Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry

A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585–591, 1988) was developed for the separation of inositol hexakisphosphate (Ins
P6
, phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (
PP
Ins
P5
or Ins
P7
) and bis-diphosphoinositol tetrakisphosphate (bis
PP
Ins
P4
or Ins
P 8
). With an acidic elution, the anion-exchange separation led to the resolution of four separable
PP
Ins
P 5
isomers (including pairs of enantiomers) into three peaks and of nine separable bis
PP
Ins
P 4
isomers into nine peaks. The whole separation procedure was completed within 20–36 min after optimization. Reference standards of all bis
PP
Ins
P 4
isomers were generated by a nonenzymatic shotgun synthesis from Ins
P 6
. Hereby, the phosphorylation was brought about nonenzymatically when concentrated Ins
P 6
bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated Ins
P 6
derivatives containing all theoretically possible isomers of
PP
Ins
P 5
, bis
PP
Ins
P 4
, and also some isomers of tris
PP
Ins
P 3
, isomers were separated by anion-exchange chromatography and fractions served as reference standards of bis
PP
Ins
P 4
isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or
NMR
-characterized purified protozoan reference compounds and partly by limited hydrolysis to
PP
Ins
P 5
isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bis
PP
Ins
P 4
in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.