JPS62116597A - Production of human insulin - Google Patents

Abstract

PURPOSE:To obtain human insulin useful as a remedy for diabetes, by eliminating protecting groups of a specific insulin derivative wherein arginine at a specific position is modified with a protecting group. CONSTITUTION:Protecting groups of insulin derivative shown by formula I (R<1> is OH-protecting group; R<2> is COOH-protecting group; R<3> is guanidiono- protecting group) wherein arginite at the 22-position of chain B is modified with a protecting group are eliminated, for example, R<3> [e.g., N<7>,N<8>-(1,2- dihydroxycyclohexylene-1,2)group(DHCH, for short), etc.,] is eliminated by allowing the derivative to stand in the presence of 1M hydroxyamine (pH 7) at 37 deg.C for 24hr and R<1> and R<2> (e.g., t-butyl group, etc.,) are eliminated by treating the derivative with trifluoroacetic acid in the presence of a cationic scavenger, to give the aimed substance, The compound shown by the formula I can be obtained by condensing [B22-Arg(DHCH)]des-B30-insulin shown by formula II derived from swine with an L-threonine derivative shown by formula III in the presence of an anzyme such as trypsin, etc.

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 the Bu422 position 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 is a valuable drug with no substitutes for the treatment of glycosymptoms, and bovine insulin and porcine insulin are currently used for treatment. However, because some of the amino acids in these insulins are different from human insulin, antibodies may be produced in the body, and once antibodies are produced, the therapeutic effect of insulin will be significantly reduced. Causes head closure. 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.
The present invention was established by discovering a method of substituting the amino acid at position 0 with threonine, which is the amino acid at position 30 of human innulin.

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

M.N. Ruttenberg CM, A. has already attempted to obtain human insulin from pig desoctapeptide insulin and human insulin octapeptide.

Ruttenberg) by Science (5cie
nce) Vol. 177, p. 623 (1972), by R. Obermeier et al.

Physiol. do not have. In the latter case, the reaction is non-specific and many side reactions occur, making purification complicated and difficult and resulting in extremely low yields. Therefore, this cannot be done on an industrial scale.

The method of the present inventors is an enzyme-based insulin synthesis method,
Unlike known chemical methods, this method has the advantage that the reaction is specific, no side reactions occur, no racemization occurs, and unreacted raw materials can be recovered without damage.

The above method produces insulin (represented by the general formula 1 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. The resulting compound (
The method consists of removing the protecting group of a compound represented by the following general formula III (hereinafter referred to as compound III).

(Left below) (In the formula, R3 represents a protecting group for the guanidino group.) CH
.

CH-OR' HN-CH-COR2I (In the formula, R' represents hydrogen and R2 represents a protecting group for a carboxyl group.) (Left below is a blank space) (In the formula, R', R' and R8 each have the same meaning as above. ) Note that all 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 Glutamic acid c1y Glycine His Histine 11a Isoleucine Leu Leucine Lys Lysine Pro Proline Phe Phenylalanine Ser Ser Serine Ih
r Threonine Tyr Tyrosine Val
Valine Cys Cystine The above compound II is obtained by (1) first using insulin obtained from pigs to introduce a protecting group into the guanidino group of the alkynine located at position 22 of the B chain, and then applying an enzyme to the amino acid at position 30. (2) Des-B30-yne>niline can also be produced by modifying the guani-7 group of furquinine at position 22 by cleaving it or (2) allowing enzymes to act on insulin obtained from pigs.

Introducing a protecting group for guanidi 7 group is 2,3-butanoone, 1
, 2-cyclohexaneone, phenylglyoxal, 2,4-pentadione, etc. are reacted in the presence of borate ions depending on the requirements. Details are described in Biochemistry Experiment Course 1, Chemistry of Kumbaku Substances IV (edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin), pages 34-41.

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 was developed by M Rosenblatt et al.
) Vol. 17, p. 3188 (1978).

That is, 1,2-nochlorohexaneone was added to a solution of insulin as a raw material, and 0.2 Mm acid salt (pH 9) was added.
After reacting for 1 to 4 hours at 40°C or overnight at room temperature, the product was purified by dialysis, dialysis, etc. to obtain [N7°N'-(1,2-hydroxychlorohexylene-1, 2) (hereinafter abbreviated as DHCH)-alkynine-B
22] - Trypsin was added to insulin in the presence of 0.2M borate (pH 8) and allowed to act at 40°C for 1 to 3 days,
Obtain Compound 1.

Further, according to the method (2), Desu B30-insulin (porcine type) can be obtained from porcine insulin using carboxypebutidase A, for example.

This method is based on E.D. Schmidt (E.
Schmitt et al.
oppe-5eyler's Zeitschrift
fiirPhysiologische Chami
e) Described in Vol. 359, p. 799 (1978). Next, in this pig-derived desu-B30-insulin, the arginine side chain at position 22 of the B chain is modified to prepare compound 11.

The one-threonine derivative (hereinafter referred to as compound ■) shown by the general formula 1 can be synthesized by ordinary chemical methods, but even if the hydroxy group, which is the side chain functional group, is fully protected or unprotected. good. In the case of protection, it is preferable to use protective groups that are commonly used in peptide synthesis reactions. Compound I
It is essential that the carboxyl group of is protected, and a commonly used carboxyne group protecting group is used. For example, 1=
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, please refer to M.
Bodanszky et al.
al, ) peptide synthesis (Peptide 5
Synthesis) 2nd edition (1976) (John
John Wilay & Sons
5ons)).

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 Compound I and Compound II is carried out under conditions suitable for the peptide bond formation reaction of trypsin or trypsin-like enzymes. pH 5 to 8, especially around 6 to 7 is preferred,
The reaction temperature is preferably 0 to 50°C, particularly 20 to 40°C. Preferably, the concentrations of Compound I and Compound II are as high as possible. Furthermore, compound ■ and compound II are 1:1 to 10
It is best to use a molar ratio of 0:1, especially 20:1.
~100; around 1 is good. A suitable organic solvent miscible with water is added to the reaction solution. Addition of an organic solvent is effective not only in lowering 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 compound II. Examples of organic solvents include:
Methanol, ethanol-Av.

Dimethylformamide, dimethylsulfoxide, 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-B 22 .

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, Dengen et al.
ys, ) Vol. 126, p. 971 (1968), Yoshida et al.
) Vol. 15, p. 129 (1971).

The enzyme used is preferably treated with tosyl-L-phenylalanine chloromethyl ketone (hereinafter referred to as IPCK) or the like for the purpose of removing contaminating chymotrypsin or chymotrypsin-like activity.

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 1 mg/m
Around 1 to 10 mg/ml is preferable. The enzyme may be used as is or as an immobilized enzyme bound to or encapsulated in a suitable insoluble carrier.

The reaction time varies depending on the reaction conditions, but it is usually sufficient to allow the time required for the enzyme reaction to reach equilibrium, and is usually 3 to 7 days.
It takes about 2 hours, and in most cases about 6 to 24 hours.

After completion of the reaction, the desired compound III can be collected from the reaction solution using various known separation means. For example, unreacted compounds are removed by gel filtration of the reaction solution! and the enzyme is isolated and recovered.The recovered Compound I and the enzyme can be reused as they are. The remainder contains Compound III and DesuB30-insulin. Compound III can be obtained by lyophilization, but in order to obtain the final target substance-containing insulin, without isolating Compound III, the remainder is subjected to removal treatment of the arginine protecting group, and then subjected to appropriate romatography or Subjected to gel filtration, 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-B2O-insulin) are separated.0 Recovered des-B30 - Insulin can be used as the raw material compound II by modifying the arginine at the 22nd position of Bfi 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, the removal of the DHCH group from the guanidino group is performed using 1M hydroxylamine (pH 7).
This is done by standing overnight at 37°C. 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 other ester, amide, substituted amide, salt, etc., the protecting group can be removed by appropriate hydrolysis treatment or desalting operation.

Compound 111 can be converted into large insulin as described above, and the human insulin obtained is useful as a therapeutic agent for diabetes having hypoglycemic action as described above.

Human insulin obtained from pig insulin has been found to have a hypoglycemic effect equivalent to that of pig insulin in mice.

Large insulin obtained from compound III is formulated and administered to humans in the same manner as commercially available porcine insulin or raw insulin. In other words, by adding zinc chloride etc. to make a stable zinc complex, by adding buffering agents such as sodium hydrogen phosphate or sodium acetate, by adding sodium chloride to make the solution isotonic, and by adding cresol, phenol, roseoxybenzoin, etc. Injections can be prepared using various preparation methods used in commercially available insulin preparations, 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 symptoms of the patient, 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 following Examples, production examples of the compounds of the present invention will be 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-butyl ester Example 1 (1) 500 mg of porcine des-830-inulin porcine insulin was mixed with a 0.1 M ammonium bicarbonate aqueous solution (
5 mg of crystalline carpoquin bebutidase A (manufactured by Wahnton Co., Ltd., treated with diisopropyl fluorophosphate, 49 u 7 mg) was added, and the mixture was allowed to react at room temperature for 8 hours. The amount of alanine produced is 0.77M/IM insulin.

After freeze-drying the reaction product, it was dissolved in 0.5M acetic acid and applied to a Sephadex G50 (ultrafine particle) column (3.5X95c).
m) and elute with 0.5M acetic acid. The 40th to 60th fractions are collected at 11.5 ml per fraction and lyophilized to collect 460 mg of the title compound. Yield 92%.

Amino acid analysis values after hydrolysis with 6N hydrochloric acid at 110°C for 124 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.

Lys 1.00 (1), His 1.91 (2)
, Arg O, 95 (1), Asp 3.21 (3)
, Thr 2.09 (2), Set 2.97 (3
), Glu 7.35 (7), Pro 1.25 (
1), Gly 4.29 (4), Ala 1.26
(1), Vat 3.86 (4), Ila 1.55
(2), Leu 6.53 (6), Tyr 4.0
8 (4), Phe3.22 (3), (2) Pig-derived [
B22-Arg(DHCH)Code-B50-Insulin 200 mg of the above product was dissolved in 0.25 M borate buffer (pH
9) Dissolve 1 in 7 ml of 1,2-cyclohexane.
Add 45 mg of dione and keep at room temperature overnight. After acidifying with glacial acetic acid, a Sephadex G50 (ultrafine grain) column (
6 x 85 cm) and elute with 0.5M acetic acid.

The absorption at 280 nm was measured, and the corresponding portions were collected and lyophilized to obtain 168 mg of the title compound. Yield 84%.

In addition, it was confirmed by high performance liquid chromatography and slab electrophoresis that alkynine was modified with x10% DHCH.

Amino acid analysis value Lys 1.00 (1), His 2.07 (2)
, Arg”0.27 (1), Asp 3.2L (3
), Thr 2.05 (2), Ser 2.98 (
3), Glu 7.19 (7), Pro 1.23
(1), cly 3.91 (4), Ala 1.29
(1), Val 3.99 (4), Ile 1.5
7 (2), Leu 6.57 (6), Tyr 3.
48 (4), Phe 3.16 (3).

During hydrolysis, y′20% of DHCH-Arg is Ar
It is thought that it was decomposed into g (Patthy
) et al., Journal of Biological Chemistry (J, Biol, Chem, ) vol. 250, 55
7 (1975)).

(3) Pig-derived (B22-Ar, g(DHCH), B5
0-Thr-OBu']-insulin (2) product 97, 8n+g (10mM) and L-threonine t-butyl ester acetate 211mg (500
mM) in ethanol/methylformamide F mixture (1:
1, v/v) 1,08 ml of 0.5 M borate buffer containing 4.2 mg of crystalline trypsin (Worthington, recrystallized three times) and TPCK to a final concentration of 0.01 mM. (pH7.5) 0.72ml
Mix and keep at 37°C overnight.

Final pH was 6.5.

After acidifying with glacial acetic acid, gel filtration was performed with a column of Sephadex G50 (ultrafine grains) to determine enzyme activity, 280 nm.
It is divided into trypsin part, insulin part, and threonine derivative part according to the ultraviolet absorbance of
(see figure). The amount of L-threonine t-butyl ester recovered was about 50%. The insulin part was freeze-dried to obtain 87 mg of the title compound.

(4) Pig-derived [B30-1hr-OBu']-insulin 85mB of the product from (3) was added to 1M hydroxyamine (
pH 7) 7, dissolved in 5 ml, purged with nitrogen gas, kept at 28°C overnight, then added to OIM Tris buffer (pH 7).
Dialyze in the cold against & (pH 7,4). Then 7M
Contains 0 O1M Tris buffer (pH 7,4)
Chromatography is carried out at 4° C. on a column of DEAE-Sephadex A25 (1.9×24.5 cm), padded with 300 ml of DEAE-Sephadex A25. After flowing 800ml of the above buffer solution, concentration gradient elution was performed to increase the salt concentration to 0.3M, and
Fraction A around 0.09 Mm degree and 0.13-0.14 M1
Fraction B near the degree is obtained. 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 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 Desu B30-insulin (porcine type) and porcine insulin.

(5) Manufacture of human insulin 2 ml of trifluoroacetic acid containing 0.2 ml of anisole is added to 28 mg of the product from (4) and kept at room temperature for 30 minutes. After removing trifluoroacetic acid in a nitrogen stream, IN acetic acid 2
ml and extract the anisole with 15 ml of ether.

The acetic acid portion was freeze-dried to obtain 26 mg of the title compound. Assuming that the purity of the pig-derived des-B30-insulin used as a food ingredient is 77%, the yield is 38%.

The ice crystals were identified as the title compound based on amino acid analysis, slab gel electrophoresis, and high performance liquid chromatogram.

Amino acid analysis value Lys 1.00 (1), His 1.85 (2)
, Arg 0.93 (1), Asp 3.21
(3), Thr 2.94 (3), Sar
2.96 (3), Glu 7.23 (7), P
ro 1.23 (1), Gly 4.27 (4>,
Alg 1.19 (1), Val 3.89 (4)
, Ila 1.56 (2), Leu 6.57
(6), Tyr 3.78 (4), Phe
3.1g (3).

[Brief explanation of drawings]

Figure 1 shows pig-derived [B 22-Arg (D)IcH);
The flow pattern by column chromatography when separating B50-Thr-OBu']-inulin is shown. Patent applicant: Shionogi & Co., Ltd. Agent: Patent attorney: Ushio 1) Yu “-〇1st Ward A280m(-)

Claims (1)

[Claims] A person who obtains human insulin 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 of manufacturing insulin. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^t represents hydrogen, R^2 represents a protecting group for a carboxyl group, and R^3 represents a protective group for a guanidino group.)