DK147437B - METHOD FOR PREPARING HUMAN INSULIN OR THREON INBESTERS OF HUMAN INSULIN OR A SALT OR COMPLEX THEREOF - Google Patents

Description

- 1 - 147437

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

In the treatment of diabetes mellitus, insulin preparations containing porcine or bovine insulin are usually used. Bovine, porcine and human insulin exhibit minor differences in their amino acid composition, the difference between human and porcine insulin being limited to a single amino acid, the B30 amino acid in human insulin 10 being threonine, whereas in porcine insulin it is alanine. However, it can be argued that the ideal insulin preparation for treating humans is insulin, which has exactly the same chemical structure as human insulin.

The required amount of human pancreatic glands for the preparation of natural human insulin is not available.

Synthetic human insulin is manufactured on a small scale at high cost, cf. Helv.Chim.Acta 5T_, 2617, and 60, 27. Semi-synthetic human insulin is produced from porcine insulin by slow pathways, cf. Hoppe-Seyler1 s 20 Z.Physiol.Chem. 357, 759.

U.S. Patent No. 3,276,961 is claimed to provide a method of producing human semi-synthetic insulin, wherein other animal insulins are reacted with threonine in the presence of an enzyme such as trypsin or carboxypeptidase 25 A. However, the yield of human insulin is extremely low ( the process is carried out in water, under which conditions trypsin causes B22 go * 3 cleavage of the Arg -Gly bond, cf. J. Biol. 236, 743.

Another known semisynthetic method for

preparation of human insulin comprises the following three steps:

In the first step of B3, porcine insulin is cleaved to porcine des (Ala) insulin by treatment with carboxypeptidase A, cf. Hoppe-Seyler's Z. Physiol.Chem. 359, 799. In the second step, 35 porcine des (Ala) insulin is subjected to a trypsin-catalyzed coupling with Thr-OBu1 ", thereby forming the ThrB30 tert-butyl ester of human insulin. In the third step, the ester in question is treated with 2- trifluoroacetic acid to give human insulin, cf.

Nature 280, 412. However, the first step results in a partial removal of AsnA21 to give des- (AlaB20, AsnA21) insulin. This derivative, after the two subsequent steps A21, gives rise to contamination with des- (Asn) insulin in the manufactured semi-synthetic human insulin, which is a contamination which cannot be easily removed with known preparative methods A21. Des- (Asn) insulin exhibits low biological activity (about 5%), cf. Amer.J.Med. £ 0, 750.

10 Surprisingly, it has now been found that insulin compounds which are different from human insulin, e.g. porcine insulin and certain impurities therein can be converted to human insulin by a process in which, in one step, the insulin compounds are directly converted to a threonine ester of human insulin. This method provides human insulin with substantially greater yield than the known methods and without by-products which are difficult to remove.

The present invention is based on the discovery that the amino acid or peptide chain linked to the car-B2-9 bonyl group, Lys, in the insulin compound of the general formula I of claim 1 can be replaced with a threonine ester in one step. The replacement in question is referred to herein as a transpept timing.

Insulin compounds of formula I are insulins and insulin-like compounds containing the human des (Thr) insulin moiety in which the B30 amino acid of the insulin is alanine (in, for example, pigs, dogs, fin whales and spermacet whales) or serine (rabbits). . The term "insulin-like compounds" as used herein includes proinsulin derived from any of the above species and primates as well as intermediates from the conversion of proinsulin to insulin. Examples of such intermediates may be split pro-insulin, desdipeptide proinsulin, desnonapeptide proinsulin and diarginine insulin, cf. R. Chance: In Proceedings of the 35th Seventh Congress of IDF, Buenos Aires 1970, 292 - 305,

Editors: R.R. Rodrigues & J.V.-Owen, Excerpta Medica,

Amsterdam.

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The method of the present invention is characterized in that an insulin compound of the general formula I and different from human insulin or several compounds of the general formula I

5 R ^ d --- A --- 21) I I? (IN)

(B 1 --- --- 29) -R

where - (1 --- A --- 21)

I I

10 (1 --- B --- 29) - denotes the human (Thr® +) insulin moiety wherein GlyA · * · is linked to the substituent designated R and Lys is connected 2 2 to the substituent designated R , R represents an amino acid or peptide chain containing not more than 36 amino acids, and Rx 15 represents hydrogen or a group of the general formula 3 R -X- wherein X represents argmin or lysine and R represents 2

a peptide chain containing not more than 35 amino acids, or R

3 together with R represents a peptide chain containing a maximum of 35 amino acids, with the proviso that the number of amino acids, 1 2

20 present in R and R is less than 37, or a salt or complex thereof which can be converted thereto is transpeptidated with an L-threonine ester of the general formula II

Thr (R5) -OR4 (II) 45 where R represents a carboxyl protecting group and R represents hydrogen or a hydroxyl protecting group, or a salt thereof in a mixture of water, a water miscible organic solvent and trypsin wherein the water content in the reaction mixture is below 50% (volume / volume) and the reaction temperature is below 50 ° C, and optionally a B3 0 30 acid is present, and the resulting threonine ester of human insulin is optionally cleaved.

Although trypsin is best known for its proteolytic properties, those skilled in the art have found that trypsin is capable of catalyzing the coupling of desboa 35 (Ala) insulin and a threonine tert-butyl ester, cf. Nature, 147437 - 4 - 280, 412. However, in the process of the present invention, trypsin is used to catalyze a transpeptidation.

According to the above, Nature 280, 412 relates to a method for peptide coupling, whereas the present invention relates to a method for transpeptidizing peptides. Disregarding the step of hydrolyzing the human insulin ester, according to Nature 280, 412, two steps must be used to prepare the human insulin ester, namely a peptide cleavage step and a peptide coupling step. By the method of the present invention, the human insulin ester is obtained directly in one step, namely a transpeptidation step.

In Nature 280, 412, it is stated that it is surprising that there is no cleavage of the Arg-Gly 15 bond in the product. It is known that trypsin in aqueous medium cleaves at both Arg -Gly and at Lys -Ala, and it is therefore to be expected, on the basis of Nature 280, 412, that B2 9 trypsin in the organic medium also does not cleave Lys B3 0

The Ala bond. In Nature 280, 412, there is no indication of where * B29 B30 20 is capable of cleavage of the Lys-Ala bond in the organic medium. Accordingly, it is very surprising that the transpeptidation of the present invention can be performed directly on swine insulin.

In Biochem.Biophys.Res.Com. j) 2, 396 discloses a method for producing human insulin, which is substantially identical to that described in Nature 280, 412, although, as an enzyme, Achromobacter lyticus proteinase is used. Accordingly, the above considerations regarding Nature 280, 412 apply by analogy to Biochem.Biophys.Res.Com. 92, 30 396.

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

_ 5 _ 147437

Preferred water-miscible organic solvents are polar solvents. As specific examples of such solvents may be mentioned methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, 5 Ν, Ν-dimethylformamide, formamide, Ν, Ν-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphorotriamide and acetonitrile. .

Depending on the water-miscible organic solvent used, the reaction temperature chosen and the presence of an acid in the reaction mixture, the content of water in the reaction mixture is preferably between 43 and 10% (volume / volume). In a preferred embodiment of the process of the present invention, the content of water in the reaction mixture is between 43 and 10% (volume / volume), thereby obtaining a product having a relatively low content of impurities.

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

In practicing the present invention, it is not essential what kind of trypsin is used. Trypsin is a well-characterized enzyme that is commercially available in high purity, especially from bovine and porcine sources. Furthermore, trypsin of microbial origin can be used. In practicing this invention, the trypsin information, e.g. crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (as long as trypsin activity is maintained), furthermore not essential. The term trypsin used herein is intended to include trypsins of any origin and all trypsin information which have retained the transpeptiding activity, including proteases with trypsin-like specificity, e.g. Achromobacter lyticus protease, cf.

35 Agric.Biol.Chem. j42, 1443.

_ 6_ 147437

As examples of active trypsin derivatives may be cited acetylated trypsin, succinylated trypsin, glutaraldehyde-treated trypsin and immobilized trypsin derivatives.

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

To obtain a proper buffer system, organic or inorganic acids such as hydrochloric, formic, acetic, propionic and butyric and bases such as pyridine, TRIS, N-methylmorpholine and N-ethylmorpholine can be added. 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%. 10 equivalents per equivalent L-threonine ester. The amount of acid is preferably between 0.5 and 5 equivalents per minute. equivalent L-threonine ester. A preferred embodiment of the process of the present invention is that the concentration of L-threonine ester in the reaction mixture exceeds 0.1 molar and that the reaction mixture contains from 0 to 10 equivalents of one acid per liter. equivalent L-threonine ester.

Ions may be added which stabilize trypsin such as calcium ions.

In order to obtain a sufficient reaction rate, enzymatic reactions with trypsin are usually carried out at ca. 37 ° C. The process of the present invention can be carried out at a temperature in the range of 50 ° C to the freezing point of the reaction mixture. However, to avoid inactivation of trypsin, it is advantageous to carry out the method of the present invention at a temperature below room temperature. In practice, reaction temperatures above about 30 degrees are preferred. 0 ° C. Accordingly, a preferred embodiment of the process of the present invention is that the reaction temperature is below 37 ° C, preferably below room temperature, and above 0 ° C.

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

_7. The weight ratio of trypsin to the insulin compound in the reaction mixture is usually above about 10%. 1: 200, preferably above ca. 1:50, and under approx. 1: 1.

rjo A

The threonine esters of human insulin obtained by the transpeptidation may be represented by the general formula III

(Thr (R5) -OR4) B30-h-In (III) where h-In represents the human des- (ThrB30) insulin moiety and R4 and R5 are as defined above.

Useful L-threonine esters of formula II are thus in which R is a carboxyl protecting group which can be removed from the threonine ester of human insulin (formula III) under conditions which do not cause significant, irreversible change in the insulin molecule. Examples of such carboxyl protecting groups may be lower alkyl, e.g.

Methyl, ethyl and tert-butyl, substituted benzyl such as p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenylmethyl, and five groups of the general formula -CH 2 CH 2 SO 2 R where R represents lower alkyl such as methyl, ethyl, propyl and n butyl. Suitable hydroxyl protecting groups R5 are those which can be removed B3 π 20 from the threonine ester of human insulin (Formula III) under conditions which do not cause significant, irreversible change in the insulin molecule. As an example of such a group 5 (R) may be indicated tert.butyl.

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

Additional, commonly used protecting groups are described by Wunsch: The Method of Organizational Chemistry (Houben-Weyl), Vol. XV / 1, editor: Eugen Muller, Georg Thieme Verlag, Stuttgart 1974.

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

The 8- threonine ester of formula II may be the free bases or suitable organic or inorganic salts thereof, preferably acetates, propionates, butyrates and hydrohalogens such as hydrochlorides.

It is desirable to use the reactants, viz.

the insulin compound and the L-threonine ester (Formula II), in high concentrations. The molar ratio of the L-threonine ester to the insulin compound is preferably above ca. 5: 1.

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

Human insulin can be obtained from threonine esters of human insulin (Formula III) by removal of protective groups.

pen R and any protecting group present R

15 in a known manner or in a manner known per se. If R4

C

is methyl, ethyl or a group of the formula -CCC ^SC ^R wherein R ^ is as defined above, the protecting group concerned may be removed under mild basic conditions in an aqueous medium, preferably at a pH of between about 8 20 and approx. 12, e.g. at about. 9.5. As the base may be used ammonia, triethylamine or hydroxides of alkali metals such as sodium hydroxide. If R 4 is tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, the group in question can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoroacetic acid may be non-aqueous or may contain some water or it may be diluted with an organic solvent such as dichloromethane. if RD is tert-butyl, that group may be removed by acidolysis, cf. above.

Preferred threonine esters of human insulin of Formula III are compounds wherein R is hydrogen and these can be prepared from L-threonine esters of Formula II wherein R 1 is hydrogen.

9 - 147437

Thus, in the transpeptidation of the present invention, any of the above insulin compounds are converted to human insulin threonine esters (Formula III) which can then be cleaved to form human insulin.

A further advantage of the present invention is the fact that in the transpeptide ring of the present invention, insulin-like compounds present in crude insulin and in some commercial insulin preparations covered by formula I are converted to B3 g of threonine. human insulin esters, which compounds can then be cleaved to form human insulin. Examples of insulin-like compounds of formula I are as follows: 1 2 15 Swine dietsulin insulin (R is hydrogen and R is 2 3 -Ala-Arg-Arg), porcine proinsulin (R is together with R -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-Lys-, where the terminal alanyl is attached to B2 9 2 3 20 Lys), canine proinsulin (R is together with R -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-, where the terminal B2 9 2 alanyl is linked to Lys), pig split proinsulin (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, and R 3 is Al

Leu-Glu-Gly-Pro-Pro-Gln-Lys-), porcine dipeptide proinsulin 1 2 (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 2 3 30 (R is 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-Gly-Ser-Leu-Gln-Lys-, where the B2 9 terminal threonyl is linked to Lys) and monoproinsulin 2 3 (R is together with R -Thr-Arg -Arg-Glu-Ala-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-, where the B2 9 terminal threonyl is linked to Lys).

In all these impurities covered by Formula I, 1 3, the R substituent designated R-X- is thus replaced by hydrogen.

As swine insulin is relatively readily available, as a starting material in a preferred embodiment of the invention, swine insulin is used. A preferred embodiment of the method of the present invention is to react raw pig insulin containing insulin-like compounds or a salt or complex thereof with an L-threonine ester (formula II) or a salt thereof under the above conditions, whereupon the protecting group 45

R and any protecting group present R

removed. By this method, swine insulin and insulin-like compounds therein are converted to human insulin.

As examples of a complex or salt of an insulin compound (formula I), a zinc complex or zinc salt may be disclosed.

When the reaction conditions are selected according to the above explanation and taking into account the results obtained in the following examples, it is possible to obtain a 333 0 yield of human insulin threonine ester above 60% and even above 80% and under certain conditions. preferred conditions above 90%. In a preferred embodiment of the process according to the present invention, the content of water in the reaction mixture is between 43 and 20% (volume / volume). A further preferred embodiment of the process of the present invention is that the insulin compound is porcine insulin, that the L-threonine ester is Thr-OMe or Thr-OBu *, that the water-miscible organic solvent is Ν, Ν- dimethylformamide or N, N-dimethylacetamide, the reaction temperature being about 37 ° C, the content of water in the reaction mixture is between 41 and 43%, the weight ratio of trypsin to the insulin compound is approx. 1: 8 that the molar ratio of the L-threonine ester to the insulin compound is about 35 120: 1 and that between 1.2 and 1.5 equivalents of acetic acid per liter is added. equivalent L-threonine ester.

_ n _ 147437

Therefore, the process of the present invention has the following advantages over the prior art: a) The enzymatic hydrolysis to remove DO Λ 5 Ala, e.g. with carboxypeptidase A, is omitted.

b) The isolation of an intermediate such as swine B3O des- (Ala) insulin is unnecessary.

A21 c) Contamination with des (Asn) insulin derivatives is avoided.

D) Proinsulin and other insulin-like impurities present in raw swine insulin are converted by the method of this invention - via the threonine ester of human insulin - to human insulin, thereby increasing the yield.

e) Antigenic insulin-like compounds, cf.

British Patent No. 1,285,023 is converted to human insulin.

A preferred method of producing human insulin is the following: 1) The starting material used for transpeptidation is raw pig insulin, e.g. crystalline insulin prepared using a citrate buffer, cf.

U.S. Patent No. 2,626,228.

2) If there is trypsin activity remaining after transpeptidation, it is preferred to remove it, e.g.

25 under conditions in which trypsin is inactive, e.g. in acidic medium at a pH below 3. Trypsin can be removed by separation by molecular weight, e.g. by gel filtration on "Sephadex G-50" or "Bio-gel P-30" in 1 M acetic acid, cf. Nature 280, 412.

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

B30 4) Then the threonine ester is cleaved by human insulin and human insulin is isolated, e.g. crystallized, in a manner known per se.

In this way, human insulin can be obtained with an acceptable pharmaceutical purity and, if desired, the human insulin can be further purified.

Abbreviations used are in accordance with the 5 rules approved (1974) by the IUPAC-IUB Commission on Biochemical Nomenclature, cf. Specifically, the meaning of the abbreviations used herein is as follows: Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Gin is glutamine, Glu is glutamic acid, Gly is glycine, Leu is leucine, Lys is lysine, Pro is proline, Ser is serine, Thr is threonine, and Val is valine. Bu * is tert-butyl and Me is methyl.

15 Sephadex and Bio-gel are trademarks.

Analytical studies:

The conversion of porcine insulin and porcine insulin 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 a rate 75% of the swine insulin rate. Pig proinsulin, which migrates at 55% of the rate of porcine insulin, is converted into the same product by the method of this invention.

The identification of the conversion product as compounds of formula III is due to the following criteria: a) The electrophoretic migration of human insulin esters of formula III relative to swine insulin corresponds to the loss of one negative charge.

B) The amino acid composition of the stained protein bands in the gel representing compounds of formula III is identical to the amino acid composition of human insulin, ie. 3 moles of threonine and 1 mole of alanine per and the composition of porcine insulin is 2 moles of threonine and 2 moles of alanine per mole. moles of insulin. The technique for analyzing amino acid compositions of protein bands in polyacrylamide gels is described in Eur.J.Biochem. 2, i, 147.

(C) The evidence that the incorporated threonine is positioned as C-terminal amino acid in the B chain can be obtained by oxidative sulfitolysis of the SS bridges of insulin in 6 M guanine dinohydrochloride followed by separation of A and B chains by ion exchange chromatography on "SP Sephadex". By digesting the B-chain S-sulfonate with carboxypeptidase A, only the C-terminal amino acid is released. The technique is described by Markussen in Proceedings of the Symposium on Proinsulin, Insulin and C-peptide, Tokushima, July 12-14, 1978 (publisher:

Baba, Kaneko and Yaniahara) Int.Congress Series No. 468, 15 Excerpta Medica, Amsterdam-Oxford. The assay is performed after the ester group is cleaved from compounds of formula III.

The 3 analyzes clearly show that human insulin has been converted.

The conversion of porcine insulin and porcine insulin to 20 human insulin esters can be quantitatively monitored by reverse phase high pressure liquid chromatography (HPLC). A 4 x 300 mm Bondapak Cg column (Waters Ass.) Is used and the elution is performed with a buffer containing 0.2 M ammonium sulfate (adjusted to a pH of 3.5 with sulfuric acid) and containing 26 - 50% acetonitrile.

vibration concentration depends on which ester of formula III

<. 4 one wants to separate from swine insulin. If R is methyl and is hydrogen, separation is obtained in 26% (volume / B30 volume) of acetonitrile. Swine insulin and (Thr-OMe) -h-ln (Me 30 are methyl) elute by 4.5 and 5.9 column volumes, respectively, as clearly separated symmetric peaks. Prior to application to the HPLC column, the proteins are precipitated in 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.

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The process for preparing human insulin esters and human insulin is illustrated in more detail by the following examples which illustrate some preferred embodiments of the process of the invention.

5 µBondapak is a trademark.

Example 1 200 g crude pig insulin once crystallized is 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 are added. After standing for 18 hours at 37 ° C, the proteins are precipitated by the addition of 40 ml of acetone and the precipitate is isolated by centrifugation. The supernatant is discarded. Analysis of the precipitate by HPLC using 26% acetonitrile (cf. Analytical studies) B30 15 shows a 60% conversion of swine insulin to (Thr-OMe) h-In. The precipitate is dissolved in 8 ml of freshly deionized 8 M urea, the pH is adjusted to 8.0 with 1 M ammonia, the solution is passed on a 2.5 x 25 cm column filled with "QAE A-25 Sephadex" which is equilibrated with a 0.1 M ammonium-chloride buffer containing 60% (v / v) ethanol and adjusted to pH 8.0 with ammonia. The elution is performed with the same buffer and 15 ml fractions are collected. (Thr-OMe) -h-In is found in Fractions Nos. 26-46, and unreacted porcine insulin in Fractions Nos. 90-120.

Fractions Nos. 26-46 are combined, the ethanol is evaporated in vacuo B3 µm, and (Thr-OMe) -h-In is crystallized in citrate buffer as described by Schlichtkrull et al., Handbuch der inneren Medizin, 7/2A, 96, Berlin, Heidelberg, New York 1975. The yield is 95 mg crystals of the same rhombic form as porcine insulin, crystallized in an analogous manner. The amino acid composition is found to be identical to the human insulin amino acid composition. Further analytical studies described in the above section: "Analytical Investigation-B3 q ser" show that the resulting product is (Thr-OMe) -h- m.

Example 2 100 mg of swine insulin meeting the purity criteria set forth in British Patent Specification No. 1,285,023 is dissolved in 0.9 ml of 3.33 M acetic acid and then 1 ml of a 2 M solution of threonine methyl ester is added. in N, N-dimethylformamide and 12 mg of TPCK-treated (TPCK is tosyl-10-phenylalanine chloromethyl ketone) trypsin, which is dissolved in 0.1 ml of water. After incubation for 24 hours at 37 ° C, the reaction is quenched by the addition of 4 ml of 1 M phosphoric acid. The obtained (Thr-B 30 OMe) -h-In is separated from unreacted porcine insulin by ionic

exchange chromatography on a 2.5 x 25 cm column of "SP

Sephadex "with an eluent containing 0.09 M sodium chloride and 0.02 M sodium dihydrogen phosphate (pH of buffer: 5.5) in B3 0 60% ethanol. Fractions containing (Thr-OMe) -h-In are collected, the ethanol in vacuo and the product is crystallized in an analogous manner as described in Example 1. The yield is 50 mg of 20 (Thr-OMe) B30-h-In.

Example 3 100 mg of swine proinsulin is dissolved in 0.9 ml of 3.33 M acetic acid and converted to (Thr-OMe) h-In and purified by analogy as described for swine insulin in Example 1. The conversion of proinsulin to (Thr OMe} B3 ^ -h-In is found to be 73% by HPLC analysis of the acetone precipitate The yield of crystalline tao λ (Thr-OMe) -h-In is 54 mg.

EXAMPLES 4 - 16 - 147497 100 mg of porcine insulin are dissolved in 0.9 ml of 2.77 M acetic acid in water and reacted in an analogous manner as described in Example 2. After completion of the reaction, the proteins 5 are precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE shows a conversion to (Thr-OMe) -h-In of 70%.

Example 5 100 mg of pig insulin is dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml of 2 M Thr-OMe is added in N-methylpyrrolidone.

The reaction is carried out in an analogous manner as described in Example B3 0 4 and the conversion to (Thr-OMe) -h-In is 20%.

Example 6 100 mg of swine insulin is dissolved in 0.9 ml of 2.77 M acetic acid in water and 1 ml of 2 M Thr-OMe is added in HMPA (hexamethylphosphorus triamide). The reaction is carried out in an analogous manner as B3O described in Example 4. The conversion to (Thr-OMe) -h-In is 80%.

Example 7 100 mg of porcine insulin is dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml of 2 M Thr-OMe is added in N, N-dimethylacetamide. The reaction is carried out in an analogous manner as described in DO Λ Example 4. The conversion to (Thr-OMe) -h-In is 80%.

Example 8 100 mg of porcine insulin is dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml of 2 M Thr-OMe is added in N, N-dimethylacetamide. Then, 200 U (units = units) of trypsin inactivity (determined with the BAEE substrate) immobilized on 1 g of glass beads is added and, after incubation at 37 ° C for 24 hours, is filtered off. The trypsin bound to the glass is treated. After completion of the reaction, the proteins are precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE shows a conversion to (Thr-OMe) B30-h-In of 40%.

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

Example 10

The procedure of Example 7 is repeated with the proviso that the ester used is 2 M Thr-OBu (Bu is tert-butyl) in Ν, Ν-dimethylacetamide. The conversion to (Thr-OBut) B30-h-In is 80%.

Example 11

The procedure of Example 8 is repeated with the proviso that the ester used is 2 M Thr-OBu1 "in N, N- t B30 dimethylacetamide. The conversion to (Thr-OBu) -h-In is 30%. Example 12 The procedure of Example 9 is repeated with the proviso that the ester used is 2 M Thr-OBu · i, Ν-dimethylacetamide The conversion to (Thr-OBu * ·) B 3--h-In is 70%.

_ 18. 147437

Example 13 Dissolve 100 mg of porcine insulin in 0.9 ml of 3.33 M acetic acid and add 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide. The reaction is carried out in an analogous manner as described in con. Example 4. The conversion to (Thr-OMe) -h-In is 80%.

Example 14 100 mg of pig proinsulin is dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml of 2 M Thr-OMe is added in N, N-dimethylacetamide. The mixture is treated with immobilized trypsin in an analogous manner as described in Example 8. The conversion to (Thr-OMe) B30-h-In is 40%.

Example 15 Dissolve 100 mg of porcine insulin in 0.9 ml of 3.33 M acetic acid and add 1 ml of 2 M Thr-OMe in N, N-dimethyl-acetamide. The mixture is treated with immobilized trypsin in an analogous manner as described in Example 9. The conversion to

Dl n (Thr-OMe) -h-In is 70%.

Example 16 Dissolve 100 mg of porcine insulin in 0.9 ml of 3.33 M acetic acid and add 1 ml of 2 M Thr-OBut in N, N-dimethylacetamide. The reaction is carried out in an analogous manner as described in Example 4. The conversion to (Thr-OBu * ") B3 ^ -h-In is 80%.

Example 17 Dissolve 100 mg of porcine insulin in 0.9 ml of 3.33 M acetic acid and add 1 ml of 2 M Thr-OBu1 * in N, N-dimethylacetamide. The mixture is treated with trypsin immobilized on glass beads in an analogous manner as described in Example 8. The conversion to (Thr-OBut) B3 + -h-In is 40%.

_19- 147437

Example 18 100 mg of porcine insulin is dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml of 2 M Thr-OBu1 "is added to N, N-dimethylacetamide. The mixture is treated with trypsin immobilized to CNBr -activated "Sephadex G-150", by analogy as described in Example 9. The conversion to (Thr-OBu *) B3

Example 19 100 mg of porcine insulin is dissolved in 0.5 ml of 6 M acetic acid, 10 and 1 ml of 1 M Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) in Ν, Ν-dimethylacetamide is added. An additional 0.5 ml of Ν, Ν-dimethylacetamide and 5 mg of TPCK-treated trypsin are added in 0.1 ml of water. The reaction mixture is stored at 32 ° C for 44 hours.

After completion of the reaction, the proteins are precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE shows B30 o a conversion to (Thr-OTmb) -h-In of 50%.

Example 20 100 mg of pig insulin is dissolved in 0.9 ml of 3 M acetic acid and 1 ml of 2 M Thr-OMe is added in dioxane. The reaction is carried out in an analogous manner as described in Example 4 and the conversion to (Thr-OMe) 03 ° -h-In is 10%.

Example 21 100 mg of pig insulin is dissolved in 0.9 ml of 3 M acetic acid and 1 ml of 2 M Thr-OMe is added in acetonitrile. Reaction 25 is carried out in an analogous manner as described in Example 4, and convert R The yield for (Thr-OMe) -h-In is 10%.

Examples 22 - 20 - 147437 B3 0 250 mg of crystalline (Thr-OMe) -h-In, prepared as described in Examples 1-9, 13 - 15, 20 or 21, are dispersed in 25 ml of water and dissolved by adding 1 N 5 sodium hydroxide solution to a pH of 10.0. The pH is kept constant at 10.0 for 24 hours at 25 ° C. The human insulin formed is crystallized by the addition of 2 g of sodium chloride, 350 mg of sodium acetate trihydrate and 2.5 mg of zinc acetate dihydrate followed by addition of 1N hydrochloric acid to give a pH of 5.52. The rhombic crystals are isolated by centrifugation after storage for 24 hours at 4 ° C, washed with 3 ml of water, isolated by centrifugation and dried in vacuo. Yield: 220 mg of human insulin.

Example 23 B3 0 15 100 mg (Thr-OTmb) -h-In, prepared as described in Example 19, is dissolved in 1 ml of ice-cold trifluoroacetic acid and the solution is stored for 2 hours at 0 ° C. The human insulin formed is precipitated by the addition of 10 ml of tetrahydrofuran and 0.97 ml of 1.03 M hydrochloric acid in tetrahydrofuran. The 20 precipitate formed is isolated by centrifugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation and dried in vacuo. The precipitate is dissolved in 10 ml of water and the pH of the solution is adjusted to 2.5 with 1 N sodium hydroxide solution. The human insulin is precipitated by the addition of 1.5 g of sodium chloride and isolated by centrifugation. The precipitate is dissolved in 10 ml of water and the human insulin is precipitated by the addition of 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 obtain a pH of 5.52. The precipitate is isolated by centrifugation after standing for 24 hours at 4 ° C, washed with 0.9 ml of water, isolated by centrifugation and dried in vacuo. Yield: 90 mg of human insulin.

EXAMPLES 24 - 21 - 100 mg of porcine insulin are dissolved in 0.5 ml of 10 M acetic acid and 1.3 ml of 1.54 M Thr-OMe is added in N, N-dimethylacetamide. The mixture is cooled to 12 ° C. 10 mg of trypsdn 5 dissolved in 0.2 ml of 0.05 M calcium acetate is added. After 48 hours at 12 ° C, the proteins are precipitated by the addition of 20 ml of acetone. converted

The donation of swine insulin to (Thr-OMe) -h-In is 97% by HPLC. Example 25

20 mg of swine insulin is 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 is added

Thr-OMe in Ν, Ν-dimethylacetamide and the mixture is cooled to -10 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added. After 72 hours at -10 ° C, the proteins are precipitated by the addition of 5 ml of acetone. The conversion of pig insulin

Don 15 to (Thr-OMe) -h-In is 64% by HPLC.

Example 26 20 mg of swine insulin is dispersed in 0.1 ml of water. By the addition of 0.6 ml of 2 M Thr-OMe in Ν, Ν-dimethylacetamide, the insulin dissolves. The mixture is cooled to 7 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added.

After 24 hours at 7 ° C, the proteins are precipitated by the addition of

Add 5 ml of acetone. The conversion of porcine insulin to (Thr-OMe) h-In is 62% by HPLC.

Example 27 25 mg of porcine insulin is dissolved in 0.135 ml of 4.45 M propionic acid. 0.24 ml of 1.67 M Thr-OMe in Ν, Ν-dimethylacetamide is added. 2 mg of trypsin is added in 0.025 ml of 0.05 M calcium acetate and the mixture is stored at 37 ° C for 24 hours. The proteins are precipitated by the addition of 10 volumes of 2-propanol. The conversion of porcine insulin to (Thr-OMe) -h-In is 75% by HPLC.

Example 28 20 mg of swine insulin is dispersed in 0.1 ml of water. 0.4 ml of 2 M Thr-OMe is added in Ν, dim-dimethylacetamide, followed by 0.04 ml of 10 N hydrochloric acid to dissolve the insulin. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added and the mixture is stored at 37 ° C for 4 hours.

10 HPLC analysis shows a 46% conversion of swine insulin to (Thr-OMe) B30-h-In.

Example 29 20 mg of porcine insulin is dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of 2 M Thr-OMe in Ν, Ν-dimethylacetamide 15 is added followed by the addition of 0.025 ml of 0.05 M calcium acetate.

10 mg of a crude preparation of Achromobacter lyticus protease is added and the mixture is stored for 22 hours at 37 ° C. The proteins are precipitated by the addition of 10 volumes of ace-B3 η tone. The conversion of porcine insulin to (Thr-OMe) -h-In is 20% by HPLC.

Example 30 20 mg of porcine insulin is dispersed in 0.1 ml of 0.5 M acetic acid. By adding 0.2 ml of 0.1 M Thr-OMe in Ν, Ν-dimethylacetamide, the insulin is dissolved. The mixture is cooled to 12 ° C and 2 mg of trypsin is added in 0.025 ml of 0.05 M calcium acetate.

After 24 hours at 12 ° C, the HPLC analysis shows a 42% conversion of porcine insulin to (Thr-OMe) -h-In.

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Example 31 Dissolve 2 mg of rabbit insulin in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe is added in N, N-dimethylacetamide, followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate. The mixture is stored at 37 ° C for 4 hours. Analysis by HPLC shows an 88% conversion of rabbit B30 insulin to (Thr-OMe) -h-In. Rabbit insulin elutes before swine insulin from the HPLC column, with the ratio of eluate B3 q volume of rabbit insulin to (Thr-OMe) -h-In being 0.72.

Example 32 B31 B32 Dissolve 2 mg of swine diarrhea insulin (Arg-Arg-insulin) in 0.135 ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe in Ν, Ν-dimethylacetamide is added followed by addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M calcium acetate.

The mixture is stored at 37 ° C for 4 hours. HPLC analysis shows B3 π a 91% conversion of diarginine insulin to (Thr-OMe) -h-

In. Diarginine insulin elutes before swine insulin from the HPLC column, with the ratio of the elution volume of diarginine B 30 insulin to (Thr-OMe) -h-In being 0.50.

Example 33 2 mg of "pig intermediates" (i.e. mixture of desdipeptide (Lys0 -Arg J) proinsulin and desdipeptide- (Arg 32

Arg) proinsulin) is reacted in an analogous manner as described in Example 32. HPLC analysis shows 88% (Thr-OMe) B ^-h-In.

Example 34 1 mg of swine insulin is dissolved in 0.1 ml of 2 M acetic acid.

0.2 ml of 2 M Thr-OMe is added in Ν, Ν-dimethylacetamide and the mixture is cooled to -18 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added and the mixture is stored for 120 hours at -18 ° C. HPLC analysis shows that the conversion to (Thr-OMe) B3 ° -h-In is 83%.

Example 35 The procedure of Example 34 is repeated with the proviso that the reaction is carried out at 50 ° C for 4 hours.

B30 HPLC analysis shows that the conversion to (Thr-OMe) -h-In is 23%.

Example 36 10 20 mg of swine insulin are dissolved in 0.1 ml of 3 M acetic acid.

0.3 ml of 0.33 M Thr-O (CH 2) 2-SO 2 -CH 3, CH 3 COH (threonine-2- (methylsulfonyl) ethyl ester hydroacetate) in N, N-dimethylacetamide are added. The mixture is cooled to 12 ° C and 2 mg of trypsin is added in 0.025 ml of 0.05 M calcium acetate. HPLC shows, after 24 hours at 12 ° C, a 77% conversion to (Thr-O (CH 2) - SO 2 -CH 2, B 3 3 -h-In. The product elutes approximately at the ^^^^ B30 position of (Thr-OMe) -h-In.

Example 37 20 mg of swine insulin is dissolved in 0.1 ml of 10 M acetic acid.

0.2 ml of 2 M Thr-OEt (Et is ethyl) is added in N, N-

dimethylacetamide and the mixture is cooled to 12 ° C. 2 mg of trypsin is added in 0.025 ml of 0.05 M calcium acetate. HPLC

after 24 h at 12 ° C shows a 75% conversion to (Thr-30 OEt) -h-In. The product elutes in a position just after 25 (Thr-OMe) B30-h-In.

Example 38 20 mg of swine insulin is dissolved in 0.1 ml of 6 M acetic acid.

0.3 ml of 0.67 M Thr (Bufc) -OBu * 1 is added in N, N-dimethylacetamide. The mixture is cooled to 12 ° C and 2 mg of trypsin is added in 0.025 ml of 0.05 M calcium acetate. After 24 hours at 12 ° C, the conversion to (Thr (Bu +) - OBu +) B3 + -h-In is 77%. The product is eluted from the HPLC column using a gradient in acetonitrile from 27% to 40%.

Example 39 20 mg of porcine insulin is dissolved in 0.1 ml of 4 M acetic acid.

0.2 ml of 1.5 M Thr-OMe dissolved in tetrahydrofuran is added and the mixture is cooled to 12 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added. After 4 hours at 10 12 ° C, HPLC analysis shows a conversion to (Thr-OMe) B3 + -h-In of 75%.

Example 40 20 mg of porcine insulin is dissolved in 0.1 ml of 4 M acetic acid.

0.8 ml of 2 M Thr-OMe dissolved in 1,2-ethanediol is added and the mixture is cooled to 12 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added. After 4 hours at 12 ° C

B30 shows HPLC analysis a 48% conversion to (Thr-OMe) -h-In.

Example 41 20 20 mg of porcine insulin are dissolved in 0.1 ml of 4 M acetic acid.

0.2 ml of 2 M Thr-OMe dissolved in ethanol is added and the mixture is cooled to 12 ° C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate is added and the reaction is carried out for 4 hours at 12 ° C. HPLC analysis shows a 46% conversion to 25 (Thr-OMe) B30-h-In.

Claims (8)

1 B29 is linked to the substituent designated R, and Lys is linked 2 2 to the substituent designated R, R represents an amino acid or peptide chain containing no more than 36 amino acids, and R 1 represents hydrogen or a group of the general formula 3 R-X-, where X represents arginine or lysine and R represents. . 2 represents a peptide chain containing at most 35 amino acids, or R 3 together with R represents a peptide chain containing at most 35 amino acids, with the proviso that the number of amino acids,. 1 2 present in R and R is less than 37, or a salt or complex thereof convertible thereto, is transpeptidated with an L-threonine ester of the general formula II - 27 - 147437 Thr (R5) -OR4 (II) 45 wherein R represents a carboxyl protecting group and R represents hydrogen or a hydroxyl protecting group, or a salt 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 optionally a B30 acid is present and the resulting threonine ester of human insulin is optionally cleaved. A process for the preparation of human insulin B30 or threonine esters of human insulin or a salt or complex thereof, characterized in that an insulin compound of the general formula I and different from human insulin or several compounds of the general formula I R1- (1- -A --- 21) II n (I) 15 (1 --- B --- 29) -R where - (1 --- A --- 21) II (1 --- B --- 29 ) - denotes the human des (ThrB30) insulin moiety in which GlyA1 is present. Process according to claim 1, characterized in that the concentration of L-threonine ester in the reaction mixture exceeds 0.1 molar and that the reaction mixture contains from 0 to 10 equivalents of one acid per liter. equivalent L-threonine ester. Method according to any of the preceding claims, characterized in that the insulin compound is of swine origin. Method according to claim 3, characterized in that relatively crude insulin is used as an insulin compound. Process according to any one of the preceding claims, characterized in that the reaction temperature is below 37 ° C, preferably below room temperature, and above 0 ° C. Process according to any one of the preceding claims, characterized in that the content of water in the reaction mixture is between 43 and 10% (volume / volume). Process according to claim 6, characterized in that the content of water in the reaction mixture is between 43 and 20% (volume / volume). Process according to any one of claims 1-5, characterized in that the insulin compound is porcine insulin, that the L-threonine ester is Thr-OMe or Thr-OBu *, that the water-miscible organic solvent is Ν, Ν-dimethylformamide or Ν, Ν-dimethylacetamide, the reaction temperature is about 37 ° C, the content of water in the reaction mixture is between 41 and 43%, the weight ratio of