Talk Session: SESSION 9: INNOVATIONS IN PEPTIDE AND PROTEIN CHEMISTRY
Date: Tuesday, June 14, 2022
Talk Time: 11:35 am - 11:55 am
Talk Title: An Underreported Side Reaction in Maleimide Bioconjugation: Thiazine Rearrangement of Maleimido N-Terminal Cys Peptides
Isaiah Gober is a Research Scientist at Bachem. In 2017, Isaiah obtained his Ph.D. in Chemistry from the University of North Carolina at Chapel Hill under the supervision of Dr. Marcey Waters, working on the design of synthetic small molecule receptors for the molecular recognition of methylated lysine.
After his graduate studies, he worked as an Applications Scientist at CEM Corporation in Matthews, NC, where he helped develop new methods in microwave-assisted peptide synthesis. In 2020, Isaiah moved to Bachem in Torrance, CA. His current research focuses on the development of improved methods for solid-phase peptide synthesis and manufacturing of peptide APIs.
Thiol-maleimide chemistry is widely used in the preparation of covalently modified peptides and proteins due to the rapid kinetics and selectivity of the reaction. Although thiol-maleimide conjugation has considerable utility, various side reactions are possible which may complicate the preparation of bioconjugates.
In this work, we report on the formation of a thiazine impurity during the conjugation of N-terminal Cys peptides to maleimides. NMR spectroscopy and mass spectrometry were used to characterize the structure of the thiazine byproduct. The pH dependence and sequence specificity were investigated using a tripeptide model system wherein the amino acid adjacent to the N-term Cys and the N-substitution of the maleimide were varied to generate a small library of conjugates.
Our findings indicate that the rearrangement to the thiazine isomer is general and that the thiazine impurity is the primary byproduct formed when succinimidyl thioether conjugates are incubated at neutral or basic pH. With this knowledge in hand, we demonstrated that the thiazine isomer can also be prepared on the sidechain of a peptide rather than the N-terminus by coupling Cys to a Lys residue prior to the maleimide conjugation and subsequent rearrangement.