GB2069502A - Process for Preparing Insulin Esters - Google Patents
Process for Preparing Insulin Esters Download PDFInfo
- Publication number
- GB2069502A GB2069502A GB8104114A GB8104114A GB2069502A GB 2069502 A GB2069502 A GB 2069502A GB 8104114 A GB8104114 A GB 8104114A GB 8104114 A GB8104114 A GB 8104114A GB 2069502 A GB2069502 A GB 2069502A
- Authority
- GB
- United Kingdom
- Prior art keywords
- leu
- insulin
- process according
- ala
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Forklifts And Lifting Vehicles (AREA)
Abstract
Insulin and insulin-like compounds from certain species can in the presence of an L-threonine ester, water, a water miscible organic solvent, and trypsin be transpeptidized into a threonine<B30> ester of human insulin in which the protecting group(s) can be removed whereby human insulin is formed.
Description
SPECIFICATION
A Process for Preparing Insulin Esters
This invention relates to the conversion of insulin and insulin-like compounds into human insulin.
In the treatment of diabetes mellitus insulin preparations derived from porcine or bovine insulin have generally been used. Bovine, porcine, and human insulins exhibit minor differences with respect to their amino acid composition, the difference between human and porcine insulin being confined to a single amino acid in that the
B30 amino acid of human insulin is threonine whereas that of porcine insulin is alanine.
However, it could be argued that the ideal insulin preparation for treatment of human beings would be an insulin having exactly the same chemical structure as that of human insulin.
For the production of natural human insulin the necessary amount of human pancreas glands is not available.
Synthetic human insulin has been prepared on a small scale at great expense, vide Helv. Chem.
Acta 57, 2617, and 60, 27. Semisyntetic human insulin has been prepared from porcine insulin by tedious pathways, vide Hoppe-Seyler's Z. Physiol.
Chem. 356, 1631, and nature 280, 412.
However, the present invention relates to a process which on an industrial scale can be used for preparing human insulin.
U.S. patent specification No. 3,276,961 purports to relate to a process for preparing semisynthetic human insulin. However, the yield of human insulin is poor because the process is performed in water, under which conditions trypsin causes a splitting of the ArgB22-GlyB23 bond, vide J. Biol. Chem. 236, 743.
A known semisynthetic process for preparing human insulin comprises the following three steps: First, porcine insulin is converted into porcine des-(Ala830)-insulin by treatment with carboxypeptidase A, vide Hoppe-Zeyier's A.
Physiol. Chem. 359, 799. In the second step porcine des-(AlaB30)-insulin is subjected to a trypsin-catalysed coupling with Thr-OBut, whereby human insulin ThrB30-tert-butyl ester is formed.
Finally, said ester is treated with triflouroacetic acid yielding human insulin, vide Nature 280, 412. The first step, however, results in a partial removal of As21, yielding des-(AlaB30, AsnA21)- insulin. This derivative gives, after the two subsequent reactions, rise to a contamination of des-(AsnA2')-insulin in the semisyntetic human insulin prepared, a contamination which cannot easily be removed with known preparative methods. Des-(AsnA21)-insulin possesses low biological activity (about 5%), vide Amer. J. Med.
40, 750.
One object of this invention is to provide a process which converts a non-human insulin into human insulin.
A second object of this invention is to provide a process which converts porcine insulin and certain impurities therein into human insulin via a threonine630 ester of human insulin.
Further objects and the advantages of this invention will be apparent in this description.
This invention is based upon the discovery that the amino acid or peptide chain bound to the carbonyl group of Lyes529 in the insulin compound can be inter-changed with a threonine ester. Said interchange is herein referred to as a transpeptidation.
The term "insulin compounds" as used herein encompasses insulins and insulin-like compounds containing the human des(ThrB30) insulin moiety, the B30 amino acid of the insulin being alanine (in insulin from, e.g., hog, dog, and fin and sperm whale) or serine (rabbit). The term "insulin-like compounds" as used herein encompasses proinsulin derived from any of the above species and primates, together with intermediates from the conversion of proinsulin into insulin. As examples of such intermediates can be mentioned split proinsulin, desdipeptide proinsulins, desnonapeptide proinsulin, and diarginine insulins, vide R. Chance: In Proceedings of the
Seventh congress of IDF, Buenos Aires 1970, 292-305, Editors: R. R. Rodriques 8 J. V.-Owen,
Excerpta Medica, Amsterdam.
The process according to this invention comprises transpeptidizing 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 content of water in the reaction mixture being less than about 50 per cent (volume/volume) and the reaction temperature being below about 500C.
Although trypsin is best known for its proteolytic properties, workers in the art have recognized that trypsin is capable of catalyzing the coupling of des-(AlaB30)-insulin and a threonine tert-butyl ester, vide nature 280, 412.
In the present process the trypsin is used to catalyze transpeptidation.
1 he transpeptidation can be performed by dissolving 1) the insulin compound, 2) an Lthreonine 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 can be mentioned methanol, ethanol, 2propanol, 1,2-ethandiol, acetone, dioxane, tetrahydrofuran, N,N-dimethylformamide, formamide, N,N-dimethylacetamide Nmethylpyrrolidone, hexamethylphosphortriamide, and acetonitrile.
Depending on which water miscible organic solvent is used, on the chosen reaction temperature, and on the presence of an acid in the reaction mixture, the content of water in the reaction mixture should be less than about 50 per cent (v/v), preferably less than about 40 per cent (v/v), and more than about 10 per cent (v/v).
One advantage by decreasing the amount of water in the reaction mixture is that thereby the formation of byproducts is decreased. Similarly, by increasing the amount of acid in the reaction mixture it is possible to decrease the byproduct formation. The increase in yield is positive correlated to a high percentage of organic solvent.
The trypsin type is not material to the practice of this invention. Trypsin is a well characterized enzyme which is commercially available in high purity, notably of bovine and porcine source.
Furthermore, trypsin of microbial origin may be used. Moreover, the trypsin form, e.g., crystalline trypsin (soluble form), immobilized trypsin or even trypsin derivatives (so long as the trypsin activity is retained) is not material to practice of this invention. The term trypsin as employed herein is intended to include trypsins from all sources and all forms of trypsin that retain the transpeptidating activity, including proteases with trypsin-like specificity, e.g. Acromobacter lyticus protease, vide Agric. Biol. Chem. 42, 1443.
As examples of active trypsin derivatives can be mentioned acetylated trypsin, succinylated trypsin, giutaraldehyde treated trypsin, and immobilized trypsin derivatives.
If an immobilized trypsin is used it is suspended in the medium.
Organic or inorganic acids such as hydrochloric acid, formic acid, acetic acid, propionic acid, and butyric acid, or bases such as pyridine, TRIS, Nmethylmorpholine, and N-ethylmorpholine may be added to bring about a suitable buffer system.
Organic acids are preferred. However, the reaction can be conducted without such additions. 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 Lthreonine ester. Ions, which stabilize trypsin such as calcium ions, may be added.
The process may be performed at a temperature in the range between 500C and the freezing point of the reaction mixture. Enzymatic reactions with trypsin are usually performed at about 370C in order to give a sufficient reaction rate, however, in order to avoid inactivation of trypsin it is advantageous to perform the process according to the present invention at a temperature below ambient. In practice reaction temperatures above about OOC are preferred.
The reaction time is usually between several hours and several days, depending upon the reaction temperature, upon the amount of trypsin added, and upon other reaction conditions.
The weight ratio between trypsin and the insulin compound in the reaction mixture is normally above about 1:200, preferably above about 1:50, and below about 1:1.
The L-threonine esters contemplated for practice of this invention can be depicted by the general formula
Thr(R5)-OR4 II wherein R4 represents a carboxyl protecting group, and R5 represents hydrogen or a hydroxyl protecting group, or a salt thereof.
The threonine830 esters of human insulin resulting from the transpeptidation can be depicted by the general formula (Thr(R5)~OR4)B30~h~ln 111 wherein h-In represents the human des-(ThrB30) insulin moiety, and R4 and R5 are as defined above.
Applicable L-threonine esters of formula II are such, in which R4 is a carboxyl protecting group which can be removed from the threonine930 ester of human insulin (formula Ill) under conditions, which do not cause substantial irreversible aiteration in the insulin molecule. As examples of such carboxyl protecting groups can be mentioned lower alkyl, e.g., methyl, ethyl, and tert-butyl, substituted benzyl such as p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenylmethyl, and groups of the general formula -CH2CH2SO2R6, wherein R6 represents lower alkyl such as methyl, ethyl, propyl, and n-butyl. Suitable hydroxyl protecting groups R5 are such which can be removed from the threonineB30 ester of human insulin (formula III) under conditions which do not cause substantial irreversible alteration in the insulin molecule.As an example of such a group (R5) can be mentioned tert-butyl.
Lower alkyl groups contain less than 7 carbon atoms, preferably less than 5 carbon atoms.
Further protection groups usually used are described by Winch: Metoden der Organischen
Chemie (Houben-Weyl), Vol. XV/1, editor: Eugen
Miller, Georg Thieme Verlag, Stuttgart 1974.
Some L-threonine esters (formula II) are known compounds and the remaining L-threonine esters (formula II) can be prepared in analogy with the preparation of known compounds.
The L-threonine esters of formula II may be the free bases or suitable organic or inorganic salts thereof preferably acetates, propionates, butyrates, and hydrohalides such as hydrochlorides.
It is desired to use the reactants, i.e. the insulin compound and the L-threonine ester (formula II), in high concentrations. The molar ratio between the L-threonine ester and the insulin compound is preferably above about 5:1.
It is desired that the concentration L-threonine ester (formula 11) in the reaction mixture exceeds 0.1 molar.
Human insulin can be obtained from the threonine830 esters of human insulin (formula Ill) by removal of the protecting group R4 and any protecting group R5 by known methods or methods known per se. In case R4 is methyl, ethyl, or a groupCH2CH2SO2R6, wherein R6 is as defined above, the said protecting group can be removed at gentie basic conditions in an aqueous medium, preferably at a pH value of about 8-12, e.g. at about 9.5. As the base can be used ammonia, triethylamine, or hydroxides of alkali metals such as sodium hydroxide. In case R4 is tert-butyl, substituted benzyl such as pmethoxvbenzyl or 2,4,6-tri methylbenzyl or diphenylmethyl the said group can be removed by acidolysis, preferably with trifluoroacetic acid.The trifluoroacetic acid may be non-aqueous or may contain some water, or it may be diluted with an organic solvent such as dichloromethane. in case
R5 is tert-butyl said group can be removed by acidolysis, vide above.
Preferred threonineB30 esters of human insulin of the formula Ill are compounds, wherein R5 is hydrogen, and these are prepared from Lthreonine esters of the formula II, wherein R9 is hydrogen.
The transpeptidation of insulin compounds into threonineB30 esters of human insulin can be described in more detail on the basis of the following formula assigned to the insulin compounds:
wherein
represents the human des(ThrB30) insulin moiety wherein GlyA1 is connected to the substituent designated R1 and Lyes829 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and R1 represents hydrogen or a group of the general formula R3-X-, wherein X represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represent a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in R1 plus R2 is less than 37.
Thus, the transpeptidation of this invention converts any of the above insulin compounds into threonineB30 esters of human insulin (formula Ill), which then can be deblocked to form human insulin.
A further advantage of this invention is that insulin-like compounds present in crude insulin and present in some commercial insulin preparations and covered by the formula I by the transpeptidation of this invention are converted into threonine930 esters of human insulin, which then can be deblocked to form human insulin.
Examples of insulin-like compounds of formula I appear from the following:
Porcine diarginine insulin (R1 is hydrogen, and
R2 is -Ala-Arg-Arg), porcine proinsulin (R3 together with R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn- Pro-GI n-Ala-Gly-Ala-Val-GIu-Leu-Gly-Gly-Gly-
Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly- Pro-Pro-Gln-Lys, wherein the terminal alanyl is connected to Lyes929), dog proinsulin (R3 together with R2 is -Al a-Arg-Arg-Asp-Val-G lu-Leu-Ala-G ly- Ala-Pro-Gly-Glu-G ly-Gly-Leu-Gln-Pro-Leu-AIa- Leu-Glu-Gly-Aia-Leu-Gln-Lys-, wherein the terminal alanyl is connected to LysB29), porcine split proinsulin (R3 is Ala-Leu-Glu-Gly-Pro-Pro
Gln-Lys-, and R2 is-Ala-Arg-Arg-Glu-Ala-Glu- Asn-P ro-Gln-Ala-Gly-Ala-Va I-G lu-Leu-Gly-Gly- Gly-Leu-G iy-G Iy-Leu-G In-Ala-Leu ), porcine desdipeptide proinsulin (R1) is hydrogen, and R2 is -Ala-Arg-Arg-G I u-Ala-Glu-Asn-P ro-G In-Ala-Gly Ala-VaI-G Iu-Leu-G ly-G ly-G ly-Leu-G ly-GIy-Leu- Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln), human proinsulin (R3 together with R2 is -Thr-Arg-Arg Glu-Ala-Glu-Asp-Leu-GIn-Val-Gly-GIn-Val-GIu- Leu-Gly-G ly-G ly-Pro-G ly-Ala-G ly-Ser-Leu-GIn- Pro-Leu-Ala-Leu-Glu-G!y-Ser-Leu-Gln-Lys, wherein the terminal threonyl is connected to Lyes929), and monkey proinsulin (R3 together with
R2 is -Thr-Arg-Arg-Glu-Ala-G lu-Asp-Pro-Gln-Val- Gly-G In-Val-G lu-Leu-G Iy-Gly-G Iy-Pro-Gly-Ala-
G Iy-Ser-Leu-G In-Pro-Leu-Ala-Leu-G lu-G Iy-Ser- Leu-Gln-Lys-, wherein the terminal threonyl is connected to LysB30).
Hence, in all these impurities covered by formula I the R1 substituent designated R3-X- is exchanged with hydrogen.
A preferred embodiment of the present invention comprises reacting crude porcine insulin containing insulin-like compounds or a salt or complex thereof with an L-threonine ester (formula li) or a salt thereof under the above conditions whereafter the protecting group R4 and any protecting group R5 is removed. By this process the porcine insulin together with insulinlike compounds therein is converted into human insulin.
As examples of a complex or a salt of an insulin compound (formula 1) can be mentioned a zinc complex or zinc sait.
When selecting the reaction conditions according to the above explanation and considering the results obtained in the following examples it is possible to obtain a yield of threonineB30 ester of human insulin which is higher than 60 per cent, and even higher than 80 per cent, and under certain preferred conditions higher than 90 per cent.
The process according to the present invention has, therefore, the following advantages over the prior art:
a) The enzymatic hydrolysis to remove Alas30, e.g., with carboxypeptidase A, is omitted.
b) The isolation of an intermediate compound, such as porcine des-(Alas30)-insulin, is unnecessary.
c) Contamination with des(AsnA21)-insulin derivatives is avoided.
d) Proinsulin and other insulin-like impurities present in crude insulin are - via the threonineB30 ester of human insulin - converted into human insulin by the process of this invention, whereby the yield is increased.
e) Antigenic insulin-like compounds, vide
British Patent No. 1 285,023, are converted into human insulin.
A preferred procedure for preparing human insulin is as follows:
1) The starting material used for the transpeptidation is crude porcine insulin, e.g., crystalline insulin obtained by the use of a citrate buffer, vide Patent Specification No. U.S.A.
2,626,228.
2) If there is any trypsin activity left after the transpeptidation, it is preferred to remove it, e.g., under conditions where trypsin is inactive, e.g.. in acid medium below pH 3. Trypsin can be removed by separation according to molecular weight, e.g., by gel filtration on "Sephadex G-50" or "Bio-gel
P-30" in 1 M acetic acid, vide Nature 280, 412.
3) Other impurities such as unreacted porcine insulin may be removed by the use of anion and/or cation exchange chromatography, vide
Examples 1 and 2.
4) Thereafter, the threonineB30 ester of human insulin is deblocked and human insulin is isolated, e.g., crystallized, in a manner known per se.
By this process human insulin of an acceptable pharmaceutical purity can be obtained and be further purified, if desired.
Furthermore, the present invention relates to novel threonine930 esters of human insulin wherein the ester moiety is different from tertbutyl and methyl.
Abbreviations used are in accordance with the rules approved (1974) by the IUPAC-IUB
Commission on Biochemical Nomenclature, vide
Collected Tentative Rules s Recommendations of the Commission on Biochemical Nomenclature lUPAC-lUB, 2nd ed., Maryland 1975.
Analytical Tests
The conversion of porcine insulin and porcine proinsulin into human insulin esters can be demonstrated by DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.06 M HCI, and 8 M urea. The pH of the buffer is 8.7. Esters of the formula III migrates with a speed of 75% of that of porcine insulin. Porcine proinsulin migrating with 55% of the speed of porcine insulin is by the process according to the present invention converted into the same product. Identification of the conversion product as compounds of the formula Ill is due to the following criteria:
a) The electrophoretic migration of the human insulin esters of formula III in relation to porcine insulin corresponds to the loss of one negative charge.
b) The amino acid composition of the stained protein bonds in the gel representing compounds of the formula Ill is identical with that of human insulin, i.e. 3 mol threonine and 1 mol alanine per mol insulin, and the composition of porcine insulin is 2 mol threonine and 2 mol alanine per mol insulin. The technique for analyzing amino acid compositions of protein bonds in polyacrylamide gels has been described in Eur. J. Biochem. 25, 147.
c) The proof that the incorporated threonine is placed as C-terminal amino acid in the B chain, is proved by oxidative sulfitolysis of the S-S bridges of insulin in 6 M guanidinium 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 liberates only the C-terminal amino acid. The technique has been described by
Markussen in Proceedings of the Symposium on
Proinsulin, Insulin and C-peptide, Tokushima, 12-14 July, 1978, (Editor: Baba, Kaneko 8 Yaniahara) Int. Congress Series No. 468, Excerpta
Medica, Amsterdam-Oxford. The analysis is performed after the ester group has been split from compounds of the formula III.
Those three analyses prove unambiguously that the conversion into human insulin has taken place.
The conversion of porcine insulin and porcine proinsulin into human insulin esters can be followed quantitatively by HPLC (high pressure liquid chromatography) on reverse phase, A 4x300 mm ",u Bondapak C,8 column" (Waters
Ass.) was used and the elution was performed with a buffer comprising 0.2 M ammonium sulphate (adjusted to a pH value of 3.5 with sulphuric acid) and containing 2650% acetonitrile. The optimal acetonitrile concentration depends on which ester of the formula Ill one desires to separate from porcine insulin. In case R4 is methyl, and R5 is hydrogen, separation is achieved in 26% (v/v) of acetonitrile.
Porcine insulin and (Thr-OMe)B30-h-ln (Me is methyl) elute after 4.5 and 5.9 column volumes, respectively, as well separated symmetrical peaks. Before the application on the HPLC column the proteins in the reaction mixture were precipitated by addition of 10 volumes of acetone. The precipitate was isolated by centrifugation, dried in vacua, and dissolved in 0.02 M sulphuric acid.
The process for preparing human insulin esters and human insulin is illustrated by the following examples which, however, are not to be construed as limiting. The examples illustrate some preferred embodiments of the process according to the invention.
Example 1
200 mg crude porcine insulin, crystallized once, was dissolved in 1.8 ml 3.33 M acetic acid.
2 ml of a 2 M solution of Thr-OMe (Me is methyl) in N,N-dimethylacetamide, and 20 mg of trypsin dissolved in 0.2 ml of water were added. After storage for 18 hours at 370C the proteins were precipitated by the addition of 40 ml of acetone, and the precipitate was isolated by centrifugation.
The supernatent was discarded. Analysis of the precipitate by HPLC using 26% acetonitrile (vide
Analytical tests) showed a 60% conversion of porcine insulin into (Thr-Ome)B30-h-In. The precipitate was dissolved in 8 ml freshly deionized 8 M urea, the pH value was adjusted to 8.0 with 1
M ammonia, and the solution was applied to a 2.5x25 cm column packed with "QAE A-25
Sephadex", equilibrated with a 0.1 M ammonium chloride buffer, which contained 60% (v/v) ethanol, the pH value of which was adjusted to 8.0 with ammonia.Elution was carried out with the same buffer, and fractions of 1 5 ml were collected. (Thr-OMe)B30-h-ln was found in the fractions Nos. 26-46, and unreacted porcine insulin in the fraction Nos. 90-120. The fractions Nos. 26-46 were pooled, the ethanol evaporated in vacuo, and the (Thr-OMe)B30-h-ln was crystallized in a citrate buffer as described by
Schlichtkrull et al., Handbuch der inneren Medizin, 7/2A, 96, Berlin, Heidelberg, New York 1975. The yield was 95 mg of crystals having the same rhombic shape as porcine insulin crystallized in the same manner. The amino acid composition was found to be identical with that of human insulin.Further analytical tests described in the above section: "Analytical Tests", proved that the resulting product was (Thr-lMe)930-h-ln.
Example 2
100 mg porcine insulin fulfilling the purity requirements stated in British Patent No.
1,285,023 was dissolved in 0.9 ml 3.33 M acetic acid and, thereafter, 1 ml of a 2 M solution of threonine methyl ester in N,N-dimethylformamide and 12 mg TPCK (tosylphenylalaninchloromethylketone) treated trypsin dissolved in 0.1 ml water were added.
After an incubation for 24 hours at 370C the reaction was stopped by the addition of 4 ml 1 M phosphoric acid. The (Thr-OMe)930-h-ln obtained was separated from non-reacted porcine insulin by ion exchange chromatography on a 2.5x25 cm column of "SP-Sephadex" with an eluent comprising 0.09 M sodium chloride and 0.02 M sodium dihydrogen phosphate (pH value of the buffer: 5.5) in 60% ethanol. Fractions containing (Thr-OMe)B30-h-ln were collected, the ethanol was removed in vacuo, and the product was crystallized as described in Example 1. The yield was 50 mg of (Thr-OMe)B30-h-ln.
Example 3
100 mg porcine proinsulin was dissolved in 0.9 ml of 3.33 M acetic acid and converted into (Thr
OMe)B30-h-ln and purified as described for porcine insulin in Example 1. The conversion of proinsulin into (Thr-OMe)830-h-ln was found to be 73% by
HPLC analysis of the acetone precipitate. The yield of crystalline (Thr-OMe)B30-h-ln was 54 mg.
Example 4
100 mg porcine insulin was dissolved in 0.9 ml of 2.77 M acetic acid in water and reacted analogically to the process described in Example 2. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe)830-h-ln of 70%.
Example 5
100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in Nmethylpyrrolidone was added. The reaction was performed in a manner analogous to that described in Example 4 and the convertion into (Thr-OMe)B30-h-ln was 20%.
Example 6
100 mg porcine insulin was dissolved in 0.9 ml of 2.77 M acetic acid in water and 1 ml 2 M Thr
OMe in HMPA (hexamethylphosphortriamide) was added. The reaction was performed in a manner analogous to that described in Example 4.
The convertion into (Thr-OMe)B30-h-ln was 80%.
Example 7
100 mg porcine insulin was dissolved in 0.9 ml
of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in
N,N-dimethylacetamide was added. The reaction
was performed in a manner analogous to that
described in Example 4. The convertion into (Thr
OMe)830-h-ln was 80%.
Example 8
100 mg porcine insulin was dissolved in 0.9 ml
of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in
N,N-dimethylacetamide was added. Thereafter,
200 U trypsin activity (measured against the
substrate BAEE) immobilized on 1 g of glass
beads was added and after incubation at 370C
during 24 hours the trypsin bound to the glass
was filtered off. After completion of the reaction
the proteins were precipitated by the addition of
10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe)B30-h-ln of 40%.
Example 9
100 mg porcine insulin was dissolved in 0.9 ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in
N,N-dimethylacetamide was added. Thereafter, 300 U trypsin (activity measured with the substrate BAEE) immobilized on 200 ,ug CNBr activated "Sephadex G-1 50" was added. After incubation at 370C during 24 hours the trypsin bound to "Sephadex" was filtred off. After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OMe)B30-h-ln of 70%.
Example 10
The process described in Example 7 was repeted, provided that the ester used was 2 M Thr-OBut (But is tert-butyl) in N,Ndimethylacetamide. The convertion into (Thr OBut)B30-h-In was 80%.
Example 11
The process described in Example 8 was repeted, provided that the ester used was 2 M Thr-OBut in
N,N-dimethylacetamide. The convertion into (Thr OBut)B30-h-In was 30%.
Example 12
The process described in Example 9 was repeted, provided that the ester used was 2 M Thr-OBut in N,N-dimethylacetamide. The convertion into (Thr-OBut)B30-h-ín was 70%.
Example 13
100 mg porcine proinsulin was dissolved in 0.9 of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in
N,N-dimethylacetamide was added. The reaction was performed in a manner analogous to that described in Example 4. The convertion into (Thr OMe)930-h-ln was 80%.
Example 14
100 mg porcine proinsulin was dissolved in 0.9
ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in
N,N-dimethylacetamide was added. The mixture was treated with immobilized trypsin analogically to the process described in Example 8. Thr convertion into (Thr-OMe)930-h-ln was 40%.
Example 15
100 mg porcine proinsulin was dissolved in 0.9
ml of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethylacetam ide was added. The mixture was treated analogically to the process described in Example 9 with immobilized trypsin. The convertion into (Thr-OMe)830-h-ln was 70%.
Example 16
100 mg porcine proinsulin was dissolved in 0.9
ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in
N,N-dimethylacetamide was added. The reaction was performed in a manner analogous to that described in Example 4. The convertion into (Thr OBut)B30-h-In was 80%.
Example 17
100 mg porcine proinsulin was dissolved in 0.9
ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in
N,N-dimethylacetamide was added. The mixture was treated with trypsin immobilized to glass beads analogically to process described in
Example 8. The convertion into (Thr-OBu')83Ph-ln was 40%.
Example 18
100 mg porcine prnin,sulin was dissolved in 0.9
ml of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in
N,N-dimethylacetamide was added. The mixture
was treated with trypsin immobilized to CNBr activated "Sephadex G-1 50" analogically with the process described in Example 9. The convertion into (Thr-OBut)B30-h-ín was 70%.
Example 19
100 mg porcine insulin was dissolved in 0.5 ml of 6 M acetic acid and 1 ml 1 M Thr-OTmb (Tmb is 2.4,6-tmethylbenzyl) in N,Ndimethylacetamide was added. Furthermore, 0.5 ml N.N-dimethylacetamide and 5 mg TPCK treated trypsin in 0.1 ml water were added. The reaction mixture was stored at 320C for 44 hours.
After completion of the reaction the proteins were precipitated by the addition of 10 volumes of acetone. Analysis by DISC PAGE showed a conversion into (Thr-OTmb)B30-h-ln of 50%.
Example 20
100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 ml 2 M Thr-OMe in dioxane was added. The reaction was performed in a manner analogous to that described in Example 4 and the conversion into (Thr-OMe)B30-h-ln was 10%.
Example 21
100 mg porcine insulin was dissolved in 0.9 ml of 3 M acetic acid and 1 mi 2 M Thr-OMe in acetonitrile was added. The reaction was performed in a manner analogous to that described in Example 4 and the conversion into (Thr
OMe)B30-h-ln was 10%.
Example 22
250 mg of crystalline (Thr-OMe)B30-h-ln was dispersed in 25 ml of water and dissolved by the addition of 1 N sodium hydroxide solution to a pH value of 1 Q.O. The pH value was kept constant at 10.0 for 24 hours at 250C. The human insulin formed was crystallized by the addition of 2 g of sodium chloride, 350 mg of sodium acetate trihydrate and 2.5 mg of zinc acetate dihydrate followed by the addition of 1 N hydrochloric acid to obtain a pH value of 5.52. After storage for 24 hours at 40C the rhombohedral crystals were isolated by centrifugation, washed with 3 ml of water, isolated by centrifugation, and dried in vacuo. Yield: 220 mg of human insulin.
Example 23
100 mg (Thr-OTmb)B30-h-ln was dissolved in 1 ml of ice cold trifluoroacetic acid and the solution was stored for 2 hours at OOC. The human insulin formed was precipitated by the addition of 10 ml tetrahydrofuran and 0.97 ml of 1.03 M hydrochloric acid in tetrahydrofuran. The precipitate formed was isolated by centrifugation, washed with 10 ml of tetrahydrofuran, isolated by centrifugation, and dried in vacua The precipitate was dissolved in 10 ml of water and the pH value of the solution was adjusted to 2.5 with 1 N sodium hydroxide solution. The human insulin was precipitated by the addition of 1.5 g of sodium chloride and isolated 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 mg of zinc acetate dihydrate and 0.14 g of sodium acetate trihydrate'followed by the addition of 1 N sodium hydroxide solution to obtain a pH value of 5.52.
After storage for 24 hours at 40C, the precipitate was isolated by centrifugation, washed with 0.9 ml of water, isolated by centrifugation and dried in vacua Yield: 90 mg of human insulin.
Example 24
100 mg of porcine insulin was dissolved in 0.5 ml of 10 M acetic acid and 1.3 ml of 1.54 M Thr- OMe in N,N-dimethylacetamide was added. The mixture was cooled to 12 0C. 10 mg of trypsin dissolved in 0.2 ml of 0.05 M calcium acetate was added. After 48 hours at 12 20C the proteins were precipitated by addition of 20 ml of acetone. The conversion of porcine insulin into (Thr-OMe)930-h- In was 97% by HPLC.
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. 0.2 ml of 2 M Thr-OMe in N,Ndimethylacetamide was added and the mixture was cooled to -1 00C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 72 hours at 00C the proteins were precipitated by addition of 5 ml of acetone.
The conversion of porcine insulin into (Thr OMe)830-h-ln was 64% by HPLC.
Example 26
20 mg of porcine insulin was dispensed in 0.1 ml of water. Addition of 0.6 ml of 2 M Thr-OMe in
N,N-dimethylacetamide caused the insulin to go into solution. The mixture was cooled to 70C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 70C the proteins were precipitated by addition of 5 ml of acetone. The conversion of porcine insulin into (Thr-OMe)B8-h-ln was 62% by HPLC.
Example 27
20 mg of porcine insulin was dissolved in 0.135 ml of 4.45 m propionic acid. 0.24 ml of
1.67 M Thr-OMe in N,N-dimethylacetamide was added. 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added and the mixture was kept at 370C for 24 hours. The proteins were precipitated by addition of 10 volumes of 2propanol. The conversion of porcine insulin into (Thr-OMe)830-h-In was 75% by HPLC.
Example 28
20 mg of porcine insulin was dispersed in 0.1 ml of water. 0.4 ml 2 M Thr-OMe in N,N- dimethylacetamide was added followed by 0.04 ml of 10 N hydrochloric acid caused the insulin to go into solution. 2 ml of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the mixture was kept at 370C for 4 hours. Analysis by
HPLC showed a 46% conversion of porcine insulin into (Thr-OMe)830-h-ln.
Example 29
20 mg of porcine insulin was dissolved in 0.175 ml of 0.57 M acetic acid. 0.2 ml of 2 M
Thr-OMe in N,N-dimethylacetamide was added followed by the addition of 0.025 ml of 0.05 M calcium acetate. 10 mg of a crude preparation of A chromobacter lyticus protease was added and the mixture was kept for 22 hours at 370C. The proteins were precipitated by the addition of 10 volumes of acetone. The conversion of porcine insulin into (Thr-OMe)B30-h-In was 12% by HPLC.
Example 30
20 mg of porcine insulin was dispersed in 0.1 ml of 0.5 M acetic acid. Addition of 0.2 ml of 0.1
M Thr-OMe in N,N-dimethylacetamide dissolved the insulin. The mixture was cooled at 120C and 2 mg of trypsin in 0.025 ml af 0.05 M calcium acetate was added. After 24 hours at 120C the analysis by HPLC showed a 42% conversion of porcine insulin into (Thr-OMe)B30-h-ln, Example 31
2 mg of rabbit insulin was dissolved in 0.135
ml of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr
OMe in N,N-dimethylacetamide was added followed by the addition of 1.25 mg trypsin in
0.025 ml of 0.05 M calcium acetate. The mixture was kept at 370C for 4 hours.Analysis by HPLC
showed an 88% conversion of rabbit insulin into (Thr-OMe)B30-h-ln. Rabbit insulin elutes before
porcine insulin from the HPLC column, the ratio
between the elution volumes of rabbit insulin to (Thr-OMe)B30-h-In being 0.72.
Example 32
2 mg of porcine diarginine insulin (ArgB3e- ArgB32-insulin) was dissolved in 0.135 ml of 4.45
M acetic acid. 0.24 ml of 1.67 M Thr-OMe in N,N
dimethylacetamide was added followed by the
addition of 1.25 mg of trypsin in 0.025 ml of 0.05
M calcium acetate. The mixture was kept at 370C for 4 hours. Analysis by HPLC showed a 91% conversion of diarginine insulin into (Thr-OMe)B30- h-In. Diarginine insulin elutes before procine
insulin from the HPLC column, the ratio between the elution volumes of diarginine insulin to (Thr
OMe)B30-h-ln being 0.50.
Example 33
2 mg of porcine intermediates (i.e. a mixture of desdipeptide (LyseZ-Arge3) proinsulin and desdipeptide (Arg31-Arg32) proinsulin) was reacted analogously to the reaction described in Example 32. Analysis by HPLC showed 88% (Thr-OMe)B30h-In.
Example 34
20 mg of porcine insulin was dissolved in 0.1
ml of 2 M acetic acid. 0.2 ml of 2 M Thr-OMe in
N,N-dimethylacetamide was added and the
mixture was cooled to -1 80C. 2 mg of trypsin
dissolved in 0.025 ml of 0.05 M calcium acetate was added and the mixture was kept for 120
hours at -180C. Analysis by HPLC showed that
the conversion into (Thr-OMe)B30-h-In was 83%.
Example 35
The process described in Example 34 was
repeted with the proviso that the reaction was
carried out at 500C for 4 hours. Analysis by HPLC showed that the conversion into (Thr-OMe)930-h- In was 23%.
Example 36
20 mg of porcine insulin was dissolved in 0.1 ml of 3 M acetic acid. 0.3 ml of 0.33 M Thr O(CH2)2-SO2-CHa, CH3COOH (threonine 2- (methylsulfonyl)ethylester, hydroacetate) in N,N
dimethylacetamide was added. The mixture was
cooled to 1 20C and 2 mg of trypsin in 0.025 ml of
0.05 M calcium acetate was added. HPLC showed after 24 hours at 1 20C a 77% conversion into (Thr-O(CH2)2-SO2-CH3)B3 -h-In. The product elutes approximately at the position of (Thr-OMe)B30-h
In.
Example 37
20 mg of porcine insulin was dissolved in 0.1 ml ef 10 M acetic acid. 0.2 ml of 2 M Thr-OEt (Et is ethyl) in N,N-dimethylacetamide was added and the mixture was cooled to 1 20C. 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. HPLC showed after 24 hours at 12 0C a 75% conversion into (Thr-OEt)B30-h-ln. The product eluted in a portion slightly after that of (Thr-OMe)B30-h-In.
Example 38
20 mg of porcine insulin was dissolved in 0.1 ml of 6 M acetic acid. 0.3 ml of 0.67 M Thr(But) OBut (Bu' is tertiary butyl) in N,Ndimethylacetamide was added. The mixture was cooled to 1 20C and 2 mg of trypsin in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours at 1 20C the conversion into (Thr(Bu') OBu')B30-h-l was 77%. The product was eluted from the HPLC column by applying a gradient in acetonitrile from 27% to 40%.
Example 39
20 mg of porcine insulin was dissolved in 0.1 mi of 4 M acetic acid. 0.2 ml of 1.5 M Thr-OMe dissolved in tetrahydrofuran was added and the mixture was cooled to 12 so. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 4 hours at 12 OC the analysis by
HPLC showed a conversion of 75% into (Thr OMe)B30 h i Example 40 2G mg of porcine insulin was dissolved in 0.1 mi of 4 M acetic acid. 0.8 ml of 2 M Thr-OMe dissolved in 1,2-ethanediol was added and the mixture was cooled to 1 20C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added.After 4 hours at 1 20C the analysis by
HPLC showed a conversion of 48% into (Thr OMe)B30-h-l Example 41
20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe dissolved in ethanol was added and the mixture was cooled to 1 20C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added and the reaction was carried out for 4 hours at 1 20C. Analysis by HPLC showed a 46% conversion into Thr-OMe)93h-ln.
Example 42
20 mg of porcine insulin was dissolved in 0.1 ml of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe in acetone was added and the mixture was cooled to 1 20C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 4 hours at 125C the analysis by HPLC showed a conversion of 48% into (Thr-OMe)B30-h-In.
Claims (18)
1. A process for preparing threonine930 esters of human insulin or a salt or complex thereof, characterized by transpeptidizing an insulin compound or a salt or complex thereof convertible thereinto with an L-threonine ester or a salt thereof in a mixture of water, a water miscible organic solvent, and trypsin, the content of water in the reaction mixture being less than 50 per cent (volume/volume), and the reaction temperature being below 500C, and in the optional presence of an acid.
2. A process according to Claim 1, characterized in that the concentration of Lthreonine ester in the reaction mixture exceeds 0.1 molar, that the reaction temperature is above the freezing point of the reaction mixture, and that the reaction mixture contains from 0 (zero) to 10 equivalents of an acid per equivalent of the Lthreonine ester.
3. A process according to Claim 1 or 2, characterized in that the insulin compound is a compound selected from the group consisting of porcine, dog, finwhale, sperm-whale, and rabbit insulin, porcine diarginine insulin, porcine split proinsulin, porcine desdipeptide proinsulin, porcine, dog, monkey, and human proinsulin, and mixtures thereof.
4. A process according to any of the Claims 1-3, characterized in that the insulin compound is of porcine origin.
5. A process according to Claim 4, characterized in that relatively crude insulin is used as the insulin compound.
6. A process according to any of the-Claims 1-5, characterized in that the reaction temperature is below 37 OC, preferably below ambient, and above 0.
7. A process according to any of the Claims 1-6, characterized in that the content of water in the reaction mixture is between 40 and 10 per cent (volume/volume).
8. A process according to any of the Claims 1-7, characterized in that the molar ratio between the L-threonine ester and the insulin compound is above 5:1.
9. A process according to any of the Claims 1-8, characterized in that the organic solvent is a polar solvent.
10. A process according to Claim 9, characterized in that the solvent is methanol, ethanol,2-propanol,1,2-ethandiol, acetone, dioxane, tetrahydrofuran, formamide, N,Ndimethylformamide, N,N-dimethylacetamide, Nmethylpyrrolidone, hexamethylphosphortriamide, or acetonitrile.
11. A process according to any of the Claims 1-10, characterized in that the acid is an organic acid, preferably formic acid, acetic acid, propionic acid, and butyric acid.
1 2. A process according to any of the Claims 1-11, characterized in that the amount of acid in the reaction mixture is between 0.5 and 5 equivalent(s) per equivalent of the L-threonine ester.
13. A process according to any of the Claims 1-12, characterized in that the weight ratio between trypsin and the insulin compound in the reaction mixture is above 1:200, preferably above 1:50, and below 1:1.
1 4. A process according to any of the Claims 1-1 3, characterized in that the transpeptidation is carried out in the presence of calcium ions.
1 5. A process according to any of the Claims 1-14, characterized in that the transpeptidation is carried out in a buffer system.
16. A process according to any of the Claims 1-1 5, characterized in that the L-threonine ester is added as an acetate or a propionate, butyrate, or hydrohalide such as hydrochloride.
17. A process according to any of the Claims 1-1 6, characterized in that the reaction temperature and the content of water and acid in the reaction mixture are chosen so that the yield of threonine930 ester of human insulin is higher than 60 per cent, preferably higher than 80 per cent, and most preferred higher than 90 per cent.
18. A process according to any one of the
Claims 1, 2 and 6-17, characterized in that the insulin compound is a compound having the formula I
wherein
represents the humanides (ThrB30)-insulin moiety wherein GlyAl is connected to the substituent designated R1 and LysB29 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and R1 represents hydrogen or a group of the general formula R3-X-, wherein X represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids or R2 together with R3 represents a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in R' plus R2 is less than 37.
18. A process according to any of the Claim 1, 2, and 6-1 7, characterized in that the insulin compound is a compound having the formula I
wherein
represents the human des(ThrB30)-insulin moiety wherein GlyAl is connected to the substituent designated R' and Lyes28 is connected to the substituent designated R2, R2 represents an amino acid or a peptide chain containing not more than 36 amino acids, and R1 represents hydrogen or a group of the general formula R3-X-, wherein X represents arginine or lysine, and R3 represents a peptide chain containing not more than 35 amino acids, or R2 together with R3 represents a peptide chain containing not more than 35 amino acids, with the proviso that the number of amino acids present in R' plus R2 is less than 37.
1 9. A process according to Claim 18, characterized in that R1 is hydrogen, and R2 is -Ala, -Ser, -Ala-Arg-Arg, or-Ala-Arg-Arg-Glu- Aia-G lu-Asn-Pro-G In-Ala-Giy-Ala-Val-Glu-Leu-
Gly-G ly-Gly-Leu-Gly-G ly-Leu-Gl n-Ala-Leu-Ala- Leu-Glu-Gly-Pro-Pro-Gln, R3 is Ala-Leu-Glu-Gly Pro-Pro-Gln-Lys-, and R2 is -Ala-Arg-Arg-Glu-Ala Gl u-Asn-Pro-G In-Ala-Gly-Ala-Val-Glu-Leu-Gly-
Gly-G ly-Leu-G ly-Gly-Leu-G ln-Ala-Leu, or R3 together with R2 is -Ala-Arg-Arg-Glu-Ala-Glu
Asn-Pro-Gln-Ala-G Iy-Ala-Val-Glu-Leu-Gly-G ly G Iy-Leu-G Iy-G Iy-Leu-G In-AI a-Leu-Ala-Leu-Glu- Gly-Pro-Pro-Gln-Lys-, wherein the terminal alanyl is connected to LysB29, -Aía-Arg-Arg-Asp-Val-Glu- Leu-Ala-G ly-Ala-Pro-G ly-Glu-G ly-Gly-Leu-Gln Pro-Leu-Ala-Leu-G lu-Gly-Al a-Leu-Gln-Lys-, wherein the terminal alanyl is connected to Lyes929, -Th r-Arg-Arg-G lu-Ala-Gl u-Asp-Leu-GI n-Val-Gly- Gln-Val-Glu-Leu-G ly-G ly-Gly-Pro-Gly-Al a-Gly-Ser- Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln
Lys-, wherein the terminal threonyl is connected to Lyes929, or -Thr-Arg-Arg-G lu-Al a-G lu-Asp-Pro- Gl n-Val-G Iy-GI n-Val-G lu-Leu-G ly-G ly-G ly-Pro- Gly-Ala-G ly-Ser-Leu-Gln-P ro-Leu-Ala-Leu-Glu-Gly- Ser-Leu-Gln-Lys-, wherein the terminal threonyl is connected to Lyes930.
20. A process according to Claim 19, characterized in that R1 is hydrogen, and R2 represents alanine.
21. A process according to any of the Claims 1-20, characterized in that the L-threonine ester has the general formula 11
Thr(R5)-OR4 (11) wherein R4 represents a carboxyl protecting group, and R5 represents hydrogen or a hydroxyl protecting group.
22. A process according to Claim 21, characterized in that R4 is lower alkyl, substituted benzyl or diphenylmethyl or a group of the general formula -CH2CH2SO2R8, wherein Re represents lower alkyl.
23. A process according to Claim 22,
characterized in that R4 is methyl, ethyl, tert-butyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, or a group of the formula -CH2CH2SO2R6, wherein Re is methyl, ethyl, propyl, or butyl.
24. A process according to any of the Claims 1-6, characterized in that the insulin compound is porcine insulin, that the L-threonine ester is
Thr-OMe orThr-OBu', that the water miscible organic solvent is N,N-dimethylformamide or N,Ndimethylacetamide, that the reaction temperature is about 370C, that the content of water in the reaction mixture is between 41 and 43%, that the weight ratio between trypsin and the insulin compound is about 1 :8, that the molar ratio between the L-threonine ester and the insulin compound is about 1:120, and that there is added between 1.2 and 1.5 equivalents of acetic acid per equivalent of L-threonine ester.
25. A process according to Claim 1 or 2, characterized in that it is performed under the reaction conditions stated in any of the Examples 1-21 or 24--42, preferably those stated separately in any of the Examples 4, 7, 10, 24, 34, 36, and 39.
26. Threonine930 esters of human insulin prepared by the process according to any of the
Claims 1-25.
27. A process for preparing human insulin, characterized in that firstly the process according to any of the Claims 1-25 is performed and, thereafter, the resulting threonine930 ester of human insulin is deblocked.
28. A process according to Claim 27, characterized in that the deblocking is performed in an aqueous medium in the presence of a base, preferably at a pH value between 8 and 12.
29. A process according to Claim 27, characterized in that the deblocking is performed by acidolysis, preferably with trifluoroacetic acid.
30. A process according to Claim 27, characterized in that the deblocking is performed under the reaction conditions stated in Example 22 or 23.
31. Human insulin prepared by the process according to any of the Claims 27-30.
32. Threonine830 esters of human insulin wherein the ester group is different from tertbutyl and methyl.
33. Any novel feature or combination of features described herein.
Amendments to Claims Filed on 5 May 1981.
Superseded Claims 6, 18.
Amended Claims:
6. A process according to any of the Claims 1-5, characterized in that the reaction temperature is below 37 CC, preferably below ambient, and above OOC.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK57480A DK147109C (en) | 1980-02-11 | 1980-02-11 | METHOD FOR PREPARING HUMAN INSULIN OR HUMAN INSULINESTERS OR A SALT OR COMPLEX THEREOF |
Publications (2)
Publication Number | Publication Date |
---|---|
GB2069502A true GB2069502A (en) | 1981-08-26 |
GB2069502B GB2069502B (en) | 1984-08-08 |
Family
ID=8095095
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8104114A Expired GB2069502B (en) | 1980-02-11 | 1981-02-10 | Process for preparing insulin esters |
Country Status (19)
Country | Link |
---|---|
JP (1) | JPS56135452A (en) |
AR (1) | AR231277A1 (en) |
AU (1) | AU541528B2 (en) |
BE (1) | BE887480A (en) |
CA (1) | CA1162868A (en) |
CH (1) | CH655949A5 (en) |
DE (1) | DE3104949C2 (en) |
DK (3) | DK147109C (en) |
ES (1) | ES8205858A1 (en) |
FI (1) | FI77876C (en) |
GB (1) | GB2069502B (en) |
GR (1) | GR73844B (en) |
IT (1) | IT1195038B (en) |
NL (1) | NL178797C (en) |
NO (1) | NO156247C (en) |
NZ (1) | NZ196224A (en) |
PT (1) | PT72485B (en) |
SE (2) | SE451143B (en) |
ZA (1) | ZA81898B (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0056951A1 (en) * | 1981-01-17 | 1982-08-04 | Hoechst Aktiengesellschaft | Process for the preparation of human insulin or its derivatives from pig insulin or its derivatives |
EP0085516A2 (en) * | 1982-01-22 | 1983-08-10 | Carlsberg Biotechnology Ltd. A/S | A process for enzymatic replacement of the B-30 amino acid in insulins |
WO1983002772A1 (en) * | 1982-02-08 | 1983-08-18 | Robin Ewart Offord | An improved method for preparing human insulin from non-human insulin |
JPS58501455A (en) * | 1981-09-15 | 1983-09-01 | ノルデイスク インシユリンラボラトリユ−ム | Method for producing human insulin or its B-30 ester |
EP0092280A1 (en) * | 1982-04-21 | 1983-10-26 | Akzo N.V. | Semi-synthetic preparation of human insulin |
EP0092829A2 (en) * | 1982-04-23 | 1983-11-02 | Wako Pure Chemical Industries, Ltd. | Process for semi-synthesis of human insulin and alkaline protease for use therein |
EP0137361A2 (en) * | 1983-09-23 | 1985-04-17 | Hoechst Aktiengesellschaft | Insulin derivatives modified in position B 30, process for their preparation, their vuse and pharmaceutical agents for the treatment of diabetes mellitus |
US4639333A (en) * | 1982-03-13 | 1987-01-27 | Hoechst Aktiengesellschaft | Process for converting preproinsulin analogs into insulins |
US7658721B2 (en) | 2004-01-16 | 2010-02-09 | Biodel Inc. | Sublingual drug delivery device |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK147437A (en) | 1980-02-11 | 1900-01-01 | Process for preparing human insulin or threonine B30 esters of human insulin, or a salt or complex thereof | |
DK55183D0 (en) * | 1983-02-09 | 1983-02-09 | Nordisk Insulinlab | METHOD OF MANUFACTURING HUMAN INSULIN |
DK119785D0 (en) * | 1985-03-15 | 1985-03-15 | Nordisk Gentofte | INSULIN PREPARATION |
US5157021A (en) * | 1985-03-15 | 1992-10-20 | Novo Nordisk A/S | Insulin derivatives and pharmaceutical preparations containing these derivatives |
SE449472B (en) * | 1985-10-15 | 1987-05-04 | Mth Gruppen Ab | EQUIPMENT AT FLOOR STORES |
PT86819B (en) * | 1987-02-25 | 1992-05-29 | Novo Industri As | A process for the preparation of new derivatives of insulin and pharmaceutical compositions containing them |
DK134189D0 (en) * | 1989-03-20 | 1989-03-20 | Nordisk Gentofte | INSULIN COMPOUNDS |
FI96503C (en) * | 1994-08-26 | 1996-07-10 | Hymatic Ltd Oy | Method and apparatus for controlling a mast |
US20080090753A1 (en) | 2004-03-12 | 2008-04-17 | Biodel, Inc. | Rapid Acting Injectable Insulin Compositions |
US7713929B2 (en) | 2006-04-12 | 2010-05-11 | Biodel Inc. | Rapid acting and long acting insulin combination formulations |
US8084420B2 (en) | 2005-09-29 | 2011-12-27 | Biodel Inc. | Rapid acting and long acting insulin combination formulations |
WO2007121256A2 (en) | 2006-04-12 | 2007-10-25 | Biodel, Inc. | Rapid acting and long acting insulin combination formulations |
US9060927B2 (en) | 2009-03-03 | 2015-06-23 | Biodel Inc. | Insulin formulations for rapid uptake |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NO153061C (en) * | 1979-04-13 | 1986-01-08 | Shionogi & Co | PROCEDURE FOR THE PREPARATION OF A B30 THREON INSULIN |
DK147437A (en) | 1980-02-11 | 1900-01-01 | Process for preparing human insulin or threonine B30 esters of human insulin, or a salt or complex thereof | |
DE3101382A1 (en) * | 1981-01-17 | 1982-09-02 | Hoechst Ag, 6000 Frankfurt | "METHOD FOR PRODUCING HUMANISULIN OR ITS DERIVATIVES FROM PIG INSULIN OR ITS DERIVATIVES" |
-
0
- DK DK147437D patent/DK147437A/en unknown
-
1980
- 1980-02-11 DK DK57480A patent/DK147109C/en not_active IP Right Cessation
-
1981
- 1981-02-09 ES ES499238A patent/ES8205858A1/en not_active Expired
- 1981-02-09 DK DK54081A patent/DK147437C/en not_active IP Right Cessation
- 1981-02-10 AR AR284258A patent/AR231277A1/en active
- 1981-02-10 NL NLAANVRAGE8100624,A patent/NL178797C/en not_active IP Right Cessation
- 1981-02-10 GB GB8104114A patent/GB2069502B/en not_active Expired
- 1981-02-10 IT IT19620/81A patent/IT1195038B/en active
- 1981-02-10 NO NO810443A patent/NO156247C/en unknown
- 1981-02-10 PT PT72485A patent/PT72485B/en not_active IP Right Cessation
- 1981-02-10 FI FI810385A patent/FI77876C/en not_active IP Right Cessation
- 1981-02-10 SE SE8100928A patent/SE451143B/en not_active IP Right Cessation
- 1981-02-10 NZ NZ196224A patent/NZ196224A/en unknown
- 1981-02-10 CA CA000370483A patent/CA1162868A/en not_active Expired
- 1981-02-10 JP JP1758081A patent/JPS56135452A/en active Granted
- 1981-02-10 SE SE8100926A patent/SE427025B/en not_active IP Right Cessation
- 1981-02-11 ZA ZA00810898A patent/ZA81898B/en unknown
- 1981-02-11 AU AU67180/81A patent/AU541528B2/en not_active Ceased
- 1981-02-11 GR GR64097A patent/GR73844B/el unknown
- 1981-02-11 DE DE3104949A patent/DE3104949C2/en not_active Expired
- 1981-02-11 BE BE6/47396A patent/BE887480A/en not_active IP Right Cessation
- 1981-02-11 CH CH910/81A patent/CH655949A5/en not_active IP Right Cessation
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4601852A (en) * | 1981-01-17 | 1986-07-22 | Hoechst Aktiengesellschaft | Process for the preparation of human or the derivatives thereof from pig insulin or the derivatives thereof |
EP0056951A1 (en) * | 1981-01-17 | 1982-08-04 | Hoechst Aktiengesellschaft | Process for the preparation of human insulin or its derivatives from pig insulin or its derivatives |
JPS58501455A (en) * | 1981-09-15 | 1983-09-01 | ノルデイスク インシユリンラボラトリユ−ム | Method for producing human insulin or its B-30 ester |
US4601979A (en) * | 1981-09-15 | 1986-07-22 | Nordisk Insulinlaboratorium | Process for preparing human insulin or B-30 esters thereof |
EP0085516A2 (en) * | 1982-01-22 | 1983-08-10 | Carlsberg Biotechnology Ltd. A/S | A process for enzymatic replacement of the B-30 amino acid in insulins |
EP0085516A3 (en) * | 1982-01-22 | 1983-09-07 | De Forenede Bryggerier A/S | A process for enzymatic replacement of the b-30 amino acid in insulins |
WO1983002772A1 (en) * | 1982-02-08 | 1983-08-18 | Robin Ewart Offord | An improved method for preparing human insulin from non-human insulin |
EP0087238A1 (en) * | 1982-02-08 | 1983-08-31 | Biogen N.V. | Am improved method for preparing human insulin from non-human insulin |
US4639333A (en) * | 1982-03-13 | 1987-01-27 | Hoechst Aktiengesellschaft | Process for converting preproinsulin analogs into insulins |
EP0092280A1 (en) * | 1982-04-21 | 1983-10-26 | Akzo N.V. | Semi-synthetic preparation of human insulin |
EP0092829A3 (en) * | 1982-04-23 | 1985-08-21 | Wako Pure Chemical Industries, Ltd. | Process for semi-synthesis of human insulin and alkaline protease for use therein |
EP0092829A2 (en) * | 1982-04-23 | 1983-11-02 | Wako Pure Chemical Industries, Ltd. | Process for semi-synthesis of human insulin and alkaline protease for use therein |
JPH0526469B2 (en) * | 1982-06-07 | 1993-04-16 | Norudeisuku Inshurin Lab | |
EP0137361A2 (en) * | 1983-09-23 | 1985-04-17 | Hoechst Aktiengesellschaft | Insulin derivatives modified in position B 30, process for their preparation, their vuse and pharmaceutical agents for the treatment of diabetes mellitus |
EP0137361A3 (en) * | 1983-09-23 | 1987-05-06 | Hoechst Aktiengesellschaft | Insulin derivatives modified in position b 30, process for their preparation, their vuse and pharmaceutical agents for the treatment of diabetes mellitus |
US7658721B2 (en) | 2004-01-16 | 2010-02-09 | Biodel Inc. | Sublingual drug delivery device |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4343898A (en) | Process for preparing esters of human insulin | |
CA1162868A (en) | Process for preparing insulin esters | |
US3950517A (en) | Insulin derivatives | |
US4701440A (en) | Insulin derivatives, processes for their preparation and their use, and pharmaceutical agents for the treatment of diabetes mellitus | |
US5015728A (en) | Process for the preparation of insulin derivatives, the B chain of which is lengthened c-terminally | |
US4511505A (en) | Semi-synthesis of human insulin | |
US4430266A (en) | Process for producing an insulin precursor | |
JPH02131594A (en) | Method for producing db30-insulin and db30-insulin derivative | |
KR880001107B1 (en) | Process for the preparation of human insulin derivatives | |
US4489159A (en) | Process for preparing esters of human insulin | |
EP0017938B1 (en) | Process for preparing a b30-threonine insulin | |
FI79853B (en) | FOERFARANDE FOER FRAMSTAELLNING AV INSULINDERIVAT. | |
US4029642A (en) | Process for the manufacture of human insulin | |
EP0088117B1 (en) | Process for preparing human insulin or b-30 esters thereof | |
EP0037255B1 (en) | Process for producing an insulin precursor | |
CA1243972A (en) | Anti-diabetic compounds | |
EP0037256B1 (en) | Process for producing an insulin | |
EP0087238A1 (en) | Am improved method for preparing human insulin from non-human insulin | |
US3752798A (en) | Tris-na1,nb1,nepsilonb29 - (3-x-3-oxo - 1-y-prop-1-en-1-yl)(ndelta-(4-z-6-r-pyrimidin - 2 - yl) ornithine b22)-insulins and their preparation | |
Canova-Davis et al. | Specific activation of the arginine carboxyl group of the B-chain of bovine des-octapeptide-(B23–30)-insulin | |
Lindsay et al. | Mono-, di, and N A1, N B1, N B29-tri-acylated insulin |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19990210 |