Synthesis of an α-phosphono-α,α-difluoroacetamide analogue of the diphosphoinositol pentakisphosphate 5-InsP7

Abstract

Diphosphoinositol phosphates (PP-InsPs) are an evolutionarily ancient group of signalling molecules that are essential to cellular and organismal homeostasis. As the detailed mechanisms of PP-InsP signalling begin to emerge, synthetic analogues of PP-InsPs containing stabilised mimics of the labile diphosphate group can provide valuable investigational tools. We synthesised 5-PCF₂Am-InsP₅ (1), a novel fluorinated phosphonate analogue of 5-PP-InsP₅, and obtained an X-ray crystal structure of 1 in complex with diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2). 5-PCF₂Am-InsP₅ binds to the kinase domain of PPIP5K2 in a similar orientation to that of the natural substrate 5-PP-InsP₅ and the PCF₂Am structure can mimic many aspects of the diphosphate group in 5-PP-InsP₅. We propose that 1, the structural and electronic properties of which are in some ways complementary to those of existing phosphonoacetate and methylenebisphosphonate analogues of 5-PP-InsP₅, may be a useful addition to the expanding array of chemical tools for the investigation of signalling by PP-InsPs. The PCF₂Am group may also deserve attention for wider application as a diphosphate mimic.

Fig. 1. A. Inositol hexakisphosphate (InsP₆) is phosphorylated on P-5 by IP6K to give 5-PP-InsP₅, also known as “5-InsP₇”. Further phosphorylation at P-1 by PPIP5K gives 1,5-[PP]₂-InsP₄, (“InsP₈”). B. Stabilised analogues of 5-PP-InsPP₅ containing methylenebisphosphonate (PCP), phosphonoacetate (PA) and phosphonodifluoroacetamide (PCF₂Am) mimics of the natural PP group. IP6K, inositol hexakisphosphate kinase; PPIP5K, diphosphoinositol pentakisphosphate kinase.

Scheme 1. Synthesis of 5-deoxy-5-amino-inositol 7. Reagents and conditions: a. see ref. 22; b. i. Tf₂O, CH₂Cl₂, pyridine, –78 °C to r.t.; ii. NaN₃, DMF, r.t., 86%; c. i. LiOH·H₂O, MeOH, THF, H₂O; ii. NaH, BnBr, DMF, 93%; d. i. CH₂Cl₂, 95% aqueous TFA; ii. NaH, BnBr, DMF, 91%; e. LiAlH₄, THF, 100%. Bn, benzyl.

Scheme 2. a. (EtO)₂P(O)CF₂COOH, EDAC, CH₂Cl₂, 87%; b. H₂, 50 p.s.i., Pd(OH)₂/C, MeOH, THF, AcOH, 100%; c. i. (BnO)₂PNⁱPr₂, 5-phenyl-1H-tetrazole, CH₂Cl₂; ii. mCPBA, 66%; d. i. TMSBr, CH₂Cl₂; ii. MeOH; iii. aqueous triethylammonium bicarbonate, 85%. Bn, benzyl.

Fig. 2. A. Inhibition of human PPIP5K2 by 5-PCF₂Am-InsP₅ (1) in the presence of 100 nM 1,5-[PP]₂-InsP₄. B. Crystallographic analysis of PPIP5K2 in complex with 1 and AMPPNP (PDB code ; 6N5C). C. The 2Fo–Fc electron density map of 1, contoured at 1.7σ, is shown as light blue mesh. PPIP5K2 is shown in cartoon and 1 is shown as a stick model, with carbon atoms coloured dark grey, phosphorus atoms orange, oxygen atoms red, nitrogen atom blue and fluorine atoms cyan. D. Another view of 5-PCF₂Am-InsP₅ (1) and its overlap with 5-PP-InsP₅, which is shown as white sticks. Panels E and F show the moieties on position 5 of 1 and the natural substrate 5-PP-InsP₅ (PDB code, ; 3T9D).