Bioconjugation Strategies Through Thiol-Alkylation of Peptides and Proteins

  • Terrence Kantner

Student thesis: Doctoral ThesisPhD

Abstract

Bioconjugation chemistry generally refers to the covalent derivatisation of biomolecules. Derivatisation of cysteine’s thiol of peptides and proteins is a common method in bioconjugation chemistry as the thiolate is an excellent nucleophile in aqueous conditions.The propensity for thiols to oxidise in an aqueous environment necessitates the need for a disulfide reduction step prior to the addition of ligands derivatised with thiol alkylating linkers. Disulfide reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP) are disulfide reducing agents that are often marketed as being non-reactive with thiol alkylating reagents. The reaction of TCEP and THPP with thiol alkylation linkers was therefore investigated. Characterisation of reaction products and mechanistic studies revealed that TCEP and THPP both react with thiol alkylation reagents. A novel protocol was, therefore, developed utilising the Staudinger reaction to oxidise excess TCEP and THPP prior to the addition of thiol alkylating reagents. The protocol offers a simple “one-pot” method for effecting conjugate production via thiol alkylation, without the need for an intermediate purification step for the removal of excess disulfide reducing agents.4-Vinyl pyridine (4-VP) derivatives were developed and explored as an alternative Michael acceptor class for thiol alkylation of peptides and proteins. The 4-VP derivatives exhibited high reactivity and specificity for thiol alkylation between pH = 7 and pH = 8. A selection of 4-VP linkers were subsequently functionalised with either carbohydrates or polyethylene glycol (PEG) and successfully utilised to produce peptide or protein conjugates via thiol alkylation reactions.
Date of Award12 Jul 2015
LanguageEnglish
Awarding Institution
  • University of Bath
SupervisorAndrew Watts (Supervisor)

Keywords

  • bioconjugation
  • thiol-alkylation

Cite this

Bioconjugation Strategies Through Thiol-Alkylation of Peptides and Proteins
Kantner, T. (Author). 12 Jul 2015

Student thesis: Doctoral ThesisPhD