FI77876C - Process for the preparation of human insulin or a threonine B30 ester of human insulin or a salt or complex thereof - Google Patents

Description

! 77876 B30

The present invention relates to a process for the preparation of human insulin or human B30 insulin threonine ester or a salt or complex thereof.

Insulin preparations containing porcine and bovine insulin are commonly used to treat diabetes mellitus. There are small differences in bovine, porcine and human insulin in terms of their amino acid composition, with the difference between human and porcine insulin being limited to a simple amino acid, in human insulin the amino acid B30 is threonine, while in porcine insulin it is alanine. However, it can be said that the ideal insulin for human treatment is an insulin preparation with exactly the same structure as human insulin.

Not enough human pancreas is available to make natural human insulin.

Synthetic human insulin is produced on a small scale at the highest cost, Helv. Chim. Acta 57 ^ 2617, and 60, 27. Semisynthetic human insulin has been prepared from porcine insulin by a complex method, Hoppe-Seyler's Z. Physiol. Chem. 357, 759.

U.S. Patent No. 3,276,961 is claimed to disclose a process for preparing semisynthetic human insulin which comprises reacting other, animal-like insulins with threonine in the presence of an enzyme such as trypsin or carboxypeptidase A. However, the yield of human insulin is very low (if human insulin is obtained at all), since the process is carried out in water, under which conditions 2 77876 B22 B23 trypsin results in cleavage of the Arg-Gly bond, J. Biol. Chem. 236, 743.

Another known semisynthetic method for the preparation of human insulin comprises the following three steps: In the first step B30, the porcine insulin is cleaved into porcine des (Ala) insulin by treatment with carboxypeptidase A, Hoppe-

Seyler's Z. Physiol. Chem. 359, 799. In the second step Β3Ό—, porcine-des- (Ala) -sulin is subjected to trypsin-catalyzed coupling with Thr-OBut to give the Thr -tert-butyl ester of the human IN-B30 line. In the third step, said ester is treated with trifluoroacetic acid to give insulin human, Nature 280, 412. However, in the first a2 1 step, a partial removal of Asn, B30 A21, takes place to give des- (Ala, Asn) insulin. This derivative gives the impurity Δ 21 den des (Asn) to the semisynthetic human insulin to be prepared during the next two reaction steps, which impurity is difficult to remove by known preparative methods. Insulin Des-A21 (Asn) has low biological activity (approx.

5%), Amer. J. Med. 40, 70.

It has now surprisingly been shown that insulin compounds other than human insulin, such as porcine insulin and certain impurities contained therein, can be converted to human insulin by a process in which the insulin compounds in one B30 step are directly converted to human insulin threonine ester. This method yields human insulin in substantially better yields than known methods and without difficult-to-remove by-products.

For the method of the invention, human insulin or human B30

for the preparation of an insulin threonine ester or a salt or complex thereof, it is characterized in that the insulin compound of formula I

i 3, 77876 R - (1 --- A --- 21) II 2 (1 --- B --- 29) -R (I) where - (1 --- A --- 21) (1—- B --- 29) -, B30 A1 is a human-des (Thr) -insulin moiety in which Glv is bound to the substituent R B29 and Lys is bound to the substituent 2

wherein R, R is an amino acid or peptide chain containing up to 36 amino acids, and R is hydrogen or a group of the general formula R -X-, wherein X is arginine or lysine and R is a peptide chain containing up to 35 amino acids. amino acid, or R together with R is a peptide chain containing up to 35 amino acids, provided that the number of amino acids in R and R is less than 37, or a salt or complex thereof which can be converted into an insulin compound of formula I. , is transpeptated with an L-threonine ester of formula II

.5 4

Thr (R) -OR (II) ... 4 5 wherein R is a carboxyl protecting group and R is hydrogen or a hydroxyl protecting group, or a salt thereof in a mixture containing water, a water-miscible organic solvent and trypsin, wherein the water content of the reaction mixture is less than 50 % (v / v) and the reaction temperature is below 50 ° C, and optionally in the presence of an acid, after which the resulting ih -.... .B30 misinsulin threonine ester is cleaved if desired.

The present invention is based on the finding that an amino acid or peptide chain attached to a Lys carbonyl group in an insulin compound of formula I can be exchanged for a threonine ester in one step. Said exchange is referred to herein as transpeptidation.

4 77876

Insulin compounds of formula I are insulins and insulin-like compounds containing a human des-B30 (Thr) insulin moiety, wherein the B30 amino acid in insulin is alanine (e.g. porcine, canine and whalebone and cascade insulin) or serine (rabbit). . As used herein, the term "insulin-like compounds" also includes proinsulin obtained from any of the above species and monkeys, as well as intermediates in the conversion of proinsulin to insulin. Examples of such intermediates include cleaved proinsulin, desdipeptide diproinsulins, desnonapept-diproinsulin, and diarginins, R. Chance: In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292-305, R.R. Rodriques & J.V.-Owen, Excerpta Medica, Amsterdam.

Although trypsin is best known for its proteolytic properties, those skilled in the art have found that B304 trypsin can catalyze the coupling of des- (Ala) -insulin and threonine tert-butyl ester, Nature 280, 412. The method of the invention uses trypsin to catalyze transpeptidation.

As discussed above, Nature 280, 412 relates to a method for coupling peptides, while the present invention relates to a method for transpeptidating peptides. If the step of hydrolyzing the human insulin ester is disregarded, according to Nature 280, 412, two steps must be used to prepare the human insulin ester, namely the peptide cleavage step and the peptide coupling step. The method of the present invention provides the human insulin ester directly in one step, which is the transpeptidation step.

Nature 280, 412 discloses that it is surprising that B22 B23 does not cleave the Arg-Gly bond in the product. It is known that trypsin in the aqueous medium 77876 B22 B23 cleaves both Arg-Glv and at B29 B30 sa Lys -Ala and thus from Nature 280, 412 it is expected that trypsin in the organic medium B29 B30 does not cleave either. Lys -Ala bond.

There is no mention in Nature 280, 412 of whether B29-B30 cleavage of the Lys -Ala bond occurs in an organic medium. Thus, according to the invention, it is particularly surprising that transpeptidation can be performed directly on porcine insulin.

In Biochem. Biophys. Res. Corn. No. 9,339 discloses a process for the preparation of human insulin which is substantially identical to that described in Nature 280, 412, but which uses the protease Achromobacter lyticus as an enzyme. The above discussion regarding Nature 280, 412 therefore also applies to Biochem. Biophys. Res. Com. 92, 396.

According to the invention, the transpeptidation can be carried out by dissolving the insulin compound, L-threonine ester and trypsin in a mixture containing water and at least one water-miscible organic solvent, optionally in the presence of an acid.

Preferred water-miscible organic solvents are polar solvents. Examples of such solvents include methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, N, N-dimethylformamide, formamide, Ν, Ν-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphoric phosphorus and acetonitrile.

The amount of water in the reaction mixture depends on the water-miscible organic solvent used, the reaction temperature, and the presence of acid in the reaction mixture, and should be less than about 50% (v / v), and preferably less than about 40% (v / v), and greater than about 10% (v / v).

6 77876

One advantage of reducing the amount of water in the reaction mixture is that the generation of by-products is then reduced. By increasing the amount of acid in the reaction mixture, it is possible to correspondingly reduce the formation of by-products. The increase in yield correlates with the high concentration of organic solvent.

The type of trypsin used is not essential to the practice of this invention. Trypsin is a well-characterized enzyme that is commercially available in very pure form, especially from cattle and pigs. Microbiologically derived trypsin can still be used. In the practice of this invention, the form of trypsin, e.g., crystalline trypsin (soluble form), immobilized trypsin, or even trypsin derivatives (as long as trypsin activity is retained), is not essential. The trypsin used herein includes trypsin from all sources and all forms of trypsin having transpeptidating activity, including proteases with trypsin-like specificity, Achromobacter lyticus protease, Agric. Biol. Chem. 42, 1443.

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

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

Organic or inorganic acids such as hydrochloric acid, formic acid, acetic acid, propionic acid or butyric acid or bases such as pyridine, TRIS, N-methylmorpholine or N-ethylmorpholine may be added to provide a suitable buffer system. Organic acids are preferred. However, the reaction can be carried out without such additions. The amount of acid added is usually less than about 10 equivalents per equivalent of L-threonine ester. The amount of acid 7 77876 is preferably between 0.5 and 5 equivalents per equivalent of L-threonine ester. Ions that stabilize trypsin, such as calcium ions, can also be added.

To achieve a sufficient reaction rate, enzymatic reactions using trypsin are usually performed at about 37 ° C. To avoid trypsin inactivation, the method of the present invention is preferably performed at a temperature below ambient temperature. In practice, reaction temperatures above about 0 ° C are preferred.

Depending on the reaction temperature, the amount of trypsin added, and other reaction conditions, the reaction time is usually from several hours to several days.

The weight ratio of trypsin to insulin compound is usually greater than about 1: 200, preferably greater than about 1:50, and less than about 1: 1.

B30

The threonine esters of human insulin obtained by transpeptidation can be represented by the general formula III: 5 4 B30 (Thr (R) -OR) -h-In (III) B30 4 wherein h-In is a human-des- (Thr) -insulin moiety, and R and 5 R have the same meaning as above.

Useful L-threonine esters of formula II are those in which R is a carboxyl protecting group which can be B30 removed from the threonine ester of human insulin (formula III) under conditions in which there are no substantially irreversible changes in the insulin molecule. Examples of such carboxyl protecting groups include lower alkyls, e.g., methyl, ethyl and tert-butyl, substituted 8,787,76 benzyls such as p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenylmethyl, and groups having the general formula -CH CH SO R wherein R is lower alkyl such as methyl, 2 2 2 ethyl, propyl and n-butyl. Suitable hydroxyl protecting groups 5R are those which can be removed from the threonine ester of human insulin B30 (Formula III) under conditions in which there are no substantially irreversible changes in the insulin molecule. As an example of such a group, R is tert-butyl.

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

Commonly used protecting groups are described in Methoden der Organischen Chemie (Houben-Weyl), Vol. XV / 1, Eugen Muller, Georg Thieme Verlag, Stuttgart 1974.

Some L-threonine esters of formula II are known compounds, and other L-threonine esters (formula II) can be prepared analogously to methods for preparing known compounds.

The L-threonine esters of formula II may exist as free bases or as suitable organic or inorganic salts, preferably as acetates, propionates, butyrates and hydrohalides, such as hydrochlorides.

It is recommended to use high concentrations of the reactants, i.e. the insulin compound and the L-threonine ester (formula II). The molar ratio of L-threonine ester to insulin compound is preferably greater than about 5: 1.

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

i 9 77876 B30

Human insulin is obtained from the threonine ester of human insulin (Formula III) by removing the protecting group R and any protecting group present in a manner known per se or per se. When R is methyl, ethyl or a group of the formula -CH CH SO R wherein R is as defined above, said protecting group may be removed under mild, basic conditions in an aqueous medium, preferably at a pH of about 8 to about 12, for example, about 9.5. Ammonia, ethylamine or alkali metal hydroxides such as sodium hydroxide can be used as the base. When R is tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, said group may be removed by acidolysis, preferably using trifluoroacetic acid. Trifluoroacetic acid may be anhydrous and may contain water, or may be diluted as an organic solvent such as dichloromethane. When R is tert-butyl, said group can be removed by acidolysis as mentioned above.

B30

Preferred threonine-5 esters of human insulin of formula III are compounds wherein R is hydrogen, and these compounds can be prepared from L-threonine esters of formula II wherein R is hydrogen.

In the transpeptidation according to the invention, any of the above-mentioned insulin compounds is thus converted into the human B30 suline threonine ester (formula III), which can then be cleaved to obtain human insulin.

Yet another advantage of this invention is that the insulin-like compounds of formula I present in crude insulin and some commercial insulin formulations can be converted to B30 human threonine ester by transpeptidation of this invention, which compounds can then be cleaved to give human insulin.

10 77876

Examples of insulin-like compounds of formula I are given below: .1 .2

Porcine diarginin insulin (R is hydrogen and R is -Ala-Arg- 3 2

Arg), porcine proinsulin (R is together with R -Lower-

Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Glu

Gly-Gly-Leu-Gly-Gly-Gln-Leu-Ala-Leu-Ala-Leu-Gly-Gl ^ -Pro-Pro

Gln-Lys-, where the terminal alanyl is bound to Lys), 3 2 canine proinsulin (R together with R is -Ala-Arg-

Arg-Asp-Glu-Val-Leu-Ala-Gly-Ala-Pro-Gly-Glu-Gly-Gly-Leu-Gln

Pro-Leu-Ala-Leu-Glu-Gly-Ala-Leu-Gln-Lys-, with terminal alanyl B29%. . . , ...

bound to Lys), cleaved porcine proinsulin (R is Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, and R 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), porcine despeptide proinsu-1 2 line (R is hydrogen and R 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), human proinsulin 3 2 (R together with R -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-Gl 2 -Ser-Leu-Gln-Lys-, where the terminal threonyl is bound to Lys) and monkeyprinsu, 3 2 line (R together with R tn with -Thr-Arg-Arg-Glu-Lower-Glu-

Asp-Pro-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- (^ 3 2 ^ 3 where the terminal threonyl is bound to Lys).

In formula I, the R substituent labeled 3 R -X- is thus replaced by hydrogen.

A preferred embodiment of the present invention comprises reacting insulin-like compounds contained in crude porcine insulin or a salt or complex thereof with L-threonine ester (Formula II) or a salt thereof under the above 4 conditions, after which the protecting group R and optionally the protecting group R present are removed. In this case, this method converts insulin swine, as well as the insulin-like compounds it contains, into human insulin.

As examples of the complex or salt of the insulin compounds (Formula I), a zinc complex or a zinc salt may be administered.

When the reaction conditions are selected according to the above, taking into account the results obtained in the following examples,. ... In B30, it is possible to obtain threonine ester of human insulin in a yield of more than 60% and even 80% and in some preferred conditions the yield is more than 90%.

The method according to the present invention thus has the following advantages over the prior art:. B30 a) Enzymatic hydrolysis to remove Ala, for example carboxypeptidase A: 11a, is avoided.

X. , B30 V

b) Isolation of an intermediate such as porcine des- (Ala) -insulin is unnecessary.

x A21 c) Avoid des (Asn) insulin derivatives present as impurities.

d) Proinsulin and other insulin-like impurities present in crude insulin are converted by the method of the invention - via human threonine ester - into human insulin, thereby increasing the yield.

e) Antigenic insulin-like compounds, GB Patent 1,285,023, are converted to human insulin.

A preferred method for preparing human insulin is set forth below: 1) Crude cyanosulin, e.g., crystalline insulin prepared using citrate buffer, is used as a feedstock for transpeptidation, U.S. Patent No. 2,626,228.

i2 7 7 8 7 6 2) After transpeptidation, any residual trypsin activity is preferably removed, for example under conditions in which the trypsin is inactivated, for example in an acidic medium with a pH below 3. Trypsin can be removed by molecular weight separation, e.g. by gel filtration "Sephadex G-50 "or" Bio-gel P-30 "in 1 M acetic acid. Nature 280, 412.

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

, B30 4) The threonine ester of human insulin is then cleaved, and human insulin is isolated, for example, crystallized in a manner known per se.

In this way, human insulin of pharmaceutically acceptable purity is obtained and, if desired, human insulin can be further purified.

The abbreviations used below are in accordance with the guidelines adopted by the IUPAC-IUB Commission (1974) for the Biochemical Vocabulary, Collected Tentative Rules Se Recommendations of the Commission on Biochemical Nomenclature IUPAC-IUB, 2nd ed., Maryland 1975.

Analytical experiments:

Conversion of porcine insulin and porcine insulin to human insulin ester can be demonstrated by DISC-PAGE electrophoresis on a 7.5% polyacrylamide gel in a buffer of 0.375 M TRIS, 0.06 M HCl, and 8 M urea. The pH of the buffer is 8.7. The esters of formula III migrate at a rate of 75% of that of insulin pork. Insulin swine, which migrates at a rate of 55% compared to the rate of insulin swine, is converted by the process of the invention into the latter product. The reaction products were identified as compounds of formula III based on the following criteria: a) The electrophoretic migration of the human insulin esters of formula III corresponds to the loss of one negative charge compared to cyanosulin.

b) The amino acid composition of the colored protein bands in the gel corresponding to the compounds of formula III is identical to the amino acid composition of human insulin, i.e. it has 3 moles of threonine and 1 mole of alanine per 1 mole of insulin, and the composition of cyan insulin has 2 moles of alanine and 2 moles of threonine moles per insulin. Methods for analyzing amino acid compositions in protein bands on polyacrylamide gels are described in Eur. J. Biochem. 2J5, 147.

c) Evidence that the added threonine is located in the B chain of the C-terminal amino acid is obtained by oxidative sulfitolysis of insulin S-S bridges in 6 M guanidine hydrochloride followed by separation of the A and B chains by ion exchange chromatography on "SP Sepha-dex". When the B-chain S-sulfonate is cleaved by carboxy-peptidase A, the C-terminal amino acid is released.

This procedure is described in Markussen, Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide, Tokushima, July 12-14, 1978 (publication:

Baba, Kaneko and Yanihara) Int. Congress Series No. 468, Excerpta Medica, Amsterdam-Oxford. The analysis is performed after cleavage of the ester group by the compounds of formula III.

The above three analyzes clearly show that there has been a conversion to human insulin.

14 77876

The conversion of porcine insulin and porcine insulin to human insulin ester can be monitored quantitatively using HPLC (high pressure liquid chromatography) in reverse phase. A 4 x 300 mm "UBonda-pak C-g column" (Waters Ass.) Is used and elution is performed with a buffer containing 0.2 M ammonium sulphate (adjusted to pH 3.5 with sulfuric acid). ) and containing 2-6-50% acetonitrile. The optimal concentration of acetonitrile depends on which ester of formula III is to be separated from insulin swine. When R is methyl and R is hydrogen, separation is achieved in 26% (v / v) acetonitrile. Insulin cyano and B 3 O (Thr-OMe) -h-In (Me is methyl) elute after 4.5 and 5.9 column volumes as distinct symmetrical peaks. Prior to loading onto the HPLC column, proteins are precipitated from the reaction mixture by the addition of 10 volumes of acetone. The precipitate is collected by centrifugation, dried in vacuo and dissolved in 0.02 M sulfuric acid.

The process for the preparation of human insulin esters and human insulin is described in more detail by the following examples, which illustrate some preferred embodiments of the process according to the invention.

Example 1 200 mg of once crystallized crude porcine insulin 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. The proteins were precipitated after standing for 18 h at 37 ° C by adding 40 ml of acetone, and the precipitate was isolated by centrifugation. The solution phase was discarded. Analysis of the precipitate by HPLC using 26% acetonitrile (see analytical experiments) shows that 60% of the cyano-B30 sulfine has been converted to (Thr-OMe) -h-In. The precipitate was dissolved in 8 ml of freshly deionized 8 M urea, the pH was adjusted to 8, with 1 M ammonia, and the solution was applied to a 2.5 x 25 cm column packed with "QAE A-25 Sephadex": equilibrated with 0.1 M ammonium chloride buffer containing 60% (v / v) ethanol, and its pH was adjusted to 8 with ammonia. Elution was performed with the same buffer and 15 ml fractions were collected. (Thr-OMe) β 3 -h-In was found in fractions 26-46, unreacted porcine insulin in fractions 90-120. Fractions 26-46 were combined, the ethanol was evaporated in vacuo, and (Thr-OMe) -h-In was crystallized in citrate buffer as described in Schlichtkull et al., Handbuch der inneren Medizin, 7 / 2A, 96, Berlin, Heidelberg, New York 1975 is described. Yield 95 mg of crystals having the same rhombic form as insulin crystallized in an analogous manner. The amino acid composition proved to be identical to the amino acid composition of human insulin. In addition, the analytical experiments performed as described above, B 3 0, showed that the product obtained was (Thr-OMe) -h-In. Example 2 100 mg of insulin cyanide meeting the purity criteria described in GB Patent 1,285,023 was dissolved in 0.9 ml of 3.33 M acetic acid, to which was then added 1 ml of a 2 M solution of threonine methyl ester in Ν, Ν-dimethylformamide. and 12 mg of TPCK-treated (TPCK is tosylphenylalanine chloromethyl ketone) trypsin dissolved in 0.1 ml of water. The reaction was stopped after incubation for 24 h at 37 ° C by the addition of 4 ml of 1 M phosphoric acid. The resulting B3 O (Thr-OMe) -h-In was separated from unreacted cyanosulin by ion exchange chromatography using a 2.5 x 25 cm "SP Sephadex" column and eluent containing 0.09 M sodium chloride and 0.02 M sodium dihydrogen phosphate (buffer pH is 5.5) in 60% ethanol. Fractions of B30 containing (Thr-IMe) -h-In were collected, ethanol was removed in vacuo, and the product was crystallized as described in Example 1. The yield was 50 mg (Thr-OMe) of B 3 H-In.

Example 3

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

Β3Π acetic acid and converted to (Thr-OMe) -h-In and purified as described for example for porcine insulin in cat 3 1. The conversion of proinsulin to (Thr-OMe) -h-In was 73% as determined by HPLC analysis. acetone- B 3 0 dipping. The yield of crystalline (Thr-OMe) -h-In was 54 mg. Example 4 100 mg of insulin cyanine was dissolved in 0.9 ml of 2.77 M acetic acid in water and reacted as described in Example 2. Upon completion of the reaction, proteins were precipitated by the addition of 10 volumes of acetone. DISC-PAGE-B 3 0 analysis showed 70% conversion to (Thr-OMe) -h-In.

Example 5

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

acetic acid, and 1 ml of 2 M Thr-OMe in N-methylpyrrolidone was added. The reaction was performed as described in Example B 3 0 4 and the conversion to (Thr-OMe) -h-In was 20%.

Example 6 100 mg of porcine insulin was dissolved in 0.9 ml of 2.77 M acetic acid in water, and 1 ml of 2 M Thr-OMe in HMPA (hexamethylphosphoric triamide) was added. The reaction was performed as described in Example 4. Conversion

Dlf | The (Thr-OMe) -h-In was 80%.

Example 7

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

17 77876 acetic acid, and 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added. The reaction was carried out as described in Example 4 of Example 3. The conversion (Thr-OMe) to h-In was 80%.

Example 8 100 mg of insulin cyanine was dissolved in 0.9 ml of 3.33 M acetic acid, and 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added. Then, 200 U (units) of trypsin activity (as determined on the substrate BAEE) immobilized on 1 g of glass beads was added, and after incubation for 24 h at 37 ° C, the glass-bound trypsin was filtered off. After completion of the reaction, the proteins were mixed by adding 10 volumes of acetone. DISC-PAGE analysis showed 40% conversion (Thr-OMe) to B30-h-In.

Example 9 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid, and 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added. 300 U of trypsin activity (measured on the substrate BAEE) immobilized on 200 ug of CNBr-activated "Sephadex G-150" was then added. After incubation for 24 h at 37 ° C, trypsin bound to "Sephadex" was filtered off. After completion of the reaction, the proteins were precipitated by adding 10 volumes of acetone. DISC-PAGE analysis showed 70% conversion (Thr-OMe) to B30-h-In.

Example 10

The procedure described in Example 7 was repeated using 2 M Thr-OBut (But is tert-butyl) in N, N-dimethylacetamide as ester. The conversion (Thr-OBut) to B30-h-In was 80%.

Example 11

The procedure described in Example 8 was repeated using 18 77876 of 2 M Thr-OBut as ester in N, N-dimethylacetamide. The conversion (Thr-OBut) to B30-h-In was 30%.

Example 12

The procedure described in Example 9 was repeated using 2 M Thr-OBu 2 as ester in N, N-dimethylacetamide. The conversion to (Thr-OBu 2) B 3 <"* - h-In was 70%.

Example 13

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

acetic acid, and 1 mL of 2 M Thr-OMe in N, N-dimethylacetamide was added. The reaction was performed as described in Example B 3 0. The conversion to (Thr-OMe) -h-In was 80%.

Example 14

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

acetic acid, and 1 mL of 2 M Thr-OMe in N, N-dimethylacetamide was added. The mixture was treated with immobilized trypsin in a manner similar to that described in Example B 3 0 8. The conversion to (Thr-OMe) -h-In was 40%. Example 15

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

acetic acid, and 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added. The mixture was treated with immobilized trypsin in a manner similar to that described in Example 9 for B03. The conversion to (Thr-OMe) -h-In was 70%. Example 16 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid, and 1 ml of 2 M Thr-OBut in N, N-dimethylacetamide was added. The reaction was carried out in the same manner as described in Example 4. The conversion of (Thr-OBu * ·) to B3B-h-In was 80%.

19 77876

Example 17 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid, and 1 ml of 2 M Thr-OBut in N, N-dimethylacetamide was added. The mixture was treated with trypsin immobilized on glass beads in a manner similar to that described in Example 8. The conversion (Thr-OBut) to B3 ^ -h-In was 40%.

Example 18

100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M

acetic acid, and 1 ml of 2 M Thr-OBut in N, N-dimethylacetamide was added. The mixture was treated with trypsin immobilized on CNBr-activated "Sephadex-G-150" in a manner similar to that described in Example 9 + * B 30. The conversion to (Thr-OBu) -h-In was 70%. Example 19 100 mg of porcine insulin was dissolved in 0.5 ml of 6 M acetic acid, and 1 ml of 1 M Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) in N, N-dimethylacetamide was added. Further, 0.5 ml of Ν, Ν-dimethylacetamide and 5 mg of TPCK-treated trypsin in 0.1 ml of water were added. The reaction mixture was stored at 32 ° C for 44 h. Upon completion of the reaction, proteins were precipitated by the addition of 10 volumes of acetone. DISC-PAGE analysis showed 50% conversion (Thr-OTmb) to B30-h-In.

Example 20 100 mg of insulin pig was dissolved in 0.9 ml of 3 M acetic acid, and 1 ml of 2 M Thr-OMe in dioxane was added.

The reaction was carried out in the same manner as in Example B 3 4 in 4, and the conversion to (Thr-OMe) -h-In was 10%. Example 21 100 mg of porcine insulin was dissolved in 0.9 ml of 3 M acetic acid, and 1 ml of 2 M Thr-OMe in acetonitrile was added.

Ii. The reaction was carried out in the same manner as B3 0 in Example 4, and the conversion to (Thr-OMe) -h-In was 10 S.

Example 22 B30 250 mg of crystalline (Thr-OMe) -h-In was dispersed in 25 ml of water and dissolved by adding 1 N sodium hydroxide solution to pH 10.0. The pH was kept constant at 10.0 for 24 h at 25 ° C. The resulting human insulin 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 the addition of 1N hydrochloric acid to obtain a pH of 5.52. The rhombohedral crystals were isolated by centrifugation, after 24 h at 4 ° C, washed with 3 ml of water, isolated by centrifugation and dried in vacuo. Yield: 220 mg of human insulin.

Example 23 B 100 mg (Thr-OTmb) -h-In was dissolved in 1 ml of ice-cold trifluoroacetic acid, and the solution was stored at 0 ° C for 2 h. The resulting human insulin was separated by adding 10 ml of tetrahydrofuran and 0.97 ml of 1.03 M hydrochloric acid in tetrahydrofuran. The precipitate formed was isolated by centrifugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation and dried in vacuo. The precipitate was dissolved in 10 ml of water, and the pH of the solution was adjusted to 2.5 with 1 N sodium hydroxide solution. Human insulin was separated by the addition of 1.5 g of sodium chloride and isolated by centrifugation. The precipitate was dissolved in 10 ml of water, and human insulin was separated 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 1 N sodium hydroxide solution to reach a pH of 5.52. The precipitate was isolated by centrifugation after standing for 24 h at 21 77876 at 4 ° C, washed with 0.9 ml of water, isolated by centrifugation and dried in vacuo. Yield: 90 mg of human insulin.

Example 24 100 mg of insulin pig was dissolved in 0.5 ml of 10 M acetic acid, and 1.3 ml of 1.54 M Thr-MeM in N, N-dimethyl acetate was added. The mixture was cooled to 12 ° C.

10 mg of trypsin dissolved in 0.2 ml of 0.05 M calcium acetate was added. After standing for 48 hours at 12 ° C, the proteins were precipitated by the addition of 20 ml of acetone. The conversion of porcine B 3 0 insulin to (Thr-OMe) -h-In is 97% by HPLC.

Example 25 20 mg of insulin pig was dissolved in a mixture of 0.08 ml of 10 M acetic acid and 0.14 ml of water. 0.2 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added and the mixture was cooled to -10 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 72 h at -10 ° C, the proteins were precipitated by adding B30 to 5 mL of acetone. The conversion of insulin cyanine (Thr-OMe) to -h-In was 64% by HPLC.

Example 26 20 mg of porcine insulin was dispersed in 0.1 ml of water. By adding 0.6 ml of 2 M Thr-OMe in H, Ν-dimethylacetamide, the insulin decomposed. The mixture was cooled to 7 ° C.

2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 24 h at 7 ° C, the proteins were precipitated by the addition of 5 ml of acetone. The conversion of porcine B 3 0 insulin to (Thr-OMe) -h-In was 62% by HPLC.

Example 27

20 mg of porcine insulin was dissolved in 0.135 ml of 4.45 M

77876 propionic acid. 0.24 ml of 1.67 M Thr-OMe in N, N-dimethylacetamide was added. 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added, and the mixture was stored at 37 ° C for 24 hours. Proteins were precipitated by the addition of 10 volumes of 2-propanol. The conversion of insulin cyanine (Thr-OMe) to B3 ^ -h-I was 75% by HPLC.

Example 28 20 mg of porcine insulin was dispersed in 0.1 ml of water. 0.4 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added, followed by 0.04 ml of 10 N hydrochloric acid, whereupon the insulin decomposed. 2 ml of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added, and the mixture was stored at 37 ° C for 4 hours. HPLC analysis B 3 0 shows 46% conversion of insulin swine to (Thr-OMe) -h-In.

Example 29 20 mg of porcine insulin was dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of 2 M Thr-OMe in N, N-dimethylacetamide was added, followed by 0.025 ml of 0.05 M calcium acetate. 10 mg of a crude preparation of Achromobacter lyticus protease was added and the mixture was stored at 37 ° C for 22 h. Proteins were precipitated by adding 10 volumes of acetone. The conversion of cyanogen insulin (Thr-OMe) to B30-h-In was 12% by HPLC.

Example 30 20 mg of insulin pig was dispersed in 0.1 ml of 0.5 M acetic acid. By adding 0.2 ml of 0.1 M Thr-OMe in N, N-dimethylacetamide, the insulin disintegrated. The mixture was cooled to 12 ° C, and 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. After 24 h at 12 ° C, HPLC analysis shows conversion of 42% porcine insulin B30 to (Thr-OMe) -h-In.

23 77876

Example 31 2 mg of rabbit insulin was dissolved in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr® Me in Ν, Ν-dimethylacetamide was added, followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate. The mixture was stored at 37 ° C for 4 h. HPLC analysis showed 88% conversion of rabbit insulin to B 3 O (Thr-OMe) -h-In. Rabbit insulin elutes porcine insulin from an HPLC column with a ratio of rabbit insulin B 3 0 elution volume to (Thr-OMe) -h-In of 0.72.

Example 32 B 31 B 3 2 2 mg of insulin porcine argin (Arg-Arg) was dissolved in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe in Ν, Ν-dimethylacetamide was added, followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate. The mixture was stored at 37 ° C for 4 h. HPLC analysis showed 91% conversion of insulin diargin to B30 to (Thr-OMe) -h-In. Insulin diargin elutes porcine insulin from an HPLC column, with a ratio of elution volume of diargin-B0 0 insulin to (Thr-OMe) -h-In of 0.50.

Example 33

C O

2 mg of "porcine intermediates" (i.e., desdipeptide- (Lys -Arg) -31 32 proinsulin and desdipeptide- (Arg -Arg) -proinsulin) were reacted in a similar manner to Example 32. HPLC analysis showed 88%: n (Thr-OMe) h-In: a.

Example 34

20 mg of porcine insulin was dissolved in 0.1 ml of 2 M acetic acid. 0.2 ml of 2 M Thr-OMe in Ν, Ν-dimethylacetamide was added, and the mixture was cooled to -18 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M was added

24 77876 calcium acetate, and the mixture was stored at -18 ° C for 120 h.

B 3 0 HPLC analysis shows that the conversion to (Thr-OMe) -h - In is 83%.

Example 35

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

Example 36 20 mg of insulin pig was dissolved in 0.1 in any 3 M acetic acid. 0.3 ml of 0.33 M Thr-O (CH 2) 2 -SO 2 -CH 3, CH-COOH / threonine-2- (methylsulfonyl) ethyl ester hydroacetate / N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C and 2 mg of trypsin 0.025 in any 0.05 M calcium acetate was added. HPLC after 24 hm at 12 ° C shows 77% tn conversion of (Thr-O (CH-)) 9-SO 2 ~ -330 ^ CH-N-h. The product eluted at approximately (Thr-OMe) -h-Intn.

Example 37 20 mg of porcine insulin was dissolved in 0.1 in any 10 M acetic acid. 0.2 ml of 2 M Thr-OEt (Et is ethyl) in Ν, Ν-dimethylacetamide was added and the mixture was cooled

12 ° Cteen. 2 mg of trypsin in 0.025 ml of 0.05 M was added

calcium acetate. HPLC shows 24 to 75% conversion of (Thr-OEt) -h-Intx after 24 h at 12 ° C. The product eluted at B30, which is obtained shortly after (Thr-OMe) -h-In.

Example 38 20 mg of insulin pig was dissolved in 0.1 of any 6 M acetic acid. 0.3 ml of 0.67 M Thr (But) -Ob but N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C and 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 12 ° C, the conversion of (Thr (Bufc) -OBufc) to β 2 -h-In is 77%. The product was eluted from the HPLC column using a gradient of acetonitrile from 27% to 40%.

Example 39 20 mg of insulin pig was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 1.5 M Thr-OMe dissolved in tetrahydrofuran was added, and the mixture was cooled to 12 ° C. 2 mg of trypsin dissolved in 0.02 5 ml was added to 0.05 M calcium acetate. After 4 h at 12 ° C, B 3 O HPLC analysis showed 75% conversion to (Thr-OMe) -h-In.

Example 40 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.8 ml of 2 M Thr-OM dissolved in 1,2-ethanediol 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 was added. After 4 hours at 12 ° C HPLC analysis

SLIDE

shows 48% conversion to (Thr-OMe) -h-In. Example 41 20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of Thr-OM dissolved 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 was added, and the reaction was carried out for 4 h at 12 ° C.

Β3Π HPLC analysis shows 46% conversion (Thr-OMe) to h-Ins.

Example 42 20 mg of insulin pig was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe in acetone was added and the mixture was cooled to 12 ° C. 2 mg of trypsin 26 77876 dissolved in 0.025 of any 0.05 M calcium acetate was added.

After 4 h at 12 ° C, HPLC analysis showed 48% conversion (The-OMe) to B30-h-In.

Claims (9)

1 B29 is attached to the substituent R and Lys is attached to the substituent R, R represents an amino acid or a peptide chain containing no more than 36 amino acids and R 2 is hydrogen or a group of the general formula R-X-, where X is arginine or lysine , and R represents a pep-2. time chain containing not more than 35 amino acids, or R together with R denotes a peptide chain containing no more than 35 amino acids, with the proviso that the number of amino acids present in R and R is less than 37, or a site or complex thereof which may be converted to an insulin compound of formula I, transpeptidated with an L-threonine ester of formula II 4,% Thr (R) -OR (II) 45 where R represents a carboxyl protecting group and R represents hydrogen or a hydroxyl protecting group or a site thereof in a mixture of water, a water-miscible organic solvent and trypsin, wherein the content of water in the reaction mixture is below 50% (v / v) and the reaction temperature is below 50 ° C and possibly in the presence of a acid, B30 after which the threonine ester formed of human insulin if desired is digested. A process for the preparation of human insulin or a B30 threonine ester of human insulin or a site or complex thereof, characterized in that an insulin compound of formula IR - (1 --- A --- 21) II 2 (1 -B-29) -R (I) where - (1- A-21) (1- B-29) - B30 Al represents the human des (Thr) insulin moiety. where Gly is Process according to claim 1, characterized in that the concentration of L-threonine ester in the reaction mixture exceeds 0.1 M, that the reaction temperature is above the freezing point of the reaction mixture, and that the reaction mixture contains 0-10 equivalents of one acid per equivalent of L-threonine ester. . Method according to claim 1 or 2, characterized in that the insulin compound is porcine or rabbit insulin, porcine arginine insulin or porcine proinsulin. Method according to any of the preceding claims, characterized in that the insulin compound is of swine origin. 5. A method according to claim 4, characterized in that the insulin compound is used with relatively correct insulin. Process according to any of the preceding claims, characterized in that the reaction temperature is below 37 C, 0 preferably below room temperature and above 0 C. Process according to any of the preceding claims, characterized in that the content of water in the reaction mixture is between 40 and 10% (v / v). Process according to any of claims 1-6, characterized in that the insulin compound is porcine insulin, that the L-threotine ester is Thr-OMe or Thr-OBu, that the water-miscible organic solvent is Ν, Ν-dimethylformamide or the like.