PREPARATION OF THE CEREBROSIDES 477 



0=C(CH2)22CHj 



NH 

 H I 

 CH. CH,),,CH=CHC-C-CH- 

 I I I 

 OHH 

 I 

 HC 



HCOH 

 I 

 HOCH 



HCOH I 



HC 1 



I 

 CHzOH 



Gluoosidoperasine 



3. Preparation of the Cerebrosides 



(1) Thierf elder Method for the Preparation of Phrenosine^^^'^-^ 



After remcval of the blood and connective tissue from the brain, the 

 tissue is macerated and extracted in 2.5-kg. batches with 3 Hters of acetone. 

 This is allowed to stand at room temperature, with frequent shaking, for 24 

 hours after which it is filtered. This treatment is then repeated. The 

 residue, which is now largely dehydrated, is extracted with 1500 ml. of 

 ether for 24 hours, during which the flask is shaken frequently. This 

 extraction is repeated until the extract is no longer colored and a minimum 

 of the material is left after concentration of the filtrate. This usually 

 requires 4 or 5 successive extractions. About 350 g. of defatted tissue re- 

 mains (13-14% of the original). After this residue is pulverized, it is 

 heated with 5 volumes of 85% ethyl alcohol and is filtered hot. The crude 

 cerebroside separates on cooling. The filtrate is used for a second extrac- 

 tion of the tissue. Similar extractions are continued, making use of the 

 filtrate each time until no more cerebroside separates on cooling. The 

 precipitate is shaken with ether for the removal of any adherent ether- 

 soluble material, and is then separated by centrifugation. 



This so-called protagon is a snow-white mass which amounts to about 

 3-4% of the fresh brain (80-100 g.). For further purification it is dis- 

 solved in methanol containing 75% chloroform. It is readily soluble, and 

 100 g. will dissolve easily in 500 ml. of the solvent when it is warmed 

 slightly. After the protagon has dissolved, the solution is filtered and is 

 allowed to stand in a closed flask at room temperature. Within a day, a 

 hard thick crust forms on the surface. This is filtered off, the filtrate is 

 cooled, and the precipitate is filtered. The precipitates are recrystallized 

 from a fresh methyl alcohol-chloroform mixture. The mother liquor is 

 evaporated to dr^Tiess and the residue is crystallized from the same solvent. 

 The precipitates are combined and dissolved in 25-30 volumes of methanol 



*" H. Loening and H. Thierfelder, Z. phi/siol. Chem., 6S, 464-470 (1910). 

 «o F. Kitagawa and H. Thierfelder, Z. phi/siol. Chem., 49, 286-292 (1906). 



