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

ABSTRACT 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 CRM 197 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.