Using LDI-MS to Explore Amide Coupling Reactions with Carboxylate Terminated N-Heterocyclic Carbene Monolayers

Abstract

N-Heterocyclic carbene (NHC) monolayers on gold display great promise as a platform for biotechnology, which requires biomolecule immobilization to NHC surfaces. The most popular method to couple biomolecules is an amide linkage between a carboxylic acid functionalized NHC and an amine terminated biomolecule. A well-established carboxylic acid terminated NHC-gold system was used as a model system to explore how steric bulk, ring strain, and functionality of amine substrates impact the success of coupling reactions. Here, we deploy laser desorption/ionization mass spectrometry (LDI-MS) to monitor the amide linkage products of the NHC monolayer with amine substrates. Mass spectrometry provides significant advantages when compared to other methods, as it can quickly screen for the successful amide linkage of biomolecules to the NHC monolayer. While we expected the NHC architecture to display a low coupling efficiency with sterically bulky and high ring strain amine substrates, coupling occurred for a wide range of substrates, illustrating the promise of NHCs for biomolecule immobilization. Then, we investigated whether the NHC could effectively couple a model biomolecule, l-lysine. Surprisingly, no evidence of coupling was observed, which prompted a series of experiments exploring the functional group tolerance of coupling reactions on the NHC surfaces. Our LDI-MS results illustrate that coupling reactions of NHC monolayers are intolerant to bifunctional amines bearing a terminal carboxylic acid and that esterification is necessary for successful coupling of amino acids. These general principles of NHC monolayer reactivity will provide a guide for the future design of NHC based biotechnology applications.