CN104926923B - A kind of stealthy polypeptide and preparation method thereof with good anti-non-specific protein absorption performance - Google Patents
A kind of stealthy polypeptide and preparation method thereof with good anti-non-specific protein absorption performance Download PDFInfo
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- CN104926923B CN104926923B CN201510324672.2A CN201510324672A CN104926923B CN 104926923 B CN104926923 B CN 104926923B CN 201510324672 A CN201510324672 A CN 201510324672A CN 104926923 B CN104926923 B CN 104926923B
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Abstract
The invention discloses a kind of stealthy polypeptides and preparation method thereof with good anti-non-specific protein absorption performance, wherein the structural formula of the stealthy polypeptide of anti-non-specific protein absorption are as follows:
Description
One, technical field
The present invention relates to a kind of polypeptides and preparation method thereof, specifically a kind of to have good anti-nonspecific protein
Stealthy polypeptide of absorption property and preparation method thereof.
Two, background technique
For biomaterial, a problem not being fully solved still is the nonspecific protein of material surface
Absorption.It is to lead to one of thrombosis, inflammatory reaction or even principal element of biomaterial failure.Polyethylene glycol (PEG) exists
Field of biomedical materials has been widely used, but under conditions of contacting for a long time with physiological blood, it is easy to oxidize, even
It is degraded.It is generally believed that the stability ratio PEG of the anti-non-specific protein absorption material of zwitterionic classes is excellent, wherein most allusion quotation
Type is carboxybetaine (CBMA), sulfonic group glycine betaine (SBMA) and Phosphorylcholine (MPC) etc., they belong to methyl-prop
Olefin(e) acid esters material.But when PEG class and methacrylate based polymers are as gene or nano-medicament carrier,
In the case where long term administration, due to its biological non-biodegradable, host cell lacks corresponding metabolic mechanism to cause to gather
The accumulation of object in the cell is closed, the eubolism of host cell can be interfered in this way, place is damaged or even caused to host cell
Chief cell is dead.Polypeptide material is attracted wide public concern due to its good biocompatibility and biodegradability.
Chen S.F. etc. is opened by glutamic acid N-carboxyl inner-acid anhydride (Glu-NCA) and lysine N- carboxyl inner-acid anhydride (Lys-NCA) mixing
The method of cyclopolymerization (ROP) has obtained the polypeptide for having good anti-non-specific protein absorption performance.But due to two kinds of monomers
Reactivity difference and different batches monomer purity be difficult to control and hardly result in the equally distributed polypeptide of positive and negative charge.
Jiang S.Y. group is prepared by Solid-phase organic synthesis method by negatively charged glutamic acid (E) and positively charged lysine
(K) oligopeptides containing 4-6 EK unit constituted, it shows good anti-non-specific protein absorption performance.But the party
Method depends on amino acid synthesizers, and low efficiency expends greatly, lower particularly with preparation efficiency for macromolecule polypeptide.Chen
Etc. H. devise and relied by what few Ethylene Glycol Methyl acryloyl chloride (OEGMA-Cl) was reacted with the alpha-amido of protectiveness lysine
Propylhomoserin widow glycolmethacrylate (OEGMA-lysine), the high polymer obtained after aggregated are excellent with being provided by OEG
Good anti-non-specific protein absorption performance and by lysine side-chain provide with plasminogen (plasminogen) specificity
In conjunction with function.Although this material shows good anti-non-specific protein absorption performance, biological non-degradable
Property problem still remains.
Three, summary of the invention
The present invention is intended to provide a kind of stealthy polypeptide and its preparation with good anti-non-specific protein absorption performance
Method can solve the difficulties such as the classical biological non-biodegradable of the anti-non-specific adsorption protein material of polymethacrylate
Topic.So-called stealth refers to the non-specific adsorption that material itself can prevent protein on its surface, avoids by human immunity system
System finds and is removed, or even causes the chance of body rejection, to realize to the stealthy of human immune system.
The present invention has the structural formula of the stealthy polypeptide of good anti-non-specific protein absorption performance as follows:
Wherein, n indicates the degree of polymerization of two kinds of monomers, n=10-30.
The present invention has the preparation process of the stealthy polypeptide of good anti-non-specific protein absorption performance as follows:
1) it is condensed
Protectiveness lysine, protectiveness aspartic acid, condensing agent and racemization inhibitor are dissolved in organic solvent, in nitrogen
Confined reaction 48h at room temperature in gas atmosphere；After reaction, vacuum distillation removes organic solvent, obtains yellow oil；It will be yellow
Color grease is dissolved in ethyl acetate, successively molten with 10wt.% (mass concentration, similarly hereinafter) citric acid solution, saturated sodium bicarbonate
Organic phase is collected in liquid and saturated common salt water washing, liquid separation；Revolving removing solvent, silica gel after organic phase is dried over anhydrous sodium sulfate
Pillar layer separation (mobile phase: dichloromethane/ethyl acetate=3:1 (volume ratio)) obtains intermediate 1, is white solid, directly uses
It is reacted in next step；
The structural formula of intermediate 1 is as follows:
Wherein, R1For
R2For
Because containing a HCl in commercially available protectiveness lysine general structure, need to carry out before the reaction pre-
Processing, sloughing the HCl in structure with triethylamine (TEA) keeps the amino in lysine free out.
The protectiveness lysine is selected from
Deng；
The protectiveness aspartic acid is selected from
Deng.
Condensing agent described in step 1) is dicyclohexylcarbodiimide (DCC) or 1- ethyl-(3- dimethylaminopropyl)
Carbodiimide hydrochloride (EDCHCl) etc.；The racemization inhibitor is 1- hydroxy benzo triazole (HOBt) or 1- hydroxyl -7-
Azo benzotriazole (HOAt) etc..
Protectiveness lysine in step 1), protectiveness aspartic acid, condensing agent and racemization inhibitor molar ratio be 1:1:
2:2。
The volume difference of the ethyl acetate, 10wt.% citric acid solution, saturated sodium bicarbonate solution, saturated salt solution
It is 8-10 times of reaction solution volume.
Organic solvent described in step 1) is N, N- diformamide (DMF) or dimethyl sulfoxide.
2) Deprotection
Intermediate 1 is added to trifluoroacetic acid (TFA)/CH2Cl2(V/V=2:1) in, reaction 3h is stirred at room temperature, revolving is de-
Except solvent, residue is dissolved in CH again2Cl2In, add TEA tune pH to 7-8, again revolving removing solvent；After washing residue
It filters and dries, obtain intermediate 2, be white solid；
The structural formula of intermediate 2 is as follows:
Wherein, R1For
3) polycondensation
The ratio of intermediate 2, condensing agent and racemization inhibitor 1:2:2 in molar ratio are added in organic solvent, nitrogen
Confined reaction 48h at room temperature in atmosphere, then reaction solution is poured into ether precipitate polymer is intermediate 3, without pure
Change is directly used in reacts in next step；Organic solvent described in step 3) is DMF or dimethyl sulfoxide.
The structural formula of intermediate 3 is as follows:
Wherein, R1For
Condensing agent described in step 3) is dicyclohexylcarbodiimide (DCC) or 1- ethyl-(3- dimethylaminopropyl)
Carbodiimide hydrochloride (EDCHCl) etc.；The racemization inhibitor is 1- hydroxy benzo triazole (HOBt) or 1- hydroxyl -7-
Azo benzotriazole (HOAt) etc..
4) Deprotection
The intermediate 3 that step 3) obtains is dissolved in mixed solution and reacts 6h at room temperature, reaction solution inclines after reaction
Enter and obtains yellow oil in ether；
Work as R1ForWhen, the mixed solution is the CH of the piperidines of 50wt.%2Cl2Solution；
Work as R1ForWhen, the mixed solution be by the HBr of 33wt.% acetic acid solution and TFA by
Volume ratio 1:1 is constituted.
5) purifying of product
The yellow oil that step 4) is obtained is soluble in water, with saturated sodium bicarbonate solution tune pH to 7-8, dialyses, freezes
Do to obtain white solid, as target product.The dialysis refers to dialyses 3 days in redistilled water, and it is primary to change water every 12h.It is described
Freeze-drying refers to be lyophilized under the conditions of vacuum degree 13Pa, -45 DEG C.
On the basis of existing technology, the present invention design and be prepared for it is a kind of by poly-aspartate with the more of lysine side-chain
Peptide, due to its good anti-protein adsorption performance, it is not easy to the immune and rejection for causing body, to realize to body
The stealthy performance of immune system；Secondly based on natural amino acid polypeptide due to its good biodegradable properties solve through
The problems such as the biological non-biodegradable of the anti-non-specific adsorption protein material of polymethacrylate and PEG class of allusion quotation.Core
Magnetic Resonance Spectrum (1HNMR it) confirms successfully to obtain target polypeptides；Using attenuated total reflection Fourier transform infrared spectrometry (ATR-
FTIR) and enzyme linked immunosorbent assay analysis method (ELISA) investigated this self-assembling polypeptide monomolecular film (SAMs) structure and
Its anti-non-specific protein absorption performance.
Four, Detailed description of the invention
Fig. 1 is gel permeation chromatography (GPC) figure of target polypeptides.As can be seen from Figure 1 mesh is successfully prepared
Polypeptide is marked, weight average molecular weight (Mw) is about 3.8kDa, and polydispersity coefficient (PDI) is 1.33.
Fig. 2 be target polypeptides nuclear magnetic resonance map (1HNMR).As can be seen from Figure 2 mesh is successfully prepared
Polypeptide is marked, and corresponding hydrogen atom is pointed out.
Fig. 3 is the attenuated total reflection Fourier transform infrared spectrometry of self-assembled monolayer (SAMs) of the polypeptide on gold plaque
(ATR-FTIR) figure.As can be seen from Figure 3 the Liang Ge characteristic absorption area of second level amide: amide I band (νC=O, 1600-1700cm-1) and amide II band (δN-H, 1500-1600cm-1), show to have formd polypeptide SAMs on gold plaque surface.
Fig. 4 is relatively non-spy of the surface polypeptide SAMs to human immunoglobulins (anti-IgG) and fibrinogen (Fg)
Foreign preteins matter adsorbance.As can be seen from Figure 4 choose anti-IgG and Fg as model protein, the two it is relatively non-
Specific protein adsorbance is respectively 8.5 ± 3.6% and 12.5 ± 4.8%.
Five, specific embodiment
1, it is condensed
δ-(α-benzyl-NεBenzyloxycarbonyl group-L- lysyl-)-α-tert-butyl-n-tertbutyloxycarbonyl-L-Aspartic acid (4)
Synthesis
H-Lys (Z)-OBzlHCl (1,6.10g, 15.0mmol) is dissolved in 30mL anhydrous DMF, 2.5mL TEA is added
(18.10mmol) reacts 20min under stirring condition, filters；Then by BOC-ASP-OtBu (3,4.34g, 15.0mmol),
EDCHCl (5.75g, 30.0mmol), HOBt (4.04g, 30mmol) are added in filtrate, after nitrogen protection 30min, room temperature
Confined reaction 48h；After reaction, vacuum distillation removes major part DMF, obtains yellow oil；Yellow oil is dissolved in second
In acetoacetic ester, 10wt.% citric acid solution, saturated sodium bicarbonate solution, saturated common salt water washing, liquid separation, organic phase are successively used
Dry with anhydrous sodium sulfate, revolving removing solvent, silica gel column chromatography separates (mobile phase: dichloromethane/ethyl acetate=3:1, body
Product ratio) obtain white solid (4) (7.80g, yield 81.02%).
1HNMR(DMSO-d6, 400MHz): δ 1.21-1.4122H, 1.43-1.732H, 2.50-2.552H, 2.87-
2.982H, 4.10-4.181H, 4.19-4.291H, 4.94-5.052H, 5.05-5.132H, 6.82-6.930.94H, 7.18-
7.250.92H, 7.25-7.3910H, 8.24-8.301H
2, the removing of protecting group tertbutyloxycarbonyl (Boc)
δ-(α-benzyl-NεBenzyloxycarbonyl group-L- lysyl-)-L-Aspartic acid (5) synthesis
The protecting group tertbutyloxycarbonyl (Boc) of dipeptides uses TFA/CH2Cl2(V/V=2:1) it removes.By 4 (8.0g,
12.47mmol) it is added to 30mL TFA/CH2Cl2(V/V=2:1) in, reaction 3h is stirred at room temperature, revolving precipitation will be remained to doing
Object is dissolved in CH again2Cl2In, add TEA tune pH 7-8, then rotate precipitation to dry；Under stirring condition, residue is suspended in water
Middle washing 2-3 times, filtering are dried to obtain white solid (5) (4.60g, yield 76.03%).
1HNMR(DMSO-d6, 400MHz): δ 1.21-1.444H, 1.52-1.752H, 2.45-2.551H, 2.77-
2.871H, 2.87-2.982H, 3.40-3.501H, 4.19-4.291H, 4.94-5.052H, 5.05-5.132H, 7.20-
7.260.80H 7.27-7.4010H
3, polycondensation
Poly- (δ-(α-benzyl-NεBenzyloxycarbonyl group-L- lysyl-)-L-Aspartic acid) (6) synthesis
By 5 (0.2g, 0.4mmol), EDCHCl (0.115g, 0.6mmol), HOBt (0.081g, 0.6mmol) is added to
In the DMF of 2mL, after nitrogen protection 30min, room temperature confined reaction 48h；Then reaction solution is poured into a large amount of ether and is precipitated
Polymer (6) is directly used in reacts in next step without further purification.
4, the removing of protecting group benzyloxycarbonyl group (Cbz)
Poly- (Nδ- L- lysyl-L-Aspartic acid) (7) synthesis
The protecting group benzyloxycarbonyl group (Cbz) of polypeptide is removed using 33wt.%HBr/HOAc.By 6 be dissolved in 18mL TFA and
After reacting 6h in the mixed solution of 33wt.%HBr/HOAc (V/V=1:1), reaction solution is poured into ether and obtains yellow oil.
5, the purifying of product
Yellow oil is dissolved in water, saturated sodium bicarbonate solution tune pH to 7-8, white solid (7) is lyophilized to obtain in dialysis.
The dialysis refers to dialyses 3 days in redistilled water, and it is primary to change water every 12h.The freeze-drying refers in vacuum degree 13Pa, -45 DEG C
Under the conditions of be lyophilized.The complete removing of protecting group can completely disappear to obtain by nuclear-magnetism peak at δ 7.27-740 and 4.94-5.13
Confirmation.
1HNMR(D2O, 400MHz): δ 1.24-1.422H, 1.52-1.824H, 2.55-2.872H, 2.88-3.002H,
4.00-4.151H 4.58-4.671H
The specific reaction process of each step is as follows:
The n value of target product is 25.
6, the characterization of target product
The successful preparation of polypeptide can be obtained really by gel permeation chromatogram (Fig. 1) and nmr spectrum (Fig. 2)
Card.
The ATR-FTIR of the self-assembled monolayer (SAMs) of polypeptide is as shown in Figure 3, here it is apparent that secondary amide bonds
The characteristic absorption area amide I band and amide II band of (peptide bond), show to have formd polypeptide SAMs on gold plaque surface；It chooses anti-human
Immunoglobulin (anti-IgG) and fibrinogen (Fg) are used as model protein, and it is non-specific to investigate resisting for polypeptide SAMs
Property adsorption of protein energy, as a result as shown in Figure 4.Using the protein adsorbance on the surface tissue culturing polystyrene (TCPS) as
Control, is set as 100%, the surface polypeptide SAMs is respectively to anti-IgG and Fg relative nonspecificity protein adsorbance
8.5 ± 3.6% and 12.5 ± 4.8%.
Claims (5)
1. a kind of stealthy polypeptide of anti-non-specific protein absorption, it is characterised in that its structural formula are as follows:
Wherein, n indicates the degree of polymerization of monomer, n=25.
2. a kind of a kind of stealthy polypeptide production methods of anti-non-specific protein absorption described in claim 1, feature exist
In the following steps are included:
1) it is condensed
Protectiveness lysine, protectiveness aspartic acid, condensing agent and racemization inhibitor are dissolved in organic solvent, in nitrogen gas
Confined reaction 48h at room temperature in atmosphere；After reaction, vacuum distillation removes organic solvent, obtains yellow oil；By yellow oil
Shape object is dissolved in ethyl acetate, successively uses 10wt% citric acid solution, saturated sodium bicarbonate solution and saturated common salt water washing, point
Liquid collects organic phase；Revolving removing solvent, silica gel column chromatography separate to obtain intermediate 1 after organic phase is dried over anhydrous sodium sulfate；
The protectiveness lysine is selected from
The protectiveness aspartic acid is selected from
2) Deprotection
Intermediate 1 is added in trifluoroacetic acid and the methylene chloride mixed solution that 2:1 is constituted by volume, reaction is stirred at room temperature
3h, revolving removing solvent, adds triethylamine tune pH to 7-8 in methylene chloride for residue is molten again, and revolving removing is molten again
Agent；It is filtered after washing residue and dry, obtains intermediate 2；
3) polycondensation
The ratio of intermediate 2, condensing agent and racemization inhibitor 1:2:2 in molar ratio are added in organic solvent, nitrogen atmosphere
In confined reaction 48h at room temperature, then reaction solution is poured into ether precipitate polymer is intermediate 3；
4) Deprotection
Intermediate 3 is dissolved in mixed solution and reacts 6h at room temperature, reaction solution is poured into ether obtains yellow oil after reaction
Shape object；The mixed solution is that 1:1 is constituted by volume by the acetic acid solution of the HBr of 33wt% and trifluoroacetic acid；
5) purifying of product
The yellow oil that step 4) is obtained is soluble in water, and with saturated sodium bicarbonate solution tune pH to 7-8, dialysis is lyophilized
White solid, as target product；
Condensing agent described in step 1) and step 3) is dicyclohexylcarbodiimide or 1- ethyl-(3- dimethylaminopropyl) carbon
Diimmonium salt hydrochlorate；
Racemization inhibitor described in step 1) and step 3) is three nitrogen of 1- hydroxy benzo triazole or 1- hydroxyl -7- azo benzo
Azoles；
Protectiveness lysine in step 1), protectiveness aspartic acid, condensing agent and racemization inhibitor molar ratio be 1:1:2:2.
3. according to the method described in claim 2, it is characterized by:
Ethyl acetate in step 1), 10wt% citric acid solution, saturated sodium bicarbonate solution, saturated salt solution volume be respectively
8-10 times of reaction solution volume.
4. according to the method described in claim 2, it is characterized by:
Organic solvent described in step 1) is N, N- diformamide or dimethyl sulfoxide；
Organic solvent described in step 3) is N, N- diformamide or dimethyl sulfoxide.
5. according to the method described in claim 2, it is characterized by:
Dialysis refers to described in step 5) dialyses 3 days in redistilled water, and it is primary to change water every 12h；The freeze-drying refers in vacuum
Degree 13Pa, it is lyophilized under the conditions of -45 DEG C.
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Non-Patent Citations (3)
| Title |
|---|
| The cytocompatibility of polyhydroxyalkanoates coated with a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) polypeptide;Dong, Cui-Ling等;《Biomaterials》;20121220;第2593-2599页 |
| 抗非特异性蛋白质吸附多肽的合成、表征及体外酶降解性能研究;杨庆华;《中国博士学位论文全文数据库》;20140815;摘要，第18－20页，第37页倒数第2段，第54－57页 |
| 抗非特异性蛋白质吸附多肽的设计、合成与性能表征;杨庆华等;《2013 年全国高分子学术论文报告会》;20131012;第605页 |
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