Inositol Phosphate Synthesis and Analysis

The structures of all inositol phosphates and diphosphoinositol phosphates described in this study are numbered according to the 1d-numbering convention and are shown in Supplementary Figure S1.
InsP₆ was obtained from Merck Millipore (Product No. 407125). An acid-hydrolysate of phytate (Sigma P-8810) was prepared and used as chromatographic standard according to Madsen et al. [21 (link)]. All InsP₅ isomers used as substrate for ITPK1 were obtained from Sichem as decasodium salts. Ins(1,3,4,5,6)P₅ obtained from Sichem showed evidence of phosphate migration between cis-vicinal hydroxyls. Ins(1,3,4,5,6)P₅ used as substrate for IPK1 was synthesized according to published procedures [22 (link)] and did not show evidence of phosphate migration. InsP₄s and InsP₃s were obtained from Cayman Chemical Company or were synthesized according to published procedures [23 (link)]. Comparisons made between different enantiomers of enantiomeric pairs were performed with compounds obtained from one source only. Diphosphoinositol phosphates (Supplementary Figure S1): 1-InsP₇, 3-InsP₇, 5-InsP₇ and 5PP-Ins(1,3,4,6)P₄ were synthesized similarly to published procedures [24 (link)]. 4-InsP₇ and 6-InsP₇ were synthesized by Dr Henning Jessen, Institute of Organic Chemistry, and the Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany. Acid-catalyzed migration of phosphate on ³²P-labeled InsPs was performed according to Stephens and Downes [25 (link)]. ³²P-labeled InsPs were used directly for HPLC without processing to remove nucleotides.