CN108409831A - A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides - Google Patents
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides Download PDFInfo
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- CN108409831A CN108409831A CN201810180480.2A CN201810180480A CN108409831A CN 108409831 A CN108409831 A CN 108409831A CN 201810180480 A CN201810180480 A CN 201810180480A CN 108409831 A CN108409831 A CN 108409831A
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- cys
- asp
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- gly
- irgd
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- 0 C*(C(C(C1)NC(C(CCC2)N2C(CNC(C(CCCC*)NC(C(CC(O)=O)NC(CNC(C(CCCNC2NC2N)NC(C(CSSC2)NC(C(CS)N)=O)=*)=O)=O)=O)=O)=O)=O)NC2C(O)=O)C1=O Chemical compound C*(C(C(C1)NC(C(CCC2)N2C(CNC(C(CCCC*)NC(C(CC(O)=O)NC(CNC(C(CCCNC2NC2N)NC(C(CSSC2)NC(C(CS)N)=O)=*)=O)=O)=O)=O)=O)=O)NC2C(O)=O)C1=O 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
Abstract
The invention discloses a kind of solid phases to pinpoint the method that oxidation disulfide bond synthesizes iRGD polypeptides.The present invention is included the following steps using method：Step 1, chemical solid phase synthetic method prepare resin peptide；Resin peptide reaction obtains the resin peptide of No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl made from step 2, the supersaturated solution for configuring iodine and step 1；Step 3, washing removing iodine residual；Step 4, the polypeptide for preparing No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl；Step 5, crude product iRGD polypeptides are obtained；The object of the present invention is to provide a kind of solid phases to pinpoint the method that oxidation disulfide bond synthesizes iRGD polypeptides.Wherein solid phase fixed point aoxidizes the appropriate protection strategy of disulfide bond and can remove protection and aoxidize the specific process that disulfide bond is carried out at the same time, and has reached oxidation rate faster, oxidation efficiency higher, the more stable effect of compound.
Description
Technical field
The present invention relates to the synthesis of polypeptide and solid phase to pinpoint the method for disulfide bond more particularly to the cyclization side of iRGD class polypeptides
Formula.
Background technology
Application of the polypeptide in active targeting delivery system in recent years has received widespread attention.According to its effect
Difference is roughly divided into 3 classes:1, simple targeting peptides are only targeted to a certain organ or specific organization, not the work(of cell-penetrating
Energy；2, cell-penetrating peptide, itself does not have a target function, but when by forming hole or contacted with the cell surface of special receptor
Receptor-mediated endocytosis can be caused to generate cell membrane penetration effect；3, cell-penetrating peptide is targeted, itself there is targeting effect, and arrive
Drug can also be delivered after up to privileged site by cell surface receptor mediated cell film penetration effect.IRGD peptides belong to the above-mentioned 3rd
Class polypeptide is currently used tumour cell-penetrating peptide.
Kazuki N etc. just report tumour cell-penetrating peptide iRGD on Science in 2010 can be obviously improved chemotherapeutic
Penetrating power of the object in tumor tissues improves curative effect of medication, has thus caused the extensive concern of people.
IRGD peptide particular sequences are
CCRGDKGPDC(Disulfide Bridge:C2-C10)
The sweet ammonia of L- cysteinyl-L- cysteinyl-L- arginyl-L- glycyl-L- aspartoyl-L- lysyls-L-
(No. 2 position cysteinyls and No. 10 position cysteine sulfydryls form molecule to acyl-L- prolyls-L- aspartoyls-L-cysteine
Interior disulfide bond)
Sequence：Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys(Disulfide Bridge:C2-C10)
Molecular weight：1051.19
Molecular formula：C38H62N14O15S3
Structural formula：
In currently used disulfide bond looping technique method cyclization, this side are carried out frequently with liquid phase fixed point disulfide bond mode
The used time is longer in process of production for method, complex steps, it cannot be guaranteed that site is made clear after cyclization, while being also easy to produce side reaction shape
At dimer.
Invention content
In order to solve the deficiency of above-mentioned existing various methods, the object of the present invention is to provide a kind of solid phases to pinpoint two sulphur of oxidation
The method for being bonded to iRGD polypeptides.The present invention finds out the appropriate protection of wherein solid phase fixed point oxidation disulfide bond by many experiments
Strategy and protection can be removed and aoxidize the specific process that is carried out at the same time of disulfide bond.
The polypeptide oxidation disulfide bond method of the present invention mostly uses liquid phase cyclization mode, and the present invention breaches consistent side
Formula proposes the synthetic method of solid phase fixed point oxidation disulfide bond on the basis of chemiluminescent polypeptide synthesis in solid state.Abandon common liquid phase
Cyclic mode, directly directly fixed point aoxidizes disulfide bond under conditions of solid phase, has reached oxidation rate faster, oxidation efficiency is more
Height, the more stable effect of compound.
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides, the party are provided according to an aspect of the present invention
Method comprises the following steps：
Step 1, chemical solid phase synthetic method prepare resin peptide Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (O tbu)-
Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys(Acm)-Linker Wang resin；
Fmoc-Cys (Acm)-Arg (Pb f)-Gly-Asp made from step 2, the supersaturated solution for configuring iodine and step 1
(Otbu) what-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys (Acm)-Linker Wang resin reacted obtains No. 1 position
With the resin peptide of No. 9 already oxidised disulfide bond of position cysteine sulfydryl
Fmoc-Cys-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Link
er Wang resin；
Step 3, washing removing iodine residual；
Step 4, NH2-Cys (T the rt)-Cys- for preparing No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl
Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Linker Wan g resin；
Prepared by step 5, cracking, obtain crude product iRGD polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-
No. 2 positions of Asp-Cys-COOH, crude product iRGD polypeptide and No. 10 already oxidised disulfide bond of position cysteine sulfydryl；
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, it should
Method can also include the following steps：
Step 6, by above-mentioned rapid 5 obtained iRGD polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-
The crude product of Asp-Cys-COOH is purified；
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, it should
Method further includes following steps：
IRGD polypeptides made from step 7, step of freeze drying 6 after purification.
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, it should
Method further includes following steps：
Step 8 is detected iRGD polypeptides obtained.
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, on
It states chemical solid phase synthetic method and prepares resin peptide Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-
The method of Pro-Asp (Otbu)-Cys (Acm)-Linker Wang resin is as follows：
The Wang resin resins that substitution degree is 0.65mmol/g are weighed, are added into reactor, DCM is added, opens and protects
Shield gas rushes mixed liquid and Wang resin resins from bottom to top makes it uniformly mix, and filters solvent, excessive Fmoc- is added
Cys (Acm)-OH amino acid is added DMF dissolvings, adds excessive HBTU, excessive HOBT, excessive DIEA and DMAP, instead
It answers；
Pyridine is then added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, filters solvent, then to anti-
It is liquid of raising one's hat to answer addition piperidines/DMF solution in device, piperidines/DMF solution, is stirred to react under a shielding gas, pumps liquid, is used
DMF is washed, and is added 100ml and is raised one's hat liquid, extracts after being stirred to react, washed with DMF, and again with methanol washing is then washed with DMF again
It washs, washes away the Fmoc protecting groups taken off；The resin after answering is negated, is washed with ethyl alcohol, detection reagent is added and detects, 105 DEG C~11 0
DEG C heating 5min, change navy blue is positive reaction；
Excessive Fmoc-Asp (Otbu)-OH amino acid is added, HBTU molar excess is dissolved with DMF, and reactor is added,
DMF is added, is added immediately excessive DIEA, reacts；
Solution is pumped, DMF is washed 3 times, takes more than ten grainy resins, is washed three times with ethyl alcohol, and detection reagent is added and detects, 105 DEG C
~110 DEG C of heating 5min, colourless is negative reaction；
Subsequent amino-acid is sequentially added by way of repeating to remove Fmoc protecting groups and input amino acid reaction, until complete
At sequence Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys
(Acm)-Linker Wang resin,
Methanol is added to wash, drains.
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, on
Stating step 2, steps are as follows：
Methanol solution is taken, iodine is slowly added on a small quantity under room ambient conditions, is stirred while being added, until saturation, obtains iodine
Supersaturated solution, the supersaturated solution of iodine is poured into the resin obtained by step 1, it is full and uniform to be stirred to react, leach out iodine
Supersaturated solution；
Obtain the resin peptide of No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl
Fmoc-Cys-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Link
Er Wang resin (No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl).
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, is washed
It is as follows to wash the removing remaining method of iodine：
DMF washings are added for several times, each protective gas rushes mixed liquid and resin from bottom to top makes it uniformly mix, after washing
The front and back color contrast of observation then continues to wash if color does not shoal, until lighter.
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides, system are provided according to another aspect of the present invention
NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly-Asp of standby No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl
(Otbu) method of-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys-Linker Wang r esin is as follows：
Piperidines/the DMF solution being added into reactor is raised one's hat liquid, is stirred to react, is pumped under the protection of protective gas
Liquid, this step are repeated several times；
Liquid is pumped, is washed with DMF, then methanol washs, and then DMF washes away the Fmoc protecting groups taken off, and takes ten
Resin after several reactions, is washed with ethyl alcohol, is added detection reagent detection, 10 5 DEG C~110 DEG C heating, become navy blue be it is positive instead
It answers；
Excessive Fmoc-Cys (Trt)-OH amino acid is added, HBTU3 times of molar excess is dissolved with DMF, and reaction is added
Device adds DMF, is added immediately ten times of excess of DIEA, reaction.
Solution is pumped, DMF washings take the grainy resin after more than ten reactions, are washed three times with ethyl alcohol, and detection reagent detection is added,
105 DEG C~110 DEG C heating, colourless is negative reaction.
Pyridine is added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, it is logical to leach out solvent, then to anti-
Addition piperidines/DMF solution 100ml in device is answered, 5 is stirred to react under protective gas protection, pumps liquid, washed with DMF, then add
Enter liquid of raising one's hat, extracts after being stirred to react, washed with DMF, again with methanol washing, then the Fmoc protections for washing away and taking off are washed with DMF
Base.The resin after more than ten reactions is taken, is washed three times with ethyl alcohol, detection reagent detection is added, 105 DEG C~110 DEG C heating 5min become
Navy blue is positive reaction.
Methanol repeated washing is added to drain afterwards for several times.
Obtain resin peptide NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-
Asp(Otbu)-Cys-Linker Wang resin。
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, slightly
The preparation method of product iRGD polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-C OOH is as follows：
Configure polypeptide cleavage liquid：By trifluoroacetic acid, TIS, methyl phenyl ethers anisole, water with 90:5:3:2 proportional arrangement lysate.
By resin peptide Fmoc-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-
Cys-Linker Wang resin are placed in round-bottomed flask, and configured good lysate is added under ice bath state, removes ice bath,
Cracking is stirred under normal temperature state.After filter out resin, the ice ether of 10 times of volumes is added, settles, is placed in after stirring
Centrifugation, is cleaned several times with ether in centrifuge.
Sediment is taken out, is dried under reduced pressure and does not stop to roll, until obtaining light grey dry powder-shaped crude product iRGD polypeptides NH2-
Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH。
A kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides is provided according to another aspect of the present invention, it is pure
Change method is as follows：
Pure water and acetonitrile mixed solution (ratio 8 are added in the crude product of iRGD polypeptides：2), supersonic oscillations stirring is until complete
The solution of iRGD polypeptides is passed through the filtering with microporous membrane in the apertures 0.45um by fully dissolved；
Using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B；Preparative liquid chromatography system；Using gradient elution.
Respectively before collection peak, three sections behind summit, peak, summit collection liquid purity is more than 98%；Collection liquid before peak and behind peak is concentrated into suitable
After amount, purity is obtained using same method purified pool and is more than 98% refined solution；Merge collection of all contents 98% or more
Liquid, spin concentration remove the acetonitrile in collection liquid.
Using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparative liquid chromatography system, purifying is taken to obtain
Purpose peptide collection liquid, sample introduction；Start to terminate when collection occurs to no peak using gradient elution, when main peak occurs；Merge collection liquid,
Spin concentration removes acetonitrile and most water in collection liquid, and iRGD polypeptide refined liquids are made.
This hair is related to English substance introduction：
Disulfide Bridge---- disulfide bridges
DCM----------------- dichloromethane
DMF-----------------N.N- dimethylformamides
HBTU---------------- benzotriazole-N, N, N, N- tetramethylurea hexafluorophosphoric acid ester
HOBT---------------- para hydroxybenzene triazoles
DIEA----------------N, N- diisopropylethylamine
DMAP----------------4- dimethyl aminopyridines
TIS----------------- tri isopropyl silanes
The protective gas that the method for the present invention uses can be nitrogen.
In currently used disulfide bond looping technique method cyclization, this side are carried out frequently with liquid phase fixed point disulfide bond mode
The used time is longer in process of production for method, complex steps, it cannot be guaranteed that site is made clear after cyclization, while being also easy to produce side reaction shape
At dimer.It is difficult to form large-scale production exploitation and effectively researches and develops route.
The present invention proposes the synthetic method of solid phase fixed point oxidation disulfide bond on the basis of chemiluminescent polypeptide synthesis in solid state.It gets rid of
Common liquid phase cyclization mode is abandoned, step is saved, directly directly fixed point aoxidizes disulfide bond under conditions of solid phase, has reached oxidation
Speed faster, oxidation efficiency higher, the more stable effect of compound.Step is simple simultaneously, avoids generating not in multiple steps
Necessary impurity generates.It can be as research and development class innovation route and the important aspect of large-scale production exploitation.
The present invention breaches consistent mode, on the basis of chemiluminescent polypeptide synthesis in solid state, proposes solid phase fixed point oxidation two
The synthetic method of sulfide linkage.Common liquid phase cyclization mode is abandoned, directly directly fixed point aoxidizes disulfide bond under conditions of solid phase, reaches
Arrive oxidation rate faster, oxidation efficiency higher, the more stable effect of compound.
A kind of design of the present invention additionally provides a kind of synthetic method of iRGD Solid-phase Polypeptides fixed point oxidation disulfide bond, method packet
Include the following steps：
1, Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otb u)-Lys is carried out using chemiluminescent polypeptide solid-phase synthesis
(Boc) preparation of-Gly-Pro-Asp (Otbu)-Cys (Acm)-Linker Wang resin；
2, the supersaturated solution of iodine is configured, Acm protecting groups is removed, is carried out at the same time solid phase iodine oxidation method, aoxidizes exposed sulfydryl
Cysteine, formed disulfide bond.Obtain sequence Fmoc-Cys (sulfydryl disulfide bond)-Arg (Pbf)-Gly-Asp (Otbu)-
Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys (sulfydryl disulfide bond)-Lin ker Wang resin；
3, washing removing iodine residual；
4, Fmoc protecting groups in sequence are removed.Next amino acid is linked, and removes Fmoc protecting groups and obtains sequence；
NH2-Cys (Trt)-Cys (sulfydryl disulfide bond)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-
Pro-Asp (Otbu)-Cys (sulfydryl disulfide bond)-Linker Wang resin
5, for sequence progress special peptides cracking is completed, resin and Side chain protective group are removed, while ensureing already oxidised
The sulfydryl of disulfide bond is not reduced.Obtain sequence
NH2-Cys-Cys (sulfydryl disulfide bond)-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys (sulfydryl two sulphur
Key)-COOH；
6, the iRGD polypeptides of already oxidised disulfide bond carry out purifying crude；
7, iRGD polypeptides are lyophilized；
8, the detection of iRGD polypeptides.
A kind of design according to the present invention provides a kind of synthetic method of iRGD Solid-phase Polypeptides fixed point oxidation disulfide bond,
This method positioning oxidation disulfide bond is mainly reflected on 3 cysteines.
Description of the drawings
Fig. 1 one of which embodiment reverse chromatograms detection figures of the present invention；
Fig. 2 one of which embodiment Mass Spectrometer Method figures of the present invention.
Specific implementation mode
The present invention will be further described below with reference to the drawings.
Following embodiments is some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.
Embodiment 1
1、Fmoc-Cys(Acm)-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu) -
The preparation of Cys (Acm)-Linker Wang resin.
The Wang resin resin 10g that substitution degree is 0.65mmol/g are weighed, are added into reactor, are added 1
00mlDCM, opening nitrogen rushes mixed liquid and resin from bottom to top makes it uniformly mix 15 minutes, so that resin is fully swollen, during which
Paying attention to being added DCM on a small quantity prevents solution evaporation to dry.Solvent is leached out by husky core, the Fmoc-Cys of 3 times of molar excess is added
(Acm)-OH amino acid is added DMF dissolvings, adds the HBTU of 3 times of molar excess, the HOBT of 3 times of molar excess, 10 times moles
Excessive DIE A, the DMAP of 0.5g react 4h.
Pyridine is then added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, time 30min.Pass through
Sha Xin leaches out solvent, 20% piperidines/DMF solution (liquid of raising one's hat) 100ml is then added into reactor, in nitrogen protection
Under be stirred to react 5min, pump liquid, washed 2 times with 100ml DMF, add 100ml and raise one's hat liquid, be stirred to react 10 minutes
After extract, washed 2 times with 100ml DMF, 100ml methanol washs 2 times, and 10 0ml DMF, which wash 2 times and wash away the Fmoc taken off, to be protected
Protect base.More than ten grainy resins are taken, are washed three times with ethyl alcohol, detection reagent detection is added, 105 DEG C~110 DEG C heating 5min become dark blue
Color is sun
Fmoc-Asp (Otbu)-OH amino acid of 3 times of molar excess, HBTU3 times of molar excess is added, use lacks DMF as possible
Reactor is added in dissolving, and 100mlDMF is added, and is added immediately ten times of excess of DIEA, reacts 30min.
Solution is pumped by sand core, 100mlDMF is washed 3 times, takes more than ten grainy resins, is washed three times with ethyl alcohol, detection is added
Reagent detects, and 105 DEG C~110 DEG C heating 5min, colourless is negative reaction.
Subsequent amino-acid is sequentially added by way of repeating to remove Fmoc protecting groups and input amino acid reaction, until complete
At sequence Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys
(Acm)-Linker Wang resin are added 100ml methanol and wash three times, are drained by the way that bottle is filtered by vacuum.
2, the configuration of oxidising agent is deprotected
100ml methanol solutions are taken, iodine is slowly added on a small quantity under room ambient conditions, is stirred while being added, until saturation.It will
The methanol solution of saturation iodine pours into the resin as obtained by step 1, full and uniform to be stirred to react 40min.It is filtered by sand core
Fall to be saturated the methanol solution of iodine.
Obtain the resin peptide of No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl
Fmoc-Cys-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Linker
Wang resin (No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl).
3, removing residual.
100mlDMF is added to wash 6 times, each nitrogen rushes mixed liquid and resin from bottom to top makes it uniformly mix 5 minutes.
It observes front and back color contrast after washing 6 times then to continue to wash if color does not shoal, until lighter.
4, NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-As p are prepared
(Otbu)-Cys-Linker Wang resin (No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl)
20% piperidines/DMF solution (liquid of raising one's hat) 100ml is added into reactor, is stirred to react under nitrogen protection
2min pumps liquid.This above-mentioned step is repeated 5 times.
Liquid is pumped, is washed 2 times with 100ml DMF, 100ml methanol washs 2 times, and 1 00ml DMF washings 2 times wash away de-
Under Fmoc protecting groups.More than ten grainy resins are taken, are washed three times with ethyl alcohol, detection reagent detection, 105 DEG C~110 DEG C heating are added
5min, change navy blue is positive reaction.
Fmoc-Cys (Trt)-OH amino acid of 3 times of molar excess is added, HBTU3 times of molar excess, it is molten that use lacks DMF as possible
Reactor is added in solution, and 100mlDMF is added, and is added immediately ten times of excess of DIEA, reacts 30min.
Solution is pumped by sand core, 100mlDMF is washed 3 times, takes more than ten grainy resins, is washed three times with ethyl alcohol, and detection examination is added
Agent detects, 105 DEG C -110 plus DEG C hot 5min, and colourless is negative reaction.
Pyridine is added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, time 30min.Pass through husky core
Solvent is leached out, 20% piperidines/D MF solution (liquid of raising one's hat) 100ml is then added into reactor, stirs under nitrogen protection
Reaction 5min is mixed, liquid is pumped, is washed 2 times with 100ml DMF, 100ml is added and raises one's hat liquid, be stirred to react 10 points
It is added after methanol 100ml is washed repeatedly 6 times and drains.
Obtain resin peptide NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-
Asp (Otbu)-Cys-Linker Wang resin (No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl)
5, (No. 2 positions and 10 crude product polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH
Number already oxidised disulfide bond of position cysteine sulfydryl) cracking prepare.
Configure polypeptide cleavage liquid：By trifluoroacetic acid, TIS, methyl phenyl ethers anisole, water with 90:5:3:2 proportional arrangement 160ml cracking
Liquid.
By resin peptide Fmoc-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-
Cys-Linker Wang resin (No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl) are placed in round-bottomed flask, ice
The configured good lysates of 160ml are added under bath state, removes ice bath, cracking 3h is stirred under normal temperature state.Pass through sand core mistake afterwards
Resin is filtered, the ice ether of 10 times of volumes is added, settles 2h after stirring, is placed in 3600 revs/min of centrifuges, is used
Ether cleans 6 times.
Sediment is taken out, is dried under reduced pressure and does not stop to roll, until obtaining light grey dry powder-shaped crude product polypeptide N H2-Cys-
Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH (No. 2 positions and already oxidised two sulphur of No. 10 position cysteine sulfydryls
Key) 6.75g.
Embodiment 2
6, the iRGD polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro- Asp-Cys- of already oxidised disulfide bond
The purifying crude of COOH (No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl).It is added in the crude product of iRGD polypeptides
70ml pure water and acetonitrile mixed solution (ratio 8：2), supersonic oscillations stirring 30min is until be completely dissolved.By iRGD polypeptides
The filtering with microporous membrane that solution passes through the apertures 0.45um.
Using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B；Preparative liquid chromatography system uses 10 μm of reverse phase
C18 (100 × 650mm) filler, UV detector set 220nm, adjust flow velocity 80ml/min, with 5% acetonitrile, balance 15 points
Clock, sample introduction.Using gradient elution：0~2min, 5%~5%；2~42min, 17%~23%；42~48min, 50%~
50%.Appearance time is in 26min or so.Respectively before collection peak, three sections behind summit, peak, summit collection liquid purity is more than 98%.
After collection liquid before peak and behind peak is concentrated into right amount, purity is obtained using same method purified pool and is more than 98% refined solution.It closes
And all contents, in 98% or more collection liquid, spin concentration removes the acetonitrile in collection liquid.
Using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparative liquid chromatography system, using 10 μm of reverse phases
C18 (100 × 650mm) filled column, UV detector set 220nm, adjust flow velocity 60ml/min, first with 80% acetonitrile, balance
It 10 minutes, then with 2% acetonitrile, balances 15 minutes.Take the purpose peptide collection liquid that purifying obtains, sample introduction.
Using gradient elution：0~15min, 5%~5%；15~65min, 5%~40%.Start to collect when main peak occurs
Terminate when occurring to no peak.Merge collection liquid, spin concentration removes acetonitrile and most water in collection liquid, it is more that iRGD is made
Peptide refined liquid 55ml.
Embodiment 3
7, (No. 2 positions freeze-drying iRGD polypeptide NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-CO OH
With No. 10 already oxidised disulfide bond of position cysteine sulfydryl)
By in the multiple clean eggplant-shape bottles of the purifying refined liquids of 55mliRGD polypeptides packing, using liquid nitrogen by iR GD polypeptides
Refined liquid is purified to freeze on eggplant-shape bottle wall.It is placed in freeze dryer.Obtain the iRGD polypeptide sterling peptides that purity is 98.11%
2.41g。
Embodiment 4
8, the detection of iRGD polypeptides
Sample is taken to be dissolved using pure water iRGD polypeptide sterling polypeptides, purity assay and confirmation mass spectrum.Reverse chromatograms detect
As shown in Figure 1, the HPLC purity of iRGD polypeptides is 98.15%；Mass Spectrometer Method is as shown in Fig. 2, show iRGD Mass Spectrometric Identifications just
Really, meet theoretical molecular weight.
Conclusion：The iRGD polypeptide steps that the present invention synthesizes are simplified, and the time is short, efficient.Total yield of products 35.70%, far
Super existing market process recovery ratio.
It the above is only some embodiments of the present invention, it is noted that for the general technology personnel of this field,
Under the premise of the concept for not departing from the present invention, other modification and improvement can also be made, these belong to the guarantor of the present invention
Protect range.
Claims (10)
1. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides, which is characterized in that the method includes following step
Suddenly：
Step 1, chemical solid phase synthetic method prepare resin peptide Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys
(Boc)-Gly-Pro-Asp(Otbu)-Cys(Acm)-Linker Wang resin；
Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp made from step 2, the supersaturated solution for configuring iodine and step 1
(Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys (Acm)-Linker Wang resin reaction obtain No. 1 position and
The resin peptide of No. 9 already oxidised disulfide bond of position cysteine sulfydryl
Fmoc-Cys-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Linker
Wang resin；
Step 3, washing removing iodine residual；
Step 4, NH2-Cys (the Trt)-Cys-Arg for preparing No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl
(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Linker Wang resin；
Prepared by step 5, cracking, obtain crude product iRGD polypeptides NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-
Cys-COOH, described No. 2 positions of crude product iRGD polypeptides and No. 10 already oxidised disulfide bond of position cysteine sulfydryl.
2. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
The method can also include the following steps：
Step 6, the iRGD polypeptides NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp- for obtaining the step 5
The crude product of Cys-COOH is purified.
3. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 2, special
Sign is that the method further includes following steps：
IRGD polypeptides made from step 7, step of freeze drying 6 after purification.
4. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 3, which is characterized in that
The method further includes following steps：
Step 8 is detected iRGD polypeptides obtained.
5. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
The chemical solid phase synthetic method prepares resin peptide Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-
The method of Gly-Pro-Asp (Otbu)-Cys (Acm)-Linker Wang resin is as follows：
The Wang resin resins that substitution degree is 0.65mmol/g are weighed, are added into reactor, DCM is added, open protection gas
Body rushes mixed liquid and Wang resin resins from bottom to top makes it uniformly mix, and filters solvent, excessive Fmoc-Cys is added
(Acm)-OH amino acid is added DMF dissolvings, adds excessive HBTU, excessive HOBT, excessive DIEA and DMAP, reacts；
Pyridine is then added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, filters solvent, then to reactor
Middle addition piperidines/DMF solution, the piperidines/DMF solution are liquid of raising one's hat, and are stirred to react under a shielding gas, pump liquid, are used
DMF is washed, and is added liquid of raising one's hat described in 100ml, is extracted after being stirred to react, washed with DMF, and again with methanol washing is then used again
DMF is washed, and washes away the Fmoc protecting groups taken off；Resin is taken, is washed with ethyl alcohol, detection reagent detection is added, 105 DEG C~110 DEG C add
Hot 5min, change navy blue is positive reaction；
Excessive Fmoc-Asp (Otbu)-OH amino acid is added, HBTU molar excess is dissolved with DMF, and reactor is added, and is added
DMF is added immediately excessive DIEA, reaction；
Solution is pumped, DMF is washed 3 times, takes more than ten grainy resins, is washed three times with ethyl alcohol, and detection reagent is added and detects, and 105 DEG C~110
DEG C heating 5min, it is colourless be negative reaction；
Subsequent amino-acid is sequentially added by way of repeating to remove Fmoc protecting groups and input amino acid reaction, until completing sequence
Arrange Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys (Acm)-
Linker Wang resin；
Methanol is added to wash, drains.
6. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
Steps are as follows for the step 2：
Methanol solution is taken, iodine is slowly added on a small quantity under room ambient conditions, is stirred while being added, until saturation, obtains the iodine
Supersaturated solution, the supersaturated solution of the iodine is poured into the resin obtained by the step 1, it is full and uniform to be stirred to react,
Leach out the supersaturated solution of iodine；
Obtain the resin peptide of No. 1 position and No. 9 already oxidised disulfide bond of position cysteine sulfydryl
Fmoc-Cys-Arg(Pbf)-Gly-Asp(Otbu)-Lys(Boc)-Gly-Pro-Asp(Otbu)-Cys-Linker
Wang resin。
7. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
The washing removing remaining method of iodine is as follows：
DMF washings are added for several times, each protective gas rushes mixed liquid and resin from bottom to top makes it uniformly mix, and is observed after washing
Front and back color contrast then continues to wash if color does not shoal, until lighter.
8. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
Described NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly- for preparing No. 2 positions and No. 10 already oxidised disulfide bond of position cysteine sulfydryl
The method of Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys-Linker Wang resin is as follows：
Piperidines/the DMF solution being added into reactor is raised one's hat liquid, is stirred to react under the protection of protective gas, is pumped liquid,
This step is repeated several times；
Liquid is pumped, is washed with DMF, then methanol washs, and then DMF washes away the Fmoc protecting groups taken off, and takes more than ten
Resin is washed with ethyl alcohol, detection reagent detection is added, 105 DEG C~110 DEG C heating, change navy blue is positive reaction；
Excessive Fmoc-Cys (Trt)-OH amino acid is added, HBTU3 times of molar excess is dissolved with DMF, and reactor is added, then
DMF is added, is added immediately ten times of excess of DIEA, reacts；
Solution is pumped, DMF washings take more than ten grainy resins, are washed three times with ethyl alcohol, and detection reagent is added and detects, 105 DEG C~110 DEG C
Heating, colourless is negative reaction；
Pyridine is added and acetic anhydride presses 1：The uniformly mixed mixed solution closing of 1 ratio, it is logical to leach out solvent, then to reactor
Middle addition piperidines/DMF solution 100ml is stirred to react 5 under protective gas protection, pumps liquid, washed with DMF, added de-
Cap liquid is extracted after being stirred to react, is washed with DMF, again with methanol washing, then the Fmoc protecting groups for washing away and taking off are washed with DMF；It takes
More than ten grainy resins are washed three times with ethyl alcohol, and detection reagent detection is added, and 105 DEG C~110 DEG C heating 5min become navy blue as the positive
Reaction；
Methanol repeated washing is added to drain afterwards for several times；
Obtain resin peptide NH2-Cys (Trt)-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp
(Otbu)-Cys-Linker Wang resin。
9. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 1, which is characterized in that
The preparation method of the crude product iRGD polypeptides NH2-Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH is such as
Under：
Configure polypeptide cleavage liquid：By trifluoroacetic acid, TIS, methyl phenyl ethers anisole, water with 90:5:3:2 proportional arrangement lysate；
By resin peptide Fmoc-Cys-Arg (Pbf)-Gly-Asp (Otbu)-Lys (Boc)-Gly-Pro-Asp (Otbu)-Cys-
Linker Wang resin are placed in round-bottomed flask, and configured good lysate is added under ice bath state, removes ice bath, room temperature
Cracking is stirred under state.After filter out resin, the ice ether of 10 times of volumes is added, is settled after stirring, is placed in centrifugation
Centrifugation, is cleaned several times with ether in machine；
Sediment is taken out, is dried under reduced pressure and does not stop to roll, until obtaining light grey dry powder-shaped crude product iRGD polypeptides NH2-Cys-
Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH。
10. a kind of method of solid phase fixed point oxidation disulfide bond synthesis iRGD polypeptides according to claim 2, feature exist
In the purification process is as follows：
Pure water and acetonitrile mixed solution (ratio 8 are added in the crude product of iRGD polypeptides：2), supersonic oscillations stirring is until completely molten
Solution, passes through the filtering with microporous membrane in the apertures 0.45um by the solution of iRGD polypeptides；
Using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B；Preparative liquid chromatography system；Using gradient elution.Respectively
Before collection peak, three sections behind summit, peak, summit collection liquid purity is more than 98%；After collection liquid before peak and behind peak is concentrated into right amount,
Purity is obtained using same method purified pool and is more than 98% refined solution；Merge collection liquid of all contents 98% or more, revolves
Turn the acetonitrile in concentration removing collection liquid；
Using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparative liquid chromatography system, the purpose for taking purifying to obtain
Peptide collection liquid, sample introduction；Start to terminate when collection occurs to no peak using gradient elution, when main peak occurs；Merge collection liquid, rotation
Concentration removes acetonitrile and most water in collection liquid, and iRGD polypeptide refined liquids are made.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386962A (en) * | 2019-07-04 | 2019-10-29 | 苏州强耀生物科技有限公司 | A kind of synthetic method of adriamycin coupling target polypeptide |
| CN110658289A (en) * | 2019-10-25 | 2020-01-07 | 苏州强耀生物科技有限公司 | Purification method of RGD peptide containing multiple cysteines |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412752A (en) * | 2007-12-26 | 2009-04-22 | 杭州诺泰制药技术有限公司 | Solid phase synthesis method of ziconotide |
| CN102108093A (en) * | 2010-10-28 | 2011-06-29 | 上海强耀生物科技有限公司 | Oxidizing method for use in solid phase synthesis of polypeptide |
| CN103214550A (en) * | 2013-03-14 | 2013-07-24 | 苏州强耀生物科技有限公司 | Multiple-pair disulfide bond fixed point cyclisation solid phase synthesis new process of polypeptide |
| CN103656650A (en) * | 2012-09-02 | 2014-03-26 | 复旦大学 | pH-sensitive brain tumor two-stage targeting nano drug delivery system, and preparation method and application thereof |
| CN106518966A (en) * | 2016-10-24 | 2017-03-22 | 合肥国肽生物科技有限公司 | Synthetic method of RGD cyclopeptide |
-
2018
- 2018-03-05 CN CN201810180480.2A patent/CN108409831A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412752A (en) * | 2007-12-26 | 2009-04-22 | 杭州诺泰制药技术有限公司 | Solid phase synthesis method of ziconotide |
| CN102108093A (en) * | 2010-10-28 | 2011-06-29 | 上海强耀生物科技有限公司 | Oxidizing method for use in solid phase synthesis of polypeptide |
| CN103656650A (en) * | 2012-09-02 | 2014-03-26 | 复旦大学 | pH-sensitive brain tumor two-stage targeting nano drug delivery system, and preparation method and application thereof |
| CN103214550A (en) * | 2013-03-14 | 2013-07-24 | 苏州强耀生物科技有限公司 | Multiple-pair disulfide bond fixed point cyclisation solid phase synthesis new process of polypeptide |
| CN106518966A (en) * | 2016-10-24 | 2017-03-22 | 合肥国肽生物科技有限公司 | Synthetic method of RGD cyclopeptide |
Non-Patent Citations (1)
| Title |
|---|
| H. VILAC¸ A ET AL.: "New cyclic RGD peptides: synthesis, characterization, and theoretical activity towards avb3 integrin", 《TETRAHEDRON》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386962A (en) * | 2019-07-04 | 2019-10-29 | 苏州强耀生物科技有限公司 | A kind of synthetic method of adriamycin coupling target polypeptide |
| CN110658289A (en) * | 2019-10-25 | 2020-01-07 | 苏州强耀生物科技有限公司 | Purification method of RGD peptide containing multiple cysteines |
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