5'- N-Phenylacetyl sTn (sTnNPhAc), an unnatural derivative of sTn antigen expressed by many tumors, and its alpha-linked protein conjugates were prepared and investigated to explore glycoconjugate cancer vaccines. sTnNPhAcalpha-KLH elicited a robust T cell dependent immunity. The antiserum derived from sTnNPhAcalpha- or sTnNPhAcbeta-KLH-inoculated mice was similarly reactive to sTnNPhAcalpha and sTnNPhAcbeta but showed very little reactivity to sTn, NeuNPhAcalpha(2,3)GalNAc--a regioisomer of sTnNPhAc, isolated phenylacetyl group, and the linker employed to conjugate sTnNPhAc and carrier protein. It was concluded that the sTnNPhAc-elicited immunity was specific for the whole antigen rather than the phenylacetyl group or other partial structures of sTnNPhAc and that the reducing end configuration or linkage of sTnNPhAc did not affect its immunological identity. It was also concluded that a new linker designed to conjugate carbohydrates and proteins did not provoke any immune reaction and that the linker, as well as the associated new and convenient coupling strategy, can be safely used for the development of glycoconjugate vaccines.