NL8100624A - PROCESS FOR PREPARING INSULIN ESTERS. - Google Patents

Description

N.G. 29,770

Method of preparation of insulin esters

The present invention relates to the conversion of insulin and insulin-like compounds to human insulin.

In the treatment of diabetes aellitus, insulin 5 preparations derived from porcine or bovine insulin are generally used. Sunder, porcine and human insulins show minor differences with regard to their amino acid composition, with the difference between human and porcine insulin limited to a single amino acid, in that the B30 amino acid of human insulin is threonine, while that of pig insulin is alanine. However, it can be argued that the ideal insulin formulation for the treatment of man will be an insulin

insulin

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

Synthetic human insulin has been prepared on a small scale at a great cost, see Helv. Chim. Acta 57, 2617 and 60, 27 · Semi-20 synthetic human insulin is prepared from porcine insulin by long-winded routes, see Hoppe-Seyler's Z. Physiol. Chem. 55 °> 1β31, and Nature 280, ij-12. However, the present invention relates to a method which can be used industrially for the preparation of human insulin.

US patent 3,270,961 claims to relate to a method of preparing semisynthetic human insulin. However, the yield of human insulin is poor because the process is performed in water under which conditions trypsin cleaves the Arg-Gly-bond, see J. Biol. Chem. 236, 7k3

A known semi-synthetic method for the preparation of human insulin involves the following three steps: Τ32.Λ

First, pig insulin is converted to porcine des- (Ala ^) insulin by treatment with carboxypeptidase A, see Hoppe-Zeyler's A. Physiol. Chem. In the second stage, porcine des (Ala) insulin is subjected to a trypsin catalyzed coupling with Thr-CBu 2 to form the human Insil-330 line Thr-butyl ester. Finally this becomes

ester treated with trifluoroacetic acid to obtain human insulin) see Nature 280, 1 * 1'2. However, the first stage A21 results in partial removal of Asn to yield des-B3Qa21 (Ala) Asn) insulin. After the next two reactions, this derivative gives rise to a contamination of des- (Asn) -insulin in the prepared semi-synthetic human insulin (an impurity) which cannot be easily removed by known preparative methods. Des- (Asn) -insulin has a low biological activity (about 5%) »see 10 Amer. J. Med. 1 * 0, 750,

An object of the present invention is to provide a method which converts a non-human insulin into human insulin.

A second object of the present invention is to provide a method which converts pig insulin and certain impurities therein to human insulin via a human insulin threonine ester.

Further objects and advantages of the present invention will become apparent from the description.

The present invention is based on the discovery that the amino acid or peptide chain bound to the Lys carbonyl group B29 in the insulin compound can be exchanged with a threonine ester. This exchange is hereinafter referred to as transpeptidation.

The term "insulin compounds" as used herein includes insulins and insulin-like compounds containing the human des (Thr 3) insulin moiety, wherein the B30 amino acid of the insulin is alanine (in insulin of, for example, pig, dog and fin whale and sperm whale) or serine (rabbit). The term "insulin-like compounds" as used herein includes proinsulin from any of the aforementioned species and primates, along with intermediates from the conversion of proinsulin to insulin. As examples of such intermediates may be mentioned cleaved proinsulin, desdipeptide proinsulin, desnonapeptide proinsulin and diarginine insulins, see R. Chance: In Proceedings of the Seventh Congress of IDP, Buenos Aires 1970, 292-305, eds: S.R. Rodriques and J.V. Owen, Excerpta Medica, Amsterdam.

The method of the present invention comprises the trans-k peptidation of an insulin compound or a salt or 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 of which in the reaction mixture is less than about 50 vol.6 and the reaction temperature is below about 50 ° 0

Although trypsin is best known for its proteolytic properties, experts have recognized that trypsin is capable of

• D-ZQ

to catalyze the coupling of des- (Ala) -insulin and a threonine tert-butyl ester, see Nature 280, ifl2. In the present method, the trypsin is used to catalyze the transpeptidation.

The transpeptidation can be performed 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, optionally 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-dinethylformamide, formamide, Ν, Ν-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphoric triide and acetonitrile , to be mentioned.

Depending on which water-miscible organic solvent is used, on the selected reaction temperature and on the presence of an acid in the reaction mixture, the amount of water in the reaction mixture should be less than about pO vol.p, preferably less than about 40 vol. £ 5 and more than about 10 vol.p.

One 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 decrease by-product formation. The increase in yield is positively correlated with a high percentage of organic solvent.

The type of trypsin is not material for the practice of the present invention. Trypsin is a well-characterized enzyme, which is commercially available in a high purity, in particular of bovine and pig origin. Furthermore, trypsin of microbial origin can be used. In addition, the trypsin form, for example crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (as long as the .4 trypsin activity is retained, is not material for carrying out the present invention The term trypsin, as used herein, is intended to mean trypsins from all sources and all forms of trypsin, which retain the transpeptidation activity, including proteases with a trypsin-like specificity, for example Acromobacter lyticus protease see Agric, Biol, Chem. 42, 1443.

As examples of active trypsin derivatives, there can be mentioned acetylated trypsin, succinylated trypsin, glutaraldehyde treated trypsin and immobilized trypsin derivatives.

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

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

The process can be carried out at a temperature in the range between 50 ° C and the freezing point of the reaction mixture.

Enzymatic reactions with trypsin are usually performed at about 37 ° C to give a sufficient reaction rate. However, in order to avoid inactivation of trypsin, it is advantageous to carry out the method of the present invention at a temperature below ambient temperature. In practice, reaction temperatures above about 0 ° C are preferred.

The reaction time is usually between a few hours and a few 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 present invention can be represented by the alternative being the threonine ester of human insulin common formula 2, wherein a the carboxylic acid protecting group and R 1 represents hydrogen or a hydroxyl protecting group or a salt thereof. The threonine J esters of human insulin from the transpeptidation can be represented by the general formula 3 wherein h-In represents the human des (Thr) insulin moiety and R 1 and R 2 are as defined above. Applicable L-threonine esters of formula 2 are those in which R ** represents the carboxyl protecting group which remove B30 10 20 23 30 1.0 (formula 3) under conditions which do not significantly alter the insulin irreversibly. molecule. As examples of such carboxyl group protecting groups, alkyl with a small number of carbon atoms, for example, methyl, ethyl and tert-butyl, substituted benzyl such as p-methoxybenzyl and 2,4,0-trinethylbenzyl and diphenylmethyl, and groups of the general 6 6 formula - CHgOH 2 SO 4 R where R represents alkyl containing a small number of carbon atoms such as methyl, ethyl, propyl and n-butyl. Suitable hydroxyl group protecting groups

- TD TA

B? are those which can be removed from the threonine ester of human insulin (formula 3) under conditions which do not cause any significant irreversible change in the insulin molecule. As an example of such a group 5 R, tert-butyl can be mentioned.

Alkyl groups with a small number of carbon atoms contain less than 7 carbon atoms, preferably less than 3 carbon atoms.

Other protective green graft commonly used are described by: Chemics of Organic Chemistry (Houben-Vyl), Vol. XV / 1, editor: Sugen Mill Ier, Georg Thieme Verlag, Stuttgart 1974.

Some L-threonine esters (Formula 2) are known compounds and the remaining L-threonine esters (Formula 2) can be prepared analogously to the preparation of known compounds.

The L-threonine esters of formula 2 may be the free bases or suitable organic or inorganic salts thereof, preferably acetates, propionates, butyrates and hydrohalides such as hydrochloride.

It is desirable to use the reagents, i.e. the insulin compound and the L-threonine ester (Formula 2) in high concentrations. Preferably, the between the L-threonine ester and the insulin compound is about 5 · 1.

Desirably, the concentration of the L-threonine ester (Formula 2) in the reaction mixture is greater than 0.1 molar.

Ό7Λ 5 Human insulin can be obtained from the threonine p esters of human insulin (formula 3) by removing k 3

the protecting group R and any protecting groups R.

according to methods known per se. When R 1 methyl, ethyl or 6 6 is a group -CH 2 CH 2 SO 2 R, where R is as defined above 10, this protecting group can be removed under cautious alkaline conditions in an aqueous medium, preferably at a pH of about 8-12, for example at about 9 »5 · As base, ammonia, triethylamine or alkali metal-

L

hydroxides such as sodium hydroxide can be used. When R 15 is tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, this group can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoroacetic acid may be non-aqueous or may contain some water or may be diluted with an organic solvent such as dichloromethane. When R is tert-butyl, this group can be removed by acidolysis, see above.

Preferred threonine esters of human insulin of formula 3-5 are compounds wherein R is hydrogen and these are prepared from the L-threonine esters of formula 2, wherein R is hydrogen.

Β7Λ

The transpeptidation of insulin compounds to threonine esters of human insulin can be described more in distillation based on formula 1 attributed to the insulin compounds, wherein the group of formula k represents the human B30 A1 30 des (Thr J) insulin part, wherein Gly is compound 1 b29 with the substituent designated R and Lys connected to 2 2 the substituent designated R, R represents an amino acid or a peptide core containing more than 36 amino acids, and R hydrogen or a group of the general formula R ^ -X-, wherein X represents arginine or lysine and R 1 represents a peptide chain 2 containing no more than 35 amino acids, or R together with R 1 represents a peptide chain containing no more than 35 amino acids, provided that that the 8 1 00 62 4 7 1 2 number of amino acids present in 3 plus 5 is less than 37. Therefore, the transpeptidation of the present invention converts each of the aforementioned insulin compounds to threo-nineB esters of human insulin (formula 3) j which can then be unblocked to form human insulin

A further advantage of the present invention is that insulin-like compounds present in impure insulin and present in some commercial insulin preparations and comprising general formula 1 are converted to threonine by the transpeptidation of the present p-ZQ invention. esters of human insulin, which can then be unblocked to form human insulin. Examples of insulin-like compounds of formula 1 are the following: 15 2 20 23 30 35 ho

Pig diarginine insulin (2 is hydrogen and R is -Ala-Arg-Arg), pig-proinsilin (H3 together with 22 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly -Ala-Val-Glu-Leu-Gly- Gly-Gly-leu-Gly-Gly-ieu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln- 329 Lys-, where the terminal alanyl is linked to Lys 7), dogs proinsulin along with 32 is -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-, in which the terminal alanyl is linked with LysB2 ^), split pig proinsulin (3 ^ is Ala-Leu-2 Glu-Gly-Pro- Pro-Gln-Lys- and H 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 desdipeptide proinsulin (3 is? / Ater and 2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Fro-Gln-Ala-Giy-Ala- Val-Glu-Leu-Gly-GIy-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Giu-Gly-Pro-Pro-Gln), human proinsulin (S 'together with 3 ^ is -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly- Gly-? Ro-Gly-Ala-Gly-Ser-leu-Gln-Pro-leu-Ala-Leu-Glu-Gly-3er-Leu-Gln- 329 Lys-, in which the terminal threonyl is connected to Lys) and monkeys proinsulin cP together with 3 ^ is -Thr-Arg-Arg-Glu-Aia- 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, in which 350 the terminal threonyl is connected to Lys. Therefore, in all these impurities covered by formula 1 1 3, the 3 substituent designated 3 -2 is substituted with hydrogen. A preferred embodiment of the present invention includes the reaction of impure porcine insulin containing in insulin-like compounds or a salt or complex thereof with an L-threonine ester (Formula 2) or a salt thereof under the here-

L

for stated conditions, after which protecting group R and optionally protecting group R are removed. By this method, the porcine insulin, together with the insulin-like compounds contained therein, is converted into human insulin.

As examples of a complex or a salt of an insulin compound (formula 1), a zinc complex or a sink salt can be mentioned.

In choosing the reaction conditions according to the preceding explanation and considering the results obtained in the following examples, it is possible to obtain a yield of threonine ester of human insulin which is higher than 60% and even higher than 80% eh under certain pre-13 ksurs conditions higher than 90:, ó,

The method of the present invention therefore has the following examples relative to the prior art: a) The enzymatic hydrolysis to remove Ala'J, for example with carboxypeptidase A, is omitted.

"R nO

B) The isolation of an intermediate product such as porcine des (Ala) insulin is not necessary.

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

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

e) Antigenic insulin-like compounds, see British Patent No. 1,285,023, are converted to human insulin.

A preferred method of preparing human insulin is as follows: 1) The starting product used for transpeptidation is impure pig insulin, for example, crystalline insulin obtained using a citrate buffer, see U.S. Patent 2,626,228.

2) If any trypsin activity remains after the transpeptidation, it is preferable to remove it, for example under conditions where trypsin is inactive, for example k0 in an acidic environment below pH 3 · Trypsin can be removed before loading 81 0 0 62 4 95 by molecular weight separation, for example, by gel filtration on "Sephadex G-50” or n3-gel P-30 "in 1 molar acetic acid, see Nature 280, 412.

3) Other impurities}, such as unconverted porcine insulin, can be removed using anion and / or cation exchange chromatography, see Examples I and II.

B30 4) Thereafter, the threonine ester of human insulin is unblocked and ashes insulin is isolated, for example crystallized, in a manner known per se.

10 15 20 23 50 35

By this method, human insulin of acceptable pharmaceutical purity can be obtained and, if desired, further purified.

Furthermore, the present invention relates to novel threonine B 2 esters of human insulin in which the ester moiety is different from tert-butyl and methyl.

Abbreviations used are in accordance with accepted rules (1974) by the lïïPAC-IUB commission on biochemical nomenclature, see Collected Tentative Pules «Pecosmendations of the Commission on Biochemical Nomenclature IUPAC-IUB, 2nd ed ·,

Maryland 1975

Analytical uroes

The conversion of porcine insulin and porcine proinsulin to human insulin esters can be demonstrated DISC FAGS electrophoresis in 7 ”polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.0β M HCl and 8 M urea. The pH of the buffer is 8.7. Esters of formula 3 migrate at a rate of 75 that of porcine insulin. Pig proinsulin, which migrates at 55 the rate of porcine insulin is by the method of the present invention. converted to the same product. Identification of the circulatory product as compounds of formula 3 is due to the following criteria: a) The electrophonetic migration of the human insulin esters of formula 3 into. association with porcine insulin is equivalent to the loss of a negative charge.

b) The amino acid composition of the colored protein bonds in the gel, representing compounds of formula 3, is identical to that of human insulin, i.e. 3 moles of threonine and 1 mole of alanine per mole of insulin, and the composition of porcine insulin is 2 moles of threonine and 2 sol alanine per mole of insulin. The technique for analyzing amino acid compositions of 40 81 0 0 62 4 protein bonds in polyacrylamide gels is described in Bur. J. Biochem * 25, 147 · c) Proof that the incorporated threonine is placed as C-terminal amino acid in the B chain is provided by oxidative sulfitolysis of the SS bridges of insulin in 6 M guanidium hydrochloride followed by separation of A and B chains by ion exchange chromatography on "SP Sephadex". Digestion of the B chain S sulfonate with carboxypeptidase A only releases the C-terminal amino acid. The 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 performed after the ester group has been split from the compounds of formula 5

These three analyzes unequivocally prove that the conversion to human insulin has taken place.

Conversion to porcine insulin and porcine proinsulin to human insulin esters can be monitored quantitatively by reverse phase HPLC (high pressure liquid chromatography). A 4 x 300 mm "µ u Bondapak C ^ g column" (Waters Ass.) Was used and the elution was performed with a buffer consisting of 0.2 molar ammonium sulfate (adjusted to a pH of 3 »5 non-sulfuric acid) and contains 26 - 50% acetonitrile. The optimum acetonitrile concentration depends on which ester of formula 3 is to be separated from pig insulin. When B is methyl and B is hydrogen, separation can be achieved in 26 vol. Acetonitrile. Pig insulin and (Thr-OMe) -h-In (Me is methyl) elute after 4.5 and 5.9 column volumes by volume, as well as separated symmetrical peaks. Before application to the HPLC column, the proteins in the reaction mixture were precipitated by adding 10 parts by volume of acetone. The precipitate was separated by centrifugation, dried under reduced pressure and dissolved in 0.02 molar sulfuric acid.

The method of preparing human insulin esters and human insulin is illustrated by the following examples, which are not, however, limiting. The examples illustrate some preferred embodiments of the method of the invention.

Example I

200 mg of crude pig insulin, crystallized at one time

4 hours, were dissolved in 1.8 ml of 3 53 molar acetic acid. 2 ml of a 2 molar solution of Thr-OMe (Me is methyl) in N, N -imethyl-acetamide and 20 mg of trypsins dissolved in 0.2 ml of water were added. After storage at 37 ° G for 18 hours, the proteins were precipitated by the addition of 40 ml of acetone and the precipitate was isolated by centrifugation. The supernatant was removed. Analysis of the precipitate by HFLC using 26% acetonitrile (see analytical tests) showed a 60 # conversion of porcine insulin to Thr-OMe) J-h-In. The precipitate was dissolved in 8 ml of freshly deionized 8 molar urea, the pK was adjusted to 8.0 with 1 molar ammonia and the solution was applied to a 2.5 x 25 cm column filled with "QAE A-25 Sephadex "equilibrated with a 0.1 molar ammonium chloride buffer containing 60 vol. of ethanol, the pH of which was adjusted to 8 with ammonia.

15

The elution was performed with 15 ml each collected. (Thr-OMe) buffer and fractions var.

-In was found in fractions 26 - 48 and unreacted porcine insulin in fractions 90 - 120. Fractions 20 - 48 were collected, the ethanol was evaporated under reduced pressure and the (Thr-OMe) -h- In '20 was crystallized in a citrate buffer as described by Schlichtkrull et al, Handbuch der inneren Medizin, 7 / 2A, 96 »> 330

Berlin, Heidelberg, New York 1975 · Me yield was 95 mg of crystals of the same rhombic form as porcine insulin, which had crystallized in the same manner. The amino acid composition was found to be identical to that of human insulin. Further analytical tests described in the previous section "Analytical Tests" showed that the product obtained (Thr-OMe) was' h-In.

Example II

100 mg of pig insulin, meeting the requirements of British Pat. No. 1,285,023, were dissolved in 0.9 ml of 33 molar acetic acid and then 1 ml of a 2 molar solution of threonine methyl ester in Μ-dimethylformamide and 12 mg TPCK (tosylphenylalanine chloromethylketone) treated trypsin dissolved in 0.1 ml of water are added. After an incubation at 57 ° G for 24 hours, the reaction was stopped by the addition of 4 ml of 30 L molar phosphoric acid. The resulting (Thr-OMe)? -H-In 'was separated from unreacted porcine insulin by ion exchange chromatography on a 2.5 x 25 cm column of "SP-3ephadex" with a 40 eluent, 09 molar sodium chloride and 0.02 molar Na- 8 1 0 0 62 4 i c.

trihydrogen hydrogen phosphate (pH of the buffer 5 »5) in 60% ethanol

• D ^ ZQ

contained. Fractions containing (Thr-OMe) J-h-In were collected, the ethanol was removed under reduced pressure and the product was crystallized as described in Example 1. The yield was 50 mg (Thr-OMe ) B ^ -h-In.

Example III

100 g of pig proinsulin were dissolved in 0.9 ml of 3 33 33 molar acetic acid and converted to (Thr-OMe) --h-In and purified as described for pig insulin in Example 1. The conversion of 10 proinsulin to ( Thr-OMe) -h-In was found to be 73% by HPLC

analysis of the acetone precipitate. The yield of crystalline (Thr-OMe) B 2 -h-In was 5k mg.

Example IV

100 mg of pig insulin were dissolved in 0.9 ml of 2.77 molar acetic acid in water and converted analogously to the procedure described in Example II. After the completion of the reaction, the proteins were precipitated by the addition of 10 parts by volume of acetone. Analysis by DISC PAGE showed a conversion to (Thr-OMe) B ^ -h-In of 70% »

Example V

100 mg of pig insulin were dissolved in 0.9 ml of 3 »33 molar acetic acid and 1 ml of 2 molar Thr-OMe in N-methylpyrrolidone was added. The reaction was analogous to that described in Example IV.

B3G

and the conversion to (Thr-OMe) J-h-In was 20%

Example VI

100 mg of pig insulin were dissolved in 0.9 ml of 2.77 molar acetic acid in water and 1 ml of 2 molar Thr-OMe in HMPA (hexamethylphosphoric triamide) was added. The reaction was carried out in an analogous manner as described in Example IV. The conversion to 30 (Thr-OMe) B _:? ^ - h-In was 80%,

Example VII

100 mg pig insulin was dissolved in 0.9 ml 3> 33 molar acetic acid and 1 ml 2 mol Thr-OMe in Ν, Ν-dimethylacetamide was added. The reaction was carried out in the same manner as described in Example IV. The conversion to (Thr-OMe) -h-In was 80

Example VIII

100 mg of pig insulin were dissolved in 0.9 ml of 3 33 33 molar acetic acid and 1 ml of 2 molar T-ir-OMe in Ν, Ν-dimethylacetamide was added + 0. Then 200 units of trypsin activity (averaged 81 0 0 62 4 13 10 20 25

SS

immobilized against the substrate 3AEE) on 1 g of glass beads were added and after Zk hours incubation at 37 ° C the trypsin bound to the glass was filtered. After the completion of the reaction, the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISK PAGE showed a conversion to (Thr-OMe) 3 ^ -h-In of kO jo. EXAMPLE 9 100 mg of pig insulin were dissolved in 0.9 µl of 3333 molar acetic acid and 1 ml of 2 molar Thr-OMe in Ν, Ν-dimethylacetamide was added. Then 3 ° 0 units of trypsin (activity measured with the substrate BAEE) immobilized on ZOO µg "Sephadex G-150" with CMBr activated activated were added. After Zk hours incubation at 37 ° G, the trypsin bound to "Sephadex" was filtered. After the completion of the reaction, the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by Disc PAGE showed 70 P conversion to (Thr-OMe) 3-h -in. Example X The procedure described in Example VII was repeated except * fc that the ester used was 2 molar Thr ~ 03h (Bu tert .butyl) in 330 Ν, Ν-dimethylacetamide The conversion to (Thr-OBu1 ') was 80 µ Example XI The procedure described in Example VIII was repeated except that the ester used was 2 molar Thr-03h in Ν, Ν-dimethyl-acetamide The conversion to (Thr-0Bu ^) 3 ^ -h-In was 30 µ Example XII The procedure described in Example IX was repeated except that the ester used was 2 molar Thr-OBu in Ν, Μ-dimethylacetanide was converted to (Thr-3 u3) 3 ^ -h-In was 70. Example XIII 100 mg of pig proinsulin were dissolved in 0.9 ml of 3, 33 molar acetic acid and 1 ml 2 molar Thr-OMe in Ν, Ν-dimethylacetamide was added The reaction was carried out analogously as 3-0 described in Example IV The conversion to (Thr-OMe) y -h-In was p Example XIV 1CG mg of pig proinsul ine were dissolved in 0.9 ml 3 »33 molar acetic acid and 1 ml 2 molar Thr-OMe in N, N-dimethylacetamid 3 was added. The mixture was immobilized with tryosin-h-In pineapple O-4 a · • * CL.

ilcog to the method described in example Owl. The 8100624 '14 conversion to (Thr-OMe) ® ^ -h-In was 1 + 0%.

Example XV

100 mg of pig proinculin were dissolved in 0.9 ml of 5> 55 Mg

1 L acetic acid and 1 ml 2 molar Thr-OMe in N, N-dimethylacetamide were added. The mixture was treated with immobilized trypsin analogous to the procedure described in Example IX. The conversion to (Thr-OMe) ® ^ -h-In was 70 Example XVI

100 mg of pig proinsulin were dissolved in 0.9 ml of 5155 molar 10% acetic acid and 1 ml of 2 molar Thr-OBu in N, N-dimethylacetamide was added. The reaction was performed in an analogous manner 1/3330 as described in Example IV. The conversion to (Thr-OBu-h-In was 80 ° / o.

Example XVII

100 mg of pig proinsulin were dissolved in 0.5 ml of 5.55 molar acetic acid and 1 ml of 2 molar Thr-OBu0 in Ν, Ν-dimethylacetamide was added. The mixture was immobilized with glass beads on glass beads analogous to the method described in Example B30

acted. The conversion to (Thr-OBu) -h-In was 1 * 0 Example XVIII

100 mg of pig proinsulin were dissolved in 0.9 ml of 5.55 molar acetic acid and 1 ml of 2 molar Thr-OBu in N, N-dimethylacetamide was added. The mixture was treated with trypsin immobilized on CNBr activated "Sephadex G-150" analogous to the procedure described in Example IX. The conversion to (Thr-OBu) ^^ - h-In was 70% ·

Example XIX

50 55

100 mg of pig insulin were dissolved in 0.5 ml of 6 molar acetic acid and 1 ml of 1 molar Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) in Ν, Ν-dimethylacetamide was added. Furthermore, 0.5 ml of Ν, dim-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 1 * 1 hours. After the completion of the reaction, the proteins were precipitated by the addition of 10 parts by volume of acetone. Analysis by DISC PAGE showed a conversion to (Thr-OTmb) ^^ - h-In of 50% · Example XX

IfO

100 mg of pig insulin were dissolved in 0.9 ml of 5 molar acetic acid and 1 ml of 2 molar Thr-OMe in dioxane was added. The reaction was performed in an analogous manner as described in Example IV and the conversion to (Thr-OMe) -h-In was 10 8 1 0 0 62 4 15

Example XXI

100 ng of pig insulin was dissolved in 0.9 ml of 3 molar acetic acid and 1 µl of 2 molar Thr-OMe in acetonitrile was added. The reaction was performed in an analogous manner as described in 5 g-zn

Example IV and the enclosure to (Thr-OMe) J-h-In was 10>. Example XXII

B30 10 12 250 mg of crystalline (Thr-OMe) -h-In were dispersed in 23 ml of water and dissolved by the addition of 1 N sodium hydroxide solution to a pH of 10.0. The pE was kept constant at 10.0 at 25 ° C for 2 hours. The human insulin formed was crystallized by the addition of 2 g sodium chloride, 350 mg sodium acetate trihydrate and 2.3 mg zinc acetate dihydrate followed by the addition of 1 N hydrochloric acid to obtain a pH of 5 52. After storing for 2 hours at k ° 0, the rhomboid crystals were separated by centrifugation, washed with 3 ml of water, separated by centrifugation and dried under reduced pressure. Yield: 220 mg of human insulin.

Example XXIII

B ^ O

100 mg (Thr-CTmb) J-h-In were dissolved in 1 ml of ice cold trifluoroacetic acid and the solution was stored at 0 ° C for 2 hours. The human insulin formed was precipitated by the addition of 10 ml of tetrahydrofuran and 0.97 ml of 1.93 molar hydrogen chloride in tetrahydrofuran. The precipitate formed was separated by centrifugation, washed with 10 ml of tetrahydrofuran, separated by centrifugation and dried under reduced pressure. The precipitate was dissolved in 10 ml of water and the pH of the solution was adjusted to 2.5 with a 1N sodium hydroxide solution. The human insulin was precipitated by the addition of 1.5 g of sodium chloride and separated by centrifugation. The precipitate was dissolved in 10 ml of water and the human insulin was precipitated by the addition of 0.8 g of sodium chloride, 3.7 g of zinc acetate dihydrate and 0.1 g of sodium acetate trihydrate followed by the addition of a 1 N sodium hydroxide solution for the obtaining a pH of 5.52. After storage for 2½ hours at k ° C, the precipitate was separated by centrifugation, washed with 0.9 ml of water, centrifuged, separated and dried under reduced pressure.

dried. Yield: 90 mg of human insulin. Example XXIV

ICC mg pig-in.

uline Tierden dissolved in 0.5 ml 10 molar, N-d imethylac e tamino acetic acid and 1.3 ml 1.2 h molar Thr-OMe in 8100624 22 5 16 were added. The mixture was cooled to 12 ° C. 10 mg of trypsin dissolved in 0.2 ml of 0.05 molar calcium acetate were added. After 1 * 8 hours at 12 ° C, the proteins were precipitated by the addition of 20 ml of acetone · The conversion of porcine insulin to (Thr-OMe) ^ - h-In was 97% by HPLC.

Example XXV

20 mg of pig insulin were dissolved in a mixture of 0.08 ml of 10 molar acetic acid and 0.11 ml of water. 0.2 ml of 2 molar Thr-OMe in Ν, Ν-dimethylacetamide was added and the mixture was cooled to -10 ° G. 2 mg of trypsin dissolved in 0.025 to 0.05 molar calcium acetate were added. After 72 hours at -10 ° C, the proteins were precipitated by addition of 5 ral acetone. The conversion of porcine insulin to (Thr-OMe) J-h-In was 61% by HPLC).

15 20 25 30 35

Example XXVI 20 mg pig insulin were dispersed in 0.1 ml of water. Addition of 0.6 ml of 2 molar Thr-OMe in Ν, Ν-dimethylacetamide caused the insulin to dissolve. The mixture was cooled to 7 ° C. 2 mg of trypsin dissolved in 0.025 to 0.05 molar calcium acetate were added. After 21 hours at 7 ° C, the proteins were precipitated by the addition of 5 ral aceg2A tons. The conversion of porcine insulin to (Thr-OMe) ^ -h-In was 62% by HPLC. Example XXVII ....................... molar 20 mg pig insulin was dissolved in 0.135 ral 1 * µl * 5 / propionic acid. 0.21 * ml 1.67 molar Thr-OMe in Ν, Ν-dimethylacetamide was added, 2 mg trypsin in 0.025 mol 0.05 molar calcium acetate was added and the mixture was kept at 37 ° C for 24 hours. The proteins were precipitated by adding 10 parts by volume of B30 2-propanol. The conversion of porcine insulin to (Thr-OMe) h-In was 75% by HPLC. Example XXVIII 20 mg pig insulin were dispersed in 0.1 ml of water. in Ν, Ν-dimethylacetamide,, “0.1 * ml 2 molar Thr-OMe / was added followed by 0.01 * ml 10 N hydrochloric acid dissolving the insulin # 2 mg trypsin dissolved in 0.025 ml 0.05 molar calcium acetate was added and the mixture was kept at 37 ° C for 1 hour. Analysis by HPLC showed a 1 * 6% conversion of porcine insulin to (Thr-OMe) ® ^ -h-In.

ι * ο 8 1 0 0 62 4 17

Example XXIX

20 mg pig insulin were dissolved in 0.175 µl of 0.57 molar acetic acid. 0.2 ml of 2 molar Thr-OMe in N, N-dimethylacetanide was added followed by the addition of 0.025 ml of 0.05 molar calcium acetate. 10 mg of an impure preparation of Acnromobacter lyticus protease were added and the mixture was kept at 57 ° C for 22 hours. The proteins were precipitated by the addition of 10 volumes of acetone. The conversion of porcine insulin to (Thr-OMe) 33 µ-h-In was 12% by HP1C.

Example XXX

20 mg of pig insulin were dispersed in 0.1 ml of 0.5 molar acetic acid. Addition of 0.2 ml of 0.1 molar Thr-OMe in N, N-dimethylacetamide dissolved the insulin. The mixture was made up to o.

o cooled to 12 DEG C. and added 2 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate - further. ka 24 hours at 12 0 the analysis showed vol (Thr-OMe) 33u-h-In

in HPLC a conversion of 22% of pig insulin 330-h Example XXXI

20

2 mg of rabbit insulin was dissolved in 0.135 ml of 4 45 45 molar acetic acid. 0.2½ ml of 1.67 molar Thr-OMe in Itf, N-dimethylacetanide was added followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate. The mixture was kept at 37 ° C for 4 hours. Analysis by HPLC showed a conversion of 88 CA

O'S A

25 from rabbit insulin to (Thr-OMe) J-h-In. Rabbit insulin elutes for porcine insulin from the HPLC column, the ratio between the volumes of elution of rabbit insulin to (Thr-OMe) S3 ° -h-In being 0.72.

Example XXXII

331 2 mg porcine diarginine insulin (Arg ^ - Arg333 insulin) were dissolved in 0.135 µl of 4.45 molar acetic acid. 0.24 ml of 1.67 molar Thr-OMe in ΙΤ, ΙΤ-im -imethylacetamide was added followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate. The mixture was kept at 37 ° C for 4 hours. Analysis by HPLC showed a 91% conversion of diarginine insulin to (Thr-OMe) -h-In. Diarginine insulin elutes for pig insulin from the HPLC column, with the ratio between the egg volumes of diarginine insulin to (Thr-CMe) J-h-In 0.50

WctO

Example XXXIII

i.e. a mixture of 2 mg of pig intermediates (81 0 0 62 4 ιο des-dipeptide (Lys ^ -Arg ^) proinsulin and desdipeptide (Arg · ^ -32

Arg ^) proinsulin was analogous to that described in Example XXXIII.

T5 * 2Q

reaction reacted. Analysis by HPLC showed 88 $ (Thr-OMe) h-In.

Example XXXIV

20 mg pig insulin was dissolved in 0.1 ml 2 molar acetic acid, 0.2 ml 2 molar Thr-OMe in Ν, Ν-dimethylacetamide was added and the mixture was cooled to -18 ° C, 2 mg trypsin - dissolved in 0.025 ml of 0.05 molar calcium acetate were added

10 and the mixture was kept at -18 ° C for 120 hours. Analysis by HPLC

indicated that the conversion to (Thr-OMe) ® ^ ° -h-In was 83%.

Example XXXV

The procedure described in Example XXXIV was repeated except that the reaction was carried out at 50 ° C for 4 hours. Analysis by HPLC indicated that the conversion to (Thr-OMe) B 2 O -h-In 23 was 7 °.

Example XXXVI

20 mg pig insulin was dissolved in 0.1 ml 3 molar acetic acid. 0.3 ml of 0.33 molar Thr-O- (CH2) 2-302-CH2, CH2 COOH 20 (threonine 2- (methylsulfonyl) ethyl ester, hydroacetate) in N, N-dimethyl acetamide. The mixture was cooled to 12 ° C and 2 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate was added. HPLC indicated after 24 hours at 12 ° C a conversion of 77% to (Thr-O (CH2) 2-SO2-CH2) B ^ -h-In. The product elutes approximately at the position of (Thr-OMe) B1 -H-In.

Example XXXVII

20 mg pig insulin was dissolved in 0.1 ml 10 molar acetic acid. 0.2 ml of 2 molar Thr-OEt (Et is ethyl) in Ν, Ν-dimethylacetamide was added and the mixture was cooled to 12 ° C.

2 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate was added. HPLC indicated a 75% conversion after 24 hours at 12 ° C

D-ZQ

to (Thr-OEt) J-h-In. The product eluted in a portion B30 slightly after that of (Thr-OMe) J-h-In.

Example XXXVIII

35 mg of pig insulin were dissolved in 0.1 ml of 6 molar acetic acid t "t / t. 0.3 ml of 0.67 molar Thr (Bu) ~ 0Bu (Bu is tertiary butyl) in Ν, Ν-dimethylacetamide was added The mixture was cooled to 12 ° C and 2 mg of trypsin in 0.025 ml of 0.05 molar calcium acetate was added After 24 hours at 12 ° C, the conversion was to (Thr (Bu ^) - 0Bu ^) ^^^ hi 77 The product was eluted from the HPLC column 81 0 0 62 4 40 5 19 using a gradient in acetonitrile of 27% to ifO #.

Example XXXIX

20 mg pig insulin was dissolved in 0.1 ml if molar acetic acid. 0.2 ml of 1.3 molar Thr-OMe dissolved in tecranydrofuran was added and the mixture was cooled to 12 ° C. 2 mg of trypsin dissolved in 0.023 ml of 0.03 molar calcium acetate were added. After hours at 12 ° G, analysis by EPLC indicated a conversion of from 75 to (Thr-OMe) S 1 -H -1.

10

Example XL

20 mg pig insulin was dissolved in 0.1 ml if molar acetic acid. 0.8 ml of 2 molar Thr-OMe dissolved in 1,2-stanediol was added and the mixture was cooled to 12 ° C. 2 mg of trypsin dissolved in 0.023 ml of 0.05 molar calcium acetate are added. After if hours at 12 "analysis by IL-LG, an om> 330 setting from if3 yj to (Thr-GMe) -hI. Example XLI 20 mg of pig insulin were dissolved in 0.1 ml if molar acetic acid. 2 ml of 2 molar Thr-OMe dissolved in ethanol was added and the mixture was cooled to 12 ° G. 2 mg of trypsin dissolved in 0.025 ml of 0.05 molar calcium acetate was added and the reaction was carried out for hours at 12 ° C * Analysis by H? LC ga; conversion of if6% to (Thr-OMe) -h-In. Example XLII 20 mg pig insulin were dissolved in 0.1 ml if molar acetic acid 0.2 ml 2 mol Thr-OMe in acetone was added and the mixture was cooled to 12 ° G 2 mg trypsin dissolved in 0.025 ml 0.05 molar calcium acetate was added After hours at 12 ° G, the analysis by HPLC indicated a conversion of if8 to (Thr-0Ke ) B50-h-In.

8 1 0 0 62 4

Claims (17)

A process for the preparation of threonine esters of human insulin or a salt or complex thereof, characterized in that an insulin compound or a salt or complex thereof which can be converted therein is trans-peptidated with a L-threonine ester 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 less than 50 vol. #, The reaction temperature is below 50 ° C and if any presence of an acid. 2. Process according to claim 1, characterized in that a concentration of the L-threonine ester in the reaction mixture is used which is greater than 0.1 molar, that the reaction temperature is above the freezing point of the reaction mixture and that the reaction mixture contains 0 to 10 equivalents of an acid per equivalent of the L-threonine ester. A method according to claim 1 or 2, characterized in that the insulin compound is a compound selected from the group consisting of pig, dog, fin whale, sperm whale and rabbit insulin, pig diarginine. insulin, split pig proinsulin, pig desdipeptide proinsulin, pig, dog, monkey and human proinsulin and mixtures thereof. Method according to claims 1 to 3i, characterized in that the insulin compound is of pig origin. A method according to claim 4, characterized in that relatively impure insulin is used as the insulin compound. 6. Process according to claims 1 to 5, characterized in that a reaction temperature is used below 37 ° C, preferably below ambient temperature and above 0 ° C. Process according to claims 1 to 6, characterized in that a water content in the reaction mixture is used between 40 and 10 vol. Process according to claims 1 to 7, characterized in that a molar ratio of L-threonine ester and insulin compound is used greater than 5'1. Process according to claims 1 to 8, characterized in that a polar solvent is used as the organic solvent. 81 00 62 4 C I ΊΟ. Marking method according to Claim 9, characterized in that the solvent used is methanol, ethanol} 2-propanol, 1,2-ethanediol, acetone, dioxane, teurahydrofuran, formamide, NiN-diaethylforsamide, X} N-dimethylacetamide, n-methylpyrroliacene, hexasethylphosfortrianide or acetonitrile. 11. Method according to claims 1 to 10, characterized in that the acid used is an organic acid, preferably formic acid, acetic acid, propionic acid or butter hour. 12. Process according to claims 1 to 11, characterized in that an amount of acid in the reaction mixture is used between 0.5 and 5 equivalents per equivalent of the L-threonine ester. Marking method according to claims 1 to 12), characterized in that a weight ratio of thrypsin to insu VI MPW 1 v 1 toeuas ov oz: preferably above 1: 50 and below 1: 1. 14. 7 / method according to claims 1 to Characterized in that the transpeptidation is carried out in the presence of calcium ions. lp. marking method according to claims 1 to 14, characterized in that the transpeptidation is carried out in a buffer system. 16. Process according to claims 1 to 1p, characterized in that the L-threonine ester is added as an acetate) propionate, butyrate or hydrohalide such as hydrochloride. Process according to claims 1 to 16, characterized in that the reaction temperature and the water and acid content in the reaction mixture are chosen such that the yield 330 of the threonine ester of human insulin is greater than 60; j) preferably greater than S0 fa and hst most preferably greater than 90% · 1o. Method according to claims 15 and 6 - 17i, characterized in that the insulin compound represents a compound 3 "30 A1 des (fhr) -insulin part, wherein Oly is linked to of formula 1, wherein the group of formula 4 the human the substituent indicated by H 'and lrjs ~' ~ 'is connected to the substituent indicated by? ƒ, S- represents an amino acid or a peptide chain w, 1, which contains no more than 3 ° amino acids and H contains hydrogen or a group of the general formula B1 -X-, wherein X represents arginine or lysine and R '' represents a peptide chain. rin 329 which is 8 1 0 0 62 4 5 <LC. 10 15 20 25 30 35 ko not more than 35 amino acids, or R1 together with R1 represents a peptide chain containing no more than 35 amino acids, provided that the number of amino acids present in R1 plus R1 is less than 37. claim 18, characterized in that R is hydrogen and R is -Ala, -3er, -Ala-Arg-Arg or -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- 7 p Gin is, Ir Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys- is and R -Ala-Arg- Arg-Glu-Ala-Glu-Asn-Fro-Gln-Ala-Gly-Ala-Val-Glu-Leu -Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu is or Φ 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 alanyi is connected with Lys ^^, -. ~ 2.a-Ar =: - Arg-Asp-Val-Glu-Leu-Ala-Gly-Ala-? ro-Gly-Glu-Gly-Gly-Leu-Gln-? ro -Leu- Ala-Leu-Glu-Gly-Ala-Leu-Gln-Lys-, in which the terminal alanyi B29 is linked to Lys', -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-8u-Leu-Gln- Lys-, before the terminal B29 threonyl is linked to Lys 7 or -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 it T3-2 / -N terminal threonyl is linked to Lys 7, is, 20. The method of claim 19, characterized in that R 1 is 2, wherein R is hydrogen and R represents alanine. 21. Process according to claims 1 to 20, characterized in that the L-threonine ester has the general formula 2. k has wherein R represents the carboxyl protecting group and R represents hydrogen or a the hydroxy protecting group. Process according to claim 21, characterized in that R 1 alkyl having a small number of carbon atoms, aralkyl, wherein the alkyl group contains a small number of carbon atoms, such as substituted benzyl or diphenylmethyl or a group having the oS general formula -CH 2 CH ^ SO ^ R wherein R represents an alkyl group with a small number of carbon atoms. A process according to claim 22, characterized in that ethyl is 4, wherein R is methyl, / tert-butyl, p-methoxybenzyl, 2,4, o-triethylethylbenzyl or a group of the formula -CH 1 C 1 -C 18 SO 4 R 10, wherein 8 1 0 0 62 4 H is methyl, ethyl, propyl or n-butyl. 2q-o Marking according to claims 1 to 6, characterized in that the insuiine compound is porcine insulin, that the L-threonine ester is Thr-OMe or Thr-OBu, that the organic solvent miscible with v / ap N, 2i-dimethylformanide or 'J, N-dimethylacetamide, that the reaction temperature is about 37 ° C, that the water content in the reaction mixture is between 41 and 43 ^ that the weight ratio between trypsin and the insulin compound is about 1: 8 is that the molar ratio between the L-threonine 10 ester and the insulin compound is about 1: 120 and that between 1.2 and 1.3 equivalents of acetic acid per equivalent of the L-threonine ester are added. 25. Process according to claim 1 or 2, characterized in that it is carried out under the reaction conditions stated in each of Examples I-XXI or 1117-1LII, preferably those separately mentioned in each of Examples IV, VII, X, XXIV, XXXIV, XXXVI, and XXXIX. c m ~ - = ters of human insulin prepared according to 2o. Threonine the label of any of claims 1 to 25 27. A process for the preparation of human insulin, characterized in that the process according to one or more of claims 1 to 25 is first carried out and then deblocked the obtained p-zQ threonine ester of human insulin. 28. Process according to claim 27, characterized in that the deblocking is carried out in an aqueous medium in the presence of a base, preferably at a pH between 3 and 12. 251 Process according to claim 27, characterized in that the deblocking is carried out by acidolysis, preferably with trifluoroacetic acid. 30. Process according to claim 27, characterized in that the deblocking is carried out under the reaction conditions stated in Examples XXII or XXIII. Human Insulin prepared according to the method of 35 and or more of claims 27 to 331 32. Threonine esters of desirable insulin, in which the [ethyl. ester green differs from tert-butyl and 1 81 00 62 4 1 1 1 1 1 Thr (R5) -OR4 A (Thr (R5) -0R4) B30-h-In A - (1- 0 “—A — 21) - B --- 29) - 8 1 0 0 62 4