JP3044299B2 - Copolymer having amino acid residue in side chain and thermoresponsive polymer, amino acid discriminating agent and optical resolving agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a copolymer having an amino acid residue in a side chain, and a thermoresponsive polymer, an amino acid discriminating agent and an optical resolving agent using the same.

[0002]

2. Description of the Related Art Conventionally, poly (N-isopropylacrylamide) has been known as a thermoresponsive polymer. With respect to this poly (N-isopropylacrylamide), those obtained by copolymerizing other functional monomers have been studied. However, this is a polymer that recognizes enzyme reactions, redox reactions, metal ion species, etc., including temperature and pH, and polymers that recognize optical isomers and amino acids have not yet been developed.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel polymer having an amino acid residue in a side chain, which can be used as a thermoresponsive polymer or the like. Another object of the present invention is to provide a thermoresponsive polymer comprising the above polymer compound having an amino acid residue in the side chain. Also, an object of the present invention is to provide an amino acid discriminating agent comprising the above-mentioned polymer compound having an amino acid residue in a side chain and capable of easily discriminating and separating types of amino acids, and an optical isomer (D-form or L-form) of an amino acid An object of the present invention is to provide an optical resolving agent that can be easily identified and separated.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and have found that a monomer having an amino acid residue in a side chain and an N-isopropylacrylamide monomer are used. Based on this finding, it was found that a copolymer obtained by polymerizing and introducing a side chain having a carboxyl group and an amino group as an ionic group had a higher transition temperature, amino acid recognition, and an optical resolving agent function. The present invention has been made. The above object of the present invention has been achieved by the following copolymer, amino acid discriminating agent and optical resolving agent. (1) A copolymer represented by the following formula (I).

[0005]

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Formula (I) (wherein m and n indicate the polymerization ratio (%) of each component in the copolymer.) (2) Consisting of the copolymer represented by the above formula (I) A thermoresponsive polymer, characterized in that: (3) An amino acid discriminating agent comprising a copolymer represented by the above formula (I). (4) An optical resolving agent for amino acids, comprising the copolymer represented by the above formula (I).

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The copolymer represented by the general formula (I) of the present invention is obtained by copolymerizing a compound of the following formula (II) with N-isopropylacrylamide (hereinafter referred to as "NIPAAm"), And Y by deprotection.

[0008]

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(Wherein, Z represents a protecting group for a hydrogen atom of an amino group, and Y represents a protecting group for a hydroxyl group of a carboxyl group. Examples of the protecting group Z include benzyloxycarbonyl and t-butoxycarbonyl. Benzyl and the like are cited as the protecting group Y substituted for the hydroxyl group in the carboxyl group.) In the copolymer (I), when the molar ratio between NIPAAm and the compound (II) is 9: 1, 2.00 g / When the optical rotation α and the specific rotation [α] were measured at an optical path length of 10.0 cm using an aqueous solution of 0.1 ml, α = 0.286, respectively.
[Α] = 14.290, indicating that the lysine residue in the polymer was not racemized.

The aqueous solution of the copolymer (I) dissolves in water below the transition temperature, but above the transition temperature, causes phase separation and insolubilization in water, and the solution becomes cloudy. Can be used. Further, since this copolymer also has different affinity for amino acids, the transition temperature of the complex changes depending on the type of amino acid added. This allows
This copolymer can also be used as a reagent for identifying and resolving the type of amino acid. This copolymer can be applied to amino acid recognition and optical recognition by a ligand exchange method. More specifically, the aqueous solution of the copolymer represented by the formula (I) of the present invention dissolves in water at a transition temperature or lower, but causes phase separation and insolubilization in water at a transition temperature or higher,
The solution becomes cloudy. Therefore, when the turbidity change of the aqueous solution is measured under different conditions such as temperature and pH, the change in the phase transition behavior can be clarified. The copolymer contained in the present invention is dissolved in a buffer solution, and an amino acid solution to be discriminated is added in the presence of a complex-forming ion to form a complex.
Examples of the complex forming ions include ions of metals such as Cu, Co, Cr, and Ni, and are preferably Cu ²⁺ ions.

The buffer is preferably an acetic acid / potassium hydroxide solution. The pH of the buffer is preferably in the range of 4 to 6. The ionic strength is preferably in the range of 0.001 to 0.1.
The concentration of the copolymer in the buffer can be appropriately determined in consideration of its solubility, but is preferably 0.05 to 0.5% by weight. The amount of the amino acid to be added is preferably 3 to 10 times the molar amount of the amino acid residue of the copolymer. The complex is formed by adding the D-form and L-form of the added amino acid.
The phase transition behavior of the complex is also different because it is different in the body. Therefore, by measuring the change in transmittance of the mixture containing the copolymer, the complex-forming ion, and the amino acid due to the temperature change, the type of the amino acid, the D-form and the L-form can be identified and divided. In this case, the amino acids that can be subjected to the optical resolution of the D-form and the L-form are preferably those having a large hydrophobic group.
The amino acid recognizing agent containing the copolymer of the present invention is an amino acid L
It is considered that the reason that the L-form and the D-form can be distinguished from each other is that the L-form forms a stable complex via copper ions, whereas the L-form and the D-form make it difficult to form a complex. Such a copolymer of the present invention can also be used as a biosensor or a column filler.

[0012]

Next, the present invention will be described in more detail with reference to Reference Examples and Examples. Reference Example Compound (5) was synthesized according to the following scheme.

[0013]

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1) ZL-Lys (Boc) -OBzl
A synthetic commercial product of (2), ZL-Lys (Boc) (1), was reacted with benzyl alcohol to protect the α-carboxyl group. ZL-Lys (Boc) (1) (manufactured by Kokusan Chemical Co., Ltd.): 1.00 g (2.63 mmol), dimethylaminopyridine (DMAP): 0.0321 g (0.
263 mmol) and benzyl alcohol (BzlO)
H): Dissolve 0.34 g (3.15 mmol) in 15.0 ml of methylene chloride and stir at 0 ° C.
Cl (water-soluble carbodiimide hydrochloride): 0.552 g
(2.88 mmol) was added. After stirring at 0 ° C for 2 hours and at room temperature overnight, the mixture was concentrated under reduced pressure, and ethyl acetate-water (5:
Extracted in 1). The organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate and then with water, and then dried over anhydrous magnesium sulfate. It was added to a large excess of a hexane-acetone (9: 1) solution to precipitate crystals of ZL-Lys (Boc) -OBzl (2). After drying, a white powder was obtained. Yield 1.07
86g, 80% yield, mp 78.7-80.1 ° C. NM
R (CDCl ₃ , room temperature, δ): 1.3-1.6 (t,
_{(CH 2) 4, 8H)} , 7.3-7.4 (s, benzene .10H), Calcd (%) C, 66.3
6; H, 7.29; N, 5.95, found (%) C, 6
5.26; H, 7.19; N, 5.87.

2) The combination of ZL-Lys-OBzl (3)
Synthesis, (Removal of Boc group ) ZL-Lys (Boc) -OBzl (2): 0.80
6 g (1.70 mmol) was dissolved in 5.0 ml of ethyl acetate, and 8.5 ml of a 4N hydrochloric acid / ethyl acetate solution was added thereto.
Was added dropwise and stirred for 1 hour. After the reaction solution was concentrated under reduced pressure, ether-petroleum ether was added, and ZL-Lys-OBz was added.
l (3) was precipitated. After drying, an oily product was obtained.
Since the Z group protecting the amino group at the α-position was not removed, only the amino group at the δ-position could be made free.

3) Monomer ZL-Lys-OBzl-A
Synthesis of Am (5) ZL-Lys-OBzl (3): 0.630 g (1.
70 mmol), triethylamine: 0.378 g
(3.74 mmol) was dissolved in 5.0 ml of tetrahydrofuran, and acrylic acid chloride: 0.18 in an ice bath.
5 g (2.04 mmol) were added dropwise. After stirring for 1 hour in an ice bath, the mixture was concentrated under reduced pressure. The precipitate was dissolved in methylene chloride, washed with a 5% aqueous solution of sodium hydrogen carbonate and then with water, and then dried over anhydrous magnesium sulfate. The compound of the present invention, ZL-Lys-OBzl-AAm (5), was precipitated in a large excess of ethyl acetate-petroleum ether (1: 9) solution. After drying, a white powder was obtained. Yield 0.703
5 g, 76% yield, mp 105.3-107.7 ° C. N
MR (CDCl ₃ , room temperature, δ): 1.3-1.6 (t,
_{(CH 2) 4, 8H)} , 5.5-6.4 (m, CH 2 =
CH, 3H), 7.3-7.4 (s, benzene, 10
H) Elemental analysis: calculated (%) C, 67.90; H, 6.
66; N, 6.60; found (%) C, 66.60;
H, 6.66; N, 6.46.

Example 1 (Synthesis of NIPAAm-L-Lys Copolymer (8)) 1) Acrylamide-based monomer (Z-
L-Lys-OBzl-AAm) (5) and N-isopro
When the Z group and the OBzl group are removed in the state of the copolymerized monomer of pyracrylamide (6), the vinyl group reacts and the polymerization reaction becomes impossible. Therefore, copolymerization with NIPAAm was carried out with both protecting groups attached to obtain a polymer, and then the protecting groups were removed. ZL-Lys-OBzl-AAm (5): 0.40 g
(0.94 mmol), N-isopropylacrylamide (NIPAAm) (6): 0.9596 g (8.48)
mmol) (molar ratio; NIPAAm (6): ZLL)
ys-OBzl-AAm (5) = 9: 1)
Butyl alcohol: dissolved in 15.0 ml. Α, α'-azobisisobutyronitrile: 1 as a polymerization initiator
5.4 mg (0.0942 mmol) was added, and the mixture was stirred at 60 ° C. for 20 hours under a nitrogen atmosphere to polymerize. After completion of the reaction, the precipitate was precipitated in a large excess of hexane, and then dried under reduced pressure.
A copolymer (7) was obtained. From the NMR measurement, the constitutional ratio of (7) is a molar ratio, and NIPAAm (6): ZL-Lys-
It was revealed that OBzl-AAm (5) = 10.6: 1. From this value, the Z-L charged during the polymerization was used.
It was found that 85.0% of -Lys-OBzl-AAm (5) had reacted. Yield 1.2523 g, yield 9
2.1%

2) NIPA of the present invention by removing a protecting group
Synthesis of Am-L-Lys copolymer (8) Copolymer (7): 0.150 g dissolved in 1M trifluoromethanesulfonic acid-thioanisole (molar ratio: 1: 1) /0.880 ml of trifluoroacetic acid Then, the mixture was stirred in an ice bath under an Ar atmosphere for 90 minutes to remove the protecting group. After completion of the reaction, the product was dissolved in tert-butyl alcohol and precipitated in a large excess of hexane. After drying under reduced pressure, the obtained polymer was dissolved in water and dialyzed under a weak alkali (pH 7 to 8). The dialyzed solution is freeze-dried and the NIPA of the present invention is dried.
Am-L-Lys copolymer (8) (poly (NIPAAm
-Co-L-Lys)). NMR measurements confirmed that almost all of the protecting groups had been removed.

Example 2 The phase transition behavior of the copolymer (8) obtained in Example 1 and an aqueous solution of PNIPAAm (a homopolymer of NIPAAm) were compared using ion-exchanged water as a solvent. In both cases, the concentration was adjusted to 0.1 wt%. The measurement results are shown in FIG.
The transition temperature of the copolymer of the present invention was increased by about 20 ° C. as compared with PNIPAAm. Next, the change of the phase transition behavior with the pH change was examined. Using a buffer solution whose ionic strength was adjusted to 0.01, NIPAAm- was used in the pH range of 2 to 11.
The phase transition change of the L-Lys copolymer (8) and PNIPAAm was observed. FIG. 2 shows the measurement results. From the results of FIG. 2, at pH 2, the amino group of the lysine residue in the copolymer (8) was dissociated, and at pH 10, the carboxyl group was dissociated. It is considered that the hydrophilicity of ()) increased and the phase transition point disappeared. In addition, it can be seen that phase separation occurs around 53 ° C. near the isoelectric point of a lysine residue as shown at pH 6.

Example 3 Preparation Method of Measurement Solution The copolymer (8) was used in the experiment. The measurement was performed using an acetic acid / potassium hydroxide buffer solution having an ionic strength of 0.01 and pH: 5. The composition of the buffer solution is shown below. 0.1M acetic acid aqueous solution 15.34ml 0.1M potassium hydroxide solution 9.99ml (Note) pH5 (pH4.95; 21.9 ° C) (I = 0.
01) 4.5 mg of copolymer (8) (lysine residue: 1.7 × 10
^-6 mol) of lysine residue and equimolar amount of copper sulfate pentahydrate:
0.424 mg (1.7 × 10 ⁻⁶ mol) was dissolved in 2.0 ml of the above buffer solution to prepare solution A. Further, a 4-fold molar amount of the amino acid with respect to the lysine residue of the polymer was dissolved in 2.0 ml of the above buffer solution to prepare solution B. In the experiment, D-form and L-form amino acids, and hydrophobic amino acids such as proline, valine, phenylalanine, alanine and tryptophan were used. Solution B was added to Solution A, and the mixture was stirred at room temperature for about 30 minutes, and tested for amino acid recognition and optical isomer recognition. FIG. 3 shows the change in transmitted light intensity of the aqueous solution of the copolymer (8) when the amino acid of the L-form of the copolymer (8) was added. Regardless of which amino acid was added, the copolymer (8) caused phase separation with an increase in temperature, but it was revealed that the temperature at which phase separation was caused by the added amino acid changed. It was also found that the greater the added amino acid's hydrophobicity, the higher the phase separation temperature. The lysine residue of the copolymer (8) forms a complex with the added amino acid via a copper ion. The larger the hydrophobic group of the added amino acid, the larger the formed complex and the polymer chain (N-
It is considered that the transition temperature increased because the isopropylacrylamide portion) became hydrophobic and easily interacted. From these results, the substance containing the copolymer (8) is expected to be applied to gels and biosensors that recognize and deform various amino acids, and further to column packing materials capable of separating amino acids by temperature change. it can. Optical isomers recognition D of the copolymer (8) and showed the copolymer upon addition of proline L-form transmitted light intensity changes in (8) an aqueous solution in FIG. Compared with the L-form, the addition of the D-form becomes cloudy at a low temperature of about 1 ° C.
It can be seen that phase separation is easy. Similarly, for valine, phenylalanine, and tryptophan, a change in the phase separation temperature was observed for D and L. Based on these results, similar to amino acid recognition, it can be applied to gels and biosensors that recognize and deform optical isomers, as well as ligand-exchanged chiral stationary phases that can separate optical isomers by temperature changes. Can be expected.

[0021]

According to the present invention, the copolymer having an amino acid residue in the side chain represented by the formula (I) is heat-responsive as an aqueous solution,
It has amino acid discriminating ability and optical isomer resolving ability, and exhibits excellent effects as a thermoresponsive polymer, amino acid discriminating agent, and optical isomer resolving agent, respectively.

[Brief description of the drawings]

FIG. 1 is a graph showing the temperature dependence of the transmittance of an aqueous solution of a copolymer of the present invention in comparison with a conventional one.

FIG. 2 is a graph showing the pH dependence of the transmittance of the aqueous copolymer solution of the present invention.

FIG. 3 is a graph showing the temperature dependence of the transmittance of the aqueous copolymer solution of the present invention in the presence of various amino acids.

FIG. 4 is a graph showing the temperature dependence of the transmittance of the aqueous copolymer solution of the present invention with respect to the optical isomer of an amino acid.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Takao Sato, Inventor 1422-5, Ohira-machi, Shimotsuga-gun, Tochigi Prefecture 6-8-4 Yakushidai, Moriya-cho, Kitasoma-gun (56) References JP-A-48-23690 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C08F 220/56 C08F 220 / 60

Claims (3)

(57) [Claims] 1. A copolymer represented by the following formula (I). Embedded image (In the formula, m and n indicate the polymerization ratio of each component in the copolymer , and m: n = 9: 1. ) 2. An amino acid discriminating agent containing the copolymer represented by the formula (I) according to claim 1 and a complex-forming ion . 3. An optical resolving agent for amino acids, comprising the copolymer represented by the formula (I) according to claim 1 and a complex-forming ion .