Information stored in digital polymers are best recovered by MS/MS sequencing when the structure of these macromolecules is designed for programmed fragmentation. A byte-truncated architecture was conceived for poly(phosphodiester)s (PPDEs), with weak alkoxyamine bonds placed between each group of eight co-monomers, so that soft activation of the chains releases blocks of size small enough to ensure de novo sequencing. Yet, due to their diradical character, some blocks exhibit puzzling fragmentation behavior as a function of their charge state. Unlike their triply charged homologues, collision-induced dissociation (CID) of diradical blocks at the 2– charge state produces additional fragments, with +1 m/z shift when holding the nitroxide α termination and –1 m/z when carrying the ω carbon-centered radical. These results suggest cyclization followed by H^• transfer upon activated reopening of this cycle.

This assumption was hence investigated with ion mobility spectrometry (IMS) experiments performed with a Waters Synapt G2 HDMS instrument. Polymers ionized in negative mode electrospray were activated in-source to release blocks that can be investigated by IMS or sequenced in the post-IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions with the IMSCal software. A multi-step protocol including quantum and molecular dynamics calculations was implemented for CCS calculation.

Combining IMS experiments with molecular modeling permitted to show that, when doubly deprotonated, diradical blocks can indeed adopt a stable cyclic form, which re-opening proceeds with occurrence of H^• transfer as evidenced when recording energy-resolved mobilograms. So-produced close-shell species then dissociate into fragments with ±1 m/z shifts compared to CID products of diradical chains. Rationalizing this CID behavior provides us with new guidelines to improve MS/MS readability of these digital polymers.