Exploring the versatility of pentafulvene-maleimide cycloaddition as a ligation strategy: buffer and pH effects.

Abstract

Ligation chemistries are often required to perform under stringent conditions that preserve the integrity and function of (bio)conjugates, including specific biological buffers. To explore the versatility of the pentafulvene-maleimide ligation for (bio)conjugations, we studied the stability of the coupling partners and their Diels-Alder cycloaddition (DAC) in buffers used commonly in biological assays, protein chemistry and bioconjugates. The stability of 6,6-dialkylpentafulvene and maleimide derivatives to a panel of buffers with pH values between 3.7 and 10.1 was monitored via1H NMR spectroscopy. While the pentafulvene displayed excellent stability, hydrolysis of the maleimide was observed in several cases, although the extent of hydrolysis did not correlate with pH. For almost all buffers the pentafulvene-maleimide DAC proceeded efficiently and at a significantly faster rate than maleimide hydrolysis under the conditions studied. The buffer composition nor pH appeared to have a significant effect on the kinetics of the DAC with second-order rate constants (k2) ranging from 0.14 to 0.33 M-1 s-1 (23 ± 1 °C). This study highlights the versatility of the pentafulvene-maleimide ligation to proceed under a wide range of conditions relevant for (bio)conjugations and that maleimide hydrolysis in aqueous system may be promoted or inhibited by certain buffers.