Substrate specificity of the sialic acid biosynthetic pathway

Unnatural analogues of sialic acid can be delivered to mammalian cell surfa ces through the metabolic transformation of unnatural N-acetylmannosamine ( ManNAc) derivatives. In previous studies, mannosamine analogues bearing sim ple N-acyl groups up to five carbon atoms in length were recognized as subs trates by the biosynthetic machinery and transformed into cell surface sial oglycoconjugates [Keppler, O. T.,et al. (2001) Glycobiology 11, 11R-18R]. S uch structural alterations to cell surface glycans can be used to probe car bohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural altera tions of the N-acyl substituent of ManNAc. A panel of analogues with ketone -containing N-acyl groups that varied in the length or steric bulk was chem ically synthesized and tested for metabolic conversion to cell surface glyc ans. We found that extension of the N-acyl chain to six, seven, or eight ca rbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Qua ntitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis prov ides a target for expanding the metabolic promiscuity of mammalian cells.