As part of our ongoing weekly tradition, we are highlighting notable and unconventional bioconjugation research. Today, I would like to discuss a recent publication from Dr. Pentelute’s lab at MIT, available at DOI: 10.1002/anie.201800860.
This study bridges the gap between purely chemical bioconjugation strategies and chemoenzymatic approaches. The researchers developed a miniature enzyme, composed of just seven amino acids, which is fused to a protein of interest. This “enzymatic tag” catalyzes a reaction between the thiol group of cysteine—incorporated within the tag—and the well-established DBCO functional group.
There is DBCO but no click-chemistry! Who would have thought that this is possible?!
An intriguing feature of this approach is its selectivity. The reaction occurs exclusively in the presence of the Enzymatic Tag next to Cysteine, while other cysteine residues within the protein sequence remain unreacted. The reported conjugation efficiency is approximately 80%, demonstrating its practical utility.
A key consideration in any bioconjugation strategy is the stability of the resulting bond. Unlike maleimide-based conjugations, which are prone to hydrolysis, this cysteine-DBCO linkage exhibits remarkable stability under physiological conditions. As the authors note:
“The generated linkage was stable to exogenous thiols at physiological pH, in stark contrast to the corresponding cysteine-maleimide conjugate. We reacted P1 with N-ethylmaleimide to generate P1–7 as a comparison with the conjugate P1–1 for their stability against the exogenous thiol glutathione at pH 7.4 and 37°C. LC-MS analysis indicated that the cysteine-cyclooctyne conjugate P1–1 was intact after 4 days (Figures S25 and S26). In contrast, the thiosuccinimide linkage of P1–7 was not stable and underwent hydrolysis and maleimide elimination, leaving less than 14% of intact P1–7 after 4 days (Figures S25 and S26). The high stability of the cysteine-DBCO conjugate underpinned the potential usage of the reaction in biological applications such as drug conjugation.”
This work presents an exciting addition to the toolkit of site-selective bioconjugation methods, offering both efficiency and stability for potential applications in biomolecular engineering and drug development.
If you are interested in more details, here is the supporting materials:
https://onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2Fanie.201800860&file=anie201800860-sup-0001-misc_information.pdf