ThioLinker-DBCO is a water-soluble bis-alkylating labeling reagent that enables site-specific incorporation of DBCO moiety onto antibodies to form well-defined conjugates. This approach involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which DBCO moiety is covalently attached. During this process, irreversible denaturation of the protein, disrupting either their tertiary structure or their biological activity does not occur.
The covalent, site-specific conjugation of PEG to a polyhistidine tag (His-tag) on a protein with bis-alkylating reagents was also reported in literature (see Selected References section).
1. Khalili, H., et al. (2012). Comparative Binding of Disulfide-Bridged PEG-Fabs. Bioconjug. Chem., 23(11), 2262-77. [PubMed]
2. Badescu, G., et al. (2014). Bridging Disulfides for Stable and Defined Antibody Drug Conjugates. Bioconjug. Chem., 25(6), 1124-36. [PubMed]
3. Brocchini, S., et al. (2008). Disulfide bridge based PEGylation of proteins. Adv Drug Deliv Rev., 60, 3-12. [PubMed]
4. Balan, S., et al. (2007). Site-Specific PEGylation of Protein Disulfide Bonds Using a Three-Carbon Bridge. Bioconjugate Chem.,, 18, 61-76. [PubMed]
5. Wang, T., et al. (2014). Bis-sulfide bioconjugates for glutathione triggered tumor responsive drug release. ChemComm.,, 50, 1116-1118. [PubMed]
6. Wilbur, T., et al. (1994). Monoclonal antibody Fab’ fragment cross-linking using equilibrium transfer alkylation reagents. A strategy for site-specific conjugation of diagnostic and therapeutic agents with F(ab’)2 fragments. Bioconjugate Chem.,, 5, 220-235. [PubMed]
Site specific conjugation to a polyhistidine tag (His-tag).
7. Cong, Y., et al. (2012). Site-Specific PEGylation at Histidine Tags. Bioconjugate Chem.,, 23, 148-263. [PubMed]