492 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



member of the group referred to as ceramides. Since it gives a neutral 

 reaction, it is obvious that both the amino and the carboxyl groups must 

 be combined. The evidence indicates that a NH — CO union occurs, as in 

 the case of hgnocerylsphingosine. 



HO H 



CH3{CH2),2CH = CHC-C-CH20H 



HN-C-C CH,2,CHs 

 II I ' ' 



OH 



Cerebronj-l-iV-sphingosine 



b. Preparation of Cerebronyl- A^-Sphingosine. According to Klenk,'**^ 

 the ceramide can be prepared from phrenosine. 10 g. of the cerebroside 

 is dissolved by warming in a mixture of 270 ml. of acetic acid and 30 ml. of 

 10% sulfuric acid. The mixture is heated for 42 minutes over a boiling 

 water bath. On cooling, 500 ml. of water is added to the flask without any 

 attention being paid to the precipitate which has appeared, and enough 

 33% sodium hydroxide is introduced to render the reaction only slightly 

 acid. It is shaken with about 2 liters of diethyl ether, whereby the precipi- 

 tate is dissolved and the galactose which has been split off remains in the 

 aqueous layer. The ether solution is now shaken with an excess of dilute 

 sodium hydroxide solution, as a result of which a voluminous precipitate 

 forms. This is separated by centrifugation. There are 3 layers, i.e., the 

 aqueous layer on the bottom, the precipitate lying above, and the ether 

 layer on top. After removal of the clear ether layer (A), the precipitate 

 (B) can easily be removed from the alkali below. 



Most of the ceramide is present in the ether layer. After removal of the 

 ether by distillation, the residue (2.7 g.) is dissolved by warming in acetone. 

 The small amount of substance which fails to dissolve is removed by filtering 

 it hot. When the solution is cooled in the refrigerator, the cerebronyl-A^- 

 sphingosine crystallizes. In order to purify this product, it is dissolved in 

 15 ml. of hot methanol and a little lead acetate solution in methanol; some 

 ammoniacal methanol is added. It is heated and filtered while still hot. 

 To a great extent the ceramide separates from the methanol solution on 

 cooling. It is dissolved in acetone and the insoluble material is discarded. 

 The residue remaining after evaporation of the acetone is again dissolved 

 in methanol (15 ml.), and any remaining lead is removed with hydrogen 

 sulfide. The lead sulfide is filtered, and in case of necessity diatomaceous 

 earth (kieselguhr) is added, to remove any colloidal precipitate. The pre- 

 cipitate which originates on cooling is recrystallized from acetone. Klenk 

 obtained 0.57 g. from this fraction. About 0.9 g. was prepared from the 

 precipitate (B) by similar procedures. 



c. Properties of Cerebronyl-iV-Sphingosine. Cerebronyl- iV-sphingo- 

 sine is soluble in warm methanol and acetone; it is difficultly soluble in 



