Labeled Sphingomyelin Synthesis

Labeled Sphingomyelin SynthesisContributor: Suprya JayadevDate: Mar. 11, 1993 Synthesis reaction: 1) Mix purified DMSM with cyclohexylamine and 14CH3I at a ratio of 1/1.1/1.3 in 5 ml of methanol.2) Allow reaction to proceed at room temperature for atleast 18 hours in the dark.3) Check completion of reaction by running a few microliters of the reaction mix on TLC (solvent system = chloroform/methanol/ammonium hydroxide @ 60:35:8).4) Once reaction is complete, dry the mixture as much as possible using nitrogen gas.--> Should be left with a white precipitate in a small volume of oily, yellow residue.Washing reaction products:5) Dissolve the residue in 2 ml of chloroform and vortex.6) Add 1 ml of 5% Na2S2O3・5H2O (sodium thiosulfate) and vortex vigorously.7) Spin @ 3,000 rpm for 5 min and discard upper, aqueous phase in radioactive waste.8) Wash organic phase again with sodium thiosulfate.9) Wash organic phase from step (8) twice with 2 N HCl, discard upper phases in radioactive waste.10) Wash the acidified organic phase twice with H2O, again discard upper phases in radioactive waste.11) Dry down washed product and resuspend in 2 ml of chloroform.--> If a precipitate persists, a little methanol may be added to completely dissolve the material.Column purification of labeled SM:12) Pack a Bio-Sil A column as follows:a) Pack base of column with a small portion of glass wool.b) Suspend 20 grams of 100-200 mesh BioSil A in 100 ml chloroform.c) Pour silica/chloroform mixture into column eluting chloroform from base.- Press out any air bubbles in the glass wool as the gel is initially being poured. While pouring column matrix, stir continuously with a stir bar in order to prevent bubble formation in the column.d) Wash column through with "200 ml chloroform.- ALWAYS maintain a fluid level above the packing silica... NEVER let column dry out!!13) Carefully layer chloroform solution over packed column.14) Allow sample to run into column while maintaining a small level of chloroform above the packed silica.15) Sequentially elute with the following solvents:a) 150 ml chloroformb) 150 ml acetonec) 300 ml acetone/methanol (9:1)d) 50 ml methanole) 100 ml methanolf) 300 ml methanolg) 300 ml methanol16) Dry down fractions (d) - (g) separately and re-suspend in 4 ml of toluene/methanol (1:1).17) Spot 2 µl of each fraction and a small portion of DMSM & SM standard onto a TLC plate.18) Run TLC in chloroform/methanol/ammonium hydroxide (60:35:8) and develop spots using iodine vapor then potassium permanganate spray.--> Fraction (d) & (e) should contain unreacted DMSM, and fraction (f) should contain the labeled SM.19) Store labeled SM in -20°C freezer.