Balan, Sibu; (2007) Disulfide Bridging Poly(ethylene glycol) Reagents For Site-specific Protein Conjugation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Disulfide bonds are ubiquitous in proteins and are broadly involved with protein structure, function and stability. Most therapeutic proteins which act extracellularly have disulfides. Frequently at least one disulfide is accessible to protein disulfide reductants. Disulfide bridging-poly (ethylene glycol) (DB-PEG) reacts with the two sulfurs derived by reduction of such an accessible disulfide and inserts a 3-carbon bridge between the thiols thus attaching the PEG to the protein. This thesis investigates the site-specificity and application of the bis-functional DB-PEG for the monoPEGylation of therapeutic proteins with accessible disulfides. The hypotheses of the project were i) some protein disulfides can be modified with DB-PEG with the protein retaining its structural features and biological activity, ii) upon reaction, the 3-carbon bridge forms between the sustain thiols, iii) DB-PEGylation can be efficient with high yields of the PEGylated protein and iv) since many therapeutic proteins possess accessible disulfides that mostly maintain protein stability, this conjugation strategy will have a broad applicability. DB-PEG was prepared by a simple synthetic route and their reactivity and bis-thiol specificity was studied using reduced glutathione. The characteristics of this PEGylation approach are i) a requirement to reduce the protein disulfides and ii) reaction efficiency depended on the disulfide bond position within the protein. Molecular modelling was done to study the 3-carbon disulfide bridged protein structure. The use of the DB-PEG to conjugate PEG to proteins was studied with L-asparaginase, interferon α-2b, leptin and somatostatin. Evaluation of the biological activities of the conjugates indicated that the observed activities were predominantly due to the steric shielding of PEG. The DB-PEG was found to be stoichiometrically efficient. Disulfide reduction without loss of protein structure was possible and the DB-PEG was specific to the thiols generated from a reduced disulfide bond.

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