Analysis of isoprenoid phosphates and oligophosphates by capillary zone electrophoresis

Abstract Capillary zone electrophoresis (CZE) has been used for the first time to analyse water-soluble phosphorylated isoprenoids, key intermediates in the branched pathway of mevalonate metabolism. Following synthesis, isoprenoid phosphates and oligophosphates were isolated by flash chromatography and their purity was established using 31 P NMR spectrometry. In developing optimum conditions for CZE, several factors were considered: ionization properties of the solutes, stability of the solutes and maximum signal-to-noise ratio. At pH 8.5 in 0.3 M sodium borate buffer and monitoring UV absorbance at 200 nm, detection of farnesyl diphosphate was linear in the sub-picomol range; the limit of detection was ca. 12 fmol. For a given phosphorylated series (i.e., monophosphates, diphosphates, triphosphates), CZE cleanly resolved isoprenes of different chain length, and plots of relative mobility vs. M r were curvlinear. Remarkably, neryl phosphate (C10, α- cis -isoprene), geranyl phosphate (C10, α- trans-isoprene ) and citronellyl phosphate (C10, α-saturated isoprene) were resolved. In addition, isopentenyl monophosphate (C5, Δ 3 ) and dimethylallyl phosphate (C5, Δ 2 ) exhibited different electrophoretic mobilities. These studies pave the way for future work on determining levels of phosphorylated isoprenoids in various tissues under conditions of altered mevalonate production.