Release of protein N-glycans by effectors of a Hofmann carboxamide rearrangement.

Chemical methods for glycan release have gained traction because of their cost efficiency, accelerated reaction time and ability to release glycans not amenable to enzymatic cleavage. Oxidative chemical glycan release via hypochlorite treatment has been shown to be a convenient and efficient method that yields N-glycans similar to classical PNGase F digestion. We observed that the initial steps of the suggested mechanism for the oxidative release of glycans from glycoproteins by hypohalites showed similarities to the initiating steps of the classical Hofmann rearrangement of carboxamides. Therefore, we investigated the ability of different stable effectors of a Hofmann-type carboxamide rearrangement to efficiently and selectively release N-glycans from glycoproteins. Released glycans obtained from different experimental chemical release approaches were analyzed by HILIC-FLD, BHZ-FACE and ESI-MS and evaluated with respect to electrophoretic mobility, retention time and integrated peak area for resolved glycans. We show that the known Hoffmann catalysts 1,3-dichloro-5,5-dimethylhydantoin, the hypervalent organoiodine (III) compound diacetoxy-iodobenzene as well as hypobromite generation using Oxone and potassium bromide are all capable of releasing protein-bound N-glycans in good yield. Among the compounds investigated, diacetoxy-iodobenzene was capable of releasing glycans in the absence of alkali. Detailed investigations of the bromide/Oxone method revealed a dependence of N-glycan release efficiency from the temporal order of bromide addition to the reaction mix as well as from a molar excess of bromide over Oxone. Conclusions. These findings suggest that the oxidative release of N-glycans occurs via the initiating steps of a Hofmann carboxamide rearrangement. Hypervalent organoiodine compounds hold the promise of releasing glycans in the absence of alkali. The generation of hypobromite by bromide/Oxone produces a consistent defined amount of reagent for rapid N-glycan release for both analytical and preparative purposes.