Rapid and flexible synthesis of 1-deoxy-D-xylulose-5-phosphate, the substrate for 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
1-Deoxy-D-xylulose-5-phosphate (DXP) is a key intermediate in the non-mevalonate pathway to terpenoids in bacteria, and it is the substrate for the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP-R). In order to study the mechanism of DXP-R, we required a flexible synthesis of the substrate which would allow the incorporation of isotopic labels, and the variation of the two stereocentres. Thus 1,4-dihydroxypent-2-yne was selectively reduced to give the E-olefin, and selective phosphorylation of the primary alcohol followed by oxidation of the secondary alcohol gave a substrate suitable for dihydroxylation. Dihydroxylation using stoichiometric OsO4 in the presence of chiral ligands gave protected DXP in high ee. Final hydrogenolysis gave DXP in quantitative yield and high purity. DXP-R was produced by rapid cloning of the dxr gene from Escherichia coli through controlled expression and ion exchange chromatography. The synthetic DXP was fully active in enzyme assays catalysed by recombinant DXP-R.
Cox
Russell J
RJ
School of Chemistry, University of Bristol, Cantock's Close, Bristol, UK BS8 1TS. r.j.cox@bris.ac.uk
de Andrés-Gómez
Ana
A
Godfrey
Christopher R A
CR
D000438:Alcohols / Q000737:chemistry; D019747:Aldose-Ketose Isomerases / Q000138:chemical synthesis* / Q000737:chemistry; D002384:Catalysis; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000138:chemical synthesis* / Q000737:chemistry; D010088:Oxidoreductases / Q000138:chemical synthesis* / Q000737:chemistry; D010428:Pentosephosphates / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D013997:Time Factors
C109460:1-deoxylulose 5-phosphate; D000438:Alcohols; D009097:Multienzyme Complexes; D010428:Pentosephosphates; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases