Title: Process for the selective formation of disulfide bridges in polypeptides and therapeutic compositions

What is claimed is:

1. Process for the selective formation of at least two disulfide bridges in a polypeptide which process comprises treating a polypeptide starting material carrying at least four SH-groups to mask at least one of two of said SH-groups, intended to form a first disulfide bridge, with a p-methoxybenzyl protective group and to mask at least one SH-group of each further pair of SH-groups intended to form one or more further disulfide bridges, with an acetamido methyl protective group, then splitting off said p-methoxybenzyl protective group with pyridine-polyhydrogen fluoride in the presence of anisol to produce that first disulfide bridge, and then treating the polypeptide with iodine in an acid solution to produce said disulfide bridge-containing polypeptide by forming said one or more further disulfide bridges.

2. Process as claimed in claim 1 wherein the formation of two or more disulfide bridges is conducted in the presence of already existing disulfide bridges.

3. Process as claimed in claim 1 wherein said polypeptide is a synthetic polypeptide.

4. Process as claimed in claim 1 wherein said polypeptide is a natural polypeptide.

5. Process as claimed in claim 2, wherein an already existing disulfide bridge is formed by reductive splitting-off of two tertiary butyl mercapto protective groups, protecting two further SH-groups carried by said polypeptide starting material, with tributyl phosphane under inert gas and selective oxidation with oxygen prior to forming the said first and further disulfide bridges.

6. Process for the selective formation of at least three disulfide bridges in a polypeptide which process comprises treating a polypeptide starting material, carrying at least 6 SH-groups, to mask two of said SH-groups intended to form a first disulfide bridge with tertiary butyl mercapto protective groups, to mask at least one of two further of said SH-groups, intended to form a second disulfide bridge, with an acetamido methyl protective group, and to mask at least one SH-group of each further pair of SH-groups, intended to form one or more further disulfide bridges, with an acetamido methyl protective group, then splitting off said two tertiary butyl mercapto protective groups with tributyl phosphane under innert gas and selective oxidation with oxygen, thereby forming that first disulfide bridge, then splitting-off said p-methoxybenzyl protective group with pyridine-polyhydrogen fluoride in the presence of anisol to produce that second disulfide bridge, and then treating the polypeptide with iodine in an acid solution to produce said disulfide bridge-containing polypeptide by forming said one or more further disulfide bridges.

7. Process as claimed in claim 1 wherein active human insulin is prepared by reacting a synthetic insulin A-chain having an acetamido methyl protective group in the A⁷ -position and a p-methoxybenzyl protective group in the A²⁰ -position and with an equimolar quantity of synthetic or natural reduced insulin B-chain.

8. Process as claimed in claim 7 wherein an insulin B-chain is used having an acetamido methyl protective group in the B⁷ -position and a p-methoxybenzyl protective group in the B¹⁹ -position.

9. Process as claimed in claim 7 wherein a synthetic insulin A-chain is used in which the small interachenaric disulfide ring has been formed by reductive splitting-off of the tertiary butyl mercapto protective groups in the A⁶ - and A¹¹ -positions with tributyl phosphane under nitrogen and selective oxidation with air.

10. Process as claimed in claim 7 wherein the B-chain of bovine insulin is used as the natural insulin, modified at the C-terminus.

11. Process as claimed in claim 7 wherein A⁶ -A⁷, A¹¹ -B⁷ -cystine insulin is prepared from synthetic A⁶ -A⁷ insulin A-chain having an acetamido methyl protective group in the A²⁰ -position and a p-methoxybenzyl protective group in the A¹¹ -position.

12. Process as claimed in claim 1 wherein A⁶ -A⁷, A¹¹ -B⁷ -cystine insulin is prepared from synthetic A⁶ -A⁷ insulin A-chain having an acetamido methyl protective group in the A¹¹ -position and a p-methoxybenzyl protective group in the A²⁰ -position.

13. Process as claimed in claim 1 wherein the disulfide bridge formation is effected by oxidation with iodine in 30% acetic acid.

14. Process as claimed in claim 1 wherein the splitting off the p-methoxybenzyl protective group is carried out in pyridine polyhydrogen fluoride (HF/pyridine) as solvent and reagent.

15. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a natural or synthetic A⁶ -A¹¹ insulin A-chain having an acetamido methyl protective group in the A⁷ -position and a p-methoxybenzyl protective group in the A²⁰ -position, with a natural or synthetic insulin B-chain in reduced form or having an acetamido methyl protective group in the B¹⁹ -position and a p-methoxybenzyl protective group in the B⁷ -position.

16. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a natural or synthetic A⁶ -A¹¹ insulin A-chain having an acetamido methyl protective group in the A²⁰ -position and a p-methoxybenzyl protective group in the A⁷ -position, with a natural or synthetic insulin B-chain in reduced form having an acetamido methyl protective group in the B⁷ -position and a p-methoxybenzyl protective group in the B¹⁹ -position.

17. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ cystine insulin is produced by reacting a synthetic A⁷ -A¹¹ insulin A-chain having an acetamido methyl protective group in the A⁶ -position and a p-methoxybenzyl protective group in the A²⁰ -position with a natural or synthetic insulin B-chain in reduced form having an acetamido methyl protective group in the B¹⁹ -position and a methoxybenzyl protective group in the B⁷ -position.

18. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a synthetic A⁶ -A¹¹ insulin A-chain having an acetamido methyl protective group in the A²⁰ -position and a p-methoxybenzyl protective group in the A⁷ -position with a natural or synthetic insulin B-chain in reduced form or having an acetamido methyl protective group in the B⁷ -position and a p-methoxybenzyl protective group in the B¹⁹ -position.

19. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a synthetic A⁶ -A¹¹ insulin A-chain having an acetamido methyl protective group in the A⁷ -position and a p-methoxybenzyl protective group in the A²⁰ -position with a natural or synthetic insulin B-chain in reduced form having an acetamido methyl protective group in the B¹⁹ -position and a p-methoxybenzyl protective group in the B⁷ -position.

20. Process as claimed in claim 1 wherein antiparallel A⁶ -A⁷, A¹¹ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a synthetic A⁶ -A⁷ insulin A-chain having an acetamido methyl protective group in the A²⁰ -position and a p-methoxybenzyl protective group in the A¹¹ -position with a natural or synthetic insulin B-chain in reduced form or having an acetamido methyl protective group in the B⁷ -position and a p-methoxybenzyl protective group in the B¹⁹ -position.

21. Process as claimed in claim 1 wherein antiparallel A⁶ -A¹¹, A⁷ -B¹⁹, A²⁰ -B⁷ -cystine insulin is produced by reacting a natural or synthetic insulin A-chain, with the intrachenaric disulfide ring in the A⁶ -A¹¹ -position, the A⁷ -A¹¹ -position or in the A⁶ -A⁷ -position in a parallel or antiparallel manner with a natural or synthetic insulin B-chain in reduced form or having acetamido methyl protective groups and a p-methoxybenzyl protective groups, where the specified A-chain by a reactive anchor bond is linked to a polymeric carrier.

22. Process as claimed in claim 21 wherein polystyrene cross-linked with divinylbenzene is used as the polymeric carrier.

23. Process as claimed in claim 22 wherein a polystyrene gel is used, which is cross-linked with 0.1% to 0.8% divinylbenzene.

24. Process as claimed in claim 23 wherein 0.5 divinylbenzene is used.


Other info:


Inventors: Birr, Christian (Leimen/St. Ilgen, DE)

Application Number: 06/158021
Filing Date: 1980-06-09
Publication_date: 1982-09-28
Assignee: Max, Planck Gesellschaft

Primary Class(es): 530/336 930/DIG.621, 530/303, 930/DIG.620, 930/260
Other Classes: C07K1/06; C07K1/113; C07K1/12; C07K14/62; A61K38/00; C07K1/00; C07K14/435; C07C103/52
US Patent Ref:

B471617	February, 1976	Kamber et al.	260/112.7
3883500	May, 1975	Geiger et al.	260/112.7	Process for the manufacture of insulin, analogs and derivatives thereof
4029642	June, 1977	Obermeier	260/112.7	Process for the manufacture of human insulin

Other Refs:
Primary Examiner: Phillips, Delbert R.
Assistant Examiner:
Attorney: Felfe & Lynch