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31P and I3C magnetic resonance spectra of fructose 2,6-bisphosphate, prepared as described by Van Schaftingen and Hers (preceding paper), and of commercial fructose 1,6-bisphosphate were obtained. Analysis of these spectra has confirmed the structure of fructose 2,6-bisphosphate; this ester was present as a single anomeric form to which the fl configuration could be assigned.Fructose 2,6-bisphosphate is the structure proposed by Van Schaftingen et al. [I] for a newly recognized stimulator of phosphofructokinase, present in rat liver. This phosphoric ester has recently been prepared in millimolar amount by conversion of fructose 1,6-bisphosphate into its cyclic 1,2-phosphodiester and alkaline hydrolysis of the latter [2]. The present investigation is an NMR analysis of this compound. MATERIALS AND METHODS Muter iulsFructose 2,6-bisphosphate was prepared as described in the preceding paper [2]. Fructose 1,6-bisphosphate was purchased from Boehringer (Darmstadt, FRG). Freshly prepared 0.05 M solutions of the tetrasodium salts in 'HzO were used. NMR Spectroscopy31P NMR spectra were obtained on a Varian XL-100 spectrometer operating at 40.5 MHz in Fourier transform mode. Peak positions of both proton-coupled and decoupled spectra are referenced to external 85 % phosphoric acid. Useful spectra were obtained at pH 10 and 30°C with 230 accumulations, 1.5 kHz spectral width, 4 K data points and 30" pulse.2°C on a Varian XL-200 NMR spectrometer operating at 50.3 MHz, in 5-mm-diameter tubes spinning in a 10-mm probe. The main spectrometer parameters were: spectral width 10 kHz; data points 32 K ; acquisition time 1.6 s; pulse angle 50"; repetition time 5.6 s; accumulation took 40 h. Chemical shifts were computed relative to an external dioxane sample and converted to the tetramethylsilane scale by using 6 (dioxane) = 67.4 ppm. Both proton broadband noise-decoupled and proton-coupled spectra were recorded.

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