One-Pot Thiol-Amine Bioconjugation to Maleimides; Simultaneous Stabilisation and Dual Functionalisation

Abstract

Maleimide chemistry is widely used in the site-selective modification of proteins. However, hydrolysis of the resultant thiosuccinimides is required to provide robust stability to the bioconjugates. Herein, we present an alternative approach that affords simultaneous stabilisation and dual functionalisation in a one pot fashion. By consecutive conjugation of a thiol and an amine to dibromomaleimides, we show that aminothiomaleimides can be generated extremely efficiently. Furthermore, the amine serves to deactivate the electrophilicity of the maleimide, precluding further reactivity and hence generating stable conjugates. We have applied this conjugation strategy to peptides and proteins to generate stabilised trifunctional conjugates. We propose that this stabilisation-dual modification strategy could have widespread use in the generation of diverse conjugates. <br>

A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability

“…The reaction between 1-hexylamine (50 mM final concentration, pH 8.0) and the GFP-PD-Br species 8 (50 μM, pH 8.0) at 37 °C for 16 h did not yield any of the desired product, likely due to a large proportion of the aliphatic primary amine being protonated under bioconjugation conditions (i.e., p K a of n -hexylamine = approximately 10.5). 37 Following the recently documented reaction between aniline-derivatives and dibromomaleimide scaffolds, that occurs under biocompatible conditions to induce stability toward thiols and hydrolysis, 36 the aniline derivative p- anisidine 15 (50 mM final concentration) was reacted with the GFP-PD-Br species 8 (50 μM, pH 8.0) at 37 °C for 16 h, which quantitatively produced the GFP-PD-amino species 17 ( Figure 3 c).…”

“…Therefore, it was envisaged that if an amine could be reacted at this position postconjugation, this transformation may confer an additional level of stability toward thiol exchange (i.e., the resultant species may be stable to high concentrations of thiol). 36 …”

Intracellular Delivery of Virus-Like Particles Using a Sheddable Linker

“…We prepared a dibromomaleimide-triphenylphosphonium (DB-TPP) linker (Figure 1A&B) via direct EDC coupling. The dibromomaleimide moiety reacts with thiol groups in a two-step reaction, 34 each step having a half-life of seconds. 28,35 Qβ contains 180 disulfide bonds, each of which can be a potential site for functionalization via this approach.…”

Tailored Bioorthogonal and Bioconjugate Chemistry: A Source of Inspiration for Developing Kinetic Target-Guided Synthesis Strategies

“…Bioorthogonal reactions are defined as chemical reactions that can proceed in biological media without interfering with natural chemical functions, while bioconjugation reactions involve chemical functions (generally electrophilic systems) that react with naturally occurring functional groups (such as amines or sulfhydryl moieties). [22][23] With the advent of bioorthogonal 24 and bioconjugate chemistry, 25 in recent years, an increasing number of experimental [26][27][28] and theoretical studies, [29][30] computational analysis 31 on rate constants of such chemical reactions have been made available to the scientific community. Accordingly, in the light of reported ligation reactions for biorthogonal ligation or bioconjugation, a non-exhaustive list of new reactions amenable to KTGS strategies will be proposed (Figure 2A), as well as clues to tune the kinetics of these reaction to increase their potential to meet the requirements for use in KTGS strategies.…”