title: In situ maleimide bridging of disulfides and a new approach to protein PEGylation.
abstract: The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London,WC1H OAJ, United Kingdom.
authors.forename: Felix F
authors.initials: FF
authors.lastname: Schumacher
authors.forename: Muriel
authors.initials: M
authors.lastname: Nobles
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ryan
authors.forename: Mark E B
authors.initials: ME
authors.lastname: Smith
authors.forename: Andrew
authors.initials: A
authors.lastname: Tinker
authors.forename: Stephen
authors.initials: S
authors.lastname: Caddick
authors.forename: James R
authors.initials: JR
authors.lastname: Baker
mesh_terms: D002460:Cell Line; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008301:Maleimides / Q000737:chemistry*; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D017481:Receptors, Somatostatin / Q000737:chemistry / Q000378:metabolism; D013004:Somatostatin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D004220:Disulfides; D008301:Maleimides; D017481:Receptors, Somatostatin; C043592:maleimide; D011092:Polyethylene Glycols; D013004:Somatostatin; C093733:somatostatin receptor 2