Synthesis of inositol phosphodiesters by phospholipase C-catalyzed transesterification

Transesterification of primary alcohols with inositol 1,2-cyclic phosphate (IcP) in the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the formation of O-alkyl inositol 1-phosphates. The starting IcP was obtained in a single step by PI-PLC catalyzed cleavage of phosphatidylinositol from the soybean phospholipid. The transesterification reaction was performed with a series of 20 structurally 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, depending mainly on the solubility of alcohols in water. The rates of transesterifications, and of the competing hydrolysis of IcP to inositol 1-phosphate (IP), were relatively insensitive to the alcohol structure. With polyhydroxyl compounds such as glycerol and hexitols, the enzyme displayed 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 environments and showed low stereoselectivity with racemic alcohols featuring a chiral center at the beta-position, The O-alkyl inositol phosphates formed were readily separable from the hydrolytic product, IP, by the anion-exchange chromatography, and were fully characterized by means 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 phosphate derivatives, as illustrated by inositol phosphoesters of mono- and oligosaccharides, nucleosides and peptides. The synthetic usefulness of this reaction, however, is not limited to the examples shown. Because transesterification activity of phospholipase C has not been reported before, its mechanism is discussed in a broad context of mechanisms of phosphoesterases.