Stereoselective chemoenzymatic phytate transformations provide access to diverse inositol phosphate derivatives

Abstract

Phosphorylated myo-inositols (InsPs) are essential cytoplasmic signaling molecules, while their lipidated analogs (PtdInsPs) play a crucial role in membrane signaling. Stereoselective synthesis of these compounds has been achieved through various methods, predominantly using the meso compound myo-inositol as a starting material. However, phytate (InsP₆), also a meso compound, is the most abundant inositol derivative in plants - far more prevalent than myo-inositol itself. Despite its abundance, phytate has been rarely used in synthetic strategies for accessing a variety of chiral inositol phosphates and their derivatives through selective dephosphorylations on a preparative scale. Here, we report gram-scale (stereo)selective dephosphorylations of phytate using phytases and demonstrate the application of these products in generating modified InsPs through a transient phosphitylation approach. Notably, the bacterial effector XopH efficiently desymmetrizes meso-phytate to yield enantiomerically pure 1-OH-InsP₅. This transformation renders the 1-position accessible for further modifications, which, in biological systems, is where glycerolphosphate diesters are attached. By using selective dephosphorylations with phytases in concert with chemoselective telescoping reaction sequences, this approach greatly advances the stereoselective synthesis of inositol phosphates and their derivatives, such as glycerophosphoinositols, from abundant InsP₆.

Scheme 1. Desymmetrization of phytate via selective dephosphorylation using phytases. Previously, 1,2,6-InsP₃ was obtained by baker's yeast digest. Here, selective dephosphorylation with expressed XopH on a preparative scale was established. The resulting chiral 1-OH-InsP₅ was transformed into the corresponding chiral 1-InsP₁ chemoenzymatically with the phytase Natuphos, thus inverting the phosphorylation pattern.

Scheme 2. Synthesis of 1-OH-InsP₅2via selective dephosphorylation of InsP₆ with XopH.

Scheme 3. Synthesis of InsP₆ derivatives modified solely at the 1-position. CE-qTOF-MS (background electrolyte (in the following BGE): NH₄OAc 35 mM pH = 9.7, CE voltage: 30 kV, CE current: 23 μA, injection: 100 mbar, 15 s (30 nL)) analysis of the transient phosphitylation of 1-OH-InsP₅ using different P-amidites. The depicted structures are color coded by identical mass. (a) Using P-amidite 1 a complex mixture was obtained. (b) Reduction of components was achieved via controlled cyclization using cyclization prone P-amidite 9. The depicted cyclic pyrophosphates are just examples of possible structures. (c) The obtained cyclic intermediates were transformed into a single product in situ via subsequent acid treatment. (d) Using an optimized transient phosphitylation work-flow 1-Fm-InsP₆8 and 1-DEACM-InsP₆11 were synthesized in one telescoping reaction sequence starting from 1-OH-InsP₅.

Scheme 4. Dephosphorylation of InsP₆ derivatives with Natuphos (10500 U mL⁻¹, 3150 U was used for the digest of phytate, 2100 U was used for the digest of 8) in NH₄OAc (50 mM, pH 6.3) leads to defined InsP₁ isomers. 1,2-InsP₂ was obtained as dephosphorylation product of 1-Fm-InsP₆8 using the 6-Phytase from E. coli (7500 U mL⁻¹, 1 U phytase was used) in HEPES-buffer (50 mM HEPES, 10 mM NaCl, 5% glycerol, 2 mM DTT, 0.5 mM, MgCl₂, pH = 4.0; at 28 °C for 45 min) and subsequent basic deprotection.

Scheme 5. (a) Different InsP₆ (15 mM) were dephosphorylated with 6-phytase from Escherichia coli (7500 U mL⁻¹ at pH = 5.0, 1 U phytase was used) (used buffer: 50 mM HEPES, 10 mM NaCl, 5% glycerol, 2 mM DTT, 0.5 mM, MgCl₂, pH = 4.0; at 28 °C for 45 min). CE-qTOF-MS (BGE: NH₄OAc 35 mM pH = 9.7, CE voltage: 30 kV, CE current: 23 μA, injection: 100 mbar, 15 s (30 nL)). (b) Analysis revealed different InsPs as product mixtures, depending on the used InsP₆ derivative. 1-Fm-InsP₆8 was relatively cleanly dephosphorylated to an InsP₂ derivative. (c) The formed 1,2-InsP₂ was identified via CE-QQQ-MS (BGE: NH₄OAc 35 mM pH = 9.7, CE voltage: 30 kV, CE current: 23 μA, injection: 100 mbar, 10 s (20 nL)) spiking experiments using a [¹³C]-InsP₂ mix (obtained via decomposition of [¹³C]-InsP₆ at 100 °C), after basic deprotection (piperidine (10 vol%)) of 1-Fm-InsP₂18.

Scheme 6. Synthesis of 1-GroPIns 21 and the non-natural 2-GroPIns derivative 20via Co(iii) catalyzed epoxide ring opening of S-(−)-glycidol with InsP₁ isomers.