FACILE ENZYMATIC DENOVO SYNTHESIS AND NMR SPECTROSCOPIC CHARACTERIZATION OF D-TAGATOSE 1,6-BISPHOSPHATE

A D-tagatose 1,6-bisphosphate aldolase requiring Zn2+ for catalytic activity (class II) was purified from E. coli cells grown on galactitol. The aldolase, a homotetramer composed of subunits of mol wt approximately 28000, had a pH optimum at 7.5 and was highly selective for L-erythro as compared to D-threo stereochemistry (99: 1). This allowed its application in a coupled enzyme system together with glycerol kinase, pyruvate kinase and triose phosphate isomerase for the de novo, one-pot synthesis Of D-tagatose 1,6-bisphosphate starting from dihydroxyacetone and phosphoenolpyruvate (for the in situ regeneration of adenosine triphosphate), in quantities of 10 mmol. The expeditious process compares very favorably in simplicity and yield (40% overall) with the known multistep chemical preparation even after improvements to the latter accomplished during the present work. The classical sequence, which starts froM D-galacturonic acid, was modified at both phosphorylation steps: 1,2:3,4-di-O-isopropylidene-D-tagatofuranose was esterified by application of the trivalent phosphitylation agent dibenzyl di-N-ethyl-phosphoramidite followed by hydrogen peroxide oxidation, and a bacterial fructose 6-phosphate kinase was used for enzymic phosphorylation Of D-tagatose 6-phosphate. For the latter enzyme, which was the isoenzyme Pfk-2 from a recombinant strain of E. coli, kinetic constants were determined. NMR spectroscopic assignments are presented for D-tagatose and its phosphates.