Inositol polyphosphates and analogues: synthesis & chemical biology
Keywords: IP8
inositol pyrophosphates
IP7

In this work, myo-inositol phosphates and pyrophosphates were investigated to facilitate further understanding of their chemical biology and biological functions. The synthesis of novel carbohydrate-based polyphosphate analogues of lower inositol phosphates, including the second messenger ᴅ-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], was undertaken, and novel routes for some naturally-occurring inositol pyrophosphates were designed.
Synthetic routes for the meso myo-inositol pyrophosphates 5-PP-InsP₅ (5-IP₇) and 5- PP-InsP₄, as well as both the racemic and chiral versions of D-1,5-(PP)₂-InsP₄ (D-1,5-IP₈), were successfully executed. These approaches incorporated the hitherto little-used methylsulfonylethyl (MSE)-protecting group, allowing its versatility to be explored. Final products were then used with collaborators to investigate: substrate binding to the yeast diphosphoinositol polyphosphate phosphohydrolase through a structural biology approach; the potential scope of a novel pyrophosphate-monitoring, europium-based luminescence assay; and structure-activity (SAR) aspects of the inositol 1,4,5,6-tetrakisphosphate binding site in the histone deacetylase 3 - SMRT corepressor (HDAC3-SMRT) complex.
The SAR investigation for HDAC3-SMRT featured seven glucose-based polyphosphate compounds (two known, five novel) that were designed, synthesised and biologically evaluated. The ʟ-glucose-based ligands were found to be the first carbohydrate-based ligands able to activate the complex, while their ᴅ-glucose-based enantiomers were inactive. A binding site region suspected to be able to accommodate steric bulk was explored, and it also was concluded that inositol pyrophosphates are unlikely to be physiologically relevant endogenous activators for the HDAC3-SMRT complex.
A further six novel glucose-based polyphosphates were synthesised to investigate calcium release via the Ins(1,4,5)P₃ receptor (InsP₃R). Two ligands, α-ᴅ-glucopyranosyl 1,3,4-trisphosphate and β-ᴅ-glucopyranosyl 1,3,4-trisphosphate, were full agonists, the latter equipotent to Ins(1,4,5)P₃ itself. These two ligands were also substrates for Arabidopsis inositol tetrakisphosphate kinase-1 (ITPK1). Although unconfirmed, it is proposed that ITPK1 phosphorylates the 6-position primary hydroxyl. Both ligands were uniquely co-crystallised with Ins(1,4,5)P₃ 3-kinase. The crystallographicallysolved ternary complexes also revealed the primary hydroxyl group to be well-situated for potential phosphorylation.
Two of the InsP₃R-study ligands, methyl α-ʟ-glucopyranoside 2,3,6-trisphosphate and methyl α-ʟ-glucopyranoside 2,4,6-trisphosphate, were low-affinity, low-efficacy partial agonists. From the presumed binding mode of the latter, structurally-inspired myo-inositol pyrophosphate-containing compounds were designed, targeting a potential higher-affinity partial agonist or antagonist. Using techniques earlier developed, three novel inositol pyrophosphates were synthesized, 4-PP-Ins(5)P, 5-PPIns(4)P and 4-PP-Ins(1,5)₂, which are the first examples of an Ins(1,4,5)P₃ pyrophosphate analogue modified at the 4,5-vicinal bisphosphate pharmacophore. Biological evaluation of these compounds is currently underway.