Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.

GalNAc is the initial sugar of mucin‐type O‐glycans, and is a component of several tumor antigens. The aim of this work was to determine whether synthetic GalNAc analogs could be taken up from the medium and incorporated into complex cellular O‐glycans. The cell line employed was CHO ldlD, which can only use GalNAc and Gal present in the medium for the synthesis of its glycans. All GalNAc analogs with modified N‐acyl groups (N‐formyl, N‐propionyl, N‐glycolyl, N‐azidoacetyl, N‐bromoacetyl, and N‐chloroacetyl) were incorporated into cellular O‐glycans, although to different extents. The GalNAc analogs linked to Ser or Thr could be extended by the β3‐galactosyltransferase glycoprotein‐N‐acetylgalactosamine 3β‐galactosyl transferase 1 in vitro and in vivo and by α6‐sialyltransferase α‐N‐acetylgalactosaminide‐α‐2,6‐sialyltransferase 1. At the surface of CHO ldlD cells, all analogs were incorporated into sialylated O‐glycan structures like those present on wild‐type CHO cells, indicating that the GalNAc analogs do not change the overall structure of core‐1 O‐glycans. In addition, this study shows that the unnatural synthetic GalNAc analogs can be incorporated into human tumor cells, and that a tumor antigen modified by an analog can be readily detected by a specific antiserum. GalNAc analogs are therefore potential targets for tumor immunotherapy.