JP4480149B2 - Method for producing polypeptide - Google Patents

Description

  The present invention provides high purity and high molecular weight necessary for producing materials using high molecular weight synthetic peptides, or for studying the role of proteins, functional studies of proteins, and application as pharmaceutical materials. The present invention also relates to a method for producing a polypeptide having a narrow molecular weight distribution and excellent molecular weight reproducibility, and a method for producing a polypeptide using amino acid N-carboxyanhydride as a raw material.

  Attempts to synthesize protein models using amino acids or derivatives thereof have been attempted in various ways since the 1900s. For example, (1) two amino acid molecules or derivatives thereof (for example, carboxylic acid groups) There was a method in which each amino acid residue was bonded by repeating condensation between the acid chloride and the like, and the molecular chain was gradually lengthened. In this method, since the reaction takes time, it is almost impossible to synthesize a high molecular weight protein model. The other is called (2) Maryfield's solid-phase synthesis method, where a reactive group is attached to a polymer such as a polystyrene film, one amino acid is bound, and the amino acid is changed to a structure that is easy to react. Therefore, there was another method of binding another amino acid. This method is time-consuming and cannot produce a large amount of a high molecular weight protein model having 100 residues or more (molecular weight of 10,000 or more).

  Conventionally, as one method for synthesizing other high molecular weight protein models, amino acid N-carboxyanhydride (hereinafter also referred to as amino acid NCA) is dissolved in an organic solvent, and a base is mainly used as a polymerization initiator. A polymerization method has been carried out.

  The amino acid NCA is a compound having a 5-membered ring that is synthesized from an amino acid and phosgene or a phosgene derivative, and is extremely sensitive to moisture and difficult to handle. From the 1940s, research on the polymerization of the amino acid NCA was gradually carried out overseas all over the world, and in the 1960s, it was actively studied in Japan. If a synthetic protein having a high molecular weight (for example, 100,000 or more) and a uniform molecular weight is produced, it can be used as a polymer material for fibers, films, medical materials, biocompatible materials, nerve models, etc. This is because many uses were expected.

 However, although it has been reported that a protein model having an average molecular weight of a high molecular weight has been made, there have been problems such as lack of reproducibility in the reaction and molecular weights not being uniform (wide molecular weight distribution). The molecular weight distribution is generally expressed as a ratio of the weight average molecular weight Mw and the number weight average molecular weight Mn, and Mw / Mn = 1.0 is an ideal state where all molecular weights are aligned. Most of the Mw / Mn values of the polypeptides obtained by polymerizing the amino acid NCA by the usual method were about 3 to 10, and at least less than 2. In addition, since it is limited to solution polymerization, there is a problem that an amino acid NCA that is insoluble in any solvent cannot be polymerized.

  For example, JP-A-5-43560 discloses S-ethylcarbamoyl-N-carboxy-L-cysteine anhydride (a kind of amino acid NCA) alone or N-carboxylate for synthesizing a polypeptide containing cysteine. A method for producing a polypeptide by adding a polymerization initiator to a mixture with -L-alanine anhydride (a kind of amino acid NCA), performing a polymerization reaction, and then removing the ethylcarbamoyl group which is a protective group is disclosed, By performing the polymerization reaction at a temperature of 30 to 40 ° C. for several days, a polypeptide having a viscosity average polymerization degree of 150 is obtained. However, the average degree of polymerization was 150 and the molecular weight was too low to be used as a polymer material. There is no description of molecular weight distribution.

  JP-A-7-41467 discloses S-phenylcarbamoyl-N-carboxy-L-cysteine anhydride (a kind of amino acid NCA) alone or N-carboxylate for synthesizing a polypeptide containing cysteine. A method for producing a polypeptide by adding a polymerization initiator to a mixture with -L-alanine anhydride (a kind of amino acid NCA), performing a polymerization reaction, and then removing a phenylcarbamoyl group that is a protective group is disclosed, By carrying out the polymerization reaction at a temperature of 30 ° C. for ten days or more, a polypeptide is obtained in a yield of 70 to 80%. Again, there is no mention of molecular weight distribution.

  Also, on page 171 of “New Experimental Chemistry Lecture 19 Polymer Chemistry [I]”, L-glutamate γ-benzyl NCA and nickel acetate are separately put in a bifurcated sealed vessel, respectively, and degassed and argon. Polypeptide that is substituted and mixed with both compounds to form a solution, then cooled to -78 ° C, tri-n-butylphosphine as an initiator is added using a syringe, and polymerized at 30 ° C for 24 hours A polypeptide having a yield of 92% and a weight average molecular weight of 564,000 is obtained. However, this method involves difficult techniques such as a sealing tube technique and the use of an expensive and unstable initiator, and mass production and reproducibility are difficult.

JP-A-5-43560 (Example) JP-A-7-41467 (Example) The Chemical Society of Japan, New Experimental Chemistry Course 19 Polymer Chemistry [I] (published in 1978, Maruzen Co., Ltd., Tokyo), page 171

  In order to produce a high molecular weight protein model in large quantities and at low cost, it is considered that polymerization of amino acid NCA is suitable. However, in the conventional polymerization method of amino acid NCA, there are reports of high molecular weight, At present, it is possible to produce a polypeptide having a molecular weight of about 40,000 to 50,000 according to a commonly used method, but it is difficult to produce a polypeptide having a higher molecular weight and a narrow molecular weight distribution. In addition, the molecular weight of the produced polypeptide is not reproducible (even if the same method is used, the average molecular weight of the product is different), and the polymerization time of the polymerization reaction is long. This problem is also described in “T. J. Deming, Nature, Vol. 390, pages 386 to 389 (1997)”, and the description has a high molecular weight and a narrow molecular weight distribution in the conventional method. This suggests that it is difficult to produce a product having a good molecular weight reproducibility. In this document, Deming discloses a special nickel catalyst for producing a block copolymer having a narrow molecular weight distribution as compared with the conventional polymerization method of amino acid NCA. There are problems such as difficulty in handling and limitation of the types of amino acid NCA used.

“TJ Deming, Nature, Vol. 390, pages 386 to 389 (1997)”.

  Accordingly, an object of the present invention is to produce a polypeptide having a high molecular weight and a narrow molecular weight distribution by using an inexpensive and easily available catalyst or polymerization initiator in the production of a polypeptide using amino acid N-carboxyanhydride as a raw material. And to provide a production method with good molecular weight reproducibility. The polypeptide includes a polypeptide composed of a single amino acid residue and a polypeptide (copolymer) composed of a plurality of amino acid residues.

  As a result of intensive studies to solve the above-described problems in the prior art, the present inventor has (1) conventionally, the polymerization reaction of amino acid N-carboxyanhydride is promoted by a polymerization initiator added together with a polymerization raw material. In reality, the polymerization initiator does not contribute much to the acceleration of the polymerization reaction, and the water present in the air absorbed by the amino acid N-carboxyanhydride acts as a polymerization initiator to cause the polymerization reaction. Therefore, the polymerization reaction cannot be controlled, the reproducibility of the molecular weight is deteriorated, or the high molecular weight and narrow molecular weight distribution cannot be produced, and (2) the amino acid produced and purified by the conventional method The N-carboxyanhydride absorbs moisture and the polymerization reaction on the surface cannot be avoided; (3) the moisture absorption is the purification of the amino acid N-carboxyanhydride and the polymerization reaction vessel. Can be prevented by performing at a specific low temperature, the polypeptide thus obtained has a high molecular weight, a narrow molecular weight distribution, and a good reproducibility of the molecular weight. The inventors have found that the polymerization rate is increased, and have completed the present invention.

That is, in the present invention (1), the amino acid N-carboxyanhydride is purified by one or more recrystallizations to obtain a purified amino acid N-carboxyanhydride, and the purified amino acid N-carboxyanhydride is polymerized. A method for producing a polypeptide to obtain a polypeptide, wherein at least the last recrystallization operation of the recrystallization is carried out in dry air at a temperature of 0 ° C. or lower and an absolute humidity of 0.004 kg / kg dry air or lower. The present invention provides a method for producing a polypeptide.

Further, the present invention (2) is to provide a manufacturing method of the present invention (1) Symbol placement polypeptide to operate the last recrystallization at a temperature of -20 to-5 ° C..

Further, the present invention ( 3 ) is the method according to the present invention (1) or (2) , wherein the polymerization of the purified amino acid N-carboxy anhydride obtained by the last recrystallization is carried out at a temperature of 0 ° C. or lower. A method for producing any one of the polypeptides is provided.

The present invention ( 4 ) is a method for producing a polypeptide according to the present invention ( 3 ), wherein the polymerization is carried out in dry air at a temperature of 0 ° C. or lower and an absolute humidity of 0.004 kg / kg dry air or lower. Is to provide.

Moreover, this invention ( 5 ) provides the manufacturing method of the polypeptide of the said this invention ( 3 ) or ( 4 ) which performs the preparation of the said polymerization under the temperature of -20--5 degreeC.

The present invention (6) also provides the purified amino acid without being removed by removing the solvent adhering to the surface of the purified amino acid N-carboxyanhydride after filtration in the last recrystallization. there is provided a method of any description of the polypeptides of the present invention to perform charging of the polymerization of N- carboxyanhydride (1) to (5).

Further, in the present invention ( 7 ), the polymerization of the purified amino acid N-carboxy anhydride is carried out by using the purified amino acid N-carboxy anhydride with a solubility of the amino acid N-carboxy anhydride at 20 ° C. of 0.05 mg / cm 2. The invention provides a method for producing a polypeptide according to any one of the inventions (1) to ( 6 ), wherein the polypeptide is immersed in a dehydrated solvent of ³ or less and subjected to solid phase polymerization in the presence of a polymerization initiator. .

Further, in the present invention (8), the dehydrated solvent having a solubility of the amino acid N-carboxy anhydride at 20 ° C. of 0.05 mg / cm ³ or less is pentane, hexane, heptane, octane, nonane, decane, The present invention provides a method for producing a polypeptide according to the present invention ( 7 ), which is petroleum ether or a mixture thereof.

Further, the present invention (9), the dissolution polymerization of the purified amino acid N- carboxyanhydride is, The purified amino acid N- carboxyanhydride, a solvent capable of dissolving all or part of the purified amino acid N- carboxyanhydride Alternatively, the present invention provides a method for producing the polypeptide according to any one of the present inventions (1) to ( 6 ), wherein the polypeptide is immersed and polymerized in the presence of a polymerization initiator.

  The method for producing a polypeptide of the present invention can obtain a polypeptide having a high molecular weight, a narrow molecular weight distribution, and a good reproducibility of the molecular weight, which has been difficult in the past, using a drug that is generally easily available and safe and inexpensive. it can. Moreover, the reaction rate of the polymerization reaction can be increased.

  The amino acid NCA according to the present invention is produced by carboxylating the N atom of the amino acid, starting from an amino acid that is a constituent unit of the target polypeptide, and represented by the following formula (1):

（式中Ｒは、ポリペプチドの構成単位となるアミノ酸により適宜定まる。）の骨格を有する化合物である。

(Wherein R is appropriately determined depending on the amino acid that is a structural unit of the polypeptide).

  The amino acids that constitute the structural unit of the polypeptide include natural amino acids, L-forms or D-forms of natural amino acids, mixtures of L-forms and D-forms, derivatives of natural amino acids, or triphosgene capable of generating phosgene or phosgene, dichloromethylchloro Non-natural amino acids that can form the skeleton of the above formula (1) by reaction with formate and the like can be mentioned. In addition, when the amino acid has a functional group other than an amino group and a carboxyl group in the amino acid involved in the formation of the main chain of the polypeptide, the functional group can be an amino acid protected with a protective group, for example, L-glutamic acid-γ-benzyl or L-aspartic acid-β-benzyl in which the carboxyl group at the gamma position of L-glutamic acid or the beta position of L-aspartic acid is protected with a benzyl group; ε (epsilon) amino group of L-lysine Protected ε-carbobenzoxy-L-lysine; SH-group of L-cysteine protected with phenylcarbamoyl group; S-phenylcarbamoyl-L-cysteine; and oxygen of hydroxyl group of L-tyrosine and L-serine to O- Examples include derivatives protected with a benzyl group. In addition, the D form of amino acids or the mixture of the L form and the D form is the same as the L form.

  The method for producing amino acid NCA using the amino acid as a raw material is not particularly limited, and can be performed by a conventional method. For example, a method in which an amino acid is reacted with triphosgene at 40 ° C. in a tetrahydrofuran solvent. An example of a method for producing an amino acid NCA using benzyl L-glutamate as an amino acid is shown in the following formula. Triphosgene is easily phosgenated during the reaction, and the phosgene reacts with benzyl L-glutamate. Instead of triphosgene, it may be phosgene or a phosgene dimer, or thionyl chloride. In accordance with the general method for synthesizing amino acid NCA.

  In the present invention, the recrystallization is performed once or twice or more, and the final recrystallization operation is performed at a temperature below 0 ° C., preferably at a temperature of 0 ° C. or less and an absolute humidity of 0.004 kg / kg dry air or less. In the dry air, the temperature is particularly preferably -20 to -5 ° C, more preferably -10 to -5 ° C. In particular, it is preferable to perform the recrystallization operation at a temperature of −10 to −5 ° C. in that the operability is improved. The higher the purity of amino acid NCA crystals, the more difficult it is to avoid polymerization on the surface due to slight moisture in the air when handled in a laboratory at room temperature. Therefore, by performing the recrystallization operation in the above atmosphere, polymerization due to moisture absorption of the purified amino acid NCA after recrystallization can be prevented. When the recrystallization temperature exceeds 0 ° C., the atmosphere to which the amino acid NCA and the like are exposed becomes air containing a lot of moisture. Therefore, the amino acid NCA absorbs moisture and is affected by the temperature, and the purified amino acid NCA It is not preferable in that a polymerization reaction occurs on the surface. On the other hand, if the temperature is too low, removal of moisture in the air is sufficient, but this is not preferable in terms of reduced workability. The preferable range of the dry air is an absolute humidity of 0.003 kg / kg dry air or less, more preferably an absolute humidity of 0.002 kg / kg dry air or less. Such dry air can be obtained by a method of putting a desiccant in a low temperature chamber such as a freezer.

  In the present invention, the recrystallization operation is performed at a temperature of 0 ° C. or lower. The recrystallization performed once or twice or more is obtained by the previous crystallization, for example, a mixed solution of a solvent and a precipitant. A step of filtering the amino acid NCA crystals with a glass filter in a state where the mixture containing the amino acid NCA crystals precipitated therein is cooled to the above temperature range, and collecting the crystals, for example, a step of dissolving in a solvent such as ethyl acetate This means that a series of recrystallization operations such as a step of adding a precipitating agent such as hexane to the solution to crystallize, for example, a step of filtering the crystal, is performed at the same temperature.

  Moreover, when performing recrystallization twice or more, the effect of this invention can be show | played by performing at least the last recrystallization under the said temperature range, However, All recrystallization is performed at the said temperature. However, it is preferable in that it has a high effect of preventing polymerization due to moisture absorption.

  In the recrystallization, the method for precipitating the amino acid NCA dissolved in the solvent is not particularly limited, but the amino acid NCA is dissolved in a solvent having high solubility of the amino acid NCA, and the solubility of the amino acid NCA is obtained in the obtained solution. A method of depositing by adding a low solvent is preferable in that it is not necessary to change the temperature under a low temperature condition where the temperature control is difficult and the operation is easy. For example, as described above, there may be mentioned a method in which amino acid NCA is dissolved in ethyl acetate and hexane is added to the obtained solution to precipitate it. In addition, it is preferable to use a dehydrated solvent for the recrystallization because it can prevent moisture absorption of the amino acid NCA due to moisture present in the solvent.

  When the precipitated crystals are separated from the solvent by filtration under the above temperature range, the polymerization of the amino acid NCA is carried out with the solvent attached to the surface of the crystal after filtration without drying. It is preferable in that the surface of the amino acid NCA is not in direct contact with air and the effect of preventing moisture absorption is enhanced. In particular, N-carboxyanhydrides such as leucine or phenylalanine are highly reactive, and polymerization occurs even in the presence of a very small amount of water, so that the solvent adhering to the surface of the crystal after filtration is not dried. It is effective to perform the operation. The solvent adhering to the surface of the crystal after filtration is a mixture of the solvent used for recrystallization existing between the fine crystals and the precipitating agent. Of the total weight, the weight percentage of only the actual crystals is about 50 to 80%, and 60 to 70% is preferable from the viewpoint of ease of work. As a method of measuring the amount of solvent adhering to the surface of the crystal after filtration, a part of the crystal containing the solvent simultaneously produced in a separate batch is quickly put in a tube and connected to a vacuum pump, followed by vacuum drying. Later, by measuring the weight, the weight% of the actual crystals alone can be determined.

  In the present invention, the purification of the purified amino acid NCA obtained by the last recrystallization is performed at a temperature of 0 ° C. or lower, preferably 0 ° C. or lower, and 0.004 kg / kg of dry air. Among these, it is particularly preferable to carry out the reaction at a temperature of -20 to -5 ° C in terms of preventing polymerization before the reaction is charged. Here, the preparation of polymerization refers to a series of operations until the purified amino acid NCA obtained by the last recrystallization is charged into the polymerization reaction vessel. That is, in the present invention, in a series of operation steps from the last recrystallization to the preparation of polymerization of the purified amino acid NCA, the purified amino acid NCA is placed under the above temperature range, and the polymerization due to moisture absorption of the purified amino acid NCA is suppressed as much as possible. Is. In addition, it is possible to reliably prevent polymerization due to moisture absorption of the purified amino acid NCA by performing operations other than the preparation of the purified amino acid NCA, such as charging of a product other than the purified amino acid NCA, under the temperature range. This is preferable. Specifically, for example, in a low temperature chamber controlled at a temperature of 0 ° C. or lower, preferably −20 to −5 ° C., after adding amino acid NCA to the polymerization reaction vessel, a polymerization initiator and a solvent are added, and a mercury column is added. In a low temperature chamber controlled at a temperature of 0 ° C. or lower, preferably -20 to −5 ° C., after adding amino acid NCA to the polymerization reaction vessel, the polymerization reaction vessel is sealed. After returning to about room temperature, a polymerization initiator and a solvent are added in an atmosphere capable of preventing moisture absorption of the amino acid NCA in the dry box, etc., and a mercury column is connected to measure the pressure for measuring the polymerization rate. A method is mentioned.

As the polymerization method for purifying an amino acid NCA in the present invention, the solubility of the amino acid NCA at 20 ° C. The purified amino acid NCA is immersed in 0.05 mg / cm ³ or less of the dehydrated solvent, in the presence of a polymerization initiator A method of polymerizing (solid phase polymerization), and a method of dissolving or immersing the purified amino acid NCA in a solvent capable of dissolving all or part of the purified amino acid NCA and polymerizing in the presence of a polymerization initiator (solution or heterogeneity) System polymerization). In the present invention, the reaction in which the amino acid NCA continuously reacts to produce a polymer polypeptide is a reaction involving the production of carbon dioxide, and should be precisely called “polycondensation”. Is conventionally expressed as “polymerization of amino acid NCA” and is therefore expressed as “polymerization”. Further, solid phase polymerization means a reaction in which an amino acid N-carboxyanhydride insoluble in a solvent is polymerized in a crystalline state, and is directly converted into a solid polypeptide. In the solid phase polymerization, polymerization is performed in a solvent in which the solubility of amino acid N-carboxyanhydride crystals is extremely low before the crystals are dissolved. In the solid phase polymerization of the present invention, the molecular weight distribution can be narrowed by adjusting the crystal size of the amino acid NCA, and the higher the crystal size, the higher the molecular weight polypeptide.

Solid-phase polymerization can be carried out by using a low dehydrated solvent (hereinafter also referred to as non-solvent) whose solubility of amino acid NCA is shown by the solubility at 20 ° C. Compared with polymerization, the reaction rate is extremely fast. Therefore, even if it is N-carboxy anhydrides, such as aspartate ester, glutamate ester, alanine, or valine, with low polymerization activity, it can superpose | polymerize favorably by setting it as the said solid phase polymerization. In the solid phase polymerization, the reaction rate increases as the purity of the amino acid NCA increases. Further, when the solubility at 20 ° C. exceeds 0.05 mg / cm ³ , a part or all of the solution becomes a solution or a heterogeneous polymerization, so that the polymerization occurs, but the reaction rate becomes slow, and the polymer is controlled. It becomes difficult. The solubility is preferably a value close to 0 as much as possible.

The non-solvent used in the solid-phase polymerization is an organic compound that is liquid at room temperature and does not dissolve the target substance. For example, it dissolves when added to a solution obtained by dissolving the target substance in a solvent that can dissolve the target substance. It has the effect of depositing substances and is also called a precipitant. The non-solvent is not particularly limited as long as it is an inert solvent for the amino acid NCA. For example, linear saturated hydrocarbons such as pentane, hexane, heptane, octane, nonane, decane, undecane, and petroleum ether. Saturated hydrocarbons having side chains such as 2-methylpentane, 2-methylhexane, 3-methylhexane, 2-methyloctane, 3-methyloctane and 4-methyloctane; cyclic saturated hydrocarbons such as cyclohexane; Examples include unsaturated hydrocarbons such as hexene and 3-hexene; hydrocarbons which are solid at room temperature but become liquid at the polymerization temperature, or mixtures thereof. In addition, among the solvents in which some of hydrogen atoms of these hydrocarbons are substituted with chlorine atoms or nitro groups, the amino acid NCA is preferably used if the solubility is 0.05 mg / cm ³ or less. it can. The amount of water in the dehydrated solvent is 0.01% by weight or less, preferably 0.005% by weight or less. The solvent can be dehydrated by a known dehydration method.

  The polymerization initiator used in the solid phase polymerization is not particularly limited as long as it is a polymerization initiator usually used in the polymerization of amino acid NCA. For example, amines such as butylamine, hexylamine, dibutylamine, and triethylamine, and sodium alcoholates Or alcohols containing a small amount of water or a small amount of water. The amount used is 1/10 to 1 / 100,000 mol, preferably 1/50 to 1/10000 mol, per mol of amino acid NCA. The atmosphere in which solid-phase polymerization is performed is not particularly limited, but moisture is removed under conditions that can prevent moisture absorption, that is, in a sealed reaction vessel, in a dry box, at 0 ° C. or lower, and using a dehumidifying agent. An atmosphere such as air or inert gas can be used.

  The polymerization temperature depends on the boiling point of the dehydrated non-solvent to be used, but is usually 5 to 100 ° C., preferably 20 to 80 ° C. If it is less than 5 ° C., the reaction rate is slow, and if it exceeds 100 ° C., the reaction rate is too fast, making it difficult to control the reaction and lowering the molecular weight reproducibility. The polymerization time varies depending on the polymerization temperature and the type of amino acid NCA, but is generally 30 minutes to 24 hours.

  Next, solution or heterogeneous polymerization will be described. In solution or heterogeneous polymerization, description of the same configuration and method as in solid phase polymerization will be omitted, and only differences will be described. That is, in the solution or heterogeneous polymerization, the difference from the solid phase polymerization is that a solvent capable of dissolving all or part of the purified amino acid NCA is used instead of the dehydrated non-solvent used in the solid phase polymerization. . Although the solution or heterogeneous polymerization has a slower reaction rate than solid phase polymerization, the degree of polymerization can be controlled by the amount of the initiator, and a polypeptide having a target average molecular weight can be obtained. In solution or heterogeneous polymerization, it was theoretically believed that when the molar ratio [amino acid NCA] / [initiator] = 200, the polymerization degree of the resulting polypeptide would be 200 (living polymerization). Features) Until now, no such example has been reported. That is, in practice, only a product having a considerably low degree of polymerization was obtained. This point is also pointed out in Deming's above-mentioned Nature magazine. In the present invention, this is made possible by carrying out the operation of recrystallization of raw materials or the preparation of polymerization of purified raw materials in an atmosphere that prevents the mixing of water and polymerization before the preparation.

  Solvents that can dissolve all or part of the purified amino acid NCA used in solution or heterogeneous polymerization are those that do not react with the amino acid NCA and dissolve the anhydride at the polymerization temperature, or completely dissolve the anhydride. Although it does not do, the solvent which melt | dissolves the surface to some extent or swells is mentioned. Examples of the solvent include dioxane, N, N-dimethylformamide, benzene, toluene, tetrahydrofuran, ethyl acetate, acetonitrile and the like, and more specifically, depending on the type of amino acid, such as benzyl esters of glutamic acid and aspartic acid. Examples of NCA include dioxane and N, N-dimethylformamide. Depending on the type of amino acid, nitrobenzene, toluene and the like can be used. The solvent is preferably dehydrated. In the solution or heterogeneous polymerization, acetonitrile or other nitriles can be used as a solvent. Almost all common amino acids NCA are soluble in these solvents such as acetonitrile, but the polymerization product is insoluble in the solvent. Thus, initially, polymerization occurs in solution and the product precipitates as a solid, and subsequent polymerization proceeds between the solid precipitate and the amino acid NCA in the solution. That is, since the polymerization proceeds at the solid phase / liquid phase interface, it is called heterogeneous polymerization.

In the solution polymerization, the degree of polymerization of the amino acid NCA can be adjusted by the molar ratio of the polymerization initiator to the amino acid NCA at the time of reaction charging (number of moles of amino acid NCA / number of moles of polymerization initiator). Specifically, in the solution polymerization, a polypeptide having a degree of polymerization similar to the value of the molar ratio of the polymerization initiator is obtained (for example, when the molar ratio of the polymerization initiator is 200, the degree of polymerization is 200). A degree of polypeptide is obtained).
That is, by performing the solution polymerization, there is an effect that the degree of polymerization can be easily adjusted.

  In the solution polymerization, the purified amino acid N-carboxyanhydride used as a raw material for polymerization is dissolved in a solvent that dissolves the purified amino acid N-carboxyanhydride and does not dissolve the resulting polypeptide. It may be a purified amino acid N-carboxy anhydride solution obtained by dissolving carboxy anhydride and then performing insoluble removal operations such as centrifugation and filtration. As a solvent that dissolves the purified amino acid N-carboxyanhydride and does not dissolve the polypeptide to be produced, when the polymerization raw material is an amino acid NCA into which a protecting group has not been introduced, for example, acetonitrile, acetone, methyl acetate , Ethyl acetate, propyl acetate, dioxane, N, N-dimethylformamide, benzene, toluene, benzene derivatives, tetrahydrofuran and the like; the raw material for the polymerization is a protecting group such as benzyl group, O-acetyl group, O-benzoyloxy group, etc. Is an amino acid NCA into which is introduced, for example, acetonitrile, acetone and the like. It is also possible to use a mixed solvent in which a plurality of solvents different in solubility of the purified amino acid N-carboxyanhydride and the resulting polypeptide are mixed to adjust the solubility.

  In the solution or heterogeneous polymerization, the polymerization temperature is usually a temperature not higher than the boiling point of the solvent, preferably 5 to 100 ° C, particularly preferably 20 to 80 ° C. When the temperature is lower than 5 ° C, the reaction rate is slow, and it takes a long time for the polymerization. When the temperature exceeds 100 ° C, the solvent or the polymerization initiator tends to evaporate during the polymerization time, and the molecular weight is difficult to control. It becomes. The polymerization time varies depending on the polymerization temperature, but is generally 1 hour to 14 days.

EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, this is merely illustrative and does not limit the present invention.
In addition, the molecular weight and molecular weight distribution (Mw / Mn value) of the polypeptide produced in the present invention were determined by gel permeation chromatography analysis (GPC) according to a conventional method. As a solvent and an eluent, N, N-dimethylformamide (DMF), dioxane (lithium bromide as a solute may be added at a concentration of 0.1 mol / liter), and in the case of a water-soluble polypeptide, pH Was used.

<Solid-state polymerization of N-carboxy-γ-benzyl-L-glutamate (hereinafter also referred to as BLGNCA)>
(Manufacture of crude BLGNCA)
10 g of γ-benzyl-L-glutamate obtained by esterification reaction of L-glutamic acid and benzyl alcohol is placed in 100 ml of tetrahydrofuran and stirred for 1 hour (this mixture is referred to as mixture A). Meanwhile, 0.5 g of activated carbon and 4.6 g of triphosgene are added to 300 ml of tetrahydrofuran, and the mixture is stirred for about 2 hours (this mixture is referred to as mixture B). Mixture A and Mixture B were mixed together and reacted at about 40 ° C. for 3 hours. The reaction mixture is initially cloudy, but with the formation of the amino acid NCA, the degree of cloudiness decreases and becomes transparent at the end of the reaction. After completion of the reaction, the reaction solution was decompressed and tetrahydrofuran was removed by distillation. After the concentrated liquid of the reaction mixture was adjusted to about 20 to 40 ml, about 200 ml of hexane was added as a precipitating agent to crystallize BLGNCA. The crystals were filtered to obtain 8.0 g of crude BLGNCA crystals (yield 72%).

(Recrystallization of crude BLGNCA)
The crude BLGNCA (8.0 g) was dissolved in 50 ml of dehydrated ethyl acetate at room temperature, and 250 ml of hexane was added to the solution to recrystallize BLGNCA and separated by filtration. Recrystallization with ethyl acetate-hexane was performed again without drying the solvent attached to the surface of the obtained BLGNCA, and the recrystallization was performed 6 times.

  Further, immediately before the polymerization operation, recrystallization with ethyl acetate-hexane was performed in a low temperature chamber of −10 ° C. to obtain 2.4 g of purified BLGNCA.

(Analysis of purified BLGNCA)
Regarding the purity of the purified BLGNCA, a portion of the BLGNCA crystal is dried and weighed, and the BLGNCA chlorine is measured by ion chromatography on an aqueous solution obtained by burning BLGNCA by a general oxygen combustion flask method. The content was determined. The determined chlorine content of BLGNCA was 0.015% by weight.

The confirmation of the formation of BLGNCA crystals was confirmed by elemental analysis (C, H, N), infrared absorption spectrum (hereinafter also referred to as IR spectrum) by the potassium bromide tablet method, and crystal obtained from X-ray analysis of a single crystal. Was compared with data of known crystal structures (H. Kanazawa et al., “Bulletin of Chemical Society of Japan”, 51, 2200-2204 (1978)). In the case of BLGNCA, its elemental analysis is calculated as BLGNCA molecular formula = C ₁₃ H ₁₃ O ₅ N: C, 59.31; H, 4.98; N, 5.32 (wt%), and the actual value. : C, 57.11; H, 5.01; N, 5.29 (%). In addition, in the IR spectrum of the product (which should be BLGNCA), characteristic absorption based on two carbonyl groups of a 5-membered ring of carboxy anhydride (NCA) was observed in the vicinity of 1853 and 1840 cm ^−1. Also confirmed the production of NCA. From the X-ray analysis of the single crystal, the lattice constants are a = 7.776, b = 27.47, c = 5.948 (the unit is angstrom (Å)), and the space group = P2 ₁ 2 ₁ 2 ₁ I was asked. The crystal structure also coincided with the known crystal structure.

(polymerization)
Next, BLGNCA (purified BLGNCA) crystals obtained by recrystallization in a low temperature chamber of −10 ° C. immediately before the polymerization operation are filtered and 1.49 g (1.01 g in terms of dry weight) in a dry state. Then, together with 20 ml of dehydrated hexane, the mixture was sealed in a polymerization reaction vessel having a connecting part connectable to a manometer for pressure measurement. Thereafter, the polymerization reaction vessel was returned to room temperature, and 1 ml of a butylamine hexane solution was added in a dry box so that the molar ratio of BLGNCA to [NCA] / [amine] = 200. The weight of butylamine is 1.36 mg. The polymerization reaction vessel was sealed again, and a capillary tube containing mercury at a height of about 1 m was connected to the connecting portion as a manometer. Thereafter, a polymerization reaction was performed at 30 ° C. until carbon dioxide was not generated. The end point was determined when the change in the height of mercury disappeared. The change in pressure was completed in about 4 hours, but 8 hours was left as a precaution (polymerization time 8 hours). The produced polypeptide was filtered, washed with ethyl acetate, and dried. From the dry weight, it was judged that the polymerization rate was 100%. In addition, as a manometer for pressure measurement, a glass tube filled with mercury, which is read from its height, and a commercially available pressure gauge (Coppal Electronics Co., Ltd. Handy Manometer PG-100) are used. As a result, the same measurement was possible. The low temperature room at −10 ° C. had a dry air atmosphere with an absolute humidity of 0.002 kg / kg dry air or less.

The BLGNCA crystals used for the polymerization were columnar crystals, and the average size was 0.15 × 0.1 × 0.1 mm. The obtained polypeptide was used as a dichloroacetic acid solution, and its solution viscosity was measured. From the Doty equation by the viscosity method (New Experimental Chemistry Lecture 19, Polymer Chemistry [I], page 171 footnote), the number average degree of polymerization and The molecular weight was determined. The molecular weight was 87,300, and the number average degree of polymerization was 400. Moreover, as a standard of molecular weight distribution of the obtained polypeptide by gel permeation chromatography analysis (GPC), the ratio of weight average molecular weight to number average molecular weight, Mw / Mn was 1.19. Note that the product is a polypeptide as a polymerized product of BLGNCA that the absorption in the amide I and amide II regions indicating the characteristic absorption of the polypeptide is 1655 cm ^{− in} the results of elemental analysis and infrared absorption spectrum measurement. It was proved from the fact that it was observed at ¹ and 1525 cm ⁻¹ .

(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
Example 1 except that the polymerization temperature was changed to 30 ° C., 20 ° C., [NCA] / [amine] = 200 instead of 2000, and the polymerization time was changed to 8 hours instead of 24 hours. The same method was used. The number average molecular weight of the obtained polypeptide was about 98,700 (number average degree of polymerization = 450), Mw / Mn determined by GPC = 1.31, and the yield of the polypeptide was 100%. The number average molecular weight was similar to that obtained from GPC.

(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
The polymerization was carried out in the same manner as in Example 1, except that the polymerization temperature was changed to 30 ° C. and 40 ° C. and the polymerization time was changed to 8 hours. The number average molecular weight of the obtained polypeptide was about 90,000 (degree of polymerization = 410), and the yield of the polypeptide was 100%.

(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
The polymerization was carried out in the same manner as in Example 1 except that the polymerization temperature was changed to 30 ° C. and 50 ° C., and the polymerization time was changed to 8 hours. The number average molecular weight of the obtained polypeptide was about 93,000 (degree of polymerization = 430), and the yield of the polypeptide was 100%.

(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
In order to confirm the reproducibility of the molecular weight, it was carried out in the same manner as in Example 1 except that the polymerization temperature was changed to 30 ° C. and the polymerization time was changed to 50 ° C. and the polymerization time was changed to 1.5 hours. . The number average molecular weight of the obtained polypeptide was about 92,000 (degree of polymerization = 420), and the yield of the polypeptide was 100%.

(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
In order to confirm the reproducibility of the molecular weight, the same procedure as in Example 1 was carried out except that the polymerization temperature was changed to 30 ° C. and 20 ° C., and the polymerization time was changed to 8 hours. The number average molecular weight of the obtained polypeptide was about 85,000 (number average degree of polymerization = 390), Mw / Mn = 1.18, and the yield of the polypeptide was 100%.

<Solution polymerization of BLGNCA>
(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
The procedure was the same as in Example 1, except that 20 ml of hexane was used and 20 ml of dioxane was used, and that the polymerization time was changed to 48 hours instead of 8 hours. The number average molecular weight of the obtained polypeptide was about 42,000 (degree of polymerization = 190), and the yield of the polypeptide was 100%. Example 7 is a solution or heterogeneous polymerization, and it can be seen that the degree of polymerization 190 of the resulting polypeptide is close to a feed ratio of 200. Mw / Mn determined by GPC = 1.19.

<Solution polymerization of BLGNCA>
(Manufacture of crude BLGNCA)
In the same manner as in Example 1, crude BLGNCA was obtained.
(Recrystallization of crude BLGNCA)
The crude BLGNCA (8.0 g) was dissolved in 50 ml of dehydrated ethyl acetate at room temperature, and 250 ml of hexane was added to the solution to recrystallize BLGNCA and separated by filtration. Recrystallization with ethyl acetate-hexane was performed again without drying the solvent attached to the surface of the obtained BLGNCA, and the recrystallization was performed 6 times.

  Further, immediately before the polymerization operation, recrystallization with ethyl acetate-hexane was performed in a low temperature chamber of −10 ° C. to obtain 2.4 g of purified BLGNCA.

(polymerization)
Next, BLGNCA (purified BLGNCA) crystals obtained by recrystallization immediately before the polymerization operation in a low temperature chamber of −10 ° C. are filtered, and 1.49 g (1.01 g in terms of dry weight) is obtained without drying. And added to 2 ml of acetonitrile. At this time, the acetonitrile solution of BLGNCA was slightly cloudy. Next, only the clear supernatant solution obtained by centrifuging the acetonitrile solution through a centrifuge (rotation speed 1000 / min, time = 5 min) was pipetted and mixed with 20 ml of dioxane to obtain a reaction solution. The reaction solution was sealed in a polymerization reaction vessel having a connecting portion connectable to a manometer for pressure measurement. Thereafter, the polymerization reaction vessel was returned to room temperature, and 1 ml of a butylamine hexane solution was added in a dry box so that the molar ratio of BLGNCA to [NCA] / [amine] = 200. The weight of butylamine is 1.40 mg. The polymerization reaction vessel was sealed again, and a capillary tube containing mercury at a height of about 1 m was connected to the connecting portion as a manometer. Thereafter, the polymerization reaction was carried out at 30 ° C. for 48 hours. The produced polypeptide was filtered, washed with ethyl acetate, and dried. From the dry weight, it was judged that the polymerization rate was 70%. Further, the number average molecular weight of the obtained polypeptide was about 53,000 (degree of polymerization = about 240). When calculated from GPC, Mw / Mn = 1.18. It can be seen that the polymerization degree 240 of the produced polypeptide is close to the charging ratio 200. The low temperature room at −10 ° C. had a dry air atmosphere with an absolute humidity of 0.002 kg / kg dry air or less.

(Comparative Example 1)
(Manufacture of crude BLGNCA)
The same method as in Example 1 was used.
(Recrystallization of crude BLGNCA)
A purified BLGNCA was obtained in the same manner as in Example 1 except that all operations were performed in a room at 20 ° C. and a humidity of 40%.
(polymerization)
Next, in a room at 20 ° C. and a humidity of 40%, the obtained purified BLGNCA is filtered, and 1.49 g (1.01 g in terms of dry weight) is taken without drying, together with 20 ml of dehydrated hexane, It sealed in the polymerization reaction container which has a connection part which can be connected with the manometer for pressure measurements. Next, 1 ml of a hexane solution of butylamine in a dry box was added so that the molar ratio of BLGNCA to [NCA] / [amine] = 200. The weight of butylamine is 1.36 mg. The polymerization reaction vessel was sealed again, and a capillary tube containing mercury at a height of about 1 m was connected to the connecting portion as a manometer. Thereafter, a polymerization reaction was performed at 30 ° C. One week was required until the polymerization rate reached 100%. The produced polypeptide was filtered, washed with ethyl acetate, and dried. The number average molecular weight of the obtained polypeptide was about 30,000 (degree of polymerization = 137), and Mw / Mn = 4.

(Comparative Example 2)
For confirmation of reproducibility, the polypeptide was polymerized four times in the same manner as in Comparative Example 1. The first time, the number average molecular weight was 12,000 (degree of polymerization = 55), and Mw / Mn = 6. The second time is number average molecular weight 22,000 (degree of polymerization = 100), Mw / Mn = 5.2, and the third time is number average molecular weight 34,000 (degree of polymerization = 155), Mw / Mn. Mn = 3.8, and in the fourth round, the number average molecular weight was 28,000 (degree of polymerization = 128) and Mw / Mn = 4.6, and there was no reproducibility.

(Comparative Example 3)
(Manufacture of crude BLGNCA)
The same method as in Example 1 was used.
(Recrystallization of crude BLGNCA)
A purified BLGNCA was obtained in the same manner as in Example 1 except that all operations were performed in a room at 20 ° C. and a humidity of 60%.
(polymerization)
Next, in place of charging the polymerization in a room at 20 ° C. and a humidity of 40%, the same procedure as in Comparative Example 1 was performed except that the polymerization was charged in a room at 20 ° C. and a humidity of 60%. Two weeks were required until the polymerization rate reached 100%. The number average molecular weight of the obtained polypeptide was about 20,000 (degree of polymerization = 91), and Mw / Mn = 6.

(Comparative Example 4)
(Production of crude BLGNCA, recrystallization of crude BLGNCA, polymerization)
Instead of carrying out the polymerization reaction at 30 ° C., it was carried out in the same manner as in Comparative Example 1 except that the polymerization reaction was carried out at 50 ° C., and it took 24 hours for the polymerization rate to reach 100%. The number average molecular weight of the obtained polypeptide was about 20,000 (degree of polymerization = 91), and Mw / Mn = 8.

(Comparative Example 5)
(Manufacture of crude BLGNCA, recrystallization of crude BLGNCA)
In the same manner as in Example 1, purified BLGNCA was obtained.
(polymerization)
Using the obtained purified BLGNCA, dioxane as a solvent and hexylamine as a polymerization initiator according to the non-patent document “ER Blout and M. Idelson, Journal of American Chemical Society, 80, 4909 (1958)”. The polymerization reaction was carried out at 25 ° C. for 48 hours with [NCA] / [amine] = 200. The polymerization rate was about 80%. Next, for confirmation of reproducibility, the polymerization reaction was performed 10 times in the same manner. The number average molecular weight of the obtained polypeptide was about 20,000-35,000 (degree of polymerization = 90-180), and there was no reproducibility with Mw / Mn = 4.2-5.0.

<Solid-state polymerization of N-carboxy-L-leucine anhydride (hereinafter also referred to as L-leucine NCA)>
L-leucine NCA was synthesized from L-leucine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1. Instead of purified BLGNCA, the obtained purified L-leucine NCA was not dried and was 0.886 g (0.607 g in terms of dry weight), and the polymerization time was 8 minutes instead of 90 minutes. Except for the above, polymerization was carried out in the same manner as in Example 1. The solution viscosity of the obtained polypeptide in trifluoroacetic acid solution (TCF) at 25 ° C. was 2.5 dl / g, and the yield of the polypeptide was 100%. There is still no method for numerically indicating the molecular weight of poly (L-leucine).

Confirmation of the production of L-leucine NCA is the same as BLGNCA in that the lattice constant of the crystal determined from elemental analysis (C, H, N), infrared absorption spectrum, and X-ray analysis of the single crystal is known. This was confirmed by comparison with crystal structure data (H. Kanazawa et al., “Bulletin of Chemical Society of Japan”, 51, 2205-2208 (1978)). From the X-ray analysis of the single crystal, the lattice constants are a = 6.518, b = 23.98, c = 5.531 (the unit is angstrom (Å)), and space group = P2 ₁ 2 ₁ 2 ₁ I was asked. The crystal structure also coincided with the known crystal structure.

(Comparative Example 6)
<Solid-state polymerization of L-leucine NCA>
(Production of crude L-leucine NCA)
L-leucine NCA was synthesized from L-leucine and triphosgene in the same manner as BLGNCA in Example 1 to obtain crude L-leucine NCA.
(Recrystallization and polymerization of crude L-leucine NCA)
Except for using the obtained crude L-leucine NCA, the same procedure as in Comparative Example 1 was carried out. As a result, the solution viscosity of the obtained polypeptide in trifluoroacetic acid solution (TCF) at 25 ° C. was 0.8 dl / g, Yield of polypeptide was 100%. This solution viscosity indicates that the molecular weight of the polypeptide is small.

<Solid-state polymerization of N-carboxy-L-alanine (hereinafter also referred to as L-alanine NCA)>
L-alanine NCA was synthesized from L-alanine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1. In place of the crystals of BLGNCA, the obtained purified L-alanine NCA was not dried and was 0.581 g (0.442 g in terms of dry weight), and the polymerization time was 8 minutes instead of 90 minutes. Polymerization was carried out in the same manner as in Example 1 except that. The solution viscosity of the obtained polypeptide in dichloroacetic acid solution at 25 ° C. was 3.2 dl / g, and the yield of the polypeptide was 100%.

The confirmation of L-alanine NCA production is the same as BLGNCA in that the crystal lattice constant determined from elemental analysis (carbon, hydrogen, nitrogen), infrared absorption spectrum, and X-ray analysis of a single crystal is known. (H. Kanazawa et al., “Acta Crystallographica, Section B”, 32, 3314-3316 (1976)). From the X-ray analysis of the single crystal, the lattice constants are a = 7.749, b = 10.70, c = 6.063 (the unit is angstrom (Å)), and space group = P2 ₁ 2 ₁ 2 ₁ I was asked. The crystal structure also coincided with the known crystal structure.

<Polymerization experiment of L-alanine NCA in acetonitrile>
The polymerization of L-alanine NCA was carried out in an acetonitrile solution in the same manner as in Example 10 except that acetonitrile was used instead of the non-solvent hexane. L-alanine NCA was dissolved in acetonitrile to obtain a transparent solution. After adding butylamine as an initiator, the reaction solution became cloudy 20 minutes later, and the polymerization proceeded as it was. However, it proceeded at a much slower speed than Example 10. The polypeptide was recovered 24 hours after the polymerization time when no pressure change was observed. The solution viscosity of the obtained polypeptide in dichloroacetic acid solution at 25 ° C. was 2.2 dl / g, and the yield of the polypeptide was 80%.

(Comparative Example 7)
<Method of performing polymerization preparation of L-alanine NCA in acetonitrile at room temperature>
L-alanine NCA crystals obtained by performing all operations of recrystallization in the collection process, dissolution process, precipitation process and filtration process at room temperature were placed in a reaction tube and then vacuum-dried at room temperature. Acetonitrile was added below, and a polymerization experiment was conducted in the same manner as in Example 11. When L-alanine NCA crystals were dissolved in acetonitrile at the stage of polymerization preparation, there was a trace amount of insoluble matter, and the acetonitrile solution of L-alanine NCA crystals was slightly cloudy. The fact that this is a polymer of amino acid NCA was found from the infrared absorption spectrum of insoluble matter collected in a similar experiment conducted separately. The polymerization rate was faster than Example 11. The polypeptide was recovered 24 hours after the polymerization time when no pressure change was observed. The solution viscosity of the obtained polypeptide in dichloroacetic acid solution at 25 ° C. was 1.5 dl / g, and the yield of polypeptide was 80%. The low molecular weight product was also empirically inferred from its rapid dissolution in dichloroacetic acid.

<Solid-State Polymerization of N-Carboxy-β-Ginyl-L-Aspartate Anhydride (hereinafter also referred to as BLANCA)>
(Manufacture of coarse BLANCA)
The crude BLANCA was carried out in the same manner as in Example 1, except that 10 g of γ-benzyl-L-glutamate was replaced with β-benzyl-L-aspartate obtained by esterification of L-aspartic acid and benzyl alcohol. Got.
(Recrystallization of crude BLANCA)
A purified BLANCA was obtained in the same manner as in Example 1 except that the obtained crude BLANCA was used instead of the crude BLGNCA.
(polymerization)
Instead of purified BLGNCA, the obtained purified BLANCA was carried out in the same manner as in Example 1, except that it was 1.41 g (0.956 g in terms of dry weight) without drying. The number average molecular weight of the obtained polypeptide was about 140,000 (number average degree of polymerization = about 670), determined by GPC, Mw / Mn = 1.32, and the yield of the polypeptide was 100%. . In addition, the number average molecular weight obtained a similar value from GPC.

<BLANCA solution polymerization>
(Manufacture of coarse BLANCA)
The crude BLANCA was carried out in the same manner as in Example 1, except that 10 g of γ-benzyl-L-glutamate was replaced with β-benzyl-L-aspartate obtained by esterification of L-aspartic acid and benzyl alcohol. Got.

(Recrystallization of crude BLANCA)
A purified BLANCA was obtained in the same manner as in Example 1 except that the obtained crude BLANCA was used instead of the crude BLGNCA.
(polymerization)
Instead of purified BLGNCA, the obtained purified BLANCA was 1.41 g (0.956 g in terms of dry weight) in a dry state, 20 ml of hexane was substituted with 20 ml of dioxane, and the polymerization time was replaced with 8 hours. The same procedure as in Example 1 was conducted except that the polymerization time was 48 hours. The number average molecular weight of the obtained polypeptide was about 49,000 (determined from GPC. Degree of polymerization = about 240), and the yield of the polypeptide was 70%. It was Mw / Mn = 1.17 when calculated | required from GPC.

(Comparative Example 8)
<BLANCA solution polymerization>
(Manufacture of coarse BLANCA)
The crude BLANCA was carried out in the same manner as in Example 1, except that 10 g of γ-benzyl-L-glutamate was replaced with β-benzyl-L-aspartate obtained by esterification of L-aspartic acid and benzyl alcohol. Got.
(Recrystallization of crude BLANCA)
All operations were performed in the same manner as in Example 13 except that the operation was performed indoors at 20 ° C. and a humidity of 40%.
(polymerization)
The polymerization was conducted in the same manner as in Example 13 except that the polymerization was carried out in a room at 20 ° C. and a humidity of 40%. The number average molecular weight of the obtained polypeptide was about 20,000 (determined from GPC. Degree of polymerization = 98), and the yield of the polypeptide was 70%. When determined from GPC, Mw / Mn = 6.

<Solid-state polymerization of N-carboxy-O-benzoyl-L-serine anhydride (hereinafter also referred to as O-benzoyl-L-serine NCA)>
(Production of crude O-benzoyl-L-serine NCA, recrystallization of crude O-benzoyl-L-serine NCA)
O-benzoyl-L-serine NCA was synthesized from commercially available O-benzoyl-L-serine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1.
(polymerization)
The same method as Example 1 except that instead of purified BLGNCA, the obtained purified O-benzoyl-L-serine NCA was 1.33 g (0.902 g in terms of dry weight) in a dry state. The polymerization was carried out at The O-benzoyl group of the obtained polymer was removed by a known method (“ER Blout and M. Idelson, Journal of American Chemical Society, 80, 4909-4913 (1958)”) A polypeptide (poly (L-serine)) was obtained. The number average molecular weight of the obtained polypeptide was about 120,000 (degree of polymerization = 1380), and the yield of the polypeptide was 100%. When calculated from GPC, Mw / Mn = 1.3.

<Solid-state polymerization of N-carboxy-O-benzoyl-L-tyrosine anhydride (hereinafter also referred to as O-benzoyl-L-tyrosine NCA)>
(Production of crude O-benzoyl-L-tyrosine NCA, recrystallization of crude O-benzoyl-L-tyrosine NCA)
O-benzoyl-L-tyrosine NCA was synthesized from O-benzoyl-L-tyrosine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1.
(polymerization)
The same method as in Example 1 except that instead of purified BLGNCA, the obtained purified O-benzoyl-L-tyrosine NCA was 1.77 g (1.20 g in terms of dry weight) in a dry state. The polymerization was carried out at The O-benzoyl group of the obtained polymer was removed by a known method (method described in Example 14) to obtain a water-soluble polypeptide (poly (L-tyrosine)). The number average molecular weight of the obtained polypeptide was about 196,000 (degree of polymerization = 1200), and the yield of the polypeptide was 100%. When calculated from GPC, Mw / Mn = 1.3.

<Solid-State Polymerization of ε-N-carbobenzoxy-L-lysine anhydride (hereinafter also referred to as ε-N-carbobenzoxy-L-lysine NCA)>
(Production of crude ε-N-carbobenzoxy-L-lysine NCA, recrystallization of crude ε-N-carbobenzoxy-L-lysine NCA)
ε-N-carbobenzoxy-L-lysine NCA was synthesized from ε-N-carbobenzoxy-L-lysine and triphosgene in the same manner as BLGNCA in Example 1, and recrystallized in the same manner as in Example 1. Purified by
(polymerization)
Example 1 except that instead of purified BLGNCA, the obtained purified ε-N-carbobenzoxy-L-lysine NCA was 1.56 g (1.06 g in terms of dry weight) in a dry state. Polymerization was carried out in the same manner as described above. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide (poly (L-lysine)). The number average molecular weight of the obtained polypeptide was about 160,000 (degree of polymerization = 690), and the yield of the polypeptide was 100%. When calculated from GPC, Mw / Mn = 1.20.

<Solution polymerization of ε-N-carbobenzoxy-L-lysine NCA>
(Production of crude ε-N-carbobenzoxy-L-lysine NCA, recrystallization of crude ε-N-carbobenzoxy-L-lysine NCA)
ε-N-carbobenzoxy-L-lysine NCA was synthesized from ε-N-carbobenzoxy-L-lysine and triphosgene in the same manner as BLGNCA in Example 1, and recrystallized in the same manner as in Example 1. Purified by
(polymerization)
Instead of purified BLGNCA, the obtained purified ε-N-carbobenzoxy-L-lysine NCA was adjusted to 1.56 g (1.06 g in terms of dry weight) in a dry state, and dioxane instead of 20 ml. The procedure was the same as in Example 1, except that the volume was 20 ml and the polymerization time was 48 hours instead of 8 hours. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 58,300 (degree of polymerization = 250), and the yield of the polypeptide was 90%. It was Mw / Mn = 1.18 calculated | required from GPC.

(Comparative Example 9)
<Solid-state polymerization of ε-N-carbobenzoxy-L-lysine NCA>
(Production of crude ε-N-carbobenzoxy-L-lysine NCA)
ε-N-carbobenzoxy-L-lysine NCA was synthesized from ε-N-carbobenzoxy-L-lysine and triphosgene in the same manner as BLGNCA of Example 1, and crude ε-N-carbobenzoxy-L- Lysine NCA was obtained.
(Recrystallization of crude ε-N-carbobenzoxy-L-lysine NCA)
Purified ε-N-carbobenzoxy-L-lysine NCA was obtained in the same manner as in Comparative Example 1 except that crude ε-N-carbobenzoxy-L-lysine NCA was used instead of crude BLGNCA. .
(polymerization)
The same method as in Comparative Example 1 except that instead of purified BLGNCA, the obtained purified ε-N-carbobenzoxy-L-lysine NCA was 1.51 g (dry weight 1.02 g) without drying. I went there. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 15,400 (degree of polymerization = 120), and Mw / Mn = 3.82 determined from GPC.

(Comparative Example 10)
<Solution polymerization of ε-N-carbobenzoxy-L-lysine NCA>
(Production of crude ε-N-carbobenzoxy-L-lysine NCA, recrystallization of crude ε-N-carbobenzoxy-L-lysine NCA)
In the same manner as in Comparative Example 9, purified ε-N-carbobenzoxy-L-lysine NCA was obtained.
(polymerization)
A method similar to Comparative Example 8 except that instead of purified BLGNCA, the obtained purified ε-N-carbobenzoxy-L-lysine NCA is 1.51 g (dry weight 1.02 g) in a dry state. I went there. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 10,000 (degree of polymerization = 78), and Mw / Mn = 4.0 determined from GPC.

<Random copolymerization in solution of O-benzoyl-L-serine NCA and O-benzoyl-L-tyrosine NCA>
O-benzoyl-L-serine NCA was synthesized from commercially available O-benzoyl-L-serine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1. Subsequently, O-benzoyl-L-tyrosine NCA was synthesized from O-benzoyl-L-tyrosine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1.

(polymerization)
Next, the purified O-benzoyl-L-serine NCA crystals and the purified O-benzoyl-L-tyrosine NCA crystals obtained by recrystallization immediately before the polymerization operation in a low temperature room of −10 ° C. are filtered and not dried. And 0.685 g (0.451 g in terms of dry weight) and 0.885 g (0.600 g in terms of dry weight), respectively, and can be connected to a manometer for pressure measurement together with 30 ml of dehydrated benzene. It sealed in the polymerization reaction container which has a connection part. Then, after returning the polymerization reaction vessel to room temperature, in a dry box, 1 ml of a hexane solution of butylamine with respect to the total number of moles of O-benzoyl-L-serine NCA and O-benzoyl-L-tyrosine NCA, [NCA] / [amine] was added so as to be 200. The weight of butylamine is 1.40 mg. The polymerization reaction vessel was sealed again, and a capillary tube containing mercury at a height of about 1 m was connected to the connecting portion as a manometer. Thereafter, a polymerization reaction was carried out at 30 ° C. for 48 hours. The low temperature room at −10 ° C. had a dry air atmosphere with an absolute humidity of 0.002 kg / kg dry air or less. The O-benzoyl group of the obtained polymer was removed by a known method to obtain a polypeptide (poly (L-serine) (L-tyrosine)). The number average molecular weight of the obtained polypeptide was 160,000 (degree of polymerization = 1,280), and Mw / Mn = 1.3 determined by GPC.

<Block Copolymerization in Solution of O-Benzoyl-L-Serine NCA and O-Benzoyl-L-Tyrosine NCA>
O-benzoyl-L-serine NCA was synthesized from commercially available O-benzoyl-L-serine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1. Then, O-benzoyl-L-tyrosine NCA was synthesized from O-benzoyl-L-tyrosine and triphosgene in the same manner as BLGNCA in Example 1, and purified by recrystallization in the same manner as in Example 1.

(polymerization)
Next, the purified O-benzoyl-L-serine NCA crystals obtained by recrystallization immediately before the polymerization operation in a low temperature chamber of −10 ° C. are filtered and 1.33 g (when converted to dry weight) without drying. 0.902 g) was taken, and together with 20 ml of dehydrated benzene, it was put in a polymerization reaction vessel having a connecting part connectable to a manometer for pressure measurement and sealed. Thereafter, the polymerization reaction vessel is returned to room temperature, and 1 ml of hexane solution of butylamine in a dry box is set to a molar ratio of O-benzoyl-L-serine NCA to [NCA] / [amine] = 200. added. The weight of butylamine is 1.40 mg. The polymerization reaction vessel was sealed again, and a capillary tube containing mercury at a height of about 1 m was connected to the connecting portion as a manometer. Thereafter, a polymerization reaction was performed at 30 ° C. for 24 hours to obtain a polymer A. The low temperature room at −10 ° C. had a dry air atmosphere with an absolute humidity of 0.002 kg / kg dry air or less.

  Next, purified O-benzoyl-L-tyrosine NCA crystals obtained by recrystallization immediately before the polymerization operation in a low temperature room of −10 ° C. are filtered and 1.77 g (in terms of dry weight when not dried) 1.19 g) was taken and dissolved in 20 ml of dehydrated benzene to obtain a benzene solution. Next, the polymerization reaction vessel containing the polymer A was transferred again to a low temperature chamber of −10 ° C., and the benzene solution was placed in the polymerization reaction vessel and sealed in the low temperature chamber. Thereafter, the polymerization reaction vessel is returned to room temperature, and 1 ml of hexane solution of butylamine in a dry box is set so that the molar ratio of O-benzoyl-L-tyrosine NCA is [NCA] / [amine] = 200. added. The weight of butylamine is 1.40 mg. Thereafter, a polymerization reaction was carried out at 30 ° C. for 48 hours to obtain a polymer B. The O-benzoyl group of the obtained polymer B was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 50,000 (degree of polymerization = 400) and Mw / Mn = 1.3 determined by GPC.

<Random copolymerization of ε-N-carbobenzoxy-L-lysine NCA and BLGNCA in solution>
In the same manner as in Example 1, purified BLGNCA was obtained. Next, ε-N-carbobenzoxy-L-lysine NCA was synthesized from ε-N-carbobenzoxy-L-lysine and triphosgene in the same manner as BLGNCA in Example 1, and as in Example 1, Purified by recrystallization.

(polymerization)
In place of purified O-benzoyl-L-serine NCA and purified O-benzoyl-L-tyrosine NCA, 1.49 g (1.01 g in terms of dry weight) of purified BLGNCA obtained without purification and purified ε The same procedure as in Example 18 was performed except that -N-carbobenzoxy-L-lysine NCA was changed to 1.56 g (1.06 g in terms of dry weight) without being dried. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 34,000 (degree of polymerization = 195), and Mw / Mn = 1.28 determined from GPC.

<Block copolymerization in solution of ε-N-carbobenzoxy-L-lysine NCA and BLGNCA>
In the same manner as in Example 1, purified BLGNCA was obtained. Next, ε-N-carbobenzoxy-L-lysine NCA was synthesized from ε-N-carbobenzoxy-L-lysine and triphosgene in the same manner as BLGNCA in Example 1, and recrystallized in the same manner as in Example 1. Purified by purification.

(polymerization)
Instead of purified O-benzoyl-L-serine NCA, the obtained purified BLGNCA was 1.49 g (1.01 g in terms of dry weight) in a dry state and replaced with purified O-benzoyl-L-tyrosine NCA. The purified ε-N-carbobenzoxy-L-lysine NCA obtained in the same manner as in Example 19 except that the amount was 1.56 g (1.06 g in terms of dry weight) in a dry state. went. The protecting group carbobenzoxyl group of the obtained polymer was removed by a known method to obtain a polypeptide. The number average molecular weight of the obtained polypeptide was 36,500 (degree of polymerization = 210) and Mw / Mn = 1.18 determined from GPC.

(Comparative Example 11)
<Solid-state polymerization of O-benzoyl-L-serine NCA>
(Production of crude O-benzoyl-L-serine NCA)
O-benzoyl-L-serine NCA was synthesized from commercially available O-benzoyl-L-serine and triphosgene in the same manner as BLGNCA of Example 1 to obtain crude O-benzoyl-L-serine NCA.
(Recrystallization of crude O-benzoyl-L-serine NCA)
The same procedure as in Comparative Example 1 was carried out except that the obtained crude O-benzoyl-L-serine NCA was used instead of the crude BLGNCA.
(polymerization)
Instead of purified BLGNCA, the obtained purified O-benzoyl-L-serine NCA was adjusted to 1.33 g (0.902 g in terms of dry weight) in a dry state, and the polymerization time was changed to 1 week for 24 hours. The method was the same as in Comparative Example 1 except that. The O-benzoyl group of the obtained polymer was removed by a known method to obtain a water-soluble polypeptide (poly (L-serine)). The number average molecular weight of the obtained polypeptide was about 12,000, and the yield of the polypeptide was 70%. When determined from GPC, Mw / Mn = 5.0.

(Comparative Example 12)
<Solid-state polymerization of O-benzoyl-L-tyrosine NCA>
(Production of crude O-benzoyl-L-tyrosine NCA)
O-benzoyl-L-tyrosine NCA was synthesized from O-benzoyl-L-tyrosine and triphosgene in the same manner as BLGNCA in Example 1 to obtain crude O-benzoyl-L-tyrosine NCA.
(Recrystallization of crude O-benzoyl-L-tyrosine NCA)
The same procedure as in Comparative Example 1 was carried out except that the obtained crude O-benzoyl-L-tyrosine NCA was used instead of the crude BLGNCA.
(polymerization)
Instead of purified BLGNCA, the obtained purified O-benzoyl-L-tyrosine NCA was made 1.77 g (1.20 g in terms of dry weight) in a dry state, and the polymerization time was changed to 1 week for 1 day. The method was the same as in Comparative Example 1 except that. The O-benzoyl group of the obtained polymer was removed by a known method to obtain a water-soluble polypeptide (poly (L-tyrosine)). The number average molecular weight of the obtained polypeptide was about 25,000 (degree of polymerization = 153), and the yield of the polypeptide was 70%. When calculated from GPC, Mw / Mn = 3.2.

(Comparative Example 13)
<Solution polymerization of O-benzoyl-L-serine NCA>
(Production of crude O-benzoyl-L-serine NCA)
O-benzoyl-L-serine NCA was synthesized from commercially available O-benzoyl-L-serine and triphosgene in the same manner as BLGNCA in Example 1 to obtain crude O-benzoyl-L-serine NCA.
(Purification and polymerization of crude O-benzoyl-L-serine NCA)
The O-benzoyl thus obtained was obtained by performing all operations of recrystallization in the step of collecting, dissolving, precipitating and filtering the crude O-benzoyl-L-serine NCA in a room at 20 ° C. and a humidity of 40%. After putting -L-serine NCA in a reaction tube, it was vacuum-dried at room temperature, and acetonitrile was added in a room at 20 ° C. and a humidity of 40%, and a polymerization experiment was conducted in the same manner as in Example 11. At this time, the polymerization time was 24 hours. The O-benzoyl group of the obtained polymer was removed by a known method to obtain a water-soluble polypeptide (poly (L-tyrosine)). The number average molecular weight of the obtained polypeptide was about 9,600 (degree of polymerization = 110), and the yield of the polypeptide was 100%. When determined from GPC, Mw / Mn = 3.4.

(Comparative Example 14)
<Random copolymerization in solution of O-benzoyl-L-serine NCA and O-benzoyl-L-tyrosine NCA>
When the recrystallization and polymerization were carried out in a room at 20 ° C. and a humidity of 40% and the polymerization time was 20 hours, the number average of the obtained polypeptides was obtained in the same manner as in Example 18. The molecular weight was about 12,000 (degree of polymerization = 96), and the yield of the polypeptide was 95%. When calculated from GPC, Mw / Mn was 6.2.

(Comparative Example 15)
<Block Copolymerization in Solution of O-Benzoyl-L-Serine NCA and O-Benzoyl-L-Tyrosine NCA>
When the recrystallization and the polymerization were carried out in a room at 20 ° C. and a humidity of 40% and the polymerization time was 24 hours, the number average polypeptide obtained was obtained in the same manner as in Example 19. The molecular weight was about 1,000 (degree of polymerization = 80), and the yield of the polypeptide was 85%. When calculated from GPC, Mw / Mn was 6.0.

  According to the method for producing a polypeptide of the present invention, amino acid NCA is used as a raw material, and a polypeptide having a high molecular weight and a narrow molecular weight distribution is generally easier to obtain using only safe, inexpensive drugs that are generally available. Can be produced by a simple method, and the reproducibility of the molecular weight is improved. Therefore, using high-molecular-weight synthetic polypeptides produced by the production method of the present invention, production of materials such as high-strength protein-like fibers and films, application as biocompatible materials, protein functions It is useful for sex research and application as a pharmaceutical material.

Claims (9)

Production of polypeptide by purifying amino acid N-carboxyanhydride by one or more recrystallizations to obtain purified amino acid N-carboxyanhydride and polymerizing the purified amino acid N-carboxyanhydride to obtain polypeptide A method for producing a polypeptide, comprising performing at least the last recrystallization operation of the recrystallization in dry air at a temperature of 0 ° C. or lower and an absolute humidity of 0.004 kg / kg dry air or lower . Production method. The process according to claim 1 Symbol placement polypeptide and performing operation of the last recrystallization at a temperature of -20 to-5 ° C.. The polypeptide according to any one of claims 1 and 2 , wherein the polymerization of the purified amino acid N-carboxyanhydride obtained by the last recrystallization is performed at a temperature of 0 ° C or lower. Production method. 4. The method for producing a polypeptide according to claim 3, wherein the polymerization is carried out in a dry air at a temperature of 0 ° C. or less and an absolute humidity of 0.004 kg / kg dry air or less. The method for producing a polypeptide according to claim 3 or 4, wherein the polymerization is carried out at a temperature of -20 to -5 ° C. Preparation of polymerization of the purified amino acid N-carboxyanhydride without removing the solvent adhering to the surface of the purified amino acid N-carboxyanhydride after filtration in the last recrystallization The method for producing a polypeptide according to any one of claims 1 to 5 , wherein: Polymerization of the purified amino acid N-carboxy anhydride involves immersing the purified amino acid N-carboxy anhydride in a dehydrated solvent having a solubility of the amino acid N-carboxy anhydride at 20 ° C. of 0.05 mg / cm ³ or less. The method for producing a polypeptide according to any one of claims 1 to 6, wherein the polypeptide is solid-phase polymerized in the presence of a polymerization initiator. The dehydrated solvent having a solubility of the amino acid N-carboxyanhydride at 20 ° C. of 0.05 mg / cm ³ or less is pentane, hexane, heptane, octane, nonane, decane, petroleum ether, or a mixture thereof. The method for producing a polypeptide according to claim 7 . In the polymerization of the purified amino acid N-carboxy anhydride, the purified amino acid N-carboxy anhydride is dissolved or immersed in a solvent capable of dissolving all or part of the purified amino acid N-carboxy anhydride, and the presence of a polymerization initiator is present. The method for producing a polypeptide according to any one of claims 1 to 6, wherein the polypeptide is polymerized below.