A new strategy for the preparation of distinct N -substituted muropeptides is described. Different orthogonally N -protected disaccharide thioglycosides were designed and synthesized. Among them, compound 4 , qualified as a key intermediate, was utilized for further chemical transformations to develop a series of diverse N -substituted-glucosaminyl N -substituted-muramyl dipeptides (GMDPs). These unique muropeptides were applied for the study of human NOD2 stimulation. Intriguingly, structural modification of the MurNAc residue to N -non-substituted muramic acid (MurNH 2 ) in GMDP dramatically impaired NOD2 stimulatory activity, but GMDPs possessing the glucosamine residue with a free amino group retained NOD2 stimulation activity. This work is the first study to illustrate the impact of both N -substituents of GMDPs on immunostimulatory activities of human NOD2.