Fueling Carbodiimide Chemistry: Direct Structural Characterization of the O -Acylisourea Intermediate

Abstract Carbodiimide-fueled molecular machines, catalytic reaction cycles, and a broad spectrum of synthetic transformations from peptide coupling to Steglich esterification depend on the fate of a single transient species: the O-acylisourea intermediate. Whether this intermediate engages in productive chemistry or diverts irreversibly to its inert N-acylurea isomer determines the outcome of many carbodiimide-mediated processes. Yet direct structural characterization of the O-acylisourea generated from standard dialkylcarbodiimides has eluded the field for over four decades. Here we report the gas-phase separation and structural identification of both the O-acylisourea and the N-acylurea isomers, sampled directly from the reaction medium by nanospray ionization (nESI) by coupling high-field asymmetric waveform ion mobility spectrometry with tandem mass spectrometry (nESI-FAIMS–MSn). Using N,N′-diisopropylcarbodiimide (DIC) and Mosher’s acid as a model system, we resolve the two isomers by FAIMS and establish their assignments through three convergent lines of evidence: isomer-specific CID fragmentation patterns, real-time monitoring of isomer-specific fragment ions after mixing, and isomer-specific infrared multiple-photon dissociation (IRMPD) spectra supported by DFT calculations. These results constitute the first gas-phase separation and structural assignment of the O-acylisourea and N-acylurea isomers generated from a dialkylcarbodiimide and a small-molecule carboxylic acid, sampled directly from solution without chemical trapping or prior isolation.