Two‐step selective formation of three disulfide bridges in the synthesis of the C‐terminal epidermal growth factor‐like domain in human blood coagulation factor IX

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Abstract
The 45‐residue C‐terminal EGF‐like domain in human blood coagulation factor IX has been synthesized by a 2‐step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4‐methyl‐benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1–3), Cys12 to Cys26 (Cys 2–4), or Cys28 to Cys41 (Cys 5–6). Only the peptide having the Cys 2–4 pair blocked with Acm forms the desired disulfide isomer (Cys 1–3/5–6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF‐like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.