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 beta-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.

The branched glycerol analogs 1 and 2 were prepared. Mono-ester derivatives of these triols undergo a chromogenic or fluorogenic reaction in the presence of NaIO 4 . In contrast, both the diesters and the triols are themselves not chromogenic or fluorogenic. Diester derivatives of these triols can be used as probes for lipases. The tris-phosphate derivative of 1 is a fluorogenic substrate for various phosphatases.Introduction. ± Enzyme assays are indispensable tools in enzymology, where they are used to identify enzymes and to evaluate their purity and activity [1]. When an enzyme is discovered, one important question is always the identity of its natural substrate, which is presumably also its best substrate in terms of kinetics behavior. In the context of enzyme assays, the related problem is to find the optimal assay method for any given enzyme, in particular the most-sensitive method. Simple assays giving readily recordable spectroscopic signals are preferred in the context of high-throughput screening for new enzymes by biodiversity mining or directed evolution. Herein, we report the synthesis and evaluation of the branched triols 1 and 2 as building blocks for enzyme substrates. Triols 1 and 2 are close analogs of glycerol, and their ester derivatives can be used to prepare chromogenic and fluorogenic analogs of glycerides, the natural substrates of lipases. The synthesis of these triols and their derivatives and their applications in enzyme assays are presented.