• aldolases;
  • enzyme catalysis;
  • enzyme evolution;
  • fluorogenic assays;
  • high-throughput screening


Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate. As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose. The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E. coli transketolase for assembly of the final product. The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose. Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde. The latter primary product undergoes a β-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone. A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3. The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.