SYNTHESIS OF INOSITOL PHOSPHODIESTERS BY PHOSPHOLIPASE C-CATALYZED TRANSESTERIFICATION

Transesterification of primary alcohols with inositol 1,2-cyclic phosp hate (IcP) in the presence of phosphatidylinositol-specific phospholip ase C (PI-PLC) resulted in the formation of O-alkyl inositol 1-phospha tes. The starting IcP was obtained in a single step by PI-PLC catalyze d cleavage of phosphatidylinositol from the soybean phospholipid. The transesterification reaction was performed with a series of 20 structu rally diverse hydroxyl compounds, ranging in the structural complexity from methanol to the serine-containing Ser-Tyr-Ser-Met tetrapeptide, to give the corresponding phosphodiesters with 20-80% yields, dependin g mainly on the solubility of alcohols in water. The rates of transest erifications, and of the competing hydrolysis of IcP to inositol 1-pho sphate (IP), were relatively insensitive to the alcohol structure. Wit h polyhydroxyl compounds such as glycerol and hexitols, the enzyme dis played complete preference toward formation of the inositol phosphate derivatives of the primary hydroxyl groups. On the other hand, PI-PLC did not discriminate between primary hydroxyl groups in different envi ronments and showed low stereoselectivity with racemic alcohols featur ing a chiral center at the beta-position, The O-alkyl inositol phospha tes formed were readily separable from the hydrolytic product, IP, by the anion-exchange chromatography, and were fully characterized by mea ns of H-1 and P-31 NMR and electrospray MS. Our results provide a new, simple, and efficient two-step synthetic route to substituted O-alkyl inositol phosphates from inexpensive starting materials. The reported reaction was successfully applied to synthesis of complex inositol ph osphate derivatives, as illustrated by inositol phosphoesters of mono- and oligosaccharides, nucleosides and peptides. The synthetic usefuln ess of this reaction, however, is not limited to the examples shown. B ecause transesterification activity of phospholipase C has not been re ported before, its mechanism is discussed in a broad context of mechan isms of phosphoesterases.