong, the anthelmintic action of HS-1 is little, but shows certain anti-inflammatory effect.Document (PawelZubrzak, Karol Kociolek, Marek Smoluch, et al.Search for new syntheticimmunosuppressants II.Tetrazole analogues of hymenistatinn 1.ActaBiochimica Polonica, 2001,48:1151-1154.) in, the author is with 1,5-two replaces tetrazole rings and modifies after the HS-1, is not very big to the immunosuppressive activity influence of HS-1.
The essential characteristic of HS-1 is: be dissolved in CHCl respectively
3And Me
2Behind the SO, the Pro of compound
3-Pro
4Between the residue cis amido linkage, at Ile
2-Tyr
5There is a β VIa corner in the zone, and at Val
6-Ile
1There are a β I or β II corner in the zone.
Amino-acid residue among the HS-1 of natural structure all is a hydrophobic amino acid, and sequence is rich in the ring texture that Pro has secondary amine in addition, and this makes and very difficulty of chemosynthesis also is unfavorable for very much purifying.
Summary of the invention
The technical issues that need to address of the present invention are, synthetic and the purifying route of designing optimal, obtain the cyclic peptide compounds of the having of productive rate height, useful as immunosuppressants that purity is high-Ile-Sta-Pro-residue segment, and the immunosuppressive activity of this cyclic peptide compounds is higher.
In order to estimate peptide bond Ile
2-Pro
3, Pro
3-Pro
4, Val
6-Pro
7What influence is biological activity for HS-1 have, and the applicant uses Ile
2-Statine
3Replace Ile
2-Pro
3, synthesize the analogue of the HS-1 of a new cyclisation.The applicant also wishes by this modification strategy and then reduces this analogue hydrophobicity, improves its solvability in the aqueous solution, and the activity that is beneficial to subsequently detects and application clinically in future.
(3S is the competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA, cholesterol is synthesized rate-limiting enzyme) 4S) to Statine, has panimmunity regulating effect and cytological effect, and does not rely on the reduction of blood cholesterol levels.Recently there are some researches show, Statine not only can suppress the antigenic expression of MHC-II that IFN-γ stimulates inductive human macrophage, endotheliocyte and smooth muscle cell, and can selective exclusion integrate the expression of plain β 2, leukocyte func tional antigens, LFAs-1 (LFA-1, i.e. CD11a/CD18).These functions and Statine are irrelevant to the restraining effect of HMG-CoA.Prompting Statine also is a kind of potential immunosuppressor.Because natural Hymenistatin-1 demonstrates more weak biologic activity, this has just hindered its application as immunosuppressor.Therefore, the present invention adopts Statine to come HS-1 is modified exactly, in the hope of can effectively improving the immunosuppressive activity of HS-1.
The amino acid of being mentioned among the present invention can be that D-type amino acid also can be L-type amino acid, and as preferably, the used amino acid of the present invention is L-type amino acid.
The cyclic peptide of the having of useful as immunosuppressants of the present invention-Ile-Sta-Pro-residue segment, ring (Ile-Ile-Sta-Pro-Tyr-Val-Pro-Leu) (being called for short A1HS1), its structural formula is:
The physicochemical property of above-mentioned cyclic peptide is that white powder is insoluble in water, is soluble in ethanol, organic solvents such as DMSO.
It is generally acknowledged that linear peptides often has good biological activity and stability external, the back activity tends to very fast disappearance in the body but enter, and this is the unfavorable aspect of linear peptides as polypeptide drugs.So people attempt to transform linear peptides as cyclic peptide, utilize the fixedly characteristics of conformation that have of cyclic peptide, make it can be better and receptors bind, improve biological activity, prolong half-life, increase receptor-selective; And, make cyclic peptide greatly reduce to the susceptibility of aminopeptidase and carboxypeptidase because the intramolecularly of cyclic peptide does not exist free aminoterminal and carboxyl terminal.In general, the metabolic stability of cyclic peptide and bioavailability will be far above linear peptides.
But the chemosynthesis of cyclic peptide is often very difficult, especially end to end cyclic peptide synthesizes the difficulty maximum, because being the peptide bond of linear peptides, the precursor of cyclic peptide has very strong P key feature, molecule is easier to form transoid conformation, be and unfold state, spatially distance is far away to cause the carboxyl of the end group that belongs to reactive center and amino, is unfavorable for taking place the intramolecular condensation reaction, but helps intermolecular condensation.Four kinds of new polypeptide of synthetic of the present invention all are that assorted poly-peptide all is end to end cyclic peptide, and these peptides all have the Pro of secondary amine, and all are to have the extremely strong characteristics of hydrophobic interaction, and therefore synthetic difficulty is very big.In the peptide chain that the present invention studied, 8 amino-acid residues all are hydrophobic amino acids, and this not only causes the synthetic difficulty, also makes purge process more difficult.Because therefore the water insoluble solution of these cyclic peptide in purifying, must be dissolved in the non-aqueous solvent, or special damping fluid, and these solvents or damping fluid are not suitable for being applied to the Bioexperiment system probably.Need special solid support in the Chang Gui solid phase synthesis in addition.With common solid support the time, in the dipeptides stage, because the loss that cyclisation causes is very high.Some situation even cause all peptide chains to lose from solid support, but if the C end during for amination Pro, uses the CTC resin then can avoid dipeptides " cut-outs " thus the side reaction meeting can make output greatly improve.Polypeptide again under the liquid phase situation another important factor of " from beginning to end " link coupled be to avoid racemization.From the synthetic angle, the consideration that the introducing of Statine (abbreviating Sta as) has two aspects: Sta is difficult for causing racemization; Secondly Sta is when N-holds, and linear polypeptide is purifying easily.Consider around these, so several different resin that is adopted among the present invention, promptly be respectively Fmoc-method Wang Resin and CTC Resin, and Boc method Merrifield Resin.Secondly, the present invention as novel compound condensation reagent, has reaction fast with BOP/HoBt/DIEA, and condensation condition gentleness and product are easy to characteristics such as purifying, can reach the raising efficiency of pcr product, reduce the purpose of purifying expense.
Be very easy to cause that for cyclization process the link of racemization, applicant taked shortening reaction times, multidigit point to connect a series of means such as reactive polypeptide simultaneously and suppressed racemization, the result is satisfactory.What deserves to be mentioned is that in addition the very strong HS-1 of hydrophobicity is after Statine modifies, solvability strengthens greatly, and solubleness improves greatly than HS-1 itself, and this is very beneficial for its bioactive detection and following clinically application.
Based on above research, the synthesis technique of cyclic peptide of the present invention is characterized in that,
1) removes amino protecting group: with Fmoc-AA
n-CTC Resin or Boc-AA
n-MerrifieldResin makes it abundant swelling with the DMF immersion, drains then; Add the DMF solution that contains piperidines, logical nitrogen reaction is drained again; Use the DMF thorough washing then,, obtain H-AA to remove residual deprotecting regent
n-CTC Resin or H-AA
n-Merrifield Resin; AA wherein
nBe selected from aminoacid sequence: arbitrary amino acid among Ile, Ile, Sta, Pro, Tyr, Val, Pro, the Leu, the arbitrary positive integer in n=1～8; 2) connect reactive polypeptide: adding is dissolved with Fmoc-AA in reactor
nThe DMF solution of-OH, logical nitrogen stirs, and adds HBTU or HATU then, adds NMM at last, and reaction obtains Fmoc-AA
N-1-AA
n-CTC Resin or Boc-AA
N-1-AA
n-Merrifield Resin; Drain, use the DMF thorough washing, use the methanol wash resin then,, with the DMF solution deprotection that contains piperidines, obtain H-AA then to remove amino acid solution and DMF
N-1-AA
n-CTC Resin or H-AA
N-1-AA
n-MerrifieldResin, wherein AA
N-1Be amino acid AA
nThe amino acid of anteposition, the rest may be inferred;
According to aminoacid sequence, continue to use Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Sta-OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, all the other 6 kinds connect reactive polypeptide among the Fmoc-Leu-OH more than the repetition, finally synthesize required peptide chain: Pro-Val-Tyr-Pro-Sta-Ile-Ile-Leu;
3) cutting of peptide chain: will synthesize peptide chain and carry out cleavage reaction with TFA liquid, filtration under diminished pressure is collected filtrate then, with the polypeptide of cold diethyl ether resolution of precipitate in cutting liquid, high speed centrifugation, thorough drying in vacuum drier, get the crude product of linear peptide chain, get linear purified polypeptide through the silicagel column purifying again;
4) cyclisation of peptide chain: linear purified polypeptide is dissolved with DMF, adds a certain amount of EDC, HOBt and DIEA, reaction process with the ion trap mass spectrometer all-the-way tracking till all linear peptides all change into ring type polypeptide; Evaporate to dryness DMF gets the cyclic peptide crude product.
In the synthesis technique of back examples of implementation, resin is Fmoc-AA
n-CTC Resin, amino acid AA
nBe Pro, AA
N-1Be Val.
As preferably, the purification step that the cyclic peptide crude product is arranged after the cyclisation step of described peptide chain: above-mentioned cyclic peptide crude product is dissolved among the DMSO, carry out separation and purification with high performance liquid chromatograph, chromatographic column is the C18 reversed-phase column, elutriant: A liquid is the aqueous solution of TFA, B liquid is the acetonitrile solution that contains %TFA, and the detection wavelength is 220nm.
The present invention adopts solid phase method to close, and the HS-1 with Statine modification natural structure synthesizes its analogue A1HS1, detects through spectrometer analysis, has verified the exactness of sintetics.Product is through the RP-HPLC purifying, and purity all reaches more than 90% behind the high and purifying of productive rate.The product physico-chemical property is stable, and the purity height is for biological activity assay is subsequently laid a good foundation.
Description of drawings
Fig. 1 is the MS mass spectrum of synthetic cyclic peptide A1HS1;
Fig. 2 is the RP-HPLC figure of synthetic cyclic peptide A1HS1.
Embodiment
Below by embodiment, technical scheme of the present invention is described in further detail.
1 composite part
1.1 instrument and reagent
(ABI 433 for Peptide synthesizer, American AB I company), half preparative high performance liquid chromatography instrument (WatersDelta Prep 4000, U.S. Waters company), (Agilent 1100 for the analysis mode high performance liquid chromatograph, U.S. Agilent company), freeze drier (Christ Alpha, Germany CHRIST company), ion trap mass spectrometer (LCQ Deca, U.S. Thermo-Finnigan company), ultraviolet spectrophotometer (BeckmanDU7400, U.S. Beckman company), the anti-phase analytical column of C18 (Waters XTerra, 3.5um
4.6 * 150mm).
Test used Boc-Pro-Merrifield Resin (substitution value 0.22mmol/g, degree of crosslinking 1%, 100-200mesh), Boc-Val-Wang Resin (substitution value 0.52mmol/g, degree of crosslinking 1%, 100-200mesh), Fmoc-Pro-CTC Resin (substitution value 0.25mmol/g, degree of crosslinking 1%, 200-400mesh), Boc-Tyr (tBu)-OH, Boc-Pro-OH, Boc-Ile-OH, Boc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Boc-Stmine (3S, 4S)-OH, HATU, HBTU, HOBt, BOP, DIEA, TFA, DMF, DMSO, DCM, ACN and thioanisole are all available from peptide in Hangzhou, piperidines uses after heavily steaming, and water is secondary deionized water.
1.2 a kind of analogue A1HS1's of HS-1 is synthetic
1.2.1 the synthesis technique of A1HS1 and purifying
1.2.1.1 remove amino protecting group: Boc-Pro-Merrifield Resin 8.0g (synthetic scale is 2.0mmol) is soaked 30min with DMF, make it abundant swelling, drain then.Add the DMF solution 20mL that contains 20% piperidines, logical nitrogen reaction 30min drains again.Then with DMF washing 5 times, to remove residual deprotecting regent.Triketohydrindene hydrate detects, if resin is mazarine, shows that then the Fmoc protecting group removes.Obtain H-Pro
7-Merrifield Resin.
1.2.1.2 connect reactive polypeptide: add the DMF solution that is dissolved with 6.6g Boc-Val-OH in reactor, logical nitrogen stirs several minutes, adds HBTU (or HATU) then, adds NMM at last, reaction 30min.Obtain Boc-Val
6-Pro
7-Merrifield Resin.The amount that adds DMF is as the criterion with complete submergence resin.Drain, with DMF washing 5 times.Use the methanol wash resin then, to remove amino acid solution and DMF, detect so that carry out triketohydrindene hydrate, detected result is colourless if resin presents, and then the explanation reaction is comparatively complete.And then, obtain H-Val with the DMF solution deprotection that contains 20% piperidines
6-Pro
7-MerrifieldResin.Aminoacid sequence by peptide chain continues to connect reactive polypeptide with Boc-Tyr (tBu)-OH, Boc-Pro-OH, Boc-Sta-OH, Boc-Ile-OH, Boc-Ile-OH, Boc-Leu-OH more than repeating successively, until finally synthesizing required peptide chain.Complete synthetic order is: Pro, Val, Tyr, Pro, Sta, Ile, Ile, Leu.
1.2.1.3 the cutting of peptide chain: will synthesize peptide chain and cut with HF liquid, the reaction times is 3.0h, filtration under diminished pressure then, collect filtrate, with the polypeptide of cold diethyl ether resolution of precipitate in cutting liquid, the centrifugal 10min of 8000rpm, dry 12h in vacuum drier, the crude product of linear peptide chain, purity 56%.Crude product is through the crude product purity 92% (yield 85%) of silicagel column purifying.
1.2.1.4 the cyclisation of peptide chain: linear purified polypeptide 0.5g is dissolved with 200mL DMF, add a certain amount of EDC, HOBt and DIEA, reaction process with the ion trap mass spectrometer all-the-way tracking till all linear peptides all change into ring type polypeptide, about 30-40 of total time minute.Evaporate to dryness DMF gets the cyclic peptide crude product, crude product purity 75%.Crude product carries out Analysis and Identification with ion trap mass spectrometer.
1.2.1.5 the purifying of cyclic peptide crude product: above-mentioned cyclisation crude product peptide is dissolved among an amount of DMSO, carry out separation and purification with high performance liquid chromatograph, chromatographic column is the C18 reversed-phase column, elutriant: A liquid is the aqueous solution of 0.1%TFA, B liquid is the acetonitrile solution that contains 0.1%TFA, and the detection wavelength is 220nm.The liquid that purifying is good carries out freeze-drying and gets final cyclisation polypeptide 244mg, purity 95.1% (yield 65.2%).
1.2.1.6 analysis mode HPLC detects purity: chromatographic column be Waters XTerra reversed-phase column (3.5u, 4.6 ± 150mm,
Elutriant: A liquid is the aqueous solution of 0.1%TFA, and B liquid is the acetonitrile solution that contains 0.1%TFA, and the detection wavelength is 220nm.
1.2.1.7 MS identifies: ion trap mass spectrometer detects the molecular weight of target product.
1.3 during the stratographic analysis purifying behind the synthetic peptide purification be exchange ion with TFA to, be elutriant, adopt the linear gradient elution mode that target peptide is carried out purifying with the acetonitrile solution that contains 0.1%TFA.Stratographic analysis result behind the purifying shows that the main peak area is bigger, and other impurity peak area shows the synthetic crude product behind the RPLC purifying than main peak area much smaller (Fig. 2), and the purity of target peptide has obtained improving greatly.
1.4 the mass spectrum behind the synthetic peptide purification identifies that the result that ESI-MS analyzes shows, the actual molecules quality and the theoretical molecular mass of actual synthetic polypeptide or assorted poly-peptide analogs are very identical.Simultaneously also as can be seen, main peak is obvious, and impurity seldom illustrates the synthetic target peptide behind the chromatographic instrument purifying, can obtain purer target peptide (Fig. 1).
Table 1: the productive rate of synthetic HS-1 and analogue thereof, molecular weight and purity
| Product | Productive rate (%) | Molecular weight (Calc/found) | Purity (%) |
| A1HS1 | 63.0 | 953.0/954.0 | 92.9 |
2 activity experiment parts
2.1 instrument and reagent
Biohazard Safety Equipment (U.S. FORMA company), CO2gas incubator (Japanese SANYO company) low-temperature and high-speed whizzer (Japanese KUBOTA company), enzyme-linked immunosorbent assay instrument (China), electronic analytical balance (Mettler-Toledo company), plastic culture dish (96,24 holes, Denmark Nunc company), 721 spectrophotometers, CsA, Viscotrol C, RPMI-1640 substratum (GIBCO company), calf serum (GIBCO company), glutamine, penicillin (Huabei Pharmaceutic Co., Ltd), Streptomycin sulphate (Shanghai four medicine company limiteds), CsA (Novartis company limited), DNFB, india ink, etc.
2.2 the experimental animal BALB/C mice, age in 6-8 week, 18-22g, male and female half and half.Available from Hangzhou Pedagogic University's Experimental Animal Center.
2.3 method
2.3.1 delayed type hypersensitivity (DTH) test
The about 3cm of mouse part skin unhairing
21.2%DNFB (get sesame oil 25ml, acetone 25ml, mixing adds 0.41mlDNFB, about 0.6 gram is dissolved in the above-mentioned sesame oil acetone mixture) 0.1ml is got with microsyringe then in the zone, evenly drips the sensitization in the depilation district.Began by 200ug/mouse the same day in sensitization, 20ug/mouse dosage is to the mouse stomach administration, and 1 time/d, 7d continuously.Behind the last administration 30min, be applied to two sides, every mouse auris dextra front and back with 1.2%DNFB.Experiment grouping situation is: blank group: irritate stomach 400 μ l 0.9%NaCl solution; Positive controls: irritate stomach 400 μ l CsA (500ug/ml); The polypeptide group: every kind of synthetic peptide is divided into height (500ug/ml), low (50ug/ml) two test group, irritates stomach 400 μ l.Each is organized and irritates stomach every day 1 time, totally 7 days.
Weigh behind the 24h, mouse is put to death in cervical vertebra dislocation, cuts ears respectively, lays round auricle with the 8mm punch tool in same area, scales/electronic balance weighing, with left and right auricle weight difference as the swelling degree.
2.3.2 mouse carbon is cleaned up experiment
Blank group: irritate stomach 400 μ l 0.9%NaCl solution; Positive controls: irritate stomach 400 μ l CsA (500ug/ml); The polypeptide group: every kind of synthetic peptide is divided into height (500ug/ml), low (50ug/ml) two test group, irritates stomach 400 μ l.Each is organized and irritates stomach every day 1 time, totally 7 days.
Inject the india ink (injection prepared Chinese ink with india ink stoste with physiological saline dilution 3-4 doubly) of dilution from mouse tail vein, calculate by every 10g body weight 0.1mL.Treat that prepared Chinese ink injects timing immediately.
After the last administration 24 hours, caudal vein injection india ink 0.1ml/10g got blood 40ul in the socket of the eye in 1 minute and 5 minutes behind injection prepared Chinese ink, put into respectively and contain 0.1%Na
2CO
3In the small test tube of 2ml, shake up, use the spectrophotometry optical density(OD), be calculated as follows and clean up index K value.
Clip liver spleen is weighed after drawing neck to put to death animal.Phagocytic function is represented with phagocytic index.
2.3.3 lymphocyte proliferation assay
Disconnected neck is put to death mouse, and the aseptic spleen of getting places to fill an amount of aseptic Hank ' s liquid plate, gently spleen is ground with tweezers, makes the individual cells suspension.Filter through 200 eye mesh screens, or spleen is ground, use Hank ' s liquid to wash 2 times, each centrifugal 10min (1000r/min) with 4 layers of gauze.Then with cell suspension in the complete culture solution of 1mL, with the blue dyeing counting viable count of platform phenol (should more than 95%), adjusting cell concn be 3 * 10
6Individual/mL.
In 96 well culture plates, every hole 100 μ l establish 6 multiple holes with the splenic lymphocyte suspension inoculation.The blank group: every hole adds 100 μ l RPMI1640 substratum; Positive controls: every hole add 100 μ lCsA (500ug/ml is mixed with the solution of 5mg/mL with dehydrated alcohol, filtration sterilization, in cryogenic refrigerator-20 ℃ preservation, the time spent is diluted to desired concn with complete culture solution.); The polypeptide group: (with the dehydrated alcohol is solvent to every kind of synthetic peptide, polypeptide is mixed with the solution of 5mg/ml; Be diluted to our needed concentration with complete culture solution then.) being divided into height (500ug/ml), low (50ug/ml) two test group, every hole adds 100 μ l.
96 orifice plates are put (5%CO2,37 ℃) cultivation 48h in the CO2 incubator.After cultivating end, 20ul MTT (5ug/ml) is added in every hole, at 5%CO2, cultivate 4h under 37 ℃ of conditions, centrifugal (1000r/min * 5min), inhale and abandon supernatant, methyl-sulphoxide 150ul is added in every hole, and light shaking 10min is determined at the absorbance value at 492nm wavelength place with the dissolve purple crystalline deposit.
The OD in experimental group and 6 multiple holes of control group is averaged separately, calculate inhibiting rate with following formula.
2.4 statistical analysis
Experimental data is with (x ± s) expression relatively adopts variance analysis and t check between each group.P＜0.05 expression has significant difference.
2.5 result
2.5.1 the solvability of cyclic peptide A1HS1
HS-1 becomes cyclic peptide A1HS1 after replacing by Sta, and its solvability strengthens greatly, especially the solvability in water.
2.5.2 cyclic peptide A1HS1 is to the influence of mouse DTH
A1HS1 swelling degree and thymus index when lower concentration is lower than HS-1 high density group and low concentration group, A1HS1 is stronger than HS-1 to the restraining effect of mouse delayed type hypersensitivity thus, and is approaching to the restraining effect of mouse delayed type hypersensitivity with positive controls (S-Neoral).
Table 1 cyclic peptide A1HS1 to the influence of mouse DTH (x ± s, n=12)
A:P＜0.05 is than negative control group (physiological saline group); B:P＜0.01 is than negative control group
C:P＜0.05 is than positive controls (S-Neoral group).
2.2 the influence of 1 pair of macrophage phagocytosis of mice of cyclic peptide A1HS
Negative control group is little, the detected result (seeing Table 2) of positive control S-Neoral group, HS-1 group and A1HS1 group.
Table 2 cyclic peptide A1HS1 to the influence of macrophage phagocytosis of mice (x ± s, n=12)
A:P＜0.05 is than negative control group; B:P＜0.01 is than negative control group.
The result shows that A1HS1 shows the obvious suppression effect to the mouse macrophage phagocytic activity when lower concentration, and shows the restraining effect stronger than HS-1.
2.3 cyclic peptide A1HS1 is to the influence of mouse lymphocyte propagation
The inhibiting rate that the S-Neoral (positive control) that utilizes mtt assay to record is organized mouse lymphocyte is 12.6%, the inhibiting rate of the HS-1 high and low dose group of natural structure is respectively 11.2% and 10.8%, be on close level with S-Neoral, but a little less than the S-Neoral group.Data see Table 3.
Table 3 cyclic peptide A1HS1 to lymphopoietic influence (x ± s, n=6)
A:P＜0.01 is than positive controls (S-Neoral group).
HS-1 shows certain restraining effect to lymphopoiesis when high density and lower concentration, and high density is slightly stronger than the restraining effect of lower concentration, but all than a little less than the positive controls, this is consistent with previous studies; The A1HS1 analogue is when lower concentration, and is stronger than high density to lymphopoietic inhibiting rate, and all strong than positive controls and HS-1 group.
3 conclusions
By comparing negative control group, positive controls, HS-1 and A1HS1 to the restraining effect of mouse delayed type hypersensitivity, to the influence of macrophage phagocytic ability and to lymphopoietic restraining effect, A1HS1 has all shown the immunosuppressive action stronger than HS-1, has reached or exceeded the immunosuppressive action of positive control (S-Neoral).Can be used for as immunosuppressor.
Claims (4)
1. the cyclic peptide of the having an of useful as immunosuppressants-Ile-Sta-Pro-residue segment, ring Ile-Ile-Sta-Pro-Tyr-Val-Pro-Leu, its structural formula is:
Wherein the Chinese of Sta is that (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid, molecular formula is C
8H
17NO
3
2. the cyclic peptide of according to claim 1 having-Ile-Sta-Pro-residue segment is characterized in that being white powder, is insoluble in water, is soluble in organic solvent ethanol, among the DMSO.
3. the synthesis technique of the cyclic peptide of the having an of useful as immunosuppressants as claimed in claim 1 or 2-Ile-Sta-Pro-residue segment is characterized in that having the following steps:
1) removes amino protecting group: Boc-Pro-Merrifield Resin 8.0g is soaked 30min with DMF, make it abundant swelling, drain then; Add the DMF solution 20mL that contains 20% piperidines, logical nitrogen reaction 30min drains again; Then with DMF washing 5 times, to remove residual deprotecting regent; Triketohydrindene hydrate detects, if resin is mazarine, shows that then the Fmoc protecting group removes; Obtain H-Pro
7-MerrifieldResin;
2) connect reactive polypeptide: add the DMF solution that is dissolved with 6.6g Boc-Val-OH in reactor, logical nitrogen stirs several minutes, adds HBTU or HATU then, adds NMM at last, reaction 30min; Obtain Boc-Val
6-Pro
7-Merrifield Resin; The amount that adds DMF is as the criterion with complete submergence resin; Drain, with DMF washing 5 times; Use the methanol wash resin then, to remove amino acid solution and DMF, detect so that carry out triketohydrindene hydrate, detected result is colourless if resin presents, and then the explanation reaction is comparatively complete; And then, obtain H-Val with the DMF solution deprotection that contains 20% piperidines
6-Pro
7-Merrifield Resin; Aminoacid sequence by peptide chain continues to connect reactive polypeptide with Boc-Tyr (tBu)-OH, Boc-Pro-OH, Boc-Sta-OH, Boc-Ile-OH, Boc-Ile-OH, Boc-Leu-OH more than repeating successively, until finally synthesizing required peptide chain; Complete synthetic order is: Pro, Val, Tyr, Pro, Sta, Ile, Ile, Leu;
3) cutting of peptide chain: will synthesize peptide chain and cut with HF liquid, the reaction times is 3.0h, filtration under diminished pressure then, collect filtrate, with the polypeptide of cold diethyl ether resolution of precipitate in cutting liquid, the centrifugal 10min of 8000rpm, dry 12h in vacuum drier, the crude product of linear peptide chain, purity 56%; Crude product is through the silicagel column purifying, crude product purity 92%, yield 85%;
4) cyclisation of peptide chain: linear purified polypeptide 0.5g is dissolved with 200mL DMF, add a certain amount of EDC, HOBt and DIEA, reaction process with the ion trap mass spectrometer all-the-way tracking till all linear peptides all change into ring type polypeptide, about 30-40 of total time minute; Evaporate to dryness DMF gets the cyclic peptide crude product, crude product purity 75%.
4. cyclic peptide synthesis technique according to claim 3, it is characterized in that having after the cyclisation step of described peptide chain the purification step of cyclic peptide crude product: above-mentioned cyclisation crude product peptide is dissolved among an amount of DMSO, carry out separation and purification with high performance liquid chromatograph, chromatographic column is the C18 reversed-phase column, elutriant: A liquid is the aqueous solution of 0.1%TFA, B liquid is the acetonitrile solution that contains 0.1%TFA, and the detection wavelength is 220nm; The liquid that purifying is good carries out freeze-drying and gets final cyclisation polypeptide 244mg, purity 95.1%, yield 65.2%.
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| CN107474118B (en) * | 2017-09-19 | 2020-07-28 | 中国工程物理研究院核物理与化学研究所 | Homo-cyclic peptide Cyclo- (Cys)6Preparation method of (1) |
| CN107400162B (en) * | 2017-09-19 | 2020-07-21 | 中国工程物理研究院核物理与化学研究所 | Homocyclic peptide Cyclo- [ (Asn)5-Cys]Preparation method of (1) |
| CN107602669B (en) * | 2017-09-19 | 2021-03-16 | 中国工程物理研究院核物理与化学研究所 | Preparation method of homocyclic peptide Cyclo- (Ala)4 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500805A (en) * | 2002-11-14 | 2004-06-02 | 吉尔生化（上海）有限公司 | Preparing process for Eptifibatide |
| CN101161674A (en) * | 2007-07-13 | 2008-04-16 | 中国科学院昆明植物研究所 | Cyclopeptide, preparation method and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500805A (en) * | 2002-11-14 | 2004-06-02 | 吉尔生化（上海）有限公司 | Preparing process for Eptifibatide |
| CN101161674A (en) * | 2007-07-13 | 2008-04-16 | 中国科学院昆明植物研究所 | Cyclopeptide, preparation method and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| Hu jingdan et al.Design and synthesis of statine-containing BACE inhibitors.Bioorganic Medicinal Chemistry Letters13.2003,134335-4339. * |
| Siemion I. Z. et al.Cyclolinopeptides and their analogs-a new family ofpeptideimmunosuppressants affecting the calcineurin system.Archivum Immunologiae et Therapiae Experimentalis47.1999,47143-153. * |
| 徐仲等.奥曲肽固相合成及环化的研究.哈尔滨工业大学学报40 2.2008,40(2),292-295. * |
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