KR100842114B1 - (nitrilotriacetic acid)-end-functionalized polymer, synthesis method thereof and analyzing, separating or purifing method of protein using the same - Google Patents

Abstract

A method for preparing a polymer end-capped with nitrilotriacetic end groups is provided to obtain a polymer useful in the field of bioengineering, particularly for analyzing, separating or purifying histidine-containing proteins. A method for preparing a polymer end-capped with nitrilotriacetic end groups comprises the steps of: (S1) providing an initiator containing nitrilotriacetic acid; and (S2) carrying out atom transfer radical polymerization of monomers by using the initiator. The initiator has a halogen as a functional group. The monomer is any one selected from styrene, acrylate, methacrylate, acrylonitrile, acrylamide, methacrylamide, diene, acrylic acid and methacrylic acid.

Description

Nitrilotriacetic acid-end-functionalized polymer, synthesis method, and analyzing, separating or purifing method of protein using the same }

1 is a graph showing the molecular weight change (GPC) of the α- (p-NTA) -polystyrene with time in the preparation method (No. 5 in Tables 1 and 2) according to an embodiment of the present invention.

¹ of polystyrene (3) - Figure 2 there α- (p-NTA) in the production method (No. 5 of Table 1 and 2) in accordance with one embodiment of the present invention-polystyrene (2) and α- (NTA) H NMR (300 MHz) results.

3 is a GPC of α- (p-NTA) -polystyrene (2) and α- (NTA) -polystyrene (3) in the manufacturing method according to an embodiment of the present invention (No. 5 in Tables 1 and 2 below). The result is.

Figure 4 shows nitrilotriacetic acid, N ' , N' -bis [(tert-butyloxycarbonyl) methyl] -N'' -bromoisobutyryl-L-lysine tert-butyl ester (1b), α- ( FT-IR results of p-NTA) -polystyrene (2), α- (NTA) -polystyrene (3) and α- (Ni-NTA) -polystyrene (4).

5 is N ', N' -bis [(tert-butyloxycarbonyl) methyl] -N'' -bromoisobutyryl-L-lysine tert-butyl ester (1b) according to one embodiment of the invention A graph showing the results of atomic transfer radical polymerization (ATRP) of styrene using (No. 5 in Tables 1 and 2) as an initiator. In FIG. 5, (A) is a graph showing that the first order kinetic plot is linear, and (B) is a conversion rate vs. molecular weight curve, which increases linearly close to the theoretical molecular weight.

In FIG. 6, (A) -1 and (A) -2 are SEM and EDX images of α- (Ni-NTA) -polystyrene (4), respectively, and (B) -1 and (B) -2 are α-, respectively. (NTA) -polystyrene (3) SEM and EDX image.

In FIG. 7, (A) is a TEM image of (alpha)-(p-NTA) -polystyrene (2), (B) is a TEM image of (alpha)-(NTA) -polystyrene (3).

FIG. 8 is a fluorescence micrograph showing the binding of micelles made of α- (NTA) -polystyrene (3) with histidine tagged protein (Green Fluorescent Protein, GFP).

9 is SDS-PAGE showing the binding of p (PEGMA) having a NTA terminal and histidine tag protein (GFP) according to an embodiment of the present invention.

The present invention relates to a polymer having a specific functional group at its end, a method for producing such a polymer, and a method for analyzing, separating and purifying histidine-containing proteins using such a polymer.

Since the combination of polymer and protein is widely used in medicine and biotechnology, research on this is being actively conducted, and various methods for bioconjugation have been developed. Among them, a general method is to use a polymer having a functional group terminal with an ε-amino group of lysine, an N-terminal amino group, or a cysteine thiol. It is reacted with a polypeptide having a group.

In many cases, however, there are more than one specific amino acid of the protein, in which case the bio-binding occurs nonspecifically, resulting in a heterogeneous combination of protein and polymer. This structural heterogeneity degrades the bioactivity of the product. Therefore, it is important to make specific bioconjugation, and research on such selective bioconjugation has been attempted.

Meanwhile, the binding of metal ions and histidine tags to chelates such as iminodiacetic acid and nitrilotriacetic acid has been actively studied. Chelate bonds have several advantages. First, the complexation of metal ions and histidine, such as Ni ^{2 +} is a fast reaction. And reversible reactions are possible using excess chelates or competitor ligands such as ethylenediaminetetraacetic acid (EDTA). Second, Ni ^{2 +} -NTA is a very popular immobilization system. Nickel ions occupy four of the six bond sites and 6 × His-tag bonds to the remaining two sites. Finally, His-tag can bind to both C- or N-terminus and is commercially available for many proteins.

Therefore, the technical problem to be achieved by the present invention is a polymer having a specific end group that can be usefully used in the field of biotechnology, such as the analysis and separation of a specific protein, a method for preparing such a polymer and the analysis, separation, It is to provide a method such as purification.

In order to achieve the above technical problem, the present invention provides a polymer having a nitrilotriacetic acid terminal.

The invention also comprises the steps of preparing an initiator comprising (S1) nitrilotriacetic acid; And (S2) provides a method for producing a polymer having a nitrilo triacetic acid terminal comprising the step of preparing a polymer by atom transfer radical polymerization of the monomer using the initiator.

The present invention also provides a method for analyzing, separating or purifying histidine-containing proteins using a polymer having nitrilotriacetic acid ends.

Hereinafter, a polymer having a nitrilotriacetic acid terminal of the present invention, a method for preparing the same, and a method for analyzing, separating or purifying a protein using the polymer will be described in more detail.

The present invention provides a polymer having a nitrilotriacetic acid terminal, and more preferably provides a polymer having a nitrilotriacetic acid terminal, wherein the nitriloacetic acid terminal is represented by the following formula (1).

As the monomer forming the polymer having the nitrilotriacetic acid terminal, any monomer can be used as long as it is capable of atom transfer radical polymerization. For example, styrene, acrylate, methacrylate, acrylonitrile (acrylonitrile), acrylamide, methacrylamide, diene, acrylic acid, methacrylic acid, derivatives thereof, and the like may be used, but is not limited thereto. Such monomers may be appropriately selected depending on the intended use.

Such a polymer having a nitrilotriacetic acid terminal may form a complex with a metal ion, and the metal ion bound to the complex has a property of binding to a histidine tag contained in the protein. The complexation of these metal ions with histidine is a rapid reaction, and the nitriloacetic acid terminal of the present invention can be reversible by using a strong chelate such as ethylenediaminetetraacetic acid (EDTA) or a competitor ligand such as imidazole in excess. The macromolecular polymer may be usefully used for analysis, separation and purification of a protein having a histidine tag.

Forming a polymer and a composite having a nitrilotriacetic acid terminal of the present invention it includes ^{Ni 2 +, Cu 2 +,} Co +2, Zn 2 + can be used as metal ions that can be used for analysis of the protein.

The invention also comprises the steps of preparing an initiator comprising (S1) nitrilotriacetic acid; And (S2) provides a method for producing a polymer having a nitrilo triacetic acid terminal comprising the step of preparing a polymer by atom transfer radical polymerization of the monomer using the initiator.

In the above production method, the initiator is not particularly limited as long as it contains nitrilotriacetic acid and can form organic radicals by an atomic transfer reaction. For example, the initiator may be a low molecular organic compound or a high molecular organic compound. Such initiators should include functional groups capable of forming radicals by atomic transfer reactions, with halogens such as Cl, Br, and I being preferred.

The initiator of the present invention may include various functional groups in addition to the nitrotriacetic acid and the radical-forming functional group according to various purposes such as solubility control and dispersibility improvement. For example, the hydrogen of the hydroxy group in the nitrilotriacetic acid included in the initiator may be substituted with a protecting group such as t-butyl, in which case the production method of the present invention further includes removing the protecting group. .

In another aspect, the present invention provides a method for producing a polymer having a nitrilotriacetic acid terminal, characterized in that using the initiator represented by the formula (2) as the initiator.

In Formula 2, R is hydrogen or methyl, X is any one of the above-described functional groups.

In the production method of the present invention, any monomer can be used as long as it can perform atom transfer radical polymerization, for example, styrene, acrylate, methacrylate, acrylonitrile (acrylonitrile), acrylamide, methacrylamide, diene, acrylic acid, methacrylic acid and derivatives thereof may be used, but is not limited thereto.

In addition, the preparation method of the present invention includes a catalyst M _t ^{n +} X _n and a ligand in the radical polymerization of the step (S2).

M _t ^{n +} X _n is a transition metal compound, M _t is a transition metal, X is a negative ion component combined with a transition metal, n is the formal charge of the transition metal. These transition metal (M _t ^{+ n)} is ^{Cu 1+, Cu 2 +, Fe} 2 +, Fe 3 +, Ru 2 +, Ru 3 +, Cr 2 +, Cr 3 +, Mo 0, Mo +, Mo ^{2 +} , Mo ^{3 +} , W ^{2 +} , W ^{3 +} , Rh ^{3 +} , Rh ^{4 +} , Co ⁺ , Co ^{2 +} , Re ^{2 +} , Re3 +, Ni ⁰ , Ni ⁺ , Mn ^{3 +} , Mn ^{4 +} , V ^{2 +,} V ^{3 +,} Zn ^+, Zn ^{2 +,} Au ^+, Au ^{2 +,} Ag ^+, and may be any one that is used selected from the group consisting of Ag ^{2 +,} X roneun halogen, alkoxy having 1 to 6 carbon atoms , (SO ₄ ) _1/2 , (PO ₄ ) _1/3 , (HPO ₄ ) _1/2 , (H ₂ PO ₄ ), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, SeR ¹ Any one selected from the group consisting of, CN and R ² CO ₂ can be used. Wherein R ¹ is aryl or a straight or branched alkyl group having 1 to 20 carbon atoms, R ² is H or a straight or branched alkyl group having 1 to 6 carbon atoms, and n is a formal charge of the transition metal, 0 ≦ n ≦ 7 is more preferable.

As the ligand, various ligands may be used according to the purpose according to the type of catalyst, such as nitrogen-based ligand, phosphorus ligand, monomer, etc. ("Atom Transfer Radical Polymerization", Chemical Reviews, 2001, Vol. 101, No. 9, p. 2941-2942).

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, the embodiments according to the present invention may be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> NTA  Terminal Polystyrene  Produce

Functional polystyrenes having NTA (nitrilotraacetic acid) ends were prepared according to Scheme 1 below.

N ' , N ' -Bis [( tert - Butyloxycarbonyl ) methyl ]- N '' - Bromopropionyl Synthesis of -L-lysine tert-butyl ester (1a)

N ^α , N ^α -bis [( tert -butyloxycarbonyl) methyl] -L-lysine tert -butyl ester ( p-AB NTA , 342 mg, 7.7 × 10 ^-1 mmol) and triethylamine in 50.0 ml THF (TEA, 320 ul, 2.3 mmol ) was dropped slowly with 2-bromopropionyl bromide (2-bromopropionyl bromide (90.0 ul , 8.5 × 10 -1 mmol) in a mixed solution loaded. with p- AB The synthesis of NTA proceeded according to known literature (Langmuir Vol. 19, No. 5, p. 1671-1680, 2003). The experiment was carried out in an ice bath under nitrogen, and stirred for 12 hours at room temperature one hour after the addition of 2-bromopropionyl bromide. Thereafter, THF was blown, dissolved in 100 ml CH ₂ Cl ₂ , and titrated five times using 100 ml of water. Finally, the product containing impurities was purified by column chromatography by flowing a mixed solvent of 4: 1 n-hexane: ethyl acetate to synthesize an initiator having NTA. The structure was confirmed by NMR analysis.

N ' , N ' -Bis [( tert - Butyloxycarbonyl ) methyl ]- N '' - Bromoisobutyryl Synthesis of -L-lysine tert-butyl ester (1b)

1a equal to the synthetic procedure described above and was used for 2-bromo-propionyl bromide (bromopropionyl bromide) 2-Bromo-assemble small butyryl bromide (bromoisobutyryl bromide) (103.7 ul, 8.5 × 10 -1 mmol) in place.

α- (p- NTA )- Of polystyrene (2)  synthesis

Add styrene (1.0 ml) and anisole (1.0 ml) to the Schlenk flask, replace with nitrogen, repeat the freeze-pump-thaw cycle three times, and use CuCl (36.4 mg) and 4,4'-di (5- Nonyl) -2,2'-bipyridine (dNbpy, 300.8 mg) was added and again the freeze-pump-thaw cycle was repeated twice. The flask was placed in an oil reactor at 110 ° C. and the initiator 1b (106.5 mg, 18.4 × 10 ⁻² mol) prepared previously was added. Samples were taken at regular intervals and diluted in THF to determine the degree of polymerization using GC and GPC. The results are shown in FIG. The product was then precipitated using methanol to give 573 mg of α- (p-NTA) -polystyrene (2). The structure was confirmed by NMR analysis, and the results are shown in FIG. 2.

α- ( NTA )- Of polystyrene (3)  synthesis

Put the prepared α- (p-NTA) -polystyrene (300mg, 61.2 × 10 ^-3 mmol) and 20.0ml CH ₂ Cl ₂ into the flask, trifluoroacetic acid (TFA, 136.7 ul, 18.4 × 10 ^-4 mol ) Was added. Thereafter, the mixture was stirred at room temperature for 12 hours, and precipitated in methanol to obtain 187 mg of α- (NTA) -polystyrene (3). The structure was confirmed by NMR analysis, and the results are shown in FIG. 2.

In other words, the polystyrene ( 3 ) at the NTA end was obtained by removing the t-butyl portion from the CH ₂ Cl ₂ / TFA with the polystyrene ( 2 ) at the NTA end. In ¹ H-NMR of Figure 2 it can be seen that 1.38 ppm and 1.40 ppm of t-butyl disappears. As shown in FIG. 3, it can be seen that the maximum molecular weight values in GPC are the same, but they show agglomeration at high molecular weights, which can be explained by the interaction of hydrogen bonding of carboxylic acid of styrene chain and GPC stationary phase. Can be. As shown in Figure 4, the synthesis was also confirmed through the IR results.

α- ( Ni - NTA )- Of polystyrene (4)  synthesis

The prepared α- (NTA) -polystyrene (100 mg, 21.3 × 10 ⁻³ mmol) was dissolved in 50 ml DMF, and nickel chloride (NiCl ₂ , 0.4 mmol, 54.4 mg) was added thereto. After stirring at room temperature for 12 hours, precipitation was carried out in methanol to obtain 97 mg of α- (Ni-NTA) -polystyrene (4).

Initiator , Catalysts and Ligand  Impact assessment

Similar to the method described above, a styrene atom transfer radical polymerization (ATRP) using an NTA-derived initiator was carried out at about 110 ° C., except that the initiator, catalyst and / or ligand were modified. Polystyrene was prepared, thereby evaluating the effect of initiator, catalyst or ligand on the production of polystyrene with NTA termini. The preparation conditions of each experiment are shown in Table 1 below, and the results are shown in Table 2 below.

number [Styrene] ₀ [Initiator] ₀ [Catalyst / ligand] ₀ Anisole One 7.869M 0.012M 0.012M 0.78ml 2 4.33M 0.65 × 10 ^-2 M 1.3 × 10 ^-2 M 7.76 ml 3 4.33M 2.25 × 10 ^-2 M 4.5 × 10 ^-2 M 1.5ml 4 7.9M 4.1 × 10 ^-2 M 4.1 × 10 ^-2 M 0.8ml 5 4.4M 0.92 × 10 ^-1 M 1.84 × 10 ^-1 M 1ml

number [M] ₀ / [I] ₀ Initiator [I] ₀ Catalyst / ligand Hour (hr) Conversion rate [%] M _n [10 ³ ] M _w / M _n One 672 1a CuBr / PMDETA 17 21 117 1.58 2 672 1a 2 × CuCl / PMDETA 7 21 140 1.92 3 196 1b 2 × CuCl / 2dNbpy 14 58 13.5 1.21 4 190 1b CuBr / 2bpy 20 77 18.9 1.24 5 48 1b 2 × CuCl / 2dNbpy 14 88 4.9 1.09

In Table 1, PMDETA means N, N, N ', N' ', N' '-pentamethyldiethylenetriamine (Aldrich) purified by vacuum distillation, and dNbpy is 4,4'-di ( 5-nonyl) -2,2'-bipyridine and bpy means 2,2'-dipyridyl.

Styrene was polymerized using the ATRP initiator 1a with the NTA, CuBr catalyst, and PMDETA ligand (No. 1 in Tables 1 and 2). In this case, the slow initiation of the 1a initiator resulted in a polymer having a low molecular weight and a high molecular weight and a wide molecular weight distribution. In order to speed up the initiation, a halogen exchage method was introduced using CuCl / PMDETA instead of CuBr / PMDETA. However, styrene polymerization still showed a low initiation tendency (No. 2 in Tables 1 and 2).

This low initiation tendency could be improved by using bipyridine-based ligands and 1b initiators, resulting in well controlled polystyrene (No. 3 in Tables 1 and 2). These results show that 2-bromoisobutyryl has a faster activation rate (production of radicals) than 2-bromopropionyl. In addition, the fast deactivation rate of the catalyst using the bi-pyridine series results in an improvement in the starting efficiency. The 1b initiator and CuCl / dNbpy catalyst show the behavior of typical living radical polymerizations. The first order kinetic plot is linear and the molecular weight for conversion also increases linearly close to the theoretical molecular weight, with a narrow molecular weight distribution of 1.09 (see Figure 5).

< Experimental Example  1> NTA  Terminal Polystyrene  evaluation

Gfp ( Green Fluorescence Protein Expression and Purification

Histidine-tagged GFP with hexahistidine as the N-terminus in E. coli BL21 (DE3) was grown at 37 ° C. until the OD was about 0.6 in 100 ml LB medium, followed by 0.05 mM IPTG was added and induced at 20 ° C. for 6 hours. Cells were lysed using Cell Lysis Buffer (Novagen) and His-tagged GFP protein thus obtained was bound to 5 mg Ni-NTA HisBind Resin (Novagen) at 4 ° C. for 3 hours and then packed into a column. Then wash twice with 4 ml each with wash buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl and 30 mM imidazole), and then 1 ml of elution buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl and 250 mM imidazole). The proteins to be obtained were allowed to elute. The purity of the eluted protein was confirmed using a 12% SDS-PAGE gel.

Vesicle  Solution preparation

A vesicle solution was prepared by evaporating the solvent. After dissolving α- (NTA) -polystyrene ( 3 ) and α- (Ni-NTA) -polystyrene ( 4 ) prepared in the above-described manner in 2 ml of THF, water (8 ml) was gradually dropped while stirring rapidly. Then heat was applied at 60 ° C. for 3 hours to remove THF and sonication for 30 minutes.

A drop of the vesicle solution of α- (NTA) -polystyrene ( 3 ) and α- (Ni-NTA) -polystyrene ( 4 ) was dropped and dried at 40 ° C. for 4 hours to prepare a SEM / EDX sample. The results are shown in FIG. As in Figure 6, it has a uniform spherical particles of ~ 500nm. C and O can be seen from the initiator, and Ni is chelated with NTA on the styrene vesicle surface at the nickel-bonded 4 . Si is from a silicon wiper.

NTA  Polymer with terminal Gfp Combination of

The binding of polystyrene and GFP with NTA terminus can be confirmed using Florescence microscopy. GFP solution (stock solution) was added to the prepared vesicle solution, followed by binding to imidazole. In the case of α- (Ni-NTA) -polystyrene ( 4 ) which selectively binds to GFP, it can be seen that 86% is eluted when measuring the amount of GFP that is eluted, and α- (NTA) -polystyrene (3). In the case of), non-selectively bound GFP is present, but about 10% of the material eluted when imidazole is flowed is irreversible NTA-His tag bond (non-selective bond, not selective bond using Ni). It could be confirmed that it is not removed well (see FIG. 8).

< Example  2> NTA  Terminal PEGMA Manufacture

Poly (ethylene glycol) methyl ether methacrylate (PEGAMA) was used as a monomer to prepare PEGMA having an NTA terminal according to Scheme 2 below. Because PEGMA is soluble in water, it can be useful for bio applications.

α- (p- NTA ) -p ( PEGMA Synthesis of 2

PEGMA (2.4 ml) and anisole (2.4 ml) were added to a Schlenk flask under nitrogen. After freeze-pump-thaw three times, CuBr (39.6mg) and PMDETA (56ul) were added to the flask and freeze-pump-thaw was added twice. The flask was then placed in an oil reactor at 90 ° C. And initiator ( 1 ) (159.6 mg, 2.8 × 10 ^-4 mol) was added to the flask. During the polymerization, samples of about 0.1 ml were taken, dissolved in THF, and analyzed by GC and GPC. At the end of the polymerization, diethyl ether was used for precipitation.

α- ( NTA ) -p ( PEGMA Synthesis of (3)

α- (p-NTA) -p (PEGMA) (2) (1.004 g, 2.2 mmol) was placed in a flask and dissolved in 40.0 ml of THF. Trifluoroacetic acid (TFA, 10 ml, 1.3 × 10 ⁻¹ mol) was placed in the flask. The magnetic bar was mixed at room temperature for 12 hours, and then precipitated using isopropyl ether.

In the preparation, the polymerization rate of pPEGMA was about 75%, the molecular weight determined by GPC was 26,950 g / mol, and the molecular weight distribution was 1.32. The structure of α- (p-NTA) -p (PEGMA) (2) was confirmed by ¹ H-NMR. In the α- (p-NTA) -p (PEGMA) (2), the t-butyl group was removed using TFA / THF to obtain α- (NTA) -p (PEGMA) (3), followed by removal of ¹ H. It was confirmed through -NMR, the peak of the t-butyl group at 1.38 ppm and 1.40 ppm in ¹ H-NMR disappeared.

< Experimental Example  2> NTA  Terminal PEGMA Evaluation of

Green fluorescent protein (GFP) was used as a bio-binding model because it fluoresces without other coenzymes. It was confirmed that bio-bonding occurred through SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), and the results are shown in FIG. 9. Imidazole was also used to confirm that this bio binding was reversible.

Scheme 3 below shows the biocojugation of p (PEGMA) with the NTA terminus and histidine tagged protein.

The present invention provides a method of analyzing, separating and purifying a histidine-containing protein using a polymer having a nitrilotriacetic acid terminal and a complex of such a polymer and a metal ion. The polymer having the nitrilotriacetic acid terminal of the present invention can be prepared by atom transfer radical polymerization (ATRP) of the monomer using an initiator having nitrilotriacetic acid.

Claims (9)

delete delete delete (S1) preparing an initiator comprising nitrilotriacetic acid; And (S2) preparing a polymer by atom transfer radical polymerization of the monomer using the initiator; And Method for producing a polymer having a nitrilotriacetic acid terminal, characterized in that it comprises a. The method for producing a polymer having nitrilotriacetic acid terminal according to claim 4, wherein the initiator contains a halogen as a functional group. The method of claim 4, wherein the monomer is styrene, acrylate, methacrylate, acrylonitrile, acrylamide, methacrylamide, diene, acrylic acid and Method for producing a polymer having a nitrilotriacetic acid terminal, characterized in that any one selected from the group consisting of methacrylic acid. The method of claim 4, wherein the hydrogen of the hydroxy group in the nitrilotriacetic acid contained in the initiator is substituted with a protecting group, the production method further has a step of removing the protecting group having a nitrilotriacetic acid terminal Method for producing a polymer. delete delete

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