A facile enzymatic synthesis of uridine diphospho-[C-14]galacturonic acid

Galacturonic acid (GalA) is a major component of plant cell-wall-derived pe ctins. It can be also found in the cell-surface polysaccharides of differen t microorganisms, including several symbiotic and pathogenic bacteria Uridi ne diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[C-14]GalA Commerciall y available UDP-[C-14]galactose was quantitatively oxidized (>95% conversio n) to UDP-[C-14]GalA in the presence of high levels of galactose oxidase an d catalase, at prolonged incubation times. Following this one-step enzymati c oxidation, UDP-[C-14]GalA was purified using a polyethyleneimine cellulos e column with a single-step 1 M NaCl elution. The authenticity of the purif ied UDP-[C-14]GalA was verified by its relative mobility on thin-layer chro matograms, analysis of its chemical hydrolysis products, and H-1 NMR spectr oscopy. Our yield of >90% is much higher than by previously described metho ds. The method may serve as a prototype for the preparation of other radiol abeled uronic acids and their nucleotide derivatives. (C) 2000 Academic Pre ss.