Team:Evry Paris-Saclay/HP/Annexe/Hichem

IGEM Evry Paris-Saclay



Experimental protocol


D-Fructose (125 g) in ethanol (750 mL) and triethylamine (25 mL) was boiled for 15 h under reflux, a modification of one method given by [1]. The solution was evaporated to a syrup, and this was dissolved in water (500 mL), and the solution successively washed with chloroform (500 mL) and diethyl ether (400 mL), stirred with charcoal for 1 h, the suspension was filtered, and the filtrate deionized by stirring with Dowex-50 (H+) resin (100 mL) for 1 h; the suspension was filtered, and the filtrate was evaporated to a syrup. This was dissolved in 3:7 methanol:water (150 mL), and the solution was chromatographed in three portions (50 mL each) in a column (4.5 x 40 cm) of Dowex AG-SOW X-2 (Ca2+) resin (200-400 mesh) with 3:7 methanol:water as the eluant, 50-mL fractions being collected. The fractionation was monitored by t.l.c. on plastic sheets precoated with PE1-Cellulose F, the eluent being 8:6:3:3 tert-butanol:butanone-88 %:formic acid:water, and the plates were sprayed with 1% p-anisidine hydrochloride in ethanol, and developed in an oven for 10 min at 95°C. Standards of D-psicose, D-fructose, and D-glucose were available.

Fractions containing mainly D-psicose were pooled, and evaporated to a syrup; this was dissolved in water (35 mL), and the solution chromatographed on the afore-mentioned column, using water as the eluant, the column having been well eluted with water (7 L) prior to the fractionation. The appropriate fractions were pooled, evaporated in vacuo, and freeze-dried for 24 h, to give D-psicose (10.0 g, 8 %). The 13C NMR spectrum was the same as that of D-psicose synthesized by the procedure of [2] and was essentially identical to that published by Que and Gray [3]. The positions of the resonances were the same, but the intensities of the peaks differed slightly. The equilibrium composition of D-psicose in solution contains four cyclic forms (anomers of the pyranose and the furanose) in substantial proportions, and these vary with the temperature: [α]22 D = +2,9° (c 1, water); lit. [α] D = +3,1°. Paper chromatography for 70 h on Whatman No.1 paper in 3:1:1 (v/v) lbutanol:ethanol:water, or in the eluant mentioned earlier, showed a single spot, identical with that given by standard D-psicose.

We estimated the total cost of the synthesis to 696$.


This experiment was tried in ICMMO laboratory.

The mixture was heated under reflux during 15h. The mixture was cooled, remaining a solid and a liquid layer. This 2 batches were analyzed by NMR spectroscopy and we did identified any characteristic signal of psicose but we identified in the solid and in the liquid layer mannose, glucose and fructose.

In conclusion we can suppose the chemical production of Psicose is not the solution because we have low yield or the epimerization does not work, it is perhaps for this reason why this method is not described and the research are stopped early.

References


  • [1] Doner WL. Isomerization of d-fructose by base: Liquid-chromatographic evaluation and the isolation of d-psicose. Carbohydrate Res (1979) 70, 209-216.
  • [2] Tipson et al. [2] Que and Gray

Acknowledgements


Karen Becherel1, Dr. Aurélien Alix2, Dr. Geneviève Estenne-Bouhtou1, Dr. Pierre Alexandre Driguez1, Eric Nicolaï1, Dr. Alain Fournier1 and Prof. David Bonnaffé2

  • 1 Sanofi-Aventis SA
  • 2 Institute of Molecular Chemistry and Materials of Orsay (ICMMO), Univ. Paris-Sud, University Paris-Saclay

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