CN106518966A - Synthetic method of RGD cyclopeptide - Google Patents
Synthetic method of RGD cyclopeptide Download PDFInfo
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- CN106518966A CN106518966A CN201610927542.2A CN201610927542A CN106518966A CN 106518966 A CN106518966 A CN 106518966A CN 201610927542 A CN201610927542 A CN 201610927542A CN 106518966 A CN106518966 A CN 106518966A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
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Abstract
The invention discloses a synthetic method of an RGD cyclopeptide. The method concretely comprises the following steps: sequentially connecting corresponding Fmoc-amino acid in a polypeptide sequence through a solid phase synthesis technology by using chlorine resin as an initial resin carrier in order to obtain polypeptide-2-Cl resin; carrying out an all-protected cleavage reaction on the peptide-2-Cl resin, carrying out rotary evaporation on the obtained cleavage solution, and freeze-drying the evaporated solution to obtain an all-protected peptide to be cyclized; dissolving the freeze-dried all-protected peptide to be cyclized, which is a chain sample, in DCM, and carrying out liquid cyclization to obtain an all-protected cyclized peptide; and carrying out a cleavage and sedimentation reaction on the obtained cyclized liquid, carrying out reverse phase high performance liquid chromatography purification, and lyophilizing the obtained purified reaction product to obtain the RGD cyclopeptide. The pure RGD cyclopeptide prepared in the invention is weighed, analyzed and calculated in order to obtain the purity of the target pure cyclopeptide of 95.3% and the pure product yieldof 75.2%, so the yield is greatly higher than the yield of 40-60% in the prior art; and the target product has the advantages of low racemization rate, high biological activity and simple operation process.
Description
Technical field
The invention belongs to Solid phase peptide synthssis technical field, more particularly to a kind of synthetic method of RGD cyclic peptide.
Background technology
RGD peptide is the small peptide that a class contains Arg-Gly-Asp sequences, have straight line peptide and cyclic peptide point.RGD sequence is cell
The universal identification site combined with integrin by epimatrix, cyclic peptide increased both can the biological stability of peptide, can also make the conformation of peptide
Number is reduced, and is easy to conformation to study.The research such as Tu Liuxiao finds that RGD cyclic peptide is used for liposome modification and can significantly improve administration system
The tumor-targeting of system.
Most of endogenouss linear peptides general only very short half-life in cyclic process, thus reduce which and control curative effect
Fruit and biological activity, in order to obtain acceptable anti metastasis effect, generally require high dose containing the linear of RGD sequence
Peptide, this undoubtedly increased medical expense and is likely to result in unnecessary side effect, therefore, increase peptides stability,
The biological activity of peptide is improved, is the target transformed to the peptide containing RGD by many Pharmaceutical Chemists.Numerous studies show, incite somebody to action
The main body or part-structure of peptide is changed to the cyclisation of loop configuration, i.e. peptide and is to increase one of method of linear peptides stability.
103588863 A of notification number CN disclose a kind of cyclic peptide preparation technology of RGD, by adding condensing agent dehydration contracting
The method of conjunction forms cyclic peptide, and technological process is complicated, and using first cutting the method being cyclized afterwards, often residual impurity in product is led
Cause purification not thorough.
The content of the invention
It is an object of the invention to provide a kind of synthetic method of RGD cyclic peptide, synthetic method cyclisation high income, the technique letter
Monocycle is short, low cost, with extensive market application foreground.
The present invention is achieved by the following technical solutions：
A kind of synthetic method of RGD cyclic peptide, comprises the steps：
(1), with chlorine resin as initial resin carrier, it is condensed by solid-phase synthesis successively corresponding in peptide sequence
Fmoc- aminoacid, obtains polypeptide -2-Cl Resin；
(2) polypeptide -2-Cl Resin are carried out into full guard cleavage reaction, cutting liquid carries out rotating, lyophilizing, obtains treating ring
Change full guard peptide；
(3), the full guard peptide chain sample to be cyclized of lyophilizing DCM is dissolved, liquid phase cyclisation is carried out, full guard cyclisation is obtained
Peptide；
(4), will cyclisation liquid revolving carry out cutting, sedimentation reaction, and adopt reversed phase high-performance liquid chromatography purification, lyophilizing to obtain
To RGD cyclic peptide sterling polypeptides.
Further, chlorine resin described in step (1) is 2-Cl (Trt)-Cl Resin, and substitution value is 0.8mmol/g.
Further, described in step (1) in condensation reaction with HOBT/DIC as activator and condensing agent, during condensation reaction
Between be 40-60min.
Further, the condensation order of Fmoc- protected amino acids described in step (1) is followed successively by, Fmoc-Gly-OH,
Fmoc-Arg (pbf)-OH, Fmoc-Lys (Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Asp (otBu)-OH, condensation reaction temperature
Spend for 30 DEG C, condensation reaction time is 40-60min.
Further, the cutting reagent 100ml formula of full guard cleavage reaction described in step (2) is：80ml DCM+
20ml TFE, cleavage reaction time are 30min.
Further, described in step (3) in liquid phase cyclization with DIC/HOBT as cyclizative condensation agent, cyclisation temperature be
30 DEG C, cyclization is overnight.
Further, the cutting of liquid is cyclized described in step (4), the 100mmL formula of cutting reagent is in sedimentation reaction：
95ml TFA+2.5ml H2O+2.5ml TIS, cleavage reaction time are 90min, react
Agent carries out the sedimentation of the thick peptide of polypeptide.
Further：Described in step (4), in reversed phase high-performance liquid chromatography, purification selects C18,10um to prepare post, with A
Phase：0.1%TFA/ water, B phases：0.1%TFA/ acetonitriles are mobile phase, with B.Cone/% (10 → 25), Time.0 → 45min's
Gradient program carries out the separating-purifying of polypeptide.
The invention has the advantages that：
RGD cyclic peptide sterlings prepared by the present invention, Jing analytical calculations after weighing, target pure product are cyclized the purity of peptide and are
95.3%, its sterling yield is 75.2%, and compared with the prior art only yield of 40-60%, product yield has significantly
Improve, and product racemization rate is low, biological activity is high, operating procedure is simple.
Specific embodiment
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only
The a part of embodiment of the present invention, rather than the embodiment of whole.Based on the embodiment in the present invention, those of ordinary skill in the art
The all other embodiment obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
Embodiment 1
S1, weigh 2-Cl (Trt)-Cl Resin 1g and be placed in Peptide systhesis reactor, in Peptide systhesis reactor plus
Enter DCM solution submergence 2-Cl resins, after swelling 30min, drain solvent；
Wherein, 2-Cl (Trt)-Cl Resin substitution values are 0.8mmol/g；
It should be noted that some conventional abbreviations are with following implication in the present invention：
2-Cl(Trt)-Cl Resin：2- chlorine trityl chloride resins；
DIC：DIC；
HOBT：I-hydroxybenzotriazole；
Fmoc：Fluorenylmethyloxycarbonyl；
Boc：Tertbutyloxycarbonyl；
otBu：The oxygen tert-butyl group；
DIEA：DIPEA；
DMF：DMF；
DCM：Dichloromethane；
TFA：Trifluoroacetic acid；
TFE：Trifluoroethanol；
TIS：Tri isopropyl silane；
S2, weigh 192mg protected amino acid Fmoc-Gly-OH and 140mg HOBT add 10mL centrifuge tubes in, add
5mL DCM solution dissolves, and in the centrifuge tube instills proper catalyst DIEA solution with dropper, activates 20s；
S3, will be the solution 5mL prepared in S2 swelling with S1 after 2-Cl (Trt)-Cl Resin mix after add at most
60min is reacted in peptide symthesis reactor, reaction drains solvent after terminating；
S4, prepare deprotection solution, the deprotection solution is the mixed solution of methanol and DIEA, wherein, methanol with
The volume ratio 1 of DIEA solution:1,5mL deprotection solution is added into the Peptide systhesis reactor of S3 the reaction sealed off on resin
Avtive spot, Peptide systhesis reactor is placed on the shaking table of 20-30r/min and shakes 30min, and reaction drains deprotection after terminating
Solution；
S5, add DMF solution washing resin in the Peptide systhesis reactor of S4, it is many that the DMF solution adds volume
The 1/3-1/2 of peptide symthesis reactor volume, polypeptides reactive device is placed on decolorization swinging table and shakes 60s, drain DMF solution；
Repeat S5 steps 2-4 time；
S6,20% piperidines/DMF deprotection solution removal amino protecting groups are added in the Peptide systhesis reactor of S5
FMOC, wherein, 1/3-1/2 of the 20% piperidines/DMF deprotections liquor capacity for Peptide systhesis reactor volume；
Peptide systhesis reactor is placed in into shake reaction 20min on the decolorization swinging table of 30r/min, after reaction terminates, is drained
Solvent；
S7, weigh 281mg protected amino acid Fmoc-Arg (pbf)-OH and 135mg HOBT add 10mL centrifuge tubes in, plus
Enter the dissolving of 5mL DCM solution, proper catalyst DIEA solution is instilled with dropper in the centrifuge tube, protected after activation 20s
Freamine Ⅲ；
S8, by the protected amino acid solution prepared in S7 add into S6 in Peptide systhesis reactor with resin reaction, will
40-60min is reacted in the isothermal vibration device of 30 DEG C of Peptide systhesis reactor, reaction uses DMF solution washing resin after terminating；
By method removing amino protecting group FMOC of the resin after washing in S6, washed with DMF solution afterwards；
S9, Fmoc-Lys (Boc)-OH, Fmoc-D-Phe-OH, Fmoc- are connected on resin successively according to above-mentioned steps
Asp (otBu)-OH aminoacid, obtains polypeptide -2-Cl resins；
Polypeptide -2-Cl the resins are：
Asp(otBu)-D-Phe-Lys(Boc)-Arg(pbf)-Gly-2-Cl Resin；
It should be noted that protected amino acid Fmoc-Gly-OH, Fmoc-Arg (the pbf)-OH, Fmoc-Lys
(Boc) molecular weight of-OH, Fmoc-D-Phe-OH and Fmoc-Asp (otBu)-OH be respectively 297.3,648.77,468.6,
387.4 and 411.5；
S10, preparation full guard cutting reagent, mixed solution of the full guard cutting reagent for DCM and TFE, wherein DCM
Solution is 4 with the volume ratio of TFE solution:1, polypeptide -2-Cl resins are added into full guard cutting reagent and cuts 30min, instead
Rotary evaporation full guard cutting reagent after should terminating, by resin lyophilization, obtains full guard peptide to be cyclized；
The full guard peptide to be cyclized is：
Asp(otBu)-D-Phe-Lys(Boc)-Arg(pbf)-Gly；
S11, the full guard peptide to be cyclized obtained in S10 is dissolved in appropriate DMF solution, add in solution DIC and
HOBT, in 30 DEG C of water-baths, cyclization overnight, obtains full guard cyclisation peptide, rotary evaporation cyclisation liquid；
Wherein, the addition of the DIC and HOBT is three times of naked peptide quality；
The full guard is cyclized peptide：
S12, preparation cutting reagent, the cutting reagent is TFA, TIS and H2The mixed solution of O, wherein TFA, TIS and H2O
Volume ratio be 38:1:1；
The full guard prepared in S11 cyclisation peptide is added into the cutting reagent, cleavage reaction 90min；
Reaction carries out the sedimentation of the thick peptide of polypeptide with the ether reagent of low temperature pre-cooling after terminating, vacuum lyophilization obtains crude product
Cyclic peptide；
S13, the crude product cyclic peptide prepared in S12 inverted high performance liquid chromatography purification is obtained into RGD cyclic peptide, wherein, it is pure
Change condition is, chromatographic column is C18, and 10 μm prepare post, and mobile phase is A (0.1%TFA/ water)/B (0.1%TFA/ acetonitriles), gradient
Program is
Target polypeptides solution is collected, is weighed after lyophilization, it is 74.8% to calculate RGD cyclic peptide sterlings yield, and purity is
99.2%；
The RGD cyclic peptide sterling is：
Above content is only citing made for the present invention and explanation, and affiliated those skilled in the art are to being retouched
The specific embodiment stated is made various modifications or supplements or substituted using similar mode, without departing from invention or super
More scope defined in the claims, all should belong to protection scope of the present invention.
Claims (8)
1. a kind of synthetic method of RGD cyclic peptide, it is characterised in that comprise the steps：
(1), with chlorine resin as initial resin carrier, Fmoc- corresponding in peptide sequence is condensed successively by solid-phase synthesis
Aminoacid, obtains polypeptide -2-Cl Resin；
(2) polypeptide -2-Cl Resin are carried out into full guard cleavage reaction, cutting liquid carries out rotating, lyophilizing, obtains treating that cyclisation is complete
Protection peptide；
(3), the full guard peptide chain sample to be cyclized of lyophilizing DCM is dissolved, liquid phase cyclisation is carried out, full guard cyclisation peptide is obtained；
(4), will cyclisation liquid revolving carry out cutting, sedimentation reaction, and adopt reversed phase high-performance liquid chromatography purification, lyophilizing to obtain
RGD cyclic peptide sterling polypeptides.
2. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Chlorine resin described in step (1)
For 2-Cl (Trt)-Cl Resin, substitution value is 0.8mmol/g.
3. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：It is condensed described in step (1) anti-
Should in HOBT/DIC as activator and condensing agent, condensation reaction time is 40-60min.
4. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Fmoc- described in step (1)
Protected amino acid condensation order be followed successively by, Fmoc-Gly-OH, Fmoc-Arg (pbf)-OH, Fmoc-Lys (Boc)-OH,
Fmoc-D-Phe-OH, Fmoc-Asp (otBu)-OH, setting-up point are 30 DEG C, and condensation reaction time is 40-60min.
5. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Full guard described in step (2)
The step of cleavage reaction is：Polypeptide -2-Cl resins are added 30min is cut into cutting reagent, reaction terminates rear rotary evaporation
Cutting reagent, by resin lyophilization, wherein, the cutting reagent 100ml formula is：80ml DCM+20ml TFE.
6. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Liquid phase ring described in step (3)
Change in reacting with DIC/HOBT as cyclizative condensation agent, cyclisation temperature is 30 DEG C, and cyclization is overnight.
7. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Step is cyclized liquid described in (4)
Cutting, the 100mmL formula of cutting reagent is in sedimentation reaction：95ml TFA+2.5ml H2O+2.5ml TIS, cleavage reaction
Time is 90min, and reaction carries out the sedimentation of the thick peptide of polypeptide with the ether reagent of low temperature pre-cooling after terminating.
8. the synthetic method of a kind of RGD cyclic peptide according to claim 1, it is characterised in that：Anti-phase height described in step (4)
In effect liquid phase chromatogram method, purification selects C18,10um to prepare post, with A phases：0.1%TFA/ water, B phases：0.1%TFA/ acetonitriles are stream
Dynamic phase, with B.Cone/% (10 → 25), the Gradient program of Time.0 → 45min carries out the separating-purifying of polypeptide.
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| CN201610927542.2A CN106518966A (en) | 2016-10-24 | 2016-10-24 | Synthetic method of RGD cyclopeptide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070019A (en) * | 2017-12-12 | 2018-05-25 | 安徽省国平药业有限公司 | A kind of synthetic method of polypeptide chelate metal ion |
| CN108409831A (en) * | 2018-03-05 | 2018-08-17 | 苏州强耀生物科技有限公司 | A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides |
| CN110229217A (en) * | 2018-03-06 | 2019-09-13 | 丝芙芮生医科技有限公司 | Ring wins peptide, the medicine comprising it or cosmetic composition and preparation method thereof |
| CN110256532A (en) * | 2019-07-03 | 2019-09-20 | 湖北强耀生物科技有限公司 | A kind of RGD cyclic peptide synthetic method |
| CN111939854A (en) * | 2020-08-26 | 2020-11-17 | 广州市锐博生物科技有限公司 | Solid-phase synthesis product cutting deprotection system and method |
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| US20030125243A1 (en) * | 2000-07-20 | 2003-07-03 | Jun Liu | Synthesis of cyclic peptides |
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| US20030125243A1 (en) * | 2000-07-20 | 2003-07-03 | Jun Liu | Synthesis of cyclic peptides |
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| ROLAND HAUBNER等: "Structural and Functional Aspects of RGD-Containing Cyclic Pentapeptides as Highly Potent and Selective Integrin αvβ3 Antagonists", 《J. AM. CHEM. SOC》 * |
| 刘雷明等: "AoGDW肽固相合成工艺研究", 《化学试剂》 * |
| 初虹等: "含RGD 序列环肽的设计与合成方法研究", 《2008年中国药学会学术年会暨第八届中国药师周论文集》 * |
| 王燕云: "促滲肽-药物的设计合成与活性评价", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070019A (en) * | 2017-12-12 | 2018-05-25 | 安徽省国平药业有限公司 | A kind of synthetic method of polypeptide chelate metal ion |
| CN108409831A (en) * | 2018-03-05 | 2018-08-17 | 苏州强耀生物科技有限公司 | A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides |
| CN110229217A (en) * | 2018-03-06 | 2019-09-13 | 丝芙芮生医科技有限公司 | Ring wins peptide, the medicine comprising it or cosmetic composition and preparation method thereof |
| CN110229217B (en) * | 2018-03-06 | 2022-04-05 | 丝芙芮生医科技有限公司 | Cyclic peptide, pharmaceutical or cosmetic composition comprising the same, and preparation method thereof |
| CN110256532A (en) * | 2019-07-03 | 2019-09-20 | 湖北强耀生物科技有限公司 | A kind of RGD cyclic peptide synthetic method |
| CN110256532B (en) * | 2019-07-03 | 2023-04-14 | 湖北强耀生物科技有限公司 | RGD cyclopeptide synthesis method |
| CN111939854A (en) * | 2020-08-26 | 2020-11-17 | 广州市锐博生物科技有限公司 | Solid-phase synthesis product cutting deprotection system and method |
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