Monitoring biosynthetic transformations of N-acetyllactosamine using fluorescently labeled oligosaccharides and capillary electrophoretic separation.

Capillary electrophoresis with laser-induced fluorescence detection was used to monitor biosynthetic transformations of a labeled derivative of N-acetyllac-tosamine (beta Gal (1 --> 4) beta GlcNAc, LacNAc) in crude microsomal extracts. For this purpose, six authentic standards including LacNAc-O-TMR itself (-TMR, a linker arm attached to tetramethylrhodamine), the tri-and tetrasaccharides that would form by either alpha (1 --> 2) or alpha (1 --> 3) fucosylation of LacNAc or both (i.e., H-type 2, LeX and LeY sequences) were chemically synthesized. The potential degradation products produced by galactosidase and hexosaminidase were also prepared. All of the standards were kinetically competent substrates for the enzymes present in HT-29 cells and could be baseline separated in a single run requiring 11 min. The action of competing enzymes acting on the common LacNAc sequence could thus be monitored in a single run with sensitivity routinely as low as a few thousand molecules.