JP4730938B2 - Graft copolymer and method for producing graft copolymer - Google Patents

Description

  The present invention relates to a polyvinyl alcohol-based graft copolymer having a molecular chain composed of a cationic block capable of forming an ion complex with an anionic compound or a molecular chain composed of the cationic block and a nonionic block, and graft copolymer The present invention relates to a method for manufacturing coalescence.

  In recent years, poly (ethyleneimine), a cationic polymer, can form ion complexes by electrostatic ionic interactions with anionic biopolymers such as DNA, making it useful for gene carrier applications. It attracts attention as being.

  Poly (ethyleneimine), which is a cationic polymer, is water-soluble, but when it forms an ion complex with an anionic substance such as an anionic biopolymer such as DNA, it becomes insoluble in water. Its use is very limited. Therefore, as a method for imparting water solubility or water dispersibility to an ion complex of a water-soluble cationic polymer and an anionic biopolymer, there is a method using a double hydrophilic copolymer.

  Unlike the conventional hydrophobic / hydrophilic amphiphilic copolymer, the double hydrophilic copolymer is water-soluble in both blocks, but both blocks have different functions. That is, one block interacts with the substrate in water and the other block contributes to water solubility. For example, when a water-soluble copolymer consisting of a cationic block and a nonionic block that are both water-soluble is used, the cationic block has the ability to form an ion complex with an anionic compound, and the nonionic block is water-soluble. Have sex. Then, it is uniformly dispersed in water to form nano micelles, and the nano micelles can be applied to a wide range of uses such as DNA carriers.

  As a double hydrophilic copolymer having a poly (ethyleneimine) block as a cationic block, for example, poly (ethylene glycol) having a sulfonate structure at one end of a molecular chain is used as an initiator, and 2-methyl- 2-oxazoline or 2-ethyl-2-oxazoline is subjected to cationic ring-opening polymerization to synthesize a water-soluble copolymer comprising a poly (ethylene glycol) block and a poly (N-acyl-ethyleneimine) block, and then poly (N -Acyl-ethyleneimine) blocks are hydrolyzed to form poly (ethyleneimine) blocks, and the like is known (for example, see Non-Patent Document 1 and Non-Patent Document 2).

  However, the double hydrophilic copolymer described above has a problem in that it has a low efficiency of forming an ion complex with an anionic substance such as DNA and is poor in affinity with a biopolymer.

Yoshitsugu Akiyama, 3 others, “Macromolecules”, 2000, Vol. 33, p. 5841-5845 Milos Sedlak, two others, “Macromolecular Chemistry and Physics”, 1999, Vol. 199, p. 247-254

  The problem to be solved by the present invention is a polyvinyl alcohol-based graft grafted with a molecular chain having a linear cationic block, which has a high efficiency of forming an ion complex with an anionic substance and has a good affinity with the anionic substance. It is providing the manufacturing method of a copolymer and its graft copolymer.

  The present invention has solved the above problems by providing a polyvinyl alcohol graft copolymer represented by the following formula (1).

［式（１）中、Ｘは下記式（２）

[In the formula (1), X represents the following formula (2)

（式（２）中、Ｒ_１は水素原子又は炭素数が１〜１８のアシル基、Ｒ_２は炭素数が１〜１８のアシル基を表し、ｐ_１、ｐ_２はそれぞれ２又は３であり、ｑ_１は２〜１００００、ｑ_２は０〜９９９８の範囲であり、ｑ_１＋ｑ_２が２〜１００００の範囲である。）
又は、下記式（３）

(In Formula (2), R ₁ represents a hydrogen atom or an acyl group having 1 to 18 carbon atoms, R ₂ represents an acyl group having 1 to 18 carbon atoms, and p ₁ and p ₂ are 2 or 3, respectively. , Q ₁ is in the range of _{2 to} 10000, q ₂ is in the range of 0 to 9998, and q ₁ + q ₂ is in the range of ₂ to 10000.)
Or the following formula (3)

（式（３）中、Ｒ_３は水素原子又は炭素数が１〜１８のアシル基を表し、ｐ_３、ｐ_４はそれぞれ２又は３であり、ｑ_３、ｑ_４はいずれも１以上でありｑ_３＋ｑ_４が２〜１００００の範囲である。）
で表される直鎖状ポリマー鎖の少なくとも一種を表し、ｎ_１／ｎ_２が３００／７００〜９９９／１の範囲であり、ｎ_１＋ｎ_２は１０〜１０００００の範囲である。］

(In Formula (3), R ₃ represents a hydrogen atom or an acyl group having 1 to 18 carbon atoms, p ₃ and p ₄ are each 2 or 3, and q ₃ and q ₄ are each 1 or more. q ₃ + q ₄ is in the range of ₂ to 10,000.)
At least one of the linear polymer chains represented by the formula: n ₁ / n ₂ is in the range of 300/700 to 999/1, and n ₁ + n ₂ is in the range of 10 to 100,000. ]

Moreover, the said subject was solved by the manufacturing method of the polyvinyl-alcohol-type graft copolymer which consists of the following process of this invention.
(A) Formula (i) below

（式（i）中、Ｒ_４は炭素数１〜１８のアシル基を表し、ｎ_３は１０〜１０００００の範囲である。）
で表されるポリマー中の側鎖アシル基の一部を、金属有機アニオンを用いて金属置換反応させることによりアルコキシド化する工程、

(In formula (i), R ₄ represents an acyl group having 1 to 18 carbon atoms, and n ₃ is in the range of 10 to 100,000.)
A step of alkoxideizing a part of the side chain acyl group in the polymer represented by the following by metal substitution reaction using a metal organic anion:

（ｂ）（ａ）の工程によりアルコキシド化した側鎖部位を、下記式（ii）
（式（ii）中、Ｒ_５は炭素数１〜５のアルキル基を表し、Ｘは式（１）と同様であり、ＹはＣｌ、Ｂｒ、Ｉ、スルホネートオキシ基のいずれかである。）
で表される反応末端を有する直鎖状ポリマー鎖で置換する工程、
（ｃ）残存している側鎖アシル基を加水分解する工程からなる
製造方法。

(B) The side chain moiety alkoxideized by the step (a) is represented by the following formula (ii)
(In formula (ii), R ₅ represents an alkyl group having 1 to ₅ carbon atoms, X is the same as in formula (1), and Y is any one of Cl, Br, I, and a sulfonateoxy group.)
A step of substituting with a linear polymer chain having a reaction end represented by:
(C) A production method comprising a step of hydrolyzing the remaining side chain acyl group.

  In the polyvinyl alcohol graft copolymer of the present invention, the poly (ethyleneimine) side chain, which is a cationic polymer grafted in a comb shape with polyvinyl alcohol as the main chain, has an anionic substance such as DNA. An ion complex can be formed efficiently by electrostatic ionic interaction with an anionic biopolymer. Moreover, since the polyvinyl alcohol block of the main chain exhibits an excellent affinity for biopolymers due to the interaction between many hydroxy groups and biopolymers, it can be usefully used in a wide range of applications such as gene carriers and biomaterials.

  In addition, the polyvinyl alcohol-based graft copolymer includes an alkoxidation step of some side chain acyl groups, a step of replacing the alkoxide side chain site with a linear polymer chain, and a remaining side chain. It can be produced by a simple method comprising a step of hydrolyzing an acyl group.

  The polyvinyl alcohol graft copolymer of the present invention is represented by the following formula (1).

［式（１）中、Ｘは下記式（２）

[In the formula (1), X represents the following formula (2)

（式（２）中、Ｒ_１は水素原子又は炭素数が１〜１８のアシル基、Ｒ_２は炭素数が１〜１８のアシル基を表し、ｐ_１、ｐ_２はそれぞれ２又は３であり、ｑ_１は２〜１００００、ｑ_２は０〜９９９８の範囲であり、ｑ_１＋ｑ_２が２〜１００００の範囲である。）
又は、下記式（３）

(In Formula (2), R ₁ represents a hydrogen atom or an acyl group having 1 to 18 carbon atoms, R ₂ represents an acyl group having 1 to 18 carbon atoms, and p ₁ and p ₂ are 2 or 3, respectively. , Q ₁ is in the range of _{2 to} 10000, q ₂ is in the range of 0 to 9998, and q ₁ + q ₂ is in the range of ₂ to 10000.)
Or the following formula (3)

（式（３）中、Ｒ_３は水素原子又は炭素数が１〜１８のアシル基を表し、ｐ_３、ｐ_４はそれぞれ２又は３であり、ｑ_３、ｑ_４はいずれも１以上でありｑ_３＋ｑ_４が２〜１００００の範囲である。）
で表される直鎖状ポリマー鎖の少なくとも一種を表し、ｎ_１／ｎ_２が３００／７００〜９９９／１の範囲であり、ｎ_１＋ｎ_２は１０〜１０００００の範囲である。］

(In Formula (3), R ₃ represents a hydrogen atom or an acyl group having 1 to 18 carbon atoms, p ₃ and p ₄ are each 2 or 3, and q ₃ and q ₄ are each 1 or more. q ₃ + q ₄ is in the range of ₂ to 10,000.)
At least one of the linear polymer chains represented by the formula: n ₁ / n ₂ is in the range of 300/700 to 999/1, and n ₁ + n ₂ is in the range of 10 to 100,000. ]

  The side chain X grafted in the polyvinyl alcohol-based graft copolymer represents at least one linear polymer chain represented by the above formula (2) or (3).

  The linear polymer chain represented by the formula (2) is a polymer chain composed of an ethyleneimine structural unit, an N-acylethyleneimine structural unit or an N-acylpropyleneimine structural unit which is a cationic structural unit, The linear polymer chain represented by the above formula (3) is a linear polymer chain composed of these cationic structural units and an ethylene glycol structural unit which is a nonionic structural unit.

  Specifically, the linear polymer chain represented by the formula (2) or (3) is a linear polymer chain composed only of an N-acylethyleneimine structural unit, or a linear chain composed solely of an ethyleneimine structural unit. A polymer chain, a linear polymer chain composed only of N-acylpropyleneimine structural units, a linear polymer chain composed solely of propyleneimine structural units, and an ethylene or propyleneimine structural unit and an N-acylethylene or propyleneimine structural unit A linear polymer chain comprising a combination of N-acylethylene or propyleneimine structural units and N-acylpropylene or ethyleneimine structural units as a combination of structural units having the same or different acyl groups Chain, N-acylethyleneimine structural unit, N-acylpropyleneimine Forming units, the linear polymer chains, which consist of a combination of ethylene imine structural units or propylene imine structural units and ethylene glycol structural units or propylene glycol structural units may be used.

R ₁ and R ₂ in the above formula (2) can be each a hydrogen atom or an acyl group having 1 to 18 carbon atoms, and the acyl group is preferably an acyl group having 1 to 5 carbon atoms. preferable.

Further, regarding the degree of polymerization of the linear polymer chain of the above formula (2), q ₁ may be ₂ to 10000, more preferably 10 to 1000, and q ₂ may be 0 to 9998, but 0 to 1000 is preferable. More desirable. Further, q ₁ + q ₂ may be in the range of ₂ to 10000, more preferably 10 to 2000, and further preferably 20 to 200. Q ₃ and q ₄ are both 1 or more and q ₃ + q ₄ may be in the range of 2 to 10,000, more preferably 20 to 2000, and further preferably 20 to 200. If the degree of polymerization is in the above range, an ion complex with an anionic substance can be suitably formed.

  Among them, for example, a polymer chain composed of an N-acylethyleneimine or N-acylpropyleneimine structural unit has high hydrophilicity, and a salt such as a hydrochloride thereof has higher water solubility.

  Among these linear polymer chains, the linear polymer chain composed of two or more types of structural units may be a random polymer chain or a block polymer chain, but the function of each structural unit is effective. In order to express it, it is preferable that it is a block polymer chain which consists of a block of each structural unit.

  Moreover, when using the linear polymer chain which consists of 2 or more types of structural units among the polymer chains represented by the said Formula (2), one side is utilized using the difference in the reactivity of each structural unit. By selectively cationizing the structural unit, a double hydrophilic polymer chain can be obtained. In addition, the polymer chain represented by the above formula (3) has a double hydrophilic structure. By using such a double hydrophilic polymer chain as a side chain, the degree of hydrophilicity in the copolymer of the present invention and the ability to form an ion complex can be adjusted.

  The polyvinyl alcohol-based graft copolymer of the present invention is characterized by having a cationic structural unit and a nonionic structural unit in its structure. The ratio of the cationic structural unit in the polyvinyl alcohol-based graft copolymer of the present invention is preferably in the range of 1 to 50% by mass, and more preferably in the range of 3 to 30% by mass of the total structural units. preferable. When the ratio of the cationic structural unit of the copolymer is in the above range, the ability to form an ion complex with an anionic substance is particularly excellent.

  Of the above linear polymer chains, a linear polymer chain composed of an N-acylethyleneimine structural unit and / or an N-acylpropyleneimine structural unit is a polyoxazoline generally obtained by cation living polymerization of an oxazoline monomer, A linear polymer chain composed of an ethyleneimine structural unit and a linear polymer chain composed of a propyleneimine structural unit are generally obtained by hydrolyzing the polyoxazoline obtained by the above cation living polymerization using an acid or a base. .

Among them, examples of N- acylethyleneimine structural units is poly (N- formyl ethyleneimine), poly (N- acetyl ethyleneimine), poly (N- propionylethyleneimine), poly (N- butyryl ethylenimine ), Poly (N-isobutyrylethyleneimine), poly (N-pivaloylethyleneimine), poly (N-lauroylethyleneimine), poly (N-stearoylethyleneimine), poly (N- (3- ( Polyethyleneimine acylated with aliphatic saturated carboxylic acids such as perfluorooctyl) propionyl) ethyleneimine), poly (N-acryloylethyleneimine), poly (N-methacryloylethyleneimine), poly (N-oleoylethyneimine) ) Ethyleneimine acylated with aliphatic unsaturated carboxylic acid such as Acylated with aromatic carboxylic acid such as poly (N-benzoylethyleneimine), poly (N-toluoylethyleneimine), poly (N-naphthoylethyleneimine), poly (N-cinnamoylethyleneimine) Examples include polyethyleneimine.

Examples of the N- acylpropyleneimine structural unit include, for example, poly (N-formylpropyleneimine), poly (N-acetylpropyleneimine), poly (N-propionylpropyleneimine), and poly (N-butyrylpropylene). Imine), poly (N-isobutyrylpropyleneimine), poly (N-pivaloylpropyleneimine), poly (N-lauroylpropyleneimine), poly (N-stearoylpropyleneimine), poly (N- (3- (Perfluorooctyl) propionyl) propyleneimine) and other aliphatic saturated carboxylic acid acylated polypropyleneimine, poly (N-acryloylpropyleneimine), poly (N-methacryloylpropyleneimine), poly (N-oleoylethyne) Imine) and other aliphatic unsaturated carboxylic acids Aromatics such as acylated propyleneimine, poly (N-benzoylpropyleneimine), poly (N-toluoylpropyleneimine), poly (N-naphthoylpropyleneimine), poly (N-cinnamoylpropyleneimine), etc. And polypropyleneimine acylated with carboxylic acid.

  As a combination of an N-acylethylene or propyleneimine structural unit and a N-acylpropylene or ethyleneimine structural unit, which is a grafted linear polymer chain represented by the above formula (2), have the same or different acyl groups The linear block polymer chain using the structural unit includes, for example, 2-oxazoline or 2-methyl-2-oxazoline by cationic ring-opening living polymerization, and then the resulting living polymer is further converted to 2-ethyl-2. -Obtained by polymerizing oxazoline. Similarly, a multi-block linear polymer chain can be obtained by alternately repeating polymerization of 2-oxazoline or 2-methyl-2-oxazoline and polymerization of 2-ethyl-2-oxazoline.

  A linear block polymer chain comprising a combination of an ethylene or propyleneimine structural unit and an N-acylethylene or propyleneimine structural unit, which is a grafted linear polymer chain represented by the above formula (2), includes: For example, a block having a molecular chain composed of a poly (ethyleneimine) block and a poly (N-propionylethyleneimine) block is a precursor of the poly (N-formylethyleneimine) block or poly (N-acetyl). A poly (N-formylethyleneimine) block or a poly (N-acetylethyleneimine) block having a molecular chain composed of an ethyleneimine block and a poly (N-propionylethyleneimine) block is preferentially hydrolyzed. Can be obtained.

  The preferential hydrolysis reaction of the poly (N-formylethyleneimine) block or (N-acetylethyleneimine) block must be carried out in the emulsion state. The emulsion basically consists of a water-soluble block copolymer, water, and an organic solvent that dissolves the poly (N-propionylethyleneimine) block but is incompatible with water. When the mixture is stirred, the poly (N-formylethyleneimine) block or (N-acetylethyleneimine) block of the water-soluble block copolymer is dissolved in the aqueous phase, while the poly (N-propionylethyleneimine) block is Dissolves in the organic solvent phase to form an emulsion. In this case, the water-soluble block copolymer acts as an emulsifier. The emulsion may be either O / W type or W / O type.

  Examples of the organic solvent incompatible with water include methylene chloride, chloroform, chlorobenzene, nitrobenzene, methoxybenzene, toluene, and a mixed solvent thereof.

  During the hydrolysis reaction, acid or alkali is added to the aqueous phase as a known hydrolysis catalyst. Usual inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid can be used as the acid, and ordinary inorganic alkalis such as sodium hydroxide, potassium hydroxide and ammonia can be used as the alkali.

  The hydrolysis reaction temperature is preferably 100 ° C. or lower, and may be set according to the concentration of the acid or alkali used. When the acid or alkali concentration is high, the temperature is set low, for example, about room temperature, and when the acid or alkali concentration is low, the reaction temperature is set high.

  And from the combination of the N-acylethyleneimine structural unit or N-acylpropyleneimine structural unit and the ethylene glycol structural unit or the propylene glycol structural unit, which is a grafted linear polymer chain represented by the above formula (3) Such a linear block polymer chain is generally obtained by tosylating commercially available alkoxy polyethylene glycol or alkoxy polypropylene glycol and then performing cationic living polymerization using an oxazoline monomer. Further, the acylimine block polymer thus obtained is hydrolyzed with an acid or a base to form a linear chain comprising a combination of an ethyleneimine structural unit or a propyleneimine structural unit and an ethylene glycol structural unit or a propylene glycol structural unit. A block polymer chain is obtained.

  As an initiator for the cationic ring-opening living polymerization, a low molecular compound and a high molecular compound can be used. As a polymerization initiator for low molecular weight compounds, a compound having a functional group such as an alkyl chloride group, an alkyl bromide group, an alkyl iodide group, a toluenesulfonyloxy group, or a trifluoromethylsulfonyloxy group in the molecule should be used. Can do. Specifically, for example, benzyl chloride, benzyl bromide, benzyl iodide, benzyl toluene sulfonate, benzyl trifluoromethyl sulfonate, methyl bromide, methyl iodide, methyl toluene sulfonate or toluene sulfonic anhydride, tri And fluoromethylsulfonic acid anhydride.

  Among these, alkyl bromide, alkyl iodide, alkyl toluenesulfonate, and alkyl trifluoromethylsulfonate have high polymerization initiation efficiency, and it is particularly preferable to use alkyl bromide and alkyl toluenesulfonate.

  In the case of the grafted linear polymer chain represented by the above formula (3), examples of the polymerization initiator for the polymer compound include a bromine atom or an iodine atom at the terminal carbon atom of poly (ethylene or propylene glycol). And those having a toluenesulfonyl group bonded to the terminal oxygen atom can be used.

  As the solvent used in the cationic ring-opening living polymerization reaction, known and commonly used solvents such as an aprotic inert solvent and an aprotic polar solvent can be used.

As for the degree of polymerization of the polyvinyl alcohol block and the block obtained by grafting the linear block of the polyvinyl alcohol graft copolymer represented by the above formula (1) of the present invention, n ₁ / n ₂ is 300/700 to 999. Although the range of / 1 may be sufficient, the range of 600/400 to 999/1 is more desirable, and n ₁ + n ₂ may be in the range of 10 to 100,000, but 100 to 10,000 is more desirable. By making each numerical value within the above range, the effects of the present invention can be suitably expressed.

  The polyvinyl alcohol-based graft copolymer represented by the above formula (1) of the present invention has a linear cationic block, and can be used for underwater micelle formation with a biopolymer or the like. The nano micelles are useful as clinical diagnostic agents and therapeutic agents in the nanomedicine field.

The polyvinyl alcohol-based graft copolymer represented by the above formula (1) of the present invention is:
(A) Formula (i) below

（式（i）中、Ｒ_４は炭素数１〜１８のアシル基を表し、ｎ_３は１０〜１０００００の範囲である。）
で表されるポリマー中の側鎖アシル基の一部を、金属有機アニオンを用いて金属置換反応させることによりアルコキシド化する工程、

(In formula (i), R ₄ represents an acyl group having 1 to 18 carbon atoms, and n ₃ is in the range of 10 to 100,000.)
A step of alkoxideizing a part of the side chain acyl group in the polymer represented by the following by metal substitution reaction using a metal organic anion:

（ｂ）（ａ）の工程によりアルコキシド化した側鎖部位を、下記式（ii）
（式（ii）中、Ｒ_５は炭素数１〜５のアルキル基を表し、Ｘは式（１）と同様であり、ＹはＣｌ、Ｂｒ、Ｉ、スルホネートオキシ基のいずれかである。）
で表される反応末端を有する直鎖状ポリマー鎖で置換する工程、及び
（ｃ）残存している側鎖アシル基を加水分解する工程
からなる製造方法により水溶性グラフト共重合体であるポリビニルアルコール系グラフト共重合体が容易に製造できる。

(B) The side chain moiety alkoxideized by the step (a) is represented by the following formula (ii)
(In formula (ii), R ₅ represents an alkyl group having 1 to ₅ carbon atoms, X is the same as in formula (1), and Y is any one of Cl, Br, I, and a sulfonateoxy group.)
A polyvinyl alcohol which is a water-soluble graft copolymer by a production method comprising the step of substituting with a linear polymer chain having a reaction end represented by the formula: and (c) hydrolyzing the remaining side chain acyl group A graft copolymer can be easily produced.

Regarding the degree of polymerization of the polyvinyl alkyl ester such as polyvinyl acetate or polyvinyl ethyl ester represented by the above formula (i), n ₃ may be in the range of 10 to 100,000, more preferably 100 to 10,000.

Used in said step (a), metal organic anions are, for example, sodium, lithium, or an organometallic anion used was a metal such as potassium may be used.

  Of the side chain acyl groups in the above formula (i), the ratio of the substitution reaction using the metal organic anion is (side chain acyl polymerization degree) / (side chain alkoxide polymerization degree) of 300/700 to 999 / A range of 1 is acceptable, but 600/400 to 999/1 is a more desirable range.

  In the step (ii), the side chain site subjected to the alkoxidation reaction is replaced with a linear polymer chain having a reaction end represented by the above formula (ii), whereby the above formula (2) or (3 ) Can be introduced.

  The reaction terminal represented by D in the above formula (ii) may be a halogen atom such as chlorine, bromine or iodine or a sulfonateoxy group. For these reaction ends, the above cationic ring-opening living polymerization initiator residue used when polymerizing the polymer chain represented by the above formula (2) or (3) can be used as it is.

  Subsequently, the polyvinyl alcohol graft copolymer represented by the above formula (1) of the present invention is easily obtained by hydrolyzing the remaining side chain acyl group with an acid or a base in the step (iii). Can be manufactured. As the acid and base used in the hydrolysis, acids and bases used in ordinary hydrolysis can be used.

  The polyvinyl alcohol-based graft copolymer represented by the above formula (1) of the present invention obtained by the production method of the present invention has a good affinity with biopolymers, so that it is a DNA carrier or an anionic dye, It is useful for the preparation of underwater nanodispersions with pigments. In addition to the above-mentioned clinical diagnostic agents and therapeutic agents in the nanomedicine field, nanoreactors, nanoparticle-type catalysts, etc. It can also be suitably used for inks, especially aqueous jet inks.

  Hereinafter, the present invention will be described in more detail with reference to examples. Unless otherwise specified, “part” and “%” represent “part by mass” and “% by mass”, respectively.

(Synthesis Example 1)
<Synthesis of linear polymer chain 1>
After replacing the inside of the reaction vessel having a volume of 100 ml with nitrogen gas, 0.20 g (1.07 mmol) of methyl p-toluenesulfonate as a cationic ring-opening living polymerization initiator, 40 ml of N, N-dimethylacetamide, 2-methyl- After adding 10.0 g (118 mmol) of 2-oxazoline, 2-methyl-2-oxazoline was subjected to cationic ring-opening living polymerization while stirring at 100 ° C. for 24 hours. The resulting reaction mixture was added to 300 ml of ethyl acetate, and after vigorous stirring at room temperature, the product solid was filtered. The solid was washed twice with 100 ml of ethyl acetate and then dried under reduced pressure to obtain 9.7 g of a solid polymer chain of poly (N-acetylethyleneimine).

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (CDCl3) measurement results:
δ (ppm): 7.72 (d), 7.18 (d), 3.50 to 3.30 (bs), 2.22 to 2.08 (m)

(Synthesis Example 2)
<Synthesis of linear polymer chain 2>
The linear polymer chain (5.0 g) obtained in Synthesis Example 1 was stirred at 90 ° C. for 6 hours in 24.4 g of 5N hydrochloric acid water to perform a hydrolysis reaction. After standing to cool, the reaction mixture solution containing the white precipitate formed over time is added to about 150 ml of acetone and stirred at room temperature for about 30 minutes, and then the product solid is filtered, washed twice with acetone, and dried under reduced pressure. 4.5 g of white solid was obtained. From the analysis by 1H-NMR, a linear polymer chain of poly (ethyleneimine) having no side chain acetyl hydrogen (δ: 2.22 to 2.08 ppm)] of poly (N-acetylethyleneimine) by hydrolysis reaction Got.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (DMSO) measurement results:
δ (ppm): 3.31 to 3.19 (bs)

(Synthesis Example 3)
<Synthesis of linear polymer chain 3>
After replacing the inside of the reaction vessel having a volume of 100 ml with nitrogen gas, 0.20 g (1.07 mmol) of methyl p-toluenesulfonate as a cationic ring-opening living polymerization initiator, 40 ml of N, N-dimethylacetamide, 2-methyl- After adding 2.26 g (50 mmol) of 2-oxazoline, 2-methyl-2-oxazoline was subjected to cationic ring-opening living polymerization while stirring at 100 ° C. for 24 hours. After the reaction solution temperature was lowered to 60 ° C., 4.95 g (50 mmol) of 2-ethyl-2-oxazoline was added, followed by heating to 100 ° C. and stirring for 24 hours. The temperature of the reaction mixture was lowered to room temperature, 10 ml of methanol was added, and the reaction mixture was concentrated under reduced pressure. This concentrated solution was poured into 100 ml of diethyl ether to precipitate a polymer.

  The methanol solution of the obtained polymer is poured into diethyl ether and reprecipitated. After suction filtration, the filtrate is vacuum-dried to obtain a poly (N-acetylethyleneimine) block and a poly (N-propionylethyleneimine) block. 8.95 g of a linear block polymer chain solid consisting of

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (D2O) measurement results:
δ (ppm): 7.70 (d), 7.18 (d), 3.70 to 3.40 (m), 2.50 to 2.30 (m), 2.24 to 1.97 (m) ), 1.19 to 1.00 (m)

(Synthesis Example 4)
<Synthesis of linear polymer chain 4>
0.5 g of the linear block polymer chain obtained in Synthesis Example 3 was dissolved in 20 ml of 5 mol / l hydrochloric acid, and 3 ml of chloroform was added thereto and stirred for 30 minutes to obtain an emulsion. The emulsion was heated to 50 ° C. and stirred for 10 hours. After adding 50 ml of acetone to the reaction solution to precipitate a polymer, the solution was suction filtered and washed with acetone. The obtained solid was dried to obtain 0.45 g. From the analysis by 1H-NMR, the poly (N-acetylethyleneimine) block has no side chain acetyl hydrogen (δ: 2.24 to 1.97 ppm)] due to hydrolysis reaction, and the poly (ethyleneimine) block and poly (N A linear block polymer chain composed of a -propionylethyleneimine) block was obtained.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (DMSO) measurement results:
δ (ppm): 3.70 to 3.40 (m), 2.50 to 2.30 (m), 1.19 to 1.00 (m)

(Synthesis Example 5)
<Synthesis of linear polymer chain 5>
Under a nitrogen atmosphere, 7.15 g of p-toluenesulfonic acid chloride was added to a mixed solution of 15.0 g (7.5 mmol) of methoxypolyethylene glycol [Mn = 2,000], 6.0 g (75.0 mmol) of pyridine, and 20 ml of chloroform ( A solution of 37.5 mmol) in chloroform (20 ml) was added dropwise for 30 minutes with ice-cooling and stirring. After completion of dropping, the mixture was further stirred at a bath temperature of 40 ° C. for 4 hours. After completion of the reaction, 40 ml of chloroform was added to dilute the reaction solution. Subsequently, after sequentially washing with 50 ml of 5% aqueous hydrochloric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, it was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained solid was washed several times with hexane, filtered, and dried under reduced pressure at 80 ° C. to obtain 15.1 g of a tosylated product.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (CDCl3) measurement results:
δ (ppm): 7.82 (d), 7.28 (d), 3.74-3.54 (bs), 3.41 (s), 2.40 (s)

  3.0 g (1.6 mmol) of a methoxypolyethylene glycol compound having a p-toluenesulfonyloxy group at the terminal synthesized above, 6.8 g (80.0 mmol) of 2-methyloxazoline and 40 ml of N, N-dimethylacetamide were added to nitrogen. The mixture was stirred at 100 ° C. for 24 hours under an atmosphere. To the obtained reaction mixture, 300 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) was added, and after vigorous stirring at room temperature, the product solid was filtered. The solid was washed twice with 100 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) and then dried under reduced pressure to obtain 8.7 g of a block polymer solid of polyethylene glycol and polymethyloxazoline. It was.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (CDCl3) measurement results:
δ (ppm): 7.71 (d), 7.18 (d), 3.50 to 3.30 (bs), 3.45 (s), 2.36 (s), 2.22 to 2. 08 (m)

(Synthesis Example 6)
<Synthesis of linear polymer chain 6>
A block polymer 4.3 g (0.69 mmol) of polyethylene glycol and polymethyloxazoline obtained in Synthesis Example 5 above was stirred in 90 ° C. for 6 hours in 14.0 g of 5N aqueous hydrochloric acid to conduct a hydrolysis reaction. . After standing to cool, the reaction mixture solution containing the white precipitate formed over time is added to about 150 ml of acetone and stirred at room temperature for about 30 minutes, and then the product solid is filtered, washed twice with acetone, and dried under reduced pressure. 3.9 g of a white solid was obtained. From the analysis by 1H-NMR, poly (N-acetylethyleneimine) has no side chain acetyl hydrogen (δ: 2.22 to 2.08 ppm)] by hydrolysis reaction, and the obtained solid is obtained from polyethylene glycol and polyethylene. It is recognized as a double hydrophilic block polymer composed of imine.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (D2O) measurement results:
δ (ppm): 3.49 (s), 3.31 to 3.19 (bs)

(Example 1)
<Alkoxylation reaction 1 of polyvinyl acetate>
In a nitrogen atmosphere, 2.08 g of sodium methoxide (25 wt%) was added to a solution of 16.7 g of polyvinyl acetate (Mwt: 83,000) made by Aldrich in 40 ml of N, N-dimethylacetamide at 0 to 5 ° C. The solution was added dropwise for 20 minutes with stirring. After completion of the dropping, the reaction was further continued at room temperature for 2 hours to obtain a partially alkoxy body of polyvinyl acetate.

<Substitution reaction 1 of linear polymer chain to partial alkoxy compound>
Subsequent to the above, in a nitrogen atmosphere, a reaction mixture of the resulting polyvinyl acetate partially alkoxy compound was mixed with 20.9 ml of the linear polymer chain reaction mixture obtained in Synthesis Example 1 at room temperature with stirring. The solution was gradually added dropwise over 30 minutes. After completion of the dropping, the reaction temperature was raised to 50 ° C., and the reaction was carried out while stirring for another 8 hours. After completion of the reaction, 300 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) was added to the obtained reaction mixture, and after vigorous stirring at room temperature, the product solid was filtered. The solid was washed twice with 100 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) and then dried under reduced pressure to obtain 17.7 g of a solid. The obtained solid is recognized as a polyvinyl alcohol graft copolymer having a poly (N-acetylethyleneimine) side chain.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (DMSO) measurement results:
δ (ppm): 5.20 to 4.40 (m), 3.50 to 3.25 (m), 2.05 to 1.90 (m), 1.90 to 1.40 (m)
(Example 2)
<Hydrolysis reaction of side chain acyl group 1>
17.7 g of the copolymer obtained in Example 1 was stirred at 90 ° C. for 6 hours in 100.0 g of 5N hydrochloric acid water to carry out a hydrolysis reaction. After standing to cool, the reaction mixture containing the white precipitate that has formed over time is added to about 300 ml of acetone and stirred at room temperature for about 30 minutes, and then the solid product is filtered, washed twice with acetone, and dried under reduced pressure. 13.5 g of solid was obtained. From the analysis by 1H-NMR, there was no side chain acetyl hydrogen (δ: 2.05-1.90 ppm)] of polyvinyl acetate and poly (N-acetylethyleneimine) by hydrolysis reaction, and the obtained solid was It is recognized as a polyvinyl alcohol graft copolymer having an (ethyleneimine) side chain.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (D2O) measurement results:
δ (ppm): 3.85 to 3.75 (m), 3.55 to 2.95 (m), 1.55 to 1.25 (m)

(Example 3)
<Polyvinyl acetate alkoxylation reaction 2>
In a nitrogen atmosphere, 1.97 g of sodium methoxide (25 wt%) was added to a solution of 15.8 g of polyvinyl acetate (Mwt: 12, 800) from Aldrich in 50 ml of N, N-dimethylacetamide at 0 to 5 ° C. The solution was added dropwise for 20 minutes with stirring. After completion of the dropping, the reaction was further continued at room temperature for 2 hours to obtain a partially alkoxy body of polyvinyl acetate.

<Substitution reaction of linear polymer chain to partial alkoxy compound 2>
Subsequent to the above, in a nitrogen atmosphere, the reaction mixture of the resulting polyvinyl acetate partially alkoxy compound was mixed with about 19.8 ml of the solution of the linear polymer chain reaction mixture obtained in Synthesis Example 1 at room temperature with stirring. The solution was gradually added dropwise over 30 minutes. After completion of the dropping, the reaction temperature was raised to 50 ° C., and the reaction was carried out while stirring for another 8 hours. After completion of the reaction, 300 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) was added to the obtained reaction mixture, and after vigorous stirring at room temperature, the product solid was filtered. The solid was washed twice with 100 ml of a mixed solution of ethyl acetate and hexane (V / V = 1/2) and then dried under reduced pressure to obtain 16.2 g of a solid. The obtained solid is recognized as a polyvinyl alcohol graft copolymer having a poly (N-acetylethyleneimine) side chain.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (DMSO) measurement results:
δ (ppm): 5.20 to 4.40 (m), 3.50 to 3.25 (m), 2.05 to 1.90 (m), 1.90 to 1.40 (m)

Example 4
<Hydrolysis reaction of side chain acyl group 2>
The linear polymer chain-substituted polymer 8.1 g obtained in Example 3 above was stirred in 46.0 g of 5 N aqueous hydrochloric acid at 90 ° C. for 6 hours to conduct a hydrolysis reaction. After standing to cool, the reaction mixture containing the white precipitate that has formed over time is added to about 300 ml of acetone and stirred at room temperature for about 30 minutes, and then the solid product is filtered, washed twice with acetone, and dried under reduced pressure. 5.5 g of solid was obtained. From the analysis by 1H-NMR, there was no side chain acetyl hydrogen (δ: 2.05-1.90 ppm)] of polyvinyl acetate and poly (N-acetylethyleneimine) by hydrolysis reaction, and the obtained solid was It is recognized as a polyvinyl alcohol graft copolymer having an (ethyleneimine) side chain.

1H-NMR (manufactured by JEOL Ltd., AL300, 300 MHz) measurement results are shown below.
1H-NMR (D2O) measurement results:
δ (ppm): 3.85 to 3.75 (m), 3.55 to 2.95 (m), 1.55 to 1.25 (m)

(Application 1)
In order to measure the particle size distribution of the polyvinyl alcohol-based graft copolymer in the solution, the polyvinyl alcohol-based graft copolymer obtained in Example 2 or 4 was dissolved by adding pure water and stirring. When the distribution was measured, an average particle diameter of several tens to several hundreds nm was obtained, and it was shown that micelles were well formed in water.

(Application example 2)
100 mg of the polyvinyl alcohol graft copolymer obtained in Example 2 or 4 above was dissolved in 1 ml of distilled water, and this solution was dissolved in 4 ml of a 2.2 mg / ml aqueous solution containing DNA extracted from salmon sperm. The solution was added dropwise and stirred for 1 hour. With respect to the dispersion, formation of associated particles of the polyvinyl alcohol-based graft copolymer and DNA was confirmed by a dynamic light scattering method. As a result, monodispersed nanoparticles were observed.

As is clear from the above examples and application examples, the polyvinyl alcohol-based graft copolymer represented by the above formula (1) of the present invention has a good affinity for biopolymers with polyvinyl alcohol as the main chain structure. In addition, since it has a cationic structural unit having a grafted linear polymer chain, it forms an ion complex with DNA, which is an anionic biopolymer, and is dispersed into an aqueous phase as nanoparticles. be able to.

Claims (4)

(A) The side chain acyl group of polyvinyl acetate having a polymerization degree of 100 to 10000 is obtained by using an organic anion of sodium, lithium or potassium, and (side chain acyl polymerization degree) / (side chain alkoxide polymerization degree) is 600 / A step of alkoxideizing to a range of 400 to 999/1,
(B) the side chain moiety alkoxideized by the step (a),
Cl, Br, I or sulfonateoxy group at the reaction end, and the degree of polymerization is 20 to 200
A linear polymer chain consisting only of N-acylethyleneimine structural units;
A linear polymer chain consisting only of ethyleneimine structural units,
A linear polymer chain consisting only of N-acylpropylimine structural units,
A linear polymer chain consisting only of propyleneimine structural units,
A linear polymer chain comprising a combination of ethylene or propyleneimine structural units and N-acylethylene or propyleneimine structural units;
A linear polymer chain using structural units having the same or different acyl groups as a combination of an N-acylethylene or propyleneimine structural unit and an N-acylpropylene or ethyleneimine structural unit, or
N-acylethyleneimine structural unit, N-acylpropyleneimine structural unit, ethyleneimine structural unit or propyleneimine structural unit and a combination of ethylene glycol structural unit or propylene glycol structural unit
(However, “N-acyl” in the linear polymer chain referred to here is an acyl group having 1 to 5 carbon atoms.)
Replacing with
(C) hydrolyzing the remaining side chain acyl group,
A process for producing a polyvinyl alcohol-based graft copolymer comprising: In the polyvinyl alcohol-based graft copolymer, the proportion of the cationic structural unit that is a polymer chain composed of an ethyleneimine structural unit, an N-acylethyleneimine structural unit, or an N-acylpropyleneimine structural unit is 3 to 3% of all structural units. The method for producing a polyvinyl alcohol-based graft copolymer according to claim 1, which is in a range of 30% by mass. In the linear polymer chain, the N-acylethyleneimine structural unit and / or the N-acylpropyleneimine structural unit is a polyoxazoline obtained by cation living polymerization of an oxazoline monomer,
The polyvinyl alcohol graft copolymer according to claim 1 or 2, wherein the ethyleneimine structural unit and / or the propyleneimine structural unit is obtained by hydrolyzing a polyoxazoline obtained by cation living polymerization of an oxazoline monomer using an acid or a base. Manufacturing method of coalescence. After the linear block polymer chain comprising the N-acylethyleneimine structural unit or the combination of the N-acylpropyleneimine structural unit and the ethylene glycol structural unit or the propylene glycol structural unit tosylates alkoxy polyethylene glycol or alkoxy polypropylene glycol , A polymer chain obtained by performing cation living polymerization using an oxazoline monomer,
A linear block polymer chain composed of a combination of an ethyleneimine structural unit or a propyleneimine structural unit and an ethylene glycol structural unit or a propylene glycol structural unit tosylates alkoxypolyethylene glycol or alkoxypolypropylene glycol, and then cation using an oxazoline monomer The method for producing a polyvinyl alcohol-based graft copolymer according to claim 1 or 2, which is obtained by hydrolyzing an acylimine block polymer obtained by performing living polymerization using an acid or a base.