JPH0211240B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the conversion of insulin and insulin-like compounds to human insulin. Insulin preparations obtained from porcine or bovine insulin are widely used in the treatment of diabetes. Although porcine, bovine, and human insulin show slight differences in their amino acid composition, it has been determined that the difference between human and porcine insulin lies in just one amino acid, in which Human insulin B ³⁰
The amino acid in porcine insulin is alanine, while the amino acid in pig insulin is threonine. However, it should be clear that the ideal insulin formulation for human treatment would be an insulin with a chemical structure identical to that of human insulin. For the production of natural human insulin,
The required amount of pancreas is not available. Synthetic human insulin is produced on a small scale at great expense (Helv.Chim.Acta vol. 57, 2617
(see p. and vol. 60, p. 27). Semi-synthetic human insulin was produced from porcine insulin by a laborious process (Hoppe-Seyler's Z.
Physiol.Chem.volume 357, page 759 and Naturevolume 280,
(See page 412). However, the present invention relates to a method for producing human insulin on an industrial scale. US Pat. No. 3,276,961 is directed to a method for producing semi-synthetic human insulin. However, the yield of human insulin is poor. this is,
This is because the process is carried out in water and under these conditions trypsin causes cleavage of the Arg ^B22 -Gly ^B23 bond (see J. Biol. Chem. Vol. 236, p. 743). A known semi-synthetic method for producing human insulin includes the following three steps: First, porcine insulin is treated with carboxypeptidase A to produce porcine des-
(Ala ^B30 ) - conversion to insulin (Hoppe-Seyler's Z.Physiol.Chem.Vol. 359, 799
(see page). In the second step, trypsin-catalyzed coupling of porcine des-Ala ^B30 )-insulin with Thr- ^{OBu t} forms human insulin Thr ^B30 -tert-butyl ester. Finally, the ester is treated with trifluoroacetic acid,
Obtaining human insulin (Nature 280, 412
(see page). However, the first step causes partial elimination of Asn ^A21 to give des-(Ala ^B30 , Asn ^A21 )-insulin. This derivative causes a contamination of des-(Asn ^A21 )-insulin in the semisynthetic human insulin obtained after two subsequent reactions, which contamination cannot be easily removed by known preparative methods. Death-(-
Asn ^A21 ) - Insulin has low biological activity (approximately 5%)
(see Amer. J. Med. Vol. 40, p. 750). The first object of the present invention relates to a method of converting non-human insulin into human insulin. A second object of the present invention is to provide a method for converting porcine insulin and certain impurities therein into human insulin via threonine ^B30 ester of human insulin. Other objects and advantages of the invention will become apparent in the course of the invention. The present invention provides Lys ^B29 in insulin compounds.
It is based on the discovery that the amino acid or peptide chain attached to the carbonyl group of can be exchanged with a threonine ester. This exchange is referred to as peptide rearrangement in the present invention. The term "insulin compounds" as used in the present invention is intended to encompass insulin as well as insulin-like compounds containing the human death (Thr ^B30 ) insulin moiety, where the B30 amino acid of insulin is alanine (e.g., porcine, canine and normal (in Japanese whales and Japanese whales) or serine (in domestic rabbits). The term "insulin-like compound" in the present invention includes proinsulin derived from any of the above species and primates, together with intermediates in the conversion of proinsulin to insulin. Examples of such intermediates include:
These include split proinsulin, deathday peptide proinsulin, desnonapeptide proinsulin, and dialginine insulin (R.Chance: In Proceedings of the Seventh
Congress of IDF, Buenous Aires 1970, 292
~See page 305, editor: RR Rodrigues & J.V.
Owen, Excerpta Medica, Amsterdam). It is understood that the process according to the invention comprises peptide rearrangement 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. characterized, and the water content in the reaction mixture is 50% (vol/vol)
and the reaction temperature is less than about 50°C. Although trypsin is best known for its proteolytic properties, researchers in the field have discovered that trypsin catalyzes the coupling with des-(Ala ^B30 )-insulin and threonine tert-butyl ester. (See Nature 280, p. 412). In the method of the invention trypsin is used to catalyze peptide rearrangement. Peptide rearrangement involves dissolving (1) an 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. It is done by twisting. Preferred water-miscible organic solvents are polar solvents. Examples of such solvents include methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, N-methylpyrrolidone, hexamethyl Mention may be made of phosphotriamide and acetonitrile. what water-miscible organic solvents are used;
Depending on how the reaction temperature is selected and also on the presence of acid in the reaction mixture, the water content in the reaction mixture may be less than about 50% (V/V), preferably about 40%. (V/V) and about 10
% (V/V). One advantage of reducing the amount of water in the reaction mixture is that it reduces by-product formation. Similarly, reducing the amount of acid in the reaction mixture makes it possible to reduce the formation of by-products. The increase in yield clearly correlates with the higher percentage of organic solvent. The trypsin type is not critical to the practice of this invention. Trypsin is a unique enzyme, which is highly pure and commercially available from apparently porcine and bovine sources. Additionally, trypsin of microbial origin can also be used. Furthermore, tryptic forms, such as crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (as long as trypsin activity is retained) are not raw materials for the practice of the invention. The term trypsin as used in the present invention refers to trypsin from all sources as well as proteases with trypsin-like specificity, such as Acromobacter lyticus protease (see Agric. Biol. Chem. Vol. 42, p. 1443). It is intended to include all forms of trypsin that retain peptide translocation activity. Examples of active trypsin derivatives may include acetylated trypsin, succinylated trypsin, glutaric aldehyde-treated trypsin and immobilized trypsin derivatives. If immobilized trypsin is used, it is suspended in the medium. Organic or inorganic acids such as hydrochloric acid, formic acid, acetic acid, propionic acid and butyric acid, or pyridine, TRIS, N
- Bases such as methylmorpholine and N-ethylmorpholine are added to provide a suitable buffer system. Organic acids are preferred. However, the reaction may proceed without such addition. The amount of acid added is approximately 10 per equivalent of L-threonine ester.
Less than an equivalent amount is normal. The preferred amount of acid added is L
- 0.5 to 5 equivalents per equivalent of threonine ester. Ions that stabilize trypsin, such as calcium ions, can also be added. The process is carried out at temperatures ranging from 50°C to the freezing point of the reaction mixture. The enzymatic reaction using trypsin is usually carried out at about 37°C to obtain a sufficient reaction rate. However, in order to avoid inactivation of trypsin, it is advantageous to carry out the process according to the invention at temperatures below room temperature. In fact, reaction temperatures above about 0°C are preferred. The reaction time is usually between several hours and several days depending on the reaction temperature, amount of trypsin added and other reaction conditions. The weight ratio of trypsin to insulin compound in the reaction mixture is usually about 1:200 or more, preferably about 1:50 or more, and less than about 1:1. L-threonine esters contemplated for the practice of this invention have the following general formula (): Thr( ^R5 ) ^-OR4 (), where ^R4 represents a carboxyl protecting group and ^R5
represents hydrogen or a hydroxyl-protecting group, or a salt thereof). Threonine ^B30 ester of human insulin, obtained from peptide rearrangement, has the following general formula (): (Thr( ^R5 ) ^-OR4 ) ^B30 -h-In (), where h-In (Thr ^B30 represents the insulin moiety and R ⁴ and R ⁵ have the meanings defined above) Applicable L-threonine esters of the formula are esters such as: R ⁴ is a carbonyl protecting group, and this group is
It can be removed from threonine ^B30 of human insulin under conditions that do not substantially cause irreversible exchange within the insulin molecule. Examples of such carboxyl protecting groups include lower alkyl such as methyl, ethyl and tert-butyl; p-
Substituted benzyl groups such as methoxybenzyl and 2,4,6-trimethylbenzyl as well as diphenylmethyl and also the general formula -CH ₂ CH ₂ SO ₂ R ⁶ where R ⁶
represents lower alkyl such as methyl, ethyl, propyl and n-butyl). A suitable hydroxyl protecting group R ⁵ is the threonine ^B30 of human insulin under conditions that do not cause substantial irreversible exchange within the insulin molecule.
A protecting group that can be removed from an ester (formula). An example of such a group (R ⁵ ) is a tert-butyl group. Lower alkyl groups contain less than 7 carbon atoms, preferably less than 5 carbon atoms. Furthermore, commonly used protecting groups are described by Wu¨nsch: Methoden der
Organischen Chemie (Houben-Weyl), XV/
Volume 1, editors: Eugen Mu¨ller, Georg Thime
Verlag, Stuttgart1974. Certain L-threonine esters (formula) are
It is a known compound, and other L-threonine esters (formula) can be produced by the same method as the production of known compounds. The L-threonine ester represented by the formula is
It may be the free base or a suitable organic or inorganic salt thereof, preferably a hydrochloride such as acetate, propionate, butyrate and hydrochloride. It is desirable to use high concentrations of the reactive components, insulin compound and L-threonine ester (formula). Preferably, the molar ratio between L-threonine ester and insulin compound is greater than about 5 to 1. Preferably, the concentration of L-threonine ester (formula) in the reaction mixture is greater than 0.1 molar. Human insulin can be prepared from the threonine ^B30 ester of human insulin by a method known per se or by a method known per se, protecting groups R ⁴ and R ⁵ .
obtained by removing . R ⁴ is methyl,
ethyl, or the group -CH ₂ CH ₂ SO ₂ R ⁶ , where R ⁶ has the meaning defined above, the protecting group is protected under mild basic conditions in an aqueous medium, preferably about It can be removed at a PH value of 8 to 12, for example about 9.5.
As a base, ammonia, triethylamine or an alkali metal hydroxide such as sodium hydroxide can be used. When R ⁴ is substituted benzyl or diphenylmethyl, such as tert-butyl, p-methoxybenzyl or 2,4,6-trimethylbenzyl, the protecting group is removed by acidolysis, preferably with trifluoroacetic acid. sell. Trifluoroacetic acid may be non-aqueous or water-containing, or may be diluted with an organic solvent such as dichloromethane. If R ⁵ is tert-butyl, the group can be removed by acidolysis (see above). Preferred threonine ^B30 esters of human insulin of the formula are those compounds in which R ⁵ is hydrogen; these are obtained from L-threonine esters of the formula where R ⁵ is hydrogen. The peptide rearrangement of an insulin compound to the threonine ^B30 ester of human insulin is expressed by the following formula for an insulin compound: {During the ceremony,

[Formula] is the human death (Thr ^B30 ) insulin part (here, Gly ^A1 is
It is bonded to the substituent represented by R ¹ and further Lys ^B29
is bonded to the substituent represented by ^R2 ), ^R2 represents an amino acid or a peptide chain containing up to 36 amino acids, and ^R1 is hydrogen or has the general formula ^R3 -X-( where X represents arginine or lysine and R ³ represents a peptide chain containing up to 35 amino acids, or the number of amino acids present in both R ¹ and R ² R ³ , provided that is less than 37
together with R ² represents a peptide chain containing up to 35 amino acids}. Thus, the peptide rearrangement of the present invention converts any of the above insulin compounds to the threonine ^B30 ester of human insulin (formula), which can then be unblocked to form human insulin. Another advantage of the present invention is that the insulin-like compounds present within crude insulin and within certain commercial insulin preparations and covered by the formula It is converted to the ^B30 ester, which can then be unblocked to form human insulin. Examples of insulin-like compounds of the formula are: porcine dialginine insulin (R ¹ is hydrogen and R ² is -Ala-Arg-Arg), porcine proinsulin (R ² and R ³ 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−Lys−, where the terminal alanyl is attached to Lys ^B29 ), canine proinsulin (R ³ together with R ² are −
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−, and the terminal alanyl is Lys ^B29
), porcine mitotic proinsulin (R ³ is Ala−Leu−Glu−Gly−Pro−Pro−Gln−
Lys−, and R ² 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 (R ¹ is hydrogen, and R ² 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 (R ² and R ³ are −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 terminal threonyl is attached to Lys ^B29 ), and monkey proinsulin (with R ²
R ³ is −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 terminal threonyl is attached to Lys ^B29 ). In all these impurities covered by the formula, the substituent R ¹ represented by R ³ -X is replaced with hydrogen. A preferred embodiment of the invention comprises reacting crude porcine insulin containing an insulin-like compound or a salt or complex thereof with an L-threonine ester (formula) or a salt thereof under the conditions described above; Thereafter protecting group R ⁴ and any protecting group R ⁵ are removed. This process converts porcine insulin and the insulin-like compounds therein to human insulin. Examples of complexes or salts of insulin compound (formula) include zinc complexes or zinc salts. Choosing the reaction conditions as described above and considering the results obtained in the examples below, it was found that over 60% of the threonine ^B30 ester of human insulin
It is even possible to obtain yields of 80% or higher, and under certain preferred conditions even 90% or higher. The process according to the invention thus has the following advantages over the prior art: (a) Enzymatic hydrolysis for removing Ala ^B30 , for example with carboxypeptidase, is omitted. (b) There is no need to isolate intermediate compounds such as porcine des-(Ala ^B30 )-insulin. (c) Contamination with des(Asn ^A21 )-insulin derivatives is avoided. (d) Porcine insulin as well as other insulin-like impurities present in crude insulin are converted to human insulin via the threonine ^B30 ester of human insulin by the process of the present invention, and thereby are harvested. rate increases. (e) Antigenic insulin-like compounds (UK patent no.
1285023) can be converted into human insulin. A preferred procedure for producing human insulin is as follows: (1) The starting material used for the peptide rearrangement is crude porcine insulin, such as crystalline insulin obtained using a citrate buffer. (see US Pat. No. 2,626,228). (2) If trypsin activity remains after peptide translocation, it is preferable to remove trypsin under conditions in which trypsin is inactive, for example in an acidic medium with a pH of less than 3. Trypsin can be separated by molecular weight, e.g. "Sephadex G-50" or "Bio-ge1P-30" in 1M acetic acid.
Can be removed by gel filtration (Nature280
(see Vol., p. 412). (3) Other impurities such as unreacted porcine insulin can be removed using anion and/or cation exchange chromatography (Example 1 and Example 2). (4) After that, human insulin threonine
The ^B30 ester is unblocked and human insulin is isolated, for example by crystallization in a manner known per se. This process allows human insulin of pharmaceutically acceptable purity to be obtained and further purified if necessary. Furthermore, the present invention relates to novel threonine ^B30 esters of human insulin, where the ester moiety is different from tert-butyl and methyl. The abbreviations used are related to biochemical nomenclature.
In accordance with the Regulations (1974) approved by the IUPAC-ICB Committee on Biochemical Nomenclature
1UPAC-IUB Commission Interim Rules and Recommendations, Second Edition, Maryland 1975). Analytical Testing: Conversion of porcine insulin and porcine proinsulin to human insulin esters was performed in a 7.5% polyamide gel in a buffer consisting of 0.375M Tris, 0.06M HCl, and 8M urea.
Proven by DISC PAGE electrophoresis. The pH of the buffer is 8.7. The ester of the formula migrates at a rate 75% of that of porcine insulin. Porcine proinsulin, which migrates at 55% of the rate of porcine insulin, is converted to the same product by the method of the present invention. Identification of conversion products with the compound of formula is according to the following criteria. (a) The electrophoresis of the human insulin ester, expressed by the formula for porcine insulin, is 1
corresponds to the loss of negative charges. The amino acid composition of the stained protein band corresponding to the compound of formula (b) is identical to that of human insulin, ie 3 moles of threonine and 1 mole of alanine to 1 mole of insulin. On the other hand, the composition of pig insulin is 2 moles of threonine and 2 moles of alanine per 1 mole of insulin. A technique for analyzing the amino acid composition of protein bonds in polyacrylamide gels was developed by Eur.J.
Biochem. Vol. 25, p. 147. (c) The fact that the bound threonine is substituted as the C-terminal amino acid in the B chain was demonstrated by oxidized sulfur decomposition of the S-S bond of insulin in 6M guanidine hydrochloride, followed by "SP Sephadex". This is evidenced by the decomposition of A and B chains by the ion exchange resin used. This technique, in which only the C-terminal amino acid is released when the S-sulfonate of the B chain is degraded by carboxypeptidase A, was published at the Symposium on Proinsulin, Insulin and C-Peptide (Tokushima, July 12-14, 1978). ) in the newsletter (editors: Baba.Kaneko and Yaniahara.Int.).
Congress Series No.468, Excerpta Medica,
Amsterdam-Oxford) analysis is carried out after the ester group has been separated from the compound of formula. These three analyzes clearly show that conversion to human insulin is occurring. The conversion of porcine insulin and porcine proinsulin to human insulin esters can be quantitatively followed using HPLC (high pressure liquid chromatography) with reversed phase. A4×300mm “μ
A Bondapak C ₁₈ column (Waters Ass.) was used and the elution was carried out using a 26
A buffer containing ~50% acetonitrile was used. The optimum acetonitrile concentration depends on the ester of the formula desired to be isolated from porcine insulin. If R ⁴ is methyl and R ⁵ is hydrogen, the separation is 26% (V/V)
of acetonitrile. Porcine insulin and (Thr-OMe) ^B30 -h-In (Me is methyl) elute after 4.5 and 5.9 column volumes, respectively, and after separation into symmetrical peaks.
Ten volumes of acetone are added to precipitate the proteins in the reaction mixture before applying to the HPLC column. The precipitate is isolated by centrifugation, dried under vacuum and dissolved in 0.02M sulfuric acid. The human insulin ester and the method for producing human insulin are further illustrated, but are of course not limited, by the following examples. The examples demonstrate preferred embodiments of the method according to the invention. Example 1 200mg of crude porcine insulin that has been crystallized,
Dissolved in 1.8 ml of 3.33M acetic acid. 2 ml of a 2M Thr-OMe (Me is methyl) solution in N,N-dimethylacetamide and 20 mg trypsin dissolved in 0.2 ml water were added. After storage at 37° C. for 18 hours, 40 ml of acetone was added to precipitate the proteins, and the precipitate was isolated by centrifugation. The supernatant was discarded. using 26% acetonitrile
Analysis of the precipitate by HPLC revealed that 60% of the pig insulin was converted to (Thr-OMe) ^B30 -h-In. The precipitate was dissolved in 8 ml of freshly deionized 8M urea. The pH value was adjusted to 8.0 with 1M ammonia, then the solution was
2.5×25cm filled with “QAE A-25 Sephadex”
applied to the column. The column was rated at 60% (V/
V) It is equilibrated with a 0.1M ammonium chloride buffer containing ethanol and whose pH value is adjusted to 8.0 with ammonia. Elution was performed using the same buffer and 15 ml fractions were collected. (Thr−
OMe) ^B30 -h-In was found in fraction numbers 26-46 and unreacted porcine insulin was found in fractions numbers 90-120. Fraction number 26~
46, pool and evaporate the ethanol in vacuo.
Then (Thr−OMe) ^B30 −h−In Schlichtkrull
Citrate buffer (Handbuch der inneren Medizin, 7/2A,
96, Berlin, Heiderberg, New York, 1975)
was crystallized. The yield was 95 mg of crystals with the same orthorhombic shape as porcine insulin crystallized in a similar manner. The amino acid composition was found to be identical to that of human insulin. Furthermore, the product obtained by the analytical test described above in “Analytical Test” is (Thr−OMe) ^B30 −
It was proven to be h-In. Example 2 100mg of porcine insulin meeting the purity described in UK Patent No. 1285023 was mixed with 0.9mg of 3.33M acetic acid.
ml of threonine methyl ester, then dissolved in N,N-dimethylformamide.
1 ml of 0.2 M solution and 12 mg dissolved in 0.1 ml water
TPCK (tosylphenylalanine chloromethyl ketone) treated trypsin was added. After incubation for 24 hours at 37°C, the reaction was stopped by adding 4 ml of 1M phosphoric acid. “SP-Sephadex”
By ion chromatography using a 2.5 x 25 cm column, using an eluent consisting of 0.09 M sodium chloride and 0.02 M sodium dihydrogen phosphate dissolved in 60% ethanol (pH value of buffer: 5.5),
The product (Thr-OMe) ^B30 -h-In was separated from unreacted porcine insulin. (Thr−OMe) ^B30 −h
The fractions containing -In were collected, the ethanol was removed in vacuo, and the product was purified as described in Example 1.
Crystallized. The yield is (Thr−OMe) ^B30 −h−
It was 50mg of In. Example 3 100mg of porcine proinsulin was added to 3.33M acetic acid.
Dissolve in 0.9 ml and then (Thr-OMe) ^B30 -h-In
and purified as described for porcine insulin in Example 1. The conversion of proinsulin to (Thr-OMe) ^B30 -h-In was found to be 73% by HPLC analysis of the acetone precipitate. Crystalline (Thr-OMe) ^B30 -h-In is 54mg
It was hot. Example 4 100 mg of porcine insulin was dissolved in 2.77 M acetic acid in water.
0.9 ml and then reacted in a manner analogous to that described in Example 2. After the reaction was complete, the proteins were precipitated by adding 10 volumes of acetone.
Analysis by DISC PAGE showed that 70% of the
OMe) was found to be converted to ^B30 -h-In. Example 5 100mg of porcine insulin was mixed with 3.33M acetic acid 0.9
1 ml of 2M Thr-OMe dissolved in N-methylpyrrolidone was added. Example 4
The reaction was carried out in a manner similar to that described in .
Conversion to (Thr-OMe) ^B30 -h-In was 20%. Example 6 100 mg of porcine insulin was dissolved in water.
2.77M dissolved in 0.9ml of acetic acid, and 2M dissolved in HMPA (hexamethylphosphonotriamide)
1 ml of Thr-OMe was added. The reaction was carried out in a manner similar to that described in Example 4. (Thr−OMe) ^B3
Conversion to ⁰ -h-In was 80%. Example 7 100mg of porcine insulin was mixed with 3.33M acetic acid 0.9
ml and 1 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added.
The reaction was carried out in a manner similar to that described in Example 4. Conversion to (Thr-OMe) ^B30 -h-In was 80%. Example 8 100mg of porcine insulin was mixed with 3.33M acetic acid 0.9
ml and 1 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added.
After that, 200U immobilized on 1g of glass bulb
Trypsin (activity measured against support BAEE)
was added and then the trypsin bound to the glass was filtered after incubation for 24 hours at 37°C. After the reaction was completed, the proteins were precipitated by adding 10 volumes of acetone. Analysis by DISC PAGE revealed that 40% was converted to (Thr-OMe) ^B30 . Example 9 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and then 1 ml of 2 M Thr-OMe dissolved in N,N-dimethylacetamide was added. After that, 200 μg of CNBr-activated “SephadexG-
300 U trypsin immobilized on 150' (activity determined on support BAEE) was added. 24 at 37℃
After an hour incubation, "Sephadex"
Trypsin bound to After the reaction is complete,
Ten volumes of acetone were added to add protein. As a result of analysis by DISC PAGE, it was found that 70% was converted to (Thr-OMe) ^B30 -h-In. Example 10 The procedure described in Example 7 was repeated using 2M Thr- ^OBut ( ^But is tert-butyl) dissolved in N,N-dimethylacetamide as the ester. (Thr−OBu ^t ) The conversion rate to ^B30 was 80%. Example 11 The procedure described in Example 8 was repeated using 2M Thr- ^OBut ( ^But is tert-butyl) dissolved in N,N-dimethylacetamide as the ester. (Thr−OBu ^t ) The conversion rate to ^B30 was 30%. Example 12 The procedure described in Example 9 was repeated using 2M Thr- ^OBut ( ^But is tert-butyl) dissolved in N,N-dimethylacetamide as the ester. (Thr−OBu ^t ) The conversion rate to ^B30 was 70%. Example 13 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
1 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added. The reaction was carried out in a manner similar to that described in Example 4. (Thr-OMe) Conversion rate to ^B30 -h-In is 80%
It was hot. Example 14 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
1 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added. The mixture was treated with immobilized trypsin similarly to the method described in Example 8. (Thr−OMe) ^B30 −
The conversion rate to h-In was 40%. Example 15 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
1 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added. Similar to the method described in Example 9, the mixture was treated with immobilized trypsin. (Thr−OMe) ^B30 −
The conversion rate to h-In was 70%. Example 16 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
ml and then 1 ml of 2M Thr-OBu ^t dissolved in N,N-dimethylacetamide was added. The reaction was carried out in a manner similar to that described in Example 4. The conversion rate to (Thr- ^But ) ^B30 -h-In was 80%. Example 17 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
ml and then 1 ml of 2M Thr-OBu ^t dissolved in N,N-dimethylacetamide was added. Similar to the method described in Example 8, the mixture was treated with trypsin immobilized on glass beads.
The conversion rate to (Thr-OBu ^t ) ^B30 -h-In was 40%. Example 18 100 mg of porcine proinsulin was mixed with 3.33 M acetic acid 0.9
ml and then 1 ml of 2M Thr-OBu ^t dissolved in N,N-dimethylacetamide was added. The mixture was treated with trypsin immobilized on CNBr-activated "Sephadex G-150" as described in Example 9. The conversion rate to (Thr-OBu ^t ) ^B30 -h-In was 70%. Example 19 100 mg of porcine insulin was dissolved in 0.5 ml of 6M acetic acid and then 1 ml of 1M Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) dissolved in N,N-dimethylacetamide was added. Furthermore, 5 mg of TPCK-treated trypsin dissolved in 0.5 ml of N,N-dimethylacetamide and 0.1 ml of water was added. The mixture was stored at 32°C for 44 hours. After the reaction was completed, 10 volumes of acetone were added to precipitate the protein. Results of analysis by DISC PAGE, 50
It was found that % was converted to (Thr-OTmb) ^B30 -h-In. Example 20 100 mg of porcine insulin was dissolved in 0.9 ml of 3M acetic acid and then 2M Thr- dissolved in dioxane.
Added 1 ml of OMe. The reaction was carried out in a manner similar to that described in Example 4. (Thr−OMe) ^B30 −h
-The conversion rate to In was 10%. Example 21 100 mg of porcine insulin was dissolved in 0.9 ml of 3M acetic acid and then 2M Thr dissolved in acetonitrile.
- Added 1 ml of OMe. The reaction was carried out in a manner similar to that described in Example 4. (Thr−OMe) ^B30 −
The conversion rate to h-In was 10%. Example 22 (Thr-OMe) 250 mg of crystals of ^B30 -h-In was added to 25 mg of water.
ml, and then 1N sodium hydroxide solution was added and dissolved to adjust the pH value to 10.0. PH value, 25
The temperature was kept constant at 10.0 °C for 24 hours. Add 2g of sodium chloride, 350mg of sodium acetate trihydrate and 2.5mg of zinc acetate, 2.5mg of dihydrate, then check the pH value.
By adding 1N hydrochloric acid to obtain 5.52,
The human insulin produced was crystallized.
After 24 hours of storage at 4°C, orthorhombic crystals were isolated by centrifugation, washed with 3 ml of water, isolated by centrifugation, and then dried in vacuo. Yield: 220 mg of human insulin. Example 23 (Thr-OTmb) 100 mg of ^B30 -h-In was dissolved in 1 ml of ice-cold trifluoroacetic acid, and the resulting solution was then diluted with 0
It was stored at ℃ for 2 hours. The human insulin produced was precipitated by adding 10 ml of tetrahydrofuran and 0.97 ml of 1.03M hydrochloric acid dissolved in tetrahydrofuran. The resulting precipitate was isolated by centrifugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation and dried under vacuum. Dissolve the precipitate in 10ml of water, and determine the pH value of the solution.
Adjusted to 2.5 with 1N sodium hydroxide solution. Human insulin was precipitated by adding 1.5 g of sodium chloride and then isolated by centrifugation.
Dissolve the precipitate in 10 ml of water, then add sodium chloride
The human insulin was precipitated by adding 0.8 mg of zinc acetate dihydrate, 3.7 mg of zinc acetate dihydrate and 0.14 g of sodium acetate trihydrate followed by the addition of 1N sodium hydroxide to obtain a pH value of 5.52. .
After storage for 24 hours at 4°C, the precipitate is isolated by centrifugation, washed with 0.9 ml of water, isolated by centrifugation and dried in vacuo. Yield: human insulin
90mg. Example 24 100 mg of porcine insulin was dissolved in 0.5 ml of 10 M acetic acid and then 1.3 ml of 1.54 M Thr-OMe dissolved in N,N-dimethylacetamide was added. The mixture was cooled to 12°C. 0.05M Calcium Acetate
10 mg of trypsin dissolved in 0.2 ml was added. 12
After 48 hours at °C, 20 ml of acetone was added to precipitate the proteins. Pig insulin (Thr−
OMe) The conversion rate to ^B30 -h-In was determined by HPLC to be 97
It was %. Example 25 20 mg of porcine insulin was dissolved in a mixture of 0.08 ml of 10 M acetic acid and 0.14 ml of water followed by 2 M Thr- in N,N-dimethylacetamide.
Added 0.2 ml of OMe. The mixture was cooled to -10°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 72 hours at −10°C,
Proteins were precipitated by adding 5 ml of acetone. Porcine insulin (Thr−OMe) ^B30 −h−
The conversion rate to In was 64% by HPLC. Example 26 20 mg of porcine insulin was dispersed in 0.1 ml of water and then dissolved in 2M N,N-dimethylacetamide.
0.6 ml of Thr-OMe was added to dissolve insulin. The mixture was cooled to 7°C. Trypsin 2 dissolved in 0.025 ml of 0.05 M calcium acetate
mg was added. After 24 hours at 7°C, 5 ml of acetone was added to precipitate the proteins. The conversion rate of pig insulin to (Thr-OMe) ^B30 -h-In is:
It was 62% by HPLC. Example 27 20 mg of porcine insulin mixed with 4.45 M propionic acid
Then 0.24 ml of 1.67M Thr-OMe dissolved in N,N-dimethylacetamide was added. The mixture was kept at 37°C for 24 hours. 0.05M
2 mg of trypsin dissolved in 0.025 ml of calcium acetate was added. After 24 hours at 37°C, 10 volumes of 2-propanol were added to precipitate the proteins. The conversion rate of porcine insulin to (Thr-OMe) ^B30 -h-In was 75% by HPLC. Example 28 20 mg of porcine insulin was dispersed in 0.1 ml of water.
2M dissolved in N,N-dimethylacetamide
Add 0.4ml of Thr-OMe, then add 0.04ml of 10N hydrochloric acid.
was added to make a solution. 0.05M Calcium Acetate
2 mg of trypsin dissolved in 0.025 ml was added.
The mixture was held at 37°C for 4 hours. The conversion rate of pig insulin to (Thr-OMe) ^B30 -h-In is:
It was 46% by HPLC. Example 29 20mg of porcine insulin to 0.175ml of 0.57M acetic acid
dissolved in Then 0.2 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added followed by 0.025 ml of 0.05M calcium acetate.
10 mg of a preparative preparation of Achromobacterlyticus protease was added. The mixture was kept at 37°C for 22 hours. Proteins were precipitated by adding 10 volumes of acetone. Porcine insulin (Thr−OMe) ^B30 −h
The conversion rate to -In was 12% by HPLC. Example 30 20 mg of porcine insulin was dispersed in 0.1 ml of 0.5M acetic acid. Next, 0.2 ml of 0.1M Thr-OMe dissolved in N,N-dimethylacetamide was added to dissolve insulin. The mixture was cooled to 12°C.
2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 24 hours at 12°C, the conversion of porcine insulin to (Thr-OMe) ^B30 -h-In was 42% by HPLC. Example 31 2 mg of rabbit insulin was mixed with 0.135 mg of 4.45M acetic acid.
Dissolved in ml. Add 0.24 ml of 1.67M Thr-OMe dissolved in N,N-dimethylacetamide and then
1.25 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. The mixture was held at 37°C for 4 hours. Rabbit insulin (Thr−OMe)
The conversion to ^B30 -h-In was 88% by HPLC. Rabbit insulin elutes from the HPLC column before pig insulin, but the ratio of the elution amount of rabbit insulin to (Thr-OMe) ^B30 -h-In is 0.72. Example 32 Porcine diaarginine insulin (Arg ^B31 −
Arg ^B32 - insulin) 2 mg, 4.45M acetic acid 0.135
Dissolved in ml. Add 0.24 ml of 1.67M Thr-OMe dissolved in N,N-dimethylacetamide and then
1.25 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. The mixture was held at 37°C for 4 hours. Porcine insulin (Thr−OMe) ^B30
The conversion rate to -h-In was 91% by HPLC. Diarginine insulin elutes from the HPLC column before porcine insulin, but the ratio of the elution amount of dialginine insulin to (Thr-OMe) ^B30 -h-In is 0.50. Example 33 Porcine intermediate (i.e. desdipeptide (Lys ⁶²
-Arg ⁶³ ) proinsulin and desdipeptide (Arg ³¹ -Arg ³² ) proinsulin) were reacted similarly to the reaction described in Example 32. HPLC analysis showed that the conversion to (Thr-OMe) ^B30 -h-In was 88%. Example 34 20 mg of porcine insulin was dissolved in 0.1 ml of 2M acetic acid and then 0.2 ml of 2M Thr-OMe dissolved in N,N-dimethylacetamide was added. Mixture, -
Cooled to 18°C. 0.05M Calcium Acetate 0.025
2 mg of trypsin dissolved in ml was added. The mixture was kept at -18°C for 120 hours. (Thr−OMe) ^B30
As a result of HPLC analysis, the conversion rate to -h-In was 83%. Example 35 The procedure described in Example 34 was repeated with the conditions that the reaction was carried out at 50° C. for 4 hours. As a result of HPLC analysis, the conversion rate to (Thr-OMe) ^B30 -h-In was 23
It was %. Example 36 20 mg of porcine insulin was dissolved in 0.1 ml of 3M acetic acid. Dissolved in N,N-dimethylacetamide
0.33M Thr-O( _CH2 ) ₂ - _SO2 - _CH3・
CH ₃ COOH (threonine 2-(methylsulfonyl)
0.3 ml of ethyl ester hydroacetate) was added. The mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 24 hours at 12°C, (Thr-O( _CH2 ) _2-
The conversion to _SO2 - _CH3 ) ^B30 -h-In was 77% by HPLC. The product is (Thr−OMe) ^B30
It elutes approximately at the position. Example 37 20 mg of porcine insulin was dissolved in 0.1 ml of 10M acetic acid. 0.2 ml of 2M Thr-OEt (Et is ethyl) dissolved in N,N-dimethylacetamide was added. The mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 24 hours at 12℃ (Thr−OEt) ^B30 −h
The conversion rate to -In was 75% by HPLC.
The product eluted slightly after that of (Thr-OMe) ^B30 -h-In. Example 38 20 mg of porcine insulin was dissolved in 0.1 ml of 6M acetic acid. Dissolved in N,N-dimethylacetamide
0.3 ml of 0.67M Thr(Bu ^t )OBu ^t (Bu ^t is tertiary butyl) was added. The mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 24 hours at 12°C, the conversion rate to (Thr( ^But ) ^-OBut ) ^B30 -h-In is:
It was 77% by HPLC. The product was eluted from the HPLC column by applying a gradient elution method in which the acetonitrile was changed continuously from 27% to 40%. Example 39 20 mg of porcine insulin was dissolved in 0.1 ml of 4M acetic acid. 1.5M dissolved in tetrahydrofuran
0.2 ml of Thr-OMe was added and the mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 4 hours at 12°C (Thr-OMe), the conversion rate to ^B30 -h-In was determined by HPLC
It was 75%. Example 40 20 mg of porcine insulin was dissolved in 0.1 ml of 4M acetic acid and then 2M in 1,2-ethanediol.
0.8 ml of Thr-OMe was added. The mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 4 hours at 12°C, the conversion rate to (Thr-OMe) ^B30 -h-In is:
It was 48% by HPLC. Example 41 20 mg of porcine insulin was dissolved in 0.1 ml of 4M acetic acid. 2M Thr−OMe0.2 dissolved in ethanol
Added ml. The mixture was cooled to 12°C. 0.05M
2 mg of trypsin dissolved in 0.025 ml of calcium acetate was added, and the reaction was carried out at 12°C for 4 hours. (Thr
-OMe) ^B30 The conversion rate to -h-In was 46% as analyzed by HPLC. Example 42 20 mg of porcine insulin was dissolved in 0.1 ml of 4M acetic acid. 0.2 ml of 2M Thr-OMe dissolved in acetone was added and the mixture was cooled to 12°C. 2 mg of trypsin dissolved in 0.025 ml of 0.05M calcium acetate was added. After 4 hours at 12℃, (Thr−OMe) ^B30 −h
-The conversion rate to In was 48% as determined by HPLC analysis.
It was hot.

Claims (1)

[Scope of Claims] 1. In the method for producing human insulin or threonine ^B30 ester of human insulin, a salt thereof, or a complex thereof, an insulin compound, a salt of the compound, or a complex thereof, which can be converted into threonine ^B30 ester of human insulin, a salt thereof, or a complex thereof. peptide rearrangement of a complex of the compound with L-threonine ester or a salt thereof in a mixture consisting of water, a water-miscible organic solvent, and trypsin, and the content of water in the reaction mixture is 50%. (volume/volume), the reaction temperature is less than 50°C, the reaction is optionally carried out in the presence of an acid, and then the obtained threonine ^B30 ester of human insulin is optionally unblocked. , said method. 2. the concentration of L-threonine ester in the reaction mixture exceeds 0.1 molar, the reaction temperature exceeds the freezing point of the reaction mixture, and the reaction mixture contains 0 to 10 equivalents of acid per equivalent of L-threonine ester; A method according to claim 1. 3. The insulin compound is each insulin of pig, dog, whale, normal whale, and domestic rabbit,
Claim 1, which is porcine dialginine insulin, porcine split proinsulin, porcine death peptide proinsulin, porcine, canine, monkey and human proinsulins, and mixtures thereof. Or the method described in paragraph 2. 4. The method according to any one of claims 1 to 3, wherein the insulin compound is obtained from a pig. 5. The method according to claim 4, wherein relatively crude insulin is used as the insulin compound. 6. A process according to any of claims 1 to 5, wherein the reaction temperature is below 37°C, preferably below room temperature, and above 0°C. 7. A process according to any of claims 1 to 6, wherein the content of water in the reaction mixture is between 10% (vol/vol) and 40% (vol/vol). 8. The method according to any one of claims 1 to 7, wherein the molar ratio of the L-threonine ester to the insulin compound is greater than 5:1. 9. The method according to any one of claims 1 to 8, wherein the organic solvent is a polar solvent. 10 The solvent is methanol, ethanol, 2
-Propanol, 1,2-ethanediol, acetone, dioxane, tetrahydrofuran, formamide, N,N-dimethylformamide, N,N
-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphotriamide, or acetonitrile. 11. A method according to any of claims 1 to 10, wherein the acid is an organic acid, preferably formic acid, acetic acid, propionic acid and butyric acid. 12. A method according to any of claims 1 to 11, wherein the amount of acid in the reaction mixture ranges from 0.5 to 5 per equivalent of L-threonine ester. 13. The method of claims 1 to 12, wherein the weight ratio of trypsin to insulin compound in the reaction mixture is about more than 1:200, preferably more than 1:50, and even less than 1:1. Any method described. 14. The method according to any one of claims 1 to 13, wherein the peptide rearrangement is performed in the presence of calcium ions. 15. The method according to any one of claims 1 to 14, wherein the peptide rearrangement is carried out in a buffer system. 16. A method according to any of claims 1 to 15, wherein the L-threonine ester is added as an acetate or a hydrohalide such as propionate, butyrate, or hydrochloride. 17. The reaction temperature and the content of water and acid in the reaction mixture are selected such that the yield of threonine ^B30 ester of human insulin is 60% or more, preferably 80% or more, most preferably 90% or more. , the method according to any one of claims 1 to 16. 18 The insulin compound is {In the formula, [formula] is the human death (Thr ^B30 ) insulin moiety (Gly ^A1 in this moiety
is bonded to the substituent represented by R ¹ and further
Lys ^B29 is bonded to a substituent represented by ^R2 ), ^R2 represents an amino acid or a peptide chain containing up to 36 amino acids, and ^R1 is hydrogen or the general formula ^R3 -X - (wherein X represents arginine or lysine and R ³ represents a peptide chain containing up to 35 amino acids), or an amino acid present in both R ¹ and R ² Provided that the total number of is less than 37,
R ³ and R ² together represent a peptide chain containing up to 35 amino acids} Claim 1
Or the method according to any one of Item 2 or Items 6 to 17. 19 R ¹ is hydrogen, R ² is -Ala, -Ser,
−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−Gln, and R ³ is Ala−
Leu−Glu−Gly−Pro−Pro−Gln−Lys−, and R ² 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 or R ³ and R ² together −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- (here, the terminal alanyl is bonded to Lys ^B29 ), -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 alanyl is attached to Lys ^B29 ), -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 terminal alanyl is attached to Lys ^B29 ) 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−
19. The method of claim 18, wherein Lya- (where the terminal threonyl is attached to Lys ^B29 ). 20. The method of claim 19, wherein R ¹ is hydrogen and R ² is alanine. 21 The L-threonine ester has the following general formula (): Thr(R ⁵ )-OR ⁴ () (wherein R ⁴ represents a carboxyl protecting group, R ⁵
represents hydrogen or a hydroxyl protecting group. 22 R ⁴ is lower alkyl, substituted benzyl or diphenylmethyl, or the general formula -
Claim 2, which is a group represented by CH ₂ CH ₂ SO ₂ R ⁶ (in the formula, R ⁶ represents lower alkyl)
The method described in Section 1. 23 R ⁴ is methyl, ethyl, tert-butyl,
p _- methoxy _- benzyl, 2,4,6 ^- trimethylbenzyl, or the formula _-CH2CH2SO2R6 , where ^R6
is methyl, ethyl, propyl or n-butyl).
The method described in Section 2. 24 The insulin compound is porcine insulin or porcine proinsulin, the L-threonine ester is Thr-OMe, Thr-OBu ^t or Thr-OTmb, and the water-miscible organic solvent is N,N-dimethyl formamide N,N-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphotriamide, dioxane or acetonitrile, the reaction temperature is within the range of about 32-37°C, and the water content in the reaction mixture is between 21% and 37°C. The weight ratio of trypsin to insulin compound is in the range of about 1:8 to 1:27, and the molar ratio of L-threonine ester to insulin compound is in the range of about 60:1 to 180:1. Claims 1, 2, 8, 9, 11, 1, wherein acetic acid is added in an amount of about 1.25 to 3.0 equivalents per equivalent of L-threonine ester.
2, the method according to any one of 14 to 18 or 21. 25 The insulin compound is porcine insulin, and the L-threonine ester is Thr-
OMe or Thr-OBu ^t , and the water-miscible organic solvent is N,N-dimethylamide or N,N-dimethylamide.
-dimethylacetamide, and the reaction temperature is about 37
°C and the water content in the reaction mixture is between 41% and
43%, the weight ratio of trypsin to insulin compound is approximately 1:8, and the molar ratio of L-threonine ester to insulin compound is approximately
25. The method of claim 24, wherein the ratio is 120 to 1, and furthermore, acetic acid is added in a range of 1.2 to 1.5 equivalents per equivalent of L-threonine ester. 26 Claim 1, wherein the water content in the reaction mixture is in the range of 43-21% (volume/volume)
6. The method according to any one of items 6 to 6. 27. A method according to claim 1, wherein the unblocking is carried out in an aqueous medium in the presence of a base, preferably at a pH of 8 to 12. 28. A method according to claim 1, wherein the unblocking is carried out by acidolysis, preferably with trifluoroacetic acid.