Pergamon Tetrahedron: Asymmetry 10 (1999) 1643–1646 Efficient multi-enzymatic synthesis of D-xylulose 5-phosphate Frank T. Zimmermann, a Achim Schneider, a Ulrich Schörken, b Georg A. Sprenger b and Wolf-Dieter Fessner a,*,† a Institut für Organische Chemie, RWTH Aachen, Professor-Pirlet-Str. 1, D-52056 Aachen, Germany b Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, P.O. Box 1913, D-52425 Jülich, Germany Received 20 April 1999; accepted 28 April 1999 Abstract Pure D-threo-2-pentulose 5-phosphate, a valuable substrate required for enzymatic assays, is prepared in gram- quantities (82% overall yield) by an enzymatic one-pot procedure starting from the readily available precursors hydroxypyruvate and fructose 1,6-bisphosphate. The procedure is based on a stereospecific C–C bond formation catalyzed by the recombinant transketolase A from Escherichia coli. © 1999 Elsevier Science Ltd. All rights reserved. For complex, multifunctional structures that are difficult to prepare by conventional means, asymmetric framework construction by enzymatic carbon–carbon bond formation is an attractive alternative to standard chemical methods due to an often complete stereochemical control, mild reaction conditions, and no requirement for protecting group chemistry. 1 In particular, a number of aldolases have proved invaluable for the preparation of complex carbohydrates and related targets. 2 Recently, the transketolase A from Escherichia coli (TK eco ; EC 2.2.1.1) has become available on a large scale by recombinant expression 3,4 which has stimulated investigations towards the development of practical procedures that use this enzyme as a catalyst in asymmetric synthesis. 5,6 In vivo, transketolase catalyzes the stereospecific transfer of a hydroxyacetyl nucleophile between various sugar phosphates in the presence of a thiamine diphosphate cofactor and divalent cations. For a spectrophotometric determination of the substrate specificity of native transketolase, or for the kinetic evaluation of site-directed mutant enzymes, the C 2 -donor component D-xylulose 5-phosphate (D-threo- 2-pentulose 5-phosphate, 1) is the assay substrate of choice because of its superior kinetic constants (K m 160 μM). 4 D-Xylulose 5-phosphate has also been implicated to serve as a second messenger in liver tissue for the stimulation of glycolysis by activating a regulatory protein phosphatase (PP2A). 7 However, commercial 1 is only ∼80% pure and very expensive. Preparation by phosphorylation of the free ketose requires the isolation of a non-commercial kinase, 8 while published protocols for the synthesis of 1 from * Corresponding author. Fax: +49 6151 16 6636; e-mail: fassner@tu-darmstadt.de † Present address: Department of Organic Chemistry, Darmstadt University of Technology, Petersenstr. 22, D-64287 Darm- stadt, Germany. 0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0957-4166(99)00166-4 tetasy 2816 Communication