Patent GB-1453454-A (GB)
Title: Process for the manufacture of insulin analogues and derivatives thereof
IPC: C07K1/113
Filed: 1973-10-25
Abstract: 1453454 Insulin HOECHST AG 25 Oct 1973 [25 Oct 1972] 49799/73 Heading C3H [Also in Division C2] Insulin (also insulin analogues and derivatives) are prepared by linking a natural or synthetic insulin A chain having protected cysteine mercapto groups, with a natural or synthetic insulin B chain having protected cysteine mercapto groups, by a method suitable for the formation of peptide bonds, via the amino groups N<SP>αA1</SP> and N<SP>#B29</SP> by means of a bridging group containing two terminal acyl groups and two protected α-amino groups, deprotecting the cysteine mercapto groups, cross linking the A and B chanis in the resulting compound through the appropriate disulphide bridges and subsequently removing the N<SP>αA1</SP>- N<SP>#B29</SP> link in which the amino groups have been deprotected by a method suitable for removing N-terminal residues in peptides viz. the Edman degradation. The bridging group has the general formula wherein Q represents an N-protective group capable of being split off by proton solvolysis and Z represents a group of the formula (CH 2 ) n  where n is 0, 1, 2, 3 or 4, of which one CH 2  member may be replaced by S or SO 2 . Analogues or derivatives are produced by (a) replacing one or more amino acids of the chain by other, preferably simpler, amino acids, (b) shortening the chains, or (c) substituting one or more amino acid functional groups. In the A-chain Gln<SP>5</SP> may be replaced Glu, Gln<SP>15</SP> by Glu, Cys<SP>7</SP> by Ala, Val<SP>10</SP> by Leu or another hydrophobic amino acid, Ser<SP>12</SP> by Ala, Tyr<SP>14</SP> by Ala, Asn<SP>18</SP> by Ala, Asn<SP>21</SP> by Ala and Tyr<SP>19</SP> by Phe. In the B-chain Phe<SP>1</SP>, Val<SP>2</SP>, Asn<SP>3</SP>, Gln<SP>4</SP>, His<SP>5</SP>, Cys<SP>7</SP>,<SP>9</SP>, Ser<SP>9</SP>, His<SP>10</SP>, Thr<SP>27</SP> and Pro<SP>28</SP> may be replaced by Ala. The amino acids B1 B2, B3 and B30 may be eliminated. According to the Examples (1) bovine insulin is prepared from A-chain/S-tetrasulphonate (cattle) and N<SP>α</SP>-Boc-Ala or Gly-B-chain S- disulphonate using as bridging reagent di- Boc - 2,7 - diamino - suberic acid - bis - 2,4,5- trichlorophenyl ester, (2) des-(Phe-Val)<SP>B1-2</SP>-des- Ala<SP>B30</SP> - [Ala<SP>A12,14,18,21</SP>] insulin (pig) is prepared from Ala<SP>12,14,18,21</SP>-insulin-A-chain S-tetrasulphonate (pig) and N<SP>α</SP>-Boc-des-Phe<SP>B1</SP>-des- Ala<SP>B30</SP> - insulin B - chain S - disulphonate (pig) using as bridging reagent di-Boc-L-lanthionine - bis - 4 - nitrophenyl ester, (3) des- Phe<SP>B1</SP>-insulin (cattle) is prepared from A-chain S-tetrasulphonate (cattle) and N<SP>α</SP>-Boc-B-chain S-disulphonate (cattle) using as bridging reagent di - Boc - 2,6 - diamino - meso - pimelic acid - bis - 2,4,5 - trichlorophenyl ester, (4) Bl - acetyl insulin (pig) is prepared from A-chain S-tetrasulphonate (pig) and Bl-acetyl B-chain S-disulphonate using as bridging reagent di- Boc - 2,7 - diamino - suberic acid - bis - 2,4,5- trichlorophenyl ester, (5) human insulin is prepared from A-chain S-tetrasulphonate (human) and N<SP>α</SP>-Boc-Ala-B-chain S-disulphonate (human) using as bridging reagent di-Boc-2,8-diamino - azelaic acid - bis - 2,4,5 - trichlorophenol ester, and (6) des-Phe<SP>B1</SP>-insulin (cattle) is prepared from bovine insulin A-chain tetrasulphonate and N<SP>B1</SP>-trifluoroacetyl B-chain disulphonate (cattle) using as bridging reagent di- Boc - 2,7 - diamino - suberic acid/bis - 2,4,5- trichlorophenyl ester. Other bridging reagents specifically mentioned are di - Boc - L - lanthionine and di - Boc - L- lanthionine - bis - N - hydroxy - succinimide ester. Boc. represents the tert.-butyloxy-carbonyl radical.