40 W. G. OVEREND AND M. STAGEY 



of the pentose phosphate afforded an optically active phosphoribitol, and 

 so position C-3 of the sugar molecule was excluded as the site of esterifica- 

 tion. In solution in methanolic hydrogen chloride the pentose phosphate 

 underwent mutarotation in a manner characteristic of a sugar which 

 can only form a furanoside.-'** Moreover the behavior of the pentose phos- 

 phate isolated from natural sources and a synthetic sample of D-ribose-5- 

 phosphate was identical. The synthetic material was prepared'" from d- 

 ribose by condensation with acetone and methanol in the presence of 

 hydrogen chloride and anhydrous copper sulfate (or alternatively dilute 

 sulfuric acid) followed by phosphorylation of the resultant syrupy methyl 

 2,3,-0-isopropylidene-D-ribofuranoside (XXXVI) with phosphorus oxy- 

 chloride and pyridine at —40°. Hydrolysis of the isopropylidene and 

 glycosidic groups from methyl 2,3-0-isopropylidene-D-ribofuranoside-5- 

 phosphate (XXXVII) yielded ribose-5-phosphate (XXXVIII) (as barium 

 salt). The structure of the important intermediate XXVI was demon- 

 strated by methylation and hydrolysis to an amorphous monomethylribose 

 (XXXIX) which was converted to a crystalline p-bromophenylosazone, 

 identical with the p-bromophenylosazone of authentic 5-0-methyl-D- 

 ribose which had been previously prepared--" (see p. 42). 



Using as phosphorylating agent dibenzylphosphorochloridate in drj^ 

 pyridine solution at —40°, Michelson and Todd'^° considerably improved 

 the synthesis. Protecting groups were removed from the intermediate XL 

 by hydrogenation and hydrolysis to give XXXVIII in 86% yield. 



An improved method for the preparation from muscle (horse, dog and 

 rabbit muscle are excellent sources) of inosinic acid and thence ribose-5- 

 phosphate has recently been described.--' Optimum conditions were deter- 

 mined for hydrolysis of the nucleotide to ribose phosphate which can be 

 obtained in a yield of 50-60% by use of this method. This phosphate 

 ester is also obtainable by acidic hydrolysis of cozymase.*^ Adenine and 

 nicotinamide are cleaved quantitatively while 20 % of the total phosphorus 

 is liberated. Adenine was removed as its silver salt and the ribose-5-phos- 

 phate was isolated as the barium salt. The isolation of this compound 

 served to identify the sugar moiety in cozymase and also the site at which 

 it was esterified by the phosphoric acid residue. Similar conclusions were 

 reached from less direct evidence by other investigators.^^ Ribose-5-phos- 

 phate has been obtained in a high degree of purity from adenosine tri- 

 phosphate.-^^ Purification was achieved by chromatography on ion-exchange 



218 P. A. Levene and M. L. Wolfrom, J. Biol. Chem. 77, 671 (1928). 



2" P. A. Levene, S. A. Harris, and E. T. Stiller, J. Biol. Chem. 105, 153 (1934). 



220 P. A. Levene and E. T. Stiller, J. Biol. Chem. 102, 187 (1933). 



221 J. Marmur, F. Schlenk, and R. N. Overland, Arch. Biochem. and Biophys. 34, 209 

 (1951). 



222 D. P. Groth, G. C. Mueller, and G. A. LePage, J. Biol. Chem. 199, 389 (1952). 



