JPS6312480B2 - - Google Patents
Info
- Publication number
- JPS6312480B2 JPS6312480B2 JP56175030A JP17503081A JPS6312480B2 JP S6312480 B2 JPS6312480 B2 JP S6312480B2 JP 56175030 A JP56175030 A JP 56175030A JP 17503081 A JP17503081 A JP 17503081A JP S6312480 B2 JPS6312480 B2 JP S6312480B2
- Authority
- JP
- Japan
- Prior art keywords
- insulin
- reaction
- protecting group
- compound
- group
- 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.)
- Expired
Links
- 125000006239 protecting group Chemical group 0.000 claims description 27
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims description 18
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 10
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 7
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- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical class N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 44
- 150000001875 compounds Chemical class 0.000 description 27
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- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 4
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
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- MUCZHBLJLSDCSD-UHFFFAOYSA-N diisopropyl fluorophosphate Chemical compound CC(C)OP(F)(=O)OC(C)C MUCZHBLJLSDCSD-UHFFFAOYSA-N 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229960005051 fluostigmine Drugs 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- DCKVNWZUADLDEH-UHFFFAOYSA-N sec-butyl acetate Chemical compound CCC(C)OC(C)=O DCKVNWZUADLDEH-UHFFFAOYSA-N 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003587 threonine derivatives Chemical class 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000011882 ultra-fine particle Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
Classifications
-
- 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
Description
ãçºæã®è©³çŽ°ãªèª¬æã
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é
µçŽ ã®äœçšã«ãããã¹âB30âã€ã³ã·ãŠãªã³ã«ïŒ
ã100åã¢ã«éã®ã¹ã¬ãªãã³èªå°äœãåå¿ãããŠã
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ã·ãŠãªã³ãåŸãæ¹æ³ãçºèŠãããDETAILED DESCRIPTION OF THE INVENTION The present invention relates to insulin derivatives. The present inventors have demonstrated that des-B30-insulin can be synthesized by the action of trypsin or a trypsin-like enzyme that has specificity for basic amino acid residues on the carbonyl side.
~100 times the molar amount of threonine derivative is reacted,
They then discovered a method to obtain insulin by removing the protecting group from the resulting condensate.
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ãªã貎éãªè¬ç©ã§ãããçŸåšäž»ãšããŠçã€ã³ã·ãŠ
ãªã³ããã³è±ã€ã³ã·ãŠãªã³ãæ²»çã«çšããããŠã
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é
žã®äžéšã人ã€ã³ã·ãŠãªã³ãšç°ãªã€ãŠããããã
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ã§æäœãç£çãããããšããããæäœãç£ç
ããããšãã以åŸã®ã€ã³ã·ãŠãªã³ã®æ²»çå¹æãè
ããäœäžãããªã©ã®åé¡ãçããããããã€ãŠã
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æ³ã®ç¢ºç«ã匷ãåžãŸããŠããã Insulin is a valuable drug with no substitutes for the treatment of diabetes, and currently bovine insulin and porcine insulin are mainly used for treatment. However, some of the constituent amino acids of these insulins are different from human insulin, so
Antibodies may be produced in the body, and the production of antibodies causes problems such as a marked decrease in the therapeutic effect of insulin. Therefore,
There is a strong desire to establish a method for synthesizing human insulin that can be carried out on an industrial scale.
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ãåæã«çšããŠäººã€ã³ã·ãŠãªã³ã容æã«åæãã
ããšãã§ããããã®ããã«ããŠåŸããã人ã€ã³ã·
ãŠãªã³ãçæ³çãªæ²»ççšã€ã³ã·ãŠãªã³ã§ããããš
ã¯è«ãä¿ããªãã According to the method of the present inventors, human insulin can be easily synthesized using pig insulin, which differs from human insulin only in the amino acid at position 30 of the B chain, as a raw material. There is no doubt that the human insulin obtained in this way is an ideal therapeutic insulin.
è±ãã¹ãªã¯ã¿ããããã€ã³ã·ãŠãªã³ãšäººã€ã³ã·
ãŠãªã³ãªã¯ã¿ãããããã人ã€ã³ã·ãŠãªã³ãåŸã
è©Šã¿ã¯ããšã ã»ãšãŒã»ã«ããã³ãã«ã°ïŒM.Aã»
RuttenbergïŒããµã€ãšã³ã¹ïŒScienceïŒ177å·»623
é ïŒ1972幎ïŒã«ãããã³ã¢ãŒã«ã»ãªãŒãã«ãã€ã€
ãŒïŒR.ObermeierïŒãããµã€ãã·ãŠãªããã»ããŠ
ã¢ã»ããžãªããžãã·ãšã»ãããŒïŒZeitschrift fušr
Physiologische ChemieïŒ357å·»759é ïŒ1976幎ïŒ
ã«çºè¡šããŠããããã«æ¢ã«ãªãããŠããããäœã
ãååŠçæ段ã«ãããã®ã§ãããåè
ã«ãããŠã¯
å·¥çšã®æåŸã«ã¢ã«ã«ãªåŠçãå«ã¿ãããã«äŒŽãå¯
åå¿ããããããªãããŸãåŸè
ã®å Žåã¯åå¿ãé
ç¹ç°çã§å€ãã®å¯åå¿ã§çããããã粟補ãè€é
ãã€å°é£ãšãªãåçãèããäœããåŸã€ãŠãå·¥æ¥
çèŠæš¡ã§ã¯å°åºè¡ãªãåŸãªãã Attempts to obtain human insulin from pig desoctapeptide insulin and human insulin octapeptide were conducted by M.A. Ruthtenberg (M.A.
Ruttenberg) Science vol. 177 623
(1972) and R. Obermeier et al.
Physiologische Chemie) Volume 357, Page 759 (1976)
Both methods are based on chemical means, and the former involves an alkali treatment at the end of the process, and accompanying side reactions are unavoidable. In the latter case, the reaction is non-specific and involves many side reactions, making purification complicated and difficult and resulting in extremely low yields. Therefore, it cannot be carried out on an industrial scale.
äžæ¹ãæ¬çºæè
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ãŠãªã³ãšïŒ¬âã¹ã¬ãªãã³èªå°äœãšãé
µçŽ åå¿ã«ã
ãçž®åãããŠã€ã³ã·ãŠãªã³ãåŸãæ¹æ³ã§ãå
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ååŠçæ段ã«ããæ¹æ³ãšã¯ç°ãªããåå¿ãç¹ç°ç
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æãæãªããã«ååã§ãããçã®å©ç¹ãæããã On the other hand, the method of the present inventors obtains insulin by condensing des-B30-insulin and an L-threonine derivative through an enzymatic reaction. It has advantages such as no reaction, no racemization, and the ability to recover unreacted raw materials without damaging them.
ããã«åæ¹æ³ã§ã¯ãïŒã100åã¢ã«éã®ïŒ¬âã¹
ã¬ãªãã³èªå°äœãçšããããšã«ãããéåžžã®é
µçŽ
åå¿ã§ã¯åœç¶èµ·ããé22äœã®ã¢ã«ã®ãã³ã®ã«ã«
ããã«åŽåæãé²æ¢ããããããã€ãŠéåžžã®é
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èŠã§ããã Furthermore, in the same method, by using 1 to 100 times the molar amount of L-threonine derivatives, the carbonyl side cleavage of arginine at position 22 of the B chain, which naturally occurs in normal enzymatic reactions, is prevented; Introduction of a protecting group to the arginine side chain, which is essential, is unnecessary.
åæ¹æ³ã¯ãããªãã·ã³ãŸãã¯ã«ã«ããã«åŽå¡©åº
æ§ã¢ããé
žæ®åºã«ç¹ç°æ§ã瀺ãããªãã·ã³æ§é
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žãæ¬ ãããã¹â
B30âã€ã³ã·ãŠãªã³ïŒä»¥äžååç©ãšèšãïŒã«ïŒ
ã100åã¢ã«éã®ïŒ¬âã¹ã¬ãªãã³èªå°äœïŒä»¥äžå
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ã¯äžèšã®äžè¬åŒã§è¡šããããã This method uses trypsin or a trypsin-like enzyme that has specificity for basic amino acid residues on the carbonyl side.
1 for B30-insulin (hereinafter referred to as compound)
It consists of reacting an L-threonine derivative (hereinafter referred to as compound) in a molar amount of ~100 times, and then removing the protecting group from the resulting condensate. The compound is represented by the general formula below.
ïŒåŒäžãR1ã¯æ°ŽçŽ ãŸãã¯ããããã·åºã®ä¿è·
åºãR2ã¯ã«ã«ããã·ã«åºã®ä¿è·åºãããããè¡š
ãããïŒ
äžèšã®ååç©ã¯è±èµ·æºã®ã€ã³ã·ãŠãªã³ã«ã«ã«
ããã·ãããããŒãŒïŒ¡ãäœçšãããããšã«ããåŸ
ãããäŸãã°ãã€ã»ãããªãŠãŒã»ã·ãŠãããïŒE.
W.SchmittïŒããããããŒã»ã€ã©ãŒãºã»ãµã€ãã·
ãŠãªããã»ããŠã¢ã»ããžãªããžãã·ãšã»ãããŒ
ïŒHoppeâSeylerâ²sZeitschrift fušr
Physiologische ChemieïŒ359å·»799é ïŒ1978幎ïŒ
ã«èšèŒããŠããæ¹æ³ã«ãã補é ã§ããããŸãã¢ã¯
ãã¢ãã¯ã¿ãŒã»ãªãã€ã«ã¹ïŒAchromobacter
lyticusïŒãç£çãããªãžã³ã«ç¹ç°æ§ãæãããª
ãžã³ã®ã«ã«ããã«åŽãåæããé
µçŽ ãçšããŠã容
æã«è£œé ããããšãã§ãããåé
µçŽ ã®åé¢ããã³
æ§ç¶ã«ã€ããŠã¯ãæ£æšããã¢ã°ãªã«ã«ããŠã©ã«ã»
ã¢ã³ãã»ãã€ãªããžã«ã«ã»ã±ãã¹ããªãŒ
ïŒAgricultural and Biological ChemistryïŒ42å·»
1443é ïŒ1978幎ïŒã«çºè¡šããŠãããåè«æã«ãã
ãŠã¯äžèšæ§ç¶ãæããé
µçŽ ããããã¢ãŒãŒãšç§°
ããŠããã (In the formula, R 1 represents hydrogen or a hydroxy group-protecting group, and R 2 represents a carboxyl group-protecting group.) The above compound is obtained by treating insulin of pig origin with carboxypeptidase A, and for example, E.D.
Hoppe-Seyler's Zeitschrift fušr
Physiologische Chemie) Volume 359, Page 799 (1978)
It can be manufactured by the method described in . Also, Achromobacter liteicus
lyticus), and can be easily produced using an enzyme that cleaves the carbonyl side of lysine. Regarding the isolation and properties of the enzyme, Masaki et al.
Agricultural and Biological Chemistry Volume 42
1443 (1978), in which the enzyme with the above properties is called protease.
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食ããã A compound can be subjected to a reaction even if its side chain functional group, hydroxy group, is not protected, but if a protecting group is introduced, a protecting group commonly used in peptide synthesis reactions, such as t-butyl, benzyl , acetyl, etc. may be used. Further, the carboxyl group of the compound needs to be protected, and a commonly used protecting group for carboxyl groups may be used. For example, modification in the form of alkyl and aralkyl esters such as t-butyl, benzyl, etc.
äžèšã®ä¿è·åºã®éžæã«ãããŠã¯ããããã®ä¿è·
åºã®å°å
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ãå€æ§ããã倱掻ãããããªããã®ãéžæããã
ãèæ
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åºã«ã€ããŠã¯ããšã ã»ããã³ã¹ãããŒïŒM.
BodanszkyïŒããããããã»ã·ã³ã»ã·ã¹
ïŒPeptide SynthesisïŒç¬¬ïŒçïŒ1976幎ïŒïŒãžãš
ã³ã»ãŠã€ãªãŒã»ã¢ã³ãã»ãµã³ãºïŒJohn WileyïŒ
SonsïŒïŒã«è©³ããèšèŒããŠããã When selecting the above-mentioned protecting groups, consideration should be given to selecting those that do not denature or deactivate insulin during the introduction or removal treatment of the protecting groups. Regarding protecting groups used in peptide synthesis, M. Bodansudsky (M.
Peptide Synthesis, 2nd edition (1976) (John Wiley & Sons)
Sons)).
ãªããä¿è·åºã®éžæã«ãããŠã¯ãäžåã®ä¿è·åº
è±é¢æäœã«ããåæã«é€å»ã§ãããã®ãéžã¶ã®ã
奜ãŸããããšã¯è«ãä¿ããªãã It goes without saying that when selecting a protecting group, it is preferable to select one that can be simultaneously removed by a single protecting group removal operation.
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ãã»ãã€ãªã±ãã¹ããªãŒã»ã¢ã³ãã»ãã€ãªãã€ãž
ã¯ã¹ïŒArch.Biochem.Biophys.ïŒ126å·»971é
ïŒ1968幎ïŒã«ãåç°ãããšãã€ãŒããŒãšã¹ã»ã¬ã¿
ãŒãºïŒFEBS Lett.ïŒ15å·»129é ïŒ1971幎ïŒã«èšèŒ
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ãã³ïŒTPCKïŒãªã©ã§åŠçãããšããã Enzymes used in the method include trypsin or trypsin-like enzymes that exhibit specificity for carboxyl-side basic amino acids derived from various animals, plants, and microorganisms. Examples of trypsin-like enzymes include enzymes extracted and separated from Streptomyces bacteria. Regarding the enzyme, Morihara et al. published Arch.Biochem.Biophys., Vol. 126, p. 971 (1968), and Yoshida et al. published FEBS Letters, Vol. 15. It is described on page 129 (1971). The enzyme used may be treated with tosyl-L-phenylalanine chloromethyl ketone (TPCK) or the like for the purpose of removing contaminating chymotrypsin or chymotrypsin-like activity.
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ããããã The condensation reaction between the compounds is carried out under conditions suitable for the peptide bond forming reaction of the enzyme described above. The pH is preferably 5 to 8, particularly around 6 to 7, and the reaction temperature is preferably 0 to 50°C, particularly 20 to 40°C. It is desirable that the compound and compound concentrations be as high as possible. Furthermore, the ratio of compound to compound is 1:1
It is preferable to carry out the reaction at a molar ratio of ~100:1, particularly around 20:1~100:1. A suitable organic solvent miscible with water is added to the reaction solution. Addition of an organic solvent is effective not only in reducing the concentration of water in the reaction solution and suppressing the hydrolysis reaction, which is a reverse reaction, but also in significantly increasing the solubility of the compound and the compound. As the organic solvent, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, glycerin, etc. are used alone or in combination. Especially 0-65%, especially 40-60
It is recommended to use it at a concentration of %. Generally, when using an organic solvent, the ratio to water is determined by considering the solubility of raw materials, denaturation of enzymes, hydrolysis reaction, etc. As a buffer for the reaction solution, trishydroxymethylaminomethane (Tris), carbonate, or the like is used.
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µçŽ ãšããŠçšããŠãããã The enzyme concentration in the reaction solution varies depending on the substrate concentration and enzyme activity, but when using commercially available crystalline trypsin, it is preferably around 1 mg/ml to 10 mg/ml. The enzyme may be used as it is, or may be used as an immobilized enzyme bound to or included in a suitable insoluble carrier.
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ã§ããã Although the reaction time varies depending on the reaction conditions, it is usually sufficient to allow the time required for the enzyme reaction to reach equilibrium, which is usually about 3 to 72 hours, and in most cases about 6 to 24 hours.
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ãåŸãã€ãã§ç®çãšãã人ã€ã³ã·ãŠãªã³ãåŸãã After the reaction, a combination of methods commonly used for separating peptides was used to obtain human insulin, in which the threonine at position 30 of the B chain has a protecting group on the carboxyl group and may have a protecting group on the hydroxyl group. , then the intended person gets insulin.
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ã«ä»ãã人ã€ã³ã·ãŠãªã³ãšããã For example, after the reaction is completed, the reaction solution is subjected to gel filtration,
Unreacted compounds and enzymes are isolated and recovered. The recovered compounds and enzymes can be reused as they are. The remainder is subjected to appropriate chromatography, and the generated threonine at position 30 of the B chain has a protecting group on the carboxyl group and human insulin which may have a protecting group on the hydroxyl group and unreacted des-
B30 - Separates insulin. The latter can be reused as a compound. The former is further subjected to a protecting group elimination reaction to produce human insulin.
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æ°Žå解åŠçã«ããä¿è·åºãé€å»ã§ããã The method for removing the protecting group differs depending on the protecting group used, but it can be carried out according to the usual method. It is eliminated upon treatment with trifluoroacetic acid in the presence of agents such as anisole. When a protecting group that can be removed by a single elimination treatment as described above is used to protect the side chain functional group and carboxyl group of threonine, the elimination treatment is simple and the yield is improved. Even when the carboxyl terminal is converted to another ester, the protecting group can be removed by appropriate hydrolysis treatment.
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ããã The insulin obtained from human insulin, in which the threonine at position 30 of the B chain of the present invention has a protective group on the carboxyl group and may have a protective group on the hydroxyl group, has a hypoglycemic effect as described above for the treatment of diabetes. Useful as a medicine or reagent. Human insulin synthesized from pig insulin by the method of the present inventors showed an effect equivalent to that of bovine insulin in a hypoglycemic test on mice.
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ããšããã Human insulin synthesized from the compound of the present invention is
It is formulated and administered to humans in the same way as commercially available swine and bovine insulin. That is, adding zinc chloride etc. to make a zinc complex, adding buffers such as sodium hydrogen phosphate or sodium acetate, and adding sodium chloride to make it an isotonic solution.
Furthermore, an injection is manufactured using a preparation method used for commercially available insulin preparations, such as adding a preservative such as cresol, phenol, or alkyl ester of paraoxybenzoic acid (eg, methyl, ethyl, propyl, butyl ester, etc.). The dosage of such injections varies depending on the patient's symptoms, but
It may be administered in the same manner as commercially available insulin preparations, and it is recommended that adults administer about 1 to 100 units per day.
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ãªãã In the Examples below, production examples of the compounds of the present invention are shown, but the Examples are not intended to limit the present invention in any way.
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ãã In addition, the abbreviations used in the examples represent the following meanings.
Ala ã¢ã©ãã³ Ile ã€ãœãã€ã·ã³
Arg ã¢ã«ã®ãã³ Leu ãã€ã·ã³
Asn ã¢ã¹ãã©ã®ã³ Lys ãªãžã³
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ž Pro ãããªã³
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ž Ser ã»ãªã³
Gln ã°ã«ã¿ãã³ Thr ã¹ã¬ãªãã³
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His ãã¹ããžã³ Val ããªã³
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æããŠããã€ã³ã·ãŠãªã³ããããAla Alanine Ile Isoleucine Arg Arginine Leu Leucine Asn Asparagine Lys Lysine Asp Aspartic acid
Phe Phenylalanine CySO 3 H Cysteinic acid Pro Proline Glu Glutamic acid Ser Serine Gln Glutamine Thr Threonine Gly Glycine Tyr Tyrosine His Histidine Val Valine OBu t t-Butyl ester residue Des-B30-insulin is the 30th position of insulin B chain It refers to insulin that is deficient in amino acids, such as porcine insulin, which is deficient in alanine.
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(1) ãã¹âB30âã€ã³ã·ãŠãªã³ïŒè±åïŒ
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ãExample 1 (1) Des-B30-insulin (pig type) 500 mg of porcine insulin was dissolved in 100 ml of 0.1 M ammonium bicarbonate (PH8.3), and crystalline carboxypeptidase A (manufactured by Worthington Co., Ltd., treated with diisopropyl fluorophosphate, 49 u /mg) and react at room temperature for 8 hours. The reaction was stopped when the amount of alanine produced was 0.77M/1M insulin, and the reaction product was lyophilized, dissolved in 0.5M acetic acid, and added to a column of ultrafine Sephadex G50 (3.5
95cm) and elute with 0.5M acetic acid at 11.5ml per fraction. 40-60 of fraction
The sample was collected and lyophilized to obtain 460 mg of the labeled compound. Yield 92%.
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ããŠãåæ§ã The amino acid analysis values obtained by subjecting the product to acid hydrolysis (6M hydrochloric acid, 110°C, 24 hours) are as follows. Values in parentheses indicate theoretical values. The same applies to the following.
Lys 1.00(1)ïŒ His 1.91(2)ïŒ Arg 0.95(1)ïŒ Asp 3.21(3)ïŒ Thr 2.09(2)ïŒ Ser 2.97(3)ïŒ Glu 7.35(7)ïŒ Gly 4.29(4)ïŒ Ala 1.26(1)ïŒ CySO3H 5.94(6)ïŒ Val 3.86(4)ïŒ Ile 1.55(2) Leu 6.53(6)ïŒ Tyr 4.08(4) Phe 3.22(3)ïŒ Pro 1.2(1)ãLys 1.00(1), His 1.91(2), Arg 0.95(1), Asp 3.21(3), Thr 2.09(2), Ser 2.97(3), Glu 7.35(7), Gly 4.29(4), Ala 1.26 (1), CySO 3 H 5.94(6), Val 3.86(4), Ile 1.55(2) Leu 6.53(6), Tyr 4.08(4) Phe 3.22(3), Pro 1.2(1).
(2) ãB30âThrâOButãâã€ã³ã·ãŠãªã³ïŒè±
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ååç©ã®ç²ç²æ«89mgãåŸãã(2) [B30-Thr-OBu t ]-Insulin (pig type) 100 mg (10 mM) of the product from (1) and 154 mg (500 mM) of L-threonine-t-butyl ester were mixed in an ethanol/dimethylformamide mixture (1:1). ) 1.05
ml and mixed with 0.75 ml of 0.5 M borate buffer (PH 6.5) containing 4 mg of crystalline trypsin (Worthington, recrystallized three times). This mixed solution contains tosyl-L-phenylalanine chloromethyl ketone (hereinafter referred to as TPCK) at a final concentration of
Keep it contained at 0.01mM. The mixture was kept at 37°C overnight. After confirming the production of the target compound using high performance liquid chromatography and determining the yield,
It was 75%. After making the mixture acidic with glacial acetic acid, it was added to a column of ultra-fine particle Cephadex G50 (4.2Ã
Perform gel filtration with 130 cm) to separate the trypsin-containing fraction,
Separate into a fraction containing the title insulin derivative and a fraction containing L-threonine-t-butyl ester. Measure enzyme activity and ninhydrin reaction,
It was confirmed that trypsin and threonine t-butyl ester were each recovered at 50%. The fraction containing the insulin derivative is lyophilized to obtain 89 mg of a crude powder of the title compound.
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ã確èªãããã The above product was then applied to a column (1.9 x 24.5 cm) of DEAE-Sephadex A25 buffered with 0.01M Tris buffer (PH7.6) and 7M urea at 4°C.
After flowing 800 ml of the above buffer, elution was performed with a linear concentration gradient up to a salt concentration of 0.3M, and
Fraction A with a concentration around 0.09M and fraction B with a concentration around 0.13 to 0.14 are sequentially obtained. Each fraction was immediately dialyzed against 0.01M ammonium acetate solution in a cold place for 3 to 4 days, and then freeze-dried.35mg of powder was obtained from fraction A and fraction B.
Get more than 27mg. High performance liquid chromatography and polyacrylamide gel electrophoresis confirmed that the former was the title compound and the latter was a mixture of des-B30-insulin (porcine type) and the title compound.
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ã以äžã«ç€ºãã The values measured by high performance liquid chromatography and polyacrylamide gel electrophoresis of the title compound are shown below.
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4.3cmã Polyacrylamide gel electrophoresis (PH8; 10%
Gel; 18.1mA/cm 2 ; Electrophoresis time 100 minutes): Travel distance
4.3cm.
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æµéïŒæ¯åïŒmlïŒä¿ææé13.2åã High performance liquid chromatography [Nucleosil 7C 18 (Macherey-Nagel); 4
mm x 50 mm (pre-column) and 4 mm x 250 mm (main column); 32% acetonitrile-buffer solution (2mM phosphoric acid, 5mM sodium n-butylsulfonate, 50mM sodium sulfate, PH3.0);
Flow rate: 2 ml per minute]: Retention time 13.2 minutes.
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ããReference example Human insulin: 2 ml of trifluoroacetic acid containing 0.2 ml of anisole
Add to 30 mg of the compound obtained in (2) above and keep at room temperature for 30 minutes. Remove trifluoroacetic acid and 1N in a nitrogen stream.
After extracting anisole with 15 ml of ether in the presence of 2 ml of acetic acid, the acetic acid portion was lyophilized to yield 28 mg of the title compound.
get. If the purity of the raw material is 77%, the yield is 43%.
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ããã This product has amino acid analysis values, slab gel electrophoresis,
It was identified as the title compound by high performance liquid chromatogram.
(1)ãšåæ§ã®æ¡ä»¶ã§è¡ã€ãã¢ããé
žåæã®çµæã¯
äžèšã®ãšããã§ããã The results of amino acid analysis conducted under the same conditions as (1) are as follows.
LyS 1.00(1)ïŒ His 1.89(2)ïŒ Arg 0.87(1)ïŒ Asp 3.32(3)ïŒ Thr 3.16(3)ïŒ Ser 2.99(3)ïŒ Glu 7.35(7)ïŒ Pro 1.29(1)ïŒ Gly 4.39(4)ïŒ Ala 1.26(1)ïŒ CySO3H 4.6(6)ïŒ Val 3.93(4)ïŒ Ile 1.61(2)ïŒ Leu 6.51(6)ïŒ Tyr 3.68(4)ïŒ Phe 3.19(3)ãLyS 1.00(1), His 1.89(2), Arg 0.87(1), Asp 3.32(3), Thr 3.16(3), Ser 2.99(3), Glu 7.35(7), Pro 1.29(1), Gly 4.39 (4), Ala 1.26(1), CySO 3 H 4.6(6), Val 3.93(4), Ile 1.61(2), Leu 6.51(6), Tyr 3.68(4), Phe 3.19(3).
ã¹ã©ãã²ã«é»æ°æ³³åïŒPHïŒïŒ10ïŒ
ã²ã«ïŒ
18.1mAïŒcm2ïŒæ³³åæé100åïŒïŒç§»åè·é¢6.0cmã
é«é液äœã¯ãããã°ã©ãã€ãŒããã¯ã¬ãªã·ã«
ïŒNucleosilïŒ7C18ïŒMachereyâNagel瀟補ïŒïŒïŒ
mmÃ50mmïŒãã¬ã«ã©ã ïŒããã³ïŒmmÃ250mmïŒã¡
ã€ã³ã«ã©ã ïŒïŒ32ïŒ
ã¢ã»ããããªã«âãããã¢ãŒ
溶液ïŒ5mMãªã³é
žã5mMã»ïœâããã«ã¹ã«ãã³
é
žãããªãŠã ã50mMç¡«é
žãããªãŠã ãPH3.0ïŒïŒ
æµéïŒæ¯åïŒmlãïŒä¿ææé4.97åã Slab gel electrophoresis (PH8; 10% gel;
18.1mA/cm 2 ; migration time 100 minutes): moving distance 6.0cm.
High performance liquid chromatography [Nucleosil 7C 18 (Macherey-Nagel); 4
mm x 50 mm (pre-column) and 4 mm x 250 mm (main column); 32% acetonitrile-buffer solution (5mM phosphoric acid, 5mM sodium n-butylsulfonate, 50mM sodium sulfate, PH3.0);
Flow rate: 2 ml per minute]: Retention time 4.97 minutes.
Claims (1)
åºãæããã€ããããã·åºã«ä¿è·åºãæããŠããŠ
ããã人ã€ã³ã·ãŠãªã³ã ïŒ ã«ã«ããã·ã«åºã®ä¿è·åºãã¢ã«ãã«ã§ããç¹
èš±è«æ±ã®ç¯å²ïŒã®äººã€ã³ã·ãŠãªã³ã ïŒ ããããã·åºã«ä¿è·åºãæããªãç¹èš±è«æ±ã®
ç¯å²ïŒã®äººã€ã³ã·ãŠãªã³ã ïŒ ããããã·åºã«ä¿è·åºãæããªãç¹èš±è«æ±ã®
ç¯å²ïŒã®äººã€ã³ã·ãŠãªã³ã[Scope of Claims] 1. Human insulin in which the threonine at position 30 of the B chain has a protecting group on the carboxyl group and may have a protecting group on the hydroxyl group. 2. The human insulin of claim 1, wherein the carboxyl group protecting group is alkyl. 3. The human insulin according to claim 1, which does not have a protecting group on the hydroxyl group. 4. Human insulin according to claim 2, which does not have a protecting group on the hydroxyl group.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56175030A JPS57155997A (en) | 1981-10-30 | 1981-10-30 | Insulin derivative |
JP61266447A JPS62116598A (en) | 1981-10-30 | 1986-11-07 | Production of insulin derivative |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56175030A JPS57155997A (en) | 1981-10-30 | 1981-10-30 | Insulin derivative |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4571079A Division JPS55138393A (en) | 1979-04-13 | 1979-04-13 | Semisynthesis of insulin |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61266447A Division JPS62116598A (en) | 1981-10-30 | 1986-11-07 | Production of insulin derivative |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS57155997A JPS57155997A (en) | 1982-09-27 |
JPS6312480B2 true JPS6312480B2 (en) | 1988-03-18 |
Family
ID=15988985
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP56175030A Granted JPS57155997A (en) | 1981-10-30 | 1981-10-30 | Insulin derivative |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS57155997A (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS54135789A (en) * | 1978-04-13 | 1979-10-22 | Nippon Soda Co Ltd | Preparation of human insulin |
-
1981
- 1981-10-30 JP JP56175030A patent/JPS57155997A/en active Granted
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS54135789A (en) * | 1978-04-13 | 1979-10-22 | Nippon Soda Co Ltd | Preparation of human insulin |
Also Published As
Publication number | Publication date |
---|---|
JPS57155997A (en) | 1982-09-27 |
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