PROCESS FOR PREPARING A B30-THREONINE INSULIN

Abstract: A process for preparing a B30-threonine-insulin is described which comprises reacting a des-B30-insulin with an excess amount of a threonine derivative represented by the formula Thr(R<1>)(R<2>) wherein Thr is L-threonine residue, R<1> is hydrogen or a hydroxy-protecting group; and R<2> is a carboxyl-protecting group, in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide linkages.

Description: Process for Preparing a B30-threonine Insulin Priority: April 13, 1979, Japan, No. 45710/1979 April 13, 1979, Japan, No. 45711/1979 This invention relates to a semi-synthesis of human insulin. More particularly, it consists of an enzymatic synthesis of a B30-threonine-insulin including human insulin. The synthesis is effected to react a des-B30-insulin with an excess amount of a threonine derivative in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide linkages and remove a protecting group of the threonine to yield a B30-threonine insulin. insulin is an indispensable medicament for treatment of diabetes. Bovine and porcine insulins are comme-:- ciallyavailable at present. These insulins are, however, different from human insulin in a few amino acid components, which cause formation of antibodies in patients. These antibodies are- known to decrease the effectiveness of further insulin treatment. Therefore, human insulin is favoured and its commercial availability is desirable Human insulin is different from other animal insulin in the amino acid components at the positions-8, 9, and 10 of the A chain and at the position 30 of the B chain. This invention provides a process to substitute the amino acid at 30 position of the B chain of an animal insulin fort-threonine. Accordingly, human insulin can be easily prepared by the process of this invention using porcine insulin as starting material. Porcine insulin is different from human insulin in the amino acid at B30 position. Thus-obtained human insulin is preferably used for treated ment of diabetic patients. Further, this invention provides other B30-threonine-insulins starting from other animal insulins. The resultant insulins are different the from human insulin intamino acids at 8, 9, and 10 positions the of the A chain and can be used as reagents to test lanti- genicity and medicament for diabetes. Human insulin has been chemically prepared by M.A. Ruttenberg as shown in U.S. Patent No. 3,903,068 and R. Obermeier et al. described in Hoppe-Seyler's Z. Physiol. Chem., 357, 759-767 (1976). These processes com- the priseS condensation of a desoctapeptide-(B23-30)-porcine insulin with a synthetic octapeptide corresponding to positions B23-30 of human insulin. The former process, an however, includeslalkaline hydrolysis which causes adverse side-reactions. The latter process is also a non-specific reaction which brings about many side-reactions and requires complicated purification procedures. Therefore, these processes cannot be applied on an industrial scale. M. Bodanszky et al.describe a process for preparing human insulin in U.S. Patent No. 3,276,961 wherein human insulin is prepared from other animal insulins by an action of an enzyme such as carboxy peptidase A and trypsin in the presence of threonine. This process is not likely to produce human insulin because trypsin and carboxypeptidase A hydrolyze not only the peptide bond of lYsyl-alanine (B29-B30) but also the other positions in insulin under the condition described there. Trypsin preferentially hydrolyzes the peptide bond of arginyl-glycine (B22-B23) than that of lysyl-alanine (B29-B30). . Meanwhile, carboxypeptidase A cannot release the solely the alanine at C-terminal of the 3 chain without the liberating asparagine at C-terminal of the A chain. Special conditio5, i.e. reacting in an ammonium hydrogen carbonate buffer solution,are necessary to prevent the covered release of the asparagine. These reaction conditions were dis- in 1978 (Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978)). Furthermore, peptide synthesis may hardly occur because hydrolysis ratio-is faster than synthesis ratio in the condition. Therefore, it is concluded that there has been no industrially available process for preparing human insulin until nows. This invention, however, can provide human insulin an a commercial scale. The process consists-of anenzymatic condensation of a des-B30-insulin with a threonine derivative. The process is different from any known process and has many advantages. It is a specific reaction not accompanying any adverse side reaction such as racemization. Additionally, unreacted starting mate rials 'can be easily recovered and re the utilized. The process uses an excess amount of1L-threonine derivative. The excess amount of the threonine derivative prevents the,breakage of the carbonyl bond of the arginine at 22 position of the B chain, although the bond is usual ly split by trypsin. Accordingly, the protection of the arginine residue which is indespensable in usual enzymatic reaction is not necessary in this process. The process comprises reacting a des-B30- insulin (noted as Compound II hereinafter) with an excess amount of a L-threonine derivative (noted as Compound I here ;after) in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide linkages and subsequent removal of the protecting groups of the threo nine. Compound I is represented by the following for mula: Thr(R1)(R2) wherein Thr is L-threonine residue; R is hydrogen or a hydroxy-protecting group; and R is a carboxyl-protecting group. Compound II can be prepared by reacting various animal insulins (e.g. pig, cattle, whale, sheep, rabbit, fish, monicey and the like) with carboxypeptidase A as disclosed by E.W. Schmitt et al. in Hoppe-Seyler's Z. Physiol. Chem 359, 799 (1978). It is also prepared by using a protease produced by Achromobacter lyticus. The protease has a specificity to lysine and specifically the splits lysine carbonyl bond in peptide linkages. The isolation and characteristics are described by Masaki et al. in Agric. Biol. Chem., 42, 1443 (1978)'. The enzyme is called as Protease I. The preparation of des-B30- porcine insulin is disclosed in Biochem. Biophys. Res. Commun. 92, 396-402 (1980) and Japanese Patent Publication (Not-examined) 54-135789. Des-B30-insulins of other animals can be prepared in the same manner. The hydroxy group of Compound I may be protected or not. Both compounds can be applicable to the reaction. The protecting group to be used is selected from any hydroxy-protecting group generally used in peptide synthesis such as t-butyl, benzyl, acetyl and the like. The carboxyl' group of Compound I has to be protected and any carboxyl-protecting group utilized in peptide synthesis can be employed. They are, for example, alkyl. esters (e.g. t-butyl eater), aralkyl esters (e.g. benzyl ester), amide, substituted amiss (e.g. anilide), salts (e.g. sodium) and so forth. Protecting group should be chosen considering the influence of the introduction and removal reactions to insulin because the reactions might result in.a denaturation or inactivation of insulin. If a hydroxy-protecting group and a carboxyl-protecting group of the threonine residue are suitably selected, one removal prodedure is enough to remove both the protecting groups. Protecting groups used in peptide synthesis are detailed by M. Bodanszky et al. in Peptide Synthesis, second edition (1976) published by John Wiley & Sons. The enzyme used in the process includes enzymes specifically acting on the basic-amino-acid carbonyl in peptide bondings which are obtainable from various plants and animals. They are, for example,. trypsin, trypsin-like enzymes and the like. Trypsin-like enzymes are illustrated such as the protease isolated by Morihara et al. in Arch. Biochem. Biophys., 126, 971 (1968) and the protease disclosed by Yoshida et al. in FEBS Lett., 15, 129 (1971). The enzyme to be used may be treated with tosyl L-phenylalanine chloromethylketone (abbreviated TPCK. hereinafter) to remove mixed enzymes such as chymotrypsin and chymotrypsin-like enzyme. Furthermore, Protease I produced by Achromobacter lyticus M497-l is a trypsin-like enzyme and included in the enzyme to be used. Pro tease I acts specifically on lysine carbonyl in peptide bondings and the isolation and characteristics are disclosed in Agric. Biol. Chem., 42, 1443 (1978). The condensation of Compound I with Compound II is effected under suitable conditions for the peptide synthesis of the above enzymes. The reaction is prefera- bly effected at about pH 5 to 9, preferably about pH 6 to 7. Reaction temperature is about 0 to 50 C, preferably about 20 to 400C. It is desired that both concentrat-ions of Compound I and Compound II are as high as possible. The preferred-molar - ratio of Compound I to- Compound II is about 5:l to 1000:1, especially about 25:1 to 200:1. Water-miscibie organic solvents are preferably added tothe reaction mixture. The addition of organic solvent iowers the water. concentration of the reaction mixture resulting in prevention of reverse reaction, i.e. hydrolysis of product and also remarkably increases solubility of Compounds I and II. The organic solvents to be used are, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, glycerol and the like. They can be used singly or as a mixture. The preferred concentration of the organic solvent is about 0 to 65 razz especially about 40 to 60 % of the reaction mixture. The ratio of the organic solvent should be determined by the solubility ofvstarting materials,tenden,cy'of ;t:he me to denature and its hydrolyzing activity. Examples of useful reaction media are trishydroxymethylaminomethane (abbreviated tris hereinafter), carbonate and borate buffer solutions. The enzyme concentration is determined depending on concentration of substrates and enzyme activity. For example, crystalline trypsin is used preferably in concentration from about 1 mg/ml to 10 mg/ml. Enzyme may be used intact or in a fixed form such as a combination with or an inclusion in an insoluble carrier such as cellulose, dextran (e.g. Sephadex (trade mark)), agarose (e.g. Sepharose (trade mark)), polyacrylamide gel (Bio-gel (trade mark)), porous glass and the like. The reaction time is variable and affected by other reaction conditions. The reaction may be continued until an equilibrium is reached beteeen substrate and product. It takes generally about 3 to 72 hours and in most cases about 6 to 24 hours. The product insulin may be isolated by the usual methods used in peptide chemistry. An isolation procedure is illustrated as follows: The reaction mixture is applied to gel filtration to isolate and collect unreacted Compound I and enzyme. The recovered Compound I and enzyme can be reutilized. The remaining part is applied to suitable chromatography to isolate a resultant protected-insulin and an unreacted des-B30-insulin. The latter can be reused as Compound II. The former is subjected to reaction for removal of the protecting group. The removal of the protecting groups 'is effected in - the usual manner, though the method should be chosen depending on the properties of the protecting group. The t-Butyl group is exemplified as a protecting group for both the hydroxy and carboxyl group of threonine. It may be removed by treating with trifluoroacetic acid in the presence of a cation-trapping agent, e.g. anisole. As noted above, if a single treatment may remove the hydroxy'protecting and the carboxyl-protecting groups at once, the process becomes simple and results in good yield. lfihen the C-terminal of the resultant insulin is protected with other ester, amide, substituted amide or salt, the protecting group may be removed by suitable hydrolysis or desalting technique. The insulin prepared by this invention is useful for treatment of diabetes and also as a reagent. The insulin obtained by this invention shows the same activity of lowering bloodsugar level as bovine insulin in mice as-follows: Test Method: Test compound is dissolved in 0.01 M hydrochloric acid and the solution is diluted to a suitable concentration (2.5 - 20 Fg/ml) with 20 to 60 times of physiological salt solution. The resultant solution is intravenously administered in a dose of 0.1 ml/10 g body weight to DS mice fasted for 5 hours. Blood is taken (i.e. the eye socket sinus) from crbital blood vessel/45 minutes after the administration and blood sugar is measured by glucose-oxidase method using a commercial kit produced by Boehringer Mannheim Corporation. Table 1 Insulin Activity EMI11.1 <tb> Test <SEP> Dose <SEP> Blood <SEP> Glucose <SEP> Level <tb> Compound <SEP> ( <SEP> g/10g <SEP> Body <SEP> Weight) <SEP> (mg/dl) <SEP> <tb> Pbysiological <tb> <SEP> - <SEP> 112.2 <SEP> 7.3 <SEP> (1) <tb> Salt <SEP> Solution <tb> Semi-synthetic <SEP> 0,25 <SEP> 80.2 <SEP> 2.6 <SEP> (2) <tb> Human <SEP> Insulin <SEP> 1 <SEP> 41.0 <SEP> 6.0 <SEP> (3) <tb> Bovine <SEP> Insulin <SEP> 0.25 <SEP> 73.2 <SEP> <SEP> 1.5 <SEP> (4) <tb> <SEP> l <SEP> 1 <SEP> <SEP> 41.0 <SEP> + <SEP> 2.3 <SEP> (5) <tb> Note: (2)-(4), (3)-(5) No significant difference Human insulin prepared by this invention can be administered to human beings in the same manner as porcine or bovine insulin preparations. It may be converted to conventional pharmaceutical preparations. For example, it may be made into an injectable solution by suitable procedure such as those for preparing zinc complex with zinc chloride, a buffered solution with a suitable buffer such as sodium hydrogenphosphate, sodium acetate and the like, and an isotonic solution with sodium chloride. Furthermore, suitable antiseptics may be added thereto. They are, for example, cresol, phenol, para-hydroxybenzoic acid alkyl esters (e.g. methyl, ethyl, propyl, butyl esters and the like). The dosage of the human insulin is the same as conventional insulin preparations. Namely, about 1 to 100 units may be administered to a human adult per day, though the dosage depends on the severity of diabetes. The following-examples are given to further illustrate and described the present invention. Meanwhile, each abbreviation has the meaning as follows: Ala: alanine, Arg: arginine, Asp: aspartic acid, CysO3H: cysteic acid, Glu: glutamic acid, Gly: glycine, His: histidine, Ile: isoleucine, Leu: leucine, Lys: lysine, Phe: phenylalanine, Pro: proline, Ser: serine, Thr: threonine, Tyr: tyrosine, Val: valine, OBut: t-butyl ester-residue. Example 1 (1) Desalanine-(B30)-porcine insulin To a solution of porcine insulin (500 mg) in 0.1 M ammonium hydrogencarbonate (100 ml, pH 8.3) is added crystalline carboxypeptidase A (Smg, Worthington Co., pretreated with diisopropylfluorophosphate, 49 u/mg). The mixture is incubated at room temperature for 8 hours. The incubation is stopped when alanine is released at a ratio of 0.77 M/lM insulin. The reaction mixture is lyophilized. The product is dissolved in 0.5 M acetic acid and adsorbed on a column (305 x 95 cm) filled with super fine particles of Sephadex G 50. The column is eluted with 0.5 M acetic acid. Fractions of 11.5 ml are collected. Fractions Nos. 40-60 are pooled and lyophilized to give the title compound (460 mg). Yield is 92 djo The product is hydrolyzed for amino acid analysis with 6 M hydrochloric acid at 1100C for 24 hours to give the following result. The figures in the parenthesis show the theoretical values throughout this specification. Amino acid analysis: Lys 1.00 (1), His 1.91 (2), Arg 0.95 (1), Asp 3.21 (3), Thr 2.09 (2), Ser 2.97 (3),- Glu 7.35 (-7), Gly 4.29 (4), Ala 1.26 (1), CysO3H 5.94 (6), Val 3.86 (4), Ile 1.55 (2), leu 6.53 (6), Tyr 4.08 '(4), Phe 3.22 (3), Pro 1.2 (l). (2) [B30-Thr-OBu ]-porcine insulin The above product (100 mg, 10 mM) and L-threonine t-butyl ester (205 mg, 500 SI) are dissolved in a mixture (1.05 ml) of ethanol and dimethylformamide (1:1,, v/v). To the solution is added a solution of crystalline trypsin (4 mg, Worthington Co., recrystallized three times) in 0.5 M borate buffer solution (0.75 ml, pH 6.5) and TPCK in the final concentration of 0.01 iI. The mixture is kept overnight at 37C. The product is confirmed with high pressure liquid chromatography and the calculated yield is 75 6. The mixture is acidified with galcial acetic acid and applied to gel filtration with a column of Sephadex & ommat; G 50 50 (4.2 x 130 cm) of super fine particles to give three fractions corresponding to trypsin, the titled insulin derivative and L-threonine t-butyl ester. Measurements of enzyme activity and Ninhydrin reaction reveals that trypsin and L-threonine t-butyl ester are recovered 50 dp, respectively. The fraction containing the insulin derivative is lyophilized to yield a crude powder (89 mg) of the title compound. The product is applied at 40C to a column of DE4E-Sephades A 25 (1.9 x 24.5 cm) which has been previously equilibrated with 0.01 M tris buffer solution (pH 7.6) and 7 M urea. The column is eluted with the above buffer solution (S00 ml) and then a linear Na gradient (to 0.3 M NaCl) is performed to give a fraction A near to 0.08 to O.Og M concentration and a fraction B near to 0.13 to 0.14 M. Each fraction is immediately dialyzed against 0.01 M ammonium acetate for 3 to 4 days under cooling and lyophilized to give a powder (35 mg) from the fraction A and a powder (27 mg) from the fraction B. The former is identified to be the title compound and the latter is identified to be a mixture of des alanine-(B30)-porcine insulin and intact porcine insulin by high precious liquid chromatography and polyacrylamide gel electrophoresis. (3) Human insulin A mixture of trifluoroacetic acid (2 ml) and anisole (0.2 ml) is added to the product (30 mg) obtained above and the mixture is kept at room temperature for 30 minutes. The trifluoroacetic acid is removed under nitrogen atmosphere and the mixture is extracted with ether (15 ml) after addition of lN acetic acid (2 ml) to remove the anisole. The acetic acid layer is lyophilized to give the title compound (28 mg) in 77 % purity. The yield is 43 % based on the starting compound. The product is identified to be the title compound by amino acid analysis, Slab gel electrophoresis and high pressure liquid chromatography. Amino acid analysis is performed under the same condition as in (l) and the result is as follows: Amino acid analysis: Lys 1.00 (1), His 1.89 (2), Arg 0.87 (l), Asp 3.32 (3), Thr 3.16 (3), Ser 2.99 (3), Glu 7.35 (7), Pro 1.29 (1), Gly 4.39 (4), Ala 1.26 (l), CysO3H 4.6 (6), Val 3.93 (4), Ile 1.61 (2), Leu 6.51 (6), Tyr 3.68 (4), Phe 3.19 (3). Example 2 (1) Desalanine-(B30)-bovine insulin To a solution of bovine insulin (200 mg) in 0.1 M ammonium hydgogencarbonate (40 ml) is added crystalline carboxypeptidase A (1.8 mg, 49 u/mg). The mixture is kept overnight at room temperature and lyophilized to give a crude powder (180 mg) of the - title compound. The product is assured to consist of 78 % of the title. compound and 22 dp of desasparagine-(A21)-desalanine-(B30) insulin. (2) [B30-Thr-OBut]-bovine insulin The product (116 mg, 5 miM) obtained above (l) and L-threonine t-butyl ester (448 mg, 500 mM) are dissolved in a mixture (2.4 ml) of ethanol and dimethylformamide (l:l). The solution is mixed with 0.5 M borate buffer solution (1.6 ml, pH 7.0) containing crystalline trypsin (87.5 mg, 0.94 mM) and TPCK (0.27 mg, 0.2 mkfJ and incubated overnight at 370C. The production of the title compound is confirmed by high pressure liquid chromatography and the yield is calculated to 80 dp. The reaction mixture is treated in the same manner as in Example 1 (2). The resultant fraction A is rechromatographed to remove desasparagine-(A21)-desaanine- (B30)-insulin and treated in the same manner as in Example 1 (2) to give a crude powder (63 mg). The product is identified to be the title compound by high pressure liquid chromatography and Slab gel electrophoresis. (3) [B30-Thr]-bovine insulin To the product (63 mg) of the above (2) is added mixture of trifluoroacetic acid (2 ml) and anisole (0.2 ml)., The mixture is kept at room temperature for 30 minutes, then treated in the same manner as in Example 1 (3) and lyophi lized to give the title compound (60 mg) in 78 % purity. The yield is 66 % based on the starting compound. The product is identified with [B30-Thr]-bovine insulin prepared by other method buy amino acid analysis, Slab gel electrophoresis and high pressure liquid chromato graphy. Amino acid analysis is performed under the same condition as in Example 1 (1) and the result is as follows: Amino acid analysis: Lys 1.00 (1), His 1.95 (2), Arg 0.98 (1), Asp 3.00 (3), Thr 2.00(2), Ser 2.83 (3), Glu 6.69 (7), Gly 4.o6 (4), Ala 2.06 (2), CysO3H 5.38 (6), Val 4.45 (5), Ile 0.76 (1), Leu 6.14 (6), Tyr 3.92 (4), Phe 2.97 (3), Pro 1.20 (1). Example 3 ( B30-Thr-OBu ]-porcine insulin Desalanine-(B30)-porcine insulin (100 mg, 10 mM) prepared by the process in Example 1 (l) and L-threonine t-butyl ester (205 mg, 500 mM) are dissolved in a mixture (1..05 ml) of ethanol and dimethylformamide (1:1, v/v) The solution is mixed with a 0.5 M borate buffer solution (0.7 ml) of Protease I (0.35 mg) produced by Achromobacter lvticus M497-l and incubated at 370C for 2 days. The reaction mixture is acidified with glacial acetic acid and applied to gel filtration with a column (4.2 x 130 cm) filled with super fine particles of Sephadex G 50 to separate into an enzyme fraction, an insulin fraction and a threonine fraction. The enzyme and L-threonine t-butyl ester are recovered in an amount of more than-50 %. The insulin fraction is lyophilized to give a crude powder (83 mg). The powder is applied to column chromatography in the same manner as in Example 1 (2) to give a powder (48 mg) from the fraction A and another powder (20 mg) from the fraction B. The former is identified to be the title compound and the latter is identified to be a mixture of desalanine-(330) porcine insulin and a contaminated porcine insulin. (2) Human insulin The product (40 mg) obtained above is-treated with trifluoroacetic acid (2 ml) and anisole (0.2 ml). The mixture is kept at room temperature for 30 minutes and then treated in tbe same manner as in Example 1 (3) to give the title compound (37 mg) in 76 % purity. The yield is 56 % based on the starting material. The product is identified to be the title compound by amino acid analysis, Slab gel elec-trophoresis and high pressure liquid chromatography. Amino acid analy sis is performed under the same condition as in Example 1 (1) and the result is as follows: Amino acid analysis: Lys 1.00 (l), His 1.90 (2), Arg 0.93 (1), Asp 3.21 (3), Thr 3.05 (3), Ser 2.99 (3), Glu 7.23 (7), Pro 1.25 (1), Gly 4.33 (4), Ala 1.25 (1), Val 3.91 (4), Iie 1.58 (2), Leu 6.51 (6), Tyr 3.66 (4), Phe 3.15 (3).

Claims: that is claimed is: 1. Process for preparing a B30-threonine-insulin which comprises reacting a des-B30-insulin with an excess amount of threonine derivative respresented by the formula 1 2 Thr(R )(R ) wherein Thr is L-threonine residue, R1 is hydrogen or a hydroxy-protecting group; and R2is a carboxyl-protecting group in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide linkages. 2. The process according to claim 1, wherein said enzyme is trypsin or a trypsin-iike enzyme. 3.. The process according to claim 2, wherein said-enzyme is trypsin. 4. The process according to claim 2, wherein said trypsinlike enzyme is Protease I. 5. The process according to claim 1, wherein the solar ratio of the L-threonine derivative to the des-B30-insulin is 5:1 to 1000:1. 6. The process according to claim 5, wherein the molar ratio is 25:1 to 200:1. 7. The process according to claim I, wherein the reaction is effected it medium containing one or more organic solvents. 8. The process according to claim 7, wherein the organic solvent is selected from methanol, ethanol, dimethylformamide, dimethyl sulfoxide and glycerol. 9. The process according to claim 8, wherein the organic solvent is selected from ethanol and dimethylformamide. 10. The process according to claim 1, wherein the des-B30insulin is des-B30-porcine-nsulin, 11. The process according to claim 1, wherein the des-B30insulin is des-B30-bovine-insulin. 12. The process according to claim 1, wherein the threonine derivative is L-threonine t-butyl ester. 13. The process according to claim 1, wherein the reaction is effected at pH 5 to pH 9. 14. The process according to claim 13, wherein the reaction is effected at pH 6 to pH 7. 15. The process according to claim 1, wherein the reaction is effected at a temperature of O to 50 OC. 16. The process according to claim 15, wherein the reaction is effected at a temperature of 20 to 400C.

Inventors: MASAKI, TAKEHARU, MORIHARA, KAZUYUKI, OKA, TATSUSHI, SOEJIMA, MASAMI, TSUZUKI, HIROSHIGE
Applicants: SHIONOGI & CO., LTD.
Application year: 1980
Main classification: C12P21
IPC codes: C07K14