Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate

Abstract

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)