Evaluation of Substituted N-Aryl Maleimide and Acrylamides for Bioconjugation

Abstract

Novel SF5-bearing maleimide and acrylamide derivatives were synthesised as potential [18F]radio-prosthetic groups for radiolabelling peptides and proteins. The efficacy of selected prosthetic groups was first assessed through bioconjugation with protected model amino acid derivatives. These reactions were investigated on an analytical scale via LC-MS across a pH range to quantitatively evaluate this prosthetic group’s reactivity and stability. Model bioconjugate reactions were then replicated using analogous para-substituted derivatives to determine the influence of the electronic effects of -SF5. Finally, the SF5-bearing prosthetic groups were utilised for bioconjugation with cancer-targeting c-RGD peptides. N-aryl maleimides reacted extremely efficiently with the model amino acid N-acetyl-L-cysteine. The subsequent conjugates were obtained as regio-isomeric mixtures of the corresponding thio-succinamic acids in yields of 80–96%. Monitoring the bioconjugate reaction by LC-MS revealed that ring hydrolysis of the intermediate SF5–thio-succinimide conjugate occurred instantaneously, an advantageous quality in minimising undesirable thiol exchange reactions with non-targeted cysteine residues. In contrast, N-aryl acrylamides demonstrated poor solubility in semi-aqueous media (<1 mM). In turn, synthetic-scale model bioconjugations with Nα-acetyl-L-lysine were performed in methanol, affording the corresponding acrylamide conjugates in modest to high yield (58–89%). Including electron-deficient, fluorinated prosthetic groups for bioconjugation will broaden their applicability within the fields of 19F-MRI and PET imaging.