Convergent solid-phase synthesis of hirudin. |
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Authors: | Spyros Goulas Dimitrios Gatos Kleomenis Barlos |
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Institution: | Department of Chemistry, University of Patras, Patras, Greece. |
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Abstract: | Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5. |
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Keywords: | hirudin 2‐chlorotrityl resin convergent synthesis protected peptide fragments solid‐phase fragment condensation oxidative folding solid‐phase peptide synthesis |
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