CH655949A5 - METHOD FOR PRODUCING A THREONIN (B30) ESTER OF HUMANINSULIN, A SALT THEREOF OR A METAL COMPLEX THE SAME, AND THESE ESTER AND USE THEREOF. - Google Patents

Description

The invention relates to a process for the preparation of a threonine B30 ester of human insulin of the general formula III

(Thr (R5) -0 R4) B3 (1-h-I n (III)

wherein R4 is a carboxyl protecting group, R5 is hydrogen or a hydroxyl protecting group and h-In is the Des (ThrB30) -Hu-maninsulin unit, or a salt or a metal complex thereof.

Insulin compounds made from swine or bovine insulin are commonly used in the treatment of diabetes mellitus.

Pig, bovine and human insulins show slight differences in their amino acid composition, the difference between human and pig insulin being limited to a single amino acid in that the B30 amino acid of human insulin is threonine, while that of pig insulin is alanine. It can therefore be argued that the ideal insulin preparation for the treatment of humans is an insulin with exactly the same chemical structure as that of human insulin.

The necessary amount of human pancreas glands is not available for the production of human, natural insulin.

Synthetic human insulin has been produced very expensive on a small scale, see Helv. Chim. Acta, 57, p. 2617, and 60, p. 27. Semi-synthetic human insulin has been prepared from porcine insulin via time-consuming pathways as described in Hoppe-Seyler's Journal of Physiological Chemistry, 357, 759 and Nature 280,412.

The US-PS 32 76 961 relates to a method for the production of semi-synthetic human insulin. Nevertheless, the yield of human insulin is poor because the process is carried out in water and under conditions in which trypsin cleaves the ArgB22-GlyB23 bond, see Journal of Biological Chemistry, 236, p. 743.

A known semisynthetic process for the production of human insulin has the following three steps: First, pig insulin is converted into pig des (AlaB30) insulin by treatment with carboxypeptidase A, see Hoppe-Seyler's Zeitschrift für Physiologische Chemie, 359, p. 799 . In the second step, porcine des (AlaB30) insulin is subjected to trypsin-catalyzed coupling with Thr-OBu ', whereby the ThrB30 tert-butyl ester of human insulin is formed. Finally, this ester is treated with trifluoroacetic acid to provide human insulin, see Nature 280, p. 412. The first step provides partial cleavage of AsnA21, which provides des- (AlaB30, -AsnA21) insulin. After the two subsequent reactions, this descendant leads to contamination of des- (AsnA2l) -insulin in the semisynthetic human insulin produced, which cannot be easily removed using the known preparation method. Des- (AsnA2l) insulin has low biological activity (about 5%), see American Journal of Medicine 40, p. 750.

The object of the invention is therefore to provide a method with which human insulin can be produced on an industrial scale, wherein non-human insulin is to be converted into human insulin. Furthermore, a method is to be delivered which converts pig insulin and certain impurities contained therein into human insulin via a threonin B30 ester of human insulin.

The object is achieved by a method for producing threonine B30 esters of human insulin, which is characterized by the combination of the features specified in claim 1. Advantageous further developments of the method as well as a result of the method and the use of this result emerge from the dependent claims.

5

10th

15

20th

25th

30th

35

40

45

50

55

office

b5

655 949

4th

The method according to the invention thus has the following advantages over the prior art:

a) The enzymatic hydrolysis to split off AlaB3 °, for example with carboxypeptidase A, is omitted.

b) Isolation of an interconnect, such as porcine des (AlaB30) insulin, is unnecessary.

c) Contamination with des (AsnA2l) insulin derivatives is avoided.

d) Proinsulin and other insulin-like impurities which are present in the crude insulin are converted into human insulin by the process according to the invention via the threonine B30 ester of human insulin, as a result of which the yield is increased.

e) Antigen-like insulin-like compounds, see GB-PS 12 85 023, are converted into human insulin.

Further features and advantages of the invention result from the claims and from the following description in which exemplary embodiments are explained.

The invention is based on the discovery that the amino acid or peptide chain bound to the carbonyl group of LysB29 in the insulin compound can be exchanged with a threonine ester. This exchange is referred to herein as transpeptidation.

The term "insulin compounds" used herein includes insulins and insulin-like compounds which contain the human des (ThrB30 insulin unit, the B30 amino acid of the insulin being alanine (in insulin from, for example, porcine, dog and fin and sperm whale) or serine The term "insulin-like compounds," as used herein, includes proinsulin derived from any of the above species and primates, along with intermediates from the conversion of proinsulin to insulin. As examples of such intermediates cleaved proinsulin, desdipeptide-proinsulins, desnona-peptide-proinsulin and diarginine insulins can also be mentioned, see also R. Chance «In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292 to 305, publisher: RR Rodrigues & JV -Owen, Excerpta Medica, Amsterdam.

The method according to the invention comprises the transpeptidation of an insulin compound or a salt or a complex thereof with an L-threonine ester or a salt thereof in a mixture of water, a water-miscible organic solvent, and trypsin, the water content in the reaction mixture being less than is about 50% by volume and the reaction temperature is below about 50 ° C.

Although trypsin is best known for its proteolytic properties, those skilled in the art have recognized that trypsin is capable of catalyzing the coupling of des (AlaB30) insulin and a threonine tert-butyl ester, vide Nature 280, p. 412. Bei In the method according to the invention, the trypsin is used to catalyze the transpeptidation reaction.

The transpeptidation can be carried out by dissolving 1. the insulin compound, 2. an L-threonine ester and 3. trypsin in a mixture of water and at least one water-miscible organic solvent, if desired in the presence of an acid.

Preferred water-miscible organic solvents are polar solvents. As specific examples of such solvents, methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, N, N-dimethylformamide, formamide, N, N-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphoric triamide and acetoni- be called tril.

Depending on the water-miscible organic solvent used, the reaction temperature selected, and whether an acid is present in the reaction mixture, the water content in the reaction mixture should be less than about 50% by volume, preferably less than about 40% by volume. -%, and more than about 10 vol .-%.

An advantage of reducing the amount of water in the reaction mixture is that it reduces the formation of by-products. Similarly, by increasing the amount of acid in the reaction mixture, it is possible to reduce the by-product formation. The increase in yield is positively correlated with a high percentage of organic solvents.

The type of trypsin is not essential to the practice of the invention. Trypsin is a well characterized enzyme that is commercially available in high purity, namely from pigs and cattle. Trypsin of microbial origin can also be used. The trypsin form, for example crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (as long as the trypsin activity is retained) is not critical to the practice of the invention. The term "trypsin" as used herein is intended to include trypsins from all sources and all forms of trypsin which have transpeptidizing activity, including proteases with trypsin-like specificity, for example Achromobacter lyticus protease, see Agric. Biol. Chem. 42, p. 1443.

Examples of active trypsin derivatives include acetylated trypsin, succinylated trypsin, glutaraldehyde-treated trypsin and immobilized trypsin derivatives.

If an immobilized trypsin is used, it is suspended in the medium.

Organic or inorganic acids such as hydrochloric acid, formic acid, acetic acid, propionic acid and butyric acid or bases such as pyridine, TRIS (tris-hydroxymethylaminomethane), N-methylmorpholine and N-ethylmorpholine can be added to bring about a suitable buffer system. Organic acids are preferred. However, the reaction can also be carried out without such additives. The amount of acid added is usually less than 10 equivalents per equivalent of L-threonine ester. The amount of acid is preferably between 0.5 and 5 equivalents per equivalent of L-threonine ester. Ions that stabilize trypsin, such as calcium ions, can be added.

The process can be carried out in a temperature range from + 50 ° C. to the solidification point of the reaction mixture. Enzymatic reactions with trypsin are usually carried out at around 37 ° C to ensure a sufficient reaction rate. Nevertheless, in order to avoid inactivation of the trypsin, it is advantageous to carry out the method according to the invention at a temperature below the ambient temperature. In practice, reaction temperatures above about 0 ° C. are preferred.

The reaction time is usually between a few hours and several days, depending on the reaction temperature, the amount of trypsin added and other reaction conditions.

The weight ratio between trypsin and the insulin compound in the reaction mixture is usually above about 1: 200, preferably above about 1:50, and below about 1: 1.

The L-threonine esters intended for the practice of the invention can be represented by the general formula

5

10th

15

20th

25th

30th

35

40

45

50

55

fcu b5

5

655 949

Thr (R5) -OR4 II

where R4 is a carboxyl protecting group and R5 is hydrogen or a hydroxyl protecting group.

The threonine B30 esters of human insulin that are produced during transpeptidation can be represented by the general formula

(Thr (R5) -OR4) B30-h-In, III

where h-In represents the human des-ThrB30) insulin unit and R4 and R5 are the same radicals as defined above.

Usable L-threonine esters of the formula II are those in which R4 is a carboxyl protecting group which can be split off from the threo-ninB30 ester of human insulin (formula III) under conditions which do not cause any essential irreversible conversion in the insulin molecule . Examples of such carboxyl protective groups are lower alkyl groups, for example methyl, ethyl and tert-butyl, substituted benzyl such as p-methoxybenzyl, 2,4,6-trimethylbenzyl and diphenylmethyl, and groups of the general formula -CH2CH2SO2R6, where R6 is one means lower alkyl radical such as methyl, ethyl, propyl and n-butyl. Suitable hydroxyl protecting groups R5 are those which can be split off from the threonine B30 ester of human insulin (formula III) under conditions which do not cause any significant irreversible conversions in the insulin molecule. An example of such a group R5 is tert-butyl.

Lower alkyl groups contain less than 7 carbon atoms, preferably less than 5 carbon atoms.

Further protective groups that are usually used have been described by Wünsch in: Methods of Organic Chemistry (Houben-Weyl), Volume XV / 1, Editor Eugen Müller, Georg Thieme Verlag, Stuttgart 1974.

Some L-threonine esters (formula II) are known compounds and the remaining L-threonine esters (formula II) can be prepared analogously to the preparation of known compounds.

The L-threonine esters of the formula II can be free bases or salts of suitable organic or inorganic acids thereof, preferably acetates, propionates, butyrates and salts of hydrogen hologues such as hydrochlorides.

It is desirable to use the reactants, namely the insulin compound and the L-threonine ester (Formula II), in high concentrations. A molar ratio above about 5: 1 is preferably used between the L-threonine ester and the insulin compound.

It is desirable that the concentration of L-threonine ester (Formula II) in the reaction mixture be above 0.1 molar.

Human insulin can be obtained from threonine B30 esters of human insulin (Formula III) by deprotection R4 and any protective group R5 by known methods or methods known per se. In the event that R4 is methyl, ethyl or a group -CH2CH2S02R6, where R6 is defined as described above, the protective group can be split off under slightly basic conditions in the aqueous medium, preferably at a pH of about 8 to 12, for example at about 9.5. Ammonia, triethylamine or alkali metal hydroxides, such as sodium hydroxide, can be used as the base. If R4 is tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, the said group can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoroacetic acid can be anhydrous or contain some water or with an organic

Solvents such as dichloromethane must be diluted. If R5 is tert-butyl, the group can be cleaved off by acidolysis as described above.

Preferred threonine B30 esters of human insulin of formula III are compounds in which R5 is hydrogen; these are prepared from the L-threonine esters of formula II in which R5 is hydrogen.

The transpeptidation of the insulin compounds in threo-ninB30 ester of human insulin is described in more detail based on the following formula given to the insulin compounds:

R1— (1 — t-AI 21)

15 (1-l-Bl-29) -R2,

where 1

- (1 — tA-T — 21)

20 (1--29) -

human des (threonine B30) insulin unit, wherein GlyAI is linked to the substituent designated R1 and 25 LysB29 is linked to the substituent referred to as R2, where R2 represents an amino acid or peptide chain that does not exceed 36 amino acids contains and R1 represents hydrogen or a group of the general formula R3-X-where X is arginine or lysine and R3 30 represents a peptide chain which contains no more than 35 amino acids, or R2 together with R3 represent a peptide chain which no longer contains than 35 amino acids, provided that the number of amino acids present in R1 plus R2 is less than 37. 35 The transpeptidation according to this invention accordingly converts any of the above insulin compounds into threonine B30 esters of human insulin (Formula III), which are then unblocked to form human insulin.

Another advantage of the invention is that the insulin-like compounds present in crude insulin, which are present in some commercially available insulin preparations and are covered by Formula 1, are converted into threo-45 ninB30 esters of human insulin by the transpeptidation according to the invention, which can then be unblocked to produce human insulin. Examples of insulin-like compounds of the formula I result from the following: porcine arginine insulin (R1 is hydrogen and R2 is thus -Ala-Arg-Arg), porcine proinsulin (R3 together with R2 is -Ala-Arg-Arg-Glu-Ala-Glu -Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys -, where the terminal alanyl is connected to LysB29), dog proinsulin (R3 together with R2 is 55 -Ala-Arg-Arg-Asp-Val-Glu-Leu-Ala-Gly-AIa-Pro-Gly-Glu-Gly-Gly -Leu-Gin-Pro-Leu-Ala-Leu-Glu-Gly-Ala-Leu-Gln-Lys-, where the terminal alanyl is linked to LysB29), split pig proinsulin (R3 is -Ala-Leu-Glu-Gly- Pro-Pro-Gln-Lys-, and R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-60 Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly- Leu-Gly-Gly-Leu-Gln-Ala-Leu), desdipeptide-proinsulin from pigs (R1 is hydrogen and R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly- Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln), 65 human Proi nsulin (R3 together with R2 is -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala- Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, the terminal threo-

655 949

6

nyl is connected to LysB29) and monkey proinsulin (R3 together with R2 is -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-GIn-Val-Glu-Leu-Gly-Gly-Gly -Pro-Gly-AIa-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, with the terminal threonyl linked to Lys829).

In all these impurities covered by the formula I, the R1 substituent designated R3-X- is exchanged with hydrogen.

A preferred embodiment of the invention comprises reacting raw swine insulin containing insulin-like compounds or a salt or a complex thereof with an L-threonine ester (formula II) or a salt thereof under the above conditions, after which the protecting group R4 and any protecting group R5 can be split off. This process converts the swine insulin, along with insulin-like compounds therein, into human insulin.

A zinc complex or a zinc salt can be mentioned as an example of a metal complex or a salt of an insulin compound (formula I).

If the reaction conditions are selected according to the explanations above and the results obtained in the following examples are taken into account, it is possible to achieve a yield of more than 60% threonine B30 ester of human insulin, and even one which is higher than 80 %, and under particularly preferred conditions one of more than 90%.

A preferred method of making human insulin is as follows:

1. The starting material for the transpeptidation is raw porcine insulin, for example crystalline insulin, which was produced by using a citrate buffer, see US Pat. No. 2,626,228.

2. If some trypsin activity remains after the transpeptidation, this is preferably removed under conditions under which trypsin is not active, for example in an acidic medium with a pH below 3. Trypsin can be removed by molecular weight separation methods, for example by gel filtration on Sephadex G-50 or Bio-gel P-30 in 1 M acetic acid, see Nature 280, p. 412.

3. Other impurities, such as unreacted porcine insulin, can be removed by anion or cation exchange chromatography, see Examples 1 and 2.

4. The threonine B30 ester of human insulin is then unblocked and the human insulin is isolated in a manner known per se, for example by crystallization.

By this method, insulin can be obtained of acceptable pharmaceutical purity and, if desired, subjected to further purification.

Furthermore, the invention also relates to novel threonine B30 esters of human insulin, in which the ester unit is different from tert-butyl and methyl.

The abbreviations used here correspond to the rules recommended by the IUPAC-IUB Commission in Biochemical Nomenclature, 1974, see Collected Tentative Rules & Recommendations of the Commission on Biochemical Nomenclature IUPAC-IUB, 2nd ed., Maryland, 1975.

Analytical investigations:

The conversion of porcine insulin and porcine proinsulin to human insulin esters can be demonstrated by DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.06 M HCl and 8 M urea. The pH of the buffer is 8.7.

Formula III esters migrate at 75% the rate of swine insulin. Pig proinsulin migrates at 55% the speed of pig insulin and is converted into the same product by the process according to the invention. The conversion products are identified as compounds of the formula III on the basis of the following criteria:

a) The electrophoretic migration of the human insulin esters of the formula III, based on pig insulin, corresponds to the loss of a negative charge.

b) The amino acid composition of the colored protein bands in the gel, which represent the compounds of formula III, is identical to that of human insulin, namely 3 moles of threonine and 1 mole of alanine per mole of insulin, and the composition of the pig insulin is 2 moles of threonine and 2 moles of alanine per mole of insulin. The technique of amino acid analysis of protein bands in polyacrylamide gels is described in Eur. J. Biochem. 25, 147.

c) Evidence that the incorporated threonine is bound as a C-terminal amino acid in the B chain is provided by oxidative sulfitolysis of the sulfur bridges of the insulin in 6 M guanidinium hydrochloride, followed by separation of the A and B chains by ion Exchange chromatography performed on SP Sephadex. Digestion of the B-chain S-sulfonate with carboxypeptidase A only releases the C-terminal amino acid. This technique is described by Markussen in Proceedings of the Symposium on Proinsulin, Insulin and C-peptide, Tokushima, July 12-14, 1978 (Editor: Baba, Kaneko & Yaniahara) Int. Congress Series No. 468, Excerpta Medica, Amsterdam-Oxford. The analysis is carried out after the ester group has been cleaved from the compounds of the formula III.

These three analyzes clearly show that conversion to human insulin has taken place.

The conversion of porcine insulin and porcine proinsulin to human insulin esters can be followed quantitatively by reverse phase HPCL (high pressure liquid chromatography). A 4 x 300 mm u Bondapak Cig column (Waters Ass.) Was used and the elution with a 0.2 M ammonium sulphate (adjusted to pH 3.5 with sulfuric acid) and 26 to 50 vol .-% acetonitrile having buffer performed. The optimal acetonitrile concentration depends on which ester of formula III you want to separate from porcine insulin. In the event that R4 is methyl and R5 is hydrogen, the separation is achieved in 26% by volume of acetonitrile. Pig insulin and (Thr-Ome) B30-h-In (Me is methyl) eluted after 4.5 and 5.9 column volumes, respectively, as well separated, symmetrical peaks. Before applying to the HPLC column, the proteins in the reaction mixture were precipitated by adding 10 parts by volume of acetone. The precipitate was isolated by centrifugation, vacuum dried and dissolved in 0.02 M sulfuric acid.

The process for the production of human insulin esters and human insulin is explained in more detail by the following examples, which should in no way be regarded as a limitation. The examples show some preferred exemplary embodiments of the method according to the invention.

example 1

200 mg of raw pig insulin, once crystallized, was dissolved in 1.8 ml of 3.33 M acetic acid. 2 ml of a 2 M solution of Thr-OMe (Me is methyl) in N, N-dimethylacetamide and 20 mg of trypsin, dissolved in 0.2 ml of water, were added. After standing at 37 ° C for 18 hours, the proteins were precipitated to add 40 ml of acetone and the precipitate was isolated by centrifugation. The supernatant was

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

7

655 949

throw. Analysis of the precipitate by HPLC using 26% acetonitrile (see “Analytical Investigations”) shows a 60% conversion of the pig insulin to (Thr-OMe) B30-h-In. The precipitate was dissolved in 8 ml of freshly deionized 8 molar urea solution, the pH was adjusted to 8.0 with 1 molar ammonia solution, and the solution was placed on a 2.5 x 25 cm column packed with QAE A-25 Sephadex with a 0 , 1 molar ammonium chloride buffer equilibrated, which contained 60 vol .-% ethanol and whose pH had been adjusted with ammonia of 8.0, applied. Elution was carried out with the same buffer and 15 ml fractions were collected. (Thr-OMe) B30-h-In was found in fractions 26 to 46, unreacted porcine insulin in fractions 90 to 120. Fractions No. 26 to 46 were combined, the ethanol was evaporated in vacuo and (Thr-OMe) B30-h-In crystallized in a citrate buffer as described by Schlichtkrüll et al., Manual of Internal Medicine 7 / 2A, 96, Berlin, Heidelberg, New York, 1975. The yield was 95 mg of crystals with the same rhombic shape as pig insulin crystallized in the same way. The amino acid composition was found to be identical to that of human insulin. Further analytical tests, which are described in the section "Analytical tests" above, showed that the resulting product (Thr-OMe) was B30-h-In.

Example 2

100 mg of pig insulin, which met the purity requirements of GB-PS 12 85 023, were dissolved in 0.9 ml of 3.33 molar acetic acid and then 1 ml of a 2 molar solution of threonine methyl ester in N, N-dimethyl-formamide and 12 mg with TPCK Trypsin (tosylphenylalanine chloromethyl ketone) treated, dissolved in 0.1 ml of water, was added. After an incubation period of 24 hours at 37 ° C., the reaction was terminated by adding 4 ml of 1 molar phosphoric acid. The (Thr-OMe) B30-h-In obtained was recovered from the unreacted porcine insulin by ion exchange chromatography on a 2.5 x 25 cm column with SP-Sephadex using an eluent containing 0.09 M sodium chloride and 0.02 M Sodium dihydrogen phosphate (pH of the buffer: 5.5) in 60% ethanol, separated. Fractions containing (Thr-OMe) B30-h-In were combined, the ethanol removed in vacuo and the product crystallized as in Example 1. The yield was 50 mg (Thr-OMe) B3ü-h-In.

Example 3

100 mg of porcine proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid, converted into (Thr-OMe) B30-h-In and purified as described for porcine insulin in Example 1. The conversion of proinsulin to (Thr-OMe) B30-h-In was determined by HPLC analysis of the acetone precipitation to be 73%. The yield of crystalline (Thr-OMe) B30-h-In was 54 mg.

Example 4

100 mg of pig insulin are dissolved in 0.9 ml of 2.77 molar acetic acid in water and reacted analogously to the process described in Example 2. After the reaction had ended, the proteins were precipitated by adding 10 parts by volume of acetone. Analysis by DISC PAGE electrophoresis shows a 70% conversion to (Thr-OMe) B30-h-In.

Example 5

100 mg of pig insulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of 2 M Thr-OMe solution in N-

methylpyrrolidone added. The reaction was carried out analogously to that described in Example 4 and the conversion to (Thr-OMe) B30-h-In was 20%.

s Example 6

100 mg of pig insulin was dissolved in 0.9 m of 2.77 M acetic acid in water and 1 ml of a 2 M Thr-OMe solution in HMPA (hexamethylphosphoric triamide) was added. The reaction was carried out analogously to that described in Example 4. The conversion to (Thr-OMe) B30-h-In was 80%.

Example 7

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 molar i5 acetic acid and 1 ml of a 2 M Thr-Ome solution in N, N-dimethylacetamide was added. The reaction was carried out analogously to that described in Example 4. The conversion to (Thr-OMe) B3U-h-In was 80%.

20 Example 8

100 mg of pig insulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M Thr-Ome solution in N, N-dimethylacetamide was added. Trypsin with 200 U activity (measured against the substrate 25 BAEE), immobilized on 1 g of glass beads, was then added. After an incubation period of 24 hours at 37 ° C., the trypsin bound to the glass was separated off by filtration.

After the reaction had ended, the proteins were precipitated by adding 10 parts by volume of acetone. Analysis by 30 DISC PAGE electrophoresis showed a conversion to (Thr-OMe) B30-h-In of 40%.

Example 9

100 mg of swine insulin was dissolved in 0.9 ml of 3.33 molar 35% acetic acid and 1 ml of a 2 M Thr-OMe solution in N, N-dimethylacetamide was added. Trypsin corresponding to an activity of 300 U (the activity was measured against the substrate BAEE) immobilized on 200 mg of CNBr-activated “Sephadex G-150” was then added. 40 After incubation for 24 hours at 37 ° C., the trypsin bound to Sephadex was separated off by filtration. After the reaction had ended, the proteins were precipitated by adding 10 parts by volume of acetone. Analysis by DISC PAGE electrophoresis showed a conversion to 45 (Thr-OMe) B30-h-In of 70%.

Example 10

The procedure described in Example 7 was repeated, provided that the ester used was a 5o 2 M solution of Thr-OBu '(Bu1 is tert-butyl) in N, N-di-ethylacetamide. The conversion to (Thr-OBu ^^ - h-In was 80%.

Example 11

55 The example described in Example 8 was repeated, the ester used being a 2 M solution of Thr-OBu1 in N, N-dimethylacetamide. The conversion to (Thr-OBu1) B30-h-In was 30%.

60 Example 12

The procedure described in Example 9 was repeated, the ester used being a 2 M solution of Thr-OBu1 in N, N-dimethylacetamide. The conversion to (Thr-OBut) B30-h-In was 70%.

65

Example 13

100 mg of pig proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of 2 M Thr-OMe in N, N-dimethyl

655 949

acetamide added. The reaction was carried out analogously to the manner described in Example 4. The conversion to (Thr-OMe) B30-h-In was 80%.

Example 14

100 mg of porcine proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added. The mixture was treated with immobilized trypsin analogously to the process described in Example 8. The conversion to (Thr-OMe) B30-h-In was 40%.

Example 15

100 mg of porcine proinsulin were dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added. The mixture was treated with immobilized trypsin analogously to the method described in Example 9. The conversion to (Thr-OMe) B30-h-In was 70%.

Example 16

100 mg of pig proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M Thr-OBu 'solution in N, N-dimethylacetamide was added. The reaction was carried out analogously to that described in Example 4. The conversion to (Thr-OBu ') B30-h-In was 80%.

Example 17

100 mg of porcine proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M Thr-OBu 'solution in N, N-dimethylacetamide was added. The mixture was carried out using trypsin immobilized on glass beads, analogously to the process described in Example 8. The conversion to (Thr-OBu ') B30-h-In was 80%.

Example 18

100 mg of porcine proinsulin was dissolved in 0.9 ml of 3.33 molar acetic acid and 1 ml of a 2 M Thr-OBu 'solution in N, N-dimethylacetamide was added. The mixture was treated with trypsin immobilized on CNBr-activated Sephadex G-150, analogously to the process described in Example 9. The conversion to (Thr-OBu ') B30-h-In was 70%.

Example 19

100 mg of pig insulin were dissolved in 0.5 ml of 6 molar acetic acid and 1 ml of 1 M solution of Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) in N, N-dimethylacetamide was added. Furthermore, 0.5 ml of N, N-dimethylacetamide and 5 mg of TPCK-treated trypsin in 0.1 ml of water were added. The reaction mixture was left at 32 ° C for 44 hours. After the completion of the reaction, the proteins were precipitated by adding 10 parts by volume of acetone. Analysis by DISC PAGE electrophoresis showed 50% conversion to (Thr-OTmb) B30-h-In.

Example 20

100 mg of pig insulin was dissolved in 0.9 ml of 3 molar acetic acid and 1 ml of a 2 M solution of Thr-OMe in dioxane was added. The reaction was carried out analogously to that described in Example 4; the conversion to (Thr-OMe) B30-h-In was 10%.

Example 21

100 mg of pig insulin was dissolved in 0.9 ml of 3 moiar acetic acid and 1 ml of a 2 M solution of Thr-OMe in acetonitrile was added. The reaction was carried out analogously to the manner described in Example 4 and the conversion to (Thr-OMe) B30-h-In was 10%.

Example 22

250 mg of crystalline (Thr-OMe) B30-h-In were dispersed in 25 ml of water and dissolved by adding 1N sodium hydroxide solution to a pH of 10.0. The pH was kept constant at 10 at 24 ° C. for 24 hours. The human insulin formed was crystallized by adding 2 g of sodium chloride, 350 mg of sodium acetate trihydrate and 2.5 mg of zinc acetate dihydrate, followed by adding 1 N hydrochloric acid to obtain a pH of 5.52. After standing at 4 ° C for 24 hours, the rhomohedral crystals were isolated by centrifugation, washed with 3 ml of water, isolated again by centrifugation and vacuum dried. Yield: 220 mg human insulin.

Example 23

100 mg (Thr-OTmb) B30 were dissolved in 1 ml of ice-cold trifluoroacetic acid and the solution was left to stand at 0 ° C. for 2 hours. The human insulin formed was precipitated by adding 10 ml of tetrahydrofuran and 0.97 ml of 1.03 molar hydrochloric acid in tetrahydrofuran. The precipitate formed was isolated by centrifugation, washed with 10 ml of tetrahydrofuran, separated by centrifugation and vacuum dried. The precipitate was dissolved in 10 ml of water and the pH of the solution was adjusted to 2.5 using 1N sodium hydroxide solution. The human insulin was precipitated by adding 1.5 g of sodium chloride and isolated by centrifugation. The precipitate was dissolved in 10 ml of water and the human insulin was added by adding 0.8 g of sodium chloride, 3.7 mg of zinc acetate dihydrate and 0.14 g of sodium acetate trihydrate, followed by the addition of 1N sodium hydroxide solution to obtain a pH of 5. 52 canceled. After standing at 4 ° C for 24 hours, the precipitate was isolated by centrifugation, washed with 0.9 ml of water, isolated again by centrifugation and vacuum dried. Yield: 90 mg human insulin.

Example 24

100 mg of porcine insulin was dissolved in 0.5 ml of 1 molar acetic acid and 1.3 ml of a 1.54 M solution of Thr-OME in N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C. 10 mg of trypsin, dissolved in 0.2 ml of 0.05 M calcium acetate solution, was added. After standing for 48 hours at 12 ° C, the proteins were precipitated by adding 20 ml of acetone. The conversion of the pig insulin to (Thr-OMe) B30-h-In was 97% as determined by HPLC.

Example 25

20 mg of porcine insulin was dissolved in a mixture of 0.08 ml of 1 molar acetic acid and 0.14 ml of water. 0.2 ml of a 2 M solution of Thr-OMe in N, N-dimethyl-acetamide was added and the mixture was cooled to -10 ° C. 2 mg trypsin dissolved in 0.025 ml 0.05 M calcium acetate solution was added. After standing at -10 ° C for 72 hours, the proteins were precipitated by adding 5 ml of acetone. The conversion of the pig insulin to (Thr-OMe) B30-h-In was 64%, as demonstrated by HPLC.

Example 26

20 mg of pig insulin was placed in 0.1 ml of water. Addition of 0.6 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide caused the insulin to dissolve. The mixture was cooled to 7 ° C. 2 mg trypsin dissolved in 0.025 ml 0.05 M calcium acetate was added. After 24 hours, the proteins were added by adding 5 ml of acetone

8th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

9

655 949

failed. The conversion of the pig insulin to (Thr-OMe) B30-h-In was 62%, as could be demonstrated by HPLC.

Example 27

20 mg of pig insulin was dissolved in 0.135 ml of a 4.45 M propionic acid. 0.24 ml of a 1.67 M solution of Thr-OMe in N, N-dimethylacetamide was added. 2 mg of trypsin, dissolved in 0.025 ml of a 0.05 M calcium acetate solution, was added and the mixture was kept at 37 ° C. for 24 hours. The proteins were precipitated by adding 10 parts by volume of 2-propanol. The conversion of the pig insulin to (Thr-OMe) B30-h-In was 75%, as could be demonstrated by HPLC.

Example 28

20 mg of pig insulin was dispersed in 0.1 ml of water. 0.4 ml of a 2 M solution of Thr-OMe in N, N-dime-thylacetamide was added, followed by 0.04 ml of 10 N hydrochloric acid, which caused the insulin to dissolve. 2 mg of trypsin, dissolved in 0.025 ml of a 0.05 M solution of calcium acetate, was added and the mixture was kept at 37 ° C. for 4 hours. Analysis by HPLC shows a 46% conversion of the pig insulin to (Thr-OMe) B30-h-In.

Example 29

20 mg of pig insulin was dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added, followed by the addition of 0.025 ml of a 0.05 M solution of calcium acetate. 10 mg of a crude preparation of Achromobacter lyticus protease was added and the mixture was kept at 37 ° C. for 22 hours. The proteins were precipitated by adding 10 parts by volume of acetone. The conversion of the pig insulin to (Thr-OMe) B30-h-In was 12%, as could be demonstrated by HPLC.

Example 30

20 mg of pig insulin was dissolved in 0.1 ml of 0.5 molar acetic acid. Addition of 0.2 ml of a 0.1 M solution of Thr-OMe in N, N-dimethylacetamide dissolved the insulin. The mixture was cooled to 12 ° C and 2 mg trypsin in 0.025 ml 0.05 M calcium acetate solution was added. After standing at 12 ° C. for 24 hours, the HPLC analysis showed a 42% conversion of the pig insulin into (Thr-OMe) B30-h-In.

Example 31

2 mg of rabbit insulin was dissolved in 0.135 ml of 4.45 molar acetic acid. 0.24 ml of a 1.67 M solution of Thr-OMe in N, N-dimethylacetamide was added, followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate solution. The mixture was kept at 37 ° C for 4 hours. HPLC analysis showed an 88% conversion of rabbit insulin to (Thr-OMe) B30-h-In. Rabbit insulin is eluted from the HPLC column before the pig insulin, the ratio between the elution volumes of the rabbit insulin to (Thr-OMe) B30-h-In being 0.72%.

Example 32

2 mg of pig diarginine insulin (ArgB31-ArgB32 insulin) was dissolved in 0.135 ml of 4.45 molar acetic acid. 0.24 ml of a 1.67 M solution of Thr-OMe in N, N-diethylacetamide was added, followed by the addition of 1.25 mg of trypsin in 0.025 ml of a 0.05 M calcium acetate solution. The mixture was kept at 37 ° C for 4 hours. Analysis by HPLC showed 91% conversion of the diarginine insulin to (Thr-OMe) B3ü-h-In. Diarginine insulin is used

Pig insulin eluted from the HPLC column, the ratio between the elution volumes of diarginine insulin to (Thr-OMe) B30-h-In being 0.50%.

Example 33

2 mg of porcine intermediates (namely a mixture of desdipeptide (Lys62-Arg63) -proinsulin and des-dipeptide (Arg3l-Arg32-proinsulin) were reacted analogously to the reaction described in Example 32. Analysis by HPLC showed an 88% yield an (Thr-OMe) B3 ° -h-In.

Example 34

20 mg of pig insulin was dissolved in 0.1 ml of 2 molar acetic acid. 0.2 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added and the mixture was cooled to -18 ° C. 2 mg of trypsin, dissolved in 0.025 ml of 0.05 M calcium acetate solution, were added and the mixture was kept at -18 ° C. for 120 hours. HPLC analysis showed that the conversion to (Thr-OMe) B30-h-In was 83%.

Example 35

The procedure described in Example 34 was repeated except that the reaction was carried out at 50 ° C in 4 hours. HPLC analysis showed that the conversion to (Thr-OMe) B30-h-In was 23%.

Example 36

20 mg of pig insulin was dissolved in 0.1 ml of 3 molar acetic acid. 0.3 ml of a 0.33 M solution of Thr-0 (CH2) 2-SO2-CH3, CH3COOH (threonine-2- (methylsulfonyl) ethyl ester, hydroacetate) in N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C and 2 mg of trypsin in 0.025 ml of a 0.05 M calcium acetate solution was added. HPLC showed a 77% conversion to (Thr-O (CH2) 2-SO2-CH3) B30-h-In after 24 hours at 12 ° C. The product elutes at approximately the same location as (Thr-OMe) B30-h-In.

Example 37

20 mg of swine insulin was dissolved in 0.1 ml of 10 molar acetic acid. 0.2 ml of a 2 M solution of Thr-OEt (Et is ethyl) in N, N-dimethylacetamide was added and the mixture was cooled to 12 ° C. 2 mg of trypsin in 0.025 ml of a 0.05 M solution of calcium acetate were added. HPLC showed a 75% conversion to (Thr-OEt) B30-h-In after 24 hours at 12 ° C. The product eluted in a position shortly after that of (Thr-OMe) B30-h-In.

Example 38

20 mg of pig insulin was dissolved in 0.1 ml of 6 molar acetic acid. 0.3 ml of a 0.67 M solution of Th ^ Bu ^ -OBu '(Bu1 is tertiary butyl in N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C. and 2 mg trypsin in 0.025 ml of a 0.1 05 M calcium acetate solution was added and the conversion to (Thr (But) -OBu ') B30-h-In was 77% after 24 hours at 12 ° C. The product was removed from the HPLC column using an acetonitrile gradient from 27 to 40% eluted.

Example 39

20 mg of pig insulin was dissolved in 0.1 ml of 4 molar acetic acid. 0.2 ml of a 1.5 M solution before Thr-OMe, in tetrahydrofuran, was added and the mixture was cooled to 12 ° C. 2 mg of trypsin, dissolved in 0.025 ml of 0.05 molar calcium acetate, were added. After 4 hours at 12 ° C the analysis by HPLC shows a conversion of 75% into (Thr-OMe) B30-h-In.

5

10th

15

20th

25th

30th

35

40

45

50

55

bO

65

655 949

10th

Example 40

20 mg of pig insulin was dissolved in 0.1 ml of 4 molar acetic acid. 0.8 ml of a 2 M Thr-OMe solution in 1,2-ethanediol was added and the mixture was cooled to 12 ° C. 2 mg of trypsin dissolved in 0.025 ml of a 0.05 M calcium acetate solution were added. After standing at 12 ° C for 4 hours, HPLC analysis showed a 48% conversion to (Thr-OMe) B30-h-In.

Example 41

20 mg of pig insulin was dissolved in 0.1 ml of 4 molar acetic acid. 0.2 ml of a 2 M Thr-OMe solution in ethanol was added and the mixture was cooled to 12 ° C.

2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate solution was added and the reaction was carried out at 12 ° C for 4 hours. Analysis by HPLC showed a 46% conversion to (Thr-OMe) B30-h-In.

5

Example 42

20 mg of pig insulin was dissolved in 0.1 ml of 4 molar acetic acid. 0.2 ml of a 2 M solution of Thr-OMe in acetone was added and the mixture was cooled to 12 ° C. 2 mg of trypsin dissolved in 0.025 ml of a 0.05 M calcium acetate solution were added. After 4 hours at 12 ° C, HPLC analysis showed a 48% conversion to (Thr-OMe) B30-h-In.

G

Claims (32)

655 949 (1- Bl-29) -R wherein (I) R1- (I-TAT-21) (l — ßi — 29) - the Des (ThrB30) human insulin moiety, wherein GlyAI is linked to the substituent designated R1 and LysB29 linked to the substituent designated R2, R2 is an amino acid or a peptide chain containing no more than 36 amino acids, and R1 is hydrogen or a group of the general Formula R3-X-, where X is arginine or lysine and R3 is a peptide chain containing no more than 35 amino acids, or R2 and R3 together are a peptide chain containing no more than 35 amino acids, with the proviso that the number of Amino acids present in R1 plus R2 is less than 37, or a salt or a metal complex thereof, with an L-threonine ester of the formula II Thr (R5) -OR4 (II) wherein R4 and R5 are as defined above, or with a salt thereof, in a mixture of water, at least one water-miscible organic solvent and a trypsin-like protease, the water content in the reaction mixture being less than 50% by volume and the reaction temperature is below 50 ° C. 2. The method according to claim 1, characterized in that the transpeptidation is carried out in the presence of an acid. 2nd PATENT CLAIMS 1. Process for the preparation of a threonine 830 ester of human insulin of the general formula II (Thr (R5) -OR4) B30-h-In (III) wherein R4 is a carboxyl protecting group, R5 is hydrogen or a hydroxyl protecting group and h-In is the Des (ThrB30) -Hu-maninsulin unit, or a salt or a metal complex thereof, characterized by transpeptidation of an insulin compound of the formula I. R1- (l — rAT — 21) 3rd 655 949 Is butyl means. 3. The method according to claim 1, characterized in that the trypsin-like protease is trypsin. 4. The method according to claim 1, characterized in that the trypsin-like protease is Achromobacter lyticus protease. 5. The method according to any one of claims 1 to 4, characterized in that the concentration of the L-threonine ester in the reaction mixture is more than 0.1 molar and the reaction temperature is above the freezing point of the reaction mixture. 6. The method according to any one of claims 2 to 5, characterized in that the reaction mixture contains up to 10 equivalents of one acid per equivalent of L-threonine ester. 7. The method according to any one of claims 1 to 6, characterized in that the insulin compound one of the pig, dog, fin whale, sperm whale and rabbit insulin, pig diarginine insulin, split pig proinsulin, pig desdipeptidoproinsulin, pig , Canine, monkey and human proinsulin and their mixtures formed group selected compound. 8. The method according to any one of claims I to 7, characterized in that raw insulin derived from the pig is used as the insulin compound. 9. The method according to any one of claims 1 to 7, characterized 5, that the reaction temperature between 0 and 37 ° C and preferably between 0 ° C and room temperature, the water content in the reaction mixture is between 10 and 40% by volume and the molar ratio between the L-threonine ester and the insulin compound is more than 5: 1 is. 10. The method according to any one of claims 1 to 9, characterized in that the organic solvent is a polar solvent such as methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, formamide, i5 is N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-pyrrolidone, hexamethylphosphoric triamide or acetonitrile. 11. The method according to any one of claims 1 to 10, characterized in that the acid is an organic acid and preferably formic acid, acetic acid, propion- 12. The method according to any one of claims 1 to 11, characterized in that the weight ratio between 13. The method according to any one of claims 1 to 12, characterized in that the transpeptidation in 3o presence of calcium ions and / or in a buffer system is carried out. 14. The method according to any one of claims 1 to 13, characterized in that the L-threonine ester as acetate, propionate, butyrate or as a hydrogen halide compound 35 dung like hydrochloride is added. 15. The method according to claim 1, characterized in that R1 is hydrogen and R2 -Ala, -Ser, -Ala-Arg-Arg or -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-GIn-Ala-Gly -Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala- 40 means Leu-Glu-Gly-Pro-Pro-Gln, or that R3 Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys- and R2 -Ala-Arg-Arg-Gl-Ala-Glu-Asn- Pro-Gln-Ala-Val-GIu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu, or that R2 and R3 together mean -Ala-Arg-Arg-Glu-AIa- Glu-Asn-Pro-Gln-Ala-Gly-Ala- 45 Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, the terminal Ala-nyl is connected to LysB29, -Ala-Arg-Arg-Asp-Val-Glu-Leu-Ala-Gly-Ala-Pro-Gly-Glu-Gly-Gly-Leu-Gln-Pro-Leu-Ala-Leu-Glu- Gly-Ala-Leu-Gln-Lys-, the terminal alanyl 50 is connected to LysB29, -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-GIy -Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, with the terminal threonyl linked to LysB29, or -Thr-Arg-Arg-Glu-Ala-GIu -Asp-Pro-GIn-Val-Gly- 55 Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, where the terminal threonyl is linked to LysB29. 16. The method according to claim 15, characterized in that R1 is hydrogen and R2 is alanine. bo 17. The method according to any one of claims 1 to 16, characterized in that R4 is lower alkyl, substituted benzyl, substituted diphenylmethyl or a group of the general formula -CH2-CH2-SO6, in which R6 is lower alkyl. 65 18. The method according to claim 17, characterized in that R4 is methyl, ethyl, tert-butyl, p-methoxybenzyl, 2,4,6-tri-methylbenzyl or a group of the general formula -CH2-CH2-SO2-R6, wherein R6 Methyl, ethyl, propyl or n- 19. The method according to any one of claims 1 to 8, characterized in that the insulin compound is porcine insulin, the L-threonine ester Thr-O-methyl or Thr-O-tert-butyl, the water-miscible organic solvent N, N -Dimethylformamide or N, N-dimethylacetamide, the reaction temperature is about 37 ° C, the water content in the reaction mixture is between 41 and 43 vol .-%, the weight ratio between trypsin and the insulin compound is about 1: 8, the molar ratio between the L-threonine ester and the insulin compound is about 1: 120 and between 1.2 and 1.5 equivalents of acetic acid are added per equivalent of L-threonine ester. 20. The method according to any one of claims 1 to 19, characterized in that the water content in the reaction mixture is between 21 and 43% by volume. 20 is acid or butyric acid, and the amount of acid in the reaction mixture is between 0.5 and 5 equivalents per equivalent of L-threonine ester. 21. The method according to any one of claims 1 to 19, characterized in that the water content in the reaction mixture is about 38% by volume. 22. The method according to any one of claims 1 to 20, characterized in that the pH in the reaction mixture is about 4.5. 23. The method according to any one of claims 1 to 20, characterized in that the pH in the reaction mixture is about 5.1. 24. The method according to any one of claims 1 to 20, characterized in that the pH in the reaction mixture is about 5.2. 25. The method according to any one of claims 1 to 20, characterized in that the pH in the reaction mixture is about 5.4. 25 see trypsin and the insulin compound in the reaction mixture between 1: 200 and 1: 1 and preferably between 1:50 and 1: 1. 26. The method according to any one of claims 1 to 20, characterized in that the pH in the reaction mixture is 4.5 to 5.4. 27. The method according to any one of claims 1 to 26, characterized in that the weight ratio between the threonine B30 ester and the insulin compound of formula I in the reaction mixture is between 60: 1 and 180: 1. 28. The method according to any one of claims 1 to 26, characterized in that the weight ratio between the threonine β30 ester and the insuine compound of the formula I in the reaction mixture is approximately 60: 1. 29. The method according to any one of claims 1 to 26, characterized in that the weight ratio between the threonine B30 ester and the insulin compound of formula I in the reaction mixture is about 120: 1. 30. The method according to any one of claims 1 to 26, characterized in that the weight ratio between the threonine B30 ester and the insulin compound of formula I in the reaction mixture is about 180: 1. 31. Threonine B30 ester of human insulin of the general formula III, prepared by the process according to claim 1. 32. Use of the threonineB3 () ester of human insulin of the general formula III prepared by the process according to claim 1 for the preparation of human insulin by cleaving off the protective groups.