Improving the stability of thiol-maleimide bioconjugates via the formation of a thiazine structure

• Published in: Journal of peptide science Vol. 29; no. 10; p. e3495
• Main Authors: Gober, Isaiah N, Sharan, Rahul, Villain, Matteo
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.10.2023
• Subjects: Conjugates; Conjugation; Cysteine; Glutathione; Michael reaction; NMR; Nuclear magnetic resonance; Peptides; Physiology; Proteins; Reagents; Stability analysis; Stereoselectivity; Structural stability; Thiols; in vivo stability; retro-Michael reaction; N-terminal cysteine; cysteine conjugation; peptide and protein conjugates; stereoselective chemical rearrangement; thiazine
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.3495

Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol-maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement. Our preliminary work indicated that the thiazine linker has favorable stability. Here, we report the evaluation of a thiazine linker as an alternative to the widely used succinimidyl thioether linker for thiol-maleimide bioconjugation. The stability of the thiazine conjugate in comparison to the thioether conjugate was assessed across a broad pH range. Additionally, the propensity for retro-Michael reaction and cross-reactivity with other thiols was evaluated by treating conjugates in the presence of glutathione. The studies indicated that the thiazine linker degrades markedly slower than the thioether conjugate. In addition, the thiazine linker is over 20 times less susceptible to glutathione adduct formation. The NMR study of the thiazine structure confirmed that the formation of the thiazine linker is a stereoselective process that yields a single diastereomer. In summary, we propose the use of the thiazine linker obtained by conjugation of maleimide-containing reagents with peptides or proteins presenting an N-terminal cysteine as a novel approach for bioconjugation. The advantages of this approach are the formation of a linker with a well-defined stereochemical configuration, increased stability at physiological pH, and a strongly reduced propensity for thiol exchange.