JP2007056079A - Lactic acid/peptide block copolymer and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lactic acid/peptide block copolymer having good bioabsorbability and high biofunctionality and being useful for medical materials, drug delivery systems, gene delivery materials, etc., and to provide a method for producing the same. <P>SOLUTION: An amide-bond-containing polylactic acid block copolymer represented by formula (I) is obtained by reacting an N-amino acid carboxylic anhydride with an active-amino-terminated polylactic acid obtained by reacting a hydroxyl-terminated polylactic acid with a protected-amino-containing amino acid mixed acid anhydride derivative in the presence of a catalyst under mild conditions to perfectly introduce the active amino acid into the terminals of the polylactic acid and deprotect the product. In formula (I), R<SB>1</SB>is a 1 to 20C aliphatic or araliphatic group; R<SB>2</SB>and R<SB>3</SB>are each a monovalent residue bonded through a carbon atom of an amino acid side chain; and m, n, and l are each a positive integer of 10 or smaller. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a block copolymer of lactic acid and a peptide and a method for producing the same. More specifically, a multi-reactive polylactic acid and peptide block copolymer having a plurality of functional groups excellent in bioabsorbability, suitable as a medical material such as a drug delivery system or a gene delivery material, and a method for producing the same It is about.

  Chemically synthesized bioabsorbable plastics are mainly aliphatic polyesters. Among them, there are polylactic acid made from lactic acid as well as polycaprolactone and polybutylene succinate. This polylactic acid is decomposed and absorbed in the body without causing harm to the living body, and finally it is a natural product that exists in the human metabolic pathway. In addition to being excellent in mechanical, physical and chemical performance, it has recently attracted attention as a non-polluting material and a medical material (Patent Documents 1 to 3).

  However, since the polylactic acid molecule does not have a reactive functional group in the main chain, it is restricted in terms of modification of materials and introduction of bioactive components. Therefore, in order to overcome such restrictions, an active ingredient such as a peptide is introduced into polylactic acid to form a polymer having reaction activity, and a drug or DNA is covalently bonded or ionically bonded to this reaction active portion. It is considered to be.

However, in order to introduce a peptide structure into a polylactic acid molecule, it is indispensable to introduce an active amino group having reactive activity into polylactic acid. Conventionally, as a method for introducing an active amino group into polylactic acid, polylactic acid obtained by ring-opening polymerization of lactide starting from a compound having a protected amino group and hydroxy in the presence of a catalyst. A method for producing a polylactic acid having an amino group terminal by deprotecting is known. However, this method has a drawback that only monofunctional polylactic acid can be produced, and difunctional polylactic acid or multi-reaction polylactic acid having a plurality of functional groups cannot be produced. As an improved method, polylactic acid having a hydroxy terminal group is synthesized by ring-opening polymerization of lactide using an alcohol compound as a raw material, and the amino acid is protected with amino acid and N, N′-dicyclohexylcarbodiimide (DCC). An attempt has been made to produce a polylactic acid having an amino acid terminal by a condensation reaction and to synthesize a polylactic acid having a terminal active amino group by deprotecting it. However, due to the low reactivity of the second hydroxy end group of polylactic acid and the steric hindrance of the huge polymer chain, the progress of the condensation reaction by DCC is difficult and the hydroxy end group of polylactic acid having a hydroxy end group is difficult. Cannot be completely changed to an amino terminal group.
JP 2001-31762 A Japanese Patent Laid-Open No. 2002-300898 JP 2005-154514 A

  From the background as described above, the present invention can produce a polylactic acid block copolymer having a peptide structure by completely changing a hydroxy terminal group to an amino terminal group without decomposing a polylactic acid derivative, It is an object to provide a new technical means that not only enables the production of multi-reacting polylactic acid but also enables control of the molecular weight and properties of lactic acid and amino acid segments.

  In order to solve the above problems, the present invention firstly provides a polylactic acid block copolymer having an amide bond represented by the following general formula (I).

（式中、Ｒ₁は炭素数１以上２０以下の脂肪族基または芳香脂肪族基を示し、Ｒ₂およびＲ₃はアミノ酸側鎖の炭素で結合されている１価の残基を示し、ｍ、ｎは整数を示し、ｌは１０以下の正の整数を示す。）
第２には、Ｒ₂がフェニルアラニン、アラニン、グリシン、ロイシン、バリンおよびイソロイシンのうちの少くともいずれかの残基であることを特徴とする上記のポリ乳酸ブロック共重合体を提供する。

(In the formula, R ₁ represents an aliphatic group or an araliphatic group having 1 to 20 carbon atoms, R ₂ and R ₃ represent a monovalent residue bonded by carbon of an amino acid side chain, m , N represents an integer, and l represents a positive integer of 10 or less.)
Second, there is provided the above polylactic acid block copolymer, wherein R ₂ is a residue of at least one of phenylalanine, alanine, glycine, leucine, valine and isoleucine.

Thirdly, the polylactic acid as described above, wherein R ₃ is a residue of at least one of L-lysine, L-aspartic acid, L-glutamic acid, L-serine, and L-tyrosine A block copolymer is provided. Fourth, the polylactic acid derivative having a terminal active amino group formed from a polylactic acid derivative having a hydroxy terminal group is reacted with an amino acid-N-carboxyanhydride. Provided is a method for producing a block copolymer comprising lactic acid and a peptide.

  Fifth, a first step of synthesizing a polylactic acid derivative having a hydroxy terminal group by ring-opening polymerization of lactide in the presence of alcohols, and a catalyst is present in the hydroxy terminal group of the polylactic acid derivative having the hydroxy terminal group A second step of synthesizing a polylactic acid derivative having an active amino terminal group by reacting a mixed acid anhydride derivative of an amino acid protected with an amino group and then deprotecting the protected amino group; The present invention provides a method for producing a block copolymer of lactic acid and a peptide, comprising the third step of reacting —N-carboxyanhydride.

  Sixth, the method for producing a block copolymer of lactic acid and a peptide as described above, wherein the polylactic acid derivative having a hydroxy terminal group has one hydroxy terminal group or a plurality of hydroxy terminal groups I will provide a.

  According to the present invention, various polyfunctional groups having a plurality of functional groups excellent in bioabsorbability suitable for medical materials such as drug delivery systems and gene delivery materials in which the hydroxy terminal group of polylactic acid is completely changed to an amino terminal group. A block copolymer of reactive polylactic acid and a peptide can be provided.

  Moreover, according to the production method of the present invention, polylactic acid having a hydroxy terminal group is produced, and the polylactic acid block having a peptide structure is obtained by completely changing the hydroxy terminal group to an amino terminal group without decomposing the polylactic acid. A block copolymer consisting of lactic acid and a peptide that can produce a copolymer and can control the molecular weight and properties of each segment of the block copolymer by adjusting the type and amount of lactic acid and amino acid used as starting materials. A manufacturing method can be provided.

  As described above, the polylactic acid block copolymer having a peptide structure provided by the present invention is represented by the following general formula (I). In this formula (I), when l is 1, it is an AB type block copolymer, and when 1 is 2, it is an ABA type block copolymer, and l is 3 or more. In this case, it becomes a multi-reaction type block copolymer.

そして、式中Ｒ₁は炭素数１以上２０以下の脂肪族基または芳香脂肪族基を、Ｒ₂およびＲ₃はアミノ酸側鎖の炭素で結合されている１価の残基を示す。ｍ、ｎは正の整数で、ｍが７〜１４０、望ましくは１４〜７０であり、ｎが１０〜１００、望ましくは１５〜５０である。この範囲を超えると、正確にブロック共重合体を製造できるには限らない。ｌ（エル）は１０以下の正の整数であれば、特に限定したものではない。

In the formula, R ₁ represents an aliphatic group or an araliphatic group having 1 to 20 carbon atoms, and R ₂ and R ₃ represent a monovalent residue bonded by carbon in an amino acid side chain. m and n are positive integers, m is 7 to 140, preferably 14 to 70, and n is 10 to 100, preferably 15 to 50. Beyond this range, the block copolymer cannot be accurately produced. l (El) is not particularly limited as long as it is a positive integer of 10 or less.

  As described above, the block copolymer of the present invention is produced by reacting a polylactic acid derivative having a terminal active amino group formed from a polylactic acid derivative having a hydroxy terminal group with N-aminocarboxy anhydride. Can do. This method of manufacture is novel.

This manufacturing method will be described in more detail. The following procedure is preferably considered.
<First step>
In the presence of a catalyst, lactide is subjected to ring-opening polymerization in the presence of an alcohol that gives an R ₁ residue to synthesize a polylactic acid derivative having a hydroxy end group represented by the following general formula (II).

＜第二工程＞
生成された式（II）のポリ乳酸誘導体のヒドロキシ末端基を、アミノ基が保護されたアミノ酸の混合酸無水物誘導体と触媒の存在下で反応させ、次の一般式（III）で表されるアミノ基が保護されたアミノ酸末端基を有するポリ乳酸誘導体を合成し、次いで脱保護反応させて一般式（IV）で表される活性アミノ末端基を有するポリ乳酸誘導体を合成する。

<Second step>
The resulting hydroxy terminal group of the polylactic acid derivative of formula (II) is reacted with a mixed acid anhydride derivative of an amino group-protected amino acid in the presence of a catalyst, and is represented by the following general formula (III) A polylactic acid derivative having an amino acid-protected amino acid terminal group is synthesized and then deprotected to synthesize a polylactic acid derivative having an active amino terminal group represented by the general formula (IV).

なお、式（III）中における符号Ｘはアミノ酸のアミノ基の保護基を示しており、たとえば、ベンジルオキシカルボニル（Ｚ）基，t−ブトキシカルボニル（Ｂｏｃ）基、もしくは９−フロオレニルメトキシカルボニル（Ｆｍｏｃ）基を示している。
＜第三工程＞
生成された式（IV）の活性アミノ末端基を有するポリ乳酸誘導体に一般式（V）で表わされるアミノ酸−Ｎ−カルボキシ無水物を加えて次の反応式に従って開環重合反応させる。

The symbol X in the formula (III) represents a protecting group for the amino group of the amino acid. For example, benzyloxycarbonyl (Z) group, t-butoxycarbonyl (Boc) group, or 9-fluorenylmethoxycarbonyl (Fmoc) group is shown.
<Third step>
The amino acid-N-carboxy anhydride represented by the general formula (V) is added to the produced polylactic acid derivative having an active amino terminal group of the formula (IV) and subjected to a ring-opening polymerization reaction according to the following reaction formula.

以上の反応工程についてさらに詳しく説明すると、まず、第一工程において用いられる、Ｒ₁残基を与えるアルコールについては、Ｒ¹が、炭素数１以上２０以下の脂肪族基または芳香脂肪族基を表わすものであれば各種のアルコール類の１種以上のものであってよい。そしてＲ¹残基は許容される各種の置換基を有していてもよいし、アルコール類は１価もしくは多価のアルコールであってもよい。たとえばこのようなアルコール類の具体例としては、メタノール、エタノール、プロパノール、イソプロパノール、ブタノール、ペンタノール、ノナノール、デカノール、ラウリルアルコール、ミリスチルアルコ−ル、セチルアルコール、ステアリルアルコール、ベンジルアルコール、乳酸メチル、乳酸ブチル等のモノアルコール、エチレングリコール、プロパンジオール、ブタンジオール、ヘキサメチレングリコール、ノナンジオール、テトラメチレングリコール、ジエチルグリコール、等のジアルコール、トリメチルオルプロパネ、ペンタエリトリトール（pentaerythritol）：Ｃ（ＣＨ₂ＯＨ）₄、グリセロール、ソルビトール、キシリトール、リビトール（ribitol）、エリスリトール（erythritol）等の多価値アルコール等が挙げられるがこれらに限定されるものではない。

To be more specific about the above reaction steps, first used in the first step, the alcohol to give R ₁ residues, R ¹ is an aliphatic group or an aromatic aliphatic group of 1 to 20 carbon atoms Any one or more of various alcohols may be used. The R ¹ residue may have various permissible substituents, and the alcohol may be a monovalent or polyvalent alcohol. For example, specific examples of such alcohols include methanol, ethanol, propanol, isopropanol, butanol, pentanol, nonanol, decanol, lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, benzyl alcohol, methyl lactate, and lactic acid. Monoalcohols such as butyl, ethylene alcohol, propanediol, butanediol, hexamethylene glycol, nonanediol, tetramethylene glycol, diethyl glycol, and other dialcohols, trimethylolpropane, pentaerythritol: C (CH ₂ OH ) _4, glycerol, sorbitol, xylitol, ribitol (ribitol), although the multi-value alcohols such as erythritol (erythritol) and the like which The present invention is not limited to.

  And as a lactide used for reaction of a 1st process, any of L-lactide, D-lactide, DL-lactide, and meso-lactide may be used, and it does not matter even if it uses individually or in multiple combinations.

Ring-opening polymerization of lactide for the formation of a polylactic acid segment in the presence of an alcohol that gives an R ₁ residue in the formation of a polylactic acid derivative of formula (II) in the reaction in the first step is a known solution polymerization or Although it is carried out by melt polymerization, any known catalyst can be used as the catalyst at that time, and specific examples thereof include tin, antimony, zinc, titanium, iron, and aluminum compounds. It is not limited to these.

  The molecular weight of the polylactic acid derivative having a hydroxy end group produced in the first step is 1000 to 20000, preferably about 2000 to 10,000. When the polylactic acid having a hydroxy end group with a molecular weight exceeding 20000 is used in the reaction in the second step, the hydroxy end group may not be completely changed to an amino end group, and the effect of the present invention is obtained. There is a fear that you will not

  In the second step of the invention of this application, a mixed acid anhydride derivative of an amino group-protected amino acid is reacted with the hydroxy terminal group of the polylactic acid derivative having a hydroxy terminal group of the formula (II) in the presence of a catalyst. Thus, a polylactic acid having a protected amino acid end group represented by the formula (III) is synthesized. In this case, a mixed acid anhydride of an amino group protected amino acid and a polylactic acid having a hydroxy end group are synthesized. The ratio of hydroxy end groups is 1.1-20, preferably 3-15, more preferably 8-12.

The mixed acid anhydride derivative of an amino acid in which the amino group is protected can be represented, for example, by the following general formula (VI). This compound is easily produced from an amino acid which provides R ₂ with an amino group protected and a carboxylic acid, acid chloride, ester or carbonate.

ここでＲ²は、前記のとおり、アミノ酸側鎖の１価の残基を表すが、これを与えるアミノ酸は分子中に少なくとも１個のアミノ基と１個のカルボキシル基を有するものであれば特に限定されるものではない。製造過程の便利性からは２官能タイプが好ましい。

Here, as described above, R ² represents a monovalent residue of the amino acid side chain, and the amino acid providing this is particularly suitable if it has at least one amino group and one carboxyl group in the molecule. It is not limited. The bifunctional type is preferred from the convenience of the production process.

  From the viewpoint that a commercially available product is easy and can be obtained at low cost, the bifunctional type includes phenylalanine, alanine, glycine, leucine, valine, isoleucine and the like.

  The amino-protecting group X may be various. For example, as described above, the known benzyloxycarbonyl (Z) group, t-butoxycarbonyl (Boc) group, 9-fluorenylmethoxycarbonyl (Fmoc) Any of the groups can be utilized.

And the code | symbol W in Formula (VI) has shown the mixed acid anhydride residue. For example, (CH ₃ ) ₃ C—, (CH ₃ CH ₂ ) ₂ CH—, i-BuO group and the like are exemplified.

  In the second step of the reaction, the polylactic acid derivative having an amino acid-protected amino acid end group is converted to a polylactic acid derivative having an active amino terminal group of the above formula (IV) by deprotection. The deprotection reaction varies depending on the protecting group, and any deprotection reaction is carried out by a known method, but preferably under anhydrous conditions. More desirably, the reaction is carried out under mild reaction conditions around room temperature or below neutral. For example, when a benzyloxycarbonyl (Z) group is used, a catalytic reduction reaction method using hydrogen using Pd as a catalyst can be mentioned. When a t-butoxycarbonyl (Boc) group is used, a deprotection method using trifluoroacetic anhydride can be mentioned. When a 9-fluorenylmethoxycarbonyl (Fmoc) group is used, a deprotection method using piperidine is exemplified.

  In the third step of the production method of the present invention, the polylactic acid derivative having an active amino terminal group represented by the above formula (IV) produced in the above second step is converted into the amino acid-N represented by the above formula (V). -To react with carboxyanhydride. By this reaction, the block copolymer represented by the above formula (I) is produced.

R ₃ in the formula (V) represents a monovalent residue of the amino acid side chain, and the amino acid providing this is particularly limited as long as it has at least one amino group and one carboxyl group in the molecule. Is not to be done. R ₃ preferably has a hydroxyl group, an amino group, or a carboxy group from the viewpoint of imparting functionality to the copolymer, but in order not to interfere with the copolymerization reaction, these hydroxyl group, amino group, or carboxy group It is desirable to protect the in advance. The amino acid that gives it is N ^ε -ZL-lysine, L-aspartic acid-β-benzyl ester (L-aspartic acid-β-benzyl) from the viewpoint that commercial products are easily available at low cost. ester), L-glutamic acid-γ-benzyl ester, O-benzyl-L-serine, O-benzyl-L-tyrosine (O- benzyl-L-tyrosine).

  In order to describe the present invention more specifically, examples will be described below, but the present invention is not limited thereto.

<First step>
10 g of purified L-lactide was placed in the dried polymerization tube, the polymerization tube was degassed several times to fill with dry argon, and then 50 mg of tin 2-ethylhexanoate was added as a 1M toluene solution. Toluene was evaporated in vacuum and the polymerization tube was again degassed several times and filled with dry argon, and then 0.37 ml or 0.26 ml of butyl alcohol was injected with a syringe.

Finally, the tube was sealed under vacuum, and the polymerization tube was placed in an oil bath at 120 ° C. for polymerization for 48 hours. The obtained white polymer packing was dissolved in chloroform, poured into cooled methanol and reprecipitated and purified. After centrifugation, the solid was air-dried at room temperature and further dried in vacuo. A polylactic acid derivative having a powdery hydroxy end group was obtained.
<Second step>
On the other hand, 1.33 g of BOC-L-phenylalanine and 0.70 ml of TEA were put into a flask degassed several times and filled with dry argon, and dissolved in 10 ml of dry ethyl acetate. When 0.61 ml of trimethylacetyl chloride cooled to −10 ° C. was added to this solution with a syringe, a white precipitate immediately formed.

  The mixture was kept at 0 ° C. for 2 hours and then stirred at room temperature for 1 hour. After removing the precipitate by filtration, the solvent was evaporated and the residue was dried in vacuo to obtain a BOC-L-phenylalanine mixed acid anhydride. The white solid was dissolved in 5 ml of dry chloroform to obtain a BOC-L-phenylalanine mixed acid anhydride solution.

  Dissolve 2 g of the polylactic acid derivative having a hydroxy group terminal generated in the first step in 10 ml of dry chloroform, add TEA (0.70 ml) and 4- (1-pyrrolidinyl) pyridine (74.1 mg), After cooling to 0 ° C., the above BOC-L-phenylalanine mixed acid anhydride solution was added, kept at 0 ° C. for 2 hours, and then stirred at room temperature for 36 hours. The reaction solution was poured into 200 ml of cooled methanol, and the purified precipitate was recovered by centrifugation to obtain a polylactic acid derivative having a BOC-L-phenylalanine end.

2 g of the above polylactic acid derivative having a BOC-L-phenylalanine end is dissolved in 20 ml of dry dichloromethane under an argon atmosphere, cooled to 0 ° C., 6 ml of trifluoroacetic anhydride is added and reacted for 2 hours to remove the solvent in vacuo. After evaporation, the polymer was remelted with dichloromethane, washed with 2% sodium hydrogen carbonate solution and water, and the organic phase was dehydrated with sodium sulfate. Finally, it was poured into chilled methanol for precipitation, which was collected by centrifugation and dried in vacuum to obtain a polylactic acid derivative having an active amino end group.
<Third step>
0.5 g of the polylactic acid derivative having an active amino terminal group and 1.0 g or 2.0 g of N ^ε -Z-lysine-N carboxyanhydride represented by the following formula are put into two flasks and removed several times. After filling with dry argon, 20 ml each of dry dichloromethane was added to dissolve the solid.

各々の溶液を混合して室温で４８時間撹拌して、回転蒸発器で溶媒を半分蒸発させ、得られた溶液を冷却したジエチルエーテルに注入して、ポリ乳酸とポリ（Ｎ^ε−Ｚ−Ｌ−リシン）（ＰＬＬＡ−ｂ−ＰＺＬｙs）のブロック共重合体を沈殿させ、ろ過により回収し真空で乾燥した。

Each solution was mixed and stirred at room temperature for 48 hours, the solvent was half evaporated on a rotary evaporator, the resulting solution was poured into cooled diethyl ether, and polylactic acid and poly (N ^ε -Z-L -A block copolymer of lysine (PLLA-b-PZLys) was precipitated, recovered by filtration and dried in vacuo.

  Place 0.2 g of the above PLLA-b-PZLys in a flask, degas several times, fill with dry argon, add dry dichloromethane to dissolve the solid, cool to 0 ° C., and then 25% bromide After adding 2.5 ml of hydrogen acid / acetic acid solution and stirring at 0 ° C. for 30 minutes, the residue precipitated by pouring into cooled diethyl ether was collected by filtration, washed repeatedly with diethyl ether, It was dried in vacuum to obtain a block copolymer of polylactic acid and poly (L-lysine) (PLLA-b-PZLys).

  In Table 1, it has shown about the copolymer 1-4 at the time of changing raw material usage-amount, a in a table | surface is the polymerization degree computed from the usage-amount of the raw material at the time of superposing | polymerizing, b is It is the result actually measured by NMR.

  From Table 1, it can be seen that the polymerization degree and molecular weight of the synthesized copolymer can be controlled by the charging ratio of lactide, butyl alcohol, and amino acid-N-carboxyanhydride as raw materials.

Claims (6)

A polylactic acid block copolymer having an amide bond represented by formula (I).

(In the formula, R ₁ represents an aliphatic group or an araliphatic group having 1 to 20 carbon atoms, R ₂ and R ₃ represent a monovalent residue bonded by carbon of an amino acid side chain, m , N represents an integer, and l represents a positive integer of 10 or less.) The polylactic acid block copolymer according to claim 1, wherein R ₂ is a residue of at least one of phenylalanine, alanine, glycine, leucine, valine and isoleucine. The polylactic acid block according to claim 1 or 2, wherein R ₃ is at least one of L-lysine, L-aspartic acid, L-glutamic acid, L-serine, and L-tyrosine. Copolymer.   A process for producing a block copolymer comprising lactic acid and a peptide comprising reacting a polylactic acid derivative having a terminal active amino group formed from a polylactic acid derivative having a hydroxy terminal group with an amino acid-N-carboxyanhydride .   A first step of synthesizing a polylactic acid derivative having a hydroxy terminal group by ring-opening polymerization of lactide in the presence of an alcohol; and an amino group in the presence of a catalyst in the hydroxy terminal group of the polylactic acid derivative having a hydroxy terminal group. A second step of reacting a mixed acid anhydride derivative of a protected amino acid and then synthesizing a polylactic acid derivative having an active amino terminal group by deprotection of the protected amino group, and further an amino acid-N-carboxyanhydride The method for producing a block copolymer of lactic acid and a peptide according to claim 4, comprising a third step of reacting the product.   The method for producing a block copolymer of lactic acid and a peptide according to claim 4 or 5, wherein the polylactic acid derivative having a hydroxy terminal group has one hydroxy terminal group or a plurality of hydroxy terminal groups.