Labeled Sphingomyelin Synthesis

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.

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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.

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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 chloroform
b) 150 ml acetone
c) 300 ml acetone/methanol (9:1)
d) 50 ml methanol
e) 100 ml methanol
f) 300 ml methanol
g) 300 ml methanol

16) 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.