Synthesis of Inositol Phosphodiesters by Phospholipase C-Catalyzed Transesterification

Abstract

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 β-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 1H and 31P 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.

Mechanism of Phosphatidylinositol-Specific Phospholipase C: A Unified View of the Mechanism of Catalysis,

“…The degree of alcohol deprotonation in the reverse reaction (which should be the same as the degree of protonation in the forward reaction) should depend on the alcohol pK a and the strength of the enzymic base. The externally added alcohol undergoes an exchange with an alcohol of a ternary complex of the products ( Figure 9) and uses the binding site formerly occupied by the leaving group alkoxide (10). The WT PI-PLC showed a small Brønsted coefficient of nuc ) -0.10 ( 0.02, somewhat lower than that obtained for reactions of RNase A with phenyl esters of uridine 3′-phosphate (-0.17 ( 0.03; 53).…”

“…Conversion of PI to IcP is 100-1000-fold faster than hydrolysis of IcP to IP depending on the conditions used (8,9). On the basis of the stereochemical course of the reaction and the X-ray structure of PI-PLC complex with myo-inositol, a mechanism involving general base-general acid catalysis, reminiscent of that of ribonuclease A, has been proposed for both steps (8,(10)(11)(12)(13) as …”

Phospholipases: Occurrence and production in microorganisms, assay for high‐throughput screening, and gene discovery from natural and man‐made diversity

“…The PI cleavage of bacterial PI-PLC constitutes an intramolecular phosphotransferase step that cleaves PI to inositol-1,2-cyclic phosphate and a cyclic phosphodiesterase step that converts inositol-1,2-cyclic phosphate to inositol-1-phosphate (62). Bruzik et al (57) reported the first enzymatic synthesis of inositol phosphodiesters starting from alcohols and inexpensive, readily available soybean phospholipids. The PI-PLC reactions were also successfully applied to the preparation of a number of complex inositol phosphoesters of mono-and oligosaccharides, nucleosides, and peptides.…”

Phospholipases as Synthetic Catalysts

“…PLC which hydrolyses phosphatidyl inositol (PLC PI ) [EC 3.1.4.10] can also be obtained from B. cereus and B. thuringiensis [92]. Application in the production of inositol phosphates has recently been reported [93].…”

“…Interestingly, it has recently been observed that Ca 2+ dependence in cabbage PLD is pH dependent [99]. In a recent work Bruzik et al [93] compare the mechanism of different phosphoesterases and their tendency to catalyze efficiently the transesterification reaction by dividing them into three groups (Scheme 4).…”