pH and temperature dual-responsive polymer

Abstract

To provide a polymer that switches the UCST type / LCST type according to pH and shows phase transition of sharp temperature responsiveness.SOLUTION: A dendritic polymer double-responsible to pH and temperature of the invention has a plurality of branch parts and a plurality of terminal groups. The plurality of branch parts is a tertiary amine. At least a part of the plurality of terminal groups contains a phenylalanine residue and a strong acid group or its salt of the amino group side of the residue. The terminal group containing the phenylalanine residue and the strong acid group or its salt links to the branch part at the carbonyl group side of the phenylalanine residue.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to pH- and temperature-responsive polymers. More specifically, the present invention relates to polymers (particularly, end-modified dendritic polymers) that exhibit a more sensitive temperature-responsive phase transition.

Background Art

[0002] Temperature-responsive polymers are classified into those that exhibit a lower critical solution temperature (LCST)-type phase transition in which they become turbid upon heating and those that exhibit an upper critical solution temperature (UCST)-type phase transition in which they dissolve upon heating. A polyamideamine (PAMAM) dendrimer (terminal group: -NH-CO-CH(CH2-C6H5)-NH2) having its terminal portion modified with an amino acid such as phenylalanine and an amino group at the outermost end exhibits an LCST-type phase transition under high pH (basic) conditions (Non-Patent Document 1). Further, a PAMAM dendrimer (terminal group: -NH-CO-R-CO-NH-CH(CH2-C6H5)-COOH; R = ethylene group; 1,2-phenylene group; 1,2-cyclohexene group) in which an amino group and a carboxy group are interchanged and modified with phenylalanine using a linker exhibits a UCST-type phase transition under low pH (acidic) conditions (Non-Patent Document 2). Furthermore, in the end-modified dendrimer described in Non-Patent Document 2, a PAMAM dendrimer (terminal group: -NH-CO-CH(CH2-C6H5)-NH-CO-CH2-CH2-COOH) in which phenylalanine and carboxylic acid (succinic acid) in the terminal group are interchanged shows switching between UCST type / LCST type depending on pH, exhibits a UCST-type phase transition under high pH (basic) conditions, and exhibits an LCST-type phase transition under low pH (acidic) conditions (Non-Patent Documents 3 to 6).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

[0004] However, the end-modified dendrimers described in Non-Patent Documents 3 to 6 have slow temperature responsiveness (i.e., the temperature change required for phase transition is wide), so their applications have been limited. Therefore, the development of a polymer that switches between UCST type and LCST type depending on pH and exhibits a more sensitive temperature-responsive phase transition has been demanded. [Means for Solving the Problems]

[0005] Accordingly, the present invention provides a pH- and temperature-responsive dendritic polymer having a plurality of branched moieties and a plurality of terminal groups, wherein the plurality of branched moieties are tertiary amines, and at least a part of the plurality of terminal groups contains a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the residue, and the terminal group containing the phenylalanine residue and the strong acid group or a salt thereof is linked to the branched moiety on the carbonyl group side of the phenylalanine residue. The present invention also provides a method for separating a substance from a solution containing a soluble substance, the method comprising: supporting the soluble substance on a solvate of the pH- and temperature-responsive dendritic polymer formed at a temperature lower than the lower critical solution temperature (LCST) or higher than the upper critical solution temperature (UCST) of the pH- and temperature-responsive dendritic polymer in the solution; and insolubilizing the dendritic polymer carrying the soluble substance at a temperature higher than the LCST or lower than the UCST.

Advantages of the Invention

[0006] According to the present invention, there is provided a pH- and temperature-responsive dendritic polymer in which the UCST type / LCST type is switched by pH and which exhibits a more sensitive temperature-responsive phase transition.

Brief Description of the Drawings

[0007]

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Mode for Carrying Out the Invention

[0008] <Dendritic Polymer> The dendritic polymer of the present invention is characterized in that a plurality of its branched portions are tertiary amines, at least a part of its plurality of terminal groups contains a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the residue, and the terminal group containing the phenylalanine residue and the strong acid group or a salt thereof is linked to the branched portion on the carbonyl group side thereof, and it has pH and temperature dual responsiveness. In the present invention, the "dendritic polymer" includes a "dendritic polymer" composed of a "core" (if present), a plurality of "branched portions" having three or more branches, and a plurality of "terminal groups". More specifically, the dendritic polymer of the present invention includes dendrimers, dendrons, and hyperbranched polymers. Among dendritic polymers, those with a very high degree of branching regularity are called "dendrimers", and those having a partial structure constituting a dendrimer and having a structure in which at least one functional group of the core portion is not branched (p = 1 in the following formula (I)) are particularly also called "dendrons". On the other hand, among dendritic polymers, those with a relatively low degree of branching regularity in the dendritic structure include "hyperbranched polymers".

[0009] (Dendrimer) The "Dendrimer" of the m-th generation (see below for "generation") is represented by formula (I): Z s (X 1 (X 2 (X 3 (...(X m-1 (X m Y m (qm)-1 ) (qm-1)-1-(rm-1) Y m-1 (rm-1) )...) q3-1-r3 Y 3 r3 ) q2-1-r2 Y 2 r2 ) q1-1-r1 Y 1 r1 ) p and can be represented as such. In the above formula,[[]] if present, Z is the core; X 1 , X 2 , X 3 ,..., X m-1 , X m are the branching structural units of the 1st, 2nd, 3rd,..., (m - 1)-th, and m-th generations respectively, and all of them may be the same or different, and any combination may be the same. Also, for multiple X 1 , X 2 , X 3 ,..., X m-1 , X m all of them may be the same or different, and any combination may be the same; Y 1 , Y 2 , Y 3 ,..., Y m-1 , Y m are the end groups bonded to the branching structural units of the 1st, 2nd, 3rd,..., (m - 1)-th, and m-th generations respectively, and all of them may be the same or different, and any combination may be the same. Also, for multiple Y 1 , Y 2 , Y 3 ,..., Y m-1 , Y mmay be different from each other (i.e., for example, when there are a plurality of Y 1 s, not all Y 1 need to be the same group, and some Y 1 may be another group);

[0010] s is 0 or 1, p is 1 or 2, preferably 2 when s is 0, and when s is 1, it is the number of bonds with the branched part of the core, which is an integer of 1 or more. p is an integer of, for example, 1 to 6, preferably 1 to 4, more preferably 2 to 4 (even more preferably 4); q1, q2, q3,..., qm-1, qm are the numbers of branches of the branched structural units of the 1st, 2nd, 3rd,..., m-1th, mth generations respectively, and generally are integers of 3 or more (for example, 3 to 10, preferably 3 to 8, more preferably 3 to 6, even more preferably 3 or 4). They may all be the same or different, and any combination may be the same. Also, a plurality of q1, q2, q3,..., qm-1, qm may be different from each other; r1, r2, r3,..., rm-1 are the numbers of branches that are bonded to the terminal groups among the branches of the branched structural units of the 1st, 2nd, 3rd,..., m-1th generations (without bonding to the next-generation branched units). rm-1 is an integer from 0 to (qm-1)-1, and a plurality of r1, r2, r3,..., rm-1 may be different from each other.

[0011] In the present invention, a dendrimer or dendron is a structure in which a branched structural unit is bonded to a functional group of a core (when a core exists) or a structure in which two (central) branched structural units are bonded to each other (when no core exists). It is called a "dendrimer or dendron of the mth generation (Gm)" according to the number of repetitions of the branched structural unit (m; or the number of layers of the branched structural unit). For example, a structure in which a branched structural unit is further bonded to the end of the branched structural unit of the first generation (G1) is called the second generation (G2). The dendrimer or dendron is not particularly limited as long as the generation number m is 2 or more, but is preferably 2 to 10, more preferably 3 to 8, even more preferably 3 to 5.

[0012] (Hyperbranched polymer) A "hyperbranched polymer" is generally a polymer with branches developed by polymerizing AB2-type monomers. Here, A and B each represent a combination of functional groups capable of undergoing a polymerization reaction, and examples of such combinations include a hydroxyl group and a carboxyl group, an amino group and a carboxyl group, etc. Alternatively, a substance that functions as a core of the branch may be used in combination. Also, a hyperbranched polymer may be produced by ring-opening polymerization of glycidol, ethyleneimine, etc. A hyperbranched polymer has a branched portion similar to a dendrimer, but a core is not essential. Also, the branched portion of the hyperbranched polymer may have some defects and irregular or discontinuous portions. The lower limit of the molecular weight of the hyperbranched polymer can be, for example, 2500, 5000, or 10000, and the upper limit of the molecular weight can be, for example, 100,000, 80,000, or 50,000. The molecular weight of the hyperbranched polymer is, for example, 5000 to 100,000, preferably 10,000 to 50,000.

[0013] Hereinafter, the "core", "branched portion", and "terminal group" in the "dendritic polymer" of the present invention will be described. (Core) When present, the core is derived from a compound having one or more functional groups. Examples of the functional group include a primary amino group, a secondary amino group, a hydroxy group, a carboxylic acid group, a thiol group, an ester group, an amide group, a ketone group, an aldehyde group, etc., and preferably a primary amino group and a secondary amino group. Specific examples of the core include, for example, C1-C 12 alkyl (preferably C1-C8 alkyl) diamine, and more specifically, ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,10-diaminodecane, 1,12-diaminododecane. An alkyl diamine (cystamine) containing an SS bond may also be used.

[0014] (Branched portion) In the present invention, the branching moiety is a tertiary amine and may have a structure as shown below.

Chemical formula

[0015] When the dendritic polymer of the present invention is made water-soluble, the branching moiety is preferably hydrophilic. Therefore, it preferably contains an amide group, an ester group, a (secondary) amino group, a carbonyl group, an ether group, or a thioether group, and particularly preferably contains an amide group, an ester group, an amino group, and / or a carbonyl group. The branching moiety is, for example, of the formula: (-R 1 XR 2 N<) n (wherein X represents a bond, -CONH-, -CO-, -NH-, -COO-, or -O-, and R 1 and R 2 each independently represent C1-C9, more preferably C1-C4, and even more preferably C2-C3 alkylene).

[0016] The branching moiety is preferably an amidoamine or an alkyleneamine, more preferably a linear amidoamine or a linear alkyleneamine. When the branching moiety is an amidoamine, a suitable branching moiety is, for example, of the formula: (-R 1 CONHR 2 N<) n (wherein R 1 and R 2 each independently represent C1-C9, more preferably C1-C4, and even more preferably C2-C3 alkylene). Specific examples of the branching moiety are (-CH2CH2CONHCH2CH2N<) n or (-CH2CH2CH2CONHCH2CH2CH2N<) n . When the branching moiety is an alkyleneamine, a suitable branching moiety is, for example, of the formula: (-R 1 N<) n (wherein R 1Each independently represents C1-C9, more preferably C1-C4, and even more preferably C2-C3 alkylene), and is represented by). Specific examples of the branched portion are (-CH2CH2N<) n , (-CH2CH2NHCH2CH2N<) n or (-CH2CH2CH2N<) n It is.

[0017] (Terminal group) At least a part of the plurality of terminal groups of the dendritic polymer of the present invention contains a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the phenylalanine residue. Examples of the strong acid group include a sulfonic acid group, a phosphonic acid group, a phosphoric acid group, and a sulfuric acid group. Preferably, the strong acid group is a sulfonic acid group. The terminal group is -(phenylalanine residue)-R 3 -SO3H, -(phenylalanine residue)-R 3 -OSO3H, -(phenylalanine residue), -R 3 -P(O)(OH)2 and -(phenylalanine residue)-R 3 It is a group selected from the group consisting of -OP(O)(OH)2 or a salt thereof. Here, R 3 is bonded to the amino group of the phenylalanine residue and is C1-C6 (preferably C2-C5, more preferably C2-C4) alkylene, C3-C6 cycloalkylene or phenylene, which may be substituted with one or more of halogen, C1-C4 alkyl and C1-C4 alkoxy.

[0018] The salt can be in the form of a monovalent metal salt, such as an alkali metal salt (more specifically, a sodium salt or a potassium salt), or a divalent metal salt, such as an alkaline earth metal salt (more specifically, a magnesium salt or a calcium salt). The terminal group containing a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the residue is directly or indirectly linked to the branched portion on the carbonyl group side of the phenylalanine residue. The dendritic polymer of the present invention does not necessarily have all the same end groups and may have two or more types of end groups. When there are two or more types of end groups, at least one of them may be an end group containing a phenylalanine residue and a strong acid group on the amino group side of the residue or a salt thereof. More than 50%, preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70% of the total number of end groups of the dendritic polymer (for example, 64 in the case of a G4 dendrimer and 128 in the case of a G5 dendrimer) are end groups containing a phenylalanine residue and a strong acid group on the amino group side of the residue or a salt thereof.

[0019] The end groups that the dendritic polymer of the present invention may have and that do not contain a phenylalanine residue and a strong acid group on the amino group side of the residue or a salt thereof are not particularly limited, but may be end groups that contain a phenylalanine residue but do not contain a strong acid group or its salt, end groups that do not contain a phenylalanine residue but contain a strong acid group or its salt, or end groups that do not contain a phenylalanine residue, a strong acid group, or its salt. The end group that does not contain a phenylalanine residue, a strong acid group, or its salt may have the structure of the last repeating unit of the branched portion, or the end group may have a structure different from the branched portion. Examples of the end group that does not contain a phenylalanine residue, a strong acid group, or its salt include an amino group, an amino (C1-C4) alkyl group, a hydroxy group, a hydroxy (C1-C4) alkyl group, a carboxyl group, a carboxyl (C1-C4) alkyl group, etc., preferably an amino group, a carboxyl group, and more preferably an amino group. The end group may combine with the group at the branched end of the branched portion to form a group as described above.

[0020] The dendritic polymer of the present invention exhibits responsiveness (i.e., dual responsiveness) to both pH and temperature. The aqueous solution of this dendritic polymer exhibits LCST-type phase transition behavior, for example, under lower pH conditions (e.g., pH 3 - 8, more specifically pH 4 - 8, more specifically pH 5 - 8), and exhibits UCST-type phase transition behavior under higher pH conditions (e.g., pH 4 - 9, more specifically pH 5 - 9, more specifically pH 6 - 9). "LCST-type phase transition" means that it is water-soluble below a specific temperature (lower critical solution temperature: LCST), while it becomes water-insoluble (i.e., phase-separates) above LCST. "UCST-type phase transition" means that it is water-soluble above a specific temperature (upper critical solution temperature: UCST), while it becomes water-insoluble below UCST. This dendritic polymer has both LCST-type and UCST-type properties, for example, between pH 4 - 8, more specifically between pH 5 - 7, more specifically between pH 5 - 6. In addition, this dendritic polymer shows a more sensitive temperature responsiveness compared to those containing a weak acid group or its salt as the terminal group instead of a strong acid group or its salt. For example, the dendritic polymer of the present invention undergoes phase transition at about 5 - 70°C, preferably about 10 - 70°C, preferably about 20 - 70°C, more preferably about 20 - 60°C, more preferably about 20 - 60°C, more preferably about 20 - 50°C, and the phase transition (change in light transmittance from 20% to 80%) occurs within a temperature range of 20°C or less, preferably within 10°C.

[0021] The dendritic polymer of the present invention is preferably a dendrimer. The branched part of the dendrimer can be, for example, the structures of (1), (2), (3), and (4) above. More specifically, the dendrimer is a dendrimer with an amidoamine as the branched part (referred to as "polyamidoamine (PAMAM) dendrimer") or a dendrimer with an alkyleneamine as the branched part (referred to as "polyalkyleneimine dendrimer"). Specific examples of amidoamine and alkyleneamine are as described above. In addition, the dendritic polymer of the present invention can be a hyperbranched polymer that is a polyalkyleneimine. Specific examples of polyalkyleneimine are polyethyleneimine and polypropyleneimine.

[0022] <Manufacturing Method> The dendritic polymer of the present invention can be produced by adding a terminal group containing a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the residue to a dendritic polymer prepared by a known production method (or by modifying the dendritic polymer with the terminal group). For example, methods for producing dendritic polymers having branching portions as described in the above (1) to (4) are described in the following documents: (1): D.A. Tomalia et al., Polym. J. 17, 117 (1985); D.A. Tomalia et al., Angew. Chem. Int. Ed. Engl. 29, 138 (1990); (2): E.M.M. de Brabander-van den Berg et al., Angew. Chem. Int. Ed. Engl. 32, 1308 (1993); E.M.M. de Brabander-van den Berg et al., Macromol. Symp. 77, 51 (1994); J.C. Hummelen et al., Chem. Eng. J. 3, 1489 (1997); C. Waner et al., Angew. Chem. Int. Ed. Engl. 32, 1300 (1993); (3): K.E. Uhrich et al., J. Chem. Soc. Perkin Trans. 1, 1623 (1992); (4): S.R. Rannard et al., J. Am. Chem. Soc. 122, 11729 (2000).

[0023] When the dendritic polymer of the present invention is a generation m PAMAM dendrimer, for example, a Michael addition reaction (e.g., reacting an acrylic acid ester) followed by an ester amide exchange reaction (e.g., using a diaminoalkane) with a primary amine (e.g., ethylenediamine) as the core is repeated m times (see Tomalia, D. A. et al., Polym. J. 17, 117 (1985); Frechet, J. M. J., Tomalia, D. A. eds., (2001) Dendrimers and other dendritic polymers, J. Wiley & Sons, West Sussex), and it can be produced by adding (or modifying with) a terminal group containing a phenylalanine residue and a strong acid group or its salt on the amino group side of the residue. When the dendritic polymer of the present invention is a hyperbranched polymer, for example, it can be produced by polymerizing an AB2 type monomer or ring-opening polymerizing glycidol, ethyleneimine, etc., and adding (or modifying with) a terminal group containing a phenylalanine residue and a strong acid group or its salt on the amino group side of the residue.

[0024] The addition (or modification with) the terminal group containing a phenylalanine residue and a strong acid group or its salt on the amino group side of the residue can be appropriately selected from known techniques according to the type of the terminal part of the generation m branched structural unit. For example, when the terminal part is an amino group or a hydroxyl group, the reaction can be carried out by reacting the group with the free carboxylic acid (i.e., carboxyl group) in a compound containing a phenylalanine residue having a free carboxylic acid and a strong acid group or its salt on the amino group side of the residue (after protecting, if necessary, the functional groups other than the free carboxylic acid in the compound). Alternatively, it can be obtained by reacting the amino group or hydroxyl group with the carboxyl group of phenylalanine with the amino group protected, and then reacting with a compound capable of imparting a strong acid group to the amino group of phenylalanine (e.g., a sulfonating agent, a phosphonating agent).

[0025] Examples of the compound capable of imparting a sulfonic acid group or a sulfuric acid group include ω-haloalkyl sulfonic acid or sulfuric acid or a salt thereof, sultone compounds, and the like. Specific examples of the sultone compound are, for example, 1,2-ethane sultone, 1,3-propane sultone, 1,4-butane sultone, 2,4-butane sultone, and 2,5-pentane sultone. Examples of the compound capable of imparting a phosphonic acid group or a phosphoric acid group include ω-haloalkyl phosphonic acid or phosphoric acid or a salt thereof, orthophosphoric acid, dioxaphospholane-2-oxide, dioxaphosphorinane-2-oxide, and the like.

[0026] <Method for Separating Substances> The method for separating substances of the present invention is characterized in that in a solution containing a soluble substance, the soluble substance is supported on the dendrimer in an insolubilized state formed at a temperature higher than the lower critical solution temperature (LCST) or lower than the upper critical solution temperature (UCST) of the dendritic polymer according to the present invention described above, thereby separating the soluble substance from the solution. The soluble substance to be separated is preferably a substance that exhibits anionic properties or does not dissociate ions in the solution. It may also be a transition metal ion that can interact with the dendritic polymer. For example, it is known that PAMAM dendrimer can retain Cu2+, Ag+, Pt2+, Pd2+, Ru3+, Ni2+ inside (Mingqi Zhao & Richard M. Crooks, Adv. Mater. 1999, 11, 217). Examples include recovery of the target substance and removal of unwanted substances from a mixture of organic reactions, separation and recovery of proteins from an aqueous solution, removal and recovery of metal ions from wastewater, and the like. Separation of the insolubilized dendrimer from the suspension containing the insolubilized soluble substance-supported dendrimer can be carried out, for example, by centrifugation or sedimentation. According to the method for separating substances of the present invention, the soluble substance can be separated from the solution containing the soluble substance simply and / or efficiently.

Examples

[0027] 1. Synthesis of G4-Phe-SO3H The dendrimer "G4-Phe-SO3H" is a fourth-generation (G4) dendrimer with an ethylenediamine core, a -CH2CH2CONHCH2CH2N< branching part, and a -phenylalanine residue-SO3H at the terminal group, and has the structure shown in the following schematic diagram.

Chemical formula

Chemical formula

[0028] ·Synthesis of G4-Phe-Boc 462 mg (1.74 mmol) of Boc-Phe was dissolved in 4.8 mL of dimethylformamide (DMF), 241 mg (2.09 mmol) of N-hydroxysuccinimide, 466 mg (2.26 mmol) of N,N'-dicyclohexylcarbodiimide (DCC), and 364 μL (2.61 mmol) of triethylamine were added, and the mixture was stirred in an ice bath (solution a). 255 mg (17.9 μmol) of the fourth-generation PAMAM dendrimer (terminal group: NH2 at the end of the branching part) was dissolved in 4.8 mL of dimethyl sulfoxide (DMSO), and after mixing this with solution a, the mixture was stirred at room temperature for 7 days. The salt was removed by filtration and applied to a Sephendex LH-20 column. By vacuum drying, G4-(Phe-Boc) was obtained (transparent film; yield: 437.1 mg; yield: 93%).

[0029] Figure 1(a) shows the 1 1H NMR spectrum of the obtained G4-(Phe-Boc). 1 The 1H NMR assignment was carried out using the symbols shown in Figure 1(f). Based on the integral value 248 of the peak at 2.15 ppm in Figure 1(a), the integral value of the peak of the Boc group at 1.16 - 1.28 ppm was calculated, and from the obtained value, the number of bound Boc-Phe was estimated to be 49. This value is the H of Pheα This was consistent with the value estimated from the integrated value of the peak at 4.08 ppm. Therefore, the G4-(Phe-Boc) obtained above has (Phe-Boc) at the 49 terminus, i.e., G4-(Phe-Boc). 49 It was found to be as follows.

[0030] ·Synthesis of G4-Phe 66.4 mg of Boc-Phe-G4 was dissolved in 2.8 mL of trifluoroacetic acid (TFA) and allowed to stand in an ice bath (4 °C or lower) for 4 hours. TFA was distilled off under reduced pressure using an evaporator. The operation of mixing with distilled water and distilling off under reduced pressure was performed until the smell of acetic acid disappeared (4 times). Vacuum drying was carried out overnight to obtain G4-Phe 49 (Yield: 92.2 mg; Yield rate: 127%; Transparent film form).

[0031] ·Synthesis of G4-Phe-SO3H With reference to the method of Chen, H.-T. et al. (J. Am. Chem. Soc., 2004, 126 (32), 10044-10048), the terminal of G4-Phe was sulfonated. 142.3 mg (1.17 mmol) of 1,3-propanesultone was dissolved in acetonitrile (2.8 mL). 92.2 mg (3.20 μmol) of G4-Phe was dissolved in 125 mM aqueous NaHCO3 solution (2.8 mL). These two solutions were mixed, and after nitrogen bubbling, the mixture was stirred at room temperature. On the third day, since the pH became 1 or lower, 4M aqueous NaOH solution was added to make it basic, and after nitrogen bubbling, the mixture was stirred at room temperature. On the fourth day, since the pH reached 7-8, the pH was adjusted to about pH9 with 4M aqueous NaOH solution. On the fifth day, since the pH was about pH8, the pH was once lowered by adding aqueous HCl solution and then adjusted to about pH8 with 4M aqueous NaOH solution. Then, after slightly reducing the volume of the solution using an evaporator, 2.1 mL of distilled water was added and dialysis was performed with distilled water. After lyophilization, G4-Phe-SO3H was obtained (Yield: 60.9 mg; Yield rate: 64%).

[0032] Figure 1(b) shows 1 the 1H NMR spectrum. 1The ¹H NMR assignments were made using the symbols shown in Fig. 1(f). Based on the peak value 245 of phenylalanine in the range of 7 ppm, the peak of the methylene (2) of the sulfonic acid bonded to the end of G4-Phe was calculated to be 115. Dividing this by 2 gives 57.5, and it was estimated that 58 terminal groups had SO₃H groups bonded. Therefore, G4-Phe-SO₃H obtained by the above synthesis method is G4-Phe 49 -SO₃H 58 and was found to be so.

[0033] 2. Synthesis of G4-Phe-Suc The dendrimer “G4-Phe-Suc” has an ethylene diamine core, a branching part of (-CH₂CH₂CONHCH₂CH₂N<) n and a terminal group of -phenylalanine residue-CO-CH₂CH₂-COOH, and is a fourth-generation (G4) dendrimer having the structure shown in the following schematic diagram (that is, having a terminal group containing a salt of a weak acid group).

Chemical formula

[0034] The synthesis of G4-Phe-Suc was carried out as follows. Up to the synthesis of G4-Phe, it was carried out as described in “1. Synthesis of G4-Phe-SO₃H”. 121.4 mg (4.23 μmol) of G4-Phe was dissolved in 3 mL of 125 mM aqueous NaHCO₃ solution, and 0.3331 g of an excess amount of succinic anhydride was added. While monitoring the pH, it was adjusted to pH 8 - 10 using 4M aqueous NaOH solution on an ice bath and stirred at 4 °C overnight. 0.1M aqueous HCl solution was added dropwise, and after confirming the turbidity of the solution (pH 5), dialysis with distilled water was carried out for 3.5 hours. Further, after dialysis of the outer layer with 125 mM aqueous NaHCO₃ solution for 3.5 hours, dialysis with distilled water was carried out over 1 day to obtain G4-Phe-Suc (yield: 67.6 mg; yield rate: 56%).

[0035] In Fig. 1(c) 1The 1H NMR spectrum is shown. 1 The 1H NMR assignments were made using the symbols shown in Fig. 1(f). From Fig. 1(c), based on the integral value (248) of the (A), (A') peaks (2.55 ppm) derived from the starting PAMAM dendrimer (terminal group: NH2), the number of bound Suc(-CO-CH2CH2-COOH) was estimated by subtracting the portion derived from the starting dendrimer from the total integral value (475) of the methylene protons derived from succinic acid and the (B), (B') peaks (2.19 ppm) derived from the starting dendrimer. The theoretical value of the methylene protons derived from succinic acid was 256, and the experimental value was 246. Therefore, it was estimated that Suc was bound to the 57 terminals of the dendrimer (i.e., G4-Phe 49 -Suc 57 ).

[0036] 3. Synthesis of QG4-Phe-Suc The dendrimer "QG4-Phe-Suc" has an ethylene diamine core, a branching part of (-CH2CH2CONHCH2CH2N<) n (where -N< is quaternized; that is, the branching part is a quaternary amine), and a terminal group of -phenylalanine residue -CO-CH2CH2-COOH, and is a fourth-generation (G4) dendrimer having the structure shown in the following schematic diagram.

Chemical formula

[0037] The synthesis of QG4-Phe-Suc was carried out as follows. The synthesis up to G4-Phe-Suc was carried out as described above. G4-Phe 49 -Suc 62(34.0 mg, 1.20 μmol) was dissolved in 3 mL of DMF, and 9-fold equivalent of methyl iodide (MeI; 43.2 μL, 693 μmol) was added to the internal tertiary amine, followed by stirring at 37 °C for 25 h (after stirring, the color of the solution changed to yellow). 30 mL of diethyl ether was added to the reaction mixture, and the mixture was left to crystallize in an ice bath for 1 h 20 min, and then the suspension was concentrated to about 3 mL using an evaporator (yellow suspension). When the obtained suspension was dialyzed against 2 M aqueous NaCl solution for one and a half days, it changed from yellowish white (opaque) to transparent. Then, dialysis was carried out with distilled water for 1 day. By freeze-drying, QG4-Phe 49 -Suc 62 was obtained (yield: 29.2 mg; yield rate: 77%).

[0038] Figure 1(d) shows 1 the 1H NMR spectrum. 1 The assignment of 1H NMR was carried out using the symbols shown in Figure 1(f). In Figure 1(d), 2.98 ppm was considered to be the peak of N + CH3. Assuming the integral value of H α to be 49, the integral value of the peak of N + CH3 was calculated to be 215, suggesting that 71 tertiary amines were quaternized. Also, the number of quaternized amines was estimated to be 67 by dividing the value obtained by subtracting the integral value of 2.2 - 3.1 ppm in (c) (excluding the integral value of free Suc at 2.24 ppm) from the integral value of 2.2 - 3.5 ppm in (d) by 3. From these results, it was considered that all the internal tertiary amines were quaternized.

[0039] 4. Synthesis of QG4-Phe-SO3H The dendrimer “QG4-Phe-SO3H” has an ethylene diamine core, a branching part of (-CH2CH2CONHCH2CH2N<) n (wherein -N< is quaternized; that is, the branching part is a quaternary amine), and a terminal group of -phenylalanine residue -SO3H, and is a fourth-generation (G4) dendrimer having the structure shown in the following schematic diagram. [Chem.]

[0040] The synthesis of QG4-Phe-SO3H was carried out as follows. The synthesis up to G4-Phe-SO3H was carried out as described above. G4-Phe-SO3H (17.8 mg, 0.63 μmol) was dissolved in 2 mL of DMF, 4.5-fold equivalent of MeI (11.0 μL, 177 μmol) was added with respect to the internal tertiary amine, and after stirring at 37 °C for 3 hours, MeI (11.0 μL, 177 μmol) was further added and stirred for 21 hours (the color of the solution changed to yellow). When the reaction mixture was dialyzed against 2 M aqueous NaCl solution for one and a half days, the inner phase changed from yellow immediately after the start of dialysis to colorless and transparent. When the outer phase was replaced with distilled water and dialyzed for 2 days, it changed from a colorless and transparent liquid to a turbid liquid. This was lyophilized to obtain QG4-Phe-SO3H (white solid; yield: 21.3 mg; yield rate: 91%). However, the QG4-Phe-SO3H obtained at this stage 1 had a quaternization rate of 57% as determined by 1H NMR measurement (Q 57% G4-Phe-SO3H), so a subsequent reaction was carried out. Q 57% G4-Phe-SO3H (12.8 mg, 0.43 μmol) was dissolved in 1 mL of DMF, 4.3-fold equivalent of MeI (20.3 μL, 326 μmol) was added with respect to the internal tertiary amine and the primary amino group adjacent to the sulfonic acid, and stirred at 37 °C for 24 hours (the color of the solution changed to yellow). It was purified in the same manner as above to obtain QG4-Phe-SO3H (white solid; yield: 4.5 mg; yield rate: 20%).

[0041] Figure 1(e) shows 1 the 1H NMR spectrum. 1 The assignment of 1H NMR was carried out using the symbols shown in Figure 1(f). In Figure 1(e), taking the peak of the phenyl ring of Phe as a reference (245), the integrated value from the peaks of (A) and (A') to the peak of (2) was determined to be 2097. Before quaternization1 The same operation was also performed on the 1H NMR spectrum. When the integrated values from (C), (C'), (D) to the peak of (2) were determined, the result was 1501. Therefore, it can be concluded that 596 hydrogens were increased by the above quaternization reaction. Since 596 divided by 3 is 198, it is estimated that 198 amino groups were quaternized. Since the number of hydrogens required to completely quaternize all 62 internal tertiary amino groups and 57 secondary amino groups (derived from phenylalanine) adjacent to the sulfo groups in the G4-Phe-SO3H dendrimer is 176, it was considered that all the internal tertiary amino groups and secondary amino groups adjacent to the sulfo groups in the dendrimer were quaternized.

[0042] 5. Measurement of Temperature-Dependent Transmittance (Turbidity) (1) 5.1. Measurement Procedure For the dendrimer sample with a concentration of 1 mg / mL (buffer concentration: 20 mM), the transmittance was measured as a function of temperature. After adjusting the pH with acetic acid buffer (pH 5.5 or lower) and phosphate buffer (pH 6), the temperature dependence of the transmittance was measured using a UV / Vis Spectrophotometer (V-630, JASCO). The measurement conditions were as follows: measurement wavelength: 500 nm, heating rate: 1 °C / min, measurement interval: 0.1 °C, bandwidth: 1.5 nm. A Peltier holder (ETC-717, JASCO) was used for temperature adjustment.

[0043] 5.2. Measurement Results (1) The measurement results are shown in Figure 2. G4-Phe-SO3H showed an LCST-type phase transition (LCST = 52 °C) at pH 5.0 and a UCST-type phase transition (UCST = 36 °C) at pH 6.5 (Figure 2(A)). Both phase transitions responded sensitively to temperature changes (i.e., the phase transitions occurred within a narrow temperature range), indicating high temperature responsiveness. Although this high responsiveness is not intended to be restricted by theory, it is considered to be the result of the interaction between dendrimers in solution changing depending only on the increase or decrease of cations inside due to the presence of strong acid groups at the terminal groups.

[0044] G4-Phe-Suc exhibited an LCST-type phase transition at pH 4 and a UCST-type phase transition at pH 6, but both phase transitions were inferior in terms of temperature responsiveness compared to that of G4-Phe-SO3H (Figure 2(B)). Also, at pH 3.5, it remained transparent within the measured temperature range without becoming turbid and did not exhibit temperature responsiveness. Although not intending to be restricted by theory, this is presumably due to the fact that as a result of all the tertiary amino groups inside the dendrimer being protonated at pH 3.5, the inside of the dendrimer became hydrophilic.

[0045] QG4-Phe-Suc remained dissolved at both pH 4 and pH 5 and did not exhibit a temperature-dependent phase transition (Figure 2(C)). During pH adjustment, since it was transparent without becoming turbid between pH 5.7 and pH 4, it is considered that the pH responsiveness of G4-Phe-Suc was lost due to the quaternization of the internal tertiary amines. Although not intending to be restricted by theory, this is presumably due to the fact that as a result of the quaternization of the internal tertiary amines, the entire dendrimer became cationic at any pH and was hydrophilic.

[0046] Since the QG4-Phe-SO3H solution remained transparent when the pH was changed from 2 to 10, it is considered that QG4-Phe-SO3H lost its pH responsiveness. QG4-Phe-SO3H remained transparent and did not exhibit temperature responsiveness at pH 5 (Figure 2(D)). Although not intending to be restricted by theory, this is presumably due to the fact that there are more quaternary amino groups, which are positively charged, compared to the number of sulfonic acid groups, which are negatively charged, so the dendrimer became cationic and hydrophilic at any pH.

[0047] The pH and temperature dependencies of the four dendrimers synthesized above are summarized in Table 1.

Table 1

[0048] Dendrimers having a quaternary amino group, which is a strong base, inside did not show temperature responsiveness and dissolved regardless of pH, regardless of whether they had a strong acid group or a weak acid group at the terminal. On the other hand, dendrimers having a tertiary amino group, which is a weak base, inside and a strong acid group or a weak acid group at the terminal switched between LCST-type / UCST-type temperature phase transitions depending on pH. In addition, the temperature responsiveness of the temperature phase transition of the dendrimer having a strong acid group at the terminal was more sensitive than that of the dendrimer having a weak acid group at the terminal. From the above results, it was shown that the internal tertiary amino group (protonation) and the terminal strong acid group of the dendrimer were involved in the pH- and temperature-dual responsiveness.

[0049] 5.3. Measurement Results (2) Measurements were also made for G4-Phe-SO3H at pH 4.0, 5.0, 5.1, 5.2, 5.5, 5.7, 6.0, 6.2, 6.5, and 7.0. The results are shown in Fig. 3. In Fig. 3, (A) shows the temperature responsiveness at pH 4.0 to 5.2, (B) shows the temperature responsiveness at pH 5.5 to 5.7, and (C) shows the temperature responsiveness at pH 6.0 to 7.0. As is clear from Fig. 3(A), at pH 4.0, it was transparent within the measurement temperature range and did not show temperature responsiveness. On the other hand, at pH 5.0 to 5.2, it showed an LCST-type phase transition of becoming turbid upon heating. When the pH increased by 0.1, the temperature response region rose by about 10 °C. Although not intended to be restricted by theory, the reason for showing solubility within the measurement temperature range at pH 4.0 is considered to be the result that the internal tertiary amino group was protonated, making the inside cationic and increasing the hydrophilicity. As the reason for showing LCST-type temperature responsiveness at pH 5.0 to 5.2, it is considered to be the result that the protonated internal quaternary amine acts as a counter cation of the terminal strong acid group, taking a surface structure similar to G4-Phe. An increase in pH by 0.1 is considered to increase the number of cations, resulting in an increase in the temperature response region.

[0050] As is apparent from Fig. 3(B), a U-shaped temperature response curve showing an LCST-type phase transition in the low temperature region and a UCST-type phase transition in the high temperature region was obtained at pH 5.5 to 5.7. As is apparent from Fig. 3(C), at pH 6.0 to 6.5, it showed UCST-type temperature responsiveness and showed more sensitive temperature responsiveness at higher pH. On the other hand, at pH 7.0, it was transparent within the measured temperature range and did not show temperature responsiveness. Although not intending to be bound by theory, the reason for showing UCST-type temperature responsiveness at pH 6.0 to 6.5 is considered to be that the dendrimer has a zwitterionic structure, and the reason for showing more sensitive temperature responsiveness at higher pH is considered to be that the interaction becomes stronger as the number of quaternized tertiary amino groups increases. On the other hand, the reason for showing solubility within the measured temperature range at pH 7.0 is considered to be that the tertiary amino group is deprotonated and the dendrimer has a negative charge, so it dissolves due to charge repulsion.

[0051] The temperature responsiveness of G4-Phe-SO3H at various pH values is summarized in Table 2 below.

Table 2

[0052] 6. Microscopic Observation 6.1. Observation Procedure For a 1 mg / mL dendrimer (G4-Phe-SO3H) solution, microscopic observations were carried out at pH 7.0, 6.5, 6.2, 6.0, and 5.2 at room temperature (25 °C), 40 °C, and 60 °C. For microscopic observation, an inverted fluorescence microscope (ECLIPSE Ti-U, Nikon Corp., Tokyo, Japan) equipped with a glass heater unit for cell culture (C-140A, BLAST Inc., Kanagawa, Japan) was used.

[0053] 6.2. Results The microscopic image is shown in Fig. 4. Under dissolution conditions at any pH, no droplets were observed, while under turbidity conditions, droplets (inside the dotted circle in the figure) were observed. Therefore, the LCST-type and UCST-type phase transitions of G4-Phe-SO3H are considered to be due to liquid-liquid phase separation (i.e., coacervation formation).

[0054] 7. Temperature-responsive separation ability of G4-Phe-SO3H 7.1. Temperature responsiveness of RB-loaded G4-Phe-SO3H dendrimer Rose bengal (RB) as a model substance:

Chemical formula

[0055] (1) Experimental procedure Samples (pH 6.5) were prepared by adding 1 mM RB aqueous solution to the dendrimer (G4-Phe-SO3H) at 1 mg / mL (35 μM, phosphate buffer concentration 20 mM) so that RB was 10 equivalents (350 μM) or 5 equivalents (175 μM). For this sample, temperature change transmittance measurement was performed using a UV / Vis Spectrophotometer (V-630, JASCO). The measurement conditions were as follows: measurement wavelength: 700 nm, measurement temperature: 20°C → 60°C, bandwidth: 1.5 nm. A Peltier holder (ETC-717, JASCO) was used for temperature adjustment.

[0056] (2) Measurement results The results are shown in Fig. 5. The RB-loaded dendrimers (RB 5 eq and RB 10 eq) had a temperature response curve shifted to the high-temperature side compared to the dendrimer alone (RB 0 eq). This is considered to be because RB is a hydrophobic molecule, resulting in a decrease in the solubility of the RB-loaded dendrimer. It was found that the G4-Phe-SO3H dendrimer exhibited sharp temperature responsiveness even when loaded with RB.

[0057] 7.1 Separation Ability Experiment (1) Experimental Procedure 100 μL of the dendrimer aqueous solution (pH 6.5) containing 5 equivalents of the above RB (1 mg / mL) was taken into an Eppendorf tube and left standing at 4°C or 60°C for 30 minutes or more. Centrifugation was performed at 4°C or 40°C for 5 minutes (11,000 rpm). The supernatant of the sample before centrifugation and the sample after centrifugation was taken and diluted with 20 mM phosphate buffer (pH 6.5) so that the RB concentration became 4 μM. After dilution, the absorbance was measured using a UV / Vis Spectrophotometer (V-630, JASCO) (measurement wavelength: 400 - 800 nm). The sample at 40°C was kept warm at 50°C from after dilution until immediately before measurement. The measurement was performed 3 times and the standard deviation was calculated.

[0058] (2) Results Figure 6(A) shows the results of spectral measurement of the RB / G4-Phe-SO3H dendrimer. (a) is the result before centrifugation at 40°C, (b) is the result after centrifugation at 40°C, and (c) is the result after centrifugation at 4°C. From the spectra shown in Figure 6, the absorbance at the peak top wavelength (549 nm) was substituted into the following formula Residual ratio = ([Absorbance after centrifugation at 4°C or 40°C] / [Absorbance before centrifugation at 40°C]) × 100 - [Absorbance at the peak top wavelength (549 nm)] to calculate the residual ratio in the solution. The results are shown in Figure 6(B). The RB residual ratio in the sample was 4% at 4°C and 82% at 40°C. From this result, it was shown that the dendrimer of the present invention can be used for efficient substance separation utilizing temperature change.

[0059] Synthesis of G4-Phe-SO3Na, G3-Phe-SO3Na, G5-Phe-SO3Na and G4-SO3Na The dendrimers "G4-Phe-SO3Na", "G3-Phe-SO3Na" and "G5-Phe-SO3Na" are the 4th generation (G4), 3rd generation (G3) and 5th generation (G5) dendrimers respectively, with an ethylenediamine core, a branching part of -CH2CH2CONHCH2CH2N<, and a -Phe residue-(CH2)3-SO3Na at the terminal group. The dendrimer "G4-SO3Na" is a 4th generation (G4) dendrimer with an ethylenediamine core, a branching part of -CH2CH2CONHCH2CH2N<, and -SO3Na at the terminal group without containing a Phe residue, having the structure shown in the following schematic diagram. [Chemical formula]

[0060] G4-Phe-SO3Na was synthesized as follows. Up to the synthesis of G4-Phe, it was carried out as described in "1. Synthesis of G4-Phe-SO3H" to obtain G4-Phe. 56 and obtained. G4-Phe 56 92.2 mg (2.27 μmol) was dissolved in 125 mM aqueous NaHCO3 solution (2.8 mL). 142.3 mg (1.17 mmol) of 1,3-propanesultone was dissolved in acetonitrile (2.8 mL). These two solutions were mixed, and after nitrogen bubbling, they were stirred at room temperature under pH 8 - 9. After 5 days, aqueous HCl solution was added to lower the pH once, and then NaOH was added to raise the pH to about 8. After dialysis with distilled water, dialysis was carried out with 125 mM aqueous NaHCO3 solution. After further dialysis with pure water for one and a half days, it was freeze-dried to obtain G4-Phe-SO3Na (yield: 61 mg; yield rate: 95%). Figure 7(a) shows the 1 1H NMR spectrum of the obtained G4-Phe-SO3Na. 1 The assignment of 1H NMR was carried out using the symbols shown in Figure 1(f) (the same applies hereinafter unless otherwise specified). Based on the integrated value of 280 for the peak of Phe near 7 ppm, the integrated value of the methylene (2) peak of the sulfonic acid bonded to the end of G4-Phe was calculated to be 128. Thus, it could be estimated that SO3Na groups were bonded to all 64 ends. Therefore, the obtained G4-Phe-SO3Na was G4-Phe 56 -(SO3Na) 64 as found out.

[0061] G3-Phe-SO3Na was synthesized as follows. Up to the synthesis of G3-Phe, the method described in "1. Synthesis of G4-Phe-SO3H" was followed using the third-generation PAMAM dendrimer (terminal group: NH2 at the end of the branched part), and G3-Phe 28 was obtained. G3-Phe-SO3Na was obtained in the same manner as for G4-Phe-SO3Na using 117.0 mg (7.97 μmol) of G3-Phe and 313.8 mg (2.57 mmol) of 1,3-propanesultone (yield: 78 mg; yield rate: 88%). 28 Figure 7(b) shows the 1 1H NMR spectrum of the obtained G3-Phe-SO3Na. In the same way as for G4-Phe-SO3Na, it could be estimated that SO3Na groups were bonded to 30 out of all 32 ends. Therefore, the obtained G3-Phe-SO3Na was G3-Phe 28 -(SO3Na) 30 as found out.

[0062] G5-Phe-SO3Na was synthesized as follows. Up to the synthesis of G5-Phe, the method described in "1. Synthesis of G4-Phe-SO3H" was followed using the fifth-generation PAMAM dendrimer (terminal group: NH2 at the end of the branched part), and G5-Phe 112 was obtained. G5-Phe-SO3Na was 112 ​Using 138.1 mg (1.94 μmol) and 381.3 mg (3.12 mmol) of 1,3 - propane sultone, it was obtained in the same manner as for G4 - Phe - SO3Na (yield: 82 mg; yield rate: 97%). Figure 7(c) shows the 1 1H NMR spectrum of the obtained G5 - Phe - SO3Na. Similar to G4 - Phe - SO3Na, it was estimated that the SO3Na group was bound to 121 out of 128 terminals in total. Therefore, the obtained G5 - Phe - SO3Na was found to be G5 - Phe 112 -(SO3Na) 121 as a result.

[0063] G4 - SO3Na was obtained in the same manner as for G4 - Phe - SO3Na using 36.4 mg (2.56 μmol) of the fourth - generation PAMAM dendrimer (terminal group: NH2 at the end of the branched part) and 168.4 mg (1.38 mmol) of 1,3 - propane sultone (yield: 45 mg; yield rate: 93%). Figure 7(d) shows the 1 1H NMR spectrum of the obtained G4 - SO3Na. Based on the integrated value of 248 of the peak at 2.27 ppm, when calculating the integrated value of the peak of the methylene (1) of the sulfonic acid (1.75 ppm) bound to the terminal of G4, it was 172. Thus, the number of bound sulfonic acids was estimated to be 86.

[0064] 9. Measurement of temperature - dependent transmittance (turbidity) (2) 9.1. Measurement procedure For a dendrimer sample of 1 mg / mL (buffer concentration 20 mM), the change in transmittance with temperature was measured using a UV / Vis Spectrophotometer (V - 630, JASCO). A Peltier holder (ETC - 717, JASCO) was used for temperature adjustment. The measurement conditions were as follows: measurement wavelength: 500 nm, heating rate: 1 °C / min, measurement interval: 0.1 °C, bandwidth: 1.5 nm. The 1 mg / mL sample was prepared as follows. 100 μL of a 10 mg / mL dendrimer aqueous solution was mixed with 700 μL of distilled water, and 200 μL of buffer solutions with different pH values was added to achieve the target pH. As the buffer solution, 100 mM acetate buffer solution (aqueous acetic acid + aqueous sodium acetate) was used when the target pH was 4 or more and 5 or less, 100 mM phosphate buffer solution (aqueous sodium dihydrogen phosphate + aqueous disodium hydrogen phosphate) was used when the target pH was 6 or more, glycine - hydrochloride buffer solution was used when the target pH was 3 or less, and phosphate buffer solution was used together with 100 mM NaOH aqueous solution when the target pH was 11 or more. For G4 - Phe - SO3Na, G3 - Phe - SO3Na, and G5 - Phe - SO3Na, for comparison, measurements were also carried out on samples with an equimolar concentration (32 μM).

[0065] 9.2. Measurement Results (1) The measurement results for G4 - SO3Na are shown in Figure 8. The sample of G4 - SO3Na had a transmittance of 100% within the measurement temperature range at both pH 5 and 6.5, showing neither temperature responsiveness nor pH responsiveness. From this fact and the results shown in "5.2. Measurement Results (1)", it can be understood that the internal tertiary amino groups of the dendrimer, as well as the terminal Phe residues and strong acid groups, play important roles in the expression of pH responsiveness and temperature responsiveness.

[0066] 9.3. Measurement Results (2) For G4 - Phe - SO3Na, G3 - Phe - SO3Na, and G5 - Phe - SO3Na, the measurement results for the 1 mg / mL samples are shown in Figure 9 and Table 3, and the measurement results for the 32 μM samples are shown in Figure 10 and Table 3.

Table 3

[0067] From Figs. 9 and 10 and Table 3, it can be seen that G4-Phe-SO3Na, G3-Phe-SO3Na, and G5-Phe-SO3Na all exhibit LCST-type phase transitions at pH 5 and UCST-type phase transitions at pH 6.5. From this, it can be understood that regardless of the generation number of the dendrimer, the LCST-type / UCST-type temperature phase transition switches according to the pH. In the 32 μM sample, at pH 5 showing an LCST-type phase transition, G4-Phe-SO3Na and G5-Phe-SO3Na underwent phase transitions at 45 °C, while G3-Phe-SO3Na underwent a phase transition at a higher 63 °C. This result indicates that G3-Phe-SO3Na is less hydrophobic compared to G4-Phe-SO3Na and G5-Phe-SO3Na. Although not intending to be restricted by theory, it is considered that this is because G3-Phe-SO3Na is not spherical, making it difficult for effective interactions to occur between Phe molecules. On the other hand, at pH 6.5 showing a UCST-type phase transition, the phase transition temperature shifted to the higher temperature side for G3-Phe-SO3Na and G5-Phe-SO3Na. This result indicates that G3-Phe-SO3Na and G5-Phe-SO3Na are less soluble compared to G4-Phe-SO3Na. Although not intending to be restricted by theory, for G3-Phe-SO3Na, it is considered that because it is not spherical, its surface area becomes larger and the intermolecular interaction becomes stronger, and for G5-Phe-SO3Na, it is considered that as the number of action points of intermolecular interaction increases, it becomes easier to aggregate.

[0068] 10. Synthesis of G4-Phe-BSO3Na, PEI-Phe-SO3Na, and DGL-Phe-SO3Na The dendrimer "G4-Phe-BSO3Na" is a fourth-generation (G4) dendrimer with an ethylene diamine core, a -CH2CH2CONHCH2CH2N< branching part, and -Phe residue-(CH2)4-SO3Na at the terminal group, and has the structure shown in the following schematic diagram.

Chemical formula

Chemical formula

[0069] G4-Phe-BSO3Na is G4-Phe 62 120 mg (3.95 μmol) and 303 mg (2.98 mmol) of 1,4-butanesultone were used and obtained according to the method described for G4-Phe-SO3Na in 8 above (yield: 27 mg; yield: 33%).

Chemical formula

[0070] The synthesis of PEI-Phe-SO3Na was carried out as follows.

Chem.

[0071] Next, for the synthesis of G4-Phe, deprotection of the Boc group was carried out using TFA in the same manner as the method described in 1 above to obtain PEI-Phe (yield: 104 mg; yield: 132%) (Fig. 12(b)). Furthermore, for G4-Phe-SO3Na, in the same manner as the method described in 8 above, 390.9 mg (3.20 mmol) of 1,3-propanesultone was used to obtain PEI-Phe-SO3Na (yield: 10 mg; yield: 9%). In Fig. 12(c), the 1 1H NMR spectrum of the obtained PEI-Phe-SO3Na is shown. Based on the integrated value of 5 for the peak of Phe near 7 ppm, when calculating the integrated value of the peak derived from the methylene (2) of the sulfonic acid side chain, it was 0.7. Thus, it was estimated that 35% of the terminals had a sulfo group bonded.

[0072] The synthesis of DGL-Phe-SO3Na was carried out as follows.

Chemical formula

[0073] Next, in the same manner as the method described in 1 above for the synthesis of G4-Phe, the Boc group was deprotected using TFA to obtain DGL-Phe 123It was obtained (yield: 56 mg; yield rate: 86%) (Figure 13(b)). Furthermore, at 37 °C, DGL-Phe-SO3Na was obtained using 131.4 mg (1.07 mmol) of 1,3-propanesultone in the same manner as the method described in 8 above for G4-Phe-SO3Na (yield: 19 mg; yield rate: 36%). Figure 13(c) shows the 1 1H NMR spectrum of the obtained DGL-Phe-SO3Na. The integrated value of the peaks from 2.6 ppm to 4.4 ppm was 1088. Subtracting the integrated values of the peaks derived from poly-L-lysine and Phe from this value and calculating the integrated values of the peaks corresponding to methylene (1) and (3), it was estimated that SO3Na was bound to the 85 terminals (binding rate 69%). Therefore, the obtained DGL-Phe-SO3Na was DGL-Phe 123 -(SO3Na) 85 It was found to be.

[0074] 11. Measurement of temperature-dependent transmittance (turbidity) (3) 11.1. Measurement procedure The measurement procedure is as described in 9.1 above. The measurement was performed on samples at 1 to 5 mg / mL (buffer concentration 20 mM). 11.2. Measurement results Figure 14 shows the measurement results when the concentration of G4-Phe-BSO3Na was changed in addition to the pH. G4-Phe-BSO3Na was dissolved within the measurement temperature range at pH 5 and pH 9 and did not show temperature responsiveness. On the other hand, at pH 7, it showed an LCST-type phase transition at a concentration of 1 to 5 mg / mL. Also, at pH 8, it showed UCST-type temperature responsiveness at a high concentration (5 mg / mL). Although not intended to be restricted by theory, it is considered that at high concentrations, intermolecular interactions become easier, resulting in a significantly lower transmittance in the low-temperature region.

[0075] Figure 15 shows the measurement results for PEI-Phe-SO3Na. PEI-Phe-SO3Na was dissolved within the measured temperature range at pH 5 and did not exhibit temperature responsiveness. On the other hand, the transmittance at pH 6.5 and 7 showed a U-shaped temperature responsiveness where it once decreased and then increased as the temperature rose. From this, it can be understood that PEI-Phe-SO3Na exhibits an LCST-type phase transition in the low-temperature region and a UCST-type phase transition in the high-temperature region when around pH 6 to 7.

[0076] The measurement results for DGL-Phe-SO3Na are shown in Fig. 16. DGL-Phe-SO3Na exhibits a UCST-type phase transition at pH 7, while at pH 4 and 6.5, it was dispersed in an aggregated state within the measured temperature range and did not show an LCST-type phase transition. From this, it is considered that the terminal group (in this case, -Phe-SO3Na) is involved in the UCST-type phase transition, and the internal tertiary amine of the dendritic polymer is necessary for the LCST-type phase transition.

[0077] The pH and temperature dependencies of the four dendritic polymers (G4-Phe-SO3Na, G4-Phe-BSO3Na, PEI-Phe-SO3Na, and DGL-Phe-SO3Na) synthesized above are summarized in Table 4.

Table 4

[0078] The above-described embodiments and examples are described as examples for facilitating the understanding of the present invention, and it should be noted that the present invention is not limited only to the specific configurations and arrangements described in this specification or the attached drawings. Those skilled in the art should understand and easily recognize that the specific configurations, means, methods, and apparatuses described in this specification can be replaced with many other known ones in the art without departing from the spirit and scope of the present invention.

Claims

1. A pH- and temperature-responsive dendritic polymer having a plurality of branching portions and a plurality of terminal groups, The dendritic polymer is a polyamidoamine dendrimer, at least a portion of the plurality of terminal groups contains a phenylalanine residue and a strong acid group or a salt thereof on the amino group side of the residue, and the terminal group containing the phenylalanine residue and the strong acid group or a salt thereof is represented by the formula -(phenylalanine residue)-R ３ -SO ３ H, -(phenylalanine residue)-R ３ -OSO ３ H, -(phenylalanine residue)-R ３ -P(O)(OH) ２ and -(phenylalanine residue)-R ３ -OP(O)(OH) ２ (In the above formula, R ３ is halogen, C １ ~C ４ Alkyl and C １ ~C ４ C optionally substituted with one or more alkoxy １ ~C ４ a group selected from the group consisting of alkylene, alkylene bonded to the amino group of a phenylalanine residue, or a salt thereof, and linked to the branching moiety at the carbonyl group side of the phenylalanine residue.

2. The dendritic polymer according to claim 1, which exhibits LCST-type phase transition behavior and UCST-type phase transition behavior, and exhibits UCST-type phase transition behavior under a pH condition higher than the pH condition that exhibits LCST-type phase transition behavior.

3. The terminal group is -(phenylalanine residue)-R ３ -SO ３ H, -(phenylalanine residue)-R ３ -OSO ３ H, -(phenylalanine residue)-R ３ -P(O)(OH) ２ and -(phenylalanine residue)-R ３ -OP(O)(OH) ２ (wherein R ３ is a group selected from the group consisting of halogen, C １ to C ４ alkyl and C １ to C ４ alkylene which may be substituted with one or more of C ２ to C ４ and is bonded to the amino group of the phenylalanine residue), or a salt thereof. The dendritic polymer according to claim 1 or 2.

4. The end group is -(phenylalanine residue)-R ３ -SO ３ H, -(phenylalanine residue)-R ３ -OSO ３ H, -(phenylalanine residue)-R ３ -P(O)(OH) ２ and -(phenylalanine residue)-R ３ -OP(O)(OH) ２ (In the above formula, R ３ is C ２ to C ４ alkylene, and is bonded to the amino group of the phenylalanine residue), or a salt thereof. The dendritic polymer according to claim 3

5. The terminal group is -(phenylalanine residue)-R ３ -SO ３ H (wherein, R ３ is halogen, C １ ~C ４ Alkyl and C １ ~C ４ C optionally substituted with one or more alkoxy ２ ~C ４ 4. The dendritic polymer of claim 3, wherein the aryl group is an alkylene and is attached to the amino group of the phenylalanine residue, or a salt thereof.

6. The terminal group is -(phenylalanine residue)-R ３ -SO ３ H (wherein R ３ is C ２ to C ４ alkylene and is bonded to the amino group of the phenylalanine residue) or a salt thereof, the dendritic polymer according to claim 5.

7. The terminal group is -(phenylalanine residue)-CH ２ -CH ２ -CH ２ -SO ３ H or a salt thereof, the dendritic polymer according to claim 6.

8. The dendritic polymer according to any one of claims 1 to 7, wherein at least 50% of the plurality of terminal groups contains a phenylalanine residue and a strong acid group on the amino group side of the residue or a salt thereof.

9. A method for separating a soluble substance from a solution containing the soluble substance, wherein in the solution, the soluble substance is supported on the dendritic polymer in an insolubilized state formed at a temperature higher than the lower critical solution temperature (LCST) or lower than the upper critical solution temperature (UCST) of the dendritic polymer according to any one of claims 1 to 8.

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