Single-Atom O/S/Se Substitution at the Glycosidic Linkage in Glycopeptide Cancer Vaccines Reveals Divergent Performance

Single-atom substitution provides an exceptionally subtle means of editing molecular structure, yet how such minimal atom-level modifications propagate into biological function remains poorly understood. Here, we report streamlined access to Se-linked Tn glycopeptides which, together with their O- and S-linked counterparts, enable systematic atom-level editing at the glycosidic linkage of MUC1 glycopeptide antigens. Surface plasmon resonance established a clear hierarchy of antigen recognition that was rationalized by molecular dynamics simulations and independently validated by STD-NMR epitope mapping, revealing that O→S→Se substitution subtly remodels glycopeptide presentation while preserving the overall 5E5 recognition epitope. Translation of these structurally defined antigens into CRM197 glycoconjugate vaccines with comparable antigen loading showed that neither antigen-binding affinity nor antibody levels alone predict therapeutic efficacy. Instead, the results demonstrate that minimal atom-level editing propagates from molecular recognition to biological function through a nonlinear structure–function relationship, highlighting that therapeutic efficacy cannot be inferred directly from antigen affinity alone.