CN106565824A - Enzyme degradation-controllable anti-nonspecific protein adsorption polypeptide and monomer and preparation method thereof - Google Patents
Enzyme degradation-controllable anti-nonspecific protein adsorption polypeptide and monomer and preparation method thereof Download PDFInfo
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- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
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Abstract
The invention discloses enzyme degradation-controllable anti-nonspecific protein adsorption polypeptide and a monomer and a preparation method thereof. By means of polycondensation of a dipeptide monomer containing protected carboxyl group and protected amino group at the side chain and subsequent deprotection, polypeptide with perfect repetitive positive and negative charges is prepared, and anti-nonspecific protein adsorption capacity of polypeptide is enhanced. Further, by polycondensation of a dipeptide monomer containing protected carboxyl group and protected amino group at the side chain and an amino acid monomer modified with protected carboxyl group and protected amino molecules simultaneously contained at the side chain at different ratios and subsequent deprotection, anti-nonspecific protein adsorption polypeptide with different enzyme degradation half-life is obtained, and regulation and control of enzyme degradation capability of the anti-nonspecific protein adsorption polypeptide is realized. By controlling the ratio of an end-capping reagent to a monomer, molecular weight of the target polypeptide is regulated and controlled. By the utilization of functional modification groups on the end-capping reagent, modification of different proteins, polypeptide and other polymer and different material surfaces can be realized.
Description
Technical field
The present invention relates to a kind of polypeptide and preparation method thereof, relates in particular to a kind of anti-non-specific protein absorption many
Peptide and preparation method thereof.
Background technology
Anti- non-specific protein absorption material (nonfouling materials) is of proposition in the nearest more than ten years
The special biocompatible materialses of class, by American Engineering institute academician Buddy D.Ratner 2004
Annu.Rev.Biomed.Eng. biocompatible materialses of new generation are classified as in.It is by the interaction with water, it is therefore prevented that egg
The absorption of white matter molecule, so as to avoid further biochemical reaction, the problems such as blood pool such as in blood.It is non-by resisting
The research that deepens continuously of specific protein adsorbing material finds that anti-non-specific protein absorption material can not only effectively be prevented
Only there is the generation of the coagulation process in contacting blood system, and may apply to prevent the biology of extensive solid liquid interface big
Human body rejection and the generation of cleaning reaction, bacterial adsorption and various biological scale formations caused by Molecular Adsorption process, wherein
Including biomembranous formation, the growth of algae, so that the growth of the shellfish of surface adsorption.Only prevent bacterial adsorption and biomembrane
Formation can think prevent biologic implant from causing infection obtain valuable time.Such as the Bausch & Lomb of 2007
Washing liquid, because of the growth for failing to prevent biomembrane on contact lenss, becomes global crisis.
Existing anti-non-specific protein absorption material is mainly with Polyethylene Glycol (PEG) as representative, but Polyethylene Glycol
There is its obvious defect, including:1) PEG meeting autoxidation, end under conditions of oxygen and transition metal ionss are present
Hydroxyl aldehyde can be oxidized to by internal ethanol dehydrogenase, and aldehyde radical is easy to enter with protein or other molecules for containing amino
Row reaction.2) adsorptivity not there are certain requirements the protein of PEG to molecules align, too closely/or molecular weight compared with metropolis lose
Deproteinization matter not adsorptivity.3) PEG molecules hydraulic radius is larger, and its apparent volume is the water soluble protein of close molecular weight
5 to 10 times or so of molecular volume, affect larger to the biomolecule volume after modification.4) PEG is fixed to behind molecule one end, separately
The functionalization of one end also brings very big difficulty.If import higher density can activated functional groups, can leading because of them
Enter and lose nonfouling characteristics, if very little, fixing another functional molecular can become extremely difficult.Therefore Polyethylene Glycol
Adsorbing material is not implanted in vivo the protein of class, and the field such as nano target medicine transmission all runs into very big difficulty.
On the other hand, from bionic angle, Phosphorylcholine is the functional group being widely present in human body, so very early
Just by the exploitation of the object of study as biomaterial, particularly application.Research finds that material surface applies last layer phosphoric acid
After the polymer coating of choline group, the blood compatibility of material is greatly improved, so as to effectively reduce blood coagulation
Occur.Just constantly there is Phosphorylcholine analog to be synthesized from the seventies and eighties, earliest is the 2- methyl in synthesis in 1977
Acrylic acid ethylene glycol ester group Phosphorylcholine (MPC), but combined coefficient is still relatively low.Therefore by the concept of Phosphorylcholine base
To other amphion.Such as sulphur-ammonium, (such as sulphonic acid betaine methacrylate (SBMA), carboxylic-ammonium is (such as carboxylic acid glycine betaine first
Base acrylate (CBMA) zwitterionic materials engender.Sulphur-ammonium, carboxylic-ammonium this two classes material, not only with Phosphorylcholine class material
Material equally has good nonfouling performances, and their monomer synthesis is simple, and efficiency high, stability is high.Particularly
Carboxylic-ammonium class nonfouling material, the ability of only not good anti-protein Molecular Adsorption, and be proved to have in a large number may be used
For the functional group of fixed ligands.On the other hand, from for the angle of synthesis, synthesized by 2 footworks, can more conveniently be avoided
The select permeability of dissolving, is easier to form copolymer with other low polar monomers.
However, either Polyethylene Glycol, or amphoteric ion polymer is in vivo, particularly there is a weight in human body
The defect wanted, that is, cannot be introduced into the homergy of human body by way of by degraded exclusion cell and in vitro, this frequently can lead to cell
Abnormal dead and internal inflammation.Therefore, in the urgent need to developing a kind of anti-non-specific protein absorption material of degradable
Material, the present invention is namely based on a kind of new anti-non-specific protein absorption material and synthetic method disclosed in the demand.
The content of the invention
The technical problem to be solved is to provide a kind of anti-nonspecific protein with controllable enzymatic degradation and inhales
Attached polypeptide and monomer and preparation method.
The present invention solves above-mentioned technical problem and is adopted the technical scheme that:
Side chain of the present invention contains by protection carboxyl and by two peptide monomers of protection amino with as shown in following formula I or Formula II
Structure:
The preparation method of above-mentioned two peptide monomer is:First reactant and the second reactant are carried out into condensation reaction, after hydrolysis
Obtain two peptide monomer;The glutamic acid that first reactant is protected for amido protecting and side chain carboxyl group, or amino guarantor
Shield and the aspartic acid of side chain carboxyl group protection;Second reactant is the lysine of carboxy protective and omega amido protectings.
Further, racemization inhibitor is included described in above-mentioned preparation method of the invention in the reaction system of condensation reaction.
Side chain of the present invention contains by protection carboxyl and by two peptide monomers of protection amino with as in following formula III to formula VI
Structure shown in any one:
Further, the preparation method of two peptide monomer of the invention is:
3rd reactant and the 4th reactant are carried out into condensation reaction, two peptide monomer is obtained after hydrolysis, the described 3rd
Reactant is the glutamic acid of alpha amido protectings and carboxy protective, or the Radix Asparagi ammonia of alpha amido protectings and carboxy protective
Acid;4th reactant is the lysine of carboxy protective and alpha amido protectings, or carboxy protective and omega amino
The lysine of protection.
Further, racemization inhibitor is included in the reaction system of condensation reaction of the present invention.
Anti- non-specific protein absorption polypeptide of the present invention with controllable enzymatic degradation has as in Formula VII to Formula X IV
Structure shown in any one:
During the above is various, m, n represent respectively the degree of polymerization of monomer, integers of the m and n respectively more than or equal to 0, and m+n >
0。
Further, the structural formula of aforementioned polypeptides of the present invention is as shown in any one in Formula X V to Formula X XX:
Wherein, R1 is that R2 is deprotection containing the carboxylic acid group of the selective reaction group for functional modification after deprotection
Afterwards containing the amino of the selective reaction group for functional modification.
Further, R1 of the present invention is thioctic acid base, or the cysteamine being connected with the carboxyl in aminothiopropionic acid
Acidic group;R2 is cysteamine base, or the cysteamine acidic group being connected with the amino in aminothiopropionic acid.
The preparation method of polypeptide of the present invention includes:First monomer and/or second comonomer are carried out into polycondensation reaction, by condensation polymer
Deprotection process is carried out, the polypeptide is obtained;
First monomer has the structure as shown in Formulas I or Formula II, and the second comonomer has such as formula III, formula IV, formula
Structure shown in V or formula VI:
Further, containing ammonia end end-capping reagent or carboxylic end end-capping reagent in the reaction system of polycondensation reaction of the present invention.
Further, ammonia end of the present invention end-capping reagent is the carboxylic acid containing sulfydryl after deprotection.
Further, ammonia end of the present invention end-capping reagent is thioctic acid or cysteine derivative, and the cysteine spreads out
The sulfydryl of the biological amino containing benzyloxycarbonyl group protection and trityl as protecting group.
Further, carboxylic end of the present invention end-capping reagent is the amine containing sulfydryl after deprotection.
Further, the cysteamine that carboxylic end of the present invention end-capping reagent is protected for mercaptotrityl, or carboxyl benzyl
The cysteine that base is protected and mercaptotrityl is protected.
Compared with prior art, the invention has the beneficial effects as follows:
(1) due to either existing Polyethylene Glycol, or the anti-non-specific protein absorption material such as amphoteric ion polymer
Material in vivo, particularly there is an important defect in human body, that is, cannot be introduced into the homergy of human body by way of by drop
Solution discharges cell with vitro, and this frequently can lead to the abnormal dead and internal inflammation of cell.Therefore, in the urgent need to development is a kind of
The anti-non-specific protein absorption material of degradable, can discharge cell and external by homergy approach.The present invention's
Anti- non-specific protein absorption polypeptide can be small peptide or aminoacid by enzymatic degradation, by normal amino acid metabolism approach
Anti- non-specific protein absorption material is eliminated in cell or internal aggregation, so as to reduce anti-non-specific protein absorption material
The side effect that material is produced during the uses such as drug delivery.
(2) due to depositing in the copolymerization product of the amino acid monomer of positively charged side chain and negative charge side chain using after deprotection
In reactivity ratio and polycondensation reaction probability problem, easily there is positively charged side chain and negative electricity in target product amphion polypeptide
The amino acid residue ratio of lotus side chain is unequal, and the amino acid residue of positively charged side chain or negative charge side chain is overlapped and occurred
Problem, prevent target product from obtaining the amphion polypeptide of repetition positive and negative charge, the present invention contains by protection carboxylic by side chain
Base and by two peptide monomer polycondensations of protection amino and follow-up deprotection, prepares and has that perfect to repeat positive and negative charge alternately more
Peptide, improves the anti-non-specific protein absorption ability of polypeptide, and the enzymatic degradation ability having with polypeptide.
(3) due to the more difficult control of normal polypeptide structure degradation in vivo speed, it is generally possible to decompose in the short period, from
And lose anti-non-specific protein absorption ability.Contain what is modified by protection carboxyl and by protection amino molecule simultaneously by side chain
Amino acid monomer polycondensation and follow-up deprotection, prepare the polypeptide with side chain positive and negative charge, are ensureing the anti-non-specific of polypeptide
It is not degraded by enzymes under property protein adsorption capacity.And, further contained by protection carboxyl and by the two of protection amino by side chain
The different ratio polycondensations simultaneously containing the amino acid monomer modified by protection carboxyl and by protection amino molecule of peptide monomer and side chain and
Follow-up deprotection, obtains the anti-non-specific protein absorption polypeptide of different enzymatic degradation half-life, realizes that antagonism is non-specific
The regulation and control of the enzymatic degradation ability of protein absorption polypeptide, reach and apply corresponding time requirement with drug delivery etc..
(4) because anti-non-specific protein absorption polypeptide is only contained by protection carboxyl and by protection amino by above-mentioned side chain
Two peptide monomers and side chain simultaneously containing the polycondensation of the amino acid monomer modified by protection carboxyl and by protection amino molecule, be condensed
The more difficult control of polypeptide molecular weight of formation, this has a certain difficulty to the concrete application that realization need to control molecular weight, therefore by plus
Entering the ratio of end-capping reagent and monomer can realize the molecular weight of goal of regulation and control polypeptide;And using the functional modification base on end-capping reagent
Group, is capable of achieving the modification to different proteins, polypeptide and other polymers, and the modification on different materials surface.
Thus, anti-non-specific protein absorption polypeptide of the present invention with controllable enzymatic degradation in medicine/gene carrier and
There is broad prospect of application in the research of degradable in vivo hydrogel.
Description of the drawings
Fig. 1 is the nuclear magnetic resonance map of the polypeptide synthesized by embodiment 1;
Fig. 2 is the finger print of the polypeptide synthesized by embodiment 1, and the enzymatic degradation time is about 20min;
Fig. 3 is the nuclear magnetic spectrum of the mixed poly- polypeptide synthesized by embodiment 2;
Fig. 4 is the enzymatic degradation collection of illustrative plates of the mixed poly- polypeptide synthesized by embodiment 2, and the enzymatic degradation time is 25min or so;
Fig. 5 is the nuclear magnetic spectrum of the mixed poly- polypeptide synthesized by embodiment 3;
Fig. 6 is the enzymatic degradation collection of illustrative plates of the mixed poly- polypeptide synthesized by embodiment 3, and the enzymatic degradation time is 35min or so;
Fig. 7 is the nuclear magnetic spectrum of the mixed poly- polypeptide synthesized by embodiment 4;
Fig. 8 is the enzymatic degradation collection of illustrative plates of the mixed poly- polypeptide synthesized by embodiment 4, and the enzymatic degradation time is 55min or so;
Fig. 9 is the nuclear magnetic spectrum of the polypeptide synthesized by embodiment 5;
Figure 10 is the 24h enzymatic degradation collection of illustrative plates of the polypeptide synthesized by embodiment 5, shows to be degraded in polymer 24h;
Figure 11 is by adjusting the different polypeptide products that monomer is obtained from the rate of charge of end-capping reagent thioctic acid in embodiment 6
Molecular weight;
Figure 12 is the anti-non-specific protein absorption the performance test results figure of polypeptide synthesized in embodiment 7;
Figure 13 is the anti-non-specific protein absorption the performance test results figure of polypeptide synthesized in embodiment 8;
Figure 14 is the anti-non-specific protein absorption the performance test results figure of polypeptide synthesized in embodiment 9;
Figure 15 is the anti-non-specific protein absorption the performance test results figure of polypeptide synthesized in embodiment 10.
Specific embodiment
Present invention utilizes the amino deprotection condition different from carboxyl of side chain and formation main chain, by amino and carboxyl
Condensation form all amino and dipeptides of the carboxyl with protection group, then remove to form the protection group on the amino of main chain and carboxyl
Group, and side chain is still within guard mode treats polycondensation glutamic acid lysine dipeptides (i.e. the first monomer and second comonomer);And profit
With the polycondensation of the amino and carboxyl for forming main chain, the polypeptide intermediate that side chain is still within guard mode, Jing deprotections are formed
With target amphion polypeptide is obtained after separation.End-capping reagent can be added or is added without in polycondensation process, to be contained or
The amphion polypeptide of the different molecular weight without the selective reaction group for functional modification;And can be by controlling end-blocking
The control to molecular weight is realized in agent with the ratio of two peptide monomers.
In the present invention, condensation, polycondensation, end groupization are the amidatioon condensation reactions that make use of amino and carboxyl.In contracting
Under mixture existence condition, condensation, polycondensation, end group have identical chemical reaction feature, also, selection involved in the present invention
Property deprotection be maturation method in Peptide systhesis chemistry, therefore, those skilled in the art understand that because of amino and carboxyl position
Put change and form different condensation intermediate and polycondensation product.For this purpose, not repeating below because of amino and carboxyl site change
Embodiment.
With 2 couples of present invention of synthetic route 1 and synthetic route there is the anti-nonspecific protein of controllable enzymatic degradation to inhale below
The synthesis of attached polypeptide carries out exemplary description.It should be noted that anti-nonspecific proteins of the present invention with controllable enzymatic degradation
The acquisition of matter absorption polypeptide is not limited to both synthetic routes.
Wherein, the structural formula of the first monomer is as shown in following formula I or Formula II:
In the structural formula of second comonomer such as following formula III to formula VI shown in any one:
Above synthetic route 1 and it is collectively referred to as in route 2, the first monomer and second comonomer can simultaneously exist or only exist wherein
One kind, i.e. in m and n can be 0, but m it is different with n when for 0.
The preparation of following exemplary ground explanation anti-non-specific protein absorption polypeptide of the present invention with controllable enzymatic degradation
Method can preferably include following components:
(1) two peptide monomers of condensation synthesis side chain protected
By the glutamic acid with different blocking groups and the condensation of lysine, such as γ-benzyl-N- tertbutyloxycarbonyl-L-
Glutamic acid and NεThe condensation of-benzyloxycarbonyl group-α-the tert-butyl group -1B, and by selectivity deprotection, obtain with side chain
For the dipeptides that benzyl and benzyloxycarbonyl group are protected, and by separating-purifying, for the polycondensation of next step.The advantage of the method is can
Avoid directly with two kinds of side chain protected aminoacid (e.g., γ-benzyl-L-Glutamic Acid and Nε- benzyloxycarbonyl group -- 1B) mixing
Band positive and negative charge amino acid residue problem pockety in polypeptide is produced during aftercondensated, so as to affect anti-non-specific egg
White matter absorbability.
(2) polycondensation reaction
In the present invention, end-capping reagent can be added or is added without during polycondensation, and two kinds of situations are correspondingly formed respectively with end
The polypeptide of position group, not band edge position group.Concrete grammar is:At ambient temperature, two peptide monomers of side chain protected (are only added
One kind in first monomer, second comonomer while adds two kinds) and condensing agent be dissolved in organic solvent, preferably can have
Racemization inhibitor is added in machine solvent, and can further add end-capping reagent, the intermediate of polypeptide is formed after confined reaction.Addition disappears
Rotation inhibitor can suppress the generation of racemization, it is ensured that the anti-non-specific protein absorption polypeptide of final product can be by enzyme identification
Degraded.If selection adds end-cap molecule when feeding intake, and by controlling the mol ratio of end-cap molecule and two peptide monomers, then can relatively hold
The molecular weight of preparation-obtained polypeptide easy to control, and make one end of polypeptide with can further chemical reaction functional group.
(3) Deprotection
Above-mentioned intermediate is dissolved in into deprotection reaction liquid, and (mixing of such as trifluoroacetic acid and 33wt.% hydrobromic acid acetic acid is molten
The volume ratio of liquid, trifluoroacetic acid and 33wt.% hydrobromic acid acetic acid is 1: in 1), room temperature reaction 2-6h carries out Deprotection, so as to
To the de- benzyloxycarbonyl group of polypeptide and benzyl protection;Then again in reactant liquor add precipitant (such as absolute ether or petroleum ether) enter
Row precipitation, so that product is easily isolated.
(4) purification of product
The polypeptide of de- side chain protected (for example taking off benzyloxycarbonyl group and benzyl protection) is dissolved in into water, saturated sodium bicarbonate solution is used
Neutralization, solution is all transferred to bag filter dialysis or separated with centrifugal separating tube that molecular cut off is 1kDa, removes little point
Son, is obtained white powdery solids product after lyophilizing.The small molecule of polypeptide is isolated and purified, removing mesh is ensure that
Solvent molecule when the unreacted small molecule outside mark polypeptide and reaction.
In specific examples below, to anti-non-specific protein absorption polypeptide of the present invention with controllable enzymatic degradation
Appraisal procedure is as follows:
(1) digest
The centrifuge tube 9 of 2mL is taken, often pipe adds 0.1M NHs of the 1mL containing 3mg/mL target polypeptides4HCO3Solution, then distinguish
Add 100 μ L tryptic 0.1M NH containing 1mg/mL4HCO3Solution, after shaking up, in 37 DEG C, 500rpm velocity of vibration conditions
Lower enzymolysis.Different time points in enzymolysis process are put into enzymolysis solution in the boiled water for having boiled boils 8-10min fully to go out
Tryptic activity.In addition, will only contain the tryptic 0.1M NH of 1mg/mL respectively4HCO3Solution and the only mesh containing 3mg/mL
The 0.1M NH of mark polypeptide4HCO3Solution is using the process of above-mentioned enzyme solution as two control samples.
(2) gel permeation chromatography (GPC) analysis
Enzyme reaction course is monitored using GPC.GPC testing conditions:Mobile phase is 0.1M NH4HCO3, flow velocity is 0.5mL/
min;Column temperature is set as 45 DEG C, the μ L of sample size 100.The Polyethylene oxide (PEO) of different molecular weight is used as standard specimen.
(3) the anti-non-specific protein absorption performance of material
Gold plaque ethanol and deionized water are cleaned, and are dried up, and repeat the process 2 times;Then place into clear in ultraviolet cleaning device
Wash 20min.Cleaned 2 times with ethanol and deionized water again, dried up, immerse the phosphorus of the sulfhydryl-containing terminated target polypeptides of 2mg/mL
Culture 48h in acid buffer (PBS pH 7.4).Further take out, deionized water is rinsed again 5 times, is dried up.Resulting is had
The goat anti-human immunoglobulin that the non-specific protein absorption of self-assembling polypeptide monomolecular film (SAMs) gold plaque passes through HRP labellings
(anti-IgG/HRP) inhale in conjunction with the surface of HRP labelled fibrinogen antibody with Fibrinogen (fibrinogen, Fg)
Attached measurement is determined.Using TCPS surfaces as control, the amount of TCPS surface adsorption protein is set as 100%.Detailed operation process is such as
Under:Testing sample cultivates 20min with anti-IgG (1 μ g/mL) or Fg (10 μ g/mL) first, then rinses to remove with PBS
All unadsorbed protein.For Fg, in addition it is also necessary to determined with Fibrinogen (anti-Fg/HRP) antibody of HRP labellings
Its adsorbance.The sample for having adsorbed Fg is cultivated again with the antibody against fibrinogen (anti-Fg/HRP) of 10 μ g/mL HRP labellings
20min, then rinses to remove all unadsorbed protein with PBS.Hereafter, all testing samples and TCPS controls is put into
24 orifice plates, citric acid solution (0.1M, the pH 5.0, containing 0.03% of the o-phenylenediamine (OPD) of 1mL 1mg/mL is injected per hole
Hydrogen peroxide).After colour developing 10-20 minutes, the 2N H of 1mL are added per hole2SO4Enzymatic activity is quenched.It is every that microplate reader determines 492nm
The absorbance in hole, and calculate protein sub-optimal fusion algorithm by unit area.
Hereinafter anti-non-specific protein absorption of the present invention with controllable enzymatic degradation is described in detail with specific embodiment
The preparation method of polypeptide.
Embodiment 1:
1) polycondensation
The first monomer (structural formula is shown in formula I) 200mg (0.4mmol) is taken, 1- ethyls-(3- dimethylaminos are added
Propyl group) carbodiimide hydrochloride (EDC.HCl) 115mg (0.6mmol) and 1- hydroxy benzo triazoles (HOBt) 81mg
(0.6mmol).The anhydrous N of 2mL are added toward system, after N- diformamides (DMF), rubber closure good seal is used, nitrogen drum is passed through
Room temperature confined reaction 48h after bubble 30min.After reaction terminates, the absolute ether that about 18mL is added into reaction system is precipitated, static
After about 20min, the alternate amphion polypeptide of glutamic acid lysine residue of the non-Deprotection of oily is obtained after the solvent that inclines.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 2h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be amphion polypeptide (target polypeptides) (structural formula as shown in Formula VII, n: m=0
∶1).The product nuclear magnetic resonance, NMR (1HNMR) collection of illustrative plates is as shown in Figure 1.1HNMR(D2O, 400MHz):δ 1.20-1.50 2H, 1.51-
1.592H, 1.65-2.06 4H, 2.08-2.36 2H, 2.90-2.94 2H, 3.95-4.23 2H.
3) degradation time of the anti-non-specific protein absorption polypeptide of synthesis is determined
According to enzyme solution above described in appraisal procedure to the polypeptide (target polypeptides) for preparing in the present embodiment 1
Digested.0 minute in enzymolysis process, 5 minutes, 10 minutes, 30 minutes, 1h, 2h, 4h, 8h, 24h equi-time point is by enzyme
Solution liquid is put in the boiled water for having boiled and boils 8-10min with abundant enzyme denaturing activity.In addition, 100 μ L only to be contained the pancreas egg of 1mg/mL
The 0.1M NH of white enzyme4HCO3The 0.1M NH of the solution and 1mL only target polypeptides containing 3mg/mL4HCO3Solution is with same process
Method carries out process and obtains two control samples.The measured gpc chromatogram of Jing gel permeation chromatographies (GPC) analysis is shown in Fig. 2, from figure
As can be seen that the synthesized polypeptide for obtaining of embodiment 1 has had Partial digestion in 5min or so in 2,10min has not also been digested
Entirely, digested substantially but in 30min or so it is complete, so, it is believed that the complete enzymatic degradation time of the polypeptide should be
20min or so.
Embodiment 2:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 100mg (0.2mmol) and second comonomer (its structural formula such as formula III
It is shown) 20mg (0.04mmol), add 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC.HCl)
(86.4mg, 0.45mmol) and 1- hydroxyls -7- azo BTAs (HOAt) (61.3mg, 0.45mmol), and end-capping reagent
Aminothiopropionic acid 0.004mmol of aminobenzyloxycarbonyl protection mercaptotrityl protection.About 2mL dry DMFs are added toward system
After, rubber closure good seal is used, it is passed through room temperature confined reaction 24h after nitrogen bubble 30min.After reaction terminates, to reaction system
The absolute ether precipitation of interior addition about 18mL, after static about 20min, after the solvent that inclines the non-Deprotection of oily polypeptide (the
The mol ratio of one monomer and second comonomer is 5: 1).
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 2h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be amphion polypeptide (target polypeptides) (structural formula as shown in Formula VII, n: m=1
: 5), the product1HNMR collection of illustrative plates is as shown in Figure 3.
3) degradation time of the anti-non-specific protein absorption polypeptide of synthesis is determined
According to enzyme solution above described in appraisal procedure to the polypeptide (target polypeptides) for preparing in the present embodiment
Digested.Reaction solution is put into and has been boiled in 0 minute, 10 minutes, 20 minutes, 30 minutes, 1 hour, each time point such as 2 hours
The abundant enzyme denaturing activity of 8-10min is boiled in the boiled water of boiling.In addition, 100 μ L only to be contained the tryptic 0.1M NH of 1mg/mL4HCO3
The 0.1M NH of the solution and 1mL only target polypeptides containing 3mg/mL4HCO3The process of solution method described above is used as control sample.Jing coagulates
The measured gpc chromatogram of glue penetration chromatograph (GPC) analysis is shown in Fig. 4, and the enzymatic degradation time is about 20min.
Embodiment 3:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 100mg (0.2mmol) and second comonomer (its structural formula such as formula III
It is shown) 100mg (0.2mmol), add dicyclohexyl carbodiimide (DCC) (154.7mg, 0.75mmol) and 1- hydroxy benzos
Triazole (HOBt) (101.2mg, 0.75mmol), and the half of end-capping reagent aminobenzyloxycarbonyl protection mercaptotrityl protection
Cystine 0.004mmol.After about 2mL dry DMFs are added toward system, rubber closure good seal is used, be passed through nitrogen bubble 30min
Room temperature confined reaction 48h afterwards.After reaction terminates, the absolute ether that about 18mL is added into reaction system is precipitated, static about 20min
Afterwards, incline after solvent to obtain the non-Deprotection polypeptide of oily (mol ratio of the first monomer and second comonomer is 1: 1).
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 2h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be amphion polypeptide (structural formula as shown in Formula VII, n: m=1: 1),1HNMR
Collection of illustrative plates is Fig. 5.
3) degradation time of the anti-non-specific protein absorption polypeptide of synthesis is determined
According to the enzyme solution described in aforementioned evaluations method to the polypeptide (target polypeptides) for preparing in the present embodiment
Digested.In 0 minute, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, each time point such as 2 hours by instead
Answer solution to be put in the boiled water for having boiled and boil 8-10min with abundant enzyme denaturing activity.In addition, 100 μ L only to be contained the pancreas of 1mg/mL
The 0.1M NH of protease4HCO3The 0.1M NH of the solution and 1mL only target polypeptides containing 3mg/mL4HCO3Solution method described above
Process as control sample.The measured gpc chromatogram of Jing gel permeation chromatographies (GPC) analysis is shown in Fig. 6, and the enzymatic degradation time is about
35min。
Embodiment 4:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 100mg (0.2mmol) and second comonomer (its structural formula such as formula III
It is shown) 200mg (0.4mmol) (mol ratio 1: 2), add dicyclohexyl carbodiimide (DCC) (233mg, 1.13mmol) and
1- hydroxyls -7- azo BTAs (HOAt) (153.8mg, 1.13mmol), and end-capping reagent aminobenzyloxycarbonyl protection mercapto
Aminothiopropionic acid 0.004mmol of base trityl as protecting group.After about 3mL dry DMFs are added toward system, sealed with rubber closure
It is good, it is passed through room temperature confined reaction 36h after nitrogen bubble 30min.After reaction terminates, add that about 25mL's is anhydrous into reaction system
Ether precipitates, after static about 20min, after the solvent that inclines the non-Deprotection polypeptide of oily (the first monomer and second comonomer rub
You are than being 1: 2).
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 2h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be amphion polypeptide (structural formula as shown in Formula VII, n: m=1: 2),1HNMR
Collection of illustrative plates is Fig. 7.
3) degradation time of the anti-non-specific protein absorption polypeptide of synthesis is determined
According to enzyme solution above described in appraisal procedure to the polypeptide (target polypeptides) for preparing in the present embodiment
Digested.Boil in reaction solution to be put into the boiled water for having boiled for 0 minute, 50 minutes, 1 hour, each time point such as 2 hours
8-10min is with abundant enzyme denaturing activity.In addition, 100 μ L only to be contained the tryptic 0.1M NH of 1mg/mL4HCO3Solution and 1mL
The 0.1M NH of the target polypeptides of 3mg/mL4HCO3The process of solution method described above is used as control sample.Jing gel permeation chromatographies
(GPC) analyze measured gpc chromatogram and see Fig. 8, the enzymatic degradation time is about 55min.
Embodiment 5:
1) polycondensation
Take second comonomer (its structural formula is as shown in formula III) 200mg (0.4mmol), add EDC.HCl (153mg,
0.8mmol) with HOBt (108mg, 0.8mmol).After about 2mL dry DMFs are added toward system, rubber closure good seal is used, led to
Enter room temperature confined reaction 40h after nitrogen bubble 30min.After reaction terminates, the absolute ether of about 18mL is added into reaction system
Precipitation, after static about 20min, obtains the polypeptide that the non-Deprotection of oily is polymerized by second comonomer after the solvent that inclines.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 3h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be amphion polypeptide (structural formula as shown in Formula VII, n: m=1: 0),1HNMR
Collection of illustrative plates is Fig. 9.1HNMR(D2O, 400MHz):δ 1.20-1.50 2H, 1.51-1.59 2H, 1.65-2.06 4H, 2.08-2.36
2H, 2.90-2.94 2H, 3.95-4.23 2H.Proof has obtained the polypeptide structure shown in Fig. 9.
3) degradation time of the anti-non-specific protein absorption polypeptide of synthesis is determined
According to enzyme solution above described in appraisal procedure to the polypeptide (target polypeptides) for preparing in the present embodiment
Digested., reaction solution is put into into what is boiled in 0 hour, 1 hour, 4 hours, 8 hours, each time point such as 24 hours
8-10min is boiled in boiled water with abundant enzyme denaturing activity.In addition, 100 μ L only to be contained the tryptic 0.1M NH of 1mg/mL4HCO3It is molten
The 0.1M NH of the liquid and 1mL only target polypeptides containing 3mg/mL4HCO3The process of solution method described above is used as control sample.Jing gels
The measured gpc chromatogram of permeation chromatography (GPC) analysis is shown in Figure 10, it can be seen that its finger print is not appointed in 24h
What changes, and obvious enzymatic degradation is had no in 24 hours.
Embodiment 6:
The first monomer (its structural formula is shown in formula I) 200mg (0.4mmol) is taken, it is separately added into end-capping reagent thioctic acid
Molar ratio is 1: 1,5: 1,9: 1,19: 1,45: 1,96: 1 and 166: 1 thioctic acid, add EDC.HCl (150mg) and
HOBt(110mg).After about 2mL dry DMFs are added toward system, rubber closure good seal is used, be passed through nitrogen bubble 30min rear chambers
Warm confined reaction 6h.After reaction terminates, the absolute ether that about 18mL is added into reaction system is precipitated, and after static about 20min, is inclined
Go after solvent to respectively obtain the alternate amphion polypeptide of glutamic acid lysine residue that thioctic acid blocks non-Deprotection.
Jing DMF are respectively for above-mentioned polycondensation product molecular weight obtained by gel permeation chromatography (GPC) analysis of mobile phase:
5K, 7K, 8K, 9.5K, 11.6K, 16.1K and 18.1K dalton (Figure 11).It is thioctic acid base ammonia end seal end two Jing after Deprotection
Property ion polypeptide (structural formula such as Formula X V, n: m=0: 1), molecular weight is respectively:2.5K, 3.5K, 4.2K, 5K, 6K, 8.3K and
9.3K.The embodiment proves that the mol ratio that can be fed intake by regulation monomer and end-capping reagent realizes the tune to polypeptide molecular weight
Control.
Embodiment 7:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 200mg (0.4mmol), add EDC.HCl (115mg,
0.6mmol) and HOBt (81mg, 0.6mmol), then respectively according to the ratio of the first monomer and thioctic acid be 20: 1,40: 1,
200: 1 mol ratio adds end-capping reagent thioctic acid.The anhydrous N of about 2mL are added toward system, after N- diformamides (DMF), rubber is used
Leather plug good seal, is passed through room temperature confined reaction 48h after nitrogen bubble 30min.After reaction terminates, add about into reaction system
The absolute ether precipitation of 18mL, after static about 20min, respectively obtains not taking off for thioctic acid three kinds of molecular weight of end-blocking after the solvent that inclines
The alternate amphion polypeptide of glutamic acid lysine residue of protection group.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33 wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.After reaction about 3h
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL
Washed once again.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and is dialysed 2-3 days, the solution for obtaining is chilled,
Vacuum drying, obtains white solid product, and product is thioctic acid base ammonia end seal end amphion polypeptide (structural formula such as Formula X V institute
Show, n: m=0: 1), molecular weight is respectively 2.8kDa, 5.4kDa, 12.0kDa.
3) the anti-non-specific protein absorption performance of material
Gold plaque ethanol and deionized water are cleaned, and are dried up, and repeat the process 2 times;Then place into clear in ultraviolet cleaning device
Wash 20min.Cleaned 2 times with ethanol and deionized water again, dried up, three kinds of thioctic acid base end-blocking of 2mg/mL are immersed respectively not
With culture 48h in the phosphate buffer (PBS pH 7.4) of molecular weight polypeptide.Further take out, deionized water is rinsed again 5 times, is blown
It is dry.The resulting non-specific protein absorption with self-assembling polypeptide monomolecular film (SAMs) gold plaque is passed through into HRP labellings
Goat anti-human immunoglobulin (anti-IgG/HRP) and Fibrinogen (fibrinogen, Fg) in conjunction with HRP labeled fibers
The surface excess of albumen original antibody is determined.Using TCPS surfaces as control, set the amount of TCPS surface adsorption protein as
100%.Detailed operation process is as follows:Testing sample is cultivated first with anti-IgG (1 μ g/mL) or Fg (10 μ g/mL)
20min, then rinses to remove all unadsorbed protein with PBS.For Fg, in addition it is also necessary to the fibrin of HRP labellings
Former (anti-Fg/HRP) antibody is determining its adsorbance.Adsorbed the sample of Fg again with the fiber egg of 10 μ g/mL HRP labellings
White original antibody (anti-Fg/HRP) culture 20min, then rinses to remove all unadsorbed protein with PBS.Hereafter, institute
There are testing sample and TCPS controls to be respectively put into 24 orifice plates, the Fructus Citri Limoniae of the o-phenylenediamine (OPD) of 1mL 1mg/mL is injected per hole
Acid buffering solution (0.1M, pH 5.0, containing 0.03% hydrogen peroxide).After colour developing 20 minutes, the 2N H of 1mL are added per hole2SO4
Enzymatic activity is quenched.Microplate reader determines absorbances of the 492nm per hole, and calculates protein sub-optimal fusion algorithm by unit area.
Test result is as shown in figure 12.From the figure, it can be seen that as molecular weight increases to 12.0kDa from 2.8kDa, it is right
Increase to 11.6 ± 5.6%, 15.2 also respectively from 5.1 ± 1.6%, 7.3 ± 1.8% in the sub-optimal fusion algorithm of anti-IgG and Fg
± 3.2%.And, from figure it is clear that for all molecular weight identical test samples, the adsorbance ratio of Fg
The adsorbance of anti-IgG will be high, but on the whole, the polypeptide of three kinds of different molecular weights has more outstanding anti-non-specificity
Protein adsorption capacity.
Embodiment 8:
1) polycondensation
Take second comonomer (its structural formula is as shown in formula III) 200mg (0.4mmol), add EDC.HCl (153mg,
0.8mmol) and HOBt (108mg, 0.8mmol), according to the ratio of second comonomer and thioctic acid it is respectively then 20: Isosorbide-5-Nitrae 0: 1,
250: 1 mol ratio adds end-capping reagent thioctic acid.After about 2mL dry DMFs are added toward system, rubber closure good seal is used, led to
Enter room temperature confined reaction 40h after nitrogen bubble 30min.After reaction terminates, the absolute ether of about 18mL is added into reaction system
Precipitation, after static about 20min, after the solvent that inclines the non-Deprotection of oily lipoic acid base by second comonomer be polymerized it is many
Peptide.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 3h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be thioctic acid base ammonia end seal end amphion polypeptide (structural formula as shown in Formula X V, n
: m=1: 0), molecular weight is respectively 3.5kDa, 5.6kDa and 13.7kDa.
3) the anti-non-specific protein absorption performance of material
Gold plaque ethanol and deionized water are cleaned, and are dried up, and repeat the process 2 times;Then place into clear in ultraviolet cleaning device
Wash 20min.Cleaned 2 times with ethanol and deionized water again, dried up, three kinds of thioctic acid base end-blocking of 2mg/mL are immersed respectively not
With culture 48h in the phosphate buffer (PBS pH 7.4) of molecular weight polypeptide.Further take out, deionized water is rinsed again 5 times, is blown
It is dry.The resulting non-specific protein absorption with self-assembling polypeptide monomolecular film (SAMs) gold plaque is passed through into HRP labellings
Goat anti-human immunoglobulin (anti-IgG/HRP) and Fibrinogen (fibrinogen, Fg) in conjunction with HRP labeled fibers
The surface excess of albumen original antibody is determined.Using TCPS surfaces as control, set the amount of TCPS surface adsorption protein as
100%.Detailed operation process is as follows:Testing sample is cultivated first with anti-IgG (1 μ g/mL) or Fg (10 μ g/mL)
20min, then rinses to remove all unadsorbed protein with PBS.For Fg, in addition it is also necessary to the fibrin of HRP labellings
Former (anti-Fg/HRP) antibody is determining its adsorbance.Adsorbed the sample of Fg again with the fiber egg of 10 μ g/mL HRP labellings
White original antibody (anti-Fg/HRP) culture 20min, then rinses to remove all unadsorbed protein with PBS.Hereafter, institute
There are testing sample and TCPS controls to be respectively put into 24 orifice plates, the Fructus Citri Limoniae of the o-phenylenediamine (OPD) of 1mL 1mg/mL is injected per hole
Acid buffering solution (0.1M, pH 5.0, containing 0.03% hydrogen peroxide).After colour developing 20 minutes, the 2N H of 1mL are added per hole2SO4
Enzymatic activity is quenched.Microplate reader determines absorbances of the 492nm per hole, and calculates protein sub-optimal fusion algorithm by unit area.
Test result is as shown in figure 13.As can see from Figure 13, as molecular weight increases to 13.7kDa from 3.5kDa,
Sub-optimal fusion algorithm for anti-IgG and Fg increases to 11.6 ± 2.8%, 15.9 respectively from 3.3 ± 1.8%, 4.4 ± 1.6%
± 5.6%.For all of test sample, the sub-optimal fusion algorithm of Fg will be higher than anti-IgG, and this is likely due to Fg
Volume is larger, loosely organized, easy degeneration and cause extra absorption relevant.On the whole, the polypeptide of three kinds of different molecular weights
There is more outstanding anti-non-specific protein absorption ability.
Embodiment 9:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 100mg (0.2mmol) and second comonomer (its structural formula such as formula III
It is shown) 100mg (0.2mmol), EDC.HCl (115mg, 0.6mmol) and HOBt (81mg, 0.6mmol) is added, then press respectively
It is 20 according to the integral molar quantity of the first monomer and second comonomer and the mol ratio of thioctic acid: the mol ratio of Isosorbide-5-Nitrae 0: 1,200: 1 adds envelope
End agent thioctic acid.The anhydrous N of about 2mL are added toward system, after N- diformamides (DMF), rubber closure good seal is used, nitrogen is passed through
Room temperature confined reaction 36h after bubbling 30min.After reaction terminates, the absolute ether that about 18mL is added into reaction system is precipitated, quiet
Only about after 20min, three kinds of polypeptides of the non-Deprotection of oily lipoic acid base are obtained after the solvent that inclines.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 3h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, product be thioctic acid base ammonia end seal end amphion polypeptide (structural formula as shown in Formula X V, n
: m=1: 1), molecular weight is respectively 3.2kDa, 5.4kDa and 12.8kDa.
3) the anti-non-specific protein absorption performance of material
Gold plaque ethanol and deionized water are cleaned, and are dried up, and repeat the process 2 times;Then place into clear in ultraviolet cleaning device
Wash 20min.Cleaned 2 times with ethanol and deionized water again, dried up, three kinds of thioctic acid base end-blocking of 2mg/mL are immersed respectively not
With culture 48h in the phosphate buffer (PBS pH 7.4) of molecular weight polypeptide.Further take out, deionized water is rinsed again 5 times, is blown
It is dry.The resulting non-specific protein absorption with self-assembling polypeptide monomolecular film (SAMs) gold plaque is passed through into HRP labellings
Goat anti-human immunoglobulin (anti-IgG/HRP) and Fibrinogen (fibrinogen, Fg) in conjunction with HRP labeled fibers
The surface excess of albumen original antibody is determined.Using TCPS surfaces as control, set the amount of TCPS surface adsorption protein as
100%.Detailed operation process is as follows:Testing sample is cultivated first with anti-IgG (1 μ g/mL) or Fg (10 μ g/mL)
20min, then rinses to remove all unadsorbed protein with PBS.For Fg, in addition it is also necessary to the fibrin of HRP labellings
Former (anti-Fg/HRP) antibody is determining its adsorbance.Adsorbed the sample of Fg again with the fiber egg of 10 μ g/mL HRP labellings
White original antibody (anti-Fg/HRP) culture 20min, then rinses to remove all unadsorbed protein with PBS.Hereafter, institute
There are testing sample and TCPS controls to be respectively put into 24 orifice plates, the Fructus Citri Limoniae of the o-phenylenediamine (OPD) of 1mL 1mg/mL is injected per hole
Acid buffering solution (0.1M, pH 5.0, containing 0.03% hydrogen peroxide).After colour developing 20 minutes, the 2N H of 1mL are added per hole2SO4
Enzymatic activity is quenched.Microplate reader determines absorbances of the 492nm per hole, and calculates protein sub-optimal fusion algorithm by unit area.
Test result is as shown in figure 14.From the figure, it can be seen that as molecular weight increases to 12.8kDa from 3.2kDa, it is right
In anti-IgG and Fg sub-optimal fusion algorithm respectively from 3.8 ± 1.6%, 5.6 ± 1.6% increase to 11.4 ± 4.8%, 15.5 ±
3.2%.For all of test sample, the sub-optimal fusion algorithm of Fg will be higher than anti-IgG, and this is likely due to Fg bodies
Product it is larger, it is loosely organized, easy degeneration and cause extra absorption relevant.On the whole, the polypeptide of three kinds of different molecular weights is equal
There is more outstanding anti-non-specific protein absorption ability.
Embodiment 10:
1) polycondensation
Take the first monomer (its structural formula is shown in formula I) 100mg (0.2mmol) and second comonomer (its structural formula such as formula III
It is shown) 100mg (0.2mmol), EDC.HCl (115mg, 0.6mmol) and HOBt (81mg, 0.6mmol) is added, then press respectively
It is 20 according to the integral molar quantity and the mol ratio of the cysteamine of mercaptotrityl protection of the first monomer and second comonomer: Isosorbide-5-Nitrae 0: 1,
200: 1 mol ratio adds the cysteamine of end-capping reagent mercaptotrityl protection.The anhydrous N of about 2mL, N- diformazans are added toward system
After amide (DMF), rubber closure good seal is used, be passed through room temperature confined reaction 36h after nitrogen bubble 30min.After reaction terminates, to
The absolute ether precipitation of about 18mL is added in reaction system, after static about 20min, mercaptotrityl is obtained after the solvent that inclines respectively
The cysteamine base of protection blocks the polypeptide of the non-Deprotection of three kinds of molecular weight.
2) Deprotection
After with about 2mL trifluoroacetic acids (TFA) above-mentioned grease is completely dissolved, about 2mL mass fractions 33% are added
(33wt.%) hydrobromic acid acetic acid (HBr/HOAc) solution deprotection, about 1h or so reaction systems will become cloudy.About 3h is backward for reaction
The absolute ether precipitation that 25mL is added in reaction system obtains product.Incline after solution, add the absolute ether of about 10mL again
Washed once.After the polypeptide distilled water of Deprotection is completely dissolved, saturation NaHCO is used3Solution is neutralized to neutrality, then will
Solution is transferred in the bag filter that molecular cut off is 1000 (MWCO=1000) and dialyses 2-3 days, and the solution for obtaining is chilled, true
Sky is dried, and obtains white solid product, and product is cysteamine base carboxylic end seal end amphion polypeptide (structural formula such as Formula X XIII institute
Show, n: m=1: 1) molecular weight is respectively 3.4kDa, 5.6kDa and 13.1kDa.
3) the anti-non-specific protein absorption performance of material
Gold plaque ethanol and deionized water are cleaned, and are dried up, and repeat the process 2 times;Then place into clear in ultraviolet cleaning device
Wash 20min.Cleaned 2 times with ethanol and deionized water again, dried up, three kinds of thioctic acid base end-blocking of 2mg/mL are immersed respectively not
With culture 48h in the phosphate buffer (PBS pH 7.4) of molecular weight polypeptide.Further take out, deionized water is rinsed again 5 times, is blown
It is dry.The resulting non-specific protein absorption with self-assembling polypeptide monomolecular film (SAMs) gold plaque is passed through into HRP labellings
Goat anti-human immunoglobulin (anti-IgG/HRP) and Fibrinogen (fibrinogen, Fg) in conjunction with HRP labeled fibers
The surface excess of albumen original antibody is determined.Using TCPS surfaces as control, set the amount of TCPS surface adsorption protein as
100%.Detailed operation process is as follows:Testing sample is cultivated first with anti-IgG (1 μ g/mL) or Fg (10 μ g/mL)
20min, then rinses to remove all unadsorbed protein with PBS.For Fg, in addition it is also necessary to the fibrin of HRP labellings
Former (anti-Fg/HRP) antibody is determining its adsorbance.Adsorbed the sample of Fg again with the fiber egg of 10 μ g/mL HRP labellings
White original antibody (anti-Fg/HRP) culture 20min, then rinses to remove all unadsorbed protein with PBS.Hereafter, institute
There are testing sample and TCPS controls to be respectively put into 24 orifice plates, the Fructus Citri Limoniae of the o-phenylenediamine (OPD) of 1mL 1mg/mL is injected per hole
Acid buffering solution (0.1M, pH 5.0, containing 0.03% hydrogen peroxide).After colour developing 20 minutes, the 2N H of 1mL are added per hole2SO4
Enzymatic activity is quenched.Microplate reader determines absorbances of the 492nm per hole, and calculates protein sub-optimal fusion algorithm by unit area.
Test result is as shown in figure 15.From the figure, it can be seen that as molecular weight increases to 13.1kDa from 3.4kDa, it is right
In anti-IgG and Fg sub-optimal fusion algorithm respectively from 4.2 ± 1.2%, 6.0 ± 1.7% increase to 12.0 ± 3.8%, 16.2 ±
3.2%.For all of test sample, the sub-optimal fusion algorithm of Fg will be higher than anti-IgG, and this is likely due to Fg bodies
Product it is larger, it is loosely organized, easy degeneration and cause extra absorption relevant.On the whole, the polypeptide of three kinds of different molecular weights is equal
There is more outstanding anti-non-specific protein absorption ability.
The specific embodiment of the present invention is described above in association with accompanying drawing, it is apparent that the invention is not restricted to above enforcement
Example, any change and deformation on the basis of the claims in the present invention, all within the scope of the present invention.
Claims (15)
1. a kind of side chain contains by protection carboxyl and two peptide monomers by protection amino, it is characterised in that:With such as with following formula I or formula
Structure shown in II:
2. the preparation method of two peptide monomers described in a kind of claim 1, it is characterised in that:By the first reactant and the second reaction
Thing carries out condensation reaction, and two peptide monomer is obtained after hydrolysis;First reactant is that amido protecting and side chain carboxyl group are protected
Glutamic acid, or amido protecting and side chain carboxyl group protection aspartic acid;Second reactant be carboxy protective and
The lysine of omega amido protectings.
3. preparation method according to claim 2, it is characterised in that:Racemization is included in the reaction system of the condensation reaction
Inhibitor.
4. a kind of side chain contains by protection carboxyl and two peptide monomers by protection amino, it is characterised in that:With such as following formula III extremely
Structure shown in any one in Formula IV:
5. the preparation method of two peptide monomers described in a kind of claim 4, it is characterised in that:
3rd reactant and the 4th reactant are carried out into condensation reaction, two peptide monomer, the 3rd reaction are obtained after hydrolysis
Thing is the glutamic acid of alpha amido protectings and carboxy protective, or the aspartic acid of alpha amido protectings and carboxy protective;
4th reactant is the lysine of carboxy protective and alpha amido protectings, or carboxy protective and omega amido protectings
Lysine.
6. preparation method according to claim 5, it is characterised in that:Racemization is included in the reaction system of the condensation reaction
Inhibitor.
7. a kind of anti-non-specific protein absorption polypeptide with controllable enzymatic degradation, it is characterised in that with such as Formula VII to formula
Structure shown in any one in XIV:
During the above is various, m, n represent respectively the degree of polymerization of monomer, integers of the m and n respectively more than or equal to 0, and m+n > 0.
8. polypeptide according to claim 7, it is characterised in that its structural formula is as shown in any one in Formula X V to Formula X XX:
Wherein, R1 is that, containing the carboxylic acid group of the selective reaction group for functional modification after deprotection, R2 is to contain after deprotection
For the amino of the selective reaction group of functional modification.
9. polypeptide according to claim 8, it is characterised in that:R1 is thioctic acid base, or with aminothiopropionic acid on carboxylic
The cysteamine acidic group of base connection;R2 is cysteamine base, or the cysteamine acidic group being connected with the amino in aminothiopropionic acid.
10. the preparation method of the polypeptide described in a kind of claim 7, it is characterised in that include:By the first monomer and/or second
Monomer carries out polycondensation reaction, and condensation polymer is carried out into Deprotection process, obtains the polypeptide;
First monomer has a structure as shown in Formulas I or Formula II, the second comonomer have such as formula III, formula IV, Formula V or
Structure shown in Formula IV:
11. preparation methoies according to claim 10, it is characterised in that:Contain ammonia in the reaction system of the polycondensation reaction
End end-capping reagent or carboxylic end end-capping reagent.
12. preparation methoies according to claim 11, it is characterised in that:Ammonia end end-capping reagent is to contain sulfydryl after deprotection
Carboxylic acid.
13. preparation methoies according to claim 12, it is characterised in that:Ammonia end end-capping reagent is thioctic acid or half Guang ammonia
Acid derivative, the cysteine derivative contains the amino of benzyloxycarbonyl group protection and the sulfydryl of trityl as protecting group.
14. preparation methoies according to claim 11, it is characterised in that:Carboxylic end end-capping reagent is to contain sulfydryl after deprotection
Amine.
15. preparation methoies according to claim 14, it is characterised in that:Carboxylic end end-capping reagent is mercaptotrityl guarantor
The cysteamine of shield, or the cysteine of carboxybenzyl protection and mercaptotrityl protection.
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CN109609333A (en) * | 2018-10-12 | 2019-04-12 | 深圳市瀚海基因生物科技有限公司 | A kind of biochip and preparation method thereof |
CN111560049A (en) * | 2020-05-17 | 2020-08-21 | 浙江大学 | Zwitterionic polypeptide and derivative thereof and nano-drug based on zwitterionic polypeptide and derivative |
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CN104926923A (en) * | 2015-06-11 | 2015-09-23 | 合肥工业大学 | Invisible polypeptide with good non-specific protein absorption resistance performance and preparation method thereof |
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CN104926923A (en) * | 2015-06-11 | 2015-09-23 | 合肥工业大学 | Invisible polypeptide with good non-specific protein absorption resistance performance and preparation method thereof |
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BY R. LEDGE ET AL.: "SEQUENTIAL POLYPEPTIDES CONTAINING y-BENZYL-L-GLUTAMYI,", 《AUST. J. CHEM.》 * |
IVANOV, V. T. ET AL.: "Synthetic studies of -Bungarotoxin", 《TOXICON》 * |
QINGHUA YANG ET AL.: "Investigation of nonfouling polypeptides of poly(glutamic acid) with lysine side chains synthesized by EDC•HCl/HOBt chemistry", 《JOURNAL OF BIOMATERIALS SCIENCE,POLYMER EDITION》 * |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109609333A (en) * | 2018-10-12 | 2019-04-12 | 深圳市瀚海基因生物科技有限公司 | A kind of biochip and preparation method thereof |
CN111560049A (en) * | 2020-05-17 | 2020-08-21 | 浙江大学 | Zwitterionic polypeptide and derivative thereof and nano-drug based on zwitterionic polypeptide and derivative |
CN111560049B (en) * | 2020-05-17 | 2022-05-17 | 浙江大学 | Zwitterionic polypeptide and derivative thereof and nano-drug based on zwitterionic polypeptide and derivative |
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