JPH0150718B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing human insulin, and more specifically, an insulin derivative in which arginine at position 22 of the B chain is modified with a protecting group is subjected to a protective group removal treatment to produce human insulin. The present invention relates to a manufacturing method for obtaining insulin.

Insulin is a valuable drug with no substitutes for the treatment of diabetes, and currently bovine insulin and porcine insulin are mainly used for treatment. However, some of the constituent amino acids of these insulins are different from human insulin, so
Antibodies may be produced in the body, and the production of antibodies causes problems such as a marked decrease in the therapeutic effect of insulin. Therefore,
There is a strong desire to establish a method for synthesizing human insulin that can be carried out on an industrial scale.

Porcine insulin differs from human insulin in the amino acid at position 30 of the B chain, and the present inventors compared this different amino acid at position 30 of the B chain to human insulin.
The present invention was established by discovering a method for substituting the position amino acid threonine.

According to the above method, human insulin and B chain 30
Human insulin can be easily synthesized using porcine insulin as a raw material, which differs only in the amino acid position. There is no doubt that the human insulin obtained in this way is an ideal therapeutic insulin.

Attempts to obtain human insulin from pig desoctapeptide insulin and human insulin octapeptide have already been made by MA Ruthtenberg (M.A.
Science 177 by Ruttenberg
Volume 623 (1972), R. Obermeier et al.
Physiol.
The former involves an alkali treatment at the end of the process, and accompanying side reactions are unavoidable. Furthermore, in the latter case, the reaction is nonspecific and many side reactions occur, making purification complicated and difficult, and yields are extremely low. Therefore, this cannot be done on an industrial scale.

The method of the present inventors is an enzymatic insulin synthesis method, which differs from known chemical methods.
It has the advantage that the reaction is specific, no side reactions occur, racemization does not occur, and unreacted raw materials can be recovered without damage.

The above method uses insulin (represented by the general formula below) in which the arginine at position 22 of the B chain is modified with a protecting group and the amino acid at position 30 of the B chain is missing by the action of trypsin or a trypsin-like enzyme. A L-threonine derivative (hereinafter referred to as a compound) represented by the following general formula is condensed with a compound (hereinafter referred to as a compound), and the protecting group of the obtained compound (represented by the following general formula and hereinafter referred to as a compound) is removed. By doing so.

(In the formula, R ³ represents a protecting group for the guanidino group.) (In the formula, R ¹ represents hydrogen and R ² represents a protecting group for the carboxy group.) (In the formula, R ¹ , R ² and R ³ each have the same meaning as defined above.) All of the amino acids used in the above formula are L-type and have the following meanings.

Ala Alanine Arg Arginine Asp Aspartic acid Asn Asparagine Gln Glutamine Glu Glutamate Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Pro Proline Phe Phenylalanine Ser Serine Thr Threonine Tyr Tyrosine Val Val Val Valine Cys Cysteine After introducing a protective group into the guanidino group of arginine located at position 22 of the B chain using insulin obtained from
(2) It can be produced by treating insulin obtained from pigs with an enzyme to produce des-B30-insulin, and then modifying the guanidino group of arginine at position 22.

To introduce a protecting group for the guanidino group, use 2,3-butadione, 1,2-cyclohexanedione, phenylglyoxal, 2,4-pentadione, etc.
The reaction is carried out in the presence of borate ions, if necessary. For details, see Biochemistry Experiment Course 1 Chemistry of Substances (edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin) 34
- Described on page 41.

When performing method (1), it is recommended to cleave the amino acid at position 30 of the B chain with trypsin. The modification method of guanidino group is described by M.Rosenblatt.
Biochemistry by et al.
It is shown in Volume 17, page 3188 (1978). That is, 1,2-cyclohexanedione was added to a solution of insulin as a raw material, and 0.2M borate (pH 9) was added.
After reacting for 1 to 4 hours at 40°C or overnight at room temperature in the presence of [ ^N7 , ^N8- (1,2-dihydroxycyclohexylene-1, 2) (hereinafter abbreviated as DHCH) -Arginine-B22] Trypsin was added to insulin in the presence of 0.2M borate (pH 8) and incubated at 40°C.
The compound is obtained by allowing it to act for 1 to 3 days.

Alternatively, by method (2), for example, porcine insulin can be desiccated using carboxypeptidase A.
B30-insulin (pig type) is obtained. This method is based on the E.D.
Hoppe-Seyler's Zeitschrift fu¨r
Physiologische Chemie) Volume 359, Page 799 (1978)
It is described in. Next, this pig-derived death-
In B30-insulin, the arginine side chain at position 22 of the B chain is modified to prepare a compound.

The L-threonine derivative (hereinafter referred to as a compound) represented by the general formula is synthesized by a conventional chemical method, and the hydroxy group as a side chain functional group may be protected or unprotected. In the case of protection, it is preferable to use protective groups that are commonly used in peptide synthesis reactions. It is essential that the carboxy group of the compound is protected, and a commonly used carboxy group protecting group is used. For example, t-
It may be modified in the form of an alkyl or aralkyl ester such as butyl or benzyl, an amide, a substituted amide such as anilino, or a salt.

When selecting the above-mentioned protecting groups, consideration should be given to selecting a protecting group that does not denature or deactivate insulin with respect to the introduction or removal treatment of the protecting group. Regarding protecting groups used in peptide synthesis, see M. Bodanszky et al.'s Peptide Synthesis, 2nd edition (1976) (John Wiley & Sons).
& Sons)).

In addition, in selecting a protecting group, it is natural that it is preferable to select one that can be simultaneously removed by a single protecting group removal treatment.

The condensation reaction between compounds is carried out under conditions suitable for the peptide bond forming reaction of trypsin or trypsin-like enzymes. pH5~8, especially 6~
7 is preferable, and the reaction temperature is 0 to 50℃, especially
20-40℃ is good. It is desirable that the compound and compound concentrations be as high as possible. Furthermore, the compounds are preferably reacted at a molar ratio of 1:1 to 100:1, particularly around 20:1 to 100:1. A suitable organic solvent miscible with water is added to the reaction solution. Addition of an organic solvent is effective not only in reducing the concentration of water in the reaction solution and suppressing the hydrolysis reaction, which is a reverse reaction, but also in significantly increasing the solubility of the compound and the compound. As the organic solvent, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, glycerin, etc. are used alone or in combination. It is particularly preferable to use it at a concentration of 0 to 65%, particularly 40 to 60%. Generally, when using an organic solvent, the ratio to water is determined by considering the solubility of the raw material, denaturation of enzymes, hydrolysis reaction, etc. As a buffer for the reaction solution, borate is suitable for stabilizing DHCH-arginine-B22.

Enzymes used in the present invention include trypsin and trypsin-like enzymes of various animal and microbial origins. An example of the latter is a trypsin-like enzyme extracted and isolated from Streptomyces. Regarding the enzyme, Morihara et al. published Archives of Biochemistry and Biophys (Arch.Biochem.Biophys.), Vol. 126, p. 971 (1968).
, Yoshida et al. published a FEBS letter (FEBS).
Lett.) Vol. 15, p. 129 (1971). The enzyme used is tosyl-L to remove contaminating chymotrypsin or chymotrypsin-like activity.
- Phenylalanine chloromethyl ketone (hereinafter
It is preferable to process the data using a method such as TPCK).

The enzyme concentration in the reaction solution varies depending on the substrate concentration and enzyme activity, but when using commercially available crystalline trypsin, it is preferably around 1 mg/ml to 10 mg/ml. The enzyme may be used as it is, or may be used as an immobilized enzyme bound to or included in a suitable insoluble carrier.

Although the reaction time varies depending on the reaction conditions, it is usually sufficient to allow the time required for the enzyme reaction to reach equilibrium, which is usually about 3 to 72 hours, and in most cases about 6 to 24 hours.

After completion of the reaction, the desired compound can be collected from the reaction solution using various known separation means. For example, the reaction solution is subjected to gel filtration to isolate and recover unreacted compounds and enzymes. The recovered compounds and enzymes can be reused as is. The remainder contains compounds and des-B30-insulin. The compound can be obtained by freeze-drying, but in order to obtain the final target substance human insulin, the compound is not isolated, but the remainder is subjected to removal treatment of the arginine protecting group, followed by appropriate chromatography or gel filtration. Attach to. The produced insulin derivative in which the carboxyl group of the terminal threonine of the B chain is protected and the insulin lacking the amino acid at position 30 in the B chain (hereinafter referred to as des-B30-insulin) are separated. Recovered Death-B30
- Insulin can be used as a raw material compound by modifying the arginine at position 22 of the B chain again. Finally, the protecting group of the carboxy group of the B chain terminal threonine is removed to obtain the final target compound, insulin.

The method for removing the protecting group varies depending on the protecting group used, but it can be carried out according to the usual method. For example, to remove the DHCH group from the guanidino group, leave it overnight at 37°C in the presence of 1M hydroxylamine (pH 7). and do it. Further, the t-butyl group used as a protecting group for a hydroxy group or a carboxyl group is removed when treated with trifluoroacetic acid in the presence of a cation scavenger (eg, anisole). When a protecting group that can be removed by a single elimination process is used to protect the side chain functional group and the carboxy group of threonine as described above, the elimination process is simple and the yield is improved. Even when the carboxyl terminal is converted into another ester, amide, substituted amide, salt, etc., the protecting group can be removed by appropriate hydrolysis treatment or desalting operation.

The compound can be converted into human insulin as described above, and the obtained human insulin is useful as a therapeutic drug for diabetes having hypoglycemic action as described above.

Human insulin obtained from porcine insulin has been found to have the same hypoglycemic effect as porcine insulin in mice.

Human insulin obtained from the compound is formulated and administered to humans in the same manner as commercially available porcine or bovine insulin. In other words, by adding zinc chloride etc. to make a stable zinc complex, by adding a buffer such as sodium hydrogen phosphate or sodium acetate, by adding sodium chloride to make an isotonic solution, and by adding cresol, phenol, paraoxybenzoic acid, etc. Injections can be prepared using various preparation methods used in commercially available insulin formulations, such as adding preservatives such as acid alkyl esters (eg, methyl, ethyl, propyl, butyl esters, etc.).

Although the dosage of such an injection varies depending on the patient's symptoms, it may be administered in the same manner as the administration of commercially available insulin preparations, and it is recommended that adults administer about 1 to 100 units per day.

In the Examples below, production examples of the compounds of the present invention are shown, but these Examples are not intended to limit the present invention in any way.

In addition, the abbreviations used in the examples represent the following meanings.

OBu ^t t-Butyl ester Example 1 (1) Porcine des-B30-insulin 500 mg of porcine insulin was dissolved in 100 ml of 0.1 M ammonium bicarbonate aqueous solution (pH 8.3), and crystalline carboxypeptidase A (manufactured by Worthington Co., Ltd., diisopropyl Add 5 mg of fluorophosphate treatment (49u/mg) and react at room temperature for 8 hours. The amount of alanine produced is 0.77M/1M insulin.

The reaction product was lyophilized and then dissolved in 0.5M acetic acid.
Apply the column (3.5 x 95 cm) to Sephadex G50 (trade name) (ultra fine particles) and elute with 0.5M acetic acid. 11.5 ml per fraction, from the 40th
Collect the 60th fraction and lyophilize to collect 460 mg of the title compound. Yield 92%.

Amino acid analysis values after hydrolysis with 6N hydrochloric acid at 110°C for 24 hours are as follows. The numbers in parentheses represent theoretical values. The above hydrolysis conditions and display method are the same in the following examples.

Lys1.00(1), His1.91(2), Arg0.95(1), Asp3.21(3), Thr2.09(2), Ser2.97(3), Glu7.35(7), Pro1 .25(1), Gly4.29(4), Ala1.26(1), Val3.86(4), Ile1.55(2), Leu6.53(6), Tyr4.08(4), Phe3. 22(3), (2) Pig-derived [B22-Arg(DHCH)]-Des-B30
-Insulin Dissolve 200 mg of the above product in 1.7 ml of 0.25 M borate buffer (pH 9), add 45 mg of 1,2-cyclohexanedione, and keep overnight at room temperature. After acidifying with glacial acetic acid, it was applied to a column (6 x 85 cm) of Cephadex G50 (trade name) (ultrafine particles) and eluted with 0.5M acetic acid.

Measure the absorption at 280 nm, collect the corresponding portions, and lyophilize to obtain 168 mg of the title compound. Yield 84%. Furthermore, high performance liquid chromatography and slab electrophoresis confirmed that almost 100% of arginine was modified with DHCH.

Amino acid analysis Lys1.00(1), His2.07(2), Arg ^* 0.27(1), Asp3.21(3), Thr2.05(2), Ser2.98(3), Glu7.19(7) ), Pro1.23(1), Gly3.91(4), Ala1.29(1), Val3.99(4), Ile1.57(2), Leu6.57(6), Tyr3.48(4) , Phe3.16(3).

*During hydrolysis, approximately 20% of DHCH-Arg becomes Arg
(Patthy et al., Journal of Biological Chemistry (J.Biol.Chem.) 250
Volume 557 (1975)).

(3) Pig-derived (B22−Arg(DHCH), B30−Thr−
97.8 mg (10 mM) of the product of insulin (2) and 211 mg (500 mM) of L-threonine t-butyl ester acetate ^.
was dissolved in 1.08 ml of ethanol/dimethylformamide mixture (1:1, v/v), and crystalline trypsin (manufactured by Worthington Co., recrystallized three times) was added.
4.2mg and final concentration 0.01mM
0.5M borate buffer containing TPCK (pH 7.5)
Mix with 0.72ml and keep at 37℃ overnight. The final pH is
It was 6.5.

After acidifying with glacial acetic acid, Sephadex G50
Gel filtration is performed using a column of (trade name) (Ultra Fine Particles), and the product is separated into trypsin, insulin, and threonine derivatives based on enzyme activity, ultraviolet absorbance at 280 nm, and ninhydrin reaction (see Figure 1). The amount of L-threonine t-butyl ester recovered was about 50%. The insulin portion was freeze-dried to obtain 87 mg of the title compound.

(4) Pig-derived [B30−Thr−OBu ^t ]-insulin 85 mg of the product from (3) above was dissolved in 7.5 ml of 1M hydroxyamine (pH 7), and the mixture was replaced with nitrogen gas.
After keeping overnight at ℃, it is dialyzed against 0.01M Tris buffer (pH 7.4) in a cold place. Then 7M
DEAE-Sephadex A25 (trade name) buffered with 0.01M Tris buffer (pH 7.4) containing urea
Chromatography is performed at 4°C using a column (1.9 x 24.5 cm). After flowing 800 ml of the above buffer solution, concentration gradient elution was performed to raise the salt concentration to 0.3M, and fraction A with a concentration of 0.08 to 0.09M and fraction A with a concentration of 0.13
Obtain fraction B around a concentration of ~0.14M. Each fraction is immediately dialyzed in the cold against 0.01M ammonium acetate solution for 3-4 days and then lyophilized. 30 mg of powder was obtained from fraction A and 25 mg of powder was obtained from fraction B. High performance liquid chromatography and polyacrylamide gel electrophoresis confirmed that the former was the title compound and the latter was a mixture of des-B30-insulin (porcine type) and porcine insulin.

(5) Production of human insulin Add 2 ml of trifluoroacetic acid containing 0.2 ml of anisole to 28 mg of the product from (4) and keep at room temperature for 30 minutes.
After removing trifluoroacetic acid in a nitrogen stream
Add 2 ml of 1N acetic acid and extract anisole with 15 ml of ether. Lyophilize the acetic acid part to obtain the title compound 26.
Get mg. If the purity of the raw material, pig-derived des-B30-insulin, is 77%, the yield is 38%. This product was identified as the title compound based on amino acid analysis, slab gel electrophoresis, and high performance liquid chromatogram.

Amino acid analysis value Lys1.00(1), His1.85(2), Arg0.93(1), Asp3.21(3), Thr2.94(3), Ser2.96(3), Glu7.23(7), Pro1.23(1), Gly4.27(4), Ala1.19(1), Val3.89(4), Ile1.56(2), Leu6.57(6), Tyr3.78(4), Phe3.18(3).

[Brief explanation of drawings]

Figure 1 shows pig-derived [B22-Arg (DHCH), B30-
FIG. 3 shows the flow pattern by column chromatography when separating Thr-OBu ^t ]-insulin.

Claims (1)

[Scope of Claims] 1. Human insulin is obtained by subjecting an insulin derivative represented by the following general formula in which arginine at position 22 of the B chain is modified with a protecting group to removal treatment of the protecting group. Method for producing human insulin. (In the formula, R ¹ is hydrogen, R ² is a carboxyl group protecting group,
R ³ each represents a protecting group for the guanidino group. )